Family Of E3 Ubiquitin Ligases Research Articles

Transcriptome-Wide Analysis and Functional Verification of RING-Type Ubiquitin Ligase Involved in Tea Plant Stress Resistance.

The ubiquitin/26S proteasome pathway is a critical protein-degradation pathway in plant growth and development as well as in nearly all biological and abiotic stress processes. Although as a member of the ubiquitin/26S proteasome pathway, the E3 ubiquitin ligase family has been shown to be essential for the selective degradation of downstream target proteins, it has been rarely reported in tea plants (Camellia sinensis). In this study, through database searches and extensive manual deduplication, 335 RING finger family proteins were selected from the Tea Plant Information Archive. These proteins were divided into six categories by the difference of RING finger domain: RING-H2, RING-HCa, RING-HCb, RING-C2, RING-v, and RING-G. Stress-induced differential gene expression analysis showed that 53 proteins in RING finger family can respond to selected exogenous stress. In vitro ubiquitination assays indicated that TEA031033, which was named CsMIEL1, exhibited the activity of E3 ubiquitin ligases. CsMIEL1-overexpressing transgenic Arabidopsis thaliana seedlings were resistant to some exogenous abiotic stresses, such as salt and drought stress but sensitive to exogenous methyl jasmonate treatment. Furthermore, CsMIEL1 reduced the accumulation of anthocyanin in transgenic plants in response to low temperature treatment. The results of this article provide basic date for studying the role of ubiquitin/26S proteasome pathway in tea plants response to stresses.

The CRL4DCAF1 cullin‐RING ubiquitin ligase is activated following a switch in oligomerization state

The cullin‐4‐based RING‐type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the ˜29 CRL4 substrate receptors reported, the DDB1‐ and CUL4‐associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4DCAF1 ligase. In the 8.4 Å cryo‐EM map of CRL4DCAF1, four CUL4‐RBX1‐DDB1‐DCAF1 protomers are organized into two dimeric sub‐assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C‐terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin‐conjugating enzymes. Upon CRL4DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization‐deficient CRL4DCAF1 mutant has higher ubiquitin ligase activity compared to the wild‐type. This study identifies a novel mechanism by which unneddylated and substrate‐free CUL4 ligases can be maintained in an inactive state.

A metabolic daylength measurement system mediates winter photoperiodism in plants.

Plants have served as a preeminent study system for photoperiodism due to their propensity to flower in concordance with the seasons. A nearly singular focus on understanding photoperiodic flowering has prevented the discovery of other photoperiod measuring systems necessary for vegetative health. Here, we use bioinformatics to identify photoperiod-induced genes in Arabidopsis. We show that one, PP2-A13, is expressed exclusively in, and required for, plant fitness in short, winter-like photoperiods. We create a real-time photoperiod reporter, using the PP2-A13 promoter driving luciferase, and show that photoperiodic regulation is independent of the canonical CO/FT mechanism for photoperiodic flowering. We then reveal that photosynthesis combines with circadian-clock-controlled starch production to regulate cellular sucrose levels to control photoperiodic expression of PP2-A13. This work demonstrates the existence of a photoperiod measuring system housed in the metabolic network of plants that functions to control seasonal cellular health.

The F-box E3 ubiquitin ligase BAF1 mediates the degradation of the brassinosteroid-activated transcription factor BES1 through selective autophagy in Arabidopsis.

Brassinosteroids (BRs) regulate plant growth, development, and stress responses by activating the core transcription factor BRI1-EMS-SUPPRESSOR1 (BES1), whose degradation occurs through the proteasome and autophagy pathways. The E3 ubiquitin ligase(s) that modify BES1 for autophagy-mediated degradation remain to be fully defined. Here, we identified an F-box family E3 ubiquitin ligase named BES1-ASSOCIATED F-BOX1 (BAF1) in Arabidopsis thaliana. BAF1 interacts with BES1 and mediates its ubiquitination and degradation. Our genetic data demonstrated that BAF1 inhibits BR signaling in a BES1-dependent manner. Moreover, BAF1 targets BES1 for autophagic degradation in a selective manner. BAF1-triggered selective autophagy of BES1 depends on the ubiquitin binding receptor DOMINANT SUPPRESSOR OF KAR2 (DSK2). Sucrose starvation-induced selective autophagy of BES1, but not bulk autophagy, was significantly compromised in baf1 mutant and BAF1-ΔF (BAF1 F-box decoy) overexpression plants, but clearly increased by BAF1 overexpression. The baf1 and BAF1-ΔF overexpression plants had increased BR-regulated growth but were sensitive to long-term sucrose starvation, while BAF1 overexpression plants had decreased BR-regulated growth but were highly tolerant of sucrose starvation. Our results not only established BAF1 as an E3 ubiquitin ligase that targets BES1 for degradation through selective autophagy pathway, but also revealed a mechanism for plants to reduce growth during sucrose starvation.

A tripartite motif protein (CgTRIM1) involved in CgIFNLP mediated antiviral immunity in the Pacific oyster Crassostrea gigas

Tripartite motif (TRIM) proteins are a large family of E3 ubiquitin ligases involved in many biological processes, such as inflammation and antiviral immunity. In the present study, a novel TRIM protein homolog named CgTRIM1 was identified from Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgTRIM1 was of 1914 bp encoding a putative polypeptide of 637 amino acid residues. There were three classical domains in the predicted CgTRIM1 protein, including one RING domain, two b-box domains and one coiled-coil domain in N-terminal. For the lack of C-terminal domains, the CgTRIM1 was classified as the member of C–V TRIM subfamily. The mRNA transcripts of CgTRIM1 were detected in all the tested tissues and haemocytes, with the highest expression level in gill. The mRNA and protein levels of CgTRIM1 in gill were significantly up-regulated at 6 h after poly (I:C) stimulation. Moreover, the nuclear translocation of CgTRIM1 was observed in haemocytes of oysters after poly (I:C) stimulation. After IFN-like protein (CgIFNLP) was knocked down by RNA interference (RNAi), the expression of CgTRIM1 in gill was markedly inhibited in both mRNA (0.14-fold, p < 0.001) and protein levels after poly (I:C) stimulation. Furthermore, after knocking down of CgTRIM1, the mRNA expression levels of IFN-stimulated genes, including myxovirus resistance of oyster (CgMx) and Interferon-induced protein 44 (CgIFI44) were significantly down-regulated post poly (I:C) stimulation, while no significant change of the CgIFNLP expression was observed. These results indicated that CgTRIM1 participated in the antiviral response of C. gigas by regulating the mRNA expressions of IFN-stimulated genes.

Expression and purification of functional recombinant CUL2\u2022RBX1 from E. coli

Cullin-2 (CUL2) based cullin-RING ligases (CRL2s) comprise a family of ubiquitin E3 ligases that exist only in multi-cellular organisms and are crucial for cellular processes such as embryogenesis and viral pathogenesis. CUL2 is the scaffold protein that binds one of the interchangeable substrate receptor modules, which consists of adaptor proteins and the substrate receptor protein. The VHL protein is a substrate receptor known to target hypoxia-inducible factor α (HIF1α) for ubiquitination and degradation. Because of its critical role in the ubiquitination of important cellular factors such as HIF1α, CRL2s have been investigated for their biological functions and the development of novel therapeutics against diseases. Given the importance of CRL2s in biological and biomedical research, methods that efficiently produce functional CUL2 proteins will greatly facilitate studies on the mechanism and regulation of CRL2s. Here, we report two cost-effective systems for the expression and purification of recombinant human CUL2 from E. coli cells. The purified CUL2 proteins were ~ 95% pure, could bind their substrate receptor modules, and were enzymatically active in transferring ubiquitin or ubiquitin-like protein to the corresponding substrate in in vitro assays. The presented methodological advancements will help advance research in CRL2 function and regulation.

Role of NEDD8 in CUL2‐dependent ubiquitination of substrates with distinct sizes

As the largest family of ubiquitin E3 ligases in eukaryotes, cullin-RING ligases (CRLs) play essential roles in a myriad of biological processes by catalyzing the ubiquitination of key regulatory proteins. Neddylation, the conjugation of NEDD8 to cullins, is one of the most important mechanisms regulating the activity of CRLs. Biochemical studies have demonstrated that neddylation promotes the activity of CUL1-based CRL1s, leading to increased rates of substrate ubiquitination. Structural studies further suggested that neddylation triggered a conformational change that potentially bridged a gap between the donor ubiquitin and the acceptor substrate. These findings led to a hypothesis that the ubiquitination of larger CRL substrates may depend less on neddylation because the substrates could close the gap by themselves. Here, we explored the effect of neddylation on CRL2VHL-dependent ubiquitination of substrates with distinct sizes, through ex vivo degradation and in vitro ubiquitination assays. We first found that treating HeLa or HEK293 cells with pevonedistat, a small molecule drug inhibiting neddylation, reduced the degradation rates of small-sized p27 or the ODD domain of HIF1α, but not the full-length HIF1α. Though results from these initial trials support our hypothesis, we further found that inhibiting neddylation did reduce the degradation rate of the full-length HIF1α in RCC4 cells expressing exogenous VHL, in which CRL2VHL-dependent ubiquitination is the primary mechanism for HIF1α degradation. This result suggests that the degradation of HIF1α we observed in HEK293 cells in the presence of pevonedistat was CRL2VHL-independent. To directly test our hypothesis, we reconstituted CRL2VHL substrate ubiquitination in vitro and found that neddylation promotes the ubiquitination of the full-length HIF1α and the degron peptide of HIF1α to similar extents, demonstrating that neddylation activates CRL2VHL-dependent ubiquitination regardless of the substrate sizes. Our findings are consistent with the recently updated structural model of the mechanism for NEDD8-induced CRL activation, suggesting that neddylation not only brings the acceptor substrate in close proximity to the donor ubiquitin, but also tunes the CRL conformation to ensure efficient ubiquitination of different-sized substrates.

Expression and purification of recombinant human CUL2 from Escherichia coli cells

Cullin-2 (CUL2) based cullin RING ligases (CRL2) comprise a family of ubiquitin E3 ligases that exist only in multi-cellular organisms and are crucial for cellular processes such as embryogenesis and viral pathogenesis. In the CRL2 complex, CUL2 serves as a docking platform for the substrate receptor module, which consists of the adaptor proteins Elongin B and Elongin C (EloB/C) as well as the tumor suppressor protein VHL, where VHL is known to target hypoxia-inducible factor α (HIF1α) for ubiquitination and degradation. Abnormalities in HIF1α degradation can result in vascular tumor cell proliferation and cause a rare disease known as von-Hippel-Lindau (VHL) syndrome. Because of its critical role in HIF1α ubiquitination, CRL2VHL has been investigated for the development of novel therapeutics and treatments against disease. Given the importance of CRL2s in biological and biomedical research, a method that efficiently produces functional recombinant human CUL2 proteins will greatly facilitate studies on CRL2-dependent protein ubiquitination and degradation. Previous studies have generated recombinant human CUL2 using a baculovirus expression system. However, this method is very costly and time consuming for optimal protein expression in insect cells. To achieve a more cost-effective way of producing the human CUL2 protein, we propose to generate recombinant CUL2 in E. coli cells. We suggest that removal of unstructured regions in CUL2 would increase its solubility in E. coli. Additionally, we explored and applied the “Split-n-Co-express” strategy as previously used for CUL1 and CUL3. We report that each of our strategies resulted in soluble CUL2 expressed in E. coli cells, and following a series of purification by chromatography, the purity of the recombinant protein reached ~95%. Using in vitro assays, we found that our purified CUL2 could conjugate to NEDD8, a key activator of CRLs, and that the neddylated CUL2 could bind to VHL•EloB/C to ubiquitinate the HIF1α degron peptide. The binding and enzymatic activity of our recombinant protein demonstrate that we have established a new system for the expression and purification of functional human CUL2 from E. coli cells.

Arginyltransferase1 mediates a pVHL‐independent oxygen sensing pathway in mammalian cells

The cellular response to oxygen availability is a fundamental process in all eukaryotes that regulates energy metabolism and death/survival decisions.The mechanisms for oxygen sensing likely co-evolved with the domestication of mitochondria as an adaptation to the oxygenation of the earth atmosphere. However, in mammalian cells, the only known oxygen sensing mechanism depends on the Von Hippel–Lindau tumor suppressor protein (pVHL), which is only found in metazoans but not in other species. This discrepancy points to a potential missing link in oxygen sensing pathways. In this study, we found that oxygen sensing in mammalian cells is also regulated by Arginyltransferase 1 (ATE1), which is ubiquitously conserved in eukaryotes and influences the degradation of many proteins by posttranslational arginylation via the principle of N-end rule. We report that ATE1 centrally controls the hypoxic response and glycolysis in mammalian cells by arginylating Hypoxia-inducible factor 1a (HIF1a), and subsequently regulating HIF1a signaling pathways in a transcription-independent manner. Moreover, we find that ATE1-mediated N-arginylation of HIF1a is sensitive to its oxygen-dependent hydroxylation mediated by the oxygen sensor prolyl hydroxylase (PHD). However, ATE1 promotes HIF1a degradation independently of the conventional pVHL E3 ubiquitin ligase pathway, but depending on typical N-end degradation machineries, the UBR E3 Ubiquitin ligase family. Bioinformatic analysis of human tumor data reveal that the ATE1/UBR and the pVHL pathway jointly regulate oxygen sensing in vivo with tissue specificities. Finally, a phylogenetic analysis suggests that ATE1 was derived from mitochondrial gene transfer and involved in oxygen-sensing in multiple lineages of eukaryotes. Our findings present an unexplored role of ATE1-mediated arginylation as a novel pathway for cellular oxygen sensing and adaptation in mammalian cells.

F-Box Protein FBXW17-Mediated Proteasomal Degradation of Protein Methyltransferase PRMT6 Exaggerates CSE-Induced Lung Epithelial Inflammation and Apoptosis.

Chronic obstructive pulmonary disease (COPD) is a chronic debilitating lung disease, characterized by progressive airway inflammation and lung structural cell death. Cigarette smoke is considered the most common risk factor of COPD pathogenesis. Understanding the molecular mechanisms of persistent inflammation and epithelial apoptosis induced by cigarette smoke would be extremely beneficial for improving the treatment and prevention of COPD. A histone methyl modifier, protein arginine N-methyltransferase 6 (PRMT6), is reported to alleviate cigarette smoke extract (CSE)-induced emphysema through inhibiting inflammation and cell apoptosis. However, few studies have focused on the modulation of PRMT6 in regulating inflammation and cell apoptosis. In this study, we showed that protein expression of PRMT6 was aberrantly decreased in the lung tissue of COPD patients and CSE-treated epithelial cells. FBXW17, a member of the Skp1-Cullin-F-box (SCF) family of E3 ubiquitin ligases, selectively bound to PRMT6 in nuclei to modulate its elimination in the proteasome system. Proteasome inhibitor or silencing of FBXW17 abrogated CSE-induced PRMT6 protein degradation. Furthermore, negative alteration of FBXW17/PRMT6 signaling lessened the proapoptotic and proinflammatory effects of CSE in lung epithelial cells. Our study, therefore, provides a potential therapeutic target against the airway inflammation and cell death in CS-induced COPD.

039 IL-7 regulates the PD-1 signaling pathway via degradation by E3 ubiquitin ligase F-Box Protein 38

Alopecia areata (AA) is a T cell-mediated autoimmune disease attacking the hair follicle (HF). The IL-7 signaling pathway plays an important role in T cell survival and it has been therapeutically targeted in several T cell-dependent autoimmune disease models. Here, we showed that C3H/HeJ mice with AA exhibited hair regrowth after anti-IL7Ra treatment. Mechanistically, we observed that IL-7Ra blockade significantly reduced the number of alopecic effector CD8+ T cells. We also found that C3H/HeJ mice treated with anti-IL-7Rα showed a significant increase in the frequency of PD-1+CD44+ T cells within SDLNs compared to controls. Our previous results indicated that IL-7 may antagonize the function of PD-1 by downregulating the expression of PD-1 in T cells, however, the mechanism of this downregulation remains unclear. Recently, the F-box protein FBXO38, a member of the SKP1–CUL1–F-box protein family of E3 ubiquitin ligases, has shown to interact with PD-1 and decrease PD-1 cell-surface expression via degradation. Based on this findings, we postulated that IL-7 might decrease PD-1 expression through upregulation of FBXO38. We found that IL-7 significantly increased the transcription levels of FBXO38 and concurrently decreased the expression of PD-1 on the surface of T cells in vitro. We further observed that FBXO38 knockdown using siRNA increased the PD-1 expression on the surface of T cells compared to control, and moreover, this effect could not be rescued by IL-7 treatment. Our results indicate FBXO38 as a critical mediator of PD-1 degradation and suggests that targeting IL-7-mediated regulation of FBXO38 expression may represent a potential strategy to enhance PD-1 signaling in T cell-mediated autoimmune diseases including AA.

091 MARCH family E3 ubiquitin ligases selectively target cadherin family proteins for degradation

Cadherins play a central role in the assembly and adhesive function of adherens junctions (AJ). Adhesion and signaling functions of cadherins regulate epidermal and vascular barrier function, development, and tumor metastasis. Our group previously showed that a viral ubiquitin ligase K5, which is encoded in Human Herpesvirus 8 genome and involved in Kaposi Sarcoma, causes endocytosis and degradation of VE-cadherin (VE-cad). K5 is homologous to endogenous human membrane associated RING-CH-type ubiquitin ligase (MARCH) proteins. However, the contribution of endogenous MARCH family proteins to cadherin regulation is unclear. To assess the effect of MARCH family protein expression on epithelial and endothelial cadherins, MARCH proteins were expressed in epithelial and endothelial cells and the effect on E-cadherin (E-cad), VE-cad, and N-cadherin (N-cad) was monitored by western blot and immunofluorescence microscopy. MARCH1 had no discernable impact on E-cad, VE-cad or N-cad. MARCH8 downregulated E-cad and slightly downregulated VE-cad but not N-cad. In contrast, MARCH2, MARCH3 and MARCH4 transfection markedly downregulated VE-cad. Interestingly, MARCH2 and MARCH3 did not downregulate E-cad and N-cad, but MARCH4 downregulated E-cad, VE-cad and N-cad. These results revealed that different MARCH proteins have different cadherin specificities. To determine the amino-acid sequences within VE-cad that dictate this specificity, we generated VE-cad/N-cad chimeras by swapping the cadherin transmembrane and juxta-membrane domains (VE-cad-NcadTMDJMD, N-cad-VEcadTMDJMD). Interestingly, MARCH2 downregulated N-cad-VEcadTMDJMD but not VE-cad-NcadTMDJMD. These results indicate that MARCH proteins exhibit selectivity for different cadherins and that the amino acid sequence within the transmembrane and juxta-membrane domains is essential for this selectivity. These findings suggest that MARCH proteins may differentially regulate cadherins in epithelial and endothelial cells to modulate cell migration and barrier function during development and inflammation.

Functional domain studies uncover novel roles for the ZTL Kelch repeat domain in clock function.

The small LOV/F-box/Kelch family of E3 ubiquitin ligases plays an essential role in the regulation of plant circadian clocks and flowering time by sensing dusk. The family consists of three members, ZEITLUPE (ZTL), LOV KELCH PROTEIN 2 (LKP2), and FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1 (FKF1), which share a unique protein domain architecture allowing them to act as photoreceptors that transduce light signals via altering stability of target proteins. Despite intensive study of this protein family we still lack important knowledge about the biochemical and functional roles of the protein domains that comprise these unique photoreceptors. Here, we perform comparative analyses of transgenic lines constitutively expressing the photoreceptor LOV domain or the Kelch repeat protein-protein interaction domains of ZTL, FKF1, and LKP2. Expression of each domain alone is sufficient to disrupt circadian rhythms and flowering time, but each domain differs in the magnitude of effect. Immunoprecipitation followed by mass spectrometry with the ZTL Kelch repeat domain identified a suite of potential interacting partners. Furthermore, the ZTL Kelch repeat domain can interact with the ZTL homologs, LKP2 and FKF1, and the LOV domain of ZTL itself. This suggests a hypothesis that the Kelch repeat domain of ZTL may mediate inter- and intra-molecular interactions of the three LOV/F-box/Kelch proteins and provides added insight into the composition of the protein complexes and an additional role for the Kelch repeat domain.

Mechanism of Viral Glycoprotein Targeting by Membrane-Associated RING-CH Proteins.

An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP, and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins.IMPORTANCE Viral envelope glycoproteins are an important structural component on the surfaces of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.

The Roles of TRIMs in Antiviral Innate Immune Signaling.

The Tripartite motif (TRIM) protein family, which contains over 80 members in human sapiens, is the largest subfamily of the RING-type E3 ubiquitin ligase family. It is implicated in regulating various cellular functions, including cell cycle process, autophagy, and immune response. The dysfunction of TRIMs may lead to numerous diseases, such as systemic lupus erythematosus (SLE). Lots of studies in recent years have demonstrated that many TRIM proteins exert antiviral roles. TRIM proteins could affect viral replication by regulating the signaling pathways of antiviral innate immune responses. Besides, TRIM proteins can directly target viral components, which can lead to the degradation or functional inhibition of viral protein through degradative or non-degradative mechanisms and consequently interrupt the viral lifecycle. However, new evidence suggests that some viruses may manipulate TRIM proteins for their replication. Here, we summarize the latest discoveries on the interactions between TRIM protein and virus, especially TRIM proteins’ role in the signaling pathway of antiviral innate immune response and the direct “game” between them.

A proteomics approach for the identification of cullin-9 (CUL9) related signaling pathways in induced pluripotent stem cell models.

CUL9 is a non-canonical and poorly characterized member of the largest family of E3 ubiquitin ligases known as the Cullin RING ligases (CRLs). Most CRLs play a critical role in developmental processes, however, the role of CUL9 in neuronal development remains elusive. We determined that deletion or depletion of CUL9 protein causes aberrant formation of neural rosettes, an in vitro model of early neuralization. In this study, we applied mass spectrometric approaches in human pluripotent stem cells (hPSCs) and neural progenitor cells (hNPCs) to identify CUL9 related signaling pathways that may contribute to this phenotype. Through LC-MS/MS analysis of immunoprecipitated endogenous CUL9, we identified several subunits of the APC/C, a major cell cycle regulator, as potential CUL9 interacting proteins. Knockdown of the APC/C adapter protein FZR1 resulted in a significant increase in CUL9 protein levels, however, CUL9 does not appear to affect protein abundance of APC/C subunits and adapters or alter cell cycle progression. Quantitative proteomic analysis of CUL9 KO hPSCs and hNPCs identified protein networks related to metabolic, ubiquitin degradation, and transcriptional regulation pathways that are disrupted by CUL9 deletion in both hPSCs. No significant changes in oxygen consumption rates or ATP production were detected in either cell type. The results of our study build on current evidence that CUL9 may have unique functions in different cell types and that compensatory mechanisms may contribute to the difficulty of identifying CUL9 substrates.

Structural and Mechanistic Insights into TRIM E3 Ubiquitin Ligase Activities

Protein ubiquitination is a post-translational modification process that regulates diverse physiological activities. The Tripartite Motif proteins (TRIM) are a family of E3 ubiquitin ligases that catalyze protein ubiquitination through multi-domain coordination. Structurally, how various domains are orientated to fulfill their E3 ligase activities remains largely enigmatic. Here we solved the crystal structure of the CC domains of TRIM29 and revealed a tetrameric structure formed by two dimers orientated in an anti-parallel fashion, which allows the substrates to directly access the ubiquitin-loaded E2. Similar structure was also observed for TRIM45 and TRIM20. We further demonstrated that the full-length TRIM proteins indeed form tetramers under physiological conditions via the CC domain and this tetrameric structure is required for their E3 activities. Thus, our results provide structural insights into the functional attributes of the TRIM family E3 ligases and offer potential therapeutic strategies in manipulating their functions.

Sirtuin 5 Is Regulated by the SCFCyclin F Ubiquitin Ligase and Is Involved in Cell Cycle Control.

The ubiquitin-proteasome system is essential for cell cycle progression. Cyclin F is a cell cycle-regulated substrate adapter F-box protein for the Skp1, CUL1, and F-box protein (SCF) family of E3 ubiquitin ligases. Despite its importance in cell cycle progression, identifying cyclin F-bound SCF complex (SCFCyclin F) substrates has remained challenging. Since cyclin F overexpression rescues a yeast mutant in the cdc4 gene, we considered the possibility that other genes that genetically modify cdc4 mutant lethality could also encode cyclin F substrates. We identified the mitochondrial and cytosolic deacylating enzyme sirtuin 5 (SIRT5) as a novel cyclin F substrate. SIRT5 has been implicated in metabolic processes, but its connection to the cell cycle is not known. We show that cyclin F interacts with and controls the ubiquitination, abundance, and stability of SIRT5. We show SIRT5 knockout results in a diminished G1 population and a subsequent increase in both S and G2/M. Global proteomic analyses reveal cyclin-dependent kinase (CDK) signaling changes congruent with the cell cycle changes in SIRT5 knockout cells. Together, these data demonstrate that SIRT5 is regulated by cyclin F and suggest a connection between SIRT5, cell cycle regulation, and metabolism.

Targeting USP13-mediated drug tolerance increases the efficacy of EGFR inhibition of mutant EGFR in non-small cell lung cancer.

In non‐small cell lung cancer (NSCLC), activating mutations in the epidermal growth factor receptor (EGFR) induce sensitivity to EGFR tyrosine kinase inhibitors. Despite impressive clinical responses, patients ultimately relapse as a reservoir of drug‐tolerant cells persist, which ultimately leads to acquired resistance mechanisms. We performed an unbiased high‐throughput siRNA screen to identify proteins that abrogate the response of EGFR‐mutant NSCLC to EGFR‐targeted therapy. The deubiquitinase USP13 was a top hit resulting from this screen. Targeting USP13 increases the sensitivity to EGFR inhibition with small molecules in vitro and in vivo. USP13 selectively stabilizes mutant EGFR in a peptidase‐independent manner by counteracting the action of members of the Cbl family of E3 ubiquitin ligases. We conclude that USP13 is a strong mutant EGFR‐specific cotarget that could improve the treatment efficacy of EGFR‐targeted therapies.

Mutational Impact on "in-Between-Ring" (IBR) Domain of PARKIN on Protein Stability and Function.

Mutations in parkin, which is encoded by the PARK2 gene, are associated with a rare form of Parkinson's disease called autosomal recessive juvenile parkinsonism (ARJP). Parkin is a member of RBR family of E3 ubiquitin ligase. Parkin contains a RING1-In-Between-Ring (IBR)-RING2 motif. The IBR domain is located at the C-terminal end of the parkin protein. Two zinc-binding sites are present in the IBR domain which shows zinc ion-dependent folding and maintains the orientation and geometry of RING domains. So, mutation in a zinc-binding region can be responsible for improper folding of parkin protein, which eventually affects the protein structure and function. Abnormalities in parkin protein increase the aggregation of mis-folded proteins in the brain cell. As a consequence, cellular toxicity occurs. The IBR domain also interacts with UbcH7 and UbcH8 proteins belonging to E2 protein family and facilitates synphilin-1, Sept5, and SIM2 protein ubiquitination. It is reported that missense mutation in parkin protein are responsible for autosomal recessive juvenile Parkinson disease. In this work, we first collected the missense mutations in the IBR domain from literature and sequence databases. Then, using various computational tools, we predicted their pathogenicity and involvements in causing possible changes in various protein properties. Evolutionary conservation of amino acids, solvent accessible surface areas, the physico-chemical properties, and changes of protein structure were analyzed. We, for the first time, analyzed the effects of these mutations in parkin to decipher the plausible molecular mechanism of Parkinson's disease.