antioxidants

Article The Antioxidant Enzyme Methionine Sulfoxide Reductase A (MsrA) Interacts with Jab1/CSN5 and Regulates Its Function

Beichen Jiang 1, Zachary Adams 2 , Shannon Moonah 3, Honglian Shi 1, Julie Maupin-Furlow 2 and Jackob Moskovitz 1,* 1 Department of Pharmacology and Toxicology, School of Pharmacy, University of Kansas, Lawrence, KS 66045, USA; [email protected] (B.J.); [email protected] (H.S.) 2 Department of Microbiology and Cell Science, College of Agricultural and Life Sciences, University of Florida, Gainesville, FL 32611, USA; zachadams@ufl.edu (Z.A.); jmaupin@ufl.edu (J.M.-F.) 3 Division of Infectious Diseases, University of Virginia, 345 Crispell Drive, MR-6, Room 2715, Charlottesville, VA 22908, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-785-864-3536



Received: 3 May 2020; Accepted: 22 May 2020; Published: 24 May 2020


Abstract: Methionine sulfoxide (MetO) is an oxidative posttranslational modification that primarily occurs under oxidative stress conditions, leading to alteration of protein structure and function. This modification is regulated by MetO reduction through the evolutionarily conserved methionine sulfoxide reductase (Msr) system. The Msr type A enzyme (MsrA) plays an important role as a cellular antioxidant and promotes cell survival. The ubiquitin- (Ub) like neddylation pathway, which is controlled by the c-Jun activation domain-binding protein-1 (Jab1), also affects cell survival. Jab1 negatively regulates expression of the cell cycle inhibitor cyclin-dependent kinase inhibitor 1B (P27) through binding and targeting P27 for ubiquitination and degradation. Here we report the finding that MsrA interacts with Jab1 and enhances Jab10s deneddylase activity (removal of Nedd8). In turn, an increase is observed in the level of deneddylated Cullin-1 (Cul-1, a component of E3 Ub ligase complexes). Furthermore, the action of MsrA increases the binding affinity of Jab1 to P27, while MsrA ablation causes a dramatic increase in P27 expression. Thus, an interaction between MsrA and Jab1 is proposed to have a positive effect on the function of Jab1 and to serve as a means to regulate cellular resistance to oxidative stress and to enhance cell survival.

Keywords: methionine oxidation; posttranslational modification; neddylation; ubiquitin; brain; oxidative stress

1. Introduction The methionine sulfoxide reductase (Msr) system consists of Msr type A (MsrA) and Msr type B (MsrB) that reduce S-MetO and R-MetO, respectively [1,2]. This basic system is highly conserved in evolution and is designed to “repair” oxidatively damaged proteins containing MetO residues [1,2]. Methionine oxidation is a common phenomenon that can alter protein function through allosteric changes of a protein [1], reduce or increase protein stability by changing its hydrophobic properties [1], and regulate protein function via changing the redox status of a pivotal Met residue [1]. A bioinformatics tool is recently developed to model the contribution of MetO residues to protein structure and function, strengthening knowledge of how global and important this modification is to biological processes [1]. A compromised expression or activity of MsrA is found to lead to various cellular malfunctions. The disruption of the msrA gene in Saccharomyces cerevisiae causes hypersensitivity to oxidative stress [3]. Likewise, MsrA knockout (KO) mice are more vulnerable to oxidative stress and demonstrate

Antioxidants 2020, 9, 452; doi:10.3390/antiox9050452 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 452 2 of 17 several molecular phenotypes that can be linked to age-associated diseases when compared to wild type (WT) [4]. For example, MsrA KO mice exhibit many of the neuropathological traits associated with Alzheimer’s disease (AD) [5] and Parkinson’s disease (PD) [6–8]. The crossed MsrA KO x AD model showed stronger phenotypes with respect to mitochondrial malfunction and the distribution of beta-amyloid forms compared with the AD model [9]. A compromise in MsrA activity can cause other organ or cellular malfunctions that are not directly linked to neurodegeneration. These include, for example, mental health disorders [8], heart disease [10], liver toxicity [11], and cancer [12]. Additionally, MsrA is involved in maintaining the basic cochlea structure of the inner ear, and its deficiency may contribute to hearing loss [13]. We also find MsrA regulates the Ub-like modification of proteins in Archaea and the ubiquitination of 14-3-3 ζ in a mouse brain [14,15], suggesting a deep evolutionary association of MsrA with Ub/Ub-like systems. Neddylation is a posttranslational modification system that adds the ubiquitin-like neural precursor cell expressed developmentally down-regulated 8 (Nedd8) to substrate proteins [16] (Figure1). Nedd8 is covalently ligated to a limited number of cellular proteins in a manner analogous to ubiquitination. In a canonical neddylation process, Nedd8 is activated by the Nedd8 activating enzyme (NAE) [17]. Nedd8 is then transferred from the NAE via the Nedd8 conjugating enzyme (NCE) and the RING-box protein RBX1 to the Cullin subunit of Cullin/RING ubiquitin ligases (CRL) [18]. RBX1 serves as the E3 ligase for Nedd8 and as an E3 ligase for subsequent ubiquitination reactions [19]. The Cullin subunits of CRLs are the best-studied neddylation substrates. Neddylation loosens the interaction of RBX1 with the WHB domain and RBX1 can subsequently promote E2-dependent ubiquitination and protein degradation [20]. CRLs are the largest family of multisubunit E3 ubiquitin ligases, controlling the degradation of about 20% of the proteasome-regulated proteins that are involved in many aspects of important biological processes [21–23]. Removal of Nedd8 from proteins is mediated by c-Jun activation domain-binding protein-1 (Jab1) (synonym CSN5), which is the fifth subunit of the constitutive photomorphogenic-9 signalosome (COP9). Jab1 was initially identified as c-Jun activation domain-binding protein-1, hence the nomenclature [24]. The COP9 signalosome (CSN) is evolutionarily conserved among all eukaryotes and has a canonical composition of eight subunits (CSN1–8) found in all multicellular organisms. CSN regulates the activity of the CRLs, the largest family of ubiquitin E3 ligases. Regulation of CRLs by the CSN involves the removal of Nedd8 from Cul-1, the cullin scaffold subunit of CRLs, through the hydrolytic activity of a metalloprotease MPN+/JAMM motif (the c-Jun binding domain) within the catalytic Jab1 subunit of CSN. In short, CSN promotes deneddylation of Cul-1, and Jab1 provides the catalytic center to execute this isopeptidase activity [25–28]. Interestingly, although Jab1 only exhibits deneddylase activity when it interacts with the other CSN components [29,30], a large portion of the free Jab1 is detected in both cytoplasm and nucleus [31] suggesting Jab1 may have a CSN-independent function. The deneddylation of Cul-1 by the Jab1 active site of CSN acts as an upstream regulator of Skp1/Cul-1/F-box (SCF)-dependent ubiquitination of numerous substrates, including P27 and IκBα [32]. P27 is a universal cyclin-dependent kinase (CDK) inhibitor that directly inhibits the enzymatic activity of cyclin-CDK complexes, resulting in cell cycle arrest at G1 [33]. Jab1 promotes cell proliferation and inactivates P27 by inducing translocation of P27 from the nucleus to the cytoplasm, which accelerates P27 degradation through the Ub-dependent proteasome pathway and promotes cell cycle progression [34]. Thus, although transcriptional regulation is possible, the cellular expression of P27 is primarily regulated at the posttranslational level by Jab1 and by the Ub-proteasome pathway [34]. Here we show that MsrA interacts with Jab1 and consequently, the Jab1 deneddylase activity (removal of Nedd8) is enhanced, causing an increased deneddylation of Cul-1. Furthermore, the action of MsrA enhances the binding affinity of Jab1 to P27, causing a reduction in P27 level (presumably through its degradation by the Ub system). This novel character of MsrA as a positive regulator of Jab1 function will have implications for the general role of MsrA in mediating protein ubiquitination/ neddylation and regulation of cell survival. Antioxidants 20202020, 9, 452x FOR PEER REVIEW 33 of of 18 17

Figure 1. Post-translational modification of proteins by neddylation. The ubiquitin-like protein FigureNedd8 is1. covalentlyPost-translational bound to modification substrate proteins of proteins by a cascade by neddylatio of E1 activating,n. The ubiquitin E2 conjugating-like protein and Nedd8E3 ligase is enzymescovalently through bound to a processsubstrate termed proteins neddylation. by a cascade Deneddylation of E1 activating, regulates E2 conjugating this process and and E3 ligaseis catalyzed enzymes by thethrough c-Jun a activation process termed domain-binding neddylation. protein-1 Deneddylation (Jab1)/Csn5 regulates subunit this active process site and of the is catalyzedCOP9-signalosome, by the c-Jun which activ cleavesation domain the isopeptide-binding linkage protein to-1 release (Jab1)/Csn5 neural subunit precursor active cell site expressed of the COP9developmentally-signalosome, down-regulated which cleaves 8the (Nedd8) isopeptide from linkage the substrate to release protein. neural ~,precursor thioester cell intermediate. expressed —,developmentally isopeptide linkage. down-regulated 8 (Nedd8) from the substrate protein. ~, thioester intermediate. —, isopeptide linkage. 2. Material and Methods 2. Material and Methods 2.1. Ethics Statement 2.1. Ethics Statement The use of wild-type (WT) and MsrA knockout (KO, MsrA−/−) mice were followed according to an approvedThe use of Animal wild-type protocol (WT) by and the MsrA Institutional knockout Animal (KO, MsrA Care− and/−) mice Use were Committee followed (IACUC) according of the to anUniversity approved of Animal Kansas. protocol The protocol by the was Institutional implemented Animal in conjunction Care and with Use theCommittee Service Policy (IACUC) (PHS) of andthe AnimalUniversity Welfare of Kansas. Act (AWA) The protocol for the humane was implemented care and use in of conjunction laboratory animals. with the The Service identification Policy (PHS) code andof IACUC Animal protocol Welfare is Act 144-01 (AWA) and for the the Animal humane Welfare care and Assurance use of laboratory Number animals. is A3339-01. The Theidentification approval codedate of of the IACUC committee protocol was is 7 July 144-01 2018. and the Animal Welfare Assurance Number is A3339-01. The approval date of the committee was 7 July 2018. 2.2. The Yeast Two-Hybrid System (Y2H) 2.2. TheA yeastYeast ActivationTwo-Hybrid Domain System (Y2H) (AD) two-hybrid (Y2H) library (“prey”) was screened in a haploid strain by mating with the opposite mating strain expressing the DNA binding domain (BD)-bait A yeast Activation Domain (AD) two-hybrid (Y2H) library (“prey”) was screened in a haploid fusion protein (“bait”). Interaction of the BD-bait and AD-prey proteins was assessed based on the strain by mating with the opposite mating strain expressing the DNA binding domain (BD)-bait expression of the reporter genes in conditional autotrophs; this approach allowed capture of the AD fusion protein (“bait”). Interaction of the BD-bait and AD-prey proteins was assessed based on the plasmid(s) expressing the interacting proteins. The AD library and BD-bait were transformed into expression of the reporter genes in conditional autotrophs; this approach allowed capture of the AD the yeast strain, Y187, and Y2HGold, respectively. The Y2HGold strain with full-length MsrA as plasmid(s) expressing the interacting proteins. The AD library and BD-bait were transformed into bait was screened against an adult human brain cDNA library (Clontech, Mountain View, CA, USA) the yeast strain, Y187, and Y2HGold, respectively. The Y2HGold strain with full-length MsrA as bait by Next Interactions Inc. (Richmond, CA, USA). The Y2H procedures were performed similarly was screened against an adult human brain cDNA library (Clontech, Mountain View, CA, USA) by to Clontech’s protocol of the Matchmaker Gold Yeast Two-Hybrid System. Constructs were made Next Interactions Inc. (Richmond, CA, USA). The Y2H procedures were performed similarly to using Clontech’s pGBKT7 and pGADT7 plasmids that are components of the Matchmaker Gold Yeast Clontech’s protocol of the Matchmaker Gold Yeast Two-Hybrid System. Constructs were made using Two-Hybrid System. The MsrA clone was purchased from DNASU Plasmid Repository (The Biodesign Clontech’s pGBKT7 and pGADT7 plasmids that are components of the Matchmaker Gold Yeast Two- Institute, Tempe, AZ, USA). Constructs encoding the MsrA protein in pGBKT7 (“bait”, Trp1 selection Hybrid System. The MsrA clone was purchased from DNASU Plasmid Repository (The Biodesign marker of Y2H Gold yeast strain) and pGADT7 (“prey”, Leu2 election marker, Y187 yeast strain) were Institute, Tempe, AZ, USA). Constructs encoding the MsrA protein in pGBKT7 (“bait”, Trp1 selection mated using the following procedure. An YPD-agar plate was used to streak the S. cerevisiae strains marker of Y2H Gold yeast strain) and pGADT7 (“prey”, Leu2 election marker, Y187 yeast strain) and then the strains were incubated in YPD medium overnight at 30 C with rotation at 225 rpm. were mated using the following procedure. An YPD-agar plate was used◦ to streak the S. cerevisiae The S. cerevisiae/YPD culture was diluted 1:10 with fresh YPD medium and incubated for an additional strains and then the strains were incubated in YPD medium overnight at 30 °C with rotation at 225 2 h at 30 C with rotation (cell density: between 107 and 3.0 107 cells per mL). The cells were rpm. The ◦S. cerevisiae/YPD culture was diluted 1:10 with fresh ×YPD medium and incubated for an centrifuged at 580 g and the pelleted cells were resuspended in YPD medium and streaked onto additional 2 h at 30× °C with rotation (cell density: between 107 and 3.0 × 107 cells per mL). The cells agar plates, containing the standard selective media (SD) lacking tryptophan (Trp) and leucine (Leu). were centrifuged at 580× g and the pelleted cells were resuspended in YPD medium and streaked Theonto platesagar plates, were incubatedcontaining for the five standard days at selective 30 ◦C. Then, media colonies (SD) lacking from eachtryptophan plate were (Trp) resuspended and leucine in H O and spotted onto agar plates lacking Trp and Leu or agar plates containing selective media (Leu).2 The plates were incubated for five days at 30 °C. Then, colonies from each plate were lacking Trp, Leu, and histidine (His). Selection for positive clones was done for HIS3 reporter activation resuspended in H2O and spotted onto agar plates lacking Trp and Leu or agar plates containing selective media lacking Trp, Leu, and histidine (His). Selection for positive clones was done for HIS3 Antioxidants 2020, 9, 452 4 of 17

[low stringency, SD-Leu,-Trp, and -His (LTH)] and for HIS3 and ADE2 reporter activation [high stringency, SD-Leu, -Trp, -His, and -Adenine (LTHA)]. The initial selection was performed for 5 days. The equivalent of 1.07 106 diploid cells was selected on SD-LTH and the equivalent of 7.4 106 on × × SD-LTHA. Isolated colonies were picked and re-streaked for 2- to 3-day growth on high stringency medium (SD-LTHA) to ensure robust growth. Fifty-three colonies were finally obtained and prey cDNAs were amplified with colony PCR using a set of flanking primers. The resulting PCR products were sequenced in the forward direction as follows. Colonies from SD-LTH and SD-LTHA agarose plates were picked into a grid to grow larger quantities of cells. After regrowth, each colony was transferred into a 20 mM NaOH suspension and then boiled for 20 min. After cooling, lysates were clarified by centrifugation at >3000 g for 5 min. The supernatants were amplified by PCR using Mango × Polymerase buffer, nucleotides mix, primers [NIXO-1838 (50-gatGAAGATACCCCACCAAACC-30) and NIXO-1839 (50-acgatgcacagttgaAGTGAA-30)], Mango Tag DNA polymerase (Bioline, London, UK), and HiFi Tag DNA polymerase (Clontech, Mountain View, CA, USA). Aliquots from each amplified sample were analyzed for DNA bands by electrophoresis using a 1% (w/v) TAE agarose gel and sequenced. DNA sequences were compared to the human proteome database (UniProtKB). Hits that did not map to the coding sequence of known protein sequences or were of bad quality were not analyzed further.

2.3. GST-MsrA “Pull-Down” Experiments Brain extracts of WT and MsrA KO mice (n = 3 per strain, 6 months of age) were made by homogenization of postmortem brains in PBS in the presence of protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) on ice. The insoluble fraction of each extract was removed by high-speed centrifugation (10,000 g for 10 min). The supernatants were collected, and protein concentration of each preparation was determined using the Bradford reagent and BSA as the protein standard (Bio-Rad, Hercules, CA, USA). The MsrA KO supernatant (20 µg protein) was incubated with Sepharose glutathione-S-transferase (GST)-MsrA or Sepharose-GST resin, serving as control. The resins were synthesized as previously described with the MsrA derived from bovine liver cDNA expressed in Escherichia coli [35]. The incubation of the resins with the lysates was carried out for two hours at room temperature followed by overnight incubation at 4 ◦C. Thereafter, the resins were washed with PBS by multiple low-speed centrifugations until no protein was detected in the washing buffer (PBS). The proteins bound to the washed resins were eluted by 2 reducing SDS-gel-electrophoresis × buffer, boiled, and separated by 4–20% reducing SDS-gel-electrophoresis. One lane containing the GST-MsrA protein was stained along with the molecular standards for protein with Coomassie Blue R-250 stain. Additional gel-separated protein lanes were subjected to Western blot analysis using anti-Jab1 antibody (1:1000 dilution) as the primary antibody (Thermo-Fisher Scientific, Waltham, MA, USA), and the appropriate secondary HRP-conjugated antibody (Bio-Rad, Hercules, CA, USA).

2.4. Immunoprecipitation (IP) Experiments Antibodies used included: anti-MsrA antibody (Proteintech Group, Rosemont, IL, USA), anti-Jab1 antibody (Thermo-Fisher Scientific, Waltham, MA, USA), anti-Cul-1 antibody (Novus, Littleton, CO, USA), anti-Nedd8 antibody (Boster Bio, Pleasanton, CA, USA), anti-P27 antibody (Proteintech Group, Rosemont, IL, USA), anti-β actin antibody (Abcam, Cambridge, UK), and HRP-conjugated secondary antibodies for Western blot analyses (Rabbit anti-mouse and Goat anti-rabbit) (Bio-Rad, Hercules, CA, USA). WT and MsrA KO mouse brain supernatants were incubated with anti-MsrA antibody (Proteintech Group, Rosemont, IL, USA) in the presence of protein-G Sepharose at room temperature for the first two hours, followed by overnight incubation at 4 ◦C. Thereafter, the resins were washed with PBS by multiple low-speed centrifugations until no protein was detected in the washing buffer (PBS). The proteins that bound to the washed resins were eluted and subjected to Western blot analysis as earlier described. Antioxidants 2020, 9, 452 5 of 17

2.5. In Vivo Met Oxidation of Jab1 in Mouse Brain Six-month-old WT mice (n = 5 males) were exposed to normoxia (20% oxygen) or max hyperoxia (100% oxygen) for 24 h, as previously described [4]. The max hyperoxia conditions foster the formation of MetO while ensuring the welfare of the animals [4]. Thereafter, the mice were euthanized and brain extracts were made in PBS, as previously described [4]. Immunoprecipitation with rabbit anti-MetO antibody [36] was performed using 500 µg of protein extracted from each brain, followed by Western blot analysis probed with the mouse primary anti-Jab1 antibody (Thermo-Fisher Scientific, Waltham, MA, USA).

2.6. Deneddylase Activity of Jab1 on Cul-1 The in vivo deneddylation activity of Jab1 on its substrate (Cul-1) was determined as a function of the presence or absence of MsrA in mouse brain and liver extracts. To achieve this goal, IP experiments were performed using the anti-Cul-1 antibody (Novus, Littleton, CO, USA) as the IP antibody and the anti-Nedd8 (Boster Bio, Pleasanton, CA, USA) as the primary antibody in the subsequent Western blot analysis and vice-versa. Furthermore, based on the supposition that Jab1 would bind to the Nedd8 moiety of Cul-1 or other neddylated proteins, an anti-Jab1 antibody was used in an IP experiment. In this IP experiment, the anti-Jab1 antibody was used as a “fishing” probe to isolate neddylated protein complexes, such as Cul-1, that may be bound to Jab1 from biological extracts (prior to the removal of Nedd8 by Jab1 and its associated cofactors). These anti-Jab1 immunoprecipitants were thereafter probed by Western blot analysis using anti-Cul-1 antibody.

2.7. Recombinant Nedd8 (FHN8) Conjugate Synthesis and Purification

An N-terminal tandem Flag-His6-tag was added to the coding sequence of the mature Nedd8 protein (Uniprot accession P29595, residues 1-76), with the removal of the original initiator methionine. The recombinant coding sequence was codon-optimized for translation in Haloferax volcanii using the Codon Optimization On-Line (COOL) web platform. NdeI/BlpI restriction sites were added to the sequence 50 and 30 ends, and the entire sequence was synthesized by GenScript and ligated into pUC57 plasmid to create a pJAM3518 plasmid. Flag-His6-Nedd8 was removed from the pJAM3518 by NdeI/BlpI digestion and then ligated into a pJAM202 plasmid to create the pJAM3519 plasmid. Finally, the pJAM3519 was transformed into an E. coli TOP10, and isolated transformants were patched on LB supplemented with ampicillin. PCR screening by primers HvFW/RV confirmed the presence of the plasmid and the correct size of the FLAG-His6-Nedd8 (FHN8) insert. Several patches were selected, and the integrity of the insert sequence was verified by Sanger DNA sequencing. Plasmid pJAM3519 was passaged through a methylation deficient strain of E. coli (GM2163) prior to transformation into H. volcanii. Note that several strains of H. volcanii expressing FHN8 were tested for conjugate formation at a small scale; HM1096 was used as it produced the highest levels observed. Accordingly, H. volcanii HM1096 cells expressing pJAM3519 were inoculated in ATCC974 medium, supplemented with 50 mM DMSO and 0.2 µg/mL novobiocin. Flasks were incubated at 42 ◦C for 93 h with rotary shaking at 200 rpm. Cultures were pooled and centrifuged at 4000 g for 15 min and resuspended in TBS-2M × (2M NaCl, 50 mM Tris-Cl, pH 7.4) supplemented with protease inhibitor cocktail (Thermo-Fisher Scientific, Waltham, MA, USA). Thereafter, the cell suspension was thrice passed through a French pressure cell (Laboratory Supply Network, Atkinson, NH, USA) (2000 psi). The resulting lysate was supplemented with imidazole [30 mM] and clarified by centrifugation (13,000 g). Clarified × supernatant was applied to a 1 mL HisTrap HP column equilibrated in TBS-2M supplemented with 30 mM imidazole. After loading of the lysate, the column was extensively washed in the same buffer and the column-bound protein was eluted using TBS-2M supplemented with 500 mM imidazole. Peak fractions were supplemented with 1 mM EDTA and analyzed in a reducing 4–20% SDS-PAGE followed by subsequent Coomassie Blue R-250 staining and analysis by Western blotting. Selected fractions were pooled and dialyzed at 4 ◦C against TBS-2M. Antioxidants 2020, 9, 452 6 of 17

2.8. Deneddylation Activity Assay Equal amounts of protein (500 µg) extracted from the brains of WT and MsrA KO mice were incubated with recombinant Nedd8 (FHN8) conjugates (8 µg) in PBS at 37 ◦C. After the designated time of incubation, equal volumes of 2 sample buffer were added and immediately heated at 100 C × ◦ for 5 min to terminate the reaction. The reaction solution of different time points was subjected to Western blot using mouse anti-His antibody (Qiagen, Hilden, Germany) as the primary antibody. The neddylation levels of the lowest molecular mass of the His-detected Nedd8 conjugates (L.M. Nedd8) were quantified by NIH Image-J program.

2.9. Jab1 Binding to P27 and P27 Expression in Brain P27 from WT and MsrA KO brain extracts was immunoprecipitated with anti-P27 and the corresponding immunoprecipitates were probed with anti-Jab1 antibody by Western blot analyses.

3. Results

3.1. MsrA Interacting Proteins Identified by Yeast Two-Hybrid (Y2H) MsrA interacting proteins of the Homo sapiens proteome were identified by a yeast-two hybrid (Y2H) approach. Using MsrA as the “bait” and human brain proteins as the “prey”, 36 hits to protein sequences were identified and are summarized in Table1. The most prominent prey hits were Jab1 (CSN5) (n = 19), cyclin-I (n = 6), and succinate dehydrogenase [ubiquinone] iron-sulfur subunit (n = 6). These prominent hits were recurring suggesting specificity in the selection. In addition, five unique hits were identified, i.e., only one copy. The high number of hits obtained for Jab1, and the involvement of MsrA and Jab1 in Ub/Ub-like systems, prompted us to focus on this MsrA interacting target (Jab1).

Table 1. Msr type A enzyme (MsrA) interacting proteins identified by yeast two-hybrid system analysis.

No. of Hits GenBank No. Description [Homo Sapiens] Met Cys COP9 signalosome complex subunit 5 19 a NP006828 3.9% b 1.2% (Jab1/CSN5) 6 NP006826.1 Cyclin-I (Cyc-1) 2.9% 2.9% Succinate dehydrogenase [ubiquinone] 6 NP002991.2 3.6% 5.0% iron-sulfur subunit, mitochondrial precursor 1 NP689854.2 AT-rich interactive domain-containing protein 2 1.9% 1.6% 1 NP004757.1 Coatomer subunit β’ 2.0% 1.7% 1 NP055093.2 Heat shock 70 kDa protein 4L 2.7% 2.1% 1 NP005678.3 Phenylalanyl-tRNA synthetase β chain 2.0% 1.9% 1 NP001171895.1 WD repeat-containing protein 44 isoform 3 1.7% 1.0% a Hits that mapped to protein sequence (36 total) were identified from 18 positive clones from the initial SD-LTHA selection and others from the SD-LTH selection. Forty-three hits were identified using BlastN program (nucleotide) and 36 hits were identified using BlastX program (protein). b % total of deduced protein sequence.

3.2. GST-MsrA “Pull-Down” of Jab1 To further probe the interaction of MsrA and Jab1, brain extracts were analyzed for pull-down of Jab1 using GST-MsrA as ‘bait’. MsrA was fused to glutathione-S-transferase (GST) and then bound to glutathione coated Sepharose resin. Thus, MsrA protein could be used as the “bait”, while GST resin alone served as a control (the relevant procedures are described in Section2). Following gel-electrophoresis of the pulled-down proteins, one lane containing the GST-MsrA protein was stained for protein with Coomassie blue stain to observe the intensity of the major GST-MsrA protein band and the other minor bound proteins (Figure2A). Additional gel-separated protein lanes were subjected to Western blot analysis using anti-Jab1 antibody as the primary antibody. As shown in Figure2A, Antioxidants 2020, 9, x FOR PEER REVIEW 7 of 18 lane harboring GST alone showed no reaction. Thus, MsrA and Jab1 appeared to interact in a complex based on analysis by GST-MsrA pull down as well as Y2H.

3.3. Jab1 Co-Immunoprecipitates with MsrA To further analyze this MsrA-Jab1 association, WT, and MsrA KO mouse brain extracts were subjected to immunoprecipitation using the anti-MsrA antibody. The resulting immunoprecipitates wereAntioxidants analyzed2020 ,for9, 452 the presence of Jab1 by Western blotting using an anti-Jab1 antibody as the primary7 of 17 antibody. As this primary antibody was mouse generated, reactions with the mouse immunoglobulin light (L) and heavy (H) chains were anticipated and observed in both sample types (Figure 2B). However,only the lane a protein harboring band the specific GST-MsrA to the showed WT mouse a signal extract corresponding (and not toin Jab1, the whileMsrA theKO lane mouse) harboring was detectedGST alone that showed migrated no at reaction. a molecular Thus, mass MsrA comparable and Jab1 appearedto Jab1 (Figure to interact 2B), thus, in a providing complex based further on evidenceanalysis that by GST-MsrA MsrA and pull Jab1 down associate as well in a as complex. Y2H.

Figure 2. Interaction between MsrA and Jab1 in mouse brain extracts. (A) Pull-down experiments in which recombinant glutathione-S-transferase-MsrA (GST-MsrA) fused protein is used as a “bait” for Figurebinding 2. ofInteraction brain proteins. between (B) MsrAImmunoprecipitation and Jab1 in mouse (IP) brain experiments extracts. using(A) Pull mouse-down brain experiments extracts and in whichanti-MsrA recombinant antibody glutathione (IP antibody),-S-transferase followed- byMsrA Western (GST blot-MsrA analysis) fused using protein mouse is used anti-Jab1 as a “bait” primary for bindingantibody. of braiC. Quantificationn proteins. (B) ofImmunoprecipitation the Jab1 band detected (IP) byexperiments Western blot using analysis mouse in brain Panel extracts B, by using and antithe-MsrA NIH Image-Jantibody program. (IP antibody), N.D, followed not detected; by Western kDa, molecular blot analysis mass using markers mouse in anti kilo-Dalton-Jab1 primary units; antibody.WT, wild-type C. Quantification mouse; MT, MsrAof theKO Jab1 mouse; band detected L, light chain by Western of mouse blot immunoglobulin analysis in Panel detected B, by using in the theextract; NIH H,Image heavy-J program chain of. mouse N.D, not immunoglobulin detected; kDa, detectedmolecular in themass extract. markers Protein in kilo stain,-Dalton Coomassie units; WT, blue wildfor protein-type mouse; staining. MT, This MsrA experiment KO mouse; was repeated L, light chain at least of 3 mouse times and immunoglobulin one set of data detectedis represented in the in extract;this figure. H, heavy chain of mouse immunoglobulin detected in the extract. Protein stain, Coomassie blue for protein staining. This experiment was repeated at least 3 times and one set of data is 3.3.represented Jab1 Co-Immunoprecipitates in this figure. with MsrA To further analyze this MsrA-Jab1 association, WT, and MsrA KO mouse brain extracts were 3.4. In Vivo Met Oxidation of Jab1 in Mouse Brain subjected to immunoprecipitation using the anti-MsrA antibody. The resulting immunoprecipitates wereTo analyzed determine for thewhether presence Jab1 of is Jab1 susceptible by Western to methionine blotting using oxidation an anti-Jab1 in the antibody brain, we as exposed the primary 6- monthantibody.-old AsWT this mice primary (n = 5 males) antibody to wasnormoxia mouse (20% generated, oxygen) reactions or hyperoxia with the (100% mouse oxygen) immunoglobulin for 24 h as previouslylight (L) and described heavy [4] (H) and chains then wereprobed anticipated for MetO- andJab1. observed As shown in in both Figur samplee 3, following types (Figurethe 24 h2 B).of hyperoxia,However, a Jab1 protein contained band specific MetO to residues, the WT asmouse judged extract by (and the MetO not in-immunoprecipitation the MsrA KO mouse) and was subsequentdetected that anti migrated-Jab1 Western at a molecular blot analysis. mass In comparable comparison, to no Jab1 MetO (Figure-Jab12 wasB), thus, detected providing in the control further miceevidence (n = 5 that males) MsrA (Figure and Jab1 3). These associate obser invations a complex. suggest that Jab1 is prone to methionine oxidation upon exposure of the animal to hyperoxic conditions [4]. 3.4. In Vivo Met Oxidation of Jab1 in Mouse Brain To determine whether Jab1 is susceptible to methionine oxidation in the brain, we exposed 6-month-old WT mice (n = 5 males) to normoxia (20% oxygen) or hyperoxia (100% oxygen) for 24 h as previously described [4] and then probed for MetO-Jab1. As shown in Figure3, following the 24 h of hyperoxia, Jab1 contained MetO residues, as judged by the MetO-immunoprecipitation and subsequent anti-Jab1 Western blot analysis. In comparison, no MetO-Jab1 was detected in the control mice (n = 5 males) (Figure3). These observations suggest that Jab1 is prone to methionine oxidation upon exposure of the animal to hyperoxic conditions [4]. Antioxidants 2020, 9, 452 8 of 17 Antioxidants 2020, 9, x FOR PEER REVIEW 8 of 18

FigureFigure 3. 3. MetMet oxidation oxidation of of Jab1 Jab1 in in mice mice exposed exposed to to hyperoxia hyperoxia (100% (100% oxygen). oxygen). WT WT mice mice (6 (6 months months old, old, nn = 55)) were exposedexposed totohyperoxia hyperoxia for for 24 24 h. h. Thereafter, Thereafter, the the mice mice were were euthanized euthanized and and brain brain extracts extracts were weremade made in PBS in as PBS described as described under Section under 2Section.( A) Immunoprecipitation 2. (A) Immunoprecipitation with rabbit with anti-MetO rabbit anti antibody-MetO antibodywere performed were performed using 500 usingµg of 500μg extracted of extracted protein from protein each from brain, each followed brain, followed by Western by Western blot analyses blot analysprobedeswith probed the primary with the mouse primary anti-Jab1 mouse antibody anti-Jab1 (see antibody details under (see details Section under2). ( B )Section Quantification 2). (B) Quantificationof the Jab1 band of the detected Jab1 band by Western detected blot by Western analysis blot in Panel analysis A, byin Panel using A, the by NIH using Image-J the NIH program. Image- JN.D, program. not detected; N.D, not H, detected; heavy chain H, ofheavy brain chain mouse of IgG;brain L, mouse light chain IgG; of L, brain light mouse chain IgG.of brain A representative mouse IgG. Ablot representative is shown. blot is shown. 3.5. MsrA-Mediated Deneddylase Activity of Jab1 on Cul-1 3.5. MsrA-Mediated Deneddylase Activity of Jab1 on Cul-1 To investigate the possibility that MsrA activates Jab1 function, we followed the abundance of To investigate the possibility that MsrA activates Jab1 function, we followed the abundance of deneddylated Cul-1 (a substrate of Jab1) as function of the presence or absence of MsrA in mouse brain deneddylated Cul-1 (a substrate of Jab1) as function of the presence or absence of MsrA in mouse and liver extracts. The added use of liver extracts was followed since MsrA is highly expressed in brain and liver extracts. The added use of liver extracts was followed since MsrA is highly expressed this organ; this approach also enabled us to investigate whether the effect of MsrA on Jab1 function in this organ; this approach also enabled us to investigate whether the effect of MsrA on Jab1 function was general in nature. To achieve this goal, proteins in both extract-types were immunoprecipitated was general in nature. To achieve this goal, proteins in both extract-types were immunoprecipitated with anti-Cul-1 antibody and then probed with anti-Nedd8 by Western blot analysis and vise-versa. with anti-Cul-1 antibody and then probed with anti-Nedd8 by Western blot analysis and vise-versa. As shown in Figure4A (a,b), compared to WT brain, MsrA KO brain showed higher level of neddylated As shown in Figure 4A (a,b), compared to WT brain, MsrA KO brain showed higher level of Cul-1. Higher Cul-1 neddylation level was also observed in MsrA KO liver extracts compared to WT neddylated Cul-1. Higher Cul-1 neddylation level was also observed in MsrA KO liver extracts (Figure4A (c,d). Higher neddylation levels of Cul-1 in the MsrA KO brain and liver suggested that compared to WT (Figure 4A (c,d). Higher neddylation levels of Cul-1 in the MsrA KO brain and liver the Jab1 deneddylation activity was impaired in the absence of MsrA. This phenomenon was also in suggested that the Jab1 deneddylation activity was impaired in the absence of MsrA. This agreement with our hypothesis that MsrA reduction of specific MetO residue/s of Jab1 lead to increase phenomenon was also in agreement with our hypothesis that MsrA reduction of specific MetO of Jab1 activity, as measured by deneddylation of Cul-1. In the absence of MsrA (i.e., as demonstrated residue/s of Jab1 lead to increase of Jab1 activity, as measured by deneddylation of Cul-1. In the in the MsrA KO tissue extracts) the activity of Jab1 was compromised presumably due to its enhanced absence of MsrA (i.e., as demonstrated in the MsrA KO tissue extracts) the activity of Jab1 was Met oxidation, resulting in more neddylation (or less deneddylation) of Cul-1. compromised presumably due to its enhanced Met oxidation, resulting in more neddylation (or less deneddylation)3.6. MsrA Impact of on Cul Jab1-1. Binding to Cul-1

3.6. MsrAAn active Impact Jab1 on Jab1 presumably Binding to binds Cul-1 to the Nedd8 moiety of Cul-1 prior to removing this Ub-like modification from Cul1 (or any other target protein). Thus, we used the anti-Jab1 antibody in an IP experimentAn active as aJab1 “fishing” presumably probe tobinds isolate to the protein Nedd8 complexes moiety of Cul neddylated-1 prior to Cul-1 removing bound this to Jab1 Ub- fromlike modificationbiological extracts from andCul1 determine (or any other whether target MsrA protein). affected Thus, this binding.we used Indeed,the anti- followingJab1 antibody this approach, in an IP experimentthe WT brain as showeda “fishing” much probe higher to isolate neddylated protein Cul-1 complexes than the ofMsrA neddylatedKO (MT), Cul as-1 judgedbound byto Jab1 anti-Cul-1 from biologicalWestern blot extracts analysis and of samplesdetermine IP withwhether anti-Jab1 MsrA antibody affected (Figure this binding.4A (e)). These Indeed, data following suggest that this approach,Jab1 binds the Nedd8 WT onbrain Cul-1 showed more emucffectivelyh higher in theneddylated presence Cul of MsrA,-1 than supporting the MsrA theKO hypothesis(MT), as judged stated byabove anti and-Cul suggesting-1 Western thatblot MetO-Jab1analysis of issamples inactive IP in with this anti binding.-Jab1 antibody We then performed(Figure 4A another(e)). These Western data suggestblot analysis that Jab1 to investigate binds Nedd8 whether on Jab1Cul-1 levels more changed effectively in the in MsrAthe presenceKO compared of MsrA, with supporting the WT mouse the hypothesisbrain extracts. stated The resultsabove showed and suggesting that the presence that MetO/absence-Jab1 of is MsrA inactive had inno e thisffect binding. on the level We of then Jab1 performed(Figure4D). another Similar Western results were blot obtainedanalysis to using investigate liver extracts whether (data Jab1 not levels shown). changed These in data the indirectlyMsrA KO comparedsupport our with hypothesis, the WT mouse that MsrAbrain extracts. regulates The Jab1 results deneddylase showed activitythat the throughpresence/absence post-translational of MsrA hadprotein no effect modifications on the level (i.e., of reductionJab1 (Figure of MetO-Jab14D). Similar to results Jab1 forms) were obtained and not throughusing liver alteration extracts of (data Jab1 notexpression shown). levels. These data indirectly support our hypothesis, that MsrA regulates Jab1 deneddylase activity through post-translational protein modifications (i.e., reduction of MetO-Jab1 to Jab1 forms) and not through alteration of Jab1 expression levels. AntioxidantsAntioxidants2020 20, 920, 452, 9, x FOR PEER REVIEW 9 of9 17 of 18

Figure 4. In vivo regulation of Jab1 activity by MsrA through monitoring Cul-1 neddylation levels in Figure 4. In vivo regulation of Jab1 activity by MsrA through monitoring Cul-1 neddylation levels in mouse brain and liver extracts. (A), a–d. Mouse extracts from 6 months old mice (n = 3) were made mouse brain and liver extracts. (A), a–d. Mouse extracts from 6 months old mice (n = 3) were made in in PBS and in the presence of protease inhibitors cocktail (Sigma-Aldrich). Immunoprecipitation (IP) PBS and in the presence of protease inhibitors cocktail (Sigma-Aldrich). Immunoprecipitation (IP) experiments were performed on brain and liver extracts (500 µg of protein per extract) using either experiments were performed on brain and liver extracts (500 μg of protein per extract) using either anti-Cul-1 antibody or anti-Nedd8 antibody followed by Western blot (WB) analyses using anti-Nedd8 anti-Cul-1 antibody or anti-Nedd8 antibody followed by Western blot (WB) analyses using anti- antibody or anti-Cul-1 antibody as the primary antibody, respectively. Ae. In a unique experiment, Nedd8 antibody or anti-Cul-1 antibody as the primary antibody, respectively. Ae. In a unique Jab1 was used as a “fishing” probe in an IP experiment using brain extracts, followed by Western blot experiment, Jab1 was used as a “fishing” probe in an IP experiment using brain extracts, followed by analysis using anti-Cul-1antibody. Only the 100-kDa neddylated Cul-1 protein was identified due to the specificWestern pull-down blot analysis of the neddylated using anti form-Cul- of1antibody. Cul-1 by Jab1.Only ( theB) Quantification 100-kDa neddylated of each band Cul-1 identified protein was by Westernidentified blot due analyses to the describedspecific pull in Panel-down ( Aof) a–e,the neddylated as determined form by of using Cul- the1 by NIH Jab1. Image-J (B) Quantification program. All theof each observed band diidentifiedfferences inby the Western observed blot band analyses levels described between thein Panel WT and (A) MT a–e, pairs as determined were statistically by using significantthe NIH as Image judged-J byprogram. student Allt-test the analysis observed (* pdifferences< 0.01, n = in3 perthe strain).observed (C )band Loading levels controls between for the the WT liverand and MT brain pairs protein were levels, statistically following significant Coomassie as judged blue staining by student (to confirm t-test analysis the use ( of* p equal < 0.01, amount n = 3 per of proteinsstrain). ( fromC) Loading each mouse controls strain for the per liver tissue and in brain the IP protein experiments. levels, following WT, wild Coomassie type; M, MsrA blue staining KO. (D)(to Western confirm blot theanalysis use of of the equal mouse amount brain extractsof protein usings from anti Jab1 each antibody mouse strain as the primaryper tissue antibody in the IP andexperiments. quantified by WT, the wild NIH type; Image-J M, program.MsrA KO. WT, (D) wildWestern type; blot MT, analysisMsrA KO. of the Arrows mouse shown brain extracts in (A) a–e using indicateanti Jab1 the position antibody of as the the neddylated primary antibody 100-kDa and Cul-1 quantified (deneddylated by the formNIH runsImage as-J ~90 program kDa protein,. WT, wild nottype; detected). MT, MsrA The shown KO. Arrows WB and shown Coomassie in (A) bluea–e indicate staining the experiments position of are the representatives neddylated 100 of-kDa three Cul-1 independent(deneddylated experiments. form runs as ~90 kDa protein, not detected). The shown WB and Coomassie blue staining experiments are representatives of three independent experiments. 3.7. MsrA Impact on Jab1 Deneddylation Activity 3.7. MsrA Impact on Jab1 Deneddylation Activity To examine the impact of MsrA on Jab1 deneddylation activity, equal amounts of brain extracted protein ofTo WT examine and MsrA the impact KO mice of MsrA were incubatedon Jab1 deneddylation with Nedd8 (Flag-His-Nedd8,activity, equal amounts FHN8) of conjugates brain extracted at 37 ◦proteinC. After of the WT designated and MsrA time KO of mice incubation, were incubated the reactions with wereNedd8 terminated (Flag-His and-Nedd8, subjected FHN8) to Westernconjugates blotat analysis 37 °C. After using the mouse designated anti-His time antibody of incubation, (that recognizes the reactions FHN8). were The terminated neddylation and levels subjected of the to lowestWestern molecular blot analysis mass of using the His-detected mouse anti-His Nedd8 antibody conjugates (that recognizes (L.M.Nedd8) FHN8). were The quantified neddylation by NIH levels Image-Jof the program. lowest molecul The rationalear mass forof the this His focused-detected imaging Nedd8 was conjugates that the (L.M.Nedd8) accumulation were of L.M.Nedd8 quantified by wouldNIH increase Image-J as program. a function The of the rationale incubation for thistime focused due to the imaging Jab1 activity. was that Indeed, the accumulation at time zero, of theL.M.Nedd8 neddylation would levels ofincrease L.M.Nedd8 as a function in the presence of the ofincubation WT and MsrA time due KO wereto the identical Jab1 activity. (Figure Ind5A,B).eed, at However,time zero, the the MsrA neddylation KO group levels exhibited of L.M.Nedd8 much lower in levelsthe presence of released of WT L.M.Nedd8 and MsrA compared KO were toidentical the WT(Figure as the incubation 5A,B). However, time progressed. the MsrA The KO deneddylationgroup exhibited level much of WT lower strain levels rapidly of released reached theL.M.Nedd8 peak aftercompared 10 min of to incubationthe WT as the time incubation that was time much progressed. higher than The the de sameneddylation peak observed level of WT for strain the MsrA rapidly KOreached strain (Figure the peak5A,B). after This 10 min large of diincubationfference intime the that deneddylase was much activityhigher than between the same the twopeak strains observed for the MsrA KO strain (Figure 5A,B). This large difference in the deneddylase activity between the Antioxidants 2020, 9, 452 10 of 17 Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 18 tworemained strains remaine up to thed up end to of the the end incubation of the incubation time (30 time min). (30 It min). is noteworthy, It is noteworthy, that the that higher the higher molecular molecularNedd8 conjugate Nedd8 conjugate levels also levels decreased also decreased proportionally proportionally to the increase to the increase of the Low of the M.W. Low Nedd8 M.W. levels Nedd8(data notlevels shown). (data not Accordingly, shown). Accordingly, the MsrA KOthe brainMsrA isKO suggested brain is suggested to exhibit to much exhibit more much Met-oxidized more Metforms-oxidized of Jab1 forms protein of Jab1 (MetO-Jab1) protein (MetO that are-Jab1) inactive that are compared inactive withcompared the WT with brain. the WT This brain. phenomenon This is phenomenon is in agreement with our hypothesis that lack of MsrA compromises the deneddylase in agreement with our hypothesis that lack of MsrA compromises the deneddylase activity of Jab1. activity of Jab1.

Figure 5. MsrA alters the kinetics of Jab1 deneddylase activity in vitro. (A) Western blot pattern of Figure 5. MsrA alters the kinetics of Jab1 deneddylase activity in vitro. (A) Western blot pattern of control group (time 00). Total protein neddylation and Nedd8 levels are shown using the primary control group (time 0′). Total protein neddylation and Nedd8 levels are shown using the primary anti- anti-His antibody. We focused on lowest molecular weight Nedd8 positive bands (Low M.W.Nedd8) His antibody. We focused on lowest molecular weight Nedd8 positive bands (Low M.W.Nedd8) as as they represent the best end-product of the Nedd8 conjugates through the deneddylation process. they represent the best end-product of the Nedd8 conjugates through the deneddylation process. (B) (B) Quantification of lowest molecular weight Nedd8 positive bands at different time points following Quantification of lowest molecular weight Nedd8 positive bands at different time points following incubation with WT and MsrA KO mouse brain extracts (n = 3, females, 6 months old). (C) Time curve incubation with WT and MsrA KO mouse brain extracts (n = 3, females, 6 months old). C. Time curve of lowest molecular weight Nedd8 levels. All data were normalized to 0 min time. The shown WB are of lowest molecular weight Nedd8 levels. All data were normalized to 0 min time. The shown WB are representatives of three repeated experiments. WT, Wild-type; KO, MsrA knockout; The difference representatives of three repeated experiments. WT, Wild-type; KO, MsrA knockout; The difference between the two animal groups was statistically significant from the 5–30 min time points (t-test, between the two animal groups was statistically significant from the 5–30 min time points (t-test, p < 0.001).p < 0.001). 3.8. MsrA-Dependent Jab1 Binding to and Expression of P27 3.8. MsrA-Dependent Jab1 Binding to and Expression of P27 One of the functions of Jab1 is to bind P27 and to facilitate its translocation from the One of the functions of Jab1 is to bind P27 and to facilitate its translocation from the nucleus to nucleus to the cytosol for Ub-dependent degradation. We propose that this function of Jab1 is the cytosol for Ub-dependent degradation. We propose that this function of Jab1 is strengthened by thestrengthened function of by MsrA. the function Here, we of provide MsrA. Here,the first we supporti provideve the evidence first supportive for this possibility evidence by for this immunoprecipitationpossibility by immunoprecipitation of P27 from WTof P27 and from MsrA WT and KO MsrA brain KO extracts brain extracts and probing and probing the P27 the P27 immunoprecipitatesimmunoprecipitates for for Jab1 Jab1 by by applying applying Western Western blot blot analyses. analyses. As shown As shown in Figure in Figure 6, the6 expression, the expression levellevel of of P27 P27 was was much much lower lower in inthe the WT WT compared compared with with the theMsrA MsrA KO brain, KO brain, suggesting suggesting enhanced enhanced degradationdegradation and and stability stability of of P27 P27 in in WT WT and and MsrA MsrA KO KO mice, mice, respectively. respectively. Furthermore, Furthermore, Jab1 was Jab1 was detecteddetected in in the the immunoprecipitates immunoprecipitates of P27 of P27 in the in theWT WTbrain brain but was but wasnot detected not detected in the in MsrA the MsrAKO KO (Figure(Figure 66).). This This observation observation suggests suggests that that the the increased increased binding binding of of Jab1 Jab1 to to P27 P27 is isindeed indeed mediated mediated by by the theMsrA’s MsrA’s eff ecteffect on Jab1;on Jab1; that that in-turn in-turn causes causes an increasean increase of P27 of degradationP27 degradation in the in WTthe brainWT brain (as suggested (as suggestedby the dramatic by the dramatic decrease decrease of P27 proteinof P27 protein expression expression in the in WT the strain). WT strain). These These observations observations strongly stronglysupport support the general the general idea that idea Jab1 that function Jab1 function is compromised is compromised by the by level the level of oxidized of oxidized MetO MetO residue /s residue/sthat accumulate that accumulate on this on protein this protein in the absencein the absence of MsrA. of MsrA. The levels The levels of Jab1 of proteinJab1 protein were were similar in similarboth mouse in both strains, mousesuggesting strains, suggesting that the that observed the observed changes changes in Jab1-P27 in Jab1 complex-P27 complex levels arelevels not are due to a notdiff dueerence to a indifference Jab1 expression in Jab1 expression in the two in mouse the two strains mouse (Figure strains6 ).(Figure 6). Antioxidants 2020, 9, x FOR PEER REVIEW 11 of 18 Antioxidants 2020, 9, 452 11 of 17

Figure 6. P27 binding to Jab1 and expression is mediated by MsrA. Mouse extracts were made in PBS and in the presence of protease inhibitors cocktail (Sigma-Aldrich, St. Louis, MO, USA) (6 months old mice, n = 3). (A) Immunoprecipitation (IP) experiments were performed on brain extracts (500 µg of proteinFigure per extract) 6. P27 binding using anti-P27to Jab1 and antibody expression followed is mediated by Westernby MsrA. blotMouse (WB) extracts analyses were usingmade in anti-Jab1 PBS antibodyand asin the presence primary of antibody. protease inhibitors WB analyses cocktail using (Sigma the-Aldrich, specific St. primary Louis, MO and, USA suitable) (6 months secondary old antibodymice, for n each= 3). ( probedA) Immunoprecipitation protein determined (IP) experiments the expression were levels performed of P27, on Jab1,brain andextractsβ-actin (500 (loadingμg of control).protein (B). Quantificationper extract) using of anti the-P27 each antibody detected followed protein-band by Weste inrn the blot Western (WB) analyses blot analyses using anti described-Jab1 antibody as the primary antibody. WB analyses using the specific primary and suitable secondary in Panel A, by using the NIH Image-J program. N.D, not detected; The same extracts used for the antibody for each probed protein determined the expression levels of P27, Jab1, and β-actin (loading experiments performed in Figure4 were used here. Western blot analysis for determining Jab1 and P27 control). (B). Quantification of the each detected protein-band in the Western blot analyses described expressionin Panel levels A, by in theseusing brainthe NIH extracts Image- wereJ program. analyzed N.D, usingnot detected; the same The gel. same Thus, extracts for clarityused for reasons, the the dataexperiments shown for performed Jab1 expression in Figure in 4 these were extractsused here was. Western duplicated blot analysis from Figure for de4terminingD. Jab1 and P27 expression levels in these brain extracts were analyzed using the same gel. Thus, for clarity 4. Discussion reasons, the data shown for Jab1 expression in these extracts was duplicated from Figure 4D. In the studies described in this report, we showed that MsrA interacts with brain Jab1. We first 4. Discussion demonstrated this interaction through Y2H screening (Table1), and this interaction was validated by GST pull-downIn the andstudies immunoprecipitation described in this report, experiments we showed (Figure that MsrA2). Methionineinteracts with oxidation brain Jab1. of We Jab1 first was much greaterdemonstrated in mouse this braininteraction following through exposure Y2H screening to hyperoxia (Table compared1), and this withinteraction normoxia, was validated strengthening by the hypothesisGST pull- thatdown Jab1 and Metimmunoprecipitation residues are oxidized experiments under (Figure oxidative 2). stressMethionine conditions oxidationin vivoof Jab1(Figure was 3). much greater in mouse brain following exposure to hyperoxia compared with normoxia, Furthermore, the ability of MsrA to increase Jab1 function (presumably through the reduction of critical strengthening the hypothesis that Jab1 Met residues are oxidized under oxidative stress conditions MetO residue/s of Jab1) was further supported by the increase of deneddylation of Cul-1 in WT in in vivo (Figure 3). Furthermore, the ability of MsrA to increase Jab1 function (presumably through comparisonthe reduction to MsrA ofKO critical brains MetO (Figure residue/s4). The of positive Jab1) was e ff ect further of MsrA suppor onted the by deneddylase the increase activity of of Jab1deneddylation was also monitored of Cul-1 inin WT vitro in, comparison using our novelto MsrA recombinant KO brains (Figure Nedd8 4). conjugatesThe positive as effect substrate of (FigureMsrA5). on the deneddylase activity of Jab1 was also monitored in vitro, using our novel recombinant InNedd8 addition conjugates to its as deneddylase substrate (Figure activity, 5). Jab1 is known for its function as a modulator of P27 degradationIn byaddition binding to its to deneddylase P27 in the activity, nucleus Jab1 and isfacilitating known for its its function translocation as a modulator to the cytosol of P27 for Ub-dependentdegradation degradation by binding [to34 ].P27 Indeed, in the nucleus this function and facilitating of Jab1 wasits translocation positively a toffected the cytosol by the for presence Ub- of MsrAdependent in the brain, degradation as the [34]. binding Indeed, of this Jab1 function to P27 of was Jab1 mostly was positively detected affected in the by WT the brains, presence while of a MsrA in the brain, as the binding of Jab1 to P27 was mostly detected in the WT brains, while a higher higher expression of P27 was observed in the MsrA KO compared with the WT brain (Figure6). expression of P27 was observed in the MsrA KO compared with the WT brain (Figure 6). Overall, the following scenario of events is suggested to occur in the brain upon oxidative stress Overall, the following scenario of events is suggested to occur in the brain upon oxidative stress conditionsconditions and areand summarized are summarized in Figurein Figure7. 7 Briefly,. Briefly, due due to to enhanced enhanced oxidative stress, stress, an an elevated elevated level oflevel MetO of MetO (both (both as protein-bound as protein-bound and and free free MetO) MetO) is is produced, produced, causing causing an an upregula upregulationtion of MsrA of MsrA throughthrough signal signal transduction transduction pathways pathways [1 ,[1,3737].]. Consequently, Consequently, the binding binding of of MsrA MsrA to toMetO MetO-Jab1-Jab1 is is increased,increased, leading leading to an to activation an activation of two of two major major functions functions of Jab1:of Jab1: (a) (a) binding binding to to P27 P27 and and facilitating facilitating P27 degradation via the Ub-system, and (b) increasing the deneddylase activity of Jab1 towards neddylated proteins, in general, and of neddylated Cul-1, in particular. The sum of these events is expected to protect against oxidative-stress mediated abnormalities that are associated with Ub-dependent cell cycle progression and transcription. The overall implication of these MsrA-dependent phenomena is Antioxidants 2020, 9, 452 12 of 17 abolishing the expression of biomarkers that are associated with neurodegenerative diseases, such as Antioxidants 2020, 9, x FOR PEER REVIEW 13 of 18 AD [5,38–40] and Parkinson’s disease (PD; through an enhanced ubiquitination of 14-3-3 ζ [6–8,14,41]).

Figure 7. A summary of the proposed events and their link to neurodegeneration diseases. The details Figure 7. A summary of the proposed events and their link to neurodegeneration diseases. The details of the figure are presented under the Section4. The blue arrows indicate an “upregulation” and the red of the figure are presented under the “Discussion” section. The blue arrows indicate an arrows indicate a “downregulation” phenomenon. “upregulation” and the red arrows indicate a “downregulation” phenomenon. Our recent novel discovery demonstrates that MsrA is involved in the regulation of Ub-like 5. Conclusions modification of proteins in Archaea and ubiquitination of 14-3-3 ζ in the mouse brain [14,15]. Thus, the interactionThe studies of and MsrA data presented with Jab1, in whichthis article is playingclearly advocate a role infor thethe independent ubiquitination and combined and neddylation processes,importance is of an Jab1 additional and MsrA intriguingin regulating component. cellular regulation Establishing posttranslational the role modifications. of MsrA as Both a positive methionine oxidation and neddylation of proteins are well described as key protein modifiers that regulator of Jab1 will have implications for the general function of MsrA in mediating protein can affect protein structure and function. A major question that remains and needs further ubiquitinationinvestigation / is:neddylation, how does MsrA as well interaction as to enhancing with Jab1 cell affect survival, Jab1 activityespecially and in the neurodegenerative resulting diseases.deneddylation For example, levels of under proteins? enhanced One oxidativepossibility stress is that conditions, the Jab1 active cells may site has be vulnerable Met residues to reactive oxygensusceptible species-mediated to oxidation and damage. inactivation To circumvent that can this be repaired situation, by the MsrA. cells Consistent upregulate with MsrA this to rescue andpossibility,/or increase the Jab1 active function site has through a Met78 the residue reduction within of the its putative MetO residue substrate/s.- Inbinding turn, pocket the protected that Jab1 activityis in close can proximity prevent the to degradationthe active site of formed Cullin-RING by residues E3 ligases that coordinate by the Ub a/ caUb-liketalytic dependentZn2+ ion (Figure degradation pathways.8A). Furthermore, Furthermore, the Met78 the residue function of Jab1 of MsrAis evolutionary prevents conserved the accumulation across a diverse of undesirable spectrum of levels of P27organisms that are from better H. regulatedsapiens to methanogenic via the MetO-reduced archaea suggesting forms of it Jab1has an that important drive P27 biological for Ub-dependent role (Figure 8B). Understanding the relationships between MsrA and Jab1-dependent protein degradation. These phenomena have implications for cell survival under oxidative stress conditions deneddylation and P27 degradation will provide important knowledge on Jab1 regulation and its that are commonly present in aging, age-associated diseases, such as neurodegenerative diseases mechanism of action. Future new data is expected to fill up the gap in knowledge related to the (e.g.,function ADand of Jab1 PD). within the Ub/Ub-like system and related diseases. In addition, overexpression of MsrAAD in is the characterized brain, through by novelthe degeneration treatments, is of anticipated neuronal topopulations protect against mainly the in expression the hippocampus, of leadingbiomarkers to progressive associated with neurological neurodegenerative malfunction diseases. [42 ]. Up-to-date, the causes and pathogenesis of AD are not clear. Accumulative evidence suggests that dysregulation of several cell cycle factors is contributing to the pathophysiology of AD [43]. Consequently, the neurons of the AD brain display various cell cycle markers prior to degeneration [44]. Accordingly, cell cycle proteins are suggested to play an important and vital role in neuronal cell death. For example, cell cycle proteins are required for the neuronal death induced by amyloid-beta, the major peptide component of senile plaques. Alteration in the expression of cell cycle factors has also been linked to apoptosis of neurons. With respect to AD specifically, both P27 and phosphorylated P27 showed elevated levels in the cytoplasm of vulnerable neuronal populations in AD disease vs. control subjects [39]. Importantly, the phosphorylated P27 showed association with tau-positive neurofibrillary pathology, including neurofibrillary tangles, dystrophic neurites, and neuropil threads [39]. Likewise, higher levels of P27 were observed in a mouse model of AD [38]. Thus, these data suggest that dysregulation of the cell cycle plays a crucial role in the pathogenesis of AD that may be controlled by the interaction of Jab1 and MsrA. Cell cycle Antioxidants 2020, 9, 452 13 of 17 plays an important role in the development of cancer and relatively higher levels of Jab1 are associated with malignancy in specific cancerous cells [45]. The role of MsrA in regulating Jab1 function in cancer is yet to be determined. For example, according to the acquired knowledge on MsrA role in the above processes, developing or identifying small molecules that can penetrate the brain and upregulate MsrA could be beneficial for protecting against the development of AD [19]. In addition to the need to acquire new knowledge on the functional role of MsrA within the neddylation and ubiquitination pathways, determining the sequence region of Jab1 that binds MsrA and enables this interaction will be important. Availability of such data will provide fundamental information that can be used in follow up structure-function studies. For example, protein crystallization experiments, aimed to define the structural complex of these two proteins, can expand our knowledge with respect to these proteins’ interaction and hint on the possibilities to enhance their binding to each other. Accordingly, determining the conditions that affect the complex stability of MsrA-Jab1 may be used as potential tools to modulate Jab1 activity when it is strongly associated with the development and progression of a specific disease such as AD.

5. Conclusions The studies and data presented in this article clearly advocate for the independent and combined importance of Jab1 and MsrA in regulating cellular regulation posttranslational modifications. Both methionine oxidation and neddylation of proteins are well described as key protein modifiers that can affect protein structure and function. A major question that remains and needs further investigation is: how does MsrA interaction with Jab1 affect Jab1 activity and the resulting deneddylation levels of proteins? One possibility is that the Jab1 active site has Met residues susceptible to oxidation and inactivation that can be repaired by MsrA. Consistent with this possibility, the Jab1 active site has a Met78 residue within the putative substrate-binding pocket that is in close proximity to the active site formed by residues that coordinate a catalytic Zn2+ ion (Figure8A). Furthermore, the Met78 residue of Jab1 is evolutionary conserved across a diverse spectrum of organisms from H. sapiens to methanogenic archaea suggesting it has an important biological role (Figure8B). Understanding the relationships between MsrA and Jab1-dependent protein deneddylation and P27 degradation will provide important knowledge on Jab1 regulation and its mechanism of action. Future new data is expected to fill up the gap in knowledge related to the function of Jab1 within the Ub/Ub-like system and related diseases. In addition, overexpression of MsrA in the brain, through novel treatments, is anticipated to protect Antioxidants 2020, 9, x FOR PEER REVIEW 14 of 18 against the expression of biomarkers associated with neurodegenerative diseases.

Figure 8. Cont. Antioxidants 20202020, ,99, ,x 452 FOR PEER REVIEW 15 of 1814 of 17

Figure 8. Proposed importance of Met78 in regulation of Jab1 function by MsrA. (A) Jab1 (blue ribbon) Figurein complex 8. Propose with inhibitord importance (orange) of Met78 revealing in regulation the close of proximityJab1 function of methionineby MsrA. (A 78) Jab1 (M78) (blue to theribbon) substrate inbinding complex cleft with of the inhibitor Zn2+ metalloprotease (orange) revealing active the closesite. Stick proximity diagram of methionine is used to indicate 78 (M78) the to inhibitor the 2+ substrate(CSN5i-3), binding residues cleft that of coordinate the Zn metalloprotease Zn2+ (His138, His140active site. and Stick D151) diagr andam other is used residues to indicate in the active the site 2+ inhibitorregion (M78, (CSN5i T154,-3), residues N158, W136 that coordinate and C145). Zn Crystal(His138, structure His140 [PDB:and D151) 5JOG, and PMID: other residues 27774986] in [the46] was active site region (M78, T154, N158, W136 and C145). Crystal structure [PDB: 5JOG, PMID: 27774986] visualized using Chimeria v1.14 [PMID: 15264254] [47]. (B) Multiple amino acid sequence alignment of [46] was visualized using Chimeria v1.14 [PMID: 15264254] [47]. (B) Multiple amino acid sequence the central domain of representative Jab1 homologs selected from diverse organisms. Coloring and * or: alignment of the central domain of representative Jab1 homologs selected from diverse organisms. indicates regions of 100% identity or similarity, respectively. Residues that coordinate Zn2+ (His138, Coloring and * or: indicates regions of 100% identity or similarity, respectively. Residues that His140 and D151) and other residues in the active site region that may be susceptible to oxidation (M78 coordinate Zn2+ (His138, His140 and D151) and other residues in the active site region that may be and C145) are indicated by down arrows. Protein identifier indicated on left with Homo sapiens denoted susceptible to oxidation (M78 and C145) are indicated by down arrows. Protein identifier indicated by a red box and Archaea indicated by blue boxes (see Table S1 for complete listing of the proteins in on left with Homo sapiens denoted by a red box and Archaea indicated by blue boxes (see Table S1 for the alignment). complete listing of the proteins in the alignment).

Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3921/9/5/452/s1, Table S1: Uniprot-numbers-Jab1-homologs. Antioxidants 2020, 9, 452 15 of 17

Author Contributions: Conceptualization, J.M.; funding acquisition and project administration, J.M., J.M.-F.; investigation, J.M., J.M.-F., B.J., Z.A., S.M., H.S.; data analyses, J.M., J.M.-F., B.J., Z.A., S.M., H.S.; visualization, J.M., J.M.-F., B.J.; Writing—original draft preparation, J.M., J.M.-F., B.J., S.M., H.S.; writing—review and editing, J.M., J.M.-F., B.J., S.M., H.S. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Hedwig Miller Fund for Aging Research of the University of Kansas to Dr. Moskovitz. J.M.F’s work on this project was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DOE DE-FG02-05ER15650) to advance applications of archaea in biotechnology and the National Institutes of Health (NIH R01 GM57498) to reveal systems important to human health through study of archaea. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations: Jab1: c-Jun activation domain-binding protein-1; P27, cyclin-dependent kinase inhibitor 1B; Cul-1, Cullin-1; Msr, methionine sulfoxide reductase; MsrA, A-type Msr; MetO, methionine sulfoxide; Nedd8, neural precursor cell expressed developmentally down-regulated 8; Ub, ubiquitin; NAE, Nedd8 activating enzyme; NCE, Nedd8 conjugating enzyme; RBX1, RING-box protein 1; CRL, Cullin/RING ubiquitin ligase; SCF, Skp1/Cul-1/F-box.

References

1. Oien, D.B.; Moskovitz, J. Substrates of the methionine sulfoxide reductase system and their physiological relevance. Curr. Top. Dev. Biol. 2008, 80, 93–133. [PubMed] 2. Jiang, B.; Moskovitz, J. The Functions of the Mammalian Methionine Sulfoxide Reductase System and Related Diseases. Antioxidants 2018, 7, 122. [CrossRef][PubMed] 3. Moskovitz, J.; Flescher, E.; Berlett, B.S.; Azare, J.; Poston, J.M.; Stadtman, E.R. Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress. Proc. Natl. Acad. Sci. USA 1998, 95, 14071–14075. [CrossRef][PubMed] 4. Moskovitz, J.; Bar-Noy, S.; Williams, W.M.; Requena, J.; Berlett, B.S.; Stadtman, E.R. Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals. Proc. Natl. Acad. Sci. USA 2001, 98, 12920–12925. [CrossRef][PubMed] 5. Pal, R.; Oien, D.B.; Ersen, F.Y.; Moskovitz, J. Elevated levels of brain-pathologies associated with neurodegenerative diseases in the methionine sulfoxide reductase a knockout mouse. Exp. Brain Res. 2007, 180, 765–774. [CrossRef] 6. Oien, D.B.; Osterhaus, G.L.; Latif, S.A.; Pinkston, J.W.; Fulks, J.; Johnson, M.A.; Fowler, S.C.; Moskovitz, J. MsrA knockout mouse exhibits abnormal behavior and brain dopamine levels. Free Radic. Biol. Med. 2008, 45, 193–200. [CrossRef] 7. Oien, D.B.; Ortiz, A.N.; Rittel, A.G.; Dobrowsky, R.T.; Johnson, M.A.; Levant, B.; Fowler, S.C.; Moskovitz, J. Dopamine D(2) receptor function is compromised in the brain of the methionine sulfoxide reductase A knockout mouse. J. Neurochem. 2010, 114, 51–61. [CrossRef] 8. Moskovitz, J.; Walss-Bass, C.; Cruz, D.A.; Thompson, P.M.; Bortolato, M. Methionine sulfoxide reductase regulates brain catechol-O-methyl transferase activity. Int. J. Neuropsychopharmacol. 2014, 17, 1707–1713. [CrossRef] 9. Moskovitz, J.; Du, F.; Bowman, C.F.; Yan, S.S. Methionine sulfoxide reductase a affects β-amyloid solubility and mitochondrial function in a mouse model of Alzheimer’s disease. Am. J. Physiol. Endocrinol. Metab. 2016, 310, E388–E393. [CrossRef] 10. Erickson, J.R.; Joiner, M.L.; Guan, X.; Kutschke, W.; Yang, J.; Oddis, C.V.; Bartlett, R.K.; Lowe, J.S.; O’Donnell, S.E.; Aykin-Burns, N.; et al. A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation. Cell 2008, 133, 462–474. [CrossRef] 11. Singh, M.P.; Kim, K.Y.; Kim, H.Y. Methionine sulfoxide reductase a deficiency exacerbates acute liver injury induced by acetaminophen. Biochem. Biophys. Res. Commun. 2017, 484, 189–194. [CrossRef][PubMed] 12. De Luca, A.; Sanna, F.; Sallese, M.; Ruggiero, C.; Grossi, M.; Sacchetta, P.; Rossi, C.; De Laurenzi, V.; Di Ilio, C.; Favaloro, B. Methionine sulfoxide reductase A down-regulation in human breast cancer cells results in a more aggressive phenotype. Proc. Natl. Acad. Sci. USA 2010, 107, 18628–18633. [CrossRef][PubMed] 13. Alqudah, S.; Chertoff, M.; Durham, D.; Moskovitz, J.; Staecker, H.; Peppi, M. Methionine Sulfoxide Reductase A Knockout Mice Show Progressive Hearing Loss and Sensitivity to Acoustic Trauma. Audiol. Neurotol. 2018, 23, 20–31. [CrossRef] Antioxidants 2020, 9, 452 16 of 17

14. Deng, Y.; Jiang, B.; Rankin, C.L.; Toyo-Oka, K.; Richter, M.L.; Maupin-Furlow, J.A.; Moskovitz, J. Methionine sulfoxide reductase A (MsrA) mediates the ubiquitination of 14-3-3 protein isotypes in brain. Free Radic. Biol. Med. 2018, 129, 600–607. [CrossRef][PubMed] 15. Fu, X.; Adams, Z.; Liu, R.; Hepowit, N.L.; Wu, Y.; Bowmann, C.F.; Moskovitz, J.; Maupin-Furlow, J.A. Methionine Sulfoxide Reductase A (MsrA) and Its Function in Ubiquitin-Like Protein Modification in Archaea. MBio 2017, 8.[CrossRef] 16. Zhou, L.; Jiang, Y.; Luo, Q.; Li, L.; Jia, L. Neddylation: A novel modulator of the tumor microenvironment. Mol. Cancer 2019, 18, 77. [CrossRef] 17. del Pozo, J.C.; Dharmasiri, S.; Hellmann, H.; Walker, L.; Gray, W.M.; Estelle, M. AXR1-ECR1–dependent conjugation of RUB1 to the Arabidopsis cullin At CUL1 is required for auxin response. Plant Cell 2002, 14, 421–433. [CrossRef] 18. Schwechheimer, C.; Giovanna, S.; Deng, X.W. Multiple ubiquitin ligase-mediated processes require COP9 signalosome and AXR1 function. Plant Cell 2002, 14, 2553–2563. [CrossRef] 19. Scott, D.C.; Sviderskiy, V.O.; Monda, J.K.; Lydeard, J.R.; Cho, S.E.; Harper, J.W.; Schulman, B.A. Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein Nedd8. Cell 2014, 157, 1671–1684. [CrossRef] 20. Duda, D.M.; Borg, L.A.; Scott, D.C.; Hunt, H.W.; Hammel, M.; Schulman, B.A. Structural insights into Nedd8 activation of cullin-RING ligases: Conformational control of conjugation. Cell 2008, 134, 995–1006. [CrossRef] 21. Petroski, M.D.; Deshaies, R.J. Function and regulation of cullin-RING ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 2005, 6, 9–20. [CrossRef][PubMed] 22. Deshaies, R.J.; Joazeiro, C.A.P. RING domain E3 ubiquitin ligases. Ann. Rev. Biochem. 2009, 78, 399–434. [CrossRef][PubMed] 23. Nakayama, K.I.; Nakayama, K. Ubiquitin ligases: Cell-cycle control and cancer. Nat. Rev. Cancer 2006, 6, 369–381. [CrossRef][PubMed] 24. Claret, F.X.; Hibi, M.; Dhut, S.; Toda, T.; Karin, M. A new group of conserved coactivators that increase the specificity of AP-1 transcription factors. Nature 1996, 383, 453–457. [CrossRef][PubMed] 25. Lyapina, S.; Cope, G.; Shevchenko, A.; Serino, G.; Tsuge, T.; Zhou, C.; Wolf, D.A.; Wei, N.; Shevchenko, A.; Deshaies, R.J. Promotion of Nedd8-CUL1 conjugate cleavage by COP9 signalosome. Science 2001, 292, 1382–1385. [CrossRef][PubMed] 26. Schwechheimer, C.; Serino, G.; Callis, J.; Crosby, W.L.; Lyapina, S.; Deshaies, R.J.; Gray, W.M.; Estelle, M.; Deng, X.W. Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIR1 in mediating auxin response. Science 2001, 292, 1379–1382. [CrossRef][PubMed] 27. Tran, H.J.; Allen, M.D.; Löwe, J.; Bycroft, M. Structure of the Jab1/MPN domain and its implications for proteasome function. Biochemistry 2003, 42, 11460–11465. [CrossRef] 28. Ning, W.; Serino, G.; Deng, X.W. The COP9 signalosome: More than a protease. Trends Biochem. Sci. 2008, 33, 592–600. 29. Cope, G.A.; Deshaies, R.J. COP9 signalosome: A multifunctional regulator of SCF and other cullin-based ubiquitin ligases. Cell 2003, 114, 663–671. [CrossRef] 30. Cope, G.A.; Suh, G.S.; Aravind, L.; Schwarz, S.E.; Zipursky, S.L.; Koonin, E.V.; Deshaies, R.J. Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 2002, 298, 608–611. [CrossRef] 31. Ning, W.; Deng, X.W. The COP9 signalosome. Ann. Rev. Cell Dev. Biol. 2003, 19, 261–286. 32. Deshaies, R.J. SCF and Cullin/Ring H2-based ubiquitin ligases. Ann. Rev. Cell Dev. Biol. 1999, 15, 435–467. [CrossRef][PubMed] 33. Kouvaraki, M.A.; Korapati, A.L.; Rassidakis, G.Z.; Tian, L.; Zhang, Q.; Chiao, P.; Ho, L.; Evans, D.B.; Claret, F.X. Potential role of jun activation domain–binding protein 1 as a negative regulator of p27kip1 in pancreatic adenocarcinoma. Cancer Res. 2006, 66, 8581–8589. [CrossRef][PubMed] 34. Tomoda, K.; Kubota, Y.; Arata, Y.; Mori, S.; Maeda, M.; Tanaka, T.; Yoshida, M.; Yoneda-Kato, N.; Kato, J.Y. The cytoplasmic shuttling and subsequent degradation of p27Kip1 mediated by Jab1/CSN5 and the COP9 signalosome complex. J. Biol. Chem. 2002, 277, 2302–2310. [CrossRef] 35. Moskovitz, J.; Weissbach, H.; Brot, N. Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins. Proc. Natl. Acad. Sci. USA 1996, 93, 2095–2099. [CrossRef] Antioxidants 2020, 9, 452 17 of 17

36. Oien, D.B.; Canello, T.; Gabizon, R.; Gasset, M.; Lundquist, B.L.; Burns, J.M.; Moskovitz, J. Detection of oxidized methionine in selected proteins, cellular extracts and blood serums by novel anti-methionine sulfoxide antibodies. Arch. Biochem. Biophys. 2009, 485, 35–40. [CrossRef] 37. Moskovitz, J.; Maiti, P.; Lopes, D.H.; Oien, D.B.; Attar, A.; Liu, T.; Mittal, S.; Hayes, J.; Bitan, G. Induction of methionine-sulfoxide reductases protects neurons from amyloid β-protein insults in vitro and in vivo. Biochemistry 2011, 50, 10687–10697. [CrossRef] 38. Wang, X.; Xu, Y.; Zhu, H.; Ma, C.; Dai, X.; Qin, C. Downregulated microRNA-222 is correlated with increased p27Kip1 expression in a double transgenic mouse model of Alzheimer’s disease. Mol. Med. Rep. 2015, 12, 7687–7692. [CrossRef] 39. Ogawa, O.; Lee, H.G.; Zhu, X.; Raina, A.; Harris, P.L.; Castellani, R.J.; Perry, G.; Smith, M.A. Increased p27, an essential component of cell cycle control in Alzheimer’s disease. Aging Cell 2003, 2, 105–110. [CrossRef] 40. Chen, Y.; Neve, R.L.; Liu, H. Neddylation dysfunction in Alzheimer’s disease. J. Cell. Mol. Med. 2012, 16, 2583–2591. [CrossRef] 41. Liu, F.; Hindupur, J.; Nguyen, J.L.; Ruf, K.J.; Zhu, J.; Schieler, J.L.; Bonham, C.C.; Wood, K.V.; Davisson, V.J.; Rochet, J.C. Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson’s disease-related insults. Free Biol. Med. 2008, 45, 242–255. [CrossRef][PubMed] 42. DeTure, M.A.; Dickson, D.W. The neuropathological diagnosis of Alzheimer’s disease. Mol. Neurodegener. 2019, 14, 32. [CrossRef][PubMed] 43. Arendt, T. Synaptic plasticity and cell cycle activation in neurons are alternative effector pathways: The ‘Dr. Jekyll and Mr. Hyde concept’ of Alzheimer’s disease or the yin and yang of neuroplasticity. Prog. Neurobiol. 2003, 71, 83–248. [CrossRef][PubMed] 44. Bowser, R.; Smith, M.A. Cell cycle proteins in Alzheimer’s disease: Plenty of wheels but no cycle. J. Alzheimers Dis. 2002, 4, 249–254. [CrossRef] 45. Guo, Z.; Wang, Y.; Zhao, Y.; Shu, Y.; Liu, Z.; Zhou, H.; Wang, H.; Zhang, W. The pivotal oncogenic role of Jab1/CSN5 and its therapeutic implications in human cancer. Gene 2019, 687, 219–227. [CrossRef] 46. Schlierf, A.; Altmann, E.; Quancard, J.; Jefferson, A.B.; Assenberg, R.; Renatus, M.; Jones, M.; Hassiepen, U.; Schaefer, M.; Kiffe, M.; et al. Targeted inhibition of the COP9 signalosome for treatment of cancer. Nat. Commun. 2016, 7, 13166. [CrossRef] 47. Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.R. UCSF Chimera—A Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004, 25, 1605–1612. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).