A Novel Program of Ubiquitination Remodels the Erythroid Proteome During Terminal Differentiation
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Citation Nguyen, Anthony Tuan. 2016. A Novel Program of Ubiquitination Remodels the Erythroid Proteome During Terminal Differentiation. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
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A novel program of ubiquitination remodels the erythroid proteome
during terminal differentiation
A dissertation presented
by
Anthony Tuan Nguyen
to
The Division of Medical Sciences
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
in the subject of
Cell Biology
Harvard University
Cambridge, Massachusetts
April 2016
© 2016 Anthony Tuan Nguyen
All rights reserved.
Dissertation Advisor: Professor Daniel Finley Anthony Tuan Nguyen
A novel program of ubiquitination remodels the erythroid proteome
during terminal differentiation
Abstract
The ubiquitin-proteasome system was initially discovered in reticulocytes, which undergo massive and rapid proteome remodeling. During terminal differentiation, hundreds of generic constituents of the cell undergo programmed elimination. However, the mechanisms that drive the turnover of normally stable proteins remain largely unknown. Two decades ago, an unusually large ubiquitin-conjugating enzyme, Ube2O, was found to be strongly and specifically upregulated in terminally differentiating reticulocytes, contemporaneously with the induction of globin. A null mutation in the murine Ube2O gene, known as hem9, resulted in a hypochromic, microcytic anemia, suggesting that Ube2O may be a major ubiquitinating factor in erythropoiesis.
To understand the role of Ube2O in terminal differentiation, we first found that all major low molecular weight ubiquitin-protein conjugate bands are greatly reduced in levels in hem9 reticulocytes. When null reticulocyte lysates were treated with recombinant Ube2O, ribosomal proteins were overwhelmingly the major class of targets.
Accordingly, hem9 reticulocytes have elevated ribosomal protein levels and 80S ribosomes. This phenotype of elevated ribosome abundance was accounted for by a defect in the elimination of ribosomes. Furthermore, overexpression of Ube2O was sufficient to drive ribosomal degradation in non-erythroid 293 cells. Quantitative mass spectrometry on these cells confirmed the destabilization of ribosomal proteins and indicated that Ube2O has a specific, yet broad ubiquitination program.
iii Interestingly, the hem9 defect was phenocopied by treating wild-type reticulocytes with proteasome inhibitors. To confirm this finding, we reconstituted the degradation of ribosomal proteins in a cell-free reticulocyte lysate system. We also reconstituted the ubiquitination of purified ribosomes by recombinant Ube2O in vitro, and the degradation of several specific ribosomal proteins with purified proteasomes. Next, we found that the initiation factor eIF2α is hyperphosphorylated in hem9 reticulocytes, suggesting a global inhibition of protein synthesis. This was independent of HRI, a dominant regulator of translation in reticulocytes; instead, another eIF2α kinase, GCN2 was activated in the null mutant. Consistent with these findings, null reticulocytes were deficient in free amino acids, a phenotype that was recapitulated by proteasome inhibition.
In summary, Ube2O selectively ubiquitinates ribosomal proteins and targets them to the proteasome for degradation, thus playing a central role during terminal erythroid differentiation.
iv Table of Contents
Abstract iii
Acknowledgements vii
Chapter 1: Introduction 1
1.1 The ATP-dependent proteolytic system of reticulocytes 2
1.2 Selective upregulation of Ube2O during terminal erythroid differentiation 6
1.3 Elimination of mitochondria from reticulocytes by autophagy 12
1.4 Interrelated nature of the ubiquitin pathway and the ribosome 15
1.5 Limitations in our understanding of the UPS in the reticulocyte 22
1.6 Hypochromic, microcytic anemic phenotype of the Ube2O null mouse 24
1.7 Summary and significance 26
Chapter 2: Ubiquitination of ribosomal proteins by Ube2O 29
Background 30
Results 32
Discussion 50
Materials and Methods 55
Chapter 3: Ube2O drives the elimination of 80S ribosomes from cells 63
Background 64
Results 66
Discussion 85
Materials and Methods 89
Chapter 4: Ribosomal degradation is proteasome-dependent 96
Background 97
Results 99
Discussion 117
Materials and Methods 122
v Chapter 5: Crosstalk between ribosomal degradation and translation in reticulocytes 127
Background 128
Results 130
Discussion 145
Materials and Methods 150
References 154
vi Acknowledgements
First, I would like to thank my thesis advisor, Dr. Daniel Finley, for his mentorship and support during my graduate studies. Through Dr. Finley’s guidance, I have developed the necessary skills of an independent scientist: rigorous scientific pursuit, effective oral and written communication, and mentorship of students. This would not have been possible without the scientific freedom that Dr. Finley has afforded me with this project.
I would also like to thank my co-sponsor, Dr. Mark Fleming, for his guidance and support during these last four years. Dr. Fleming has served as the consummate physician scientist role model. Dr. Fleming’s expertise on inherited anemias and mouse models to interrogate these diseases have been uniquely complementary to Dr. Finley’s expertise on the biochemistry and cell biology of ubiquitination.
I would like to thank Miguel Prado, a postdoctoral fellow who joined the Finley lab at the same time as I did. Miguel performed the mass spectrometry analysis for this project, and has served as a trusted and supportive friend during my graduate studies.
I also extend my appreciation to Mingwei Min, a postdoctoral fellow, and Geng Tian, an instructor in the lab, who have helped with the in vitro biochemistry of this project. I greatly appreciate the help that they have provided over the years.
I would like to thank Verena Dederer and Mona Kawan, who were my two diploma
(Master’s) students from the University of Stuttgart. I look forward to seeing their progress as they now pursue their PhDs.
I would like to especially thank Stefanie de Poot, Monica Boselli, and Sharon Hung, who are members of the Finley lab, for their careful proofreading and thoughtful comments on my thesis.
vii I would also like to thank all of the current and past members of the Finley lab, including
Byung-Hoon Lee and Suzanne Elsasser, who have contributed to the positive lab environment throughout my stay.
I would like to thank the members of the Fleming lab, including Dean Campagna, Paul
Schmidt, Anoop Sendamarai, and Daniel Lichtenstein. They have served as an important reservoir of knowledge about animal procedures and mouse genetics.
I would like to thank my dissertation advisory committee (DAC), Professor David Golan,
Professor Randy King, and Professor Benjamin Ebert. Even with their busy schedules, they have offered their time to read my DAC reports and to provide insightful suggestions during my DAC meetings.
Lastly, I would like to express special gratitude to my family. I would like to thank my fiancé, Vina Pulido, who has been a constant source of support and enthusiasm throughout. One unexpected benefit of my time away from medicine was meeting Vina, and I could not be happier. Finally, I would like to thank my parents, Tanh and Tuyetmai, and my siblings, Catherine and Andrew, for their support and encouragement.
viii
CHAPTER ONE
Introduction
1.1 Discovery of the ubiquitin proteasome system in the reticulocyte
1.1.1 The ATP-dependent proteolytic system of reticulocytes
The ubiquitin-proteasome system (UPS) is among the most complex in nature, known to be involved in cell-cycle control, innate immunity, DNA repair, and signal transduction
(Komander and Rape, 2012). The UPS was initially discovered in the reticulocyte, an enucleated precursor of the mature red blood cell (Ciechanover et al., 1980; Ciehanover et al., 1978). It was first observed that rabbit reticulocytes rapidly degrade both abnormal amino acid-analog containing hemoglobin (Rabinovitz and Fisher, 1964) and mutated or unassembled hemoglobin chains in sickle cell anemia and thalassemias (Carrell and
Lehmann, 1969; Huehns and Bellingham, 1969). Since reticulocytes do not contain any lysosomes, the degradation of hemoglobin was hypothesized to occur via a nonlysosomal pathway. This novel pathway was first isolated and characterized in a cell- free system, demonstrating the existence of an ATP-dependent proteolytic system in the reticulocyte (Etlinger and Goldberg, 1977).
Further studies in the reticulocyte resolved the UPS into two complementary components: Fraction I containing a small, heat-stable protein, which was later identified as ubiquitin (Wilkinson et al., 1980), and Fraction II containing the proteolytic activity
(Ciehanover et al., 1978). Ubiquitin is a highly conserved, 76 amino acid eukaryotic protein that had been previously purified during the isolation of thymopoietin (Goldstein,
1974). In the reticulocyte, multiple ubiquitin moieties are covalently conjugated onto the proteolytic substrate in an ATP-dependent manner, prior to substrate degradation
(Ciechanover et al., 1980; Hershko et al., 1980). Thus, in sharp contrast to lysosomal degradation, the UPS appeared to be highly specific and selective in its nature. At the time, histone 2A (H2A) was the first substrate known to be modified with ubiquitin;
2 however, H2A was modified with only one ubiquitin moiety, and the ubiquitination did not affect protein stability (Goldknopf and Busch, 1977). Characterization of this isopeptide bond showed that it was between the C-terminal glycine of ubiquitin and the ε-NH2 group of lysine 119 on H2A (Hunt and Dayhoff, 1977). Though the monoubiquitination of histones would be later implicated in the epigenetic control mechanisms of transcription
(Weake and Workman, 2008), understanding of the chemical structure of the ubiquitin bond allowed for further dissection of the ubiquitin conjugation pathway and ubiquitin- mediated protein degradation. Finally, this degradative system was highly active in the breakdown of endogenous proteins in reticulocytes, independently of lysosomal function
(Boches and Goldberg, 1982; Muller et al., 1980).
1.1.2 The Ubiquitination Cascade
Ubiquitination of substrate proteins is mediated by the E1-E2-E3 cascade of enzymes
(Finley et al., 2012). First, the E1 ubiquitin-activating enzyme forms a high-energy thioester bond with the C-terminal glycine of ubiquitin upon ATP hydrolysis. E1 was first purified from reticulocyte lysate using an affinity-based approach with ubiquitin-
Sepharose; moreover, E1 was found to bind ubiquitin through a covalent thioester linkage (Ciechanover et al., 1982; Ciechanover et al., 1981). The activated ubiquitin is then transferred from the E1 to an E2 ubiquitin-conjugating enzyme by transesterification. The first E2s were also purified from reticulocyte lysate using a ubiquitin column in the presence of E1 and ATP, and could be eluted using a thiol compound (Hershko et al., 1983). Finally, E3 ubiquitin ligases catalyze the transfer of the activated ubiquitin from the E2 to the substrate protein. The first E3 was also isolated using a ubiquitin affinity column, but was found to bind non-covalently, in contrast to E1 and E2s (Hershko et al., 1983).
3 There are three families of E3 ubiquitin ligases: the RING E3s, HECT E3s, and RBR
E3s. The most common family of E3 ligases, the RING E3s, contain a RING domain, which binds to an activated E2 and catalyzes the transfer of ubiquitin onto a substrate protein (Deshaies and Joazeiro, 2009). In contrast, HECT E3s contain an active site cysteine that forms an obligate and transient thioester intermediate with ubiquitin prior to substrate ubiquitination. Finally, RBR E3s function as a RING-HECT hybrid with a canonical RING domain (RING1, which docks an E2) and a HECT-like RING-2 domain with an active site cysteine (Spratt et al., 2014). In a cell, there are typically one or two
E1s, multiple E2s, and a large family of E3s (Finley et al., 2012); this hierarchical organization confers increasing selectivity and specificity to the ubiquitination machinery of the cell.
Ubiquitin is most commonly conjugated to lysine residues on the substrate protein, though ubiquitination has been also reported on the N-terminal amino group (Ben-
Saadon et al., 2004), and on cysteine, threonine or serine residues (Cadwell and
Coscoy, 2005; Ravid and Hochstrasser, 2007; Wang et al., 2007b). Substrate proteins can be ubiquitinated on a single residue, known as monoubiquitination, monoubiquitinated on several residues, known as multi-monoubiquitination, or polyubiquitinated on a single lysine residue, forming a polyubiquitin chain. In a polyubiquitin chain, all seven lysine residues on ubiquitin (K6, K11, K27, K29, K33, K48, and K63) can accept the ubiquitin isopeptide bond, resulting in varying chain topologies
(Komander and Rape, 2012). Moreover, multiple lysines on the same ubiquitin molecule can accept a ubiquitin isopeptide bond, forming branched polyubiquitin chains (Meyer and Rape, 2014). While mono- and multi-monoubiquitination are thought to mediate proteasome-independent functions like subcellular localization or intracellular trafficking
(Ziv et al., 2011), there is increasing evidence that multi-monoubiquitination also serves
4 as a signal for canonical proteasomal degradation (Dimova et al., 2012; Kravtsova-
Ivantsiv et al., 2009).
1.1.3 The Proteasome
Upon ubiquitination, substrate proteins are targeted to a multi-subunit protease complex known as the proteasome for degradation (Finley et al., 2016). The 26S proteasome is the most complex protease in nature with hundreds of substrates and regulatory functions. This 2.5 MDa complex is comprised of a 19-subunit regulatory particle (RP or
19S subunit) that recognizes ubiquitinated proteins, removes the ubiquitin, and translocates the substrate protein into the proteolytic chamber of the 28-subunit core particle (CP or 20S subunit). The RP contains ubiquitin receptors to recognize substrate proteins and AAA-family ATPases that translocate the target protein upon ATP hydrolysis. This ATP-dependence was initially observed when characterizing the protease found in Fraction II of reticulocyte lysate; ATP was required not only for ubiquitin conjugation, but also for substrate degradation (Hershko et al., 1984; Tanaka et al., 1983). Another key component of the RP is Rpn11, an ATP-dependent deubiquitinating enzyme that cleaves ubiquitin chains en bloc, which recycles ubiquitin prior to substrate degradation. The mammalian proteasome also contains two other
ATP-independent deubiquitinating enzymes, Usp14 and Uch37, which have been shown to modulate proteasome function.
The 26S proteasome was first purified from rabbit reticulocytes and was shown to degrade ubiquitin conjugates (Waxman et al., 1987). Subsequently, the 20S CP was purified along with the 26S complex (Hough et al., 1987), and was found to be similar to the “multicatalytic proteinase complex” previously identified in the bovine pituitary gland
(Wilk and Orlowski, 1980). This barrel-shaped chambered 20S subunit consists of four
5 stacked heteroheptameric rings with α subunits on the outer rings and β subunits on the inner rings (Finley et al., 2016). Three of the seven β subunits, β1, β2, and β5, are threonine proteases and contain the catalytic activity of the CP. β1 cleaves after acidic residues, β2 after basic residues, and β5 after hydrophobic residues. The breadth of specificities ensures that substrates are rapidly degraded upon translocation into the CP.
To prevent aberrant degradation, the highly conserved N-termini of the α subunits occlude the translocation chamber of free CP. RP binding to CP opens the gate and relieves autoinhibition of the CP (Finley et al., 2016).
1.2 Selective upregulation of Ube2O during terminal erythroid differentiation
1.2.1 Identification of an unusually large E2 in reticulocytes
Ube2O is an E2 ubiquitin-conjugating enzyme that is highly and selectively upregulated in the reticulocyte stage of erythroid terminal differentiation (Wefes et al., 1995). Ube2O was first identified in reticulocytes in a study of declining E2 levels during erythroid maturation. Initially termed E2L for its unexpectedly large molecular weight (Pickart and
Vella, 1988), this protein was later renamed E2-230K, referring to its apparent size of
230 kDa on SDS-PAGE (it is in fact 141 kDa) (Pickart et al., 1989). Enzymes of the E2 class are defined by the ability to form a labile ubiquitin adduct in the presence of E1 ubiquitin-activating enzyme (Hershko et al., 1983). Accordingly, Ube2O was first identified in a ubiquitin adduct formation assay with the addition of E1 and ATP (Pickart and Vella, 1988); moreover, purified Ube2O from rabbit reticulocytes could monoubiquitinate cytochrome c with the assistance of only E1 (Klemperer et al., 1989).
Analysis of the human Ube2O cDNA also predicted a conserved 56 amino acid residue
UBC domain with an active-site cysteine that is shared by all E2 enzymes (Yokota et al.,
2001).
6 Like other E2s, Ube2O was able to ubiquitinate small, basic proteins including histone
H3, lysozyme, human and bovine α-lactalbumin, and α-casein without an E3 ligase
(Klemperer et al., 1989). However, Ube2O was only slowly inactivated by iodoacetamide, which rapidly inactivates other E2s by alkylating the active site cysteine
(Klemperer et al., 1989). Moreover, Ube2O did not cross-react with antibody to the yeast
RAD6, which recognized several other reticulocyte E2s (Berleth and Pickart, 1990).
Though Ube2O shared many features of the canonical E2, it was also fascinatingly unusual in its size and biochemical behavior.
Additional mechanistic studies further differentiated Ube2O from other E2 enzymes. E2 enzymes serve as the ubiquitin donor in E3-catalyzed ubiquitination. At the time, E2-14K was the only erythroid E2 known to act in protein degradation (Haas and Bright, 1988;
Pickart and Rose, 1985). Unlike E2-14K, Ube2O was unable to effectively reconstitute
E3-catalyzed degradation of human α-lactalbumin (Klemperer et al., 1989), suggesting that Ube2O does not function in ubiquitin-dependent protein degradation of the model substrates under study, but possibly in a regulatory role. To further study its mechanism of action, histone H2B was established as a gold-standard model substrate for Ube2O
(Berleth and Pickart, 1996). In a pulse-chase assay, the entire pool of Ube2O-ubiquitin adduct efficiently transferred its loaded ubiquitin onto histone H2B; in the absence of substrate, Ube2O also showed prominent autoubiquitination activity (Berleth and Pickart,
1996). At longer times in the chase, histone H2B was progressively monoubiquitinated, suggesting that product dissociation is rate-limiting for steady-state turnover, leading to increased processivity of this E2 (Berleth and Pickart, 1996).
Moreover, Ube2O was inactivated by arsenite in a time- and concentration-dependent manner (Klemperer et al., 1989). Since arsenite is specific for vicinal sulfhydryl sites in proteins (Stevenson et al., 1978), this observation suggested that Ube2O may contain
7 two essential cysteine sites. Arsenite sensitivity was not observed in other E2s, which characteristically have only one active site cysteine. In addition, phenylarsenoxides, which also bind to vicinal thiols, were shown to inhibit Ube2O-ubiquitin adduct formation without affecting turnover of preformed Ube2O-ubiquitin adducts (Berleth and Pickart,
1996). This led to the model that Ube2O functions as an E2-E3 hybrid with two cysteine residues that form sequential thiol esters with ubiquitin. Finally, though Ube2O was slowly resistant to iodoacetamide inhibition, Ube2O was rapidly inactivated by another hydrophobic alkylating agent, N-ethylmaleimide (NEM) (Berleth and Pickart, 1996), suggesting a relatively hydrophobic active site.
1.2.2 Ube2O is highly and selectively induced in the reticulocyte lineage
The temporal expression of Ube2O was first studied in murine erythroleukemia (MEL) cells, which are stably transformed with the Friend erythroleukemia virus, arresting the cells at a proerythroblast-like stage (Friend et al., 1971). Upon addition of DMSO or other inducers, MEL cells undergo erythroid differentiation, allowing access to stages of differentiation between the proerythroblast and early reticulocyte (Parker and Housman,
1985; Patel and Lodish, 1987; Volloch and Housman, 1982). However, very low levels of
Ube2O were detected in undifferentiated MEL cells; moreover, the behavior of Ube2O during MEL cell differentiation varied in a qualitative manner dependent on FBS batches, unlike the behavior of other E2 proteins (Haldeman et al., 1995). This variability of
Ube2O expression suggested that Ube2O induction is specific to a late stage of erythropoiesis that is not routinely accessed in differentiating MEL cells, and that MEL cells were likely unable to differentiate into actual reticulocytes.
Consequently, murine splenic erythroblasts infected with the anemia-inducing strain of
Friend virus (FVA) cells were used to better study ubiquitination in the terminal stages of
8 erythropoiesis. FVA cells provide a more homogenous population of erythroid progenitors that can differentiate to the reticulocyte stage after 48 hours of erythropoietin treatment (Koury et al., 1984; Sawyer et al., 1987). Using FVA cells, Ube2O mRNA was induced ~16- to 17-fold after 48 hours of erythropoietin treatment (Wefes et al., 1995). In fact, the rate of Ube2O mRNA induction closely paralleled that of globin reaching equivalent levels to that in reticulocytes after 48 hours of erythropoietin treatment (Wefes et al., 1995). GATA1, a key erythroid transcription factor, binds the promoter of the
Ube2O gene (Li et al., 2013), consistent with the finding that it is induced in parallel with globin. Interestingly, another E2, Ube2H (a.k.a E2-20K), was also sharply upregulated
(~16-fold) during erythropoietin treatment of FVA cells. In contrast, two other E2s, E2-
25K and UbcH5, were downregulated during terminal differentiation (Wefes et al., 1995).
The tissue specificity of Ube2O has also been studied. Ube2O was found at very high levels in reticulocytes, and absent or in very low levels in other cell types (Wefes et al.,
1995). This was consistent with Northern blotting for Ube2O mRNA in various tissues;
Ube2O was preferentially detected in reticulocytes compared to other cell types including bone marrow and spleen, which contain early erythroid progenitors (Wefes et al., 1995). Interestingly, Ube2O could not be detected in mature erythrocytes, indicating the transient nature of Ube2O induction. As such, Ube2O appears to function solely in terminal differentiation rather than in circulating red blood cells. Another study reported the high expression of Ube2O mRNA in skeletal muscle and heart (Yokota et al., 2001), which are two highly perfused tissues; however, reticulocytes were not used in this study as a positive control. Ube2O was also reported as a highly abundant protein in the vitreous proteomes of idiopathic epiretinal membranes, though Western blots of Ube2O in these tissues did not correlate with the high abundance observed by mass spectrometry (Yu et al., 2014).
9 As previously mentioned, Ube2H is another E2 that is highly induced during FVA cell induction with erythropoietin (Wefes et al., 1995). As such, Ube2H protein and mRNA were also found specifically in differentiating reticulocytes, but not in mature erythrocytes or other various tissues. Similar to Ube2O, Ube2H was also non-reactive to antibody to yeast RAD6 (Berleth and Pickart, 1990). In contrast, E2-25K mRNA was downregulated during FVA cell differentiation. Consistent with this, while E2-25K was present in high levels in most tissues, E2-25K was found in low levels in reticulocytes and mature erythrocytes. Decades later, global transcriptome analyses of human and mouse terminal erythropoiesis confirmed that both Ube2O and Ube2H are highly induced during late stages of erythroid differentiation (An et al., 2014; Shi et al., 2014; Wong et al.,
2011). Moreover, while the vast majority of ubiquitin-proteasome related genes are downregulated, there are a few specific E2s and E3s that are upregulated. These dynamic changes in gene expression suggested a programmed global rewiring of the specificity of the UPS during terminal erythroid differentiation.
1.2.3 Possible functions of Ube2O
Studies on Ube2O were published sparingly for nearly two decades until Sanderson and colleagues published a paper on the human E2 ubiquitin conjugating enzyme protein interaction network (Markson et al., 2009). Yeast two-hybrid screens were used to determine the specificity and combinatorial nature of the human E2/E3-RING network.
First, to validate their approach, 39 annotated human E2 proteins including Ube2O, and five E2-like proteins and five transcriptional variants, were screened against two high- complexity human libraries from fetal brain and from K562 cells (Markson et al., 2009).
15 reproducible positive interactions were identified for Ube2O; of note, four ribosomal proteins were identified, RPL29, RPL12, RPS7, and RPS20 (Markson et al., 2009).
Recently, high-throughput affinity-purification mass spectrometry further identified
10 ribosomal proteins RPL35, RPL15, and RPL18 as novel interactors of Ube2O (Huttlin et al., 2015). Of note, all previous studies on Ube2O were performed in post-ribosomal
Fraction II cell lysates, and Ube2O was assayed for in vitro degradation of only human
α-lactalbumin (Klemperer et al., 1989).
Next, ~5700 potential E2/E3-RING combinations were assayed in yeast two-hybrid screens, identifying 557 reproducible positive interactions (Markson et al., 2009). The
E3-RING proteins TRIM27 and RNF10 were identified as the only positive interactors with Ube2O. TRIM27 was recently shown to bind the ubiquitin ligase enhancer MAGE-
L2, and localize to endosomes containing the retromer protein complex (Hao et al.,
2013). On the endosome, MAGE-L2-TRIM27 was shown to ubiquitinate the regulatory
WASH complex. Knockdown of Ube2O impaired retrograde endosomal transport with similar penetrance as knockdown of TRIM27 (Hao et al., 2013). Though Ube2O appears to function with MAGE-L2-TRIM27 in supporting retrograde endosomal transport, ubiquitination of the regulatory WASH complex was not directly Ube2O-dependent.
Three additional studies were recently published on possible functions of Ube2O. First,
Ube2O was identified as a negative regulator of NF-κB activation by inhibiting polyubiquitination of the tumor necrosis factor-associated factor 6 (TRAF6) (Zhang et al.,
2013b). In HEK293T cells, overexpressed Ube2O co-immunoprecipitated with TRAF6.
Moreover, overexpression of Ube2O inhibited TRAF6 K63-polyubiquitination, blocked the interaction between TRAF6 and the adaptor protein MyD88, and impaired NF-κB activation upon LPS stimulation (Zhang et al., 2013b). Notably, TRAF6 regulation was independent of Ube2O catalytic activity. Second, Ube2O was shown to potentiate bone morphogenetic protein 7 (BMP7) signaling by monoubiquitinating the inhibitory SMAD6
(Zhang et al., 2013a). Ube2O co-immunoprecipitated with and monoubiquitinated
11 SMAD6. Since SMAD6 is inhibitory, Ube2O potentiated BMP7-induced SMAD signaling, and promoted BMP6-induced osteoblast-like cell differentiation in C2C12 cells (Zhang et al., 2013a). However, the role of SMAD6 ubiquitination by Ube2O in physiological adipogenesis is unclear. Finally, overexpressed Ube2O was shown to multi- monoubiquitinate the nuclear localization signal of BAP1, which promoted its cytoplasmic sequestration (Mashtalir et al., 2014). Overexpression of Ube2O also promoted adipocyte differentiation in the 3T3-L1 cell line, and resulted in the exclusion of a fraction of BAP1 from the nucleus (Mashtalir et al., 2014). While Ube2O and BAP1 appear to antagonize each other, Ube2O and BAP1 are located in entirely different cellular compartments, cytoplasmic and nuclear, respectively. Though these studies have elucidated some functions of Ube2O, the physiological role of Ube2O, especially in terminal erythropoiesis, remains unknown.
1.3 Elimination of mitochondria from reticulocytes by autophagy
1.3.1 Nix is essential for mitophagy in reticulocytes
During the switch from oxidative to glycolytic metabolism in terminal erythroid differentiation, mitochondria are eliminated from reticulocytes. The presence of mitochondria in mature erythrocytes would compromise the oxygen-carrying function of the red cell. The elimination of mitochondria from differentiating reticulocytes has been characterized in some detail. Initial work on mitochondrial elimination looked at the induction of the BCL2 family of proteins in response to erythropoietin stimulation, namely, the induced expression of the antiapoptotic protein BCX-XL (Socolovsky et al.,
1999) and the concurrent transcriptional upregulation of the proapoptotic protein Nix
(also known as BNIP3L) (Aerbajinai et al., 2003). Since BCX-XL can bind to and inhibit
Nix (Imazu et al., 1999), Nix was hypothesized to be a negative regulator of
12 erythropoiesis. Accordingly, Nix-/- mice had profound reticulocytosis and thrombocytosis, massive splenomegaly, and erythroid hyperplasia with no impact on hematopoietic progenitor cell numbers (Diwan et al., 2007). On blood smear, Nix-/- mice had increased polychromatophils and abnormally shaped erythrocytes (Schweers et al., 2007). Most interestingly, about half of the circulating Nix-/- erythrocytes stained positively for
Mitotracker Red on flow cytometry, suggesting a defect in mitochondrial elimination.
Indeed, isolated Nix-/- reticulocytes failed to eliminate their mitochondria after three days of culture (Schweers et al., 2007).
Nix-/- mice were also independently generated using embryonic stem cells with a gene trap insertion between exons 3 and 4 of Nix (Sandoval et al., 2008). As in the previous study, Nix-/- reticulocytes from these mice were also severely deficient in mitochondria elimination by flow cytometry and immunohistochemistry (Sandoval et al., 2008). In all,
Nix-/- mice are severely defective in mitochondrial elimination in the erythroid lineage. As a consequence of excess mitochondria, Nix-/- erythrocytes had a shorter lifespan in vivo due to increased caspase activation (Sandoval et al., 2008), likely due to reactive oxygen species production (Raha and Robinson, 2001). Notably, ribosome elimination was unperturbed in Nix-/- mice.
1.3.2 Nix is a ubiquitin-independent autophagy receptor for mitochondria
How does Nix function in the programmed elimination of mitochondria? In the previous study, Nix-dependent elimination of mitochondria was shown to be independent of the downstream apoptotic proteins, Bax and Bak, and the apoptotic activators, Bim and
Puma (Schweers et al., 2007). Moreover, the induction of autophagy was unperturbed in
Nix-/- mice (Schweers et al., 2007). Notably, under transmission electron microscopy,
Nix-/- reticulocytes showed incomplete incorporation of mitochondria into
13 autophagosomes (Sandoval et al., 2008; Schweers et al., 2007). Furthermore, disruption of the mitochondrial membrane potential with FCCP or the BCL2 homology domain 3
(BH3) mimetic, ABT-737, was able to rescue mitochondrial autophagy in Nix-/- reticulocytes (Sandoval et al., 2008). Thus, Nix-dependent loss of the mitochondrial membrane potential was sufficient to selectively drive mitochondria into autophagosomes. To date, there is a large literature describing the specific elimination of damaged mitochondria by autophagy, termed mitophagy (Elmore et al., 2001; Kim et al.,
2007; Narendra et al., 2008; Priault et al., 2005).
In selective autophagy, autophagy receptors bind to membrane-anchored, autophagy- specific ubiquitin-like proteins, Atg8-family proteins LC3/GABARAP (Kirkin and Dikic,
2007) through their W/YxxL/I motif, known as the LC3-interacting region (LIR) (Komatsu et al., 2007). Multiple sequence alignment of Nix predicted several putative LIRs in both the N-terminal domain and adjacent to an atypical BH3-like domain (Novak et al., 2010).
Nix was also shown to interact with Atg8/LC3 by yeast two-hybrid screening, and in vitro and in vivo co-immunoprecipitation assays (Novak et al., 2010). Finally, Nix was able to recruit LC3/GABARAP-L1 to damaged mitochondria, and disruption of the
Nix:GABARAP interaction by mutagenesis of the LIR sequence impaired mitochondrial elimination in murine reticulocytes (Novak et al., 2010). As such, Nix serves as a selective autophagy receptor for mitochondria during terminal erythropoiesis. Unlike many selective autophagy receptors, Nix does not appear to be ubiquitin-dependent.
1.3.3 Other autophagy components required for mitochondrial elimination
Other components of the mitophagy pathway were also identified in addition to Nix. For example, Ulk1 is a serine threonine kinase homologous to yeast Atg1p, which initiates autophagosome formation in response to changes in cellular nutrients (Mizushima,
14 2010). In FVA cells, Ulk1 protein and mRNA are highly induced upon erythropoietin treatment (Kundu et al., 2008). Analysis of Ulk-/- mice showed a similar phenotype to
Nix-/- mice; namely, a fraction of mature erythrocytes stained positively for mitochondria on flow cytometry, and Ulk-/- reticulocytes were deficient in ribosome elimination in ex vivo culture (Kundu et al., 2008). Consistent with the erythroid phenotype, Ulk-/- mouse embryonic fibroblasts also showed an increase in mitochondrial mass.
Further mechanistic work showed that Ulk1 phosphorylates Atg13, allowing Atg13 to localize to damaged mitochondria (Joo et al., 2011). Moreover, Ulk1 is stabilized and activated by the chaperone Hsp90 and the kinase-specific co-chaperone Cdc37 complex, which are essential for eliminating depolarized mitochondria (Joo et al., 2011).
Ulk1-dependent reticulocyte mitophagy is also independent of Atg5, which is an essential component of conventional macroautophagy (Honda et al., 2014). Finally, another important component of autophagosome formation is the E1-like enzyme Atg7, which conjugates Atg12 to Atg5, and Atg8 (LC3/GABARAP) to phosphatidyl- ethanolamine (PE) (Komatsu et al., 2005). Similarly to Nix-/- and Ulk-/- reticulocytes,
Atg7-null reticulocytes had diminished mitochondrial elimination; however, the mitochondria of Atg7-/- reticulocytes remained polarized compared to wild-type cells
(Zhang et al., 2009). In summary, the selective autophagy pathway drives the programmed elimination of mitochondria during terminal erythropoiesis.
1.4 Interrelated nature of the ubiquitin pathway and the ribosome
1.4.1 Ubiquitin is expressed as a fusion gene with ribosomal proteins
The ubiquitin pathway and the ribosome are deeply intertwined with each other. In yeast, ubiquitin is generated by the cleavage of precursor proteins in which ubiquitin is joined to itself or an unrelated, highly conserved ‘tail’ amino-acid sequence (Ozkaynak et al.,
15 1987). In fact, three of these four yeast ubiquitin genes, ubi1-3, encode hybrid proteins with ubiquitin fused to ribosomal proteins that are incorporated into 40S and 60S ribosomal subunits after deubiquitination (Finley et al., 1989). Similar ubiquitin gene fusions to ribosomal proteins are present in humans and plants (Bishoff and Schwartz,
1990; Muller-Taubenberger et al., 1989; Redman, 1994). The ‘tail’ protein of ubi3 was identified as ribosomal protein S27a in eukaryotic cells (Redman and Rechsteiner,
1989). Moreover, ubiquitin also plays an important role in ribosome biogenesis. For example, the ubiquitin portion of these hybrid proteins appears to serve as a chaperone in ribosome biogenesis. Deletion of the ubi3 gene resulted in a defect in pre-rRNA processing that could be rescued by supplementing the ubi3 gene without the ubiquitin- coding element (Finley et al., 1989). Accordingly, ubiquitinated proteins of the pre-rRNA processing complex were identified in the nucleolus where pre-rRNA processing occurs
(Stavreva et al., 2006). In addition to pre-rRNA processing, ubiquitin was shown to mediate nonfunctional rRNA decay through an E3 ubiquitin ligase complex (Fujii et al.,
2009). Thus, not only is ubiquitin post-translationally fused to ribosomal proteins, it also serves a regulatory role in ribosome biogenesis and quality control.
1.4.2 Regulatory function of ribosomal ubiquitination
Ribosomal proteins were also shown to be a direct, physiological target of ubiquitination.
Ribosomal ubiquitination was first identified in studies of ubiquitin chain linkages. Yeast mutants deficient in K63 ubiquitination, the ubiK63R strain, were found to be depleted in a low molecular weight ubiquitin conjugate, which was subsequently identified as yeast ribosomal protein L28 (Spence et al., 2000; Spence et al., 1995). Both yeast RPL28 and the human ortholog, RPL27a, were modified with a polyubiquitin chain in translationally competent monosomes and polysomes, in a cell-cycle-dependent manner (Spence et al., 2000). Moreover, ubiK63R polysomes were unstable in low MgCl2 conditions,
16 demonstrating that K63-linked polyubiquitin chains are required for proper functioning of the ribosome (Spence et al., 2000). Later studies showed that K63 ubiquitination functions in quality control during stalled translation (Saito et al., 2015). Interestingly,
K63 polyubiquitination was independently identified as a modulator of the oxidative stress response in yeast (Silva et al., 2015). In response to hydrogen peroxide treatment, the E2 Rad6 and E3 Bre1 were shown to mediate K63 ubiquitination, which primarily targeted ribosomal proteins and ribosomal elongation factors (Silva et al.,
2015). Consistent with previous studies, ribosomal K63 ubiquitination stabilized 80S monosomes and polysomes under oxidative stress, rendering the cell more resistant to insult (Silva et al., 2015).
Ribosome ubiquitination was also identified as a direct response to endoplasmic reticulum stress (Higgins et al., 2015). Activation of the unfolded protein response (UPR) led to the early and specific ubiquitination of 40S ribosomal proteins, including RPS2,
RPS3, and RPS20, found in monosomes and polysomes (Higgins et al., 2015).
Moreover, activation of the integrated stress response through PERK was necessary but not sufficient to induce RPS2 and RPS3 ubiquitination upon UPR activation (Higgins et al., 2015). Ribosomal ubiquitination was also observed in broad surveys of the human ubiquitome (Kim et al., 2011; Matsumoto et al., 2005). Finally, ribosomal proteins were also shown to be modified by the ubiquitin-like molecule, NEDD8, and the absence of
NEDDylation led to ribosomal protein instability (Xirodimas et al., 2008).
1.4.3 Ribophagy: starvation-induced autophagy of ribosomes
Can ribosome ubiquitination serve as a signal for its elimination? Under nutrient starvation, mature ribosomes were shown to be degraded by both non-selective and a novel selective autophagy, which was termed ribophagy (Kraft et al., 2008). Instead of
17 being dependent on an E2 or E3, ribophagy was dependent on the deubiquitinating enzyme complex, Ubp3p/Bre5p (Kraft et al., 2008). Cells deficient in Ubp3p/Bre5p accumulated 60S ribosomes and had increased ubiquitination of ribosomal subunits or ribosome-associated factors (Kraft et al., 2008). Further work on the Ubp3p/Bre5p complex showed that it interacts with Cdc48, a ubiquitin-dependent chaperone-like AAA-
ATPase, and its ubiquitin-binding cofactor, Ufd3 (Meyer et al., 2012; Ossareh-Nazari et al., 2010). Moreover, Cdc48 and Ufd3 were required for ribophagy under nutrient starvation in yeast (Ossareh-Nazari et al., 2010). Since ribophagy is driven by the autophagic pathway, proteasome inhibition did not impair ribophagy despite involvement of Cdc48 (Ossareh-Nazari et al., 2010). Thus, under nutrient starvation, ribosome ubiquitination appears to actually play a protective role against ribophagy given its dependence on Ubp3p/Bre5p. If Ubp3p/Bre5p activity is required for ribophagy, then an unknown E3 ligase must ubiquitinate the ribosome prior to deubiquitination.
One possible E3 was Listerin, Ltn1/Rkr1, a RING-domain-type ubiquitin ligase, which associates with the 60S ribosomal subunit (Bengtson and Joazeiro, 2010). Rpl25 could be directly ubiquitinated by Listerin, and deubiquitinated by Ubp3p (Ossareh-Nazari et al., 2014). Moreover, deletion of Ltn1 was able to rescue ribophagy in the absence of
Ubp3p. As such, Listerin-dependent ubiquitination appears to protect 60S ribosomes from starvation-induced ribophagy, which is promoted by Ubp3p. Finally, a ribophagy- like process was also described in plants; ribosomal RNA (rRNA) decay in A. thaliana was shown to be dependent on RNS2, an intracellular RNase T2 that localizes to the endoplasmic reticulum and vacuoles (Hillwig et al., 2011). RNS2 mutant cells have accumulated RNA and are defective in clearing out rRNA. In contrast to yeast, the plant phenotype is also observed in nutrient-rich conditions, suggesting a constitutive process of autophagy and ribophagy (Hillwig et al., 2011). In all, the selective autophagy of
18 ribosomes, or ribophagy, is a recognized phenomenon in both yeast and plants.
1.4.4 Protein quality control by co-translational ubiquitination
Ubiquitination can also occur cotranslationally on newly synthesized peptides as a form of protein quality control (Wang et al., 2015). Early studies showed that up to 30% of newly synthesized proteins were immediately degraded via the proteasome (Schubert et al., 2000), However, a subsequent study argued that the fraction of cotranslational degradation was much lower, but the process was still thought to be proteasome- dependent (Vabulas and Hartl, 2005). Though the cell has evolved a family of ribosome- bound chaperones to assist in the folding of nascent chains, several studies have demonstrated cotranslational ubiquitination. For example, this was specifically demonstrated during in vitro translation of two substrates, the cystic fibrosis transmembrane conductance regulator (CFTR) and apolipoprotein B100 (Sato et al.,
1998; Zhou et al., 1998). Cotranslational protein degradation was demonstrated in vivo in yeast using a “ubiquitin sandwich” technique; using an N-terminal degradation signal, more than 50% of nascent peptide chains could be degraded cotranslationally before reaching their mature size (Turner and Varshavsky, 2000).
Ubiquitination of ribosome-associated nascent polypeptides was recently characterized in vivo in yeast (Duttler et al., 2013). In this case, cotranslational ubiquitination occurs for a small fraction, approximately 1-6%, of nascent chains, typically for longer and more hydrophobic proteins (Duttler et al., 2013). This cotranslational folding is mediated through a complex network of E3 ubiquitin ligases including the ribosome-bound ligase,
Ydr266c/Hel2, and in part due to Ltn1, and targets the nascent chains for proteasomal degradation (Duttler et al., 2013). This was also studied in mammalian cells, in which 12-
15% of nascent chains were reported to be cotranslationally ubiquitinated in vivo (Wang
19 et al., 2013). These polyubiquitinated nascent chains were primarily K48-linked chains, and occurred within active translation complexes (Wang et al., 2013). Also, cotranslational ubiquitination was enhanced to about 50% under conditions of translational error or protein misfolding. Thus, the ubiquitin pathway functions during active translation in quality control.
1.4.5 Listerin is an E3 ligase that participates in cotranslational ubiquitination
Another form of cotranslational ubiquitination occurs on stalled translational complexes as a form of mRNA quality control. One such pathway is in the regulation of mRNAs missing a stop codon (“non-stop mRNAs”). Initially identified in a forward screen for neurodegeneration in mice (Chu et al., 2009), the RING-type E3 ligase Ltn1 was found to function specifically in quality control of non-stop proteins rather than general protein quality control (Bengtson and Joazeiro, 2010; Brandman et al., 2012). Cotranslational ubiquitination by Ltn1 rapidly targeted newly synthesized non-stop proteins upon recognition of the polylysine tract as a result of translating through the poly(A) tail without a stop codon (Bengtson and Joazeiro, 2010). Moreover, Ltn1 was found to associate specifically with 60S ribosomes, but not 80S monosomes or polysomes (Bengtson and
Joazeiro, 2010; Brandman et al., 2012). Accordingly, Ltn1 requires ribosome subunit dissociation by recycling factors such as Pelota, Hbs1, and ABCE1 to generate a 60S- nascent chain complex to which it can bind (Shao et al., 2013).
This was confirmed by a reconstitution of ribosome-associated ubiquitination with purified components in vitro (Shao and Hegde, 2014). Cryo-EM structural studies of the
60S-Listerin complex showed that the N-terminus of Ltn1 binds a surface on the 60S subunit that contacts the 40S subunit (Lyumkis et al., 2014; Shao et al., 2015; Shao and
Hegde, 2014). Thus, steric hindrance by the 40S subunit prevents Ltn1 binding until the
20 ribosome is fully dissociated. Also from the cryo-EM work, the C-terminal RING domain of Ltn1 was found to be oriented towards the nascent chain exit tunnel of the 60S ribosome, allowing access to emerging polypeptides (Lyumkis et al., 2014; Shao et al.,
2015). In summary, Ltn1 is a well-studied E3 ligase that associates with 60S ribosomes and ubiquitinates nascent chain non-stop proteins as a form of protein quality control.
1.4.6 Ribosome deficiencies in human disease
Finally, another fascinating intersection between the ubiquitin pathway and the ribosome is through p53 ubiquitination and free ribosomal proteins. Mdm2 is the E3 ligase for the p53 tumor suppressor and an inhibitor of p53 transcriptional activity (Momand et al.,
1992; Oliner et al., 1993). Free ribosomal protein RPL11 was found to bind the MDM2 human ortholog, HDM2, which prevents HDM2-mediated p53 ubiquitination and degradation, and restores p53 transactivation (Zhang et al., 2003). Interfering with ribosome biogenesis increased HDM2-RPL11 interaction, suggesting that this pathway functions as a ribosomal stress checkpoint (Zhang et al., 2003). MDM2 was also found to bind other ribosomal proteins including RPL23 and RPL5, which all interfere with p53 ubiquitination (Dai and Lu, 2004; Jin et al., 2004). However, RPL11, and not RPL5 or
RPL23, binding led to increased autoubiquitination, but decreased proteasomal degradation of MDM2 (Dai et al., 2006). Subsequent work found that MDM2 bound many other ribosomal proteins including RPS3, RPS7, and RPL26, among others
(Yadavilli et al., 2009; Zhang et al., 2010; Zhu et al., 2009).
Upon impairment of ribosomal biogenesis, RPL5 and RPL11 are protected from proteasomal degradation and imported into the nucleolus (Bursac et al., 2012). These observations are clinically relevant to the pathophysiology of Diamond Blackfan Anemia, in which ribosomal proteins are mutated in approximately 50% of patients (Boria et al.,
21 2010). Consistent with this, knockdown of RPS19, which is mutated in about 25% of patients, led to an increase in p53 and mRNA levels for p21, a transcriptional target of p53 (Dutt et al., 2011; Sieff et al., 2010). As such, aberrant ribosome biogenesis or ribosomal protein haploinsufficiency can contribute to p53 activation and erythropoietic failure as seen in Diamond Blackfan Anemia and other ribosome-related diseases, known as ribosomopathies.
1.5 Limitations in our understanding of the UPS in the reticulocyte
1.5.1 Globin as a possible target of the UPS
Reticulocytes are known to rapidly degrade hemoglobin that contains abnormal amino acid-analogs (Rabinovitz and Fisher, 1964) and mutated or unassembled globin chains
(Carrell and Lehmann, 1969; Huehns and Bellingham, 1969). Could unstable hemoglobin chains be degradative substrates of the reticulocyte UPS? The pathways of globin degradation were elucidated around the study of hemin, which is the oxidation product of heme. Early work showed that globin production in reticulocytes depends on the supply of hemin (Bruns and London, 1965). In fact, hemin also enhanced the synthesis of non-globin proteins, such as carbonic anhydrase, in reticulocyte lysates
(Beuzard et al., 1973). Though increased synthesis seemed to account for the accumulation of globin upon hemin treatment, Etlinger and Goldberg found that hemin actually inhibited the rapid ATP-dependent degradation of abnormal globins, apohemoglobin (after heme extraction), and apomyoglobin (Etlinger and Goldberg,
1980). Further work by Haas and Rose showed that hemin specifically inhibited ubiquitin conjugate degradation, later defined as the UPS (Haas and Rose, 1981).
In β-thalassemia, a very common hemoglobinopathy, β-globin gene mutations result in the accumulation and precipitation of excess α-globin subunits. Early work in β-
22 thalassemic hemolysates showed that unpaired α-globin chains were degraded in an
ATP- and ubiquitin-dependent manner (Shaeffer, 1988). Further analysis showed that α- globin is predominantly and heterogenously monoubiquitinated in β-thalassemic hemolysates (Shaeffer, 1994a). In support of these findings, ultrastructural immunocytochemical studies showed that precipitated globin chains from bone marrow sections of patients with β-thalassemia and hemoglobin H disease cross-reacted with anti-ubiquitin antibodies (Wickramasinghe and Lee, 1998). Accordingly, monoubiquitinated α-globin was shown to be specifically degraded by direct interaction with 26S proteasomes (Shaeffer and Kania, 1995). Since α-globin degradation appeared to be ubiquitin- and proteasome-dependent, inhibition of deubiquitinating enzymes with ubiquitin aldehyde accelerated the degradation of exogenous and endogenous α-globin
(Shaeffer and Cohen, 1996, 1997).
Similarly, using a murine model of β-thalassemia, α-globin was found to be ubiquitinated in β-thalassemic erythroid cells and accumulated upon proteasome inhibition (Khandros et al., 2012). Moreover, β-thalassemic erythroblasts had increased expression of proteasomal components mediated by the transcription factor Nrf1. However, in vivo treatment of β-thalassemic mice with proteasome inhibitor did not enhance the accumulation of α-globin; instead, proteasome inhibitor treatment activated compensatory stress-response mechanisms including autophagy (Khandros et al.,
2012). In all, accumulation of excess α-globin subunits appears to trigger various protein quality control mechanisms including, but not limited to, the upregulation of the UPS.
23 1.5.2 The exosome pathway remodels the reticulocyte cytoskeleton
Finally, the exosome pathway was discovered in the mammalian reticulocyte in the selective removal of membrane proteins (Johnstone et al., 1987). When sheep reticulocytes were cultured in vitro, they were found to release vesicles that were absent when culturing mature erythrocytes. Early characterization of reticulocyte exosomes showed the presence of the transferrin receptor (TFR1) and other plasma membrane proteins. One plasma membrane protein that is partitioned into exosomes is the water channel, aquaporin-1 (AQP-1) (Blanc et al., 2009). AQP-1, which regulates cell volume in response to rapid changes in plasma tonicity, was found to be selectively released into exosomes during in vitro maturation and in vivo. Interestingly, culturing reticulocytes with the proteasome inhibitor MG132 inhibited the partitioning of AQP-1 and TFR1 into exosomes, suggesting that ubiquitination serves an important role in signaling through the exosome pathway. Lastly, the UPS may play a role in membrane and cytoskeleton remodeling during terminal erythropoiesis (Liu et al., 2010). The cytoskeleton proteins, tubulin and actin, were degraded during terminal differentiation of reticulocytes into erythrocytes. When reticulocytes were cultured with MG132, the degradation of tubulin and actin was inhibited, as was the degradation of TFR1 (Liu et al., 2010). As a negative control, β-spectrin levels do not change during differentiation and with proteasome inhibition. Thus, the UPS helps mediate membrane and membrane cytoskeleton remodeling during terminal erythroid differentiation.
1.6 Hypochromic, microcytic anemic phenotype of the Ube2O-null mouse
A null mutation in the murine Ube2O gene, termed hem9, was identified in the lab of
Mark Fleming at Boston Children’s Hospital (unpublished data). hem9/hem9 homozygotes (hereafter referred to as hem9) have a hypochromic, microcytic anemia
24 associated with an erythrocytosis and an increase in reticulocytes. Peripheral blood smears show mildly hypochromic RBCs as well as basophilic RBC inclusions.
Precipitated hemoglobin inclusions (Heinz bodies) are not detectable on supravital stain or by electron microscopy. hem9 was mapped to chromosome 11 and proved to be a nonsense mutation in exon 18 (E1121X) of the gene encoding Ube2O (Tian et al.,
2008). Immunoblot analysis of reticulocyte-rich blood from hem9 mice demonstrated a complete absence of the full-length protein, indicating that hem9 is likely a null allele. A second allele obtained via gene-trapping also showed no detectable full-length protein and produced a phenotype comparable to that of hem9. Based on detailed study, the hem9 phenotype appears at present to be specific to the erythroid lineage, where
Ube2O is found at markedly elevated levels (Wefes et al., 1995).
As will be described in Chapter Two, immunoblotting reticulocyte lysates with an antibody to ubiquitin revealed significant differences between wild-type (WT) and hem9 samples. Notably, a set of low molecular weight ubiquitin conjugates is depleted, and among these α-globin was definitively identified in its ubiquitinated form by mass spectrometry. Since excess α-globin is rapidly degraded with a half-life estimated at ~1 min or less, among the shortest known in all eukaryotes (Hunter and Jackson, 1972),
Ube2O is potentially the first known globin E3-ligase. hem9 mice were then crossed to the murine Hbbth-3/+ heterozygous β-thalassemia intermedia model in which both the b1 and b2 adult globin genes have been deleted (Yang et al., 1995). This compound mutant shows a compensatory erythrocytosis with an increase in hemoglobin, a decreased reticulocyte count, and a marked decrease in the spleen:body mass ratio.
Thus, deficiency of Ube2O unexpectedly ameliorates murine β-thalassemia intermedia.
These findings suggest that there are important physiological substrates of Ube2O, other than α-globin, that mediate disease modification.
25 1.7 Summary and Significance
The ubiquitin-proteasome system (UPS) plays a fundamental and indispensable role in the regulation of numerous cellular processes, including cell cycle progression, division, differentiation, and intracellular trafficking. Though ubiquitously found in eukaryotic cells, the UPS was initially discovered in the reticulocyte, where it is highly active in degrading abnormal proteins, particularly excess globin. Similarly, the degradation of tubulin, actin, and dynein in reticulocytes proceeds at least in part through the UPS. These processes occur in the final step of terminal erythroid differentiation from reticulocyte to erythrocyte, during which massive cellular remodeling of the proteome occurs. However, these pathways remain uncharted, and apart from our recent work, as described below, no specific ubiquitinating factor has been implicated in these important events. One aspect of reticulocyte remodeling, mitochondrial clearance, has been shown to be autophagy- dependent rather than UPS-dependent. During the initial characterization of the UPS in reticulocytes, Finley, Pickart, and colleagues cloned and characterized two ubiquitinating enzymes, Ube2O and Ube2H, that are highly and selectively upregulated during terminal erythroid differentiation. Ube2O is an unusually large E2 with a molecular mass of 141 kDa. The size of Ube2O led to the hypothesis that it functions as an E2-E3 hybrid that can both charge itself with ubiquitin (i.e. an E2, or ubiquitin conjugating enzyme) and transfer ubiquitin onto proteins (i.e. an E3, ubiquitin ligase). The encompassing hypothesis given its specificity and abundance is that Ube2O is a ubiquitin ligase that is critical for terminal erythroid differentiation. This is, in part, supported by the hypochromic, microcytic anemic phenotype of Ube2O-null mice. To this end, understanding the role of Ube2O may give significant insight into global cellular remodeling of terminally differentiating cells, and may hold significant clinical implications for many inherited anemias.
26 As will be described in Chapter Two, I have identified ribosomal proteins as a major class of physiological substrates for Ube2O. To identify substrates of Ube2O, I expressed it from a baculovirus vector in insect cells, purified it, and added it back to reticulocyte lysates from hem9 animals. The major class of identified targets was ribosomal proteins. By immunoblotting and polysome profile analysis, I observed elevated levels of ribosomal proteins and 80S ribosomes in hem9 reticulocytes, consistent with a marked global defect in ribosomal protein degradation. Finally, when cultured ex vivo, hem9 reticulocytes were defective in ribosome elimination by flow cytometry analysis. Thus, Ube2O is critical for ribosome elimination in the erythroid lineage. In Chapter Three, I showed that Ube2O is sufficient to drive ribosome elimination in non-erythroid 293 cells. To this end, I cloned the human Ube2O gene and stably integrated it into the Flp-In T-Rex 293 cell line. Overexpression of WT Ube2O destabilized ribosomal proteins by immunoblotting and drove the elimination of 80S ribosomes by polysome profile analysis. Using a specific rRNA polymerase inhibitor, I showed that Ube2O degrades pre-existing ribosomes rather than interfering with ribosome assembly. Moreover, Ube2O overexpression did not induce apoptosis or necrosis in 293 cells. Finally, we confirmed the destabilization of ribosomal proteins by mass spectrometry in 293 cells.
In Chapter Four, I showed that ribosomal degradation is proteasome-dependent.
Treating WT reticulocytes with proteasome inhibitors phenocopied the ribosome elimination defect of Ube2O deficiency. Moreover, I reconstituted the degradation of ribosomal proteins in Ube2O-null reticulocyte lysates and in an in vitro purified system, which was shown to be proteasome-dependent. Finally, in Chapter Five, I showed that
Ube2O-null reticulocytes are amino acid deficient and translationally defective. Using quantitative metabolomics profiling, I found that hem9 reticulocytes are amino acid
27 deficient, especially in L-arginine and L-lysine, a phenotype that could be recapitulated by treating WT reticulocytes with proteasome inhibitors. I subsequently showed that hem9 reticulocytes have an activated integrated stress response that is independent of
HRI kinase. Furthermore, GCN2 was activated in hem9 reticulocytes, consistent with its amino acid deficiencies. Thus, the anemic phenotype of Ube2O-null mice may be caused by an activation of the integrated stress response through amino acid deficiency.
Together, these results suggest that Ube2O is both critical and sufficient to drive ribosome elimination in a proteasome-dependent manner, and that Ube2O deficiency activates the integrated stress response through GCN2.
28
CHAPTER TWO
Ubiquitination of ribosomal proteins by Ube2O
Background
Global remodeling of the proteome is an essential aspect of terminal differentiation, consisting of the programmed elimination of many generic constituents of the cell in parallel with abundant synthesis of new, cell-type-specific proteins such as globin.
Reticulocytes are a canonical example of a proteome in massive and rapid transition.
During the final stages of erythropoiesis, nascent reticulocytes will eliminate all intracellular organelles including mitochondria and ribosomes (Geminard et al., 2002).
Though the elimination of mitochondria has been shown to proceed through selective autophagy (Sandoval et al., 2008; Schweers et al., 2007), the mechanisms of ribosomal degradation have yet to be elucidated.
The ubiquitin proteasome system (UPS) is a multienzyme proteolytic pathway that was first characterized in the reticulocyte (Ciechanover et al., 1980; Ciehanover et al., 1978).
In contrast to lysosomal degradation, the UPS is highly specific in nature. This specificity is achieved by conjugation of ubiquitin to substrate proteins by the E1-E2-E3 cascade of enzymes (Finley et al., 2012). Enzymes from this pathway were also initially purified from reticulocyte lysate (Hershko et al., 1983). While most E2 ubiquitin-conjugating enzymes were found in the low molecular weight range, a single E2 migrated with an abnormally large molecular mass of 230 kDa (Pickart and Vella, 1988). This protein, named Ube2O or E2-230K referring to its apparent size, shared many features of the canonical E2 class, but behaved like a hybrid E2-E3 enzyme (Klemperer et al., 1989).
Further studies showed that Ube2O is highly and selectively induced in terminally differentiating reticulocytes (Wefes et al., 1995). In fact, Ube2O is upregulated ~16- to
17-fold during differentiation, and is induced contemporaneously with globin. This stark induction of Ube2O occurs while the vast majority of other ubiquitinating enzymes are
30 downregulated (An et al., 2014; Shi et al., 2014; Wong et al., 2011). As such, it was hypothesized that Ube2O plays a critical role in the remodeling of the erythroid proteome during terminal differentiation. As a ubiquitinating enzyme, Ube2O may facilitate the specific, programmed elimination of the generic constituents of the reticulocyte.
To date, Ube2O may participate in the retromer complex (Hao et al., 2013), SMAD regulation (Zhang et al., 2013a; Zhang et al., 2013b), or BAP1 localization (Mashtalir et al., 2014); however, these studies do not consider the physiological context of Ube2O expression. Ube2O was also examined by global yeast two-hybrid studies (Markson et al., 2009) and high-throughput affinity-purification mass spectrometry (Huttlin et al.,
2015). In these screens, Ube2O was shown to interact with specific 60S ribosomal proteins, though these interactions were not pursued further.
Recently, a null mutation in the murine Ube2O gene was identified in the lab of Mark
Fleming at Boston Children’s Hospital. Ube2O null mice have a hypochromic, microcytic anemia with no known sequelae outside of the erythroid system. The anemic phenotype is also red cell intrinsic and transplantable. Therefore, it appears that a failure of ubiquitination during terminal erythroid differentiation can cause anemia in mice. This supports the longstanding hypothesis that Ube2O is a critical E2 in erythropoiesis. We can now address the role of Ube2O during terminal differentiation using this null mouse.
Here we report the finding that Ube2O can directly ubiquitinate ribosomal proteins in reticulocyte lysate. Moreover, Ube2O null reticulocytes have elevated levels of ribosomal proteins and 80S ribosomes, consistent with a global defect of elimination. Mutant reticulocytes also have an aberrant ubiquitin profile, deficient in globin-ubiquitin conjugates. These observations offer insight on our limited understanding of proteomic remodeling during terminal differentiation.
31 Results
Depletion of ubiquitin conjugates in Ube2O null reticulocytes
Since Ube2O is highly and selectively induced in terminal erythroid differentiation (Wefes et al., 1995), we hypothesized that Ube2O is a dominant ubiquitinating factor in the reticulocyte. To validate this hypothesis, we first sought to identify a biochemical phenotype of the Ube2O null mouse to complement its hypochromic, microcytic anemia.
If Ube2O is indeed a dominant E2, then its deficiency may manifest in the ubiquitin profile of null reticulocytes. Since there are normally very few circulating reticulocytes in peripheral blood, we induced reticulocytosis in our mice by serial retro-orbital bleeding.
We then analyzed the reticulocyte lysates by SDS-PAGE. Immunoblotting of the extracts with an anti-ubiquitin conjugate antibody revealed reduced levels of multiple ubiquitin conjugates in Ube2O null reticulocytes (Figure 2.1, lane 2). There are at least four discrete bands in the low molecular weight region that are strongly reduced in the mutant
(Figure 2.1, lane 2 and 5). Heterozygous reticulocytes showed an intermediate phenotype of conjugate depletion (Figure 2.1, lane 3), and the gene trap allele (gt) behaved as a null allele (Figure 2.1, lane 6 and 7). We further showed that these low molecular weight ubiquitin conjugates are isopeptidase T-resistant, indicating that they are not free ubiquitin chains (commonly found in certain tissues). Finally, the molecular weight of these conjugates suggests that the substrate is monoubiquitinated, and that the unmodified substrate is approximately 15 kDa in size. Of note, there do not appear to be any changes in free ubiquitin level between these reticulocyte extracts (Figure 2.1).
Since hundreds of ubiquitin ligases exist, it is unusual that a single ubiquitinating factor should produce such large changes in ubiquitin profiles. In summary, these perturbations of the ubiquitin profile confirm that Ube2O is indeed a dominant E2 in erythroid differentiation.
32
Figure 2.1
Reduced levels of multiple ubiquitin conjugates in hem9 reticulocytes.
Reticulocyte lysates were resolved by 4-12% SDS-PAGE and immunoblotted with an anti-ubiquitin conjugate antibody. +, wild-type allele. “gt,” gene trap allele, an apparent null. h9, hem9. Each sample is from a different mouse induced by serial retro-orbital bleeding.
33 Identification of depleted ubiquitin conjugates in reticulocytes
To better understand the role of Ube2O in terminal erythropoiesis, we then sought to identify these low molecular weight ubiquitin conjugates. We first fractionated WT reticulocyte lysates by cation exchange chromatography. When the ubiquitin proteasome system (UPS) was first characterized, reticulocyte lysate was resolved by anion exchange chromatography on DEAE-cellulose (Ciechanover et al., 1980). The non- absorbed protein contained hemoglobin and ubiquitin, and was termed Fraction I. To purify Fraction I, including ubiquitin and its conjugates, we used a cation exchange column to retain Fraction I, and eluted the protein from the column with a salt gradient.
We then analyzed the eluted fractions by immunoblotting for ubiquitin. We found that the complex set of low molecular weight ubiquitin conjugates eluted over multiple fractions, allowing us to carefully resolve each individual band. After pooling fractions containing similar bands of conjugates, we then purified the conjugates using ubiquitin-affinity agarose (TUBEs, or Tandem Ubiquitin Binding Entities), which contains tandem UBA1 domains of the protein ubiquilin (Hjerpe et al., 2009). The ubiquitin conjugates were eluted with SDS reducing sample buffer, and analyzed by SDS-PAGE and LC-MS/MS.
Among these lower molecular weight conjugates (around 25 kDa), α-globin was definitively identified in ubiquitinated form by mass spectrometry (Figure 2.2). α-globin was predominantly ubiquitinated on lysine 91, though an additional ubiquitination site was identified on lysine 41. The molecular weight of these ubiquitin conjugates corresponded to the monoubiquitinated form of α-globin. Ubiquitinated β-globin (on lysine 18, 60, 67, and 121) was also identified in the higher molecular weight band
(around 37 kDa). In a reticulocyte, since α-globin is translated in excess of β-globin, excess α-globin is degraded particularly rapidly, with a half-life estimated at ~1 minute or
34
Figure 2.2
α-globin was identified in its ubiquitinated form from reticulocyte lysate.
Low molecular weight ubiquitin conjugates were purified by cation exchange chromatography and with ubiquitin affinity agarose from reticulocytes from WT mice induced by serial retro-orbital bleeding. The ubiquitin profile (left) is from Figure 2.1. The identified ubiquitinated proteins are listed for each purified conjugate (right).
35 less (Hunter and Jackson, 1972). Moreover, unpaired α-chains are degraded in a ubiquitin-dependent manner (Shaeffer, 1988; Shaeffer and Cohen, 1997, 1998). Thus,
Ube2O is potentially the first known globin E3 ligase. However, residual conjugates are present in the mutant, indicating the existence of a second globin ligase. In addition to globin, we also identified multiple ubiquitin linkages in the low molecular weight region
(K6, K11, and K48). Finally, in the upper molecular weight region, we identified ubiquitinated forms of ALAD and AHSP. AHSP, or alpha hemoglobin stabilizing protein, is a well-characterized chaperone that binds free α-globin and inhibits the production of reactive oxygen species (Gell et al., 2002; Kihm et al., 2002; Kong et al., 2004; Yu et al.,
2007). As such, AHSP may be another important putative Ube2O substrate that might underlie the anemic phenotype of the null mouse.
If α-globin is the major substrate of Ube2O, then null mice may be anemic due to an excess of α-globin production, similar to the pathophysiology of β-thalassemia. To test this hypothesis, we therefore crossed Ube2O null mice to murine Hbbth-3/+ heterozygous
β-thalassemia intermedia model, expecting an exacerbation of the globin chain imbalance (Yang et al., 1995). Contrary to expectation, Ube2O deficiency ameliorated murine β-thalassemia intermedia. Thus, the ubiquitination of α-globin does not appear to drive the anemic phenotype. Could elevated levels of AHSP account for the amelioration of murine β-thalassemia intermedia? In principle, supraphysiolgical levels of AHSP, due to a deficiency of Ube2O, can bind any excess α-globin. Unfortunately, it has been previously shown that supraphysiological levels of AHSP cannot mitigate the severity of murine β-thalassemia (Nasimuzzaman et al., 2010). In all, this suggests that there are additional physiologically important substrates of Ube2O, and that the depletion of these globin conjugates may not account for the anemic phenotype.
36 Identification of Ube2O substrates in reticulocyte lysates reconstituted with recombinant Ube2O
Additional substrates of Ube2O were then identified using an activity-based approach.
To this end, we first expressed Ube2O with an N-terminal polyhistidine tag from baculovirus vector in High Five insect cells, and purified it using Ni-NTA agarose.
Recombinant Ube2O was then added back at physiological levels to reticulocyte lysates from null animals. Since ubiquitination is canonically a signal for substrate degradation,
Ube2O-dependent substrates should be found at elevated levels in null reticulocytes. As a negative control, we added back catalytically inactive Ube2O with an active site cysteine to alanine mutation (Ube2O CA) at the same concentration as the active enzyme. Prior to adding back recombinant Ube2O, we treated the reticulocyte lysate with N-ethylmaleimide (NEM), a cysteine-alkylating reagent, to quench any endogenous ubiquitinating activity. After alkylation, the lysate was then treated with excess DTT to inactivate any remaining NEM. This simple add-back approach appears to be valid because endogenous reticulocyte Ube2O is not purified as a stable complex, according to both conventional purification (Berleth and Pickart, 1996) and immunoprecipitation data (Huttlin et al., 2015).
The reaction was then incubated in the presence of biotinylated ubiquitin to distinguish newly formed ubiquitin conjugates from existing conjugates, and to allow us to purify them from the reaction. We also supplemented recombinant E1 enzyme to replenish
NEM-inactivated endogenous enzyme (Figure 2.3). To outcompete endogenous, untagged ubiquitin, biotin-ubiquitin was added in excess. Moreover, E1 was preincubated with biotin-ubiquitin to ensure efficient loading onto the E1 and transfer to
Ube2O. When Ube2O was reconstituted in reticulocyte lysate (Figure 2.3, lane 4),
Ube2O robustly formed ubiquitin conjugates after a 45 minute incubation at 37°C. In
37
Figure 2.3
Reconstitution of ubiquitination in hem9 lysates using recombinant Ube2O.
Ube2O was purified from baculovirus-infected cells. Ube1: ubiquitin activating enzyme, which transfers ubiquitin to Ube2O. Ubiquitin is supplied in a biotin-tagged form.
Endogenous ubiquitinating factors of the reticulocyte lysate (from Ube2O null mice induced by serial retro-orbital bleeding) were inactivated by NEM, followed by DTT to inactivate NEM. The ubiquitination was run for 45 min at 37°C. Note, some ubiquitin ligases are subject to autoubiquitination as seen for Ube2O in lane 3. 8% of each reaction was loaded per lane.
38 addition to a high molecular weight smear of ubiquitin conjugates, Ube2O also formed a set of low molecular weight ubiquitin conjugates that resemble the depleted conjugates in Ube2O null reticulocyte lysates (Figure 2.1). As a negative control, Ube2O CA did not have any conjugating activity (Figure 2.3, lane 5), as the only bands evident are of biotin- ubiquitin and auto-ubiquitinated E1 (similar to Lane 2). There was no endogenous ubiquitinating activity in the lysate, validating the efficacy of alkylation. In the absence of lysate, Ube2O strongly autoubiquitinated itself (Figure 2.3, lane 3) as previously reported in the initial characterization of this enzyme (Berleth and Pickart, 1996).
We then recovered the newly formed ubiquitin conjugates with high capacity agarose beads conjugated to NeutrAvidin, a deglycosylated form of streptavidin. Since the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature, the agarose beads were stringently washed with 1M NaCl to remove all non- specific binding proteins. From the conjugation reaction, the smear of high molecular weight ubiquitin conjugates suggested that Ube2O can modify substrate proteins with multiple biotin-ubiquitin moieties in reticulocyte lysate, as previously reported for in vitro substrates (Berleth and Pickart, 1996). As such, eluting conjugates that are abundantly modified by ubiquitin from the NeutrAvidin agarose would be difficult with even the most stringent conditions. To maximize the identification of putative Ube2O substrates, these ubiquitin conjugates were directly trypsinized from the NeutrAvidin agarose beads and analyzed by LC-MS/MS. Because ubiquitin modifies lysine residues, and thereby occludes the tryptic cleavage site, each ubiquitin modification results in a junctional, branched tryptic peptide. The terminal di-glycine segment from ubiquitin, with its distinct molecular weight signature, remains on the peptide after limit cleavage (Denison et al.,
2005; Kim et al., 2011). To stringently identify putative substrates, we first analyzed di- glycine junctional peptides, which likely indicate direct ubiquitination by Ube2O.
39 The major class of identified targets was ribosomal proteins, comprising 86% of all ubiquitinated peptides, other than ubiquitinated Ube2O itself (Table 2.1). The majority of the ubiquitinated ribosomal proteins were from the large 60S subunit, though ubiquitinated 40S ribosomal proteins were also identified. The four most abundantly identified proteins were Rpl29, Rpl35, Rpl23a, and Rpl37, which accounted for nearly
70% of the ubiquitinated peptides from ribosomal proteins. Rpl29 was the most frequently identified ubiquitinated protein from the reconstitution experiments, followed by Rpl35. Multiple ubiquitination sites were also identified on each ribosomal protein, including Rpl29 (most commonly on lysine 33 and 38), Rpl35 (lysine 43 and lysine 66), and Rpl37 (lysine 25 and 52). In total, we detected 12 unique ubiquitination sites on
Rpl29, suggesting that Ube2O may have a broad substrate recognition motif. In further support of this view, global yeast two-hybrid studies point to specific interaction between
Ube2O and several ribosomal proteins, notably Rpl29, our strongest hit (Markson et al.,
2009). Moreover, Rpl35 was recently identified as a novel interactor of Ube2O by high- throughput affinity-purification mass spectrometry (Huttlin et al., 2015).
The ubiquitination of the 60S subunit is likely a unique observation; to date, the majority of reported ribosomal ubiquitination occurs mainly on the 40S subunit (Higgins et al.,
2015; Silva et al., 2015; Spence et al., 2000). Similar to these previous studies, we were also able to identify ubiquitinated ribosomal proteins from the 40S subunit. These ribosomal proteins included Rps19, Rps30, and Rps15, and are distinct from the ribosomal proteins identified in previous studies. Few ubiquitinated non-ribosomal proteins were identified, apart from α- and β-globin. Multiple ubiquitinated peptides from
α- and β-globin were identified when Ube2O was added back to null reticulocyte lysate; however, these peptides also were identified when null lysates were reconstituted with the catalytically inactive enzyme. As such, globin ubiquitination does not appear to be
40 Table 2.1
Ube2O modifies ribosomal proteins efficiently in reconstituted hem9 lysates.
Immunoprecipitated ubiquitin conjugates were digested with trypsin. Peptides containing di-glycine motifs (ubiquitination sites) were identified and modified lysines mapped by
LC-MS/MS. Hits were dependent on addition of Ube2O. Spectral counts: the number of identifications over three replicate experiments. Ub sites: unique ubiquitination sites.
41 solely Ube2O-dependent. Finally, we identified multiple types of ubiquitin linkages, including K6, K11, K27, K33, K48, and K63. These results suggest that either Ube2O can directly synthesize a wide variety of ubiquitin-ubiquitin topologies, or that Ube2O works together with a cognate E3 to synthesize these chains.
In addition to analyzing ubiquitinated peptides, we also considered the enrichment of total peptides after reconstituting Ube2O in null reticulocyte lysate. In total, 634 proteins were identified with more than 10 total spectral counts, indicating sufficient identification and protein coverage. Of these 634 proteins, we focused specifically on proteins that are enriched in the WT reconstitution compared to the CA reconstitution. Using a criterion of
1.5-fold enrichment of the WT to CA reconstitution, there were 98 proteins significantly enriched in the WT experiment. Gene ontology enrichment analysis of these 98 proteins was significant for the ribosome and ribonucleoprotein complexes. Consistent with our analysis of ubiquitinated peptides, ribosomal proteins are the major class of identified targets of Ube2O. Thus, using an activity-based approach, ribosomal proteins were provisionally assigned as Ube2O substrates.
Direct ubiquitination of ribosomal proteins by Ube2O
Could these ribosomal proteins be isolated in complex with each other when pulling down the biotin-ubiquitin? To ensure that the visualized ubiquitination events are due directly to Ube2O, this experiment was repeated with conjugates isolated under high salt conditions (1M NaCl) or under denaturing conditions (6M urea). These denaturing conditions are sufficient to detach ribosomal proteins from the 80S complex (Spitnik-
Elson and Greenman, 1971), while preserving the binding of biotin-ubiquitin to the
NeutrAvidin agarose. Analysis of the identified ubiquitinated peptides yielded very similar results with respect to ribosomal proteins under denaturing conditions (Table 2.2). The
42 majority of ubiquitinated ribosomal proteins were still from the 60S ribosomal subunit, including Rpl29, Rpl35, Rpl37, and Rpl19; additional 40S ribosomal proteins were identified including Rps19, Rps15, and Rps25. Consistent with our previous data, Rpl29 proved to be our strongest hit when conjugates were isolated under denaturing and non- denaturing conditions. Multiple ubiquitination sites were also detected on Rpl29 and other ribosomal proteins, further supporting the hypothesis of a broad recognition motif by Ube2O. While other non-cysteine-based ubiquitinating enzymes (i.e. RING E3 ligases) may contribute to the ubiquitination of ribosomal proteins, we will later demonstrate the ubiquitination of ribosomal proteins by Ube2O with purified components
(requiring only E1 and ubiquitin) (Chapter Four). Under denaturing conditions, α-globin
(modified on lysine 17 and 91) was the only ubiquitinated non-ribosomal protein that was enriched besides Ube2O and ubiquitin.
Analysis of total isolated peptides also showed enrichment for ribosomal proteins. Rpl29,
Rpl23a, and Rpl5 were the most abundant proteins represented by spectral counts.
While α- and β-spectrin were identified under non-denaturing conditions, only α-spectrin was enriched under denaturing washes, suggesting that β-spectrin was pulled down non-specifically under non-denaturing conditions. This is consistent with previous studies on erythrocyte cytoskeleton remodeling showing that β-spectrin levels are not affected by proteasome inhibitors (Liu et al., 2010). Ankyrin, Hsp90-alpha, and p97/Vcp were also identified in abundance under 1M NaCl washes, but were not enriched under 6M urea washes. As such, these proteins likely represent non-covalent, non-specific interactors with Ube2O, the ribosome, or ubiquitin. Thus, this experiment suggests that the isolated ribosomal protein conjugates are directly ubiquitinated by Ube2O, and that ribosomal proteins are not being pulled down in complex with each other in the 80S ribosome.
43 Table 2.2
Ribosomal proteins are still enriched under denaturing conjugate purification.
Immunoprecipitated ubiquitin conjugates were purified under denaturing (urea) or non- denaturing (NaCl) conditions, and digested with trypsin. Di-glycine junctional peptides
(ubiquitinated peptides) were identified including the modified lysine (ubiquitination site) by LC-MS/MS. Purification of ubiquitinated ribosomal proteins was unaffected with denaturing purification, indicating direct modification of these proteins.
Urea Wash NaCl Wash
Gene No. Ub Urea No. Ub NaCl Prot Description Symbol sites WT-CA sites WT-CA Rpl29 60S ribosomal protein L29 10 25 12 24 Rps27a Ubiquitin-40S ribosomal protein S27a 5 13 4 7 Ube2o Ubiquitin-conjugating enzyme E2 O 8 8 9 10 Rpl19 60S ribosomal protein L19 5 7 5 5 Rpl35 60S ribosomal protein L35 4 5 4 6 Rpl37 60S ribosomal protein L37 4 5 7 10 Fau 40S ribosomal protein S30 2 4 3 5 Rps19 40S ribosomal protein S19 3 4 1 1 Rpl39 60S ribosomal protein L39 4 4 4 6 Rpl23a 60S ribosomal protein L23a 3 3 2 2 Uba52 Ubiquitin-60S ribosomal protein L40 3 3 2 3 Rpl5 60S ribosomal protein L5 1 2 1 2 Hba-a1 Alpha globin 1 3 2 3 4 Rpl8 60S ribosomal protein L8 1 1 0 0 Rps25 40S ribosomal protein S25 1 1 1 1 Rps6 40S ribosomal protein S6 1 1 1 1 Rps15 40S ribosomal protein S15 1 1 1 1 Itga1 Integrin alpha-1 0 0 1 0 Prdx2 Peroxiredoxin-2 0 0 1 1 Ryr3 Ryanodine receptor 3 0 0 1 -1 Uba1 Ubiquitin-like modifier-activating enzyme 1 1 0 1 1 Hbbt1 Beta-globin 2 0 3 3 Vmn1r208 Protein Vmn1r208 2 -4 2 0 Total 64 85 69 92
44 Elevated levels of ribosomal proteins in Ube2O null reticulocytes
To test whether ribosomal protein ubiquitination was faithful or spurious, WT and null reticulocyte lysates were probed with antibodies to ribosomal proteins (Figure 2.4).
However, immunoblotting for ribosomal proteins in reticulocyte lysate proved to be difficult for multiple reasons. First, most ribosomal proteins migrate within the same low molecular weight region as α- and β-globin, which constitutes 95% of the reticulocyte proteome and drastically reduces antigen detection sensitivity. Second, to increase reticulocyte counts in peripheral blood, we induced reticulocytosis by serial retro-orbital bleeding or by phenylhydrazine treatment. Reticulocyte lysates were loaded at 100 µg of protein per lane, and slowly electrophoresed on 4-12% SDS-PAGE gradient gels to finely separate low molecular weight proteins. This approach allowed for the visualization of elevated levels of all immunoblotted ribosomal proteins in Ube2O null reticulocytes, consistent with a marked global defect in ribosomal protein degradation.
These results were consistent for reticulocytes from mice induced by serial retro-orbital bleeding and by phenylhydrazine. Specifically, there were elevated levels of Rpl29, our top ribosomal protein hit. We also observed elevated levels of Rpl35, Rpl37, Rpl23a, and
Rpl36a from the 60S subunit, and elevated levels of Rps19 and Rps23 for the 40S subunit. Rpl36a and Rps23 were not identified in the Ube2O reconstitution of null reticulocyte lysate, but were found in elevated levels in null reticulocytes. GAPDH and β- spectrin were blotted as loading controls, and Ube2O was absent in mutant reticulocyte lysates. Unlike β-tubulin or actin, β-spectrin is not degraded via the ubiquitin-proteasome system (Liu et al., 2010), which makes β-spectrin an ideal loading control when probing a ubiquitinating factor mutant.
45
Figure 2.4
Ribosomal proteins and putative Ube2O substrates are present at elevated levels in hem9 reticulocytes.
Reticulocyte lysates from hem9 and WT mice (induced by phenylhydrazine) were resolved by 4-12% SDS-PAGE, and immunoblotted using antibodies to ribosomal proteins (left) and to other non-ribosomal Ube2O substrates (right). β-spectrin and
GAPDH were blotted as loading controls. Ube2O is absent in hem9 reticulocyte lysates.
100 µg of protein was loaded per lane.
46 In addition to ribosomal proteins, other putative Ube2O substrates were found in elevated levels in Ube2O null reticulocytes (Figure 2.4). We had previously identified
AHSP in its ubiquitinated form when we purified ubiquitin conjugates from reticulocyte lysate (Figure 2.2). Accordingly, we found elevated levels of AHSP in null reticulocyte lysates, consistent with a defect in elimination. With this elevation of AHSP in null lysates, we provisionally assigned AHSP as a substrate of Ube2O. From global proteomic analysis of mutant and WT reticulocytes (unpublished data), we further identified proteins that are elevated in levels in the absence of Ube2O. By immunoblotting, we found that two of these proteins, DGKZ and KLHDC4, are also strongly elevated in mutant reticulocytes and were nearly absent in WT reticulocytes.
Finally, from mass spectrometry analysis of Ube2O overexpression in HEK 293 cells
(Chapter 3, Figure 3.11), we identified DDX56 as a protein that is rapidly destabilized by
Ube2O overexpression. Immunoblotting Ube2O null and WT reticulocyte lysates for
DDX56 showed an elevated level of this protein, suggesting that it may be a Ube2O substrate. In all, these data suggest that Ube2O mediates a global program of ubiquitination and proteome remodeling in reticulocytes.
Elevated levels of 80S ribosomes in Ube2O null reticulocytes
Given that ribosomal proteins are elevated in Ube2O null reticulocytes, are 80S ribosomes also found at elevated levels in the mutant? To further test whether these immunoblotting results have physiological significance, we generated polysome profiles for WT and mutant reticulocytes by sucrose gradient centrifugation. We first treated null and WT reticulocytes with cycloheximide to freeze the ribosome on its mRNA, and lysed the cells in a high MgCl2 buffer to maintain ribosome stability on the mRNA. We then fractionated the treated samples on a 20-50% sucrose gradient to allow us to separate
80S ribosomes from higher-order polysomes, or multiple ribosomes in active translation
47 on an mRNA molecule. After centrifugation, the gradient was fractionated and the absorbance reading at 260 nm (A260) was measured as a readout of RNA. Consistent with the immunoblotting data, null reticulocytes have elevated ribosome levels, evident in a substantially exaggerated 80S monosome peak (Figure 2.5). We also observed polysomes of two to six ribosomes on an mRNA in both samples. We then precipitated the proteins from each fraction of the sucrose gradient, and analyzed the proteins by
SDS-PAGE. We were unable to observe any differences in ubiquitination of the ribosomal proteins, though ubiquitination may prevent the antibody from recognizing the protein. The effect size of the null mutant on 80S ribosomes was very substantial, supporting a marked global defect of 80S ribosome elimination.
Next, we sought to confirm that this substantially exaggerated peak is indeed 80S ribosomes. To this end, we increased the salt concentration in the sucrose gradient, which will dissociate non-translating ribosomes into 60S and 40S subunits. Under these conditions of increased ionic strength, the mutant 80S peak was dissociated into more slowly migrating 60S and 40S peaks, confirming that it is truly an 80S peak (Figure 2.5).
Moreover, it has been previously shown by ribosome profiling that high salt concentrations lead to a specific loss of reads close to the start codon and stop codon
(Becker et al., 2013). This suggests that translating ribosomes located in the middle of the transcript are more resistant to dissociation by high salt. We also observed this effect when increasing the ionic strength of our sucrose gradients. While polysomes were unaffected, the mutant 80S peak was strongly dissociated into 60S and 40S peaks, suggesting that the mutant 80S peak is largely non-translating. Moreover, polysome stability is likely unaffected in the Ube2O null mutant; as such, this class of ribosome ubiquitination appears to regulate ribosome elimination rather than stability as seen in the ubiquitin-K63R mutant of S. cerevisiae (Spence et al., 2000).
48
Figure 2.5
Aberrant polysome profile of the hem9 mutant.
Reticulocyte lysates from hem9 and WT mice (induced by serial retro-orbital bleeding) were fractionated on a 20-50% sucrose gradient. The X-axis represents the position in the sucrose gradient, the Y-axis represents OD260. hem9 shows a strong peak of non- translating ribosomes (top). Under increased ionic strength of the sucrose gradient, the
80S monosome peak was dissociated into 60S and 40S subunits (bottom).
49 Discussion
Since Ube2O null mice have a hypochromic, microcytic anemia, we first looked at the ubiquitin profile of WT and null reticulocytes. We showed that Ube2O null reticulocytes are deficient in a set of low molecular weight ubiquitin conjugates. We then purified these conjugates and definitively identified α-globin in its ubiquitinated form. From this, we also identified AHSP, a well-characterized α-globin chaperone, as a putative Ube2O substrate. Next, we reconstituted null reticulocyte lysates with recombinant Ube2O.
Using LC-MS/MS, we identified ribosomal proteins as the dominant target of Ube2O.
Finally, we observed elevated levels of ribosomal proteins and 80S ribosomes, consistent with a global defect of elimination.
First, these data confirm the longstanding hypothesis that Ube2O is a dominant E2 in erythroid differentiation. The Ube2O null phenotype indicates that a failure of ubiquitination can cause anemia in mice. Though the anemia is mild, it is an important finding in the ubiquitin field. To date, there are few diseases linked to mutations in ubiquitinating factors (i.e. Ubiquilin-2 mutants and ALS, Parkin mutations in Parkinson’s disease) (Deng et al., 2011; Lucking et al., 2000). Thus, Ube2O must play a critical role during the terminal stages of erythroid differentiation. Moreover, Ube2O is upregulated in both human and mouse erythropoiesis. This suggests that there are possible human mutations in Ube2O that cause microcytic anemia in patients. However, Ube2O deficiency anemia resembles iron deficiency anemia, which may not warrant whole exome sequencing. This may reduce the number of confirmed probands.
The aberrant ubiquitin profile from Ube2O null reticulocytes also supports the hypothesis that Ube2O is a dominant E2. That a single ubiquitinating factor should produce such large changes in ubiquitin profiles is highly unusual and likely an unprecedented
50 phenotype in the ubiquitin field due to the multiplicity of ubiquitin ligases. These ubiquitin conjugates may represent direct Ube2O substrates that are no longer ubiquitinated in the null mutant. Since there appears to be residual ubiquitin conjugates, this also suggests that there might be compensatory mechanisms of ubiquitination in the absence of Ube2O. However, these changes in the ubiquitin profile may also result from indirect effects of Ube2O deficiency. Nonetheless, in toto, these data confirm that Ube2O is a dominant E2 in erythroid differentiation.
We initially considered the possibility that Ube2O is the first identified globin ligase.
Since the UPS was discovered in the reticulocyte, it has been linked to the degradation of globin chains. Rabbit reticulocytes were shown to rapidly degrade abnormal globin using an ATP-dependent proteolytic system (Etlinger and Goldberg, 1977). In fact, excess α-globin is degraded particularly rapidly, with a half-life estimated at ~1 minute or less (Hunter and Jackson, 1972), among the shortest known in all eukaryotes. In fact, unpaired α-chains are degraded in a ubiquitin-dependent manner (Shaeffer, 1988;
Shaeffer and Cohen, 1997, 1998; Wickramasinghe and Lee, 1998). More recently, accumulation of excess α-globin in a murine model of β-thalassemia led to the upregulation of UPS components in erythroblast precursors (Khandros et al., 2012).
In this view, the pathophysiology of Ube2O deficiency (accumulated excess α-chains) should exacerbate β-thalassemia. However, contrary to expectation, Ube2O deficiency ameliorated a murine model of β-thalassemia intermedia. This surprising finding had many overarching implications. First, this suggested that there are other substrates besides globin that mediate the Ube2O phenotype. Second, inhibition of Ube2O appears to be a novel therapeutic target for treating β-thalassemia. Since Ube2O is expressed only in the erythroid system, we would not expect any side effects from inhibiting this
51 enzyme. Moreover, Ube2O has a defined active site with a catalytic cysteine that is subject to pharmacological inhibition. Therefore, we may be able to ameliorate β- thalassemia by inhibiting a ubiquitin ligase.
When reconstituting reticulocyte lysates, Ube2O formed ubiquitin conjugates with only
E1, ubiquitin, and ATP. We identified ribosomal proteins as the major target of Ube2O, which strongly supported another longstanding hypothesis that Ube2O is an E2-E3 hybrid (Berleth and Pickart, 1996). These data may also explain why Ube2O is unusually large (in order to recognize the 80S ribosome). The ubiquitination of the ribosome has been previously reported, mainly in regulatory roles. For example, K63-linked ubiquitination of 40S subunits were shown to regulated polysome stability (Spence et al.,
2000). Recently, 40S ubiquitination was demonstrated in response to oxidative stress
(Silva et al., 2015) and the unfolded protein response (Higgins et al., 2015). In contrast, the majority of Ube2O ubiquitination was mapped onto the large 60S subunit. We did not identify any ubiquitination of the 40S ribosomal proteins reported in these previous studies. Could this ubiquitination of the 60S ribosome by Ube2O serve as a signal for degradation instead? Since ribosomes are eliminated from differentiating reticulocytes, we favored the hypothesis that Ube2O ubiquitinates ribosomes to target them for degradation. We address this possibility in Chapter 3.
These observations could also elucidate the phenotype of the Ube2O null mouse. On blood smear, there are basophilic inclusions in the whole blood of the null animal; this may represent basophilic staining of ribosomal RNA that has not been eliminated from differentiating reticulocytes. Moreover, autophagosome-like structures observed by EM could possibly reflect compensatory mechanisms of ribosome elimination in the absence of Ube2O. As mentioned in Chapter One, the Ube2O null has a microcytic anemia; while
Cyclin D3 has been shown to regulate cell size through controlling the number of cell
52 divisions (Marion et al., 2011), the underlying causes of microcytosis are still not well understood. Another determinant of cell size is by splenic processing; macrophages of the spleen can remove a variety intracellular inclusions from circulating red blood cells
(Crosby, 1957; Schnitzer et al., 1972). Thus, it is possible that the inclusions of uncleared ribosomes are subjected to phagocytosis by splenic macrophages, which may reduce cell size while leaving the red blood cell intact.
Interestingly, Ube2O has been previously shown to interact with ribosomal proteins by global yeast two-hybrid studies (Markson et al., 2009) and high-throughput affinity- purification mass spectrometry (Huttlin et al., 2015). We are particularly interested in our strongest candidate Rpl29, which is a eukaryotic-specific ribosomal protein. Since Rpl29 is found on the solvent exposed surface of the 60S ribosome (Klinge et al., 2011), it is likely more accessible to direct ubiquitination by Ube2O. Moreover, deletion of Rpl29 in mice results in global growth deficiencies (Kirn-Safran et al., 2007), suggesting that
Rpl29 is a key regulator of protein synthesis. The ubiquitination of Rpl29 may explain why Ube2O mice are anemic.
40S subunits were also ubiquitinated, though at much lower efficiency than 60S ribosomal proteins. One of these subunits is Rps19, which is mutated in nearly 25% of patients with Diamond-Blackfan anemia (DBA), a ribosomopathy (Boria et al., 2010)
Interestingly, mutant forms of Rps19 are stabilized by proteasome inhibitors (Cretien et al., 2008), suggesting that ribosomal proteins may be degraded by the proteasome. We address the degradation of ribosomal proteins in Chapter 4. Moreover, since DBA is caused by a deficiency of ribosomal proteins, we entertained the hypothesis that Ube2O deficiency may be ameliorative of DBA (since Ube2O deficiency is characterized by excess ribosomes). However, DBA mutations lead to ineffective erythropoiesis at earlier stages of erythroid differentiation before the reticulocyte. Thus, inhibiting Ube2O in the
53 reticulocyte may not rescue the deleterious effects in erythroid precursors.
Could this explain why Ube2O is specifically upregulated in reticulocytes? The vast majority of proteins from precursors have already been eliminated by the reticulocyte stage; in fact, the reticulocyte proteome consists essentially of globin and ribosomes.
Once released into circulation, nascent reticulocytes eliminate their mitochondria and ribosomes within 48 to 72 hours (Geminard et al., 2002; Ney, 2011). Through multiple lines of experiments, we confirmed that the ubiquitination of ribosomal proteins by
Ube2O is direct and faithful in the physiological context of erythroid differentiation.
Moreover, this appears to be a highly specific program of ubiquitination and degradation that drives ribosome elimination. In the absence of Ube2O, both ribosomal proteins and
80S ribosomes are found at elevated levels in differentiating reticulocytes. The Ube2O null phenotype was indeed much stronger than a yeast mutant defective in the autophagy-dependent turnover of ribosomes under starvation conditions (Kraft et al.,
2008). In summary, over 20 years since its initial discovery, Ube2O was shown to directly ubiquitinate ribosomal proteins, and deficiency of Ube2O to lead to elevated levels of ribosomal proteins and 80S ribosomes in reticulocytes.
54 Materials and Methods
Antibodies
The following antibodies were used for immunoblot analysis: anti-ubiquitin-protein conjugates (Biomol, UG9510, rabbit polyclonal, used at 1:2000); anti-ubiquitin (Santa
Cruz Biotech, sc-8017, mouse monoclonal, used at 1:200); streptavidin-HRP (Fisher,
21130, used at 1:10000); anti-Rpl35 (Sigma, SAB4500233, rabbit polyclonal, used at
1:1000); anti-Rpl36a (Novus, H00006173-M02, mouse monoclonal, used at 1:500); anti-
Rpl37 (Abcam, ab103003, rabbit polyclonal, used at 1:500); anti-Rpl23a (Sigma,
SAB1300595, rabbit polyclonal, used at 1:1000); anti-Rpl29 (Santa Cruz Biotech, sc-
103166, goat polyclonal, used at 1:200); anti-Rps19 (Sigma, SAB2500898, goat polyclonal, used at 1:1000); anti-Rps23 (Novus, H00006228-M02, mouse monoclonal, used at 1:500); anti-Ube2O (Bethyl, A301-873A, rabbit polyclonal, used at 1:2000); anti-
β-spectrin (Santa Cruz Biotech, sc-374309, mouse monoclonal, used at 1:200); anti-
GAPDH (Abcam, ab8245, mouse monoclonal, used at 1:5000); anti-DGKZ (Bethyl,
A302-967A, rabbit polyclonal, used at 1:2000); anti-KLHDC4 (Santa Cruz Biotech, sc-
398467, mouse monoclonal, used at 1:200); anti-DDX56 (Origene, UM800050, mouse monoclonal, used at 1:2000); and anti-AHSP (Rockland, 100-401-E79, rabbit polyclonal, used at 1:1000).
Animal care and analysis
Ube2OE1121X/E1121X mice (Ube2Ohem9/hem9) were generated previously (Kile et al., 2003) and bred onto a C57BL/6J genetic background. Genotyping and mapping of the allele was completed as described previously (Tian et al., 2008). XL530 is a Bay Genomics
Genetrap ES cell clone (129P2/OlaHsd) of Ube2O available from the Mouse Mutant
Resource Center (MMRRC, 009349-UCD) containing a β-geo cassette inserted into
55 intron 1. All genetically modified mice were born and housed in the barrier facility at
Children’s Hospital Boston and handled according to approved protocols. Mice were maintained on the Prolab RMH 3000 diet (Lab Diet; 380 ppm iron). The facility employs a constant dark-night light cycle and all mice were provided both water and food ad libitum. Because of differences in iron metabolism between male and female mice, only females were analyzed for peripheral blood complete blood count (CBC) analysis.
Reticulocyte induction and blood analysis
Whole blood was collected retro-orbitally from mice anesthetized with intraperitoneal injections of 1.0% tribromoethanol in isoamyl alcohol (Avertin) in PBS. Reticulocytosis was induced by two methods: (1) serial retro-orbital bleeds of approximately 2% of the mouse’s body weight on days 1, 3, and 5, or (2) intraperitoneal phenylhydrazine injection at 40 mg/kg on days 0, 1 and 3. Reticulocytes were harvested on day 7 into EDTA- coated Microtainer tubes (BD Biosciences). Samples were analyzed on an Avida 120 analyzer (Bayer) in the Boston Children’s Hospital Department of Laboratory Medicine
Clinical Core Laboratories. Whole blood samples were washed with ice-cold PBS, and the buffy coat was manually removed by pipetting. The packed cells were snap-frozen with liquid nitrogen and then stored at -80°C, or used immediately.
Ubiquitin immunoblot analysis of reticulocyte lysate
Approximately 100 µl of packed blood cells enriched for reticulocytes was washed with ice-cold PBS, and total protein harvested in modified RIPA buffer (50 mM Tris pH
7.5,150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). Protein concentration was measured by Bichinchoninic acid (BCA) assay (Pierce), and samples were diluted in Laemmli buffer (0.2 M DTT final), boiled for 5 minutes, and subjected to electrophoresis through 4-12% polyacrylamide gels (Invitrogen) and immunobloted
56 analysis using a rabbit anti-ubiquitin-protein conjugates antibody (1:2000, Biomol). Blots were incubated anti-rabbit secondary antibody conjugated to horseradish peroxidase
(1:5000) (VWR) and then subjected to chemiluminescence (PerkinElmer ECL).
Fractionation of reticulocyte lysate by ion exchange chromatography
Reticulocytes were lysed in 25 mM Tris HCl pH 7.5, 10 mM N-ethylmaleimide (NEM), 1 mM AEBSF, 5 mM EDTA, 10 mM 1,10-phenanthroline, and 1x protease inhibitor cocktail. Lysate was clarified by ultracentrifugation at 50,000 rpm for 1 hour at 4°C. The total protein was precipitated by 80% saturated ammonium sulfate, and centrifuged immediately at 14,000 rpm for 5 minutes at 4°C. The pellet was resuspended in 500 µl of
50mM MES pH 6.0 and 1 mM EDTA. To remove the excess ammonium sulfate, the sample was dialyzed against 1 L of 50mM MES pH 6.0 and 1mM EDTA for 2 hours, and then an additional 1 L for 4 hours. The sample was then fractionated by cation exchange chromatography on a HiTrap SP HP column (GE) by FPLC (GE). Proteins were eluted with a linear gradient from 0 to 1 M NaCl. Fractions were analyzed by 4-12% SDS-PAGE
(Invitrogen), transferred onto 0.45 µM PVDF membranes (Amersham), and immunoblotted with a mouse monoclonal anti-ubiquitin antibody (1:200, Santa Cruz
Biotech). Fractions containing relevant ubiquitin conjugates were pooled and dialyzed overnight against 1 L of 50 mM Tris HCl pH 7.5, 5 mM EDTA, and 10% glycerol at 4°C.
Ubiquitin affinity conjugate purification
Ubiquitin conjugates were then purified using Tandem Ubiquitin Binding Entities
(TUBEs) coupled to agarose beads (LifeSensors, UM402). The slurry was incubated with wash buffer (20 mM Tris pH 8.0, 15 mM NaCl, and 0.1% Tween-20) for 5 minutes on a rocker platform, and then washed twice with wash buffer. After equilibration, the sample was incubated with the TUBEs agarose for 1 hour at 4°C. The beads were
57 washed three times with 1 mL of wash buffer. To eluted the ubiquitinated proteins, the beads were resuspended with 5x Laemmli buffer and boiled at 95°C for 5 minutes.
Eluted samples were analyzed by 4-12% SDS-PAGE (Invitrogen) and immunobloted using an anti-ubiquitin antibody (1:200, Santa Cruz Biotech), or stained using SYPRO
Ruby (Bio-Rad) for band detection. Bands of interest were excised and subjected to mass spectrometry identification.
LC-MS/MS analysis of excised gel bands
The following steps are performed at room temperature unless stated otherwise. The excised gel band was destained with 50 mM HEPES pH 8.2/acetonitrile (ACN) (70/30 v/v) by vortexing. The gel band was then dehydrated with 50 mM HEPES pH 8.2/ACN
(50/50 v/v) for 15 minutes, incubated in 100% ACN for 15 minutes, and then dried by vacuum concentration. To reduce and alkylate disulfide bonds, the sample was resuspended in 5 mM TCEP for 25 minutes, incubated with 14 mM iodoacetamide for 30 minutes in the dark, and then incubated with 10 mM DTT for 15 minutes (all solutions in
50 mM Hepes pH 8.2). Next, the samples were washed twice with 50 mM HEPES pH
8.2/ACN (50/50 v/v) for 15 minutes, washed once with 100% ACN for 15 minutes, and then dried by vacuum concentration. The dried sample was incubated in 10 ng/µl trypsin in 50 mM HEPES pH 8.2 overnight at 37°C. After trypsin digestion, the peptides were eluted from the gel with 50% ACN and 5% formic acid (FA). The eluted peptides were dried by vacuum concentration and resuspended in 5% FA. Next, the peptides were desalted using the C18 Stop and Go Extraction Tip (STAGE-Tip) (Rappsilber et al.,
2003), and resuspended with 5% FA prior to LC-MS/MS analysis.
Ube2O reconstitution of null reticulocyte lysates
Reticulocyte samples were lysed in 25 mM Tris HCl pH 7.5, 1 mM AEBSF, 5 mM EDTA,
58 10 mM 1,10-phenanthroline, and 1x protease inhibitor cocktail. After incubation on ice for
5 minutes, the lysate was clarified by ultra-centrifugation at 50,000 rpm for 1 hour at 4°C.
The supernatant was then transferred to low-protein binding tubes (Eppendorf), and treated with 10 mM NEM for 30 minutes at room temperature in the dark to inactivate all endogenous ubiquitin ligases and deubiquitinating enzymes. NEM was quenched with excess of dithiothreitol (DTT) with a final concentration of 6 mM. Protein concentration was measured by BCA assay (Pierce). Ube2O activity was reconstituted in alkylated, clarified null reticulocyte lysate. The final concentrations in the reaction were 10 mM Tris
HCl pH 7.5, 5 mM MgCl2, 2 mM ATP, 10 μM biotinylated ubiquitin (Boston Biochem, UB-
560), 200 nM His-UBE1 (Boston Biochem, E-306), 100 ng/mL recombinant Ube2O, and
25 mg/mL reticulocyte lysate. To inhibit proteasome activity, 5 μM PS-341 and
5 μM epoxomicin were added. Prior to the reaction, His-UBE1 and biotinylated ubiquitin were pre-incubated with 2mM ATP for 5 minutes at room temperature. The ubiquitination reaction was then incubated at 37°C for 45 minutes. For SDS-PAGE analysis, the reaction was boiled in 5x Laemmli buffer. When the sample was used for conjugate purification, the ubiquitination reaction was stopped by alkylation with 10 mM NEM.
Biotin-ubiquitin conjugate purification and elution
Biotinylated ubiquitin conjugates were purified using high capacity NeutrAvidin agarose
(Thermo Scientific, 29202). 100 µl of NeutrAvidin agarose was equilibrated to room temperature, and washed with 1x PBS. The ubiquitination reaction was diluted to 500 µl with 1x PBS, and incubated with the equilibrated NeutrAvidin resin for 1 hour at room temperature. The resin was washed on a spin column with 100 bed volumes of 1x PBS with either non-denaturing 1 M NaCl or denaturing 6 M urea, and then washed with 20 bed volumes of 1x PBS. After PBS wash, the resin was incubated with 5 mM TCEP for
25 minutes at room temperature, and then incubated with 14 mM NEM for 30 minutes at
59 room temperature. Next, the resin was resuspended with 50 mM HEPES pH 8.0 and 10 ng/µl trypsin and incubated at 37°C overnight. The trypsin digestion was quenched with
5% FA, and filtered with a 2 mL spin column to remove all agarose resin. The resin was eluted 2x with 200 µl of 3% FA and 3% ACN. The eluted peptides were desalted by
STAGE Tip, and dried by vacuum concentration.
LC-MS/MS analysis of eluted peptides
Prior to LC-MS/MS analysis, the dried peptides were dissolved in 5% FA. All peptides were separated on a reverse-phase C18 column and directly injected into the LTQ
Orbitrap Velos where the most intense MS1 peaks detected in the Orbitrap were selected and fragmentated for MS2 analysis in the linear ion trap. Peptide identification was performed with SEQUEST with variable modifications including diglycine lysine
(+114.0429 Da), oxidized methionine (+15.9949 Da), and static modifications including cysteine alkylation with NEM (+125.1253 Da). Using in-house software we quantified each peptide in the samples by spectral counting (number of times a peptide is identified in the sample analyzed).
Immunoblot analysis of putative Ube2O substrates
Reticulocytes were washed 5x with 5 mL of ice-cold PBS with manual removal of the buffy coat by pipetting. The packed cells were then lysed with 2x volume of urea lysis buffer (50 mM HEPES pH 7.5, 8M urea, 75 mM NaCl, 1x EDTA-free protease inhibitor cocktail (Roche), 1x PhosSTOP phosphatase inhibitor cocktail (Roche)), for 15 minutes at room temperature. The lysate was clarified by centrifugation at 13,200 rpm for 10 minutes at 4°C, and the supernatant was transferred to low-protein binding tubes
(Eppendorf). Protein concentration was measured by BCA assay (Pierce). 100 µg of protein per lane was diluted in Laemmli buffer, and analyzed by 4-12% SDS-PAGE
60 (Invitrogen) using MES running buffer. Dual color protein standards (Bio-Rad) were used to approximate molecular weight. The samples were transferred to 0.2 µM PVDF membranes (Amersham), and blocked with 5% BSA (Sigma) in 1x TBST for 1 hour at room temperature. Membranes were incubated with the respective antibodies at 4°C overnight (see Antibodies section). Finally, the membranes were incubated with IRDye secondary antibodies (LI-COR) for 1 hour at room temperature, washed 3x with TBST and 1x with TBS buffer, and imaged using the Odyssey CLx infrared imaging system (LI-
COR).
Polysome profile analysis of reticulocytes
Fresh null and WT reticulocytes were washed 5x with ice-cold PBS with 100 µg/mL cycloheximide. The reticulocytes were then lysed with ice-cold 200 mM Tris HCl pH 8.8,
25 mM MgCl2, 25 mM KCl, 1% Triton X-100, 1.3% sodium deoxycholate, 100 µg/mL cycloheximide, and 200 units/mL RNAse inhibitor (New England Biolabs). The lysate was rotated end on end for 10 minutes at 4°C, and then centrifuged at 3,000 rpm for 10 minutes at 4°C. 60 µl of the reticulocyte lysate was loaded onto a 20-50% (w/w) sucrose gradient (50 mM Tris HCl pH 8.8, 25 mM MgCl2, 25 mM KCl, and 100 µg/mL cycloheximide). For increased ionic strength, the salt concentration of the sucrose gradient was increased to 250 mM KCl. Sucrose gradients were made in 14 mL thin- walled, ultra-clear centrifuge tubes (Beckman) using a Gradient Station (Biocomp) and cooled to 4°C before use. The loaded sucrose gradient was centrifuged at 40,000 rpm for 2 hours and 30 minutes at 4°C with minimal acceleration and no active deceleration in an SW 40 Ti rotor (Beckman). Fractions were collected using a Gradient Station
(Biocomp), and absorption was measured at 260 nm and 290 nm. Collected fractions were stored at -80°C. Protein precipitation was performed with ice-cold trichloroacetic
61 acid (TCA) to a final concentration of 10%. Fractions were incubated on ice for 10 minutes, and centrifuged for 25 minutes at max speed at 4°C. Pellets were dried by vacuum concentration, resuspended in 30 µl of Laemmli buffer and analyzed by 4-12%
SDS-PAGE.
Contributions
I performed the majority of the experiments described in this Chapter. Miguel Prado performed the mass spectrometry analysis. Stefanie de Poot and Daniel Finley edited the manuscript.
62
CHAPTER THREE
Ube2O drives the elimination of 80S ribosomes from cells
Background
The mature erythrocyte is a highly specialized cell with a hemoglobin concentration of nearly 95%. In principle, this should require a specific program of cellular remodeling to saturate its proteome with globin. After reticulocytes are released into circulation, these cells will eliminate all intracellular organelles and ribosomes within 72 hours (Geminard et al., 2002; Ney, 2011). The mechanisms of mitochondrial elimination have been studied in some detail. Nix is an outer mitochondrial protein that is upregulated during terminal erythroid differentiation (Aerbajinai et al., 2003). Nix null mice were found to be severely deficient in eliminating mitochondria from nascent reticulocytes (Sandoval et al.,
2008; Schweers et al., 2007). Further work showed that Nix is actually the selective autophagy receptor for mitochondria (Novak et al., 2010); without Nix, mitochondria do not completely fuse with the autophagosome. Of note, the function of Nix is ubiquitin- independent. Though differentiating reticulocytes in Nix null mice have a clear defect in mitochondrial elimination, they have no defect in ribosomal degradation. To date, there is little known about how ribosomes are eliminated from reticulocytes. The only mechanistic understanding of this process is that erythroid pyrimidine 5’-nucleotidase participates in the breakdown of ribosomal RNA (rRNA), and that its deficiency causes a hemolytic anemia (Valentine et al., 1974).
Though ribosome elimination is not well understood in mammalian cells, the degradation of mature ribosomes has been described in yeast and plants. Under nutrient starvation, mature ribosomes were degraded in yeast by a novel type of selective autophagy, termed ribophagy (Kraft et al., 2008). In yeast, ribosomal ubiquitination by Listerin was actually protective of starvation-induced ribophagy (Bengtson and Joazeiro, 2010).
Moreover, ribophagy was actually dependent on the deubiquitinating enzyme complex,
Ubp3p/Bre5p (Kraft et al., 2008). Yeast cytoplasmic ribosomes were also rapidly
64 degraded in response to rapamycin treatment (Pestov and Shcherbik, 2012). When yeast was cultured in rich medium, rapamycin inhibition of TOR led to a rapid decrease in rRNA by up to 60%. Unfortunately, there are no additional mechanistic studies on this phenomenon. Finally, a ribophagy-like process was also reported in A. thaliana, namely the degradation of rRNA by an intracellular RNase T2 (Hillwig et al., 2011). Therefore, ribosome elimination appears to occur in yeast under various stressors and in plants; however, this process has yet to be elucidated in mammalian cells.
Ube2O is an E2 ubiquitin-conjugating enzyme that is upregulated during terminal erythroid differentiation (Wefes et al., 1995). When null reticulocyte lysates were treated with recombinant Ube2O, ribosomal proteins were identified as the main target of
Ube2O. Null reticulocytes were then shown to have highly elevated levels of ribosomal proteins and 80S ribosomes. Since ubiquitination is canonically a signal for substrate degradation, these data were consistent with a critical defect of elimination. Since mature erythrocytes do not have ribosomes, Ube2O may execute the programmed elimination of ribosomes from differentiating reticulocytes. If Ube2O does serve a critical role in ribosome elimination, is Ube2O sufficient to drive ribosome elimination?
Here we report the finding that Ube2O null reticulocytes are severely deficient in the elimination of ribosomes. Moreover, Ube2O is sufficient to drive ribosome elimination in non-erythroid 293 cells. We show that Ube2O targets pre-existing ribosomes, and that ribosome elimination does not affect cell viability. Finally, we use quantitative mass spectrometry to confirm the elimination of ribosomal proteins from 293 cells. From this approach, we identify non-ribosomal proteins that are also destabilized by Ube2O overexpression. Thus, Ube2O executes a broad program of degradation that remodels the proteome.
65 Results
Critical defect of ribosome elimination in Ube2O null reticulocytes
In Chapter Two, we showed that ribosomal proteins constitute a major class of physiological targets for Ube2O. As a consequence, there is a marked elevation of both ribosomal proteins and 80S ribosomes in fresh Ube2O null reticulocytes from induced mice. Does this phenotype reflect a major deficiency in ribosome elimination in the erythroid lineage? To this end, we studied the dynamics of ribosome elimination in nascent reticulocytes from mutant and WT mice. We cultured nascent reticulocytes ex vivo at 37°C, during which they spontaneously mature into erythrocytes over 48 to 72 hours (Sandoval et al., 2008). As discussed in Chapter One, reticulocytes will eliminate all intracellular organelles during this time frame (Geminard et al., 2002). We leveraged this powerful experimental system to systematically and quantitatively monitor of two key processes of reticulocyte maturation, mitochondrial and ribosomal elimination.
Specifically, cultured reticulocytes were analyzed by flow cytometry using the cell permeable MitoTracker dye to measure mitochondria and thiazole orange to measure
RNA and specifically rRNA, which is the most abundant species in reticulocytes. We also stained for CD71 (or Transferrin receptor 1), which is a reticulocyte-specific receptor that is lost in mature red blood cells.
Apparent after 24 hours of ex vivo culture, null CD71+ reticulocytes stained intensely for thiazole orange, but weakly for MitoTracker (Figure 3.1). In contrast, WT reticulocytes stained weakly for both after undergoing programmed elimination of mitochondria and ribosomes. By 48 and 72 hours of differentiation, null CD71+ reticulocytes still stained intensely thiazole orange-positive, in contrast to WT reticulocytes, which have completely eliminated their ribosomes and mitochondria. In fact, the difference in
66
Figure 3.1
FACS analysis showing defect in rRNA elimination in hem9 reticulocytes.
Reticulocytes were collected from WT and Ube2O null mice induced by serial retro- orbital bleeding. After 48 hours of ex vivo differentiation, hem9 CD71+ reticulocytes
(blue) retained their thiazole orange (TO) staining (which stains for RNA, and predominantly rRNA in reticulocytes) more than WT CD71+ reticulocytes retained their
TO staining (red) (top). Loss of MitoTracker staining is largely unaffected by the hem9 mutation (bottom).
67 thiazole orange signal between Ube2O null and WT reticulocytes spanned multiple orders of magnitude, indicating a severe defect in ribosome (rRNA) elimination.
Mitochondrial content was equivalent between null and WT reticulocytes during ex vivo differentiation, perhaps reflecting that mitochondrial elimination proceeds through the autophagic pathway (Sandoval et al., 2008; Schweers et al., 2007). When analyzing both CD71+ and CD7- cells, mutant samples retained a strong population that is thiazole orange-positive and MitoTracker-negative (Figure 3.2); in contrast, WT samples stained weakly for both markers. rRNA content was also compared to CD71 staining as a marker for progression from reticulocyte to erythrocyte. WT reticulocytes concurrently lost their staining for both thiazole orange and CD71, indicating normal terminal differentiation. In contrast, null reticulocytes maintained their staining for thiazole orange while losing CD71 and proceeding through terminal differentiation (Figure 3.3). Loss of
CD71 proceeds through the exosome pathway (Liu et al., 2010), which is unaffected in the null mutant (data not shown). Thus, the Ube2O null mutant is strongly defective in ribosome elimination during terminal erythroid differentiation. Moreover, the elevated levels of ribosomal proteins and 80S ribosomes in Ube2O null reticulocytes likely reflect a global defect in degradation.
The stability of individual ribosomal proteins was also followed using ex vivo cultured reticulocytes and immunoblotting (Figure 3.4). After ex vivo culture, we lysed the cells with a denaturing buffer (8M urea), and analyzed the lysate by SDS-PAGE. We observed rapid elimination of ribosomal proteins from WT reticulocytes by 24 hours of ex vivo culture, which is consistent with thiazole orange staining by flow cytometry analysis.
We stained for Rpl29, Rpl35, and Rpl23a, which were our top ribosomal candidates. In contrast, these ribosomal proteins were stabilized in null reticulocytes up to 48 to 72 hours, consistent with flow cytometry data. These immunoblotting data confirm that both
68
Figure 3.2
Bivariate analysis of MitoTracker and thiazole orange staining during ex vivo differentiation.
Reticulocytes were collected from WT and Ube2O null mice induced by serial retro- orbital bleeding. X-axis shows MitoTracker signal; Y-axis shows thiazole orange signal. hem9 reticulocytes have a severe defect in thiazole orange elimination, but minimal defect in mitochondrial elimination (bottom) compared to WT reticulocytes (top).
69
Figure 3.3
Bivariate analysis of CD71 and thiazole orange staining during ex vivo reticulocyte differentiation.
Reticulocytes were collected from WT and Ube2O null mice induced by serial retro- orbital bleeding. X-axis shows CD71 staining; Y-axis shows thiazole orange signal. hem9 reticulocytes retaining thiazole orange staining as CD71 signal is lost over time
(bottom); WT reticulocytes retain minimal thiazole orange staining (top).
70
Figure 3.4 hem9 defect in elimination of ribosomal proteins by immunoblot analysis.
WT and hem9 reticulocytes (induced by serial retro-orbital bleeding) were cultured ex vivo for the indicated times. Samples were taken for immunoblot analysis. GAPDH, which is not eliminated from reticulocytes, serves as a loading control. AHSP is also strongly stabilized in hem9 reticulocytes. 100 µg of protein was loaded per lane.
71 rRNA and ribosomal proteins are stabilized in the Ube2O null mutant beyond the normal program of elimination. In addition to immunoblotting for ribosomal proteins, we also probed our samples with an anti-AHSP antibody. We subsequently observed the stabilization of AHSP, another putative Ube2O substrate, which was markedly elevated in null reticulocytes. In total, these data indicate that AHSP is another Ube2O substrate.
Moreover, these data show that the effects of Ube2O are not limited to only ribosomal proteins during terminal differentiation. GAPDH was blotted as a loading control in these experiments.
Finally, we compared polysome profiles of WT and mutant reticulocytes after 31 hours of ex vivo culture. Similar to the analysis of nascent reticulocytes, we treated the cultured reticulocytes with cycloheximide, and analyzed them on a 20-50% sucrose gradient. We found that the elimination of ribosomes is nearly complete by 31 hours in WT reticulocytes; in contrast, the 80S peak remained very prominent in Ube2O null reticulocytes (Figure 3.5). Interestingly, despite the persistence of this peak, little translation was evident in the gradient profile. In all, we confirmed by flow cytometry, immunoblotting, and polysome profiling that 80S ribosomes are stabilized in Ube2O null reticulocytes, indicating a critical defect in elimination.
Ube2O ubiquitinates ribosomal proteins in HEK293 cell lysates
Since Ube2O mediates ribosome elimination in erythroid differentiation, could Ube2O be sufficient to drive ribosomal degradation in a non-erythroid cell? Is there an intrinsic red blood cell factor that is required for ribosome recognition by Ube2O or does Ube2O function autonomously? To test this, we first asked if Ube2O can ubiquitinate ribosomal proteins in non-erythroid cell lysates. Similar to the reconstitution of Ube2O in null reticulocyte lysate, we added back recombinant Ube2O (WT and CA) to HEK293 lysates
72
Figure 3.5
An 80S monosome peak persists in hem9 reticulocytes after 31 hours of ex vivo differentiation.
Polysome gradient analysis shows an exaggerated 80S peak in hem9 but not WT reticulocytes (induced by serial retro-orbital bleeding) after 31 hours of ex vivo differentiation.
73 at physiological levels. Prior to adding Ube2O, we alkylated the lysate with NEM and then inactivated NEM with excess DTT. After incubating the reaction with biotin-ubiquitin and E1 at 37°C, we purified the newly formed ubiquitin conjugates with NeutrAvidin agarose and analyzed the sample by LC-MS/MS. We found that the majority of ubiquitinated peptides belonged to ribosomal proteins, indicating that Ube2O can ubiquitinate the ribosome in a non-erythroid lysate (Table 3.1). Again, the majority of the ubiquitinated proteins were from the 60S ribosomal subunit with ribosomal proteins from the 40S ribosome. The most common 60S ribosomal subunits were Rpl37, Rpl19,
Rpl29, Rpl36a, Rpl35, and Rpl23a. We also identified Rps25 and Rps15 from the 40S ribosome. In this experiment, we found that other non-ribosomal proteins were ubiquitinated, though we detected far fewer peptides for these proteins. Finally, when analyzing total peptides that were enriched, gene ontology analysis was significant for proteins from the large ribosomal subunit. These data suggest that Ube2O may also drive the elimination of ribosomes when overexpressed in non-erythroid cells.
Ube2O is sufficient to drive ribosome elimination in non-erythroid cells
Next, we cloned the human Ube2O gene in untagged form into the pcDNA 5/FRT/TO expression vector, which contains a tetracycline-regulated promoter and a Flp
Recombination Target site allowing for stable integration into a specific genomic location. We also cloned the catalytically inactivate Ube2O mutant (Ube2O C1040A, CA) into the same vector. We then transfected Flp-In T-REx 293 cells with the respective
Ube2O construct and the pOG44 plasmid containing the Flp recombinase under control of the human CMV promoter. After transfection, we selected for stable transformants using hygromycin B. Using these stable transformants, we can induce the overexpression of Ube2O WT and Ube2O CA with doxycycline (Figure 3.6). Of note, expression of Ube2O was the same in both cell lines, as judged using GAPDH used as a
74 Table 3.1
Ubiquitination sites identified when Ube2O was reconstituted in HEK293 lysates.
The table summarizes the number of ubiquitination sites (modified lysines) and spectral counts of ribosomal proteins (di-glycine juncational peptides) when recombinant Ube2O was added to 293 cell lysates.
75
Figure 3.6
Ube2O induction by doxycycline treatment in Flp-In T-REx 293 cells.
Full length Ube2O WT and full-length Ube2O C1040A (CA) genes were stably integrated into Flp-In T-REx 293 cells and selected by hygromycin B. Ube2O was induced with doxycycline for 24 hr; 20 µg of cell lysate was loaded per lane for Immunoblotting with anti-Ube2O and anti-GAPDH as a loading control (top). Ube2O expression levels were compared to expression levels in reticulocytes at 20 µg of cell lysate per lane (bottom).
76 loading control. We then compared Ube2O levels to those in reticulocyte lysate. Indeed, we observed strong induction of Ube2O, reaching levels above those in reticulocytes
(Figure 3.6). As such, we validated these stable cell lines as a powerful tool to study
Ube2O function.
Next, we induced Ube2O expression for 48 to 72 hours to recapitulate the time frame of terminal erythroid differentiation. We treated the cells with doxycycline, and replenished the media every 24 hours to ensure high levels of doxycycline. We then lysed the cells with a denaturing buffer (8M urea), and analyzed the lysate by SDS-PAGE. After 48-72 hours of induction, we found reduced levels of five ribosomal proteins from the 60S and
40S subunit by immunoblotting with GAPDH as a loading control (Figure 3.7). We observed a destabilization of Rpl29 (our top ribosomal hit), as well as of Rpl35, Rpl23a,
Rps19, and Rps15 (other putative Ube2O substrates). The catalytically inactive Ube2O
CA did not affect ribosomal protein levels during this induction. Thus, it appears as though Ube2O drives the elimination of these ribosomal proteins upon simple overexpression. This observation recapitulates normal erythroid elimination of ribosomal proteins during terminal erythropoiesis.
Given that Ube2O destabilizes these ribosomal proteins, could Ube2O ultimately drive the elimination of 80S ribosomes? To answer this question, we performed polysome profile analysis on the stable transformants after 72 hours of Ube2O induction. After doxycycline treatment, the cells were treated with cycloheximide, lysed with a detergent- based buffer, and fractionated on a 20-50% sucrose gradient. The resulting polysome profiles showed a strong attenuation of the 80S monosome peak when Ube2O WT was overexpressed, indicating a relative deficiency of ribosomes versus mRNA (Figure 3.8).
In contrast, the 80S monosome peak is unaffected by the overexpression of Ube2O CA, and is unaffected in uninduced cells. We did not observe any significant changes in the
77
Figure 3.7
Ube2O drives the elimination of ribosomal proteins in Flp-In T-REx 293 cells.
After induction of Ube2O (WT or CA) for the indicated times (48 and 72 hours), ribosomal protein levels were analyzed by immunoblotting. GAPDH was blotted as a loading control. 20 µg of cell lysate was loaded per lane, and all samples were from the same experiment.
78
Figure 3.8
Ube2O induction drives the elimination of 80S ribosomes in Flp-In T-REx 293 cells by polysome profile analysis.
Polysome profile analysis shows an attenuated 80S monosome peak when Ube2O WT is induced for 72 hours by doxycycline treatment. Ube2O CA and uninduced cells have a normal 80S peak.
79 polysome region of induced or uninduced cells. Therefore, Ube2O is sufficient to drive the elimination of ribosomes in 293 cells. Moreover, Ube2O does not appear to require an erythrocyte-intrinsic factor for its function.
Ube2O eliminates pre-existing ribosomes without affecting cell viability
As a control, we wanted to confirm that we are directly eliminating pre-existing ribosomes rather than interfering with ribosome synthesis. We initially used a conventional pulse-chase approach with radiolabeled nucleotide to monitor the loss of rRNA after Ube2O overexpression. Specifically, we used radiolabeled 3H-uridine, which is readily taken up by the cell and preferentially incorporated into rRNA (Mahajan, 1994;
Schneider et al., 1974). However, the 293 cells died after prolonged incubation with radiolabeled 3H-uridine due to the extended Ube2O induction times need for this experiment. The radiation damage problem is consistent with prior studies on cell death by β-radiation from 3H-uridine uptake in HeLa cells (Marin and Bender, 1963). To circumvent this complication, we instead used a specific inhibitor of RNA polymerase I,
CX-5461, to inhibit the synthesis of new rRNA (Bywater et al., 2012; Drygin et al., 2011).
This treatment allowed us to isolate the degradation of ribosomes from their synthesis, and allowed us to “chase” pre-existing ribosomes with Ube2O. In this experiment, we first induced expression of Ube2O, and then inhibited rRNA transcription CX-5461.
Under these conditions, Ube2O drove the destabilization of ribosomal proteins after 24-
48 hour of CX-5461 treatment, with no effect observed with the catalytic null mutant
(Figure 3.9). In all, Ube2O degrades pre-existing ribosomes consistent with the previous observation that Ube2O destabilizes individual ribosomal proteins.
Finally, does the elimination of ribosomes affect cell viability? Are the effects on ribosome stability secondary to apoptosis or necrosis? As an additional control, we
80
Figure 3.9
Ube2O drives the degradation of pre-existing ribosomal proteins when Flp-In T-
REx 293 cells are treated with a RNA polymerase I inhibitor.
After Ube2O was induced for 24 hours, Flp-In T-REx 293 cells were treated with a specific inhibitor to RNA polymerase I to stop the production of new ribosomes. The cells were treated with the RNA polymerase I inhibitor for the indicated times. Immunoblot shows the destabilization of ribosomal proteins by Ube2O WT with GAPDH as a loading control. 20 µg of protein was loaded per lane.
81 wanted to make sure that Ube2O induction does not affect cell viability. To this end, we induced Ube2O expression and then stained with propidium iodide and annexin V-FITC, which assay for cell membrane permeability and phosphatidylserine expression, respectively (Vermes et al., 1995). We found that Ube2O induction did not induce apoptosis or necrosis in our stable transformants (Figure 3.10), in further support of our model.
Ube2O executes a broad program of ubiquitination
In final confirmation of ribosomal degradation, we used mass spectrometry with isobaric tandem mass tags to quantify whole proteomic changes after induction of Ube2O
(McAlister et al., 2012). We quantified over 7500 total proteins at five different time points of induction of Ube2O WT and CA (0, 12, 24, 48, and 72 hours). First, we observed the steady induction of both Ube2O WT and CA, reaching maximal expression at 72 hours. In total, we quantified 78 ribosomal proteins, the majority of which are destabilized by Ube2O WT and unaffected by Ube2O CA (Figure 3.11). Interestingly, five ribosomal proteins showed exceptional destabilization in comparison to other ribosomal proteins: Rpl35, Rpl35a, Rpl36a, Rps23, and Rps2. Thus, ribosomal proteins are not degraded as a cohort, as expected in the case of an autophagic mechanism.
Moreover, 347 proteins behaved similarly to ribosomal proteins; gene ontology analysis of these genes was enriched for 60S and 40S ribosomal proteins, and proteins involved in ribosome biogenesis and rRNA processing (Figure 3.11). In all, these data confirm the induced breakdown of ribosomes, complexes that are thought to be highly stable normally. Overall, Ube2O is sufficient to drive ribosome elimination in non-erythroid cells in the absence of reticulocyte-specific factors; as such, erythroid ribosomes are eliminated simply by the ubiquitinating activity of Ube2O. Moreover, it suggests that
Ube2O executes a very broad program of degradation and proteomic remodeling.
82
Figure 3.10
FACS analysis of Annexin V and propidium iodide staining of Flp-In T-REx 293 cells after Ube2O induction.
After Ube2O was induced for 72 hours, cells were stained with propidium iodide and
Annexin V-FITC and analyzed by flow cytometry. Ube2O overexpression did not induce frank apoptosis or necrosis in these cells.
83 Quantified Ribosomal Proteins 0.8
0.6 RPS23 RPL36A 0.4 RPL35 RPL35A 0.2 RPS2 0 log2 (72h/0h) CA (72h/0h) log2 -0.2
-0.4 -1.5 -1 -0.5 0 0.5 log2 (72h/0h) WT
Figure 3.11
Global proteomic analysis confirms ribosomal protein destabilization in 293 cells.
Using tandem mass tags, global proteomic changes were analyzed when Ube2O was induced in 293 cells at five different time points (0, 12, 24, 48 and 72 hours). In total, 78 ribosomal proteins were quantified during this time course. The majority of the quantified ribosomal proteins were destabilized when Ube2O WT was induced as indicated by a negative ratio comparing 72 hours and 0 hours on the X-axis. Ribosomal proteins do not change, or increase, when Ube2O CA was induced as indicated by a positive ratio comparing 72 hours and 0 hours on the Y-axis. Five ribosomal proteins showed the greatest response, Rps23, Rpl35, Rpl35a, Rpl36a, and Rps2; the quantified ribosomal proteins are not destabilized at the same rates.
84 Discussion
In Chapter Two, we showed that Ube2O ubiquitinates ribosomal proteins, and that
Ube2O deficiency results in elevated levels of ribosomal proteins and 80S ribosomes in reticulocytes. These data suggested that null reticulocytes have a global defect of ribosome elimination. To confirm this, we differentiated WT and null reticulocytes ex vivo and analyzed the elimination of mitochondria and ribosomes by flow cytometry.
Consistent with a major deficiency of ribosome elimination, null reticulocytes had an attenuated loss of rRNA staining, which is the dominant RNA species in reticulocytes.
We also confirmed the attenuated loss of ribosomal proteins by immunoblot and 80S ribosomes by polysome profile analysis. Next, we identified ribosomal proteins as the major class of targets when Ube2O was added to HEK293 lysates. When we overexpressed Ube2O in 293 cells, we observed the destabilization of ribosomal proteins and 80S ribosomes. As a control, we showed that Ube2O degrades pre-existing ribosomes without affecting cell viability. Finally, using quantitative proteomics with tandem mass tags (TMT), we confirmed that Ube2O destabilizes ribosomal proteins, including other non-ribosomal targets.
For the first time, we have extensive mechanistic insight into how ribosomes are eliminated from terminally differentiating reticulocytes. As we hypothesized, ubiquitination of 60S subunits by Ube2O drives the elimination of ribosomes from nascent reticulocytes. Thus, developmentally programmed ubiquitination is responsible for ribosomal degradation in erythropoiesis. Ube2O is expressed specifically during the final stages of differentiation to degrade ribosomes after the completion of globin synthesis. Since the reticulocyte synthesizes approximately 30% of total globin, early expression of Ube2O would stop protein synthesis prematurely. As previously discussed, Ube2O null mice have a hypochromic, microcytic anemia that is likely caused
85 by reduced globin synthesis. However, absence of Ube2O in the null mutant results in elevated levels of ribosomal proteins and 80S ribosomes, which would suggest prolonged protein synthesis into the mature erythrocyte. These two phenotypes appear to contradict each other, as we understand the system. In order to account for the anemia, these excess ribosomes should not be actively engaged in translation in the mature erythrocyte. This is likely to be the case since mitochondria are eliminated normally from the mutant, which stops the heme synthesis required for globin production. How, then, do elevated levels of ribosomes actually inhibit general protein synthesis? We hypothesize that there are indirect effects on translational output in the
Ube2O null mutant. We will explore the crosstalk between ribosomal degradation and protein synthesis in Chapter 5.
After Ube2O ubiquitinates the ribosome, how are they eliminated from the cell? Selective autophagy is a generally ubiquitin-dependent pathway of targeting proteins for degradation, and could be downstream of Ube2O. Selective autophagy is upregulated in terminally differentiating reticulocytes to eliminate mitochondria. Moreover, there is a literature of ribosome degradation that proceeds in part through autophagy (Kraft et al.,
2008). However, when we quantified whole proteome changes upon Ube2O induction in
293 cells, we observed five ribosomal proteins showed exceptional elimination compared to other ribosomal proteins. Moreover, not all ribosomal proteins were eliminated at the same rate. These data suggest that ribosomes are not eliminated by either selective or bulk autophagy, which would likely degrade the 80S ribosome in toto.
Instead, there appeared to be a step-wise degradation with specific subunits destabilized at a faster rate. This raises the question of how ribosomal proteins are extracted from the 80S complex, and if ubiquitination is sufficient to pry the subunit out of the ribosome.
Of these five rapidly cleared ribosomal proteins, Rpl35 was one of our strongest hits
86 from the reconstitution of null reticulocyte lysates. It is possible that Rpl35 is one of the first ribosomal proteins that is ubiquitinated and degraded, making the 80S complex more susceptible to Ube2O ubiquitination. Additional biochemical studies are required to understand the temporal distribution of ribosomal protein degradation.
The sufficiency of Ube2O to drive ribosome elimination is consistent with the restricted tissue expression of this enzyme. As discussed in Chapter 1, Ube2O is expressed almost exclusively in reticulocytes (Wefes et al., 1995), likely due the fact that the key erythroid transcription factor GATA 1 binds the promoter of Ube2O (Li et al., 2013).
Mature erythrocytes are one of the few cell types that no longer divide and that do not have ribosomes. Improper expression of Ube2O in dividing and translationally active cells could have deleterious effects on cellular function. Interestingly, Ube2O may have minor levels of expression in brain and testes (Wefes et al., 1995). This may be a false positive since brain and testes often show nonspecific cross reactivity with different antibodies. However, mature sperm are terminally differentiated cells that no longer divide, similar to erythrocytes. Mammalian sperm are also widely accepted to be translationally inactive. In fact, these cells have been shown to translate nuclear- encoded proteins by mitochondrial-type ribosomes (Gur and Breitbart, 2006). Could
Ube2O actually be expressed in sperm as a developmental program of proteome remodeling? If Ube2O is expressed in sperm, does GATA 1 also drive its expression or is it driven by another transcription factor? Since Ube2O is sufficient to drive ribosome elimination, it is likely that Ube2O serves this role in mammalian sperm as well.
Moreover, platelets are another terminally differentiated cell type that is translationally silent after dividing from megakaryocytes. Ube2O may mediate ribosome elimination from platelets, which no longer need to translate additional proteins.
87 Interestingly, Ube2O is also highly induced during lens fiber cell differentiation (Chauss et al., 2014; Hoang et al., 2014; Matsui et al., 2006). The lens fiber cell is a terminally differentiated cell type that consists mainly of one type of protein, crystallin. In this regard, the lens fiber cell is very similar to the mature erythrocyte, which contains mainly globin. During differentiation, the lens fiber cell will enucleate and eliminate all of its organelles similar to an erythrocyte. In fact, the autophagy receptor for mitochondria
(NIX) is also highly upregulated, suggesting that mitochondria are eliminated from these fiber cells through mitophagy. In parallel, Ube2O is upregulated, suggesting that it functions to drive ribosome elimination upon lens differentiation. Since Ube2O is sufficient in function, it appears likely that Ube2O can function in lens fiber cell differentiation. If ribosomes are not developmentally eliminated from lens fiber cells, does this affect the clarity of the mouse lens? Are there additional differentiated cell types that upregulate Ube2O? We will systematically analyze Ube2O expression with reticulocytes as the proper positive control. Further experimental work will clarify these questions.
Finally, from the quantitative proteomics experiment, we also detected non-ribosomal proteins that are destabilized by Ube2O overexpression. These data suggest that
Ube2O may act on other proteins beyond ribosomal proteins. In Chapter Two, we showed that some of these candidate proteins are also elevated in Ube2O null reticulocytes, indicating that they may be direct Ube2O substrates. As such, it appears as though Ube2O executes a specific, but broad program of ubiquitination and degradation. Moreover, this program requires only Ube2O overexpression as observed in reticulocytes and our 293 cell line. In summary, Ube2O is critical for ribosome elimination in the erythroid lineage, and sufficient for ribosome elimination in non- erythroid 293 cells.
88 Materials and Methods
Antibodies
The following antibodies were used for immunoblot analysis: anti-Rpl35 (Sigma,
SAB4500233, rabbit polyclonal, used at 1:1000); anti-Rpl37 (Abcam, ab103003, rabbit polyclonal, used at 1:500); anti-Rpl23a (Sigma, SAB1300595, rabbit polyclonal, used at
1:1000); anti-Rpl29 (Santa Cruz Biotech, sc-103166, goat polyclonal, used at 1:200); anti-Rps19 (Sigma, SAB2500898, goat polyclonal, used at 1:1000); anti-Rps15 (Sigma,
SAB2104122, rabbit polyclonal, used at 1:1000); anti-Ube2O (Bethyl, A301-873A, rabbit polyclonal, used at 1:2000); anti-GAPDH (Abcam, ab8245, mouse monoclonal, used at
1:5000); and anti-AHSP (Rockland, 100-401-E79, rabbit polyclonal, used at 1:1000).
Ex vivo differentiation of reticulocytes and flow cytometry analysis
Reticulocytes were cultured in IMDM supplemented with 20% BIT (serum substitute,
StemCell Technologies), 0.1% monothioglycerol (Sigma), 100 units/mL penicillin, 100
µg/mL streptomycin, and 2 mM L-glutamine. Cells were diluted 1:500 in complete medium and cultured at 37°C with 5% CO2. At the specified time of ex vivo differentiation, reticulocytes were stained with 200 nM MitoTracker Deep Red FM (Life
Technologies) for 30 minutes at 37°C. The cells were then stained with thiazole orange
(2 µg/mL in methanol, Sigma) for 40 minutes at room temperature in the dark. Finally, the cells were incubated with anti-mouse CD71 conjugated to Brilliant Violet 421 (1:300 in PBS, BioLegend) for 20 minutes at 4°C. After staining, the cells were washed 2x in ice-cold PBS, and resuspended in 500 µl of cold-PBS for flow cytometry analysis.
Reticulocytes were analyzed using an LSR-II Flow Cytometer (BD Biosciences) at the
Flow Cytometry Core Facility in the Division of Immunology at Harvard Medical School.
Data were analyzed using FACSDiva (BD Biosciences) and FlowJo software (TreeStar).
89 Immunoblot analysis of ribosomal protein degradation
Reticulocytes were cultured in complete media (see Ex vivo differentiation) at 37°C with
5% CO2. After ex vivo differentiation, reticulocytes were harvested, washed 1x with ice- cold PBS, and lysed with urea lysis buffer (50 mM Hepes pH 7.5, 8M urea, 75 mM NaCl,
1x EDTA-free protease inhibitor cocktail (Roche), 1x PhosSTOP phosphatase inhibitor cocktail (Roche)) for 15 minutes at room temperature. The lysate was clarified by centrifugation at 13,200 rpm for 10 minutes at 4°C, and protein concentration was measured by BCA assay (Pierce). 100 µg of protein per lane was diluted in Laemmli buffer, boiled, and analyzed by 4-12% SDS-PAGE (Invitrogen) and immunoblotted using anti-ribosomal protein antibodies with fluorescent secondary antibodies as described in
Chapter 2 (see Methods, Immunoblot analysis of putative Ube2O substrates).
Polysome profile analysis of differentiated reticulocytes
As described, reticulocytes were differentiated ex vivo in complete media at 37°C with
5% CO2. After 31 hours of ex vivo differentiation, reticulocytes were treated with 100
µg/mL cycloheximide, harvested, and then washed with ice-cold 1x PBS containing 100
µg/mL cycloheximide. The cells were then lysed with ice-cold 200 mM Tris HCl pH 8.8,
25 mM MgCl2, 25 mM KCl, 1% Triton X-100, 1.3% sodium deoxycholate, 100 µg/mL cycloheximide, and 200 units/mL RNAse inhibitor (New England Biolabs). Lysates were incubated for 10 minutes at 4°C with end on end rotation, and clarified by centrifugation at 3,000 rpm for 10 minutes at 4°C. Lysates were analyzed by sucrose gradient centrifugation as described in Chapter 2 (see Methods, Polysome profile analysis of reticulocytes).
90 Ube2O reconstitution of HEK293 cell lysate
Approximately 108 HEK293 cells were lysed in hypotonic buffer (50 mM Tris HCl, pH 7.4,
1x EDTA-free protease inhibitor cocktail (Roche)) through multiple freeze and thaw cycles. NaCl was added to the lysate at a final concentration of 150 mM. Debris and membrane were clear by centrifugation. The lysate was treated with 10 mM NEM for 30 min at room temperature to alkylate cysteines thereby inhibiting endogenous ubiquitination enzymes. DTT was added at 10 mM to quench NEM. Ubiquitination reactions were carried out by adding 200 nM His-UBE1 (Boston Biochem, E-306), 700 nM His-Ube2O, 3 µM biotin-ubiquitin (Boston Biochem, UB-560), 0.6 unit/mL inorganic pyrophosphatase, 5 µM PS-341, 5 mM ATP, 9 µM creatine kinase and 30 mM creatine phosphate. The reactions were kept on a rotating wheel in 37°C for 4 hours.
Recombinant Ube2O and E1 were cleared using Ni-NTA beads (Qiagen). Biotin- ubiquitin conjugates were purified using 50 µl NeutrAvidin agarose (Thermo Scientific,
29202), binding at room temperature for 1 hour, followed by washing 3x with buffer containing 8 M urea and 1% (w/v) SDS. Ubiquitin conjugates were prepared for LC-
MS/MS identification as described in Chapter 2 (see Methods, Biotin-ubiquitin conjugate purification and elution).
Generation of the Flp-In T-REx 293 cell line with Ube2O expression
The full open-reading frame of the human Ube2O gene was cloned into the pCDNA
5/FRT/TO plasmid (Invitrogen) in an untagged form. The untransfected Flp-In T-REx 293 cell line (Thermo Fisher) was cultured in DMEM supplemented with 10% FBS, 2 mM L- glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, 100 µg/mL Zeocin, and 15
µg/mL blasticidin. At 60% confluency, the media was changed to DMEM with 1% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. To transfect the
91 cell line, the pCDNA 5/FRT/TO with human Ube2O plasmid and pOG44 recombinase vector were incubated with 20 µg of polyethylenimine (PEI) at a 1:4 DNA to PEI ratio for
30 minutes at room temperature. The pCDNA 5/FRT/TO with human Ube2O plasmid and pOG44 vector were at a 1:9 ratio (0.5 µg and 4.5µg, respectively). The DNA:PEI solution was then added dropwise to the cells, and mixed gently. After 24 hours, the media was changed to DMEM with 10% FBS and 2 mM L-glutamine. After 48 hours, stable transfectants were selected using hygromycin B. The media was changed to
DMEM supplemented with 10% dialyzed FBS, 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, 100 µg/mL hygromycin B, and 15 µg/mL blasticidin.
Colonies were visible after two weeks of selection, and were transferred to complete media with hygromycin B and blasticidin to maintain selection of stable transformants.
Immunoblot analysis of Ube2O induction in 293 cells
Flp-In T-REx 293 cells with stably integrated Ube2O were grown in filtered DMEM with
10% dialyzed FBS, 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin,
100 µg/mL hygromycin B, and 15 µg/mL blasticidin. To induce Ube2O expression, doxycycline was added to the media at 10 µg/mL. The media with doxycycline was replenished every 24 hours to maintain high levels of Ube2O expression. After induction for the specific time, the cells were washed with ice-cold PBS and lysed with 150 µl of urea lysis buffer (50 mM Hepes pH 7.5, 8M urea, 75 mM NaCl, 1x EDTA-free protease inhibitor cocktail (Roche), 1x PhosSTOP phosphatase inhibitor cocktail (Roche)) for 15 minutes on ice. The cell lysate was passed 10x through a 25G syringe, and clarified by centrifugation at 13,200 rpm for 10 minutes at 4°C. Protein concentration was measured using the BCA assay (Pierce). 25 µg of protein were loaded per lane, and analyzed by 4-
12% SDS-PAGE, and immunoblotted by chemiluminescence (PerkinElmer ECL).
92 Polysome profile analysis of 293 cell line
Stable transformants of the Flp-In T-REx 293 cells with Ube2O were induced with 10
µg/mL doxycycline for 72 hours or without doxycycline. The media with or without doxycycline was replaced every 24 hours. Prior to analysis, the cells were treated with
100 µg/mL cycloheximide for 5 minutes at 37°C. The cells were then transferred to ice, and washed 2x with ice-cold PBS with 100 µg/mL cycloheximide. The cells were then lysed with 600 µl of lysis buffer (200 mM Tris HCl pH 8.8, 25 mM MgCl2, 25 mM KCl, 1%
Triton X-100, 1.3% sodium deoxycholate, 100 µg/mL cycloheximide, and 200 units/mL
RNAse inhibitor). The cells were harvested into a microcentrifuge tube, rotated end on end at 4°C for 10 minutes, and centrifuged at 3,000 x g for 15 minutes at 4°C. The supernatant was then concentrated to 100 µl using a 0.5 mL centrifugal filter with a 10 kDa molecular weight cutoff (Amicon). The concentrated cell lysates were analyzed on
20-50% (w/w) sucrose gradients as previously described in Chapter 2 (see Methods,
Polysome profile analysis of reticulocytes).
Chase experiment with RNA Polymerase I inhibitor
Flp-In T-REx 293 cells with Ube2O were induced with 10 µg/mL doxycycline for 24 hours. After the initial induction, the cells were treated with 1 µM CX-5461, a selectively inhibitor of RNA polymerase I, and 10 µg/mL doxycycline. The cells were harvested at
24 and 48 hours of CX-5461 treatment with Ube2O induction. The cells were lysed with urea lysis buffer, and 25 µg of protein was analyzed by 4-12% SDS-PAGE and immunoblotted for each respective protein by chemiluminescence (PerkinElmer ECL).
93 Annexin V and propidium iodide staining
To stain 293 cells, the medium was aspirated and transferred to a 50 mL conical tube.
The cells were washed 1x with PBS, and the wash was transferred to the same 50 mL conical tube. The cells were treated with 0.5 mL of 0.05% trypsin for 2 minutes at 37°C, resuspended with 2 mL of complete medium, and transferred to the 50 mL conical tube.
The cells were centrifuged at 1,000 rpm for 5 minutes, and the cell pellet was resuspended in 1 mL of annexin V binding buffer (20 mM Hepes, 150 mM NaCl, and
0.1% BSA). 100 µl of the resuspended cells was incubated with 5 µl of Annexin V-Alexa
Fluor 488 (Life Technologies) and 1 µg/mL of propidium iodide for 15 minutes at room temperature. After incubation, 400 µl of binding buffer was added, mixed gently, and analyzed using an LSR-II Flow Cytometer (BD Biosciences) at the Flow Cytometry Core
Facility in the Division of Immunology at Harvard Medical School.
Quantitative whole proteomic analysis of 293 cells with tandem mass tags (TMT)
Flp-In T-REx 293 cells with Ube2O WT and CA were induced with 10 µg/mL doxycycline for 12, 24, 48, and 72 hours or without doxycycline. Cells were washed 2x with ice-cold
PBS, and scraped into a 50 mL conical tubes with PBS. After centrifugation, the cell pellet was lysed with an SDS lysis buffer (50 mM Hepes pH 7.5, 2% SDS, 150 mM NaCl,
1x EDTA-free protease inhibitor cocktail (Roche), 1x PhosSTOP phosphatase inhibitor cocktail (Roche)) at room temperature. The cell lysate was then passed 10x through a
25G needle, and centrifuged at 13,200 rpm for 10 minutes at 4°C. Protein concentrations were measured by BCA assay (Pierce). To reduce and alkylate the disulfide bonds, 150
µg of protein for each sample were incubated with 5 mM TCEP for 25 minutes, and then with 14 mM iodoacetamide for 30 minutes in the dark. Proteins were precipitated with methanol/chloroform, and then resuspended with 200 mM EPPS pH 8.5. LysC digestion
94 were performed overnight at RT at a 1:100 (LysC:protein) ratio, and then with trypsin for
6 hours at 37C at a 1:75 (trypsin:protein) ratio. After digestion, all samples were labeled with the tandem mass tags using the TMT10plex reagent set (Thermo, 90110) as described in the manufacture’s protocol. After labeling, all samples were mixed at 1:1 ratios and desalted using a Sep-Pak C18 cartridge (Waters, WAT054960), and then dried by vacuum concentration.
The dried peptides were separated by chromatography using a HPLC equipped with a
C18 column under Hi pH conditions (pH 8). 96 fractions were collected and combined into 24 fractions. 12 of them were dried by vacuum concentration, desalted by STAGE-
Tip, and dried again by vacuum concentration. The dried peptides were resuspended in
5% FA, 3% ACN, and then separated on a reverse-phase chromatography coupled to an
Orbitrap Fusion. In the mass spectrometer, for every single MS1 spectra detected in the
Orbitrap, the 20 most intense peptides were selected and fragmented by CiD to generate the MS2 spectra. These fragment ions, detected in the ion trap and used for the identification of the peptides, were fragmented again, in this case by HCD, to generate the report ions that would be detected in the Orbitrap and used for the relative quantification. All RAW files obtained from the mass spectrometer were transformed using in-house software based on SEQUEST for the identification and relative quantification of the proteins presence in the samples.
Contributions
I performed the majority of the experiments described in this Chapter. Miguel Prado performed the mass spectrometry analysis. Mingwei Min performed the reconstitution of
HEK293 lysates. Stefanie de Poot and Daniel Finley edited the manuscript.
95
CHAPTER FOUR
Ribosomal degradation is proteasome-dependent
Background
Preceding the discovery of the ubiquitin proteasome system (UPS), rabbit reticulocytes were first observed to degrade abnormal or mutated hemoglobin (Carrell and Lehmann,
1969; Huehns and Bellingham, 1969; Rabinovitz and Fisher, 1964). Since mature reticulocytes do not have lysosomes (Ciechanover, 2005), an ATP-dependent proteolytic system was first isolated and characterized in the reticulocyte (Etlinger and Goldberg,
1977). The UPS was resolved into complementary components containing ubiquitin and the proteolytic machinery, later known as the proteasome (Ciechanover et al., 1980;
Ciehanover et al., 1978). Early work showed that the reticulocyte UPS can degrade reticulocyte mitochondrial proteins and hexokinase (Magnani et al., 1986; Rapoport et al., 1985). However, study of the UPS would eventually move out of the reticulocyte into more complex cell types.
Despite its discovery in the reticulocyte, the UPS is poorly understood in the erythroid system. To date, α-globin appears to be a substrate of the reticulocyte UPS. Unpaired α- globin has a half-life of approximately one minute or less (Hunter and Jackson, 1972). In
β-thalassemic hemolysates, unpaired α-globin chains were degraded in an ATP- and ubiquitin-dependent manner (Shaeffer, 1988). α-globin is monoubiquitinated on different lysine residues (Shaeffer, 1994b), which targets it for degradation by the 26S proteasome (Shaeffer and Kania, 1995). In a mouse model of β-thalassemia, components of the UPS appear to be upregulated in response to unpaired α-chains
(Khandros et al., 2012). Still, we do not know how α-globin is ubiquitinated (namely by which ubiquitin ligase and associated proteins). We also do not know the physiological substrates of the reticulocyte UPS. Understanding the substrate specificity of this system may give us insight into why the reticulocyte has a robust and processive UPS.
97 Ube2O is an E2-E3 hybrid that ubiquitinates ribosomal proteins during terminal differentiation. Consistent with this, Ube2O null reticulocytes have a critical defect in the elimination of ribosomes. When Ube2O was overexpressed in 293 cells, ribosomal proteins were degraded at varying rates, suggesting that autophagy is not involved in this process. Moreover, autophagy mutants do not appear to have a defect in ribosome elimination (Sandoval et al., 2008). Could the degradation of ribosomal proteins be proteasome-dependent? Previous studies have suggested that ribosomal proteins are degraded by the proteasome. For example, mutant forms of the 40S ribosomal protein
Rps19 were stabilized by proteasome inhibitors (Cretien et al., 2008). Since ribosome elimination is ubiquitin-dependent, we hypothesized that ribosomal degradation is also proteasome-dependent.
Here we report the finding that the elimination of ribosomes in reticulocytes relies on the proteasome. We confirm this finding with three orthogonal approaches, in intact reticulocytes, in a cell-free lysate system, and with purified components. As controls, we show that autophagy, mTOR, and p97 are not involved in ribosome elimination. We then reconstitute the ubiquitination and degradation of ribosomes with purified components.
Through these experiments, we definitively show that Ube2O is an E2-E3 hybrid, and that degradation of ribosomal proteins is proteasome-dependent.
98 Results
Proteasome inhibitor treatment attenuates ribosome elimination in reticulocytes
How are ubiquitinated ribosomes degraded? As ribosome elimination is dependent on a ubiquitinating enzyme, we considered the hypothesis that ribosomes are eliminated via the proteasome. Thus, we incubated WT reticulocytes with low concentrations of the proteasome inhibitors PS-341 and epoxomicin (50nM each). These inhibitors were selected for their coverage of the active sites on the proteasome. PS-341 (also known as bortezomib or Velcade) reversibly targets the proteasomal β5 active site and the β1 active site to a lesser extent (Berkers et al., 2005). Epoxomicin irreversibly inhibits the chymotrypsin-like activity of the proteasome (Meng et al., 1999). After 48 hours of ex vivo culture, we stained the cells with MitoTracker and thiazole orange to quantify the remaining amount of mitochondria and ribosomes, respectively. We also stained for
CD71, a reticulocyte marker, and analyzed the sample by flow cytometry.
FACS analysis of these treated reticulocytes showed retention of thiazole orange staining with low MitoTracker signal, resembling the hem9 reticulocytes that had not been exposed to proteasome inhibitor (Figure 4.1, top). In fact, the treated WT reticulocytes retained their thiazole orange staining more efficiently than untreated hem9 reticulocytes (Figure 4.1, middle); moreover, treated hem9 reticulocytes further increased the retention of thiazole orange staining over untreated reticulocytes. As such, treating WT reticulocytes with proteasome inhibitors appears to phenocopy the hem9 effect, suggesting that ribosomal proteins are degraded in part through the proteasome.
Proteasome inhibitors faintly attenuated the loss of MitoTracker signal though the effect size is orders of magnitude less than that with thiazole orange staining (Figure 4.1, bottom).
99
Figure 4.1
FACS analysis of proteasome inhibitor treatment of WT and hem9 reticulocytes.
Treatment of WT reticulocytes (induced by serial retro-orbital bleeding) with proteasome inhibitors (50 nM epoxomicin and 50 nM PS-341, 48 hours treatment) attenuates loss thiazole orange staining (top). WT reticulocytes retain thiazole orange staining at higher levels than hem9 reticulocytes when treated with proteasome inhibitors (middle). Loss of
MitoTracker signal is mildly attenuated by proteasome inhibition (bottom).
100 To see if there is a dose-dependence in the retention of rRNA, we then titrated down the concentration of proteasome inhibitors used to treat WT reticulocytes (Figure 4.2). The retention of thiazole orange signal was directly correlated with the concentration of proteasome inhibitors. There was an observable inhibition of ribosome elimination with as low as 6.26 nM of PS-341 and epoxomicin, though not as potent as with higher concentrations. Mitochondrial elimination was also attenuated in a dose-dependent manner. One caveat of this experiment is that since reticulocytes are enucleated, they are lacking in transcriptionally based compensatory pathways, and thus likely to be unusually susceptible to the ubiquitin-depleting effects of proteasome inhibitors. If the effect of proteasome inhibitors on ribosomal proteins degradation were mediated by ubiquitin depletion, the stabilizing effect of these inhibitors would be indirect, and the conclusion that ribosomal proteins are degraded via the proteasome could be incorrect.
We did indeed observe ubiquitin depletion when analyzing lysates of treated reticulocytes by SDS-PAGE. Immunoblotting for ubiquitin showed a perturbation of the ubiquitin profile upon proteasome inhibition. After 48 hours of ex vivo culture with proteasome inhibitors, the pool of free ubiquitin was depleted in the highest concentrations of treatment (Figure 4.3). Consistent with proteasome inhibition, we also observed a marked elevation of high molecular weight ubiquitin conjugates at 24 and 48 hours of incubation. Thus, as anticipated, free ubiquitin stores were somewhat reduced, though likely not to the extent to account for the attenuation of ribosome elimination.
Reconstitution of ribosomal protein degradation in reticulocyte lysates
Reconstituting the degradation of ribosomal proteins in a cell-free reticulocyte lysate system allowed us to overcome the problem of ubiquitin depletion by proteasome
101
Figure 4.2
Dose-dependence of attenuated thiazole orange elimination by WT reticulocytes.
WT reticulocytes (induced by serial retro-orbital bleeding) were cultured with varying concentrations of proteasome inhibitors, and analyzed by flow cytometry after 48 hours of ex vivo differentiation. Bivariate analysis of MitoTracker and thiazole orange staining with titrated concentrations of proteasome inhibitor (top). Retention of thiazole orange staining is dependent on concentration of proteasome inhibitor (bottom, left).
MitoTracker signal is minimally affected by proteasome inhibition (bottom, right).
102
Figure 4.3
Immunoblot analysis of ubiquitin levels in reticulocytes differentiated ex vivo with proteasome inhibitor treatment.
After ex vivo differentiation for the indicated times (24 and 48 hours), reticulocyte lysate was analyzed by 4-12% SDS-PAGE, and immunoblotted with an anti-ubiquitin antibody.
After 48 hours of culture, proteasome inhibitor treatment reduces stores of free ubiquitin in WT reticulocytes (right). 100 µg of protein was loaded per lane.
103 inhibitors. A key advantage to this system is that we can supplement free ubiquitin levels to avoid secondary effects of ubiquitin depletion. Moreover, the exosome pathway is not active in a cell-free lysate system. To generate the Ube2O null reticulocyte lysate, we washed hem9 reticulocytes with cold PBS, and then lysed the cells with cold water via hypotonic lysis. We then added excess E1 and ubiquitin to drive the reaction forward,
Ube2O WT or CA, and an ATP regenerating system. We then incubated the reaction at
37°C for 8 hours to drive the reaction forward (30°C was insufficient). Finally, we supplemented the reaction with ATP at 4 hours since the reticulocyte lysate uses high levels ATP. Using this system, we reconstituted the degradation of ribosomal proteins by adding back Ube2O to null reticulocyte lysate (Figure 4.4). This was shown for Rpl35,
Rpl29, Rpl36a, and Rps23, which are our top ribosomal hits. Consistent with this, ribosomal proteins were not destabilized by the catalytic null enzyme, Ube2O CA.
To test whether this degradation is proteasome-dependent, we then incubated the reaction with PS-341 and epoxomicin. Proteasome inhibitor treatment attenuated the degradation of ribosomal proteins in reticulocyte lysate without depleting available ubiquitin levels. However, the stabilizing effect of the inhibitors, while apparently complete for Rpl29 and Rps23, was partial in the case of Rpl35 and Rpl36a, if judging from the intensity of the major protein band representing these proteins. However, for both proteins a high molecular weight species appeared upon proteasome inhibition.
This suggested that stabilization by proteasome inhibition might in fact be complete for
Rpl35 and Rpl36a, with the high molecular weight species representing ubiquitinated forms of Rpl35 and Rpl36a. Thus, to test whether proteasome inhibitors block ribosomal protein degradation completely, we sought to concentrate Rpl35 into a single band by deubiquitinating all high molecular weight forms of the protein. To this end, we first stopped the reaction with NEM and DTT to prevent any additional ubiquitination. We
104
Figure 4.4
Reconstitution of ribosomal protein degradation in hem9 reticulocyte lysate.
Ube2O WT drives the degradation of ribosomal proteins in a cell-free lysate system.
Proteasome inhibitors can inhibit the degradation of these ribosomal proteins in the lysate system, and stabilize a monoubiquitinated form of Rpl35 and Rpl36a. 100 µg of protein was loaded per lane.
105 then treated the samples with the pan-deubiquitinating enzyme USP21 (Gong et al.,
2000; Ye et al., 2011). When WT Ube2O and proteasome inhibitors were added back to hem9 lysate, we observed that USP21 effectively concentrated Rpl35 into a single band that corresponds to complete stabilization of the protein. Therefore, these data in our cell-free reticulocyte lysate system strongly confirm that ribosomal protein degradation is proteasome-dependent (Figure 4.5).
Ribosome degradation does not appear to be autophagy- or TOR-dependent
Another major pathway of targeting proteins for degradation is selective autophagy, which is generally a ubiquitin-dependent process and might be downstream of Ube2O.
The key selective autophagy receptor of reticulocytes is NIX, which mediates breakdown of mitochondria, but not ribosomes (Novak et al., 2010; Sandoval et al., 2008; Schweers et al., 2007); however, the function of NIX is not ubiquitin-dependent. Though autophagy-dependent turnover of ribosomes has been reported (Kraft et al., 2008), it occurs primarily under starvation conditions, at least in yeast. To test whether selective autophagy mediates ribosome elimination, we treated wild-type reticulocytes with bafilomycin A1, a known inhibitor of autophagy and inhibitor of the vacuolar H+ ATPase, and analyzed ribosomal and mitochondrial content by flow cytometry. We found that bafilomycin A1 had minimal effect on the elimination of ribosomes during ex vivo differentiation of reticulocytes (Figure 4.6). In addition to this finding, mature reticulocytes contain few lysosomes, at best (Ciechanover, 2005; Veldman et al., 1984). Thus, selective autophagy does not appear to be involved in ribosome elimination. Autophagy inhibitors produced only a mild attenuation of mitochondria elimination. This may seem contrary to our understanding of mitophagy in reticulocytes, but in fact none of the studies on NIX were able to phenocopy the null effect with autophagy inhibitors
(Sandoval et al., 2008; Schweers et al., 2007).
106
Figure 4.5
USP21 treatment concentrates Rpl35 into a single band.
Proteasome inhibition stabilized a monoubiquitinated form of Rpl35. To show that proteasome inhibitor can completely inhibit ribosomal protein degradation, we treated the sample with a pan-deubiquitinating enzyme USP21 to deubiquitinate this ubiquitin adduct. This treatment concentrated Rpl35 into a single band, corresponding to complete stabilization of the protein. 100 µg of protein was loaded per lane.
107
Figure 4.6
FACS analysis of bafilomycin A1 treatment of WT reticulocytes.
Bafilomycin A1 does not attenuate loss of thiazole orange staining in ex vivo differentiated WT reticulocytes (induced by serial retro-orbital bleeding) (left).
MitoTracker and thiazole orange staining is unaffected by bafilomycin A1 treatment
(right).
108 The TOR pathway has been shown to regulate ribosome levels in S. cerevisiae (Pestov and Shcherbik, 2012). Inhibition of TOR by rapamycin drove the rapid cytoplasmic turnover of ribosomes when yeast were grown in rich media. To interrogate the role of the TOR pathway in ribosome turnover in a mammalian system, we treated our stable
293 transformants with rapamycin. We were unable to stimulate ribophagy or ribosomal protein turnover in Flp-In T-REx 293 cells with rapamycin treatment (Figure 4.7). As a positive control, we induced Ube2O overexpression and observed the degradation of ribosomal proteins, Rpl35, Rpl36a, Rps19, and Rps23. Additionally, we immunoblotted for phospho-p70 (S6 kinase, P-Thr 389), which was absent with rapamycin treatment.
Finally, though ribosome degradation appears to be proteasome-dependent, the proteasome itself is thought to be incapable of disassembling large, stable complexes such as the ribosome. p97, also known as Vcp or Cdc48 in yeast, is a ubiquitin- dependent chaperone that can disassemble such stable complexes in an ATP- dependent mechanism (Meyer et al., 2012). To test whether p97 is required for ribosome elimination, we treated WT reticulocytes with NMS-873, a potent and specific inhibitor of p97 (Magnaghi et al., 2013). We did not observe a strong effect on ribosome elimination when p97 was inhibited (Figure 4.8). As such, the mTOR pathway and p97 do not appear to be involved in ribosome elimination.
Reconstitution of ribosomal degradation in a purified system
Next, since ribosome elimination does not require a reticulocyte specific factor, we sought to reconstitute ribosome ubiquitination and degradation with purified components.
First, we used recombinant Ube2O to ubiquitinate the gold standard, model substrate for
Ube2O, histone H2B, with E1 enzyme and HA-ubiquitin (Figure 4.9). Second, we purified ribosomes from HEK 293 cells using a sucrose cushion (Belin et al., 2010). Prior to ultracentrifugation, we treated the 293 lysate with a pan-deubiquitinating enzyme,
109
Figure 4.7 mTORC1 inhibition by rapamycin does not drive ribosomal protein degradation.
Rapamycin treatment (1 hour) of Flp-In T-REx 293 cells does not drive ribosomal protein degradation. Ube2O induction (72 hours) is used as a positive control for ribosomal protein destabilization. 20 µg of protein was loaded per lane.
110
Figure 4.8
FACS analysis of p97 inhibition of WT reticulocytes.
NMS-873, a potent and specific p97 inhibitor, (48 hour treatment) does not affect loss of
MitoTracker or thiazole orange staining in WT reticulocytes (induced by serial retro- orbital bleeding) (top). Histogram plot of thiazole orange and MitoTracker signal in
CD71+ reticulocytes (bottom).
111
Figure 4.9
Ube2O directly recognizes ribosomal proteins and other substrates.
Ube2O can ubiquitinate ribosomes in a purified system with E1 and HA-ubiquitin (4 hour reaction time at 37°C). Ube2O can also ubiquitinate histone H2B and AHSP. N-terminal truncations abrogate recognition of the ribosome and H2B, but do not affect AHSP ubiquitination.
112 USP21, to remove all endogenous ubiquitination (Gong et al., 2000; Ye et al., 2011). We then added recombinant Ube2O, E1, and HA-ubiquitin to the purified ribosomes and the sample was incubated at 37°C. We observed efficient ubiquitination of ribosomal proteins, and used LC-MS/MS to identify the ubiquitination sites, which correlate with previously identified sites (Table 4.1). Specifically, we identified multiple ubiquitin sites on Rpl37, Rpl38, Rpl37a, and Rpl36a among others. We also identified ubiquitination of
Rpl35, Rpl23a, Rps23, Rps15, and Rps19, which were previously identified as Ube2O targets. Also consistent with previous data, we observed more ubiquitination of the 60S subunit components than of the 40S subunit components. We did not identify Rpl29, which may have been lost during the ribosome purification by sucrose cushion.
Ube2O was also able to efficiently ubiquitinate a non-ribosomal physiologic target,
AHSP, an α-globin chaperone (Figure 4.9). As such, Ube2O appears to directly recognize its substrates including the ribosome. Moreover, Ube2O appears to function as a ubiquitin ligase with a built-in E2. By deletion analysis, we demonstrated that the first conserved N-terminal domain is required for recognition of H2B and the ribosome.
However, N-terminal truncations of Ube2O could still ubiquitinate AHSP, suggesting that
Ube2O has multiple apparent substrate recognition domains.
Finally, we reconstituted the degradation of ribosomal proteins in vitro using recombinant
Ube2O, ubiquitin-activating E1 enzyme, HA-ubiquitin, purified human ribosomes and purified proteasomes (Lee et al., 2010; Wang et al., 2007a). Incubation for 8 hours at
37°C led to efficient degradation of ribosomal proteins. To test whether this process was proteasome-dependent, we sought to attenuate the degradation of ribosomal proteins by inhibiting the proteasome. Similar to the cell-free reticulocyte lysate system, we aimed to concentrate Rpl37 into a single band by deubiquitinating all high molecular weight forms of the protein. To confirm that proteasome inhibitors completely block ribosomal protein
113 Table 4.1
Ubiquitination sites on the ribosome from in vitro ubiquitination reaction.
Purified 80S ribosomes were ubiquitinated by Ube2O, and ubiquitinated proteins were pulled down and analyzed by LC-MS/MS. The table summarizes the number of ubiquitination sites and peptides identified for ribosomal proteins (top). Ubiquitin linkages are also quantified (bottom).
114 degradation, we first stopped reaction with a specific E1 inhibitor (Ungermannova et al.,
2012). We then treated the reaction with the pan-deubiquitinating enzyme USP21. When the reaction was treated with proteasome inhibitors, we found that USP21 was able to concentrate Rpl37 into a single band, corresponding to complete stabilization of the protein (Figure 4.10). In summary, ribosomal protein degradation is dependent on both
Ube2O and the proteasome across multiple systems.
115
Figure 4.10
Ribosomal proteins can be degraded by the proteasome in a purified system.
When incubated with recombinant Ube2O and purified proteasomes, ribosomal proteins can be degraded in a proteasome-dependent manner (after 8 hours of incubation at
37°C). USP21 is used to concentrate Rpl37 into a single band. W – Ube2O WT; C –
Ube2O CA.
116 Discussion
In Chapter 4, we sought to determine how ribosomes are degraded after being ubiquitinated by Ube2O. Could the UPS also degrade ribosomal proteins? Could ribosomal protein degradation explain why the UPS is so processive and active in reticulocytes?
A simple test of proteasome-dependence was to treat WT reticulocytes with proteasome inhibitors. Indeed, treatment with proteasome inhibitors attenuated the loss of rRNA staining, phenocopying the Ube2O null effect. We observed the depletion of free ubiquitin in these enucleated cells after treatment, though not likely enough to account for these observations. To avoid the secondary effects of ubiquitin depletion, we reconstituted the degradation of several ribosomal proteins in a cell-free reticulocyte lysate system, and inhibited their degradation with proteasome inhibitors. The lysate system allowed us to supplement exogenous ubiquitin to avoid the indirect effects of ubiquitin depletion secondary to proteasome inhibition. As controls, we ruled out selective autophagy, mTOR, and p97 involvement in ribosome elimination. Finally, we reconstituted the ubiquitination and degradation of ribosomes in a purified system.
Thus, we confirmed that ribosomal elimination is not only ubiquitin-dependent, but also proteasome-dependent. We used three orthogonal approaches to confirm that ribosomal proteins are degraded by the proteasome. When we treated WT reticulocytes with proteasome inhibitors, we observed an even stronger phenotype than the Ube2O null mutant. This is consistent with the hypothesis that there are perhaps redundant ubiquitin ligases in the cell that can ubiquitinate the ribosome in the absence of Ube2O. This may also explain why Ube2O is not completely necessary for ribosome elimination in reticulocytes. Thus, while there are multiple ubiquitin ligases that can drive ribosome
117 elimination (with Ube2O being the dominant ligase in terminal differentiation), this process relies entirely on the proteasome to degrade the ribosome. It would be mechanistically interesting to further identify these additional ligases that mediate ribosome elimination.
When we reconstituted the degradation of ribosomal proteins in the cell-free lysate system, we stabilized a monoubiquitinated form of Rpl35 and Rpl36a with proteasome inhibitors. We observed that monoubiquitinated form of Rpl35 was susceptible to deubiquitination the pan-deubiquitinating enzyme, USP21 (Gong et al., 2000; Ye et al.,
2011). Why do proteasome inhibitors stabilize these monoubiquitinated forms of Rpl35 and Rpl36a? Ube2O is a processive ubiquitin ligase that can multimonoubiquitinate its substrates (Berleth and Pickart, 1996). Since we do not observe this monoubiquitinated form in the absence of proteasome inhibitor, Ube2O may multimonoubiquitinate these proteins so that they no longer migrate at the lower molecular weight range. This is likely since multimonoubiquitination has been shown to serve as a signal for proteasomal degradation (Dimova et al., 2012; Kravtsova-Ivantsiv et al., 2009). However, stabilization of the monoubiquitinated form may suggest that these are the dominant species when lysates are treated with Ube2O. These monoubiquitinated forms may represent the initial ubiquitination of the ribosomal protein; one scenario is that subsequent ubiquitination events may require the ribosomal protein to be separated from the 80S complex to expose additional buried lysines.
Another advantage to this cell-free lysate system is that the ribosomes cannot be extruded from the cell via the exosome pathway, which requires a cell membrane. The exosome pathway, which was discovered in the reticulocyte (Johnstone et al., 1987), actually has some crosstalk with the ubiquitin proteasome system when remodeling the erythroid cytoskeleton (Blanc et al., 2009; Liu et al., 2010). Could the exosome pathway
118 be involved in eliminating ribosomes as well? In a cell free lysate system, elimination by the exosome pathway cannot occur, therefore bypassing this form of protein clearance.
We also entertained the possibility that selective autophagy was involved in ribosome elimination since it is a ubiquitin-dependent process. The autophagy-dependent turnover of ribosomes has been reported in yeast, though primarily under starvation conditions
(Kraft et al., 2008). However, there is no apparent defect in ribosome elimination when the autophagy receptor for mitochondria (NIX) is knocked out (Sandoval et al., 2008;
Schweers et al., 2007). Moreover, the function of NIX is ubiquitin-independent (Novak et al., 2010). Another critical regulator of autophagy is Ulk1, the deficiency of which produced a very mild defect in ribosomal clearance when deficient in reticulocytes
(Kundu et al., 2008). However, Ulk1 is not an autophagy receptor, but a global regulator of autophagy that may serve principally to control overall rates of autophagy rather than specificity. We did not observe any attenuation of rRNA elimination when we inhibited selective autophagy. This observation was consistent with previous studies on autophagy in reticulocytes. Moreover, the proteome of a mature erythrocyte consists of nearly 95% globin. Classic, “bulk” autophagy should not play a significant role since it would not be concentrative of globin, as it would also destroy globin. Instead, reticulocyte maturation is characterized by highly selective degradation events as highlighted by Ube2O. Finally, the mature reticulocyte has minimal lysosomes, at best, if any at all (Ciechanover, 2005; Veldman et al., 1984).
In addition to autophagy, the TOR pathway was shown to regulate ribosome levels in yeast (Pestov and Shcherbik, 2012). Specifically, inhibition of TOR by rapamycin rapidly drove the turnover of ribosomes in yeast grown in rich media. We treated the 293 cells with rapamycin to inhibit mTOR, but were unable to recapitulate this rapid turnover of ribosomes. Finally, if ribosome elimination is proteasome-dependent, it is thought that
119 the proteasome itself is unable to disassemble large, stable complexes. Instead, p97, or
Vcp or Cdc48 in yeast, is a ubiquitin-dependent chaperone that uses ATP to disassemble these complexes (Meyer et al., 2012). However, inhibiting p97 with NMS-
873 (Magnaghi et al., 2013) did not attenuate the elimination of ribosomes from reticulocytes. If ribosome elimination proceeds independently of the co-factor p97, how does it handle a large, stable complex like the ribosome? One possibility is that the ubiquitination of ribosomal proteins helps remove the subunit from the rRNA scaffold.
Certainly, ubiquitination of positively charged lysines can abolish any salt bridge interactions between ribosomal protein and rRNA. Additional biochemical experiments will have to elucidate these mechanisms.
In final confirmation of ribosomal degradation by the proteasome, we reconstituted the ubiquitination and degradation of ribosomes in a purified system. We definitively show that Ube2O is a ubiquitin ligase with a built in E2 (i.e. it can charge itself with ubiquitin and recognize its substrates). Using purified components, we also identified the specific ubiquitination sites on the ribosome. In fact, these ubiquitination sites were in close agreement with the sites obtained from Ube2O reconstitution of reticulocyte lysate. As such, Ube2O functions autonomously from cell-type to cell-type or system. That ribosomal proteins could be degraded by the simple addition of purified proteasomes is also consistent with conclusion that p97 is not involved in ribosome elimination.
Finally, by deletion analysis, we found that truncation of the first N-terminal conserved domain was sufficient to abrogate substrate recognition of the ribosome and histone
H2B. Interestingly, the N-terminal truncated Ube2O was still able to ubiquitinate AHSP.
As such, Ube2O may have multiple substrate recognition domains that impart increased substrate specificity. We plan to map out the specific binding motifs on both Ube2O and substrate proteins. Since Ube2O may have a broad program of ubiquitination, it is
120 possible that the Ube2O recognition motif is rather broad. Since E2s canonically ubiquitinate small, basic proteins, it is possible that Ube2O recognizes substrate proteins based on their charge. Unlike other E2s, Ube2O can likely ubiquitinate larger proteins or protein complexes such as the ribosome.
121 Materials and Methods
Antibodies
The following antibodies were used for immunoblot analysis: anti-ubiquitin (Santa Cruz
Biotech, sc-8017, mouse monoclonal, used at 1:200); anti-Rpl35 (Sigma, SAB4500233, rabbit polyclonal, used at 1:1000); anti-Rpl36a (Novus, H00006173-M02, mouse monoclonal, used at 1:500); anti-Rpl37 (Abcam, ab103003, rabbit polyclonal, used at
1:500); anti-Rpl29 (Santa Cruz Biotech, sc-103166, goat polyclonal, used at 1:200); anti-
Rps19 (Sigma, SAB2500898, goat polyclonal, used at 1:1000); anti-Rps23 (Novus,
H00006228-M02, mouse monoclonal, used at 1:500); anti-GAPDH (Abcam, ab8245, mouse monoclonal, used at 1:5000); anti-AHSP (Rockland, 100-401-E79, rabbit polyclonal, used at 1:1000); anti-phospho-p70 S6 kinase (Thr 389) (Cell Signaling, 9205, rabbit polyclonal, used at 1:1000); anti-p70 S6 kinase (Cell Signaling, 2708, rabbit monoclonal, used at 1:1000); and anti-HA-HRP (Sigma, 12013819001, rat monoclonal, used at 1:1000).
Flow cytometry analysis of proteasome inhibitor treatment
Reticulocytes were cultured at 1:500 in IMDM with 20% BIT (serum substitute, StemCell
Technologies), 0.1% monothioglycerol (Sigma), 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. In addition, proteasome inhibitors (PS-341 and epoxomicin) or DMSO was added to the media at the reported concentrations. After culture at 37°C with 5% CO2, the cells were stained and analyzed by flow cytometry as described in Chapter 3 (see Methods, Ex vivo differentiation of reticulocytes and flow cytometry analysis).
12 2 Immunoblot for ubiquitin levels after proteasome inhibition
In addition to flow cytometry analysis, the cultured reticulocytes were harvested at the specified time point, washed with ice-cold PBS, and lysed with urea lysis buffer (50 mM
Hepes pH 7.5, 8M urea, 75 mM NaCl, 1x EDTA-free protease inhibitor cocktail (Roche),
1x PhosSTOP phosphatase inhibitor cocktail (Roche)) for 15 minutes at room temperature. The lysate was centrifuged at 13,200 rpm at 4°C for 10 minutes, and protein concentration was measured by BCA assay (Pierce). 50 µg of protein was loaded per lane for 4-12% SDS-PAGE analysis and immunoblotted using an anti- ubiquitin antibody (see Antibodies) by chemiluminescence (PerkinElmer ECL).
Cell free reticulocyte lysate degradation reconstitution
Reticulocytes from hem9 animals were washed 5x with ice-cold PBS with manual removal of the buffy coat by pipetting. The cells were then lysed with 2x volume of LC- mass spectrometry grade water for 15 minutes on ice. The lysate was then clarified by centrifugation at 13,200 rpm for 10 minutes at 4°C. Protein concentration was measure by BCA assay (Pierce). The lysate system was prepared as follows: 2 mM DTT, 30 µL hem9 reticulocyte lysate, 60 µM ubiquitin (Boston Biochem, U-100H), 100 ug/mL E1
(Boston Biochem, E-304), 10 mM creatine phosphate, 0.1 mg/mL creatine kinase, and
100 ug recombinant Ube2O. To inhibit the proteasome, 5 µM PS-341 and 5 µM epoxomicin were used. The reaction was then incubated at 37°C for 8 hours. At 4 hours of incubation, the reaction was supplemented with 0.5 µL of 500 mM ATP. At 0 and 8 hours, 20 µL of the reaction was resuspended with 20 µL of 5x Laemlli buffer to quench the reaction. The sample was then boiled at 95°C for 3 minutes. Finally, 100 µg of protein per lane were analyzed by 4-12% SDS-PAGE, and immunoblotted with antibodies against ribosomal proteins, ubiquitin, and GAPDH.
123 Usp21 treatment of samples from cell free lysate degradation system
To concentrate the ribosomal protein into a single band for immunoblot analysis, the cell free degradation system was modified. The lysate system was prepared as previously described. After the reaction was completed at 8 hours, instead of resuspending the reaction in 5x Laemlli buffer, the reaction was quenched with 10 nM NEM for 30 minutes at room temperature in the dark. After alkylation, the reaction was treated with excess
DTT to a final concentration of 6 mM DTT for 15 minutes on ice. Next, the reaction was treated with 1.5 µM USP21 for 30 minutes at 37°C to deubiquitinate any ribosomal protein-ubiquitin conjugates. After the USP21 incubation, the samples were then quenched with 5x Laemlli buffer, and 100 µg of protein were analyzed per lane by SDS-
PAGE and immunoblotting for Rpl35, ubiquitin, and GAPDH.
Autophagy and p97 inhibitor treatment of reticulocytes
As described, reticulocytes were cultured in complete medium at 37°C with 5% CO2. The cells were incubated with bafilomycin (100 nM, Santa Cruz Biotech) or NMS-873 (1 µM,
Aobious). After 48 to 72 hours, the cells were stained and analyzed by flow cytometry as described (see Flow cytometry analysis of proteasome inhibitor treatment).
Immunoblot analysis of rapamycin treatment on ribosomal protein stability
One hour prior to rapamycin treatment, the media was replaced with fresh media to ensure that the cells are not under starvation conditions. Next, the Flp-In T-REx 293 cells with stably integrated Ube2O were treated with 2 µM rapamycin for 1 hour at 37°C.
As a positive control for ribosomal protein degradation, Flp-In T-REx 293 cells with stably integrated Ube2O were also induced for 72 hours with 10 µg/mL doxycycline.
After treatment, the cells were washed with ice-cold PBS, and lysed with urea lysis
124 buffer (50 mM Hepes pH 7.5, 8M urea, 75 mM NaCl, 1x EDTA-free protease inhibitor cocktail (Roche), 1x PhosSTOP phosphatase inhibitor cocktail (Roche)) on ice. The cells were also lysed in parallel with SDS lysis buffer which contains 2% SDS instead of 8 M urea since phospho-p70 S6 kinase antibody does not recognize samples lysed with urea. The lysate was passed 10x through a 25G syringe, and then centrifuged at 13,200 rpm for 10 minutes at 4°C. After protein quantification, 25 µg was loaded per lane for 4-
12% SDS-PAGE, and immunoblotted for ribosomal proteins and GAPDH.
In vitro ubiquitination of ribosomes by recombinant Ube2O
Ribosomes were purified from proliferating HEK293 cells as described in (Belin et al.,
2010), except that the cell lysate was incubated with 1.6 µM USP21 for 15 minutes at
37°C prior to sucrose cushion centrifugation to eliminate any endogenous ubiquitination of the ribosome. USP21 was purified as described in (Ye et al., 2011). Mouse Ube2O
WT, catalytic dead (C1037A), truncation 1 (Δ1-471) and truncation 2 (Δ1-718) were expressed using the baculovirus system (Life Technologies) following manufacturer’s instruction and purified on Ni-NTA beads (Qiagen). Based on previous work (Berleth and
Pickart, 1996; Klemperer et al., 1989), ubiquitination reactions were carried out with 700 nM Ube2O, 200 nM His-UBE1 (Boston Biochem, E-306), 10 µM ubiquitin (Boston
Biochem, U-100H), 2 µM HA-ubiquitin (Boston Biochem, UB-560) and 0.6 unit/mL inorganic pyrophosphatase (New England Biolabs) in buffer containing 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 5 mM ATP, 150 mM NaCl and 1 mM DTT. Ribosome, H2B (New
England Biolabs) and AHSP (a kind gift from Mitchell Weiss, St. Jude Children's
Research Hospital) were used at 100 nM, 7.7 µM and 90 µM, respectively. Reactions were kept on a rotating wheel in 37°C for 4 hours unless indicated otherwise. At the end of reactions, Ni-NTA resin (Qiagen) was used to clear off autoubiquitinated Ube2O before immunoblot analysis, to minimize interference from this signal.
125 LC-MS/MS analysis of ribosome ubiquitination sites
The in vitro ribosome ubiquitination assay was performed with 3 μM biotin-ubiquitin
(Boston Biochem, UB-560) instead of HA-ubiquitin. After the reaction was completed, instead of SDS-PAGE analysis, biotin-ubiquitin conjugates were purified using 50 µl
NeutrAvidin beads (Thermo Scientific), binding at room temperature for 1 hour, followed by washing trice with buffer containing 8 M urea and 1% (w/v) SDS. Ubiquitin conjugates were prepared for LC-MS/MS identification as described in Chapter 2 (see Methods,
Biotin-ubiquitin conjugate purification and elution).
In vitro degradation of ribosomal proteins with purified proteasomes
Human 26S proteasome was purified from a biotin tagged RPN11 cell line (Wang et al.,
2007a) as described before (Lee et al., 2010), and added into the in vitro ubiquitination mix at 6 nM. Proteasome inhibitors PS-341 (5 µM) and epoxomicin (5 µM) were added where indicated. Creatine kinase (9 µM) and creatine phosphate (30 mM) were used to regenerate ATP. Degradation reactions were stopped by adding E1 inhibitor
(Ungermannova et al., 2012) after 8 h, followed by USP21 treatment (1.6 µM) for 30 min at 37°C where indicated. The reaction was analysed by 12% SDS-PAGE and immunoblotted for Rpl37.
Contributions
I performed the majority of the experiments described in this Chapter. Miguel Prado performed the mass spectrometry analysis. Mingwei Min performed the in vitro ubiquitination and degradation experiments. Daniel Finley edited the manuscript.
126
CHAPTER FIVE
Crosstalk between ribosomal degradation and translation
in reticulocytes
Background
Heme-regulated eIF2α kinase (HRI) is an important regulator of translation in reticulocytes (Han et al., 2001). Under low heme concentrations, HRI phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (eIF2). Phosphorylated eIF2α inhibits the guanine nucleotide exchange function of eIF2B, which prevents the exchange of
GTP for GDP bound to eIF2 (Krishnamoorthy et al., 2001). This inhibits general protein synthesis, which is known as the integrated stress response. In reticulocytes, HRI is the dominant eIF2α kinase. When heme concentrations are low, HRI stops globin synthesis to balance the heme:globin ratio. Clinically, iron deficiency leads to reduced concentrations of heme, activating HRI and the integrated stress response. Iron deficiency manifests as a hypochromic microcytic anemia.
There are three additional eIF2α kinases in the cell, GCN2, PERK, and PKR, which respond to different stressors. PERK is activated by endoplasmic reticulum stress, and
PKR is activated by double-stranded RNA. The final eIF2α kinase is GCN2 or eIF2ak4, which senses amino acid deprivation by binding to uncharged tRNAs (Dever et al.,
1992). Interestingly, activation of GCN2 has been linked to proteasome inhibition (Jiang and Wek, 2005). Upon treatment of mouse embryonic fibroblasts with proteasome inhibitors, activated GCN2 phosphorylated eIF2α, resulting in the inhibition of general protein synthesis. This work suggested that perhaps proteasome function was required to maintain amino acid homeostasis in the cell. In fact, under acute amino acid deprivation, yeast cells were able to maintain efficient translation by proteasomal degradation of pre-exisiting proteins (Vabulas and Hartl, 2005). Accordingly, proteasome inhibition led to rapid amino acid depletion in yeast. Another study proposed that amino acid supplementation can rescue cells (yeast, mammalian cells, flies) from the
128 deleterious effects of proteasome inhibition (Suraweera et al., 2012). Thus, it appears that the proteasome may regulate amino acid homeostasis in cells, which would be especially important in terminally differentiating reticulocytes that have downregulated amino acid transporters (An et al., 2014).
In reticulocytes, ribosomes are ubiquitinated by the E2-E3 hybrid enzyme Ube2O; this ubiquitination serves as a signal for ribosomal protein degradation by the proteasome.
Therefore, Ube2O null reticulocytes have elevated levels of ribosomal proteins and ribosomes, reflecting a critical defect of elimination. Ube2O null mice also have a hypochromic, microcytic anemia. However, these mice are iron replete and have no major imbalances of globin chains. As such, it appears as though these mice are anemic from a failure of ubiquitination. Interestingly, the hypochromic, microcytic anemia is often associated with decreased rates of globin translation. Since Ube2O null reticulocytes have excess amounts of ribosomes, there is expected to be an indirect effect on overall protein synthesis in these reticulocytes.
Here we report the finding that eIF2α is hyperphosphorylated in Ube2O null reticulocytes, reflecting an activation of the integrated stress response and likely suggesting a global defect in translation. This stress response is independent of HRI function. Instead, we observe that GCN2 is activated in these reticulocytes. Accordingly,
Ube2O null reticulocytes are deficient in free amino acid pools. Finally, given that ribosomes are degraded by the proteasome, we can recapitulate these free amino acid deficits by proteasome inhibition.
129 Results
Ube2O null reticulocytes have an activated integrated stress response, independent of HRI
One possible explanation for the Ube2O-/- phenotype of hypochromic, microcytic anemia is an indirect effect on translation. To explore this possibility, we looked at the phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), which is a key and well-established mechanism of global inhibition of translation initiation. The eukaryotic initiation factor 2 (eIF2) complex is found in its active state bound to GTP or inactive state bound to GDP. After eIF2-GTP binds Met-tRNAi to form a ternary complex with the 40S subunit and mRNA, the GTP is hydrolyzed to GDP. For eIF2α to recycle to its active state, GDP must be exchanged for GTP by its guanine nucleotide exchange factor, eIF2B, which is found at limiting concentrations in the cell (Krishnamoorthy et al.,
2001). However, phosphorylation of eIF2α at serine 51 inhibits the recycling of GDP for
GTP by eIF2B, locking eIF2 in the inactive state. Since eIF2B is limiting, low levels of eIF2α phosphorylation can have profound effects on protein synthesis. We hypothesized that eIF2α phosphorylation may mediate an indirect effect on translation of the hem9 mutant. To test this hypothesis, we analyzed hem9 and WT reticulocyte lysates by SDS-
PAGE and immunoblotted for total and phosphorylated eIF2α (Figure 5.1). We observed that eIF2α is hyperphosphorylated at serine 51 in Ube2O null reticulocytes, suggesting, though not proving, a global inhibition of translation.
There are four eukaryotic eIF2α kinases that are activated by different stresses, HRI
(heme deprivation), GCN2 (amino acid starvation), PKR (double-stranded RNA in viral infection), and PERK (unfolded proteins in the ER) (Sonenberg and Hinnebusch, 2009).
Activation of any of these four arms constitutes an activation of the integrated stress
130
Figure 5.1
Increased phosphorylation of eIF2α in hem9 reticulocytes, independent of HRI.
Fresh reticulocyte lysate (from mice induced by phenylhydrazine) was resolved by 4-
12% SDS-PAGE, and immunoblotted with an antibody to phospho-eIF2α and total eIF2α. +, wild-type allele, h9, hem9; HRI, HRI allele. Each sample is from a different mouse. 100 µg of protein was loaded per lane.
131 response, which downregulates translation generally, while inducing translation of mRNA molecules encoding few specific transcription factors. A dominant regulator of translation in reticulocytes is the heme-regulated eIF2α kinase (HRI), which is activated by heme deficiency (Han et al., 2001). When hem9 mutants were crossed to hri null mice, we observed little change in the hematological phenotype (Figure 5.2). The double mutants still have a hypochromic, microcytic anemia, indicating that there is no genetic interaction between HRI and Ube2O. To confirm that HRI is not involved in ribosome elimination, we then probed the double mutant biochemically. First, we generated polysome profiles for reticulocytes from HRI-/- hem9/hem9, HRI-/-, hem9, and WT animals. After fractionating the lysate on 20-50% sucrose gradients, we observed an exaggerated 80S monosome peak in HRI-/- hem9/hem9 reticulocytes that is similar to the 80S monosome peak in hem9 reticulocytes (Figure 5.3). As such, HRI-/- hem9/hem9 reticulocytes appear to have elevated levels of 80S ribosomes independently of HRI.
This gave us further confidence that the hem9 phenotype is independent of HRI. Next, we probed for the phosphorylation state of eIF2α in HRI-/- hem9 reticulocytes.
Consistent with these data, we find that eIF2α is also hyperphosphorylated in HRI and
Ube2O null animals, indicating that this response is independent of HRI kinase (Figure
5.1). Finally, we immunoblotted WT and hem9 reticulocyte lysates for HRI to look for activation of this kinase by phosphorylation. We did not observe any hyper- phosphorylation of HRI, which would result in an upshifted banding pattern (Figure 5.2).
Thus, eIF2α may be phosphorylated by another kinase independently of HRI.
Activation of GCN2 in Ube2O null reticulocytes
Which kinase, then, is responsible for activation of the integrated stress response?
There is no longer an endoplasmic reticulum in reticulocytes, making PERK a less likely
132 HRI hem9 HGB MCV MCH +/+ h9/h9 11.4±0.5 42.0±1.1 10.3±0.5
-/- h9/h9 11.2±0.4 42.8±0.8 10.4±0.2 T-test 0.56 0.27 0.74
Figure 5.2
HRI knockout does not affect the hem9 phenotype. hem9 and HRI-/- hem9/hem9 animals have a hypochromic, microcytic anemia (top).
Immunoblot analysis does not show activation of HRI in hem9 animals induced by phenylhydrazine. +, wild-type allele; h9, hem9. 100 µg of protein was loaded per lane.
133
Figure 5.3 h9/h9 HRI-/- animals have an exaggerated 80S monosome peak.
Reticulocyte lysates (from mice induced by serial retro-orbital bleeding) were fractionated on 20-50% sucrose gradients. h9/h9 HRI-/- reticulocytes have an exaggerated 80S peak, similar to h9/h9 reticulocytes.
134 candidate. Moreover, hem9 mice are presumably not virally infected, which may rule out
PKR. eIF2α can also be phosphorylated by GCN2 (eIF2ak4), which senses amino acid deprivation through binding to uncharged tRNA (Dever et al., 1992). To see if GCN2 is involved, we probed hem9 and WT reticulocyte lysates for GCN2 activation
(phosphorylation). First we immunoprecipitated total GCN2 from hem9 and WT reticulocyte lysates, analyzed the samples by SDS-PAGE, and immunoblotted for total
GCN2, phosphorylated GCN2, and for phospho-threonine. Through this approach, we detected both phosphorylated GCN2 and phosphorylated threonine, indicating an activation of GCN2 (Figure 5.4). These data suggested that Ube2O null reticulocytes may be deficient in free amino acids, resulting in activation of the integrated stress response through GCN2. In turn, this may lead to a global inhibition of translation, resulting in a hypochromic, microcytic anemia. An interesting possibility is that an amino acid deficiency could reflect that protein degradation is required to supply raw material for the production of globin.
The importance of regulating amino acid availability for proper globin translation has been previously proposed. A recent paper reported that the mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability (Chung et al., 2015).
However, immunoblotting WT and hem9 reticulocytes lysates for phospho-S6 kinase, a downstream target of mTORC1, shows that this pathway is not activated in terminally differentiating reticulocytes (Figure 5.5). Moreover, we showed above that inhibiting the mTORC1 pathway with rapamycin does not induce ribosomal degradation, and that the mTORC1 pathway is still activated when Ube2O is induced (Chapter 4, Figure 4.7). As such, mTORC1 does not appear to act in late stage reticulocytes, which have already undergone massive cellular remodeling.
135
Figure 5.4
GCN2 is activated in hem9 reticulocytes.
GCN2 was immunoprecipitated from fresh WT and hem9 reticulocytes (from mice induced by phenylhydrazine), and analyzed by 4-12% SDS-PAGE. Immunoblot analysis with an anti-phospho-GCN2 and anti-phospho-threonine antibody showed activation of
GCN2 in hem9 reticulocytes.
136
Figure 5.5 mTORC1 pathway does not appear to be active in terminal reticulocytes.
Fresh reticulocyte lysates (from mice induced by phenylhydrazine) were immunoblotted for S6 kinase and phospho-S6 kinase. There does not appear to be any phosphorylation of S6 kinase in terminally differentiating reticulocytes. 100 µg of protein was loaded per lane.
137 Ube2O null reticulocytes and erythrocytes are deficient in free amino acids
The proteasome has been linked to amino acid homeostasis in multiple organisms
(Suraweera et al., 2012; Vabulas and Hartl, 2005). Since hem9 reticulocytes are deficient in ribosome elimination via the proteasome, we hypothesized that there are perturbations of the free amino acids pools in these reticulocytes. To this end, we used mass spectrometry to quantify ~250 endogenous polar metabolites from WT and hem9 reticulocytes. Polar metabolites were extracted from hem9 and WT reticulocytes with
80% methanol, and total metabolites were normalized by total cell volume of the sample
(given the RBC count and mean cell volume). This quantitative profiling showed a deficiency of a subset of free amino acids in the absence of Ube2O. L-arginine and L- lysine were the most strongly reduced amino acids at 61.4% and 66.2%, respectively
(Figure 5.6). Proline, valine, tryptophan, alanine, and glutamine were also deficient in hem9 reticulocytes. While the majority of amino acids are depleted or unchanged in the mutant, no amino acids were significantly increased in Ube2O null reticulocytes. Amino acids that are ostensibly somewhat increased in the mutant may be perturbed due to reduced incorporation into nascent polypeptides. In addition, 51 other polar metabolites were significantly changed in the absence of Ube2O. Many of these metabolites are involved in arginine and proline metabolism or closely related to arginine (p=0.047).
We also performed quantitative metabolomics profiling on WT and Ube2O null whole blood. Endogenous metabolites were also extracted with 80% methanol and analyzed by mass spectrometry. Consistent with the metabolomics of reticulocytes, Ube2O null blood was significantly deficient in L-arginine and L-lysine (Figure 5.7). Valine, methionine cysteine, proline, threonine, and alanine were also significant depleted in hem9 reticulocytes. Moreover, 39 significantly changed polar metabolites are involved in amino acid metabolism.
138
Figure 5.6
Quantitative metabolomics profiling shows free amino acid deficiency in hem9 reticulocytes.
Amino acids and other polar metabolites were quantified from freshly harvested reticulocytes (from mice induced by phenylhydrazine) by mass spectrometry. The histogram shows the log-2 ratio change of amino acid concentrations between hem9 and
WT reticulocytes.
139
Figure 5.7
Quantitative metabolomics profile of mature erythrocytes shows amino acid deficiencies in hem9 mutants.
Polar metabolites were extracted from mature erythrocytes from WT and hem9 animals, and analyzed by mass spectrometry. The histogram shows the log-2 ratio of amino acid concentrations between hem9 and WT animals from peripheral blood samples.
140 Proteasome inhibition can deplete free amino acid pools in reticulocytes
Can amino acid deficiency be induced by proteasome inhibition in reticulocytes? To this end, we first showed that ex vivo differentiation of reticulocytes is not affected by amino acid-free medium. We then cultured WT reticulocytes with proteasome inhibitors, PS-
341 and epoxomicin, or DMSO in amino acid-free DMEM, and after 48 hours of ex vivo differentiation, we extracted endogenous polar metabolites with 80% methanol.
Proteasome inhibition led to a significant depletion of L-arginine and L-lysine (4.5- and
5.8-fold, respectively) (Figure 5.8). We also saw a significant depletion of aspartate, histidine, tryptophan, serine, and asparagine. Of note, five amino acids accumulated significantly upon proteasome inhibition, including glycine, threonine, proline, glutamine, and leucine/isoleucine. Metabolites linked to amino acid metabolism were also significantly changed upon proteasome inhibition.
We are particularly interested in L-arginine and L-lysine, since they are over-represented in mouse ribosomal proteins to bind the negatively-charge rRNA. L-arginine and L-lysine are the two most common amino acids found in ribosomal proteins at 10.25% and
12.88%, respectively (Figure 5.9). Moreover, it has been previously reported that arginine is not actively transported during red cell maturation (Benderoff et al., 1978), and that the SLC7A1 arginine transporter is downregulated by the orthochromatic stage in both mouse and human erythropoiesis (An et al., 2014). Also, arginine deficiency was shown to cause runting in mice by activating GCN2 (Marion et al., 2011). Finally, arginine starvation also occurs in human colorectal carcinoma cells, thereby activating
GCN2 and repressing global translation (Vynnytska-Myronovska et al., 2016).
In summary, we showed that hem9 reticulocytes have an activated integrated stress response, as indicated by the hyperphosphorylation of eIF2α in nascent reticulocytes.
141
Figure 5.8
Proteasome inhibitor treatment of WT reticulocytes could deplete free amino acid pools in reticulocytes.
WT reticulocytes (from mice induced by phenylhydrazine) were treated with 50 nM PS-
341 and 50 nM epoxomicin for 48 hours; after ex vivo culture, polar metabolites were extracted and analyzed by mass spectrometry. The histogram shows the log-2 ratio of amino acid concentrations in treated versus untreated WT reticulocytes.
142
Figure 5.9
L-lysine and L-arginine are highly represented in ribosomal proteins.
Amino acid frequencies were analyzed in mouse ribosomal proteins. The histogram shows the frequency of representation for each amino acid in ribosomal proteins.
143 This suggests a global inhibition of general translation that could potentially contribute to the hypochromic, microcytic anemic phenotype of the hem9 mouse. As a parallel, iron deficiency also causes a hypochromic, microcytic anemia, presumably through activation of the integrated stress response. However, in iron deficiency anemia, eIF2α is phosphorylated by HRI, a dominant regulator of translation in reticulocytes (Han et al.,
2001). We thoroughly explored the possibility that HRI is involved in the hem9 mutant.
However, there is no genetic interaction between the two alleles: HRI does not appear to be activated in hem9 reticulocytes, and HRI does not appear to affect ribosome elimination in reticulocytes. We then showed that another eIF2α kinase, GCN2, is activated in hem9 reticulocytes. This was supported by quantitative metabolomics, which indicate a depletion of positively charged amino acids in hem9 reticulocytes. As a proof of principle, we recapitulated this phenotype of amino acid deficiency by treating reticulocytes with proteasome inhibitors. In all, these data suggest that the degradation of ribosomes provides free amino acids for the final stages of globin translation during terminal erythropoiesis.
144 Discussion
Microcytic anemias are commonly caused by either iron deficiency or globin chain imbalances (thalassemias). Interestingly, the Ube2O null mouse does not appear to have a defect related to these pathways. All of the iron parameters of these mutant mice are normal (unpublished data, Fleming lab), and there does not appear to be a massive imbalance of globin chains (which would manifest as Heinz bodies, or hemoglobin inclusions, on supravital stain). Thus, the hypochromic, microcytic anemia of the Ube2O null mouse appears to be a novel anemia due to a failure of ubiquitination. However, the anemia phenotype of the mouse appears to be paradoxical with its biochemical phenotype of high ribosome abundance. Hypochromic, microcytic anemia is often associated with global defects in translation for example caused by iron deficiency.
Based on our characterization of Ube2O null reticulocytes, there are supraphysiological levels of ribosomal proteins and 80S ribosomes. As such, Ube2O is expected to have an indirect effect on translation, especially since it ubiquitinates the ribosome.
In Chapter 5, we observed the hyperphosphorylation of eIF2α in Ube2O null reticulocytes, which constitutes an activation of the integrated stress response and corresponds to a global inhibition of general protein synthesis. We did not find any genetic interaction between HRI and Ube2O when the alleles were crossed in mice.
Consistent with this observation, we observed an exaggerated 80S peak in Ube2O null and the compound null mutant (this exaggerate 80S peak was absent in the HRI null animal). Finally, we immunoblotted WT and null reticulocyte lysates for HRI and did not observe any frank activation of this enzyme (evidenced by laddering of this enzyme due to hyperphosphorylation). Next, by immunoprecipitated of GCN2 and immunoblotting for phospho-GCN2 or phospho-threonine, we observed that GCN2 is phosphorylated, or activated, in Ube2O null reticulocytes. Consistent with an activation of GCN2, we found
145 that null reticulocytes are deficient in a variety of amino acids. Finally, we were able to recapitulate this phenotype by treating WT reticulocytes with proteasome inhibitors.
As discussed in Chapter 2, we initially identified Ube2O as a globin ligase after identifying globin in the ubiquitinated form. Thus, we anticipated that the pathophysiology of Ube2O deficiency is due to a globin imbalance that would exacerbate β-thalassemia.
Contrary to expectations, Ube2O deficiency ameliorates a murine model of β- thalassemia intermedia. This compound mutant has increased hemoglobin, decreased splenomegaly, and a more severe microcytosis than each individual mutant. How does
Ube2O deficiency improve hemoglobin production, but result in an even smaller mean cell volume? This seemingly paradoxical amelioration of β-thalassemia was reported with an RNAi therapeutic targeting Tmprss6 in murine β-thalassemia intermedia
(Schmidt et al., 2013). Tmprss6 is a membrane-bound serine protease on hepatocytes that negatively modulates the production of hepcidin, a hepatocyte peptide hormone that negatively regulates cellular iron export. Since dietary restriction of iron or moderate overexpression of hepcidin was shown to ameliorate murine β-thalassemia intermedia
(increased hemoglobin, decreased splenomegaly, decreased iron overload) (Gardenghi et al., 2010), Schmidt and colleagues knocked down Tmprss6, which increased hepcidin and diminished tissue and serum iron levels. This led to a nearly identical amelioration of
β-thalassemia as seen with Ube2O deficiency, namely an increase in hemoglobin, decreased splenomegaly, decreased mean cellular volume, and decreased reticulocyte mean cell hemoglobin. Also, when WT mice were treated with the Tmprss6 siRNA, this caused a mild hypochromic, microcytic anemia.
What do Ube2O deficiency and Tmprss6 knockdown have in common? Since siRNA for
Tmprss6 decreases serum iron levels, it likely leads to a heme deficiency, which is
146 sensed by the heme-regulated eIF2α kinase (HRI) (Han et al., 2001). When intracellular concentrations of heme decrease (i.e. from iron deficiency), HRI is activated, which causes it to phosphorylate the alpha subunit of translational initiation factor 2 (eIF2α). eIF2α is considered a master regulator of cellular translation (Krishnamoorthy et al.,
2001). When phosphorylated at serine 51, eIF2α inhibits the recycling of GDP for GTP by eIF2B. Since eIF2B is found at limiting concentrations in the cell, low levels of eIF2α phosphorylation can lead to global defects in general translation. Thus, it appears as though the integrated stress response is activated in Ube2O null reticulocytes. Even though Ube2O null reticulocytes have an excess amount of ribosomes, null mice have a hypochromic, microcytic anemia. Thus, the hyperphosphorylation of eIF2α appears to have a dominant negative effect by inhibiting general protein synthesis indirectly.
Next, we sought to identify the kinase responsible for phosphorylating eIF2α in Ube2O null reticulocytes. Contrary to expectations, HRI, a dominant eIF2α kinase in reticulocytes, does not appear to mediate the Ube2O null phenotype. Since reticulocytes do not have an endoplasmic reticulum, PERK (eIF2ak3) is a less likely candidate.
Moreover, PKR (eIF2ak2) is not very likely since Ube2O null mice are presumably not virally infected. The final eIF2α kinase is GCN2 (eIF2ak4), which sense amino acid deprivation through uncharged tRNAs (Dever et al., 1992). Could GCN2 be activated in
Ube2O null reticulocytes? GCN2 has been previously linked to proteasome function.
When mouse embryonic fibroblast (MEF) cells were treated with proteasome inhibitors, this led to a significant decrease in protein synthesis due to eIF2α phosphorylation
(Jiang and Wek, 2005); in this study, the primary eIF2α kinase activated by proteasome inhibition was GCN2. Since proteasome inhibitors could deplete free amino acid pools similarly to deficiency of Ube2O, we hypothesized that Ube2O null reticulocytes could be
147 considered as proteasome hypomorphs akin to treatment with proteasome inhibitors.
Indeed, GCN2 appears to be activated in Ube2O null reticulocytes.
Does the proteasome, then, regulate free amino acid levels during terminal erythroid differentiation? The proteasome has been implicated in regulating amino acid homeostasis in multiple organisms. For example, under an acute decrease in external amino acid supply, protein synthesis is dependent on proteasome function, presumably to supply free amino acids (Vabulas and Hartl, 2005). Moreover, it has been proposed that amino acid supplementation can rescue cells (yeast, mammalian cells, and flies) from the effects of proteasome inhibition (Suraweera et al., 2012). Therefore, we hypothesized that there are amino acid deficiencies in Ube2O null reticulocytes, and that these perturbations activate GCN2. The two most depleted amino acids in Ube2O null reticulocytes were L-arginine and L-lysine, which are positively charged amino acids that are highly enriched in ribosomal proteins (to bind the negatively charged rRNA). Analysis of the other polar metabolites that were depleted showed enrichment for metabolic intermediates of arginine and proline metabolism. Thus, it appears that Ube2O null reticulocytes are deficient in a subset of amino acids, including the two most commonly represented amino acids in ribosomal proteins. Moreover, it appears as though the proteasome can regulate free amino acid levels in terminally differentiating reticulocytes.
Finally, we recapitulated these free amino acid deficiencies by proteasome inhibitor treatment. In fact, these amino acid deficiencies (under proteasome inhibition) were quite severe. Consistent with the metabolomics profiling of reticulocytes, the strongest changes we observed were in the depletion of L-arginine and L-lysine. As such, these data suggest that free amino acid pools are sensitive to proteasome function in these cells. Since Ube2O is a dominant E2 in terminal differentiation, Ube2O deficiency can similarly deplete free amino acids compared to inhibition of the proteasome.
148 Since GCN2 is activated in mutant reticulocytes, we are testing this hypothesis genetically in mice. The GCN2-/- mouse is viable and fertile with no observable phenotype under normal growth conditions (Zhang et al., 2002). We are currently crossing the hem9 mouse to the GCN2-/- mouse. If GCN2 is the dominant activator of the integrated stress response in Ube2O null mouse, then the compound mutant should have either an ameliorated or exacerbated phenotype. We also strongly consider the scenario that eIF2α is pleiotropically phosphorylated by multiple kinases in addition to
GCN2. Though PERK and PKR are less likely to be involved in terminally differentiating reticulocytes, it is possible that these kinases contribute to eIF2α phosphorylation. In this case, this would make the null mouse even more interesting to study as multiple stress response pathways are activated during terminal differentiation.
Moreover, we are also looking at the translation rates of these mutant reticulocytes.
Conventional pulse chase assays with radiolabeled amino acids did not produce consistent results measuring incorporation rates. We now recognize that perturbed amino acid pools in null reticulocytes may contribute to variable rates of translation in these cells. Another approach to assaying translation in reticulocytes is by ribosome profiling, which analyzes the mRNAs engaged by translating ribosomes and polysomes.
We are currently using ribosome profiling to assess whether these mutant reticulocytes are deficient in translation and how Ube2O deficiency affects mRNA engagement.
In summary, Ube2O null reticulocytes have an activated integrated stress response that is independent of the dominant eIF2α kinase HRI. Instead, we observed an activation of
GCN2 likely due to amino acid depletion, a phenotype that can be recapitulated by proteasome inhibition.
149 Materials and Methods
Antibodies
The following antibodies were used for immunoblot analysis: anti-phospho-p70 S6 kinase (Thr 389) (Cell Signaling, 9205, rabbit polyclonal, used at 1:1000); anti-p70 S6 kinase (Cell Signaling, 2708, rabbit monoclonal, used at 1:1000); anti-phospho-eIF2α
(Cell Signaling, 3398, rabbit monoclonal, used at 1:1000); anti-eIF2α (Cell Signaling,
5324, rabbit monoclonal, used at 1:1000); anti-GCN2 (Cell Signaling, 3302, rabbit polyclonal, used at 1:1000); anti-phospho-threonine (Cell Signaling, 9381, rabbit polyclonal, used at 1:1000); anti-phospho-GCN2 (Thr898) (Biorbyt, orb251471, rabbit polyclonal, used at 1:100); and anti-HRI (kind gift from Jane-Jane Chen, MIT,
Cambridge, MA, rabbit polyclonal, used at 1:3000).
Immunoblot analysis of reticulocyte lysate hem9 and WT reticulocytes were washed 5x with 5 mL of ice-cold PBS, and the buffy coat was manually removed by pipetting. Next, reticulocytes were lysed with SDS lysis buffer (50 mM Hepes pH 7.5, 2% SDS, 150 mM NaCl, 1x EDTA-free protease inhibitor cocktail (Roche), 1x PhosSTOP phosphatase inhibitor cocktail (Roche)) for 15 minutes at room temperature to prevent SDS precipitation. The cell lysate was then centrifuged at 13,200 rpm for 10 minutes at 4°C, and protein concentrations were measured by BCA assay (Pierce). 100 µg of protein per lane were analyzed by 4-12% SDS-PAGE
(Invitrogen) and immunoblotting for the respective protein with fluorescent secondary antibodies as described in Chapter 2 (see Methods, Immunoblot analysis of putative
Ube2O substrates).
150 Polysome profile analysis of HRI-/- and HRI-/- hem9/hem9 reticulocytes
Reticulocytes from hem9, WT, HRI-/-, and HRI-/- hem9/hem9 mice were treated with
100 µg/mL cycloheximide, and then washed with ice-cold 1x PBS containing 100 µg/mL cycloheximide. The reticulocytes were then lysed with ice-cold 200 mM Tris HCl pH 8.8,
25 mM MgCl2, 25 mM KCl, 1% Triton X-100, 1.3% sodium deoxycholate, 100 µg/mL cycloheximide, and 200 units/mL RNAse inhibitor (New England Biolabs). The lysates were incubated for 10 minutes at 4°C, centrifuged at 3,000 rpm for 10 minutes at 4°C, and fractionated on 20-50% (w/w) sucrose gradients as described in Chapter 2 (see
Methods, Polysome profile analysis of reticulocytes).
GCN2 immunoprecipitation and immunoblot analysis
Reticulocytes were washed 5x with 5mL of ice-cold PBS, and lysed with 0.5 mL of ice- cold lysis buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1
µg/mL leupeptin, 1x EDTA-free protease inhibitor cocktail, 1x PhosSTOP phosphatase inhibitor cocktail). Lysate was incubated on ice for 5 minutes and then centrifuged at
14,000 x g for 10 minutes at 4°C. The lysate was precleared with 30 µl of 50% Protein A magnetic bead slurry (Cell Signaling, 8687), and was incubated with rotation at 4°C for 1 hour. The magnetic beads were pelleted with a magnetic separation rack, and the supernatant was transferred to a fresh tube. Anti-GCN2 antibody was added to the lysate at a 1:100 dilution, and the lysate was incubated with rotation overnight at 4°C.
Next, 30 µl of 50% protein A magnetic beads was incubated with the lysate with rotation for 30 minutes at 4°C. The beads were pelleted with a magnetic separation rack, and washed 5x with 500 µl of ice-cold lysis buffer. For immunoblot analysis, the pellet was resuspended with 30 µl of 3x SDS sample buffer (187.5 mM Tris HCl pH 6.8, 6% SDS,
151 30% glycerol, 0.03% bromophenol blue, 125 mM DTT), vortexed, then centrifuged for 30 seconds at 14,000 x g. The sample was boiled at 95°C for 4 minutes, and then centrifuged for 1 minute at 14,000 x g. 15 µl of sample was loaded per lane for SDS-
PAGE analysis (see Chapter 2, Methods, Immunoblot analysis of reticulocyte lysate) and
Immunoblotting with anti-GCN2, anti-phospho-GCN2, and anti-phospho-threonine antibodies.
Quantitative metabolomic profiling of nascent reticulocytes and erythrocytes
Reticulocytes were carefully washed 5x with 5 mL of ice-cold PBS, with manual disruption of the buffy coat. Immediately after washing, 4 mL of 80% methanol (pre- chilled to -80°C) was added to the reticulocytes. The sample was then transferred on dry ice to -80°C for 20 minutes. Next, the samples were thawed on ice until it could be resuspended into a homogenous mixture. The samples were then centrifuged at maximum speed for 5 minutes at 4°C to pellet cell debris and precipitated proteins. The supernatant was transferred to a 50 mL conical tube, pre-chilled on dry ice. Next, the cell pellet was resuspended with 500 µl of 80% methanol by vortexing, transferred to a 1.5 mL Eppendorf tube on dry ice, and centrifuged at 13,200 rpm for 5 minutes at 4°C. The supernatant was carefully transferred to the 50 mL conical tube on dry ice. Finally, the cell pellete was resuspended with another 500 µl of 80% methanol and centrifuged at
13,200 rpm for 5 minutes at 4°C. The supernatant was pooled with the other two extractions on dry ice. To dry the samples, 1.5 mL of the extracted metabolites were aliquoted to a 1.5 mL eppendorf tube, and vacuum dried for approximately 4 hours without heat. The pellets were stored at -80°C until submitted for analysis. Quantitative metabolomics profiling was performed at the BIDMC Mass Spectrometry Core Facility.
LC-MS/MS was performed on a QTRAP 5500 (SCIEX). Metabolites were normalized to total cell volume, as calculated by RBC count per µl of blood and mean cellular volume.
152 Proteasome inhibitor treatment of reticulocytes and metabolomic analysis
Reticulocytes were differentiated ex vivo in DMEM without amino acids, supplemented with 20% BIT (serum substitute, StemCell Technologies), 0.1% monothioglycerol
(Sigma), 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. The cells were treated with 200 nM epoxomicin and 200 nM PS-341, or DMSO. After 48 hours of culture at 37°C with 5% CO2, the cells were harvested and washed with ice-cold
PBS. Next, the polar metabolites were extracted with pre-chilled 80% methanol (-80°C) as previously described (see Quantitative metabolomic profiling of nascent reticulocytes and erythrocytes).
Amino acid frequency of ribosomal proteins
Murine ribosomal protein sequences were downloaded in FASTA format from UniProt.
Only reviewed and non-redundant sequences were included in the sequence analysis.
Amino acid frequencies were calculated using the Composition based Protein
Identification (COPid) web tool given the inputted FASTA file. Data analysis and representation were performed with Prism 6 (GraphPad).
Contributions
I performed the majority of the experiments described in this Chapter. Stefanie de Poot and Daniel Finley edited the manuscript.
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