דוקטור לפילוסופיה Doctor of Philosophy

עבודת גמר )תזה( לתואר Thesis for the degree דוקטור לפילוסופיה Doctor of Philosophy

מוגשת למועצה המדעית של Submitted to the Scientific Council of the מכון ויצמן למדע Weizmann Institute of Science רחובות, ישראל Rehovot, Israel

מאת By אסף בירן Assaf Biran

הקצה הקרבוקסילי של תת היחידה PSMA3 של 20S פרוטאזום לוכד IDPs

The C terminus of the 20S proteasome subunit PSMA3 traps IDPs

מנחה: :Advisor Prof. Yosef Shaul פרופ' יוסף שאול

ניסן, תשע"ה March, 2015

1 Table of contents

List of Abbreviations…………………………………………………………………3 Abstract……………………………………………………………..……………..….4 Introduction…………………………………………………………………………..5 Results In vivo visualization of 20S interacting proteins………………………...... 9 The 20S proteasome PSMA ring has two hub regions of protein-protein Interaction…………………………………………………………………...11 PSMA3 and PSMA7 are the candidates IDP trappers…………………...19 C-terminus truncated PSMA3 and PSMA7 are inefficient in p21 trapping………………………………………………..……....………...... 20 The PSMA3 trapping region is functional in the PSMA5 context……….22 The PSMA3 IDP trapper is involved in the default degradation pathway……………………………………………………………………...24 NQO1 association to 20S proteasome is a regulated process…………….28 Discussion……………………………………………………………………………32 Materials and methods………………………………………………………..…….38 References……………………………………………………………………….…..41 Declaration…………………………………………………………..……………....48 Acknowledgments………………………………………………………………..…48 Hebrew summary………………………………………………………………..….49

2 List of Abbreviations IDP - Intrinsically disordered protein BiFC - Bimolecular fluorescence complementation MS - Mass spectrometry FACS - Flourescence activated cell sorting (Flow cytometry) IP - Immunoprecipitation VFP - Venus fluorescent protein RFP - Red fluorescent protein

3 Abstract The process of proteasomal degradation by default of proteins is an important ubiquitin independent degradation pathway. The list of proteins that undergo ubiquitin independent degradation grows. The majority if not all the substrates of this pathway are intrinsically disordered proteins (IDP or IUP). The basic principle of this pathway is that IDPs are degraded by default unless they are found in a complex or in association with a nanny protein, a protein that protects IDP. The 20S proteasome was identified as the protease in degradation by default pathway. However, whether and how the IDPs are selectively targeted to the 20S is an open question. To address this question, we adopted the BiFC cell based technique for visualizing protein interactions with 20S proteasome. We found that IDPs interact with a subset of 20S proteasome alpha subunits. The results suggest that the 20S proteasome in the alpha ring has two hub subunits PSMA3 and PSMA7 for interaction with IDPs with different affinities. Next we delineated the PSMA regions involved in IDP binding by mutagenesis and domain swapping experiments. We identified that these two PSMA subunits interact with p21 through their 69 amino acids C terminus, a region exposed outside the 20S alpha ring structure. Remarkably this region is not only required but also sufficient to “trap” the substrate, as evident by the capacity of this region to convey substrate recognition to PSMA5, a refractory subunit. We refer to these regions as substrate tapper regions. For further mapping of the substrate trapper sequences we focused on the subunit PSMA3 as the BiFC indicated it has a higher affinity to IDPs. To demonstrate that the PSMA3 substrate-trapping region is active in isolation in vitro we used GST-fusion recombinant protein for pull-down experiments. Remarkably, we found that PSMA C-terminus region selectively binds p21, Fos and p53 when incubated with cellular extracts. We next used total cellular extracts pre- treated for IDP enrichment and revealed by MS analysis that vast majority of the binding proteins are either completely or partially IDPs. Moreover, we demonstrate that sequestrating the trapper in cell free system and in the cells protects IDPs from default degradation. We propose that in the process of protein degradation by default, targeting of IDPs to the proteasome through substrate trapper regions is a required step. An IDP binding partner can inhibit the process of degradation by default. We termed this type of IDP partners as nannies. NQO1 is a gatekeeper of the 20S proteasome and interacts with a large number of 20S proteasome substrates, and as

4 such it is a hub nanny. We have found that NQO1 association with the 20S proteasome differs between various tissues and cell line suggesting it is regulated. Web proteomic data suggests that NQO1 is highly modified including by S/T and Y phosphorylation. We have some preliminary data to indicate that c-Abl can phosphorylate NQO1 and the phosphorylation affects the association with the 20S proteasome. These results suggest that NQO1 modification modulates its 20S association.

Introduction Protein degradation plays a key role in the regulation of diverse cellular processes including proliferation, differentiation, death, antigen processing, DNA repair, inflammation and stress response as reviewed (Voges et al. 1999). In the cell there are two major protein destruction pathways. One involves proteolytic enzymes in the lysosomes and the other involves the multicatalytic proteasome complex. The most extensively studied proteasomal degradation pathway is the ubiquitin pathway. In this pathway proteins undergoing proteasomal degradation follow two successive steps. First the protein substrate is tagged by a covalent attachment of multiple ubiquitous molecules termed ubiquitin (polyubiquitination) in a tightly regulated process. Secondly, the tagged protein targets the 26S proteasome via the 19S subunit. Next the polyubiquitin chains are removed and the substrate is unfolded and degraded by the 26S proteasome (Hershko & Ciechanover 1998). The 26S proteasome is a complex comprised of a 20S core component with multiple peptidase activities and one or two 19S regulatory complexes consisting of a lid and a base. The lid contains the subunits that recognize and bind the ubiquitinated substrates while the base contains multiple ATPases that unfold the protein and feed it to the catalytic chamber of the 20S core particle in an ATP dependent manner. The 20S core particle is comprised of four rings of seven subunits that form the catalytic chamber (Coux et al. 1996). Experiments performed in vitro with purified 20S proteasome revealed that many proteins undergo degradation without ubiquitination. The microtubule associated protein tau (David et al. 2002; Elliott et al. 2007), α−synuclein (Tofaris et al. 2001), IκBα (Alvarez-Castelao & Castano 2005) and BIM(EL) (Wiggins et al. 2011) are a few examples. Currently it is estimated that 20S proteasome can degrade

5 more than 20% of the cellular proteins (Baugh et al. 2009). Moreover, it is currently estimated that the 20S proteasome is the most abundant specie of proteasome found in the cell emphasizing the relevance of this degradation pathway (Brooks et al. 2000; Wang et al. 2010; Tanahashi et al. 2000; Fabre et al. 2013). The proteins that are susceptible to 20S proteasomal degradation appear to share a common feature, namely a large unstructured segments in their native state. Over the last two decades many proteins have been identified as containing extensive unstructured regions, and some proteins are even completely unstructured under physiological conditions (Dyson & Wright 2005; Wright & Dyson 1999). These proteins are termed natively unfolded (Uversky et al. 2000) or intrinsically disordered proteins (IDPs) (Dyson & Wright 2005; Wright & Dyson 1999). IDPs are involved in many cellular processes, including transcription regulation and signal transduction (Ward et al. 2004). Given the fact that degradation of IDPs by 20S proteasome is a passive process, as they do not require modification prior to degradation, we termed this pathway ‘degradation by default’ (Tsvetkov et al. 2008; Asher et al. 2006). 20S proteasome can degrade natively disordered substrates at internal peptide bonds even when they lack accessible termini (Liu et al. 2003), thus suggesting that the inherent disorder of the protein has a dual rules, it serves as a signal for degradation as well as providing a domain sensitive for degradation. It is worth noting that even in the ubiquitin pathway an unstructured initiation site is required for efficient degradation (Prakash et al. 2004). One of the most extensively studied model proteins of the ubiquitin- degradation system is p53. p53 is a short-lived tumor suppressor protein that accumulates following various types of stress and induces growth arrest or apoptosis (Vogelstein et al. 2000). p53 level is tightly regulated by the rate of its proteasomal degradation. The ubquitin-dependent degradation of p53 is regulated by Mdm2, an E3 RING finger ubiquitin ligase, or by the two additional E3 ligases (COP1 and Pirh2) recently identified that bind to the amino-terminal trans-activation domain of p53 and ubiquitinate p53 (Haupt et al. 1997; Kubbutat et al. 1997; Leng et al. 2003; Dornan et al. 2004). p53 is further polyubiquitinated by p300 or Mdm2 and the polyubiquitinated p53 is eventually degraded by the 26S proteasomes (Shmueli & Oren 2004). A novel pathway of p53 degradation was characterized in our laboratory. p53 is unstructured at both N- and C-termini (Bell et al. 2002) and we have shown that it can also undergo ubiquitin and Mdm2-independent degradation by the 20S

6 proteasome and that this pathway is regulated by the enzyme NAD(P)H quinone oxidoreductase (NQO1) (Asher, Lotem, Kama, et al. 2002; Asher, Lotem, Sachs, et al. 2002; Asher et al. 2003; Asher et al. 2005; Tsvetkov et al. 2005). In this pathway p53 and p73α can undergo 20S proteasome degradation unless protected by binding to NQO1. This binding is NADH dependent and dicoumarol, a potent inhibitor of NQO1 that competes for the NADH binding site, dissociates the binding of p53 and p73α to NQO1 and by that enhancing their degradation. Several other short-lived proteins such as c-Fos (Adler et al. 2010), ornithine decarbosylase (Asher et al. 2005), eukaryotic translation initiation factor 4GI (Alard et al. 2010), p33IGB1 (Garate et al. 2008) and p21 (unpublished results) can also be regulated by NQO1 in this 20S proteasomal degradation pathway. NQO1 is a ubiquitous homo-dimeric flavo-enzyme containing one molecule of FAD per subunit. It catalyzes two-electron reduction of various quinones and aromatic nitro compounds by utilizing NAD(P)H as an electron donor. NQO1 protects cells from toxicity of various quinones such as menadione and benzo[a]pyrene quinone (Lind et al. 1990) by reducing them to stable hydroquinones and thus preventing oxidative stress (Lind et al. 1990; Ross et al. 2000). Given the fact that NQO1 binds a number of client proteins in an NADH dependent manner, it is very likely that NQO1 plays a key role in protecting IDPs from degradation by default. In addition, NQO1 is also associated with 20S proteasomes in mouse liver (Asher et al. 2005) and human red blood cells (Adler J., unpublished data), suggesting that it functions as a gatekeeper. Recently it was reported that lot6, the yeast orthologue of NQO1, is bound to the 20S proteasome (Sollner et al. 2009). Furthermore, yap4, a transcription factor, is recruited to lot6-20S proteasome complex when the flavo moiety of lot6 is in a reduced state. Binding of yap4 to lot6 protects it from ubiquitin independent 20S proteasomal degradation. NADH is the electron donor for NQO1 enzymatic activity, and it reduces the flavo moiety of NQO1. Furthermore, as mentioned above, NQO1 binding of IDPs is NADH dependent. This suggests that NQO1 binding to IDPs is dependent in the redox state of the flavo moiety. The yeast model raises the possibility that regulation of ubiquitin independent 20S proteasomal degradation by NQO1 orthologs is conserved from yeast up to mammals. Sollner also showed in-vitro that purified Lot6 and 20S proteasome form a complex when incubated together, suggesting their association does not require

7 additional factores. However we have prelinimery results suggesting that in mammales this is not the case as in tissue culture cells, NQO1 is poorly associated with 20S proteasome. How this association is regulated and changes in different physiological states in mammales has not been elucidated yet. We have perlinimery data sugguesting NQO1 is a substrate of c-Abl, We plan to invitigate if this modification regulates NQO1 binding to the 20S proteasome. IDPs are protected from degradation also when they are found in a complex (Asher et al. 2006; P Tsvetkov et al. 2008), thus it is very likely that nascent IDP and “free” proteins are degraded by the 20S proteasome. This led us to hypothesize the nanny model, in which the nanny protein protects IDPs from 20S proteasomal degradation by default, thereby ensuring their maturation into functional complexes which can be degraded only by the ubiquitin pathway (fig 1) (Tsvetkov, Reuven & Shaul 2009). Examining the degradation of pulse labeled p53 supported this hypothesis. The decay of labeled p53 has biphasic kinetics, newly synthesized p53 is degraded in a fast phase by 20S proteasome and mature p53 is degraded by 26S proteasome in a slow phase (Tsvetkov, Reuven, Prives, et al. 2009). The prevalence of default degradation becomes clearer with the identification of new substrates of this degradation pathway. In addition as the 20S proteasome is the most abundant proteasome (Brooks et al. 2000; Wang et al. 2010; Tanahashi et al. 2000; Fabre et al. 2013) implies that the default degradation pathway can not be ignored. Moreover, proteins that are degraded by this pathway are key regulators of cellular pathways and aberrant degradation is linked with diseases (van der Lee et al. 2014; Babu et al. 2011; Dyson & Wright 2005). Thus further elucidating the mechanism of the default degradation pathway is crucial. The steps leading to degradation of IDPs in the default degradation pathway are not known. In the process of ubiquitin dependent proteasomal degradation the steps leading to degradation were extensively studied. The polyubiquitin chains attached to the protein signals it is destined for degradation by targeting the protein to the 26S proteasome. In the default degradation pathway on the other hand, the protein is degraded without a prior modification that serves as a signal for degradation. This raised the question whether IDPs encounter randomly the proteasome or there is some kind of targeting to the 20S proteasome.

Figure 1: The nanny model. Once an IDP is synthesized, it is susceptible to degradation by default by the 20S proteasome (I) unless the disordered segment is masked by a nanny that binds the newly synthesized IDP (II). The binding of the nanny to the client IDP is a transient process enabling the proper maturation and formation of the functional complex (III). Once in a complex, the IDP is refractory to degradation by default and can only be degraded by the UD pathway mediated by the 26S proteasome (IV). The decay kinetics of the two degradation processes (I and IV) are biphasic, as outlined below. Adapted from Tsvetkov, Reuven & Shaul 2009

Results

In vivo visualization of 20S interacting proteins: The 20S proteasome is composed of four stacked rings in a barrel shape, two PSMA and two PSMB rings. Proteolytic activity resides in the chamber formed by the inner PSMB rings. The outer PSMA rings are identical and each has seven distinct subunits. PSMA rings form a gated orifice controlling substrate entrance into the proteasome. 20S proteasome regulatory particles (PA700, PA28) interact with the PSMA ring and modulate 20S proteasome activity by opening the entrance into the orifice (Forster et al. 2005; Rabl et al. 2008; Stadtmueller et al. 2010; Whitby et al. 2000). In addition many other cellular and viral proteins were shown to interact with subunits of the PSMA ring and these are only a fraction (Apcher et al. 2003; Touitou et al. 2005; Touitou et al. 2001b; Kehn et al. 2005; Fischer et al. 1995; Ikeda et al. 2009; Dächsel et al. 2005; Liu et al. 2006a; Sdek et al. 2005; Cho et al. 2001). Thus suggesting that interacting proteins in the default degradation pathway would interact with this ring in order to modulate the degradation. In order to identify binding partner candidates, biochemical approaches (such as co-fractionation, affinity pull-down and immunopercipitation) and genetic approaches (such as yeast two-hybrid) are used. The two general approaches have yielded a great amount of data on protein-protein interaction. However, in the biochemical approaches weak or transient interaction can be missed whereas the

9 genetic approaches tend to deal with direct interaction of two proteins disregarding interaction with a protein complex. When searching for proteins interacting with a complex such as the 20S proteasome, it is crucial to verify that the interaction occurs in the complex context and in the cells. We adopted the bimolecular fluorescence complementation (BiFC) technique to identify and explore protein interaction with the 20S proteasome in the cells. BiFC was employed to effectively identify protein-protein interaction in vivo (Hu et al. 2006; Shyu et al. 2006). In this technique, two non-fluorescent fragments of a florescent protein are fused to the interacting proteins. We use two constructs, one has the N-terminal fragment of Venus (VN173), which is fused to suspected interacting protein and the other has the C-terminal fragment of ECFP (CC155) which is fused to an alpha subunit C terminus of the 20S proteasome (fig 2a). The C termini of the alpha subunits point outside of the complex (fig 2b), thus the fluorescent fragment is expected to be accessible. Upon interaction of the two proteins the fragments are brought close enough to interact and establish the native state of the florescent protein, which restores the fluorescence (fig 2c). It is important to note, once the florescent protein has restored its native state it is stable. Thus, transient interactions are also captured and visualized in this technique, making it an ideal system for identification of 20S proteasome substrates.

Figure 2: Illustration of BiFC principal. (a) Illustration of the constructs used for BiFC assay. A fluorescent protein was split to N (VN 173) and C (CC155) terminal fragments. CC155 is fused to a 20S proteasome alpha subunit and VN 173 is fused to a protein suspected to interact with the proteasome. (b) Crystal structure of PSMA ring adapted from Unno et al. 2002. (c) Illustration of BiFC. Upon interaction of interacting proteins the two fluorescent fragments are brought into proximity interact, restore native structure and fluorescence is restored.

The 20S proteasome PSMA ring has two hub regions of protein-protein interaction: Stable expression of chimeric PSMA subunits

10 We established seven U2OS stable cell lines each expressing one of the 20S proteasome alpha subunit fused to a fluorescent protein fragment (fig 3a). Stable cell lines express a much lower level of the fused proteasome subunits in compression to transiently transfected cells. The low levels in the stable lines can diminish unspecific signals and increase their probability to be incorporated into the 20S proteasome. The cell cycle regulator, p21 is the first IDP protein that we chose to investigate its binding to the 20S proteasome. Previously it has been reported that p21 is a substrate of 20S proteasome and binds the alpha subunit PSMA3 in vitro (Touitou et al. 2001b). We added a 6xmyc tag to the N-terminus of p21 as it was shown to dramatically increase its half-life (Bloom et al. 2003) possibly by escaping its proteasomal degradation by the associated 20S proteasome. The seven cell lines were transiently transfected with 6xmyc p21 VN173 to examine the ability of the different subunits to achieve BiFC (fig 3b). Out of the seven subunits only three subunits produced BiFC signal. PSMA3 signal was the strongest one, supporting the previously reported in vitro binding (Touitou et al. 2001b). PSMA7 and to much lower level PSMA5 gave a visualized signals as well. It is noteworthy that the two major subunits (PSMA3 and PSMA7) are antipodal to each other in the PSMA ring (fig 3c).

Figure 3: Chimeric PSMA3 and PSMA7 stably expressed in U2OS cell line yield BiFC signal with p21. (a) U2OS cells were infected with lentivirus in order to create cell line stably expressing 20S proteasome alpha subunits fused to fluorescent protein fragment (CC155). Expression was verified by IB. (b) U2OS cells stably expressing chimeric 20S proteasome alpha subunits were transiently transfected with Flag p21 VN173 and H2B RFP constructs as indicated. (c) Illustration of PSMA ring indicating BiFC positive subunits with p21. Photos were taken with a fluorescent microscope, x10 objective 48h after transfection. Immunoblot (IB). This experiment was done one time.

Interestingly the pattern of HA-PSMA CC155 constructs in the stable cells is unstable and is downregulated with time. In particular the relative expression level of three chimeric subunits; PSMA4, 6 and 7, was sharply reduced (fig 3a and 4a). This raised the possibility that the chimeric PSMA subunits either cannot incorporate into proteasome and thus their expression is lost or are toxic to the cells. In order to examine the incorporation possibility, we semi-purified proteasomes from U2OS stable cell lines using the protocol described in fig 4c. Using this protocol we could detect the presence of the chimeric subunits in the 20S enriched fraction with the comparable levels detected in crude extracts (fig 4d). We further analyzed the proteasomes by native gel electrophoresis. The native gels were analyzed based on the proteasome activity (Fig 4e), which detects the total proteasomal activity or by immunobloting (Fig 4f) either selectively detecting the chimera constructs or the total proteasomal levels. Interestingly PSMA2, 3, 6 and 7 chimera proteins were detected in the 20S and as well in 26S proteasomes, thus the chimera do not hinder 19S proteasome association with the 20S proteasome (fig 4f). This may suggest that the levels of incorporated proteins are more stable, with the exception of PSMA5 subunit.

Figure 4: Stably expressed chimeric PSMA subunits incorporate into proteasomes. (a) Frozen stocks of U2OS stably expressing chimeric 20S proteasome alpha subunits were thawed and expression was verified. (b) Expression of the chimeric 20S proteasome alpha subunits was examined again after 5 passages. (c) Illustration describing semi-purification of proteasomes for native gel assay. (d-f) semi- purified proteasomes from U2OS cells were run on SDS PAGE gel to check the presence of chimeric 20S proteasome alpha subunits (d) and native gel for activity with the peptide suc-LLVY-AMC (e) and incorporation of the chimeric 20S proteasome alpha subunits into proteasome populations (f) Immunoblot (IB). Proteasomes were identified with antibody against the subunit PSMA4. This experiment was done once.

Transient expression of chimeric PSMA subunits Working with the stable cell line is not ideal as the expression of the chimeric PSMA subunits is lost overtime. Thus, we turned to a transient expression model. First we examined the incorporation of transiently transfected chimeric PSMA3 and PSMA7 into proteasomes in HEK293 cells. As described above, we enriched the proteasome fraction by employing the semi-purification protocol (fig 4c) of the cells after transfection. Both PSMA3 and 7 were detected in this fraction in a SDS-PAGE immunobloting assay (fig 5a). Interestingly, the level of the proteins in the transfected cells peaked at the first day and reduced later on. This is in particular the case with PSMA7. Similar results were obtained when native gel electrophoresis was used to quantify the 26S and 20S proteasomal complexes (Fig 5b and c). The chimeric subunits did not show an effect on the activity of total proteasome population (fig 5b). At the level of immunoblotting detection one day post transfection both PSMA subunits were incorporated into the 20S and 26S proteasomes as seen in the U2OS cell lines (fig 5c). Chimeric PSMA3 and PSMA7 were similarly incorporated into the 20S and 26S proteasomes already at the first day, however unlike the chimeric PSAM3 the incorporated chimeric PSMA7 was temporal and decreased dramatically

13 with time. It does not support the possibility that differential incorporation of the subunits in our case is responsible for differential accumulation. We did not further peruse the question of what regulates the differential behavior of these two chimeric subunits in respect to their proteasomal incorporation. We next examined incorporation of chimeric PSMA3 into proteasomes of the total extract by glycerol gradient ultracentrifugation (fig 5d). The chimeric PSMA3 subunit fractionation profile resembled the fractionation profile of the endogenous PSMA4 subunit. The transfected chimera PSMA3 was detected in the 20S fractions (Fig 5d, fractions 3-6) and in 26S fractions (fractions 7-13) and not as a free form. This implies that in total extract most if not all of the chimeric PSMA3 subunit were incorporated a large complex. Thus, it is highly unlikely that the BiFC signal derives from interaction with a free subunit.

Figure 5: Transiently expressed chimeric PSMA subunits incorporate into proteasomes one day after transfection. (a-c) HEK293 cells were transiently transfected with HA PSMA3 CC155 and HA PSMA7 CC155. Cells were harvested as indicated post transfection. Semi-purified proteasomes were run on SDS PAGE gel to check the presence of chimeric 20S proteasome alpha subunits (a), native gel for activity with the peptide suc-LLVY-AMC (b) and incorporation of the chimeric 20S proteasome alpha subunits into proteasome populations (c). (d) HEK293 cells were transiently transfected with HA PSMA3 CC155. Cells were harvested 48h after transfection, lysed, loaded on 11ml 10%-40% linear glycerol gradient and 0.5ml fractions were collected. Immunoblot (IB). Proteasomes were identified with antibody against the subunit PSMA4. This experiment was done once.

Next HEK293 cells were co-transfected with 6xmyc p21 VN173 and chimeric PSMA subunits (fig 6a). PSMA3, 6 and 7 yielded the strongest signals. In general the obtained data are similar to that observed in the U2OS stable cell lines, with the exception that the signal level of PSMA6 is much higher in HEK293 cells (fig 6b). HA-PSMA6 CC155 chimera protein is well expressed in both cell lines (Fig 6c and 3a) and therefore the observed difference at the level of fluorescence has to be cell type specific. It might indicate that in HEK293 cells p21 is targeted to PSMA6 as

14 well via an auxiliary protein that is not found in the U2OS cells. Using flow cytometry we quantified and normalized BiFC signal to co-transfected H2B RFP. We recorded BiFC signal intensity of RFP positive cells. We calculated BiFC/RFP fluorescence ratio per cell and used the medians as the ratios distribution is skewed (Hu et al. 2002). In agreement with the fluorescence images, PSMA3, 6 and 7 median ratios are the most potent p21 targets (fig 6d).

Figure 6: A subset of PSMA subunits yield BiFC signal with p21 in HEK293 cell line. (a) HEK293 cells were transiently transfected as indicated with 6xmyc p21 VN173, chimeric 20S proteasome alpha subunit and H2B RFP constructs. This experiment was done twice (b) Illustration of PSMA ring indicating BiFC positive subunits with p21 (c) Cells from panel a were harvested and expression level of the transient transfected constructs was examined. This experiment was done twice (d) HEK293 cells were transiently transfected with 6xmyc p21 VN173, chimeric 20S proteasome alpha subunit and H2B RFP constructs. Cells were harvested 48 hours post transfection and fluorescence intensities of at least 400 cells for each PSMA-p21 combination were recorded by flow cytometry. BiFC signal was normalized to RFP signal per cell. The BiFC/RFP median is used as the ratio distribution is skewed (Hu et al. 2002). Standard deviation bars represent two independent experiments. Photos were taken with a fluorescent microscope, x20 objective 48h after transfection. Immunoblot (IB).

Similar pattern of BiFC signals were obtained with transiently transfected HeLa cells (fig 7a, b and c). As in HEK293 the adjacent subunits PSMA3 and PSMA6 yielded a relatively strong signal and PSMA7 yielded a relatively weak signal. In HeLa cells as well, PSAM1 was barely expressed and PSMA3 had a higher expression level than all the other chimeric PSAMA subunits (Fig 7c). Notably, although the chimeric subunits PSMA2, 4 and 5 have a similar expression as PSMA7 in HeLa cell line they did not yield BiFC with 6xmyc p21 VN173, demonstrating the specificity of the targeting process. The fact that PSMA7 expression is similar to PSMA6 and yet PSMA6 yields a stronger BiFC signal, further indicates for the specificity of the process.

Figure 7: Three subunits yield BiFC signal with p21 in HeLa cell line. (a) HeLa cells were transiently transfected as indicated with 6xmyc p21 VN173, chimeric 20S proteasome alpha subunit and H2B RFP constructs. This experiment was done twice (b) Illustration of PSMA ring indicating BiFC positive subunits with p21. (c) Cells from panel a were harvested and expression level of the transient transfected constructs was examined. Photos were taken with a fluorescent microscope, x20 objective 24h after transfection. This experiment was done twice. Immunoblot (IB).

Our data suggest that p21 targets the proteasome not only via PSMA3, as has been reported based on cell free system analysis (Touitou et al. 2001a) but also via PSMA6 and 7. As for the PSMA6 it is possible that the signal is the results of the bystander effect of the PSMA3 juxtaposition. PSMA7 is likely to be independently targeted by the p21 IDP. c-Fos interaction with Chimeric PSMA subunits Analysis of 6xmyc p21 VN173 interaction with chimeric PSMA subunits revealed that there are two possible interaction regions within the alpha ring. Next we asked whether this is the case also with other IDPs. We have previously shown that c-Fos is a substrate of the 20S proteasome as well (Adler et al. 2010). Thus, we examined if c-Fos will also yielded BiFC with the chimeric PSMA subunits. HEK293 cells were transiently co-transfected with Flag c-Fos VN173 and chimeric PSMA subunit (fig 8a). c-Fos targeted mainly PSMA3 and 6, like p21, and to lower extent PSMA4 and 7. In the case of c-Fos the emerging picture is symmetrical targeting the two opposing pairs 3 and 6 in one side and 4 and 7 in the other side (fig 8b). These results suggest that two different IDPs, known substrates of the 20S proteasome are trapped via two regions a strong one and a weaker one positioned at opposite sites in the ring.

Figure 8: Two pairs Juxtapositioned across the PSMA ring yield BiFC signal with c-Fos in HEK293 cell line. (a) HEK293 cells were transiently transfected as indicated with 6xmyc p21 VN173, chimeric 20S proteasome alpha subunit and H2B RFP constructs. (b) Illustration of PSMA ring indicating BiFC positive subunits with c-Fos. Photos were taken with a fluorescent microscope, x20 objective 48h after transfection. This experiment was done once.

Structured proteins have a different BiFC pattern We further questioned if the two interaction sites in the alpha ring are unique for IDPs. To challenge this possibility we took the advantage of the structured protein NQO1 that is known to bind to the 20S proteasome (Gad Asher et al. 2005; Moscovitz et al. 2012). HEK293 cells were co-transfected with NQO1 VN173 and a chimeric PSMA subunit (fig 9a). NQO1 BiFC did not resemble the IDPs BiFC pattern. PSMA6 stood out and gave the strongest BiFC signal (9b). This raised the possibility that NQO1 is associated with the proteasome through binding of PSMA6.

Figure 9: NQO1 yields the strongest BiFC signal with chimeric PSMA6 in HEK293 cell line. (a) HEK293 cells were transiently transfected as indicated with NQO1 VN173, chimeric 20S proteasome alpha subunit and H2B RFP constructs. (b) Illustration of PSMA ring indicating BiFC positive subunits with NQO1. Photos were taken with a fluorescent microscope, x10 objective 48h after transfection. This experiment was done twice.

Next we asked what would be the BiFC pattern of a protein that is not a substrate or modulator of the default degradation pathway. We chose HNF- 4α as we previously shown in-vitro it is resistant to 20S proteasome degradation (Tsvetkov et al. 2008). HEK293 cells were co-transfected with HNF-4α VN173 and a chimeric PSMA subunit (fig 10a). PSMA6 gave the strongest signal with HNF- 4α. The fact that a HNF-4α which is not predicated to interact with the PSMA ring gave a signal with PSMA6 suggests this is not the binding site of NQO1.

Figure 10: HNF-4α yields the strongest BiFC signal with chimeric PSMA6 in HEK293 cell line. (a) HEK293 cells were transiently transfected as indicated with HNF-4α VN173, chimeric 20S proteasome alpha subunit and H2B RFP constructs. (b) Illustration of PSMA ring indicating BiFC positive subunits with HNF-4α. Photos were taken with a fluorescent microscope, x10 objective 48h after transfection. This experiment was done once.

18 PSMA3 and PSMA7 are the candidates IDP trappers: Throughout the BiFC experiments two subunits stand out as possible IDP trappers (fig 11a). Chimeric PSMA3 and 7 gave a strong signal when co-expressed with IDP in all the examined conditions. When examining interaction of a protein with a protein complex using the BiFC assay the bystander effect should be considered. A main requirement of BiFC is that the two fluorescent protein fragments are brought into proximity and not direct interaction of the examined protein. In the case of two proteins forming a dimer, direct interaction is required in order to bring the fluorescent protein fragments into proximity. However, in the case of a protein interacting with a protein complex, the fluorescent protein fragments can be brought into proximity by interaction of the protein with an adjacent subunit. We suspect that PSMA4 BiFC signal derives from the bystander effect. PSMA4 gave a positive BiFC signal with c-Fos, a large protein, and not with p21, a small protein. This raises the possibility that c-Fos size enabled the bystander effect. c-Fos may directly interact with PSMA7, however due to c-Fos size, its fused fluorescent fragment can interact with chimeric PSMA4 fused fluorescent fragment and BiFC signal is achieved (fig 11b). It is highly likely that chimeric PSMA6 signal also derives from the bystander effect. In HEK293 and HeLa cell lines, the chimeric subunit gave a strong signal when it was co-transfected with IDP or structured protein. On the other hand when the subunit was stably expressed in U2OS cell line, the BiFC signal was not achieved with p21. This suggests that the visualized interaction is not direct with PSMA6 and mediated through another factor, which is missing in the U2OS cell line. Remarkably, most of the reported interactions of the substrates with the 20S proteasomes are mapped to take place via the PSMA3 or PSMA7 subunits (Apcher et al. 2003; Cho et al. 2001; Dächsel et al. 2005; Fischer et al. 1995; Ikeda et al. 2009; Kehn et al. 2005; Liu et al. 2006a; Sdek et al. 2005; Touitou et al. 2001b; Touitou et al. 2005), consistent with the results of the BiFC assay.

Figure 11: PSMA3 and PSMA7 are the suspected interacting subuints. (a) Illustration summarizing the BiFC results. (b) Illustration demonstrating possible bystander effect in BiFC assay.

C-terminus truncated PSMA3 and PSMA7 are inefficient in p21 trapping: Next we aimed at mapping the PSMA3 and 7 regions mediating the p21 interaction. Structurally the alpha subunits mainly differ from each other in their termini (Apcher, Maitland et al. 2004). Thus, We suspect that the C or N termini of the alpha subunits are the sequences mediating protein-protein interactions visualized with BiFC. We constructed truncation mutants in the C termini of chimeric PSMA3 and PSMA7 (fig 12a and 13a) and tested their ability to yield BiFC with 6xmyc p21 VN173 in HEK293 cell line. Truncation of 11 residues in PSMA3 C terminus did not affect the BiFC signal. Truncation of 26 residues in the C terminus (PSMA3 1-229) partially reduced BiFC. However, the delta 26 aa failed to accumulate to the wild type level (Fig 12b) raising the possibility that the expression level and not inefficient interaction reduced the BiFC signal. Truncation of 69 residues in the C terminus (PSMA3 1-186) was expressed like the delta 26 yet was dramatically reduced at the level of BiFC signal (fig 12c). This indicates that a protein-protein interaction site, the trapper region, resides between residues 187-229.

Figure 12: Chimeric PSMA3 interacts with 6xmyc p21 VN173 through its C terminus. (a) Illustration of chimeric PSMA3 C terminus truncation constructs used to identify the interaction region with p21. Truncated regoins are colored in gray. Adapted from Unno et al. 2002. (b-c) HEK293 cells were transiently transfected as indicated with 6xmyc p21 VN173, chimeric PSMA3 subunit and H2B RFP constructs. Expression level of constructs (b) and BiFC (c) were examined. Photos were taken with a fluorescent microscope, x10 objective 72h after transfection. Immunoblot (IB). This experiment was done twice.

Similar experiments were conducted with PSMA7. The C-terminus truncation mutants, delta 12 aa and delta 27 aa (fig 13a), were expressed at the level of wild-type PSMA7 or even higher (Fig 13b). Delta 12 was as effective as wild-type in BiFC assay however further truncation (PSMA7 1-221) sharply reduced BiFC level (fig 13c). Thus, the protein-protein interaction site, namely the trapping region, resides between residues 222-236. It is interesting that truncation of PSMA7 C terminus had an opposite effect on protein accumulation. PSAM7 accumulated better in the 27 residues truncation mutant whereas PSMA3 truncations reduced accumulation. This strongly suggests that the C terminus plays an important role in PSMA subunit stability as well.

Figure 13: Chimeric PSMA7 interacts with 6xmyc p21 VN173 through its C terminus. Illustration of chimeric PSMA7 C terminus truncation constructs used to identify the interaction region with p21. Truncated regions are colored in gray. Adapted from Unno et al. 2002. (b-c) HEK293 cells were transiently transfected as indicated with 6xmyc p21 VN173, chimeric PSMA7 subunit and H2B RFP constructs. Expression level of constructs (b) and BiFC (c) were examined. Photos were taken with a fluorescent microscope, x10 objective 72h after transfection. Immunoblot (IB). This experiment was done twice.

The PSMA3 trapping region is functional in the PSMA5 context: Having delineated the PSMA3 and PSMA7 regions required for the p21 trapping we next asked whether this region is also sufficient in trapping substrates. To this end we took the advantage of the PSMA5 that has no p21 trapping sequence. We swapped the C terminus of the BiFC negative subunit PSMA5 with PSMA3 C terminus. We used two insertion mutations, one with a minimal part of PSMA3 C terminus residues 187-229 identified by the truncation mutations, which we refer to as PSMA5-3Short (fig 14a) and in the second all of PSMA3 C terminus was replaced, which we refer to as PSMA5-3Long (fig 14b). All the constructs were expressed as efficiently as the wild-type (fig 14c). In contrast the swap mutants yielded stronger BiFC signal with 6xmyc p21 VN173 relative to chimeric PSMA5 wild type as revealed by visualization (fig 14d) and validated by FACS quantification (fig 14e). In agreement with the fluorescence pictures, the median fluorescence ratio of PSMA5- 3Short was 1.5 fold higher than PSMA5 wild type and PSMA5-3Long median fluorescence ratio was 2.6 fold higher than PSMA5 wild type. Thus, the C-terminal PSMA3 trapping region is sufficient to confer a similar activity in the context of the PSMA5. Moreover, the higher BiFC signal of PSMA5-3Long compared to PSMA5- 3Short indicates that the C terminus of PSMA3 might have two trapping sites. One

22 site is found between residues 187-229 and the second is found between residues 230- 255. This result also demonstrates that BiFC is more sensitive to interaction specificity, not expression differences. The finding that PSMA3 trapping region is sufficient was further confirmed by co-immunoprecipitation experiments. To this end we used 6xmyc p21 that lacks the fluorescent protein fragment and not the 6xmyc p21 VN173. 6xmyc p21 was not brought down with the wild type PSMA5 but only with the swapped constructs (fig 12f).

Figure 14: Chimeric PSMA5 subunit containing PSMA3 C terminus gains the ability to interact with p21. (a-b) Illustration of mutant PSMA5 constructs used. Crystal structures adapted from Unno et al. 2002. (c-d) HEK293 cells were transiently transfected as indicated with 6xmyc p21 VN173, chimeric 20S proteasome PSMA5 subunit and H2B RFP constructs. This experiment was done twice. Expression level of constructs (b) and BiFC (d) were examined. Photos were taken with a fluorescent microscope, x20 objective 72h after transfection. (e) HEK293 cells were transiently transfected with 6xmyc p21 VN173, chimeric 20S proteasome PSMA5 subunit and H2B RFP constructs. Cells were harvested 48 hours post transfection and fluorescence intensities of at least 25,000 cells for each PSMA5-p21 combination were recorded by flow cytometry. BiFC signal was normalized to RFP signal per cell. The BiFC/RFP median is used as the ratio distribution is skewed (Hu et al. 2002). Standard deviation bars represent three independent experiments. *p-value=0.02 using 2 sided student t test. (f) HEK293 cells were transiently transfected as indicated with 6xmyc p21 and chimeric 20S proteasome PSMA5 subunit. Cells where harvested 48 hours post transfection lysed and subjected to immunoprecipitation (IP) with HA beads to bring down chimeric 20S proteasome PSMA5 subunit. This experiment was done once.

In addition we verified that our results are not cell line specific by examining BiFC in HeLa cell line. Expression of the chimeric PSMA5 wild type and swap mutants was similar (fig 15a). Similarly as in HEK293 both swap constructs yielded

23 higher BiFC signal compared to chimeric PSMA5 wild type (fig 15b), lending further support to the finding that the PSMA3 c-terminus region is sufficient to function as a p21 trapper in different cell lines.

Figure 15: Chimeric PSMA5 subunit containing PSMA3 C terminus gains the ability to interact with p21 In HeLa cells. (a-b) HeLa cells were transiently transfected as indicated with 6xmyc p21 VN173, chimeric 20S proteasome PSMA5 subunit and H2B RFP constructs. Expression level of constructs (a) and BiFC (b) were examined. Photos were taken with a fluorescent microscope, x20 objective 48h after transfection. This experiment was done once. Immunoblot (IB).

The PSMA3 IDP trapper is involved in the default degradation pathway: The PSMA3 trapper region in isolation binds known 20S substrates First we asked whether the PSMA3 C terminus trapping capacity is general. To this end we fused residues 187-255 and residues 188-241 of PSMA3 and PSMA5 C terminus, respectively, to GST to generate recombinant Trapper-GST fusion proteins. The PSMA5 C-terminus region serves as a negative control. First we examined the ability of the purified recombinant GST-fusion proteins to interact with p21. HEK293 cell extracts overexpressing 6xmyc p21 were incubated with the obtained GST-fusion proteins and the associated fractions were analyzed by immunobloting. We found that the 6xmyc p21 was specifically GST-PSMA3 C terminus associated (fig 16a). The trapping function of this PSMA3 region was also demonstrated by analyzing c-Fos. Untreated extracts were loaded to the GST-fusion columns and the bound fractions were immunobloted for c-Fos detection. c-Fos was detected only by the PSMA3 trapper (fig 16b). We next examined p53, which is a disordered protein at both N- and C-termini (Bell et al. 2002) and undergoes 20S proteasomal degradation (Gad Asher et al. 2005; Tsvetkov, Reuven, Prives, et al. 2009). Endogenous p53 was also specifically pulled down by GST-PSMA3 from naïve HEK293 cell lysate (fig 16c), consistent with the possibility that PSMA3 C terminus is active in trapping a variety of IDPs.

The PSMA3 trapper region in isolation protects 20S substrates from default degradation Having demonstrated that the PSMA3 trapper in isolation binds a number of known 20S substrates, we next asked whether the trapper in isolation could prevent access to the 20S proteasome by sequestering the substrates. In that case, trapper ectopic over-expression should inhibit the default degradation of the substrates. We first performed in vitro reaction using p53 as a substrate. Adding recombinant GST- PSMA3 C terminus to the digestion reaction protected p53 from default degradation (fig 16d). Next we conducted in vivo experiment using c-Fos as a substrate. Given the fact that in the cells the IDPs are protected by nannies, we looked for a condition whereby IDPs are susceptible to the default degradation. We have previously shown that in tissue culture c-Fos serum induction is elevated by NQO1 overexpression, as it serves as a nanny protecting c-Fos from default degradation pathway (Adler et al. 2010). c-Fos serum induction was dramatically higher in the presence of CFP fused to PSMA3 trapper region opposed to only CFP (fig 16e). Thus demonstrating that IDPs are targeted for default degradation through interaction with PSMA3 trapper region.

Figure 16: PSMA3 C terminus protects IDPs from default degradation. (a-c) GST, GST PSMA3 C terminus and GST PSMA5 C terminus bound to Glutathione agarose beads were incubated with HEK293 cell lysate overexpressing 6xmyc p21 (a) and naive HEK293 cell lysate (b-c). GST constructs and interacting proteins were eluted with 10mM reduced glutathione. GST constructs were visualized with ponceau. (d) p53 purified from baculovirus infected cells was incubated 1h in 370C with purified 20S proteasome, GST-PSMA3 C terminus, GST-PSMA5 C terminus and GST as indicated. (e) HCT116 cells transfected with CFP-PSMA3 C terminus or CFP as indicated were serum starved 24h. 1h after serum induction cells were harvested and accumulation of c-Fos was examined. Immunoblot (IB). This set of experiments were done once. *Panel d experiment was conducted in collaboration with Nadav Myers

PSMA3 C-terminus functions as a more general IDPs trapper Having demonstrated that the PSMA3 C terminus trapped a number of IDPs we next conducted experiments to provide a more general view of the IDPs. It has been reported that IDPs are more thermo-resistant and heated extracts are enriched with IDPs (Csizmók et al. 2006; Galea et al. 2006; Irar et al. 2006; Galea et al. 2009). We therefore used heat-treated HEK293 cell extract as an IDP enriched source to load on the GST columns. GST chimera bound fractions were eluted, separated by SDS- PAGE gel and visualized with silver staining. GST-PSMA3 trapper had a unique pull- down pattern compared to the negative controls GST-PSMA5 C terminus and GST alone, indicating it specifically interacts with a number of thermo-resistant proteins (fig 17a). Using mass spectrometry we identified protein content. In the heat-treated cell lysate we identified 1471 proteins of which 932 (63%) are predicated by IUPred (http://iupred.enzim.hu) to be at least 50% disordered (fig 17b). Thus, supporting previous reports of IDPs enrichment after heat treatment. The pull down experiment was repeated in triplicates and eluted proteins were analyzed by mass spectrometry (table 1). The eluted GST fusion proteins are too abundant to allow detailed analysis

26 of the other proteins and therefore the obtained number of hits was lower than anticipated. We divided the identified proteins into three groups: proteins enriched in GST-PSMA3 trapper pull-down triplicates, proteins enriched in GST-PSMA5 C terminus pull-down triplicates and proteins enriched in GST-PSMA3 trapper and GST-PSMA5 C terminus pull-down triplicates. Identified proteins that were not enriched compered to GST pull-down control triplicates were discarded. A protein was considered as enriched if there was a three-fold change. In GST-PSMA3 trapper pull-down we identified 25 enriched proteins out of which 17 were predict by the sequence analysis algorithms foldindex (http://bip.weizmann.ac.il/fldbin/findex) and IUPred to contain large disordered regions. In GST-PSMA5 C terminus pull-down we identified only 2 enriched proteins, which were not predicted to have large disordered regions. Only one protein was enriched in both GST-PSMA3 trapper and GST- PSMA5 C terminus pull-down and was predicted to contain large disordered regions. The GST pull-down results are in accordance to the BiFC analysis and further demonstrate the IDP trapping function of the PSMA3 C terminus.

PSMA3 C-terminus in isolation protects IDPs from 20S proteasomal degradation We examined if this interaction, as seen for individual proteins, inhibits IDPs default degradation. Heat-treated HEK293 cell lysate incubated with purified 20S proteasome was almost completely degraded (fig 17c). This result further demonstrates that the thermo-stable proteins are highly enriched for IDPs since the structured proteins are resistant to 20S proteasome degradation (Tsvetkov et al. 2008). Addition of the recombinant GST-PSMA3 trapper to the reaction prevented degradation whereas GST alone did not. These results suggest that PSMA3 trapper region interacts with multiple IDPs and this interaction is involved in 20S substrates recognition.

Figure 17: PSMA3 C terminus protects thermo-resistant proteins from default degradation. (a) HEK293 cell lysate was heated to 980C for 5 minutes and precipitates were removed by centrifugation. Heat treated lysate was incubated with GST, GST PSMA3 C terminus and GST PSMA5 C terminus bound to Glutathione agarose beads as indicated. GST constructs and interacting proteins were eluted with 10mM reduced glutathione and visualized with silver stain. This experiment was done once. (b) Proteins identified by mass spectrometry in heat-treated HEK293 cell lysate are divided into 10% increments. Proteins are grouped according to percent of disorder in their sequence predicted with IUPred. Proteins were identified from three repeats. (c) HEK293 heat-treated cell lysate was incubated 3h in 370C with purified 20S proteasome, GST-PSMA3 C terminus and GST as indicated. Proteins were visualized with InstantBlue stain. This experiment was done twice. *Panel b was experiment was conducted in collaboration with Tzachi Hagai. Panel c experiment was conducted in collaboration with Nadav Myers

Table 1:Interacting proteins identified by mass spectrometry. GST, GST PSMA3 C terminus and GST PSMA5 C terminus bound to glutathione agarose beads were incubated with boiled HEK293 cell lysate. GST constructs and interacting proteins were eluted with 10mM reduced glutathione and sent to mass spectrometry analysis. The results show enriched proteins in three independent repeats.

NQO1 association to 20S proteasome is a regulated process: NQO1 is a 20S proteasome gatekeeper that protects several IDPs from default degradation by 20S proteasome (Adamovich et al. 2013; G Asher et al. 2005; Gad Asher et al. 2005; Garate et al. 2008; Hershkovitz Rokah et al. 2010). Furthermore, we have found that the majority of NQO1 is associated with purified 20S proteasome from mouse liver (Gad Asher et al. 2005) and human red blood cells (Adler J., unpublished data). In vitro recombinant NQO1, although with low efficiency and under low temperature, associates with the 20S proteasome as well (Moscovitz et al. 2012). We purified 20S proteasome from mice brains and kidney utilizing three purification steps, differential ammonium sulfate precipitation, gel-filtration chromatography and anion exchange chromatography. In mouse brain a substantial

29 amount of NQO1 is detected, but only a small fraction is found in the 20S proteasome fraction (fig 18a-c). For the kidney samples we employed a shorter protocol that is quicker and requires less starting materials. The protocol consists of only two purification steps, ammonium sulfate precipitation to separate 20S proteasome from 26S proteasome and glycerol gradient fractionation. In similar to the brain in the mouse kidney a large fraction of the NQO1 is not in association with the 20S proteasome (fig 18d-e).

Figure 18: A minor fraction of NQO1 is associated with 20S proteasome in mouse brain and kidney. (a-c) mouse brain. (d-e) mouse kidney. (a) Mice brain extracts were precipitated with 40% ammonium sulfate followed by a second precipitation with 80% ammonium sulfate of the resulting supernatant. Pellets were analyzed with the indicated antibodies. (b) 40%-80% ammonium sulfate pellet was loaded on Superpose 6 prep grade gel-filtration column. Fraction were collected and analyzed with the indicated antibodies. (c) Fractions 10-13 from Superpose 6 prep grade gel-filtration column were pooled and loaded on Resource Q anion exchange column and eluted with different concentrations of NaCl. Fractions were collected and analyzed with the indicated antibodies. (d) Differential ammonium sulfate precipitation. Pellets were analyzed indicated antibodies. (e) 40%-80% ammonium sulfate was loaded on a 10%-40% glycerol gradient. Fractions were collected and analyzed with the indicated antibodies. Immunoblot (IB). 20S was identified with antibody against the subunit PSMA4 and 26S was identified with antibody against the 19S subunit PSMD1. This experiment was done once.

Next we examined association of NQO1 with 20S proteasome in the following human cell lines; HepG2, HeLa, HCT116 and MCF-7. In HepG2 (fig 19a) and HeLa (fig 19b) cells only a minor fraction of NQO1 was associated with 20S proteasome. In the cell lines HCT116 (fig 19c) and MCF-7 (fig 19d) we could not detect NQO1 associated with 20S proteasome. The HepG2 behavior was surprising since these cells are from liver origin and in the mouse liver tissue essentially all the NQO1 was co- fractionated with the 20S proteasome (Gad Asher et al. 2005). This might imply that there is a miss regulation of NQO1 association with the 20S proteasome in the cultured cells.

Figure 19: NQO1 association with 20S proteasome varies between different cell lines. (a-d) Left panel: differential ammonium sulfate precipitation. Pellets were analyzed by with the indicated antibodies. Right panel: 40%-80% ammonium sulfate was loaded on a 10%-40% glycerol gradient. Fractions were collected and analyzed with the indicated antibodies. (a) HepG2 (b) HeLa (c) HCT116 (d) MCF-7. Immunoblot (IB). 20S was identified with antibody against the subunit PSMA4 and 26S was identified with antibody against the 19S subunit PSMD1. This experiment was done once.

c-Abl modulates NQO1 association with 20S proteasome: Based on the website phosphosite it appears that NQO1 is heavily modified including by tyrosine phosphorylation. It has been previously reported that c-Abl binds and phosphorylates the proteasome alpha subunit PSMA7 (Liu, Huang et al. 2006). Moreover, this modification decreases the enzymatic activity of the proteasome. This led has to hypothesis that c-Abl may also phosphorylate NQO1 in order to modulate the activity of the 20S proteasome. First we explored whether NQO1 is a putative c-Abl substrate. NQO1 has eight tyrosine residues and position

Y222 was identified as the major site (fig 20).

Figure 20: c-Abl phosphorylates NQO1 at position 222Y. HEK293T cells were transiently transfected with NQO1 Flag, NQO1 Y22F Flag and c-Abl Δ1-81 as indicated. Cell lysate was subjected to immunoprecipitation (IP) with anti-flag antibody to bring down NQO1 and analyzed with the indicated antibodies. Immunoblot (IB). * The experiment was conducted by Nina Reuven

31 Next we explored if the proteasome, c-Abl and NQO1 are found in a complex. We used HEK293T cells stably expressing a flag-tagged beta subunit of the 20S proteasome in order to immunoprecipitate the proteasome. NQO1 cannot be detected by western blot in HEK293T thus we expressed it ectopically. NQO1 and endogenous c-Abl co-immunoprecipitated with the proteasome (fig 21, lane 1) and treatment with STI571, an inhibitor of c-Abl, reduced the amount of co-immunoprecipitated c-Abl and NQO1 (fig 21, lanes 2-3). Overexpression of the constitutively active c-Abl increased the co-immunoprecipitation of NQO1 and c-Abl (fig 21, lane 4), however treatment with STI571 had only a modest effect on the complex (fig 21, lane 5). Thus, supporting the possibility that c-Abl phosphorylates NQO1 and modulates its association with 20S proteasome. However, NQO1 Y222F, a mutant that is poorly phosphorylated by c-Abl, can also form a complex with the proteasome and c-Abl (fig 21, lane 6). Thus raising the possibility that c-Abl can modulate the association of NQO1 to the 20S proteasome but it is not the only regulator of this association.

Figure 21: NQO1 c-Abl and proteasome forms a complex. HEK293T cells stably expressing a Flag tagged beta subunit of the 20S proteasome (PSMB2) were transiently transfected with NQO1, NQO1 Y22F and c-Abl Δ1-81 as indicated. The cells were incubated in the presence or absence of STI571 and vehicle. Cell lysate was subjected to immunoprecipitation (IP) with anti-flag antibody to bring down the 20S proteasome and analyzed with the indicated antibodies. immunoblotting (IB) This experiment was done once.

32 Discussion The default degradation pathway of IDPs plays an important role in proteostasis. Its importance is indicated by the ever growing list of substrates and the estimation that more then 20% of cellular proteins undergo degradation by 20S proteasome (Baugh et al. 2009). In this work we focused on the regulation of the default degradation pathway, we show that IDPs are targeted to the proteasome through a trapper region found in the C terminus 20S proteasome subunit PSMA3. Moreover, sequestering the 20S accessibility by ectopic expression of the trapper region rescues IDPs from default degradation, attributing to the trapper region an important role in proteasomal default degradation. NQO1 is another regulator of the default degradation process. We examined is NQO1 association with 20S proteasome. Association of NQO1 differs between different tissues and poorly detected in tissue culture. This observation strongly suggests that the NQO1-20S association is modulated by NQO1 modification.

IDPs interact with a subset of the 20S proteasomes’ PSMA ring In this study we used the BiFC assay in order to examine the possibility that IDPs interact with the proteasomes, as a targeting process in the process of their degradation. We found two juxtapositioned subunits in the ring that yield a strong BiFC signal with IDPs in all the examined conditions. PSMA3 gave the strongest BiFC signal and PSMA7, which is found across the ring, gave a weaker signal. The intensity of the BiFC signal implies that one region has a higher affinity than the other. We searched the literature for known interactions with 20S proteasome alpha subunits. Although, by large there were mapped in a manner that cannot distinguish between the free subunits from the proteasomally incorporated one, nevertheless we considered them relevant in comparing them to our findings. Indeed as in our BiFC assay, we found that two subunits were recurring in a number of studies. PSMA3 and PSMA7 were the major PSMA subunits for which interactions with proteins were reported. Our findings and the reported cases led us to suggest that the alpha ring has at least two subunits, PSMA3 and 7, which function as hubs for protein-protein interaction. Chimeric PSMA6 and PSMA4 also gave BiFC signal in some of the examined cases. Their ability to yield a BiFC signal in only some of the cases can be explained by the bystander effect as previously described. We did not further pursue this

33 possibility and additional experiments are needed in order to determine with certainty that indeed the examined proteins interact with an adjacent subunit of the proteasome.

The 20S proteasome putative trapping sequences In order to identify the regions in PSMA3 and PSMA7 mediating IDPs interaction, we constructed C terminus deletion and swapping mutants and tested their ability to produce BiFC (fig 22a). The obtained data identified the involved regions at the C-termini. Inspection of the sequences of these regions revealed a box whose sequence and structure are relatively similar in the C termini of both subunits (fig 22b). Either deletion or insertion of the region containing this box had a significant effect on p21 trapping. It is interesting to note that the sequence of the putative boxes is highly charged and folds into an alpha helix. Moreover, it has been noted that structurally thermo-stable proteins and 20S proteasome substrates are enriched for alpha helixes (Baugh et al. 2009; Galea et al. 2009). Thus, it raises the possibility the interaction between these trapping sequences and IDPs resemble a coiled coil configuration. Further characterization of this box by mutations that designed either to change the net charge or by alpha helix braking is required to validate our model. The truncation and swapping mutants of PSMA3 indicate that a putative second trapping box resides between residues 187-229. In this region, when compared to PSMA5, a highly charged sequence stands out (fig 22a). It is reminiscent of the two other suspected boxes by its charged residues. However, there is no structure similarity, since the 187-229 box is comprised of a linker and beta strand structures (fig 22b and c). The contribution of this box, if any, has to be further experimentally validated.

Figure 22: Identification of suspected trapping sequences. (a) C terminal sequence of PSMA3, 7 and 5 with truncation sites and swap region marked as specified in panel legend. (b) PSMA7 and PSMA3 suspected similar trapping sites. (c) PSMA3 second suspected trapping site.

34 Throughout the BiFC experiments, PSMA3 was more potent in yielding BiFC compared to PSMA7 with the examined IDPs. The finding that PSMA3 has two putative trapping boxes might provide a reasonable explanation.

PSMA3 IDP trapping specificity In order to examine a broader array of IDPs and not specific examples we used a proteomic approach. We fused PSMA3 C terminus, residues 187-255, to GST, and first tested its ability to pull-down specific IDPs, validating that the trapping region retained its activity in isolation as well. Next we challenged PSMA3 trapping region with cell lysate enriched with IDPs. Enrichment was achieved by heat- treatment of cell lysate, IDPs tend to be more thermo-stable than structured proteins (Csizmók et al. 2006; Galea et al. 2009; Galea et al. 2006; Irar et al. 2006). PSMA3 trapping region specifically shows a unique pull-down band pattern. Thus indicating PSMA3 trapping region has a broad interacting ability and can trap some other IDPs. Mass spectrometry analysis identified 25 proteins pulled-down with PSMA3 trapper region. 17 proteins are predicated by algorithms to contain large disordered regions. Among the identified proteins are RNA binding proteins, which are known to have large disordered regions (Huntley & Golding 2002; Uversky 2002; Michelitsch & Weissman 2000). Thus, for some of the identified proteins, providing structural evidence that they are indeed IDPs. To date there is no high throughput strategy for identification IDPs. We propose that PSMA3 trapper region can provide such a tool. It is possible that the trapped IDPs can be uniquely categorized by their composition. At the moment the identified repertoire is too limited to reach such a conclusion.

IDP trapping by the proteasomes is required for default degradation The 20S proteasome was shown to degrade IDPs however the stages prior to degradation were not known. In this work we showed that IDPs interact with PSMA7 and 3 C termini and identified suspected trapping sequences. In in-vitro degradation assay the recombinant PSMA3 trapper region inhibited degradation. In tissue culture cells ectopic expression of tagged PSMA3 C-terminus region protected IDPs from default degradation. The simplest way to explain these data is to assume that the excess of the free trapper regions competes with the IDPs to access the proteasomes.

35 In that case trapping of IDPs by the proteasome appears to be an essential step in the default degradation pathway. In the crystal structure of the 20S proteasome the orifice is occluded by the N termini of the PSMA ring subunits thus creating a gate limiting access into the proteolytic chamber (Groll et al. 2000; Whitby et al. 2000; Groll et al. 1997). Examination of 20S proteasome by other methods revealed that the 20S gate can interchange between open and closed conformations (Osmulski & Gaczynska 2000; Latham et al. 2014; Religa et al. 2010). We suggest that the trapping of the IDP by PSMA7 and 3 serves two functions. First The IDP is recognized by the proteasome by interaction with PSMA7 and 3. Later on, the trapping keeps the IDP close the orifice, elevating the chance to encounter an open gate conformation that leads to default degradation (fig 23). It is possible that the trapping do not act only passively to improve the chance encountering an open gate. Consistent with this model is the fact that 20S regulatory particles (19S, 11S, PAN) interaction with the alpha ring induces an open gate conformation (Whitby et al. 2000; Forster et al. 2005; Forster et al. 2003; Gillette et al. 2008; Rabl et al. 2008; Stadtmueller et al. 2010). Similarly, the trapping of IDP by an alpha subunit may induce a change favoring an open gate conformation.

Figure 23: Proposed model – IDPs are targeted to the proteasome. An IDP is recognized by a subset of 20S proteasome PSMA subunits. Interaction of the IDP with the PSMA subunit enables it to remain near the 20S proteasome orifice thus increasing its chance to enter and undergo default degradation.

The 20S proteasome is considered as the proteasome responsible for default degradation. However, recently our lab reported the existence of another form of 26S proteasome. The 26S proteasome is also stable when it binds the small molecule NADH without the need for ATP (Tsvetkov et al. 2014). We have evidence 26S NADH proteasome can efficiently degrade IDPs. The chimeric PSMA subunits incorporate also into 26S proteasome. Thus, we cannot exclude the possibility that 26S proteasome can trap IDPs through PSMA7 and 3.

NQO1 association with 20S proteasome NQO1 is an important regulator of the default degradation pathway. Taken into account the reported findings on the NQO1 capacity to protect a variety of IDPs, we assume that NQO1 has a more general nanny role. Since NQO1-20S association varies between tissues and cultured cells we suggest that NQO1 association with the 20S proteasome is a regulated process. A possible effector regulating the association might be mediated via NQO1 or 20S subunits modifications. Keeping this model in mind we examined if NQO1 undergoes phosphorylation. The non-receptor tyrosine

37 kinase, c-Abl, regulates proteasomal activity (Li et al. 2015; Liu et al. 2006) and degradation of the IDP α-synuclein (Mahul-Mellier et al. 2014). We have preliminary results to show that NQO1 is phosphorylated by c-Abl. c-Abl improves NQO1 association with the 20S particle. However a mutant NQO1 that does not undergo phosphorylation by c-Abl also displays an improved 20S association. Thus, either c- Abl improves NQO1 association by modifying the 20S subunits or by modifying some unknown auxiliary component.

Regulation of default degradation by NQO1 NQO1 association with the 20S proteasome was a puzzling finding. It is not intuitive that a nanny protein protecting IDPs from default degradation is bound to the proteasome. Targeting of IDPs to the proteasome through the trapping alpha subunits may provide an explanation. IDPs trapped by the proteasome are protected from degradation by the associated NQO1. Furthermore, this may be the mechanism for NQO1 client proteins recognition. Thus, modulating NQO1 association level with 20S proteasome may regulate 20S proteasome trapping capability and as a result default degradation.

38 Materials and methods:

Tissue culture The cell lines used were: HEK293, HCT116 human colon blastoma cells, HepG2 human liver blastoma, HeLa human cervix adenocarcinoma and MCF-7 breast adenocarcinoma. Cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin and cultured at 37°C in a humidified incubator with 5.6% CO2.

Plasmids, transfection and infection Plasmids used: PSMA subunits (Kindly provided by prof. k Tanaka, Tokyo Metropolitan Institute of Medical Science, Japan) were cloned into pBiFC-VN173 (Addgene, Cambridge, MA, USA; plasmid no. 22010). pLENTI6 PSMA subunit VN173 were cloned from pBiFC PSMA subunit VN173. 6xmyc p21 (Kindly provided by prof. C Kahana, Weizmann institute of science, Israel), c-Fos, NQO1 and HNF-4a coding sequence of residues 141-464 were cloned into pBiFC-CC155 (Addgene, Cambridge, MA, USA; plasmid no. 22015). pCI 6xmyc p21, PCDNA3 CFP, PCDNA3 CFP PSMA3 187-255, PCDNA3 Flag NQO1, PCDNA3 Flag NQO1 Y222F, PCDNA3 c-Abl D1-81. HEK293 cells were transfected by the calcium phosphate method. HCT116 and HeLa cells were transfected with jetPEI (Polyplus-transfection SA, Illkirch, France). U2OS cells stably expressing chimeric PSMA subunits were created with the gateway system (Invitrogen).

Immunoblot analysis Cells were lysed with NP40 buffer (20mM Tris-HCl pH7.5, 320mM sucrose, 5mM MgCl, 1% NP40) supplemented with 1mM Dithiothreitol (DTT) and protease and phosphatase inhibitors (Sigma). Laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.125M Tris-HCl) was added to the samples, heated at 950C for 5 minutes and loaded on a polyacrylamide-SDS gel. Proteins were transferred to cellulose nitrate 0.45 mm membranes. Antibodies: Mouse anti HA, mouse anti Flag, mouse anti actin, mouse anti tubulin and mouse anti human p53 Pab1801 were purchased from Sigma. Mouse anti myc, rabbit anti cAbl, mouse anti pY20 and goat anti NQO1 C19 and R20 were purchased from Santa Cruz. Rabbit anti PSMD1

39 (Acris), a subunit of the 19S proteasome. Rabbit anti PSMA4, a subunit of the 20S proteasome (Mamroud-Kidron et al. 1994) kindly provided by prof. C. Kahana, Weizmann institute of science, Israel. Secondary antibodies were horseradish peroxidase-linked goat anti rabbit, donkey anti goat and goat anti-mouse (Jackson ImmunoResearch). Signals were detected using the EZ-ECL kit (Biological Industries).

Co-immunoprecipitation Samples were incubated with primary antibody 16h. Samples were washed 6 times with NP40. Bound and associated proteins were eluted with HA or FLAG peptide (Sigma) according standard protocol.

Nondenaturing PAGE Samples were prepared and run as described (Tsvetkov, Reuven, Prives, et al. 2009).

Flow cytometry analysis Cells were harvested two days post transfection, washed and resuspended in PBS. Samples were analyzed with BD LSR II flow cytometer using FACSDiva software (BD Biosciences). VFP and RFP intensities of RFP positive cells were recorded. Values of VFP and RFP fluorescence intensities of each cell were extracted using FlowJo software ( FlowJo, LLC )

Purification of the 20S proteasome The first step was the preparation of the tissue: mouse liver or brain were homogenized in buffer A [20mM Tris-HCl 7.5, 1mM DTT, 1mM EDTA, 250mM sucrose] using POLYTRON homogenizer and then Teflon homogenizer. The homogenate was centrifuged using a 45TI rotor at 40,000 rpm for 1 hour at 40C and the supernatant was then subjected to the first precipitation with 40% (w/v) saturated ammonium sulfate followed by a second precipitation of the resulting supernatant with 80% (w/v) saturated ammonium sulfate. The pellet that was obtained after centrifugation at 40,000 rpm, using 45TI rotor for 1/2 an hour at 40C was resuspended in a minimal volume of buffer B [20mM Tris-HCl 7.5, 1mM DTT, 20% Glycerol]. The resuspended pellet was then loaded on to a Superpose 6 prep grade gel-filtration column. Collected fractions were analyzed for the presence of 20S

40 proteasomes by Western blot analysis. The fractions containing the proteasome were combined and loaded on a resource-Q ion-exchange column. Proteins were eluted with a gradient of NaCl and fractions were analyzed for the presence of 20S proteasomes by Western blot. 20S proteasome from liver was further purified by glycerol gradient. The fractions containing the proteasome were combined and dialyzed over night against buffer C [50mM Tris-HCl pH 7.5, 150mM NaCl, 5mM MgCl, 1mM DTT]. The sample was concentrated with Amicon Ultra centrifugal filter, loaded on an 11 ml linear 10%-40% glycerol gradient and centrifuged using SW 41TI rotor at 24,000 rpm for 16 hours at 40C. 500ul fractions were collected and analyzed for the presence of 20S proteasomes by Western blot. The fractions containing the proteasome were combined and dialyzed over night against buffer C. The sample was concentrated with Amicon Ultra centrifugal filter, divided to aliquots, frozen and stored at -800C.

In-vitro degradation assay Degradation of recombinant baculovirus expressed and purified p53 (kindly provided by Prof. C. Prives, Columbia University, New York) and heat-treated cell lysate with 20S proteasome was carried out in degradation buffer [100mM Tris-HCL pH 7.5, 150mM NaCl, 5mM MgCl, 2mM DTT] at 37ºC for 1 hour and 3 hours respectively. The degradation reaction was stopped with the addition of Laemmli sample buffer to the samples. The samples were then heated at 95ºC for 5 min and fractionated by electrophoresis in polyacrylamide-SDS gel. Following electrophoresis, proteins were visualized by gel staining or transferred to cellulose nitrate membranes. Purified 20S proteasome was detected by immunobloting with rabbit anti PSMA4 antibody. Baculo expressed and purified p53 was detected by immunobloting with mouse anti human p53 (1801).

Protein identification by Mass spectrometry GST constructs and associated proteins were eluted from glutathione agarose beads with 70ul of 10mM glothathione in 50mM Tris-HCl pH 9.5. Eluted proteins were analyzed by the proteomic unit of the INCPM at the Weizmann institute of science.

41 Purified p53 Infection and purification of p53 from insect cells was done as described (Jayaraman et al. 1997).

Two step purification of 20S proteasome Tissues were homogenized in buffer E (100mM Tris-HCl 7.5, 150mM NaCl, 2mM EDTA, 1mM DTT, 1%NP40) using a POLYTRON homogenizer and then Teflon homogenizer. Tissue culture cells were lysed with buffer E. Homogenate/cell extract were centrifuged in a tabletop centrifuge at 13,000 rpm for 15 minutes at 40C and then was subjected to precipitation with 40% (w/v) saturated ammonium sulfate. The pellet that was obtained after centrifugation at 54,000 rpm, using TLA 120.1 rotor for 30 min at 40C was resuspended in a minimal volume of buffer R [100mM Tris-HCl 7.5, 150mM NaCl, 2Mm EDTA, 1mM DTT]. The resuspended pellet was dialyzed for 5 hours against buffer R at 40C. After dialysis the resuspended pellet was loaded on an 11 ml linear 10%-40% glycerol gradient and centrifuged at 28,400 rpm, using rotor SW 41TI. 500ul fractions were collected and analyzed for NQO1 and 20S proteasome presence by western blot.

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Declaration This thesis is a summery of my work. Experiments in figure 16d and figure 17c were conducted in collaboration with Nadav Myers. Experiment in figure 17b was conducted in collaboration with Tzachi Hagai. Experiment in figure 20 was conducted by Nina Reuven.

Acknowledgments

I would like to express my deepest gratitude and appreciation to my advisor prof. Yosef shaul for sharing with me is knowledge, great ideas and especially for showing me how science should be done.

Special thanks to all former and currant lab members; Dr. Nina Reuven, Dr. Yaarit Adamovich, Dr. Matan Shanzer, Dr. Inna Ricardo-Lax, Dr. Rom Keshet, Nadav Myers, Dr. Julia Adler and Lior Handler for their scientific advices and much appreciated friendship throughout the years.

I am thankful to my PhD. committee members Prof. Michal Sharon and Prof. Ami Navon for our wonderful scientific discussions.

I can never appreciate enough my mom and brother for their love and much needed support.