Degradation Of Protein Substrates Research Articles

TRIM6 promotes glioma malignant progression by enhancing FOXO3A ubiquitination and degradation

TRIM6, an E3 ubiquitin ligase with tripartite motif, directly targets protein substrates for degradation through ubiquitination. Studies have shown that TRIM6 plays a significant role in tumor development in various human malignancies. Thus, the aim of this study was to investigate the importance of TRIM6 and its associated mechanism in promoting the progression of glioma. The expression of TRIM6 and its prognostic value in glioma patients were collected from the TCGA and CGGA databases. The effects of TRIM6 on glioma were investigated in vitro by CCK8, colony formation, wound healing, and transwell assays. Co-IP and western blot analysis were used to detect the interaction between TRIM6 and FOXO3A. The effects of TRIM6 were verified in vivo in subcutaneously xenograft models, and tumor size, and immunohistochemical changes were observed. Our analysis of TRIM6 expression in glioma tissues revealed a high level of expression, and the heightened expression of TRIM6 showed a positive correlation with the unfavorable prognosis among glioma/GBM patients. Through loss-of-function and gain-of-function experiments, we observed a profound impact on the proliferation, invasion, and migration abilities of glioma cells both in vitro and in vivo upon deletion of TRIM6. Conversely, the overexpression of TRIM6 intensified the malignant characteristics of glioma. Additionally, our findings revealed a significant interaction between TRIM6 and FOXO3A, wherein TRIM6 contributed to the destabilization of FOXO3A protein by promoting its ubiquitination and subsequent degradation. Experiments conducted in the rescue study affirmed that the promotion of glioma cell proliferation, invasion, and migration is facilitated by TRIM6 through the suppression of FOXO3A protein levels. These observations imply that the TRIM6-FOXO3A axis could potentially serve as an innovative focus for intervening in glioma.

Development of Peptide Displacement Assays to Screen for Antagonists of DDB1 Interactions.

The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for protein degradation. As an adaptor protein specific to Cullin-RING E3 ligases, DDB1 binds different receptors that poise protein substrates for ubiquitination and subsequent degradation by the 26S proteasome. Examples of DDB1-binding protein receptors are Cereblon (CRBN) and the WD-repeat containing DDB1- and CUL4-associated factors (DCAFs). Cognate substrates of CRBN and DCAFs are involved in cancer-related cellular processes or are mimicked by viruses to reprogram E3 ligases for the ubiquitination of antiviral host factors. Thus, disrupting interactions of DDB1 with receptor proteins might be an effective strategy for anticancer and antiviral drug discovery. Here, we developed fluorescence polarization (FP)-based peptide displacement assays that utilize full-length DDB1 and fluorescein isothiocyanate (FITC)-labeled peptide probes derived from the specific binding motifs of DDB1 interactors. A general FP-based assay condition applicable to diverse peptide probes was determined and optimized. Mutagenesis and biophysical analyses were then employed to identify the most suitable peptide probe. The FITC-DCAF15 L49A peptide binds DDB1 with a dissociation constant of 68 nM and can be displaced competitively by unlabeled peptides at sub-μM to low nM concentrations. These peptide displacement assays can be used to screen small molecule libraries to identify novel modulators that could specifically antagonize DDB1 interactions toward development of antiviral and cancer therapeutics.

RBX1-CRLs Promote Perinatal Heart Development by Regulating Hippo-YAP Signaling

Background: During development, the heart is the first organ to form, ensuring the pumping of blood through the body. Defects in cardiac morphogenesis can lead to congenital heart diseases (CHD) or predispose the heart to cardiomyopathies in adult life. The molecular mechanisms underlying this process remain incompletely understood. Cullin-RING E3 ubiquitin ligases (CRLs) are multi-subunit complexes critical to maintaining protein homeostasis by targeting diverse protein substrates for ubiquitination and subsequent degradation. In CRLs, RBX1 serves as the catalytic subunit that promotes the transfer of ubiquitin to the target protein. Germline ablation of RBX1 in worms, flies, and mice caused embryonic lethality. Deletion of RBX1 in zebrafish resulted in cardiac malformation and dysfunction, which was rescued by endothelial, but not myocardial, RBX1 overexpression. However, the cardiomyocyte-autonomous role of RBX1 in the heart in vertebrates is unknown. Therefore, we hypothesize that cardiomyocyte RBX1 is indispensable for cardiac development. Methods and Results: We generated a cardiomyocyte-specific RBX1 knockout (RBX1CKO) mouse model via αMHCCre. RBX1-deficient mouse embryos were initially morphologically indistinguishable from the littermate controls at E14.5, but began to show peripheral edema and hemorrhage at E16.5, and eventually died around E18.5. Histological analysis of RBX1CKO hearts revealed severe myocardial hypoplasia and bi-ventricular non-compaction by E14.5. Bulk RNA sequencing and proteomic analyses of RBX1CKO hearts identified perturbations in glycolytic to oxidative transition and dysregulation of sarcomeric proteins. EdU incorporation assay showed a significant reduction in cardiomyocyte proliferation in E14.5 RBX1-deficient hearts, accompanied by the impaired nuclear translocation of YAP, a co-transcriptional factor essential for cardiomyocyte proliferation and maturation. Mechanistically, RBX1 deficiency destabilized multiple CRLs and resulted in the accumulation of Hippo kinases and adaptor proteins such as SAV1, LATS1/2, and MOB1, leading to the elevated phosphorylation and inactivation of YAP. We further demonstrated that loss of RBX1 inhibited the degradation of the Hippo adaptor MOB1, likely due to suppressed ubiquitination. Conclusion: Our study identifies RBX1 as an essential regulator of ventricular maturation and found that RBX1-mediated proteolysis of Hippo kinases serves as a critical checkpoint to ensure YAP activation and, hence, cardiomyocyte proliferation and maturation during cardiac development. AHA Predoctoral Fellowship, American Heart Association, 23PRE1012641. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mezigdomide-A Novel Cereblon E3 Ligase Modulator under Investigation in Relapsed/Refractory Multiple Myeloma.

Mezigomide is an oral cereblon E3 ligase modulator (CELMoD) that is under clinical investigation in patients with relapsed/refractory (RR) multiple myeloma (MM). Like other CELMoD compounds, mezigdomide acts by altering the conformation of cereblon within the cullin 4A ring ligase-cereblon (CRL4CRBN) E3 ubiquitin ligase complex, thereby recruiting novel protein substrates for selective proteasomal degradation. These include two critical lymphoid transcription factors, Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3), also known as Ikaros and Aiolos, which have important roles in the development and differentiation of hematopoietic cells, in MM pathobiology, and in suppressing the expression of interferon-stimulating genes and T-cell stimulation. Among the CELMoDs, mezigdomide has the greatest cereblon-binding potency, plus the greatest potency for the degradation of Ikaros and Aiolos and subsequent downstream antimyeloma effects. Preclinical studies of mezigdomide have demonstrated its anti-proliferative and apoptotic effects in MM, along with its immune-stimulatory effects and its synergistic activity with other antimyeloma agents, including in lenalidomide-/pomalidomide-resistant MM cell lines and mouse xenograft models. Early-phase clinical trial data indicate notable activity in heavily pretreated patients with RRMM, including those with triple-class-refractory disease, together with a tolerable and manageable safety profile. This review summarizes current preclinical and clinical findings with mezigdomide and its potential future roles in the treatment of MM.

Abstract 13987: RBX1-CRLs Are Essential for Perinatal Heart Development by Regulating Hippo-YAP Signaling

Defects in cardiac development can lead to congenital heart diseases (CHD) or predispose the heart to cardiomyopathies in adult life. The molecular mechanisms that regulate this process remain elusive. Cullin-RING E3 ligases (CRLs) are multi-subunit complexes that are critical to maintain protein homeostasis by targeting a variety of protein substrates for proteasomal degradation. In the CRLs, RBX1 serves as the catalytic subunit that promotes the transfer of ubiquitin to the target protein. Previous animal studies uncovered a crucial role for RBX1 in development. However, its importance in the developing heart has not been explored. Here, we report for the first time the indispensable role of RBX1 in perinatal cardiac development. We created mice with cardiac-specific deletion of RBX1 driven by αMHC Cre . Compared with littermate control mice, RBX1-deficient mouse embryos exhibited no gross morphological abnormalities at E12.5 and E14.5, displayed peripheral edema at E16.5 indicative of heart failure, and eventually died around E18.5. Histological analysis revealed myocardial hypoplasia and a substantially thinner compact layer in E16.5 mutant hearts. This event was preceded by a remarkable decrease in cardiomyocyte proliferation and diminished nuclear localization of YAP, a powerful regulator of cardiomyocyte proliferation, at E14.5. In cultured cardiomyocytes, depletion of RBX1 inhibited proliferation and induced profound transcriptomic changes like the upregulation of cardiomyopathy-related genes. Mechanistically, RBX1 deficiency destabilized multiple CRLs and resulted in the accumulation of Hippo kinases and adaptor proteins such as SAV1, LATS1/2, and MOB1 leading to the inactivation of YAP by phosphorylation. We further demonstrated that RBX1 degrades the Hippo adaptors SAV1 and MOB1. Collectively, we propose that the fine-tuned, RBX1-mediated removal of Hippo components serves as a critical checkpoint to activate YAP signaling ensuring cardiac ventricular chamber development and morphogenesis.

High selectivity of the hyperthermophilic subtilase propeptide domain toward inhibition of its cognate protease.

Microbial extracellular subtilases are highly active proteolytic enzymes commonly used in commercial applications. These subtilases are synthesized in their inactive proform, which matures into the active protease under the control of the propeptide domain. In mesophilic bacterial prosubtilases, the propeptide functions as both an obligatory chaperone and an inhibitor of the subtilase catalytic domain. In contrast, the propeptides of hyperthermophilic archaeal prosubtilases act mainly as tight inhibitors and are not essential for subtilase folding. It is unclear whether this stronger inhibitory activity of hyperthermophilic propeptides results in their higher selectivity toward their cognate subtilases, in contrast to promiscuous mesophilic propeptides. Here, we showed that the propeptide of pernisine, a hyperthermostable archaeal subtilase, strongly interacts with and inhibits pernisine, but not the homologous subtilisin Carlsberg and proteinase K. Instead, the pernisine propeptide was readily degraded by subtilisin Carlsberg and proteinase K. In addition, the catalytic domain of unprocessed propernisine was also susceptible to degradation but became proteolytically stable after autoprocessing of propernisine into the inactive, noncovalent complex propeptide:pernisine. This allowed efficient transactivation of the autoprocessed complex propeptide:pernisine through degradation of pernisine propeptide by subtilisin Carlsberg and proteinase K at mesophilic temperature. Moreover, we demonstrated that active pernisine molecules are inhibited by the propeptide that is released after pernisine-catalyzed degradation of the unprocessed propernisine catalytic domain. This highlights the high inhibitory potency of the hyperthermophilic propeptide toward its cognate subtilase and its importance in regulating subtilase maturation, to prevent the degradation of the unprocessed subtilase precursors by the prematurely activated molecules. IMPORTANCE Many microorganisms secrete proteases into their environment to degrade protein substrates for their growth. The important group of these extracellular enzymes are subtilases, which are also widely used in practical applications. These subtilases are inhibited by their propeptide domain, which is degraded during the prosubtilase maturation process. Here, we showed that the propeptide of pernisine, a prion-degrading subtilase from the hyperthermophilic archaeon, strongly inhibits pernisine with extraordinarily high binding affinity. This interaction proved to be highly selective, as pernisine propeptide was rapidly degraded by mesophilic pernisine homologs. This in turn allowed rapid transactivation of propernisine by mesophilic subtilases at lower temperatures, which might simplify the procedures for preparation of active pernisine for commercial use. The results reported in this study suggest that the hyperthermophilic subtilase propeptide evolved to function as tight and selective regulator of maturation of the associated prosubtilase to prevent its premature activation under high temperatures.

The role of the ubiquitin ligase Cul4b in B lymphocytes

Abstract Vaccination or infection induces antibody secretion by long-lived plasma cells, creating durable immune protection against secondary infections. These plasma cells develop from B cells that have undergone affinity maturation, which requires somatic hypermutation in proliferating B cells, forming single strand DNA breaks that are repaired by mismatch repair machinery. Cullin 4b (Cul4b) is an E3 ubiquitin ligase found in most cells, including B cells, but its function in B cells is unknown. Cul4b is one of two docking proteins forming the backbone of the Cullin RING Ligase 4 (CRL4) complex, which ubiquitinates substrate proteins for degradation. Previous studies have shown that Cul4b promotes DNA repair with cell proliferation, and high Cul4b is linked to aggressive colon cancer. Because proliferation and DNA repair are essential for humoral immunity, we hypothesized that Cul4b also functions in B cells. To test this, we generated mice that lack Cul4b in B cells. We immunized these mice with an mRNA vaccine against an H1N1 Influenza A virus, and measured virus-specific serum antibody. We found that Influenza-specific antibodies were dramatically reduced in mice serum that lacked Cul4b in B cells. To determine if Cul4b promotes DNA damage repair in B cells, we induced single-strand DNA breaks with UV irradiation and then stimulated the cells to proliferate in vitro. We found that Cul4b promoted B lymphocyte proliferation after UV exposure. Understanding the Cul4b-DNA damage repair pathway in B cells will identify new drug targets to transiently amplify this pathway to improve vaccines and encourage stronger antibody responses, or to inhibit this pathway to prevent tumor progression in cancer.

Pharmacologic targeting of Nedd8-activating enzyme reinvigorates T-cell responses in lymphoid neoplasia

Neddylation is a sequential enzyme-based process which regulates the function of E3 Cullin-RING ligase (CRL) and thus degradation of substrate proteins. Here we show that CD8+ T cells are a direct target for therapeutically relevant anti-lymphoma activity of pevonedistat, a Nedd8-activating enzyme (NAE) inhibitor. Pevonedistat-treated patient-derived CD8+ T cells upregulated TNFα and IFNγ and exhibited enhanced cytotoxicity. Pevonedistat induced CD8+ T-cell inflamed microenvironment and delayed tumor progression in A20 syngeneic lymphoma model. This anti-tumor effect lessened when CD8+ T cells lost the ability to engage tumors through MHC class I interactions, achieved either through CD8+ T-cell depletion or genetic knockout of B2M. Meanwhile, loss of UBE2M in tumor did not alter efficacy of pevonedistat. Concurrent blockade of NAE and PD-1 led to enhanced tumor immune infiltration, T-cell activation and chemokine expression and synergistically restricted tumor growth. shRNA-mediated knockdown of HIF-1α, a CRL substrate, abrogated the in vitro effects of pevonedistat, suggesting that NAE inhibition modulates T-cell function in HIF-1α-dependent manner. scRNA-Seq-based clinical analyses in lymphoma patients receiving pevonedistat therapy demonstrated upregulation of interferon response signatures in immune cells. Thus, targeting NAE enhances the inflammatory T-cell state, providing rationale for checkpoint blockade-based combination therapy.

Abstract 4783: ADO is essential for redox homeostasis in liver cancer

Abstract 2-Aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that plays a direct role in both metabolism and protein stability. Oxidation of the metabolite cysteamine produces hypotaurine, while oxidation of N-terminal cysteines targets protein substrates for N-degron pathway-mediated degradation. Despite these known functions, the (patho)physiological roles of ADO remain obscure. By analyzing TCGA datasets we discovered that high ADO expression is associated with poor outcome for patients with hepatocellular carcinoma (HCC) (HR 1.2, p<0.001). HCC is linked to viral hepatitis, alcohol, metabolic syndrome, non-alcoholic fatty liver disease and chronic inflammation. It is the third leading cause of cancer-related deaths worldwide and is significantly more common in males than females. With poor outcomes, there is a need to better understand HCC biology and vulnerabilities. To assess the functional roles of ADO, we created ADO knockout mice as well as two liver cancer cell line models where ADO was depleted by siRNA, doxycycline-inducible shRNA, or CRISPR/Cas9. While male ADO knockout mice were viable, fertile and healthy, depletion of ADO in liver cancer models significantly reduced cancer cell proliferation and colony formation. ADO was also essential for supporting xenograft growth when implanted subcutaneously in immune-compromised mice. Taken together, this suggested that ADO depletion represents a cancer-specific vulnerability. Comprehensive metabolomic, transcriptomic and proteomic characterization of HCC cells isogenic for ADO demonstrated that loss of ADO resulted in dysregulation of glutathione, ascorbate, polyamine and proline metabolism. Consistent with this, ADO depleted cells had high levels of reactive oxygen species (ROS) measured by CellROX flowcytometry, and an increased ratio of oxidized to reduced glutathione, indicative of oxidative stress. Since mitochondria represent a substantial source of ROS, we assessed mitochondrial mass and function using MitotrackerTM flow cytometry and Seahorse stress tests, respectively. ADO depleted cancer cells had higher mitochondrial mass and higher maximal respiration rates compared to control cells. Finally, exogenously supplied antioxidants could rescue the survival of HCC cells with ADO depletion, demonstrating that the observed loss of viability is due to oxidative stress. This work shows that ADO is essential for redox homeostasis in liver cancer models and suggests that interfering with ADO function may represent a novel targeting strategy for HCC. Citation Format: Sandy C-E Lee, Andrea H. Pyo, Helia Mohammadi, Ji Zhang, Anna Dvorkin-Gheva, Lucie Malbeteau, Stephen Chung, Shahbaz Khan, Thomas Kislinger, Julie A. Reisz, Courtney Jones, Marianne Koritzinsky. ADO is essential for redox homeostasis in liver cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4783.

The roles of E3 ubiquitin ligases in cancer progression and targeted therapy.

Ubiquitination is one of the most important post-translational modifications which plays a significant role in conserving the homeostasis of cellular proteins. In the ubiquitination process, ubiquitin is conjugated to target protein substrates for degradation, translocation or activation, dysregulation of whichis linked to several diseases including various types of cancers. E3 ubiquitinligases are regarded as the most influential ubiquitin enzyme owing to their ability to select, bind and recruit target substrates for ubiquitination. In particular, E3 ligases are pivotal in the cancer hallmarks pathways where they serve as tumour promoters or suppressors. The specificity of E3 ligases coupled with their implication in cancer hallmarks engendered the development of compounds that specifically target E3 ligases for cancer therapy. In this review, we highlight the role of E3 ligases in cancer hallmarks such as sustained proliferation via cell cycle progression, immune evasion and tumour promoting inflammation, and in the evasion of apoptosis. In addition, we summarise the application and the role of small compounds that target E3 ligases for cancer treatment along with the significance of targeting E3 ligases as potential cancer therapy.

The activated ClpP peptidase forcefully grips a protein substrate

ATPases associated with diverse cellular activities (AAA+) proteases power the maintenance of protein homeostasis by coupling ATP hydrolysis to mechanical protein unfolding, translocation, and ultimately degradation. Although ATPase activity drives a large portion of the mechanical work these molecular machines perform, how the peptidase contributes to the forceful denaturation and processive threading of substrates remains unknown. Here, using single-molecule optical trapping, we examine the mechanical activity of the caseinolytic peptidase P (ClpP) from Escherichia coli in the absence of a partner ATPase and in the presence of an activating small-molecule acyldepsipeptide. We demonstrate that ClpP grips protein substrate under mechanical loads exceeding 40 pN, which are greater than those observed for the AAA+ unfoldase ClpX and the AAA+ protease complexes ClpXP and ClpAP. We further characterize substrate-ClpP bond lifetimes and rupture forces under varying loads. We find that the resulting slip bond behavior does not depend on ClpP peptidase activity. In addition, we find that unloaded bond lifetimes between ClpP and protein substrate are on a timescale relevant to unfolding times (up to ∼160 s) for difficult to unfold model substrate proteins. These direct measurements of the substrate-peptidase bond under load define key properties required by AAA+ proteases to mechanically unfold and degrade protein substrates.

TDP-43 is a ubiquitylation substrate of the SCFcyclin F complex

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of upper and lower motor neurons in the brain and spinal cord. ALS and frontotemporal dementia (FTD) are overlapping diseases with shared pathological features. Affected neurons of people with ALS and FTD typically contain ubiquitin-immunoreactive inclusions, of which TDP-43 (Tar DNA-binding protein of 43 kDa) is a major component. However, what triggers the formation of these abnormal TDP-43 inclusions is unclear. Previously, we identified CCNF mutations in cohorts of familial and sporadic cases of ALS and FTD. CCNF encodes cyclin F, the substrate-binding component of a multiprotein E3 ubiquitin ligase complex that ubiquitylates and subsequently directs a set of protein substrates for proteasomal degradation. Here, we explored the relationship between cyclin F and TDP-43. MethodsWe used a series of complementary biochemical approaches including immunoprecipitations, in vitro ubiquitylation assays, immunofluorescence imaging and immunocytochemistry. Unpaired student t-tests were used to determine statistical significance of the results. ResultsIn this study, we demonstrate that that the SCFcyclin F complex directly mediates the poly-ubiquitylation of TDP-43. Importantly, we demonstrate that cyclin F bearing the pathogenic ALS/FTD mutation, S621G, leads to aberrant ubiquitylation of TDP-43 as well as the accumulation of K48-ubiquitylated TDP-43 in neuron-like cells. Furthermore, we demonstrate that a patient carrying the ALS/FTD cyclin FS195R mutation displayed skein-like cytoplasmic TDP-43 aggregates, implying abnormal TDP-43 degradation in a CCNF mutation bearing patient. ConclusionIn summary, this study reports a direct ubiquitylation mechanism for TDP-43, revealing important insights into the regulation of cyclin F-mediated TDP-43 turnover and clues towards understanding the molecular origins of the ubiquitylated TDP-43 inclusions that are the hallmark pathological feature in ALS and FTD.

Processive cleavage of substrate at individual proteolytic active sites of the Lon protease complex.

The Lon protease is the prototype of a family of proteolytic machines with adenosine triphosphatase modules built into a substrate degradation chamber. Lon is known to degrade protein substrates in a processive fashion, cutting a protein chain processively into small peptides before commencing cleavages of another protein chain. Here, we present structural and biochemical evidence demonstrating that processive substrate degradation occurs at each of the six proteolytic active sites of Lon, which forms a deep groove that partially encloses the substrate polypeptide chain by accommodating only the unprimed residues and permits processive cleavage in the C-to-N direction. We identify a universally conserved acidic residue at the exit side of the binding groove indispensable for the proteolytic activity. This noncatalytic residue likely promotes processive proteolysis by carboxyl-carboxylate interactions with cleaved intermediates. Together, these results uncover a previously unrecognized mechanism for processive substrate degradation by the Lon protease.

UBE3A Enzyme Hyperactivity as a Driver of Neurodevelopmental Disease

UBE3A is a HECT (homologous to E6AP C-terminus) domain E3 ubiquitin ligase that targets substrate proteins for degradation through the ubiquitin-proteasome pathway. Deletion or null mutation of the maternal UBE3A gene causes Angelman syndrome, a severe intellectual disability disorder also characterized by epilepsy, motor deficits, and an unusually happy disposition. Hundreds of single amino acid variants of UBE3A have been reported in the literature, but the biological significance for most these variants are unknown. Here, we devised a high-throughput assay to screen the functional consequence of UBE3A missense variants to identify precise mutations that drive disease. We screened over 150 UBE3A variants and identified distinct functional classes of UBE3A mutants, including over a dozen gain-of-function variants previously not known to exist. Mice carrying a specific hyperactivating mutation in UBE3A exhibited aberrant motor and communication deficits, and analysis of patient data showed that UBE3A hyperactivation causes a neurodevelopmental disorder that is distinct from Angelman syndrome. Finally, our detailed structure-function analysis revealed a novel allosteric regulatory site in UBE3A that is perturbed by both gain and loss-of-function mutations. Altogether, our study demonstrates that functional variant analysis can delineate disease subtypes and provide deep structure-functional data to uncover disease-relevant regulatory mechanisms.

Switching DCAFs: Beyond substrate receptors.

Deciphering how DCAFs (DDB1-CUL4 Associated Factors) modulate a broad spectrum of cellular processes, including cell cycle progression and maintenance of genomic integrity is critical to better understand cellular homeostasis and diseases. Cells contain more than 100 DCAFs that associate with the Cullin-Ring Ubiquitin Ligase 4 (CRL4) complex that target specific protein substrates for degradation. DCAFs are thought to act as substrate receptors that dictate the specificity of the ubiquitination machinery ("catalytic DCAFs"). However, recent studies have suggested that some DCAFs might play a different role by targeting CRL4 complexes to distinct cellular compartments ("structural DCAFs"). Once localized to their correct cellular domains, these CRLs dissociate from the structural DCAFs prior to their association with other, substrate-specific catalytic DCAFs. Thus, we propose that DCAF switches can provide a mechanistic basis for the degradation of proteins that regulate cell growth and proliferation at precise points in space and time.

A pH-Dependent Conformational Switch Controls N. meningitidis ClpP Protease Function.

ClpPs are a conserved family of serine proteases that collaborate with ATP-dependent translocases to degrade protein substrates. Drugs targeting these enzymes have attracted interest for the treatment of cancer and bacterial infections due to their critical role in mitochondrial and bacterial proteostasis, respectively. As such, there is significant interest in understanding structure-function relationships in this protein family. ClpPs are known to crystallize in extended, compact, and compressed forms; however, it is unclear what conditions favor the formation of each form and whether they are populated by wild-type enzymes in solution. Here, we use cryo-EM and solution NMR spectroscopy to demonstrate that a pH-dependent conformational switch controls an equilibrium between the active extended and inactive compressed forms of ClpP from the Gram-negative pathogen Neisseria meningitidis. Our findings provide insight into how ClpPs exploit their rugged energy landscapes to enable key conformational changes that regulate their function.

Kelch‐like Proteins Have A Sweet Spot: Site‐specific Glycosylation Influences Metabolic Regulation and Protein Homeostasis

The Kelch‐like (KLHL) family of proteins, a conserved group of 42 human members, binds to E3 ubiquitin ligase complexes to target protein substrates for proteasomal degradation. Although mutations in KLHL genes cause various human diseases—from cancer to neurodegeneration—their functional regulation remains understudied. Post‐translational modifications in general influence protein fates, yet how they modulate KLHL proteins is not fully explored. In this context, our laboratory has examined O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc), a nutrient‐sensing, intracellular glycosylation governed by O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) that decorates serines (S) or threonine (T) residues in response to cellular stimuli. Using global transcriptional responses to OGT or OGA inhibition and chemical biology approaches, we previously reported that O‐GlcNAcylation of KEAP1, a KLHL protein, is required for the ubiquitination and regulation of the transcription factor NRF2 and the cellular response to oxidative stress. Given the functional conservation of the KLHL family, we hypothesized similar roles for O‐GlcNAcylation in other KLHL proteins, such as gigaxonin that promotes the degradation of intermediate filament (IF) proteins. Human loss‐of‐function gigaxonin mutations are known to cause IF accumulation in neurons, resulting in neuronal swelling and ultimately giant axonal neuropathy, a fatal neurodegenerative disease. By mass spectrometry, immunofluorescence and co‐immunoprecipitation assays, we showed that gigaxonin glycosylation sites at residues S272 and T277 are important for clearing IF aggregates in gigaxonin−/− cells. Taken together, our findings implicate O‐GlcNAc modification of KLHL proteins in governing protein homeostasis of E3 ligase substrates, suggest O‐GlcNAcylation as a general mode of regulation for other KLHL proteins, and inspire future tool developments to detect site‐specific glycosylation changes in human health and disease.Support or Funding InformationResearch on this project in the Boyce Lab is supported by NIH grants R01GM118847 and R01NS111588 and a gift from the Hannah’s Hope Foundation

Dynamic Interaction of USP14 with the Chaperone HSC70 Mediates Crosstalk between the Proteasome, ER Signaling, and Autophagy.

SummaryUSP14 is a deubiquitinating enzyme associated with the proteasome important for protein degradation. Here we show that upon proteasome inhibition or expression of the mutant W58A-USP14, association of USP14 with the 19S regulatory particle is disrupted. MS-based interactomics revealed an interaction of USP14 with the chaperone, HSC70, in neuroblastoma cells. Proteasome inhibition enhanced binding of USP14 to HSC70, and to XBP1u and IRE1α proteins, demonstrating a role in the unfolded protein response. Striatal neurons expressing mutant huntingtin exhibited reduced USP14 and HSC70 levels, whereas inhibition of HSC70 downregulated USP14. Furthermore, proteasome inhibition or use of the mutant W58A-USP14 facilitated the interaction of USP14 with the autophagy protein, GABARAP. Functionally, overexpression of W58A-USP14 increased GABARAP positive autophagosomes in striatal neurons, and this was abrogated using the HSC70 inhibitor, VER-155008. Modulation of the USP14-HSC70 axis may represent a potential therapeutic target in HD to beneficially influence multiple proteostasis pathways.

Inhibition of Ubiquitin Receptor PSMD4/Rpn10 as Therapeutic Strategy to Overcomes Bortezomib-Resistance in Multiple Myeloma cells

and Rationale Proteasome inhibitors (PIs) represent major advance in the treatment of relapsed/refractory and newly diagnosed multiple myeloma (MM); however, PI-therapy is associated with adverse effects and the development of drug-resistance. Strategies to overcome PI-resistance include targeting ubiquitin proteasome system (UPS) upstream of 20S proteasome subunit. One such exemplary target is ubiquitin receptor PSMD4/Rpn10, which is localized on 19S regulatory lid of proteasome, and play a key role in recognizing ubiquitylated substrate proteins for degradation via downstream 20S proteasome.

URM1-Mediated Ubiquitin-Like Modification Is Required for Oxidative Stress Adaptation During Infection of the Rice Blast Fungus.

Ubiquitin is a small modifier protein which is usually conjugated to substrate proteins for degradation. In recent years, a number of ubiquitin-like proteins have been identified; however, their roles in eukaryotes are largely unknown. Here, we describe a ubiquitin-like protein URM1, and found it plays important roles in the development and infection process of the rice blast fungus, Magnaporthe oryzae. Targeted deletion of URM1 in M. oryzae resulted in slight reduction in vegetative growth and significant decrease in conidiation. More importantly, the Δurm1 mutant also showed evident reduction in virulence to host plants. Infection process observation demonstrated that the mutant was arrested in invasive growth and resulted in accumulation of massive host reactive oxygen species (ROS). Further, we found the Δurm1 mutant was sensitive to the cell wall disturbing reagents, thiol oxidizing agent diamide and rapamycin. We also showed that URM1-mediated modification was responsive to oxidative stresses, and the thioredoxin peroxidase Ahp1 was one of the important urmylation targets. These results suggested that URM1-mediated urmylation plays important roles in detoxification of host oxidative stress to facilitate invasive growth in M. oryzae.