TRIM7 ubiquitinates SARS-CoV-2 membrane protein to limit apoptosis and viral replication.

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7 -/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M-K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.

Ubiquitin system mutations in neurological diseases.

Neuronal ubiquitin balance impacts the fate of countless cellular proteins, and its disruption is associated with various neurological disorders. The ubiquitin system is critical for proper neuronal cell state transitions and the clearance of misfolded or aggregated proteins that threaten cellular integrity. This article reviews the state of and recent advancements in our understanding of the disruptions to components of the ubiquitin system, in particular E3 ligases and deubiquitylases, in neurodevelopmental and neurodegenerative diseases. Specific focus is on enzymes with recent progress in their characterization, including identifying enzyme-substrate pairs, the use of stem cell and animal models, and the development of therapeutics for ubiquitin-related diseases.

Stabilization of GTSE1 by cyclin D1-CDK4/6 promotes cell proliferation: relevance in cancer prognosis.

Cyclin D1 is the activating subunit of the cell cycle kinases CDK4 and CDK6, and its dysregulation is a well-known oncogenic driver in many human cancers. The biological function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G2 and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unknown substrate of cyclin D1-CDK4/6. The phosphorylation of GTSE1 mediated by cyclin D1-CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 also during the G1 phase of the cell cycle. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1's role in cell cycle control and oncogenesis beyond RB phosphorylation.

PD-1/LAG-3 co-signaling profiling uncovers CBL ubiquitin ligases as key immunotherapy targets.

Many cancer patients do not benefit from PD-L1/PD-1 blockade immunotherapies. PD-1 and LAG-3 co-upregulation in T-cells is one of the major mechanisms of resistance by establishing a highly dysfunctional state in T-cells. To identify shared features associated to PD-1/LAG-3 dysfunctionality in human cancers and T-cells, multiomic expression profiles were obtained for all TCGA cancers immune infiltrates. A PD-1/LAG-3 dysfunctional signature was found which regulated immune, metabolic, genetic, and epigenetic pathways, but especially a reinforced negative regulation of the TCR signalosome. These results were validated in T-cell lines with constitutively active PD-1, LAG-3 pathways and their combination. A differential analysis of the proteome of PD-1/LAG-3 T-cells showed a specific enrichment in ubiquitin ligases participating in E3 ubiquitination pathways. PD-1/LAG-3 co-blockade inhibited CBL-B expression, while the use of a bispecific drug in clinical development also repressed C-CBL expression, which reverted T-cell dysfunctionality in lung cancer patients resistant to PD-L1/PD-1 blockade. The combination of CBL-B-specific small molecule inhibitors with anti-PD-1/anti-LAG-3 immunotherapies demonstrated notable therapeutic efficacy in models of lung cancer refractory to immunotherapies, overcoming PD-1/LAG-3 mediated resistance.

An E3 Ubiquitin Ligase Localization Screen Uncovers DTX2 as a Novel ADP-Ribosylation-Dependent Regulator of DNA Double-Strand Break Repair.

DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: non-homologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response (DDR) relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DDR regulation remains incomplete. Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Amongst these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DTC domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 re-expression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.

Role of MARCH E3 ubiquitin ligases in cancer development.

Membrane-associated RING-CH (MARCH) E3 ubiquitin ligases, a family of RING-type E3 ubiquitin ligases, have garnered increased attention for their indispensable roles in immune regulation, inflammation, mitochondrial dynamics, and lipid metabolism. The MARCH E3 ligase family consists of eleven distinct members, and the dysregulation of many of these members has been documented in several human malignancies. Over the past two decades, extensive research has revealed that MARCH E3 ligases play pivotal roles in cancer progression by ubiquitinating key oncogenes and tumor suppressors and orchestrating various signaling pathways. Some MARCH E3s act as oncogenes, while others act as tumor suppressors, and the majority of MARCH E3s play both oncogenic and tumor suppressive roles in a context-dependent manner. Notably, there is special emphasis on the sole mitochondrial MARCH E3 ligase MARCH5, which regulates mitochondrial homeostasis within cancer cells. In this review, we delve into the diverse functions of MARCH E3 ligases across different cancer types, shedding light on the underlying molecular mechanisms mediating their effects, their regulatory effects on cancer and their potential as therapeutic targets.

Ubiquitination in viral entry and replication: Mechanisms and implications.

The ubiquitination process is a reversible posttranslational modification involved in many essential cellular functions, such as innate immunity, cell signaling, trafficking, protein stability, and protein degradation. Viruses can use the ubiquitin system to efficiently enter host cells, replicate and evade host immunity, ultimately enhancing viral pathogenesis. Emerging evidence indicates that enveloped viruses can carry free (unanchored) ubiquitin or covalently ubiquitinated viral structural proteins that can increase the efficiency of viral entry into host cells. Furthermore, viruses continuously evolve and adapt to take advantage of the host ubiquitin machinery, highlighting its importance during virus infection. This review discusses the battle between viruses and hosts, focusing on how viruses hijack the ubiquitination process at different steps of the replication cycle, with a specific emphasis on viral entry. We discuss how ubiquitination of viral proteins may affect tropism and explore emerging therapeutics strategies targeting the ubiquitin system for antiviral drug discovery.

GhATL68b regulates cotton fiber cell development by ubiquitinating the enzyme required for ß-oxidation of polyunsaturated fatty acids.

E3 ligases are key enzymes required for protein degradation. Here, we identified a C3H2C3 RING domain-containing E3 ubiquitin ligase gene named GhATL68b. It is preferentially and highly expressed in developing cotton fiber cells and shows greater conservation in plants than in animals or archaea. The four orthologous copies of this gene in various diploid cottons and eight in the allotetraploid G. hirsutum were found to have originated from a single common ancestor that can be traced back to Chlamydomonas reinhardtii at about 992 million years ago. Structural variations in the GhATL68b promoter regions of G. hirsutum, G. herbaceum, G. arboreum, and G. raimondii are correlated with significantly different methylation patterns. Homozygous CRISPR-Cas9 knockout cotton lines exhibit significant reductions in fiber quality traits, including upper-half mean length, elongation at break, uniformity, and mature fiber weight. In vitro ubiquitination and cell-free protein degradation assays revealed that GhATL68b modulates the homeostasis of 2,4-dienoyl-CoA reductase, a rate-limiting enzyme for the ß-oxidation of polyunsaturated fatty acids (PUFAs), via the ubiquitin proteasome pathway. Fiber cells harvested from these knockout mutants contain significantly lower levels of PUFAs important for production of glycerophospholipids and regulation of plasma membrane fluidity. The fiber growth defects of the mutant can be fully rescued by the addition of linolenic acid (C18:3), the most abundant type of PUFA, to the ovule culture medium. This experimentally characterized C3H2C3 type E3 ubiquitin ligase involved in regulating fiber cell elongation may provide us with a new genetic target for improved cotton lint production.

E3 ligases and DUBs target ferroptosis: A potential therapeutic strategy for neurodegenerative diseases.

Ferroptosis is a form of cell death mediated by iron and lipid peroxidation (LPO). Recent studies have provided compelling evidence to support the involvement of ferroptosis in the pathogenesis of various neurodegenerative diseases (NDDs), such as Alzheimer's disease (AD), Parkinson's disease (PD). Therefore, understanding the mechanisms that regulate ferroptosis in NDDs may improve disease management. Ferroptosis is regulated by multiple mechanisms, and different degradation pathways, including autophagy and the ubiquitinproteasome system (UPS), orchestrate the complex ferroptosis response by directly or indirectly regulating iron accumulation or lipid peroxidation. Ubiquitination plays a crucial role as a protein posttranslational modification in driving ferroptosis. Notably, E3 ubiquitin ligases (E3s) and deubiquitinating enzymes (DUBs) are key enzymes in the ubiquitin system, and their dysregulation is closely linked to the progression of NDDs. A growing body of evidence highlights the role of ubiquitin system enzymes in regulating ferroptosis sensitivity. However, reports on the interaction between ferroptosis and ubiquitin signaling in NDDs are scarce. In this review, we first provide a brief overview of the biological processes and roles of the UPS, summarize the core molecular mechanisms and potential biological functions of ferroptosis, and explore the pathophysiological relevance and therapeutic implications of ferroptosis in NDDs. In addition, reviewing the roles of E3s and DUBs in regulating ferroptosis in NDDs aims to provide new insights and strategies for the treatment of NDDs. These include E3- and DUB-targeted drugs and ferroptosis inhibitors, which can be used to prevent and ameliorate the progression of NDDs.

Phosphonoalamides Reveal the Biosynthetic Origin of Phosphonoalanine Natural Products and a Convergent Pathway for Their Diversification.

Phosphonate natural products, with their potent inhibitory activity, have found widespread use across multiple industries. Their success has inspired development of genome mining approaches that continue to reveal previously unknown bioactive scaffolds and biosynthetic insights. However, a greater understanding of phosphonate metabolism is required to enable prediction of compounds and their bioactivities from sequence information alone. Here, we expand our knowledge of this natural product class by reporting the complete biosynthesis of the phosphonoalamides, antimicrobial tripeptides with a conserved N-terminal l-phosphonoalanine (PnAla) residue produced by Streptomyces. The phosphonoalamides result from the convergence of PnAla biosynthesis and peptide ligation pathways. We elucidate the biochemistry underlying the transamination of phosphonopyruvate to PnAla, a new early branchpoint in phosphonate biosynthesis catalyzed by an aminotransferase with evolved specificity for phosphonate metabolism. Peptide formation is catalyzed by two ATP-grasp ligases, the first of which produces dipeptides, and a second which ligates dipeptides to PnAla to produce phosphonoalamides. Substrate specificity profiling revealed a dramatic expansion of dipeptide and tripeptide products, while finding PnaC to be the most promiscuous dipeptide ligase reported thus far. Our findings highlight previously unknown transformations in natural product biosynthesis, promising enzyme biocatalysts, and unveil insights into the diversity of phosphonopeptide natural products.

NEDD4 and NEDD4L: Ubiquitin Ligases Closely Related to Digestive Diseases.

Protein ubiquitination is an enzymatic cascade reaction and serves as an important protein post-translational modification (PTM) that is involved in the vast majority of cellular life activities. The key enzyme in the ubiquitination process is E3 ubiquitin ligase (E3), which catalyzes the binding of ubiquitin (Ub) to the protein substrate and influences substrate specificity. In recent years, the relationship between the subfamily of neuron-expressed developmental downregulation 4 (NEDD4), which belongs to the E3 ligase system, and digestive diseases has drawn widespread attention. Numerous studies have shown that NEDD4 and NEDD4L of the NEDD4 family can regulate the digestive function, as well as a series of related physiological and pathological processes, by controlling the subsequent degradation of proteins such as PTEN, c-Myc, and P21, along with substrate ubiquitination. In this article, we reviewed the appropriate functions of NEDD4 and NEDD4L in digestive diseases including cell proliferation, invasion, metastasis, chemotherapeutic drug resistance, and multiple signaling pathways, based on the currently available research evidence for the purpose of providing new ideas for the prevention and treatment of digestive diseases.

The ubiquitin-proteasome system in the regulation of tumor dormancy and recurrence.

Tumor recurrence is a mechanism triggered in sparse populations of cancer cells that usually remain in a quiescent state after strict stress and/or therapeutic factors, which is affected by a variety of autocrine and microenvironmental cues. Despite thorough investigations, the biology of dormant and/or cancer stem cells is still not fully elucidated, as for the mechanisms of their reawakening, while only the major molecular patterns driving the relapse process have been identified to date. These molecular patterns profoundly interfere with the elements of cellular proteostasis systems that support the efficiency of the recurrence process. As a major proteostasis machinery, we review the role of the ubiquitin-proteasome system (UPS) in tumor cell dormancy and reawakening, devoting particular attention to the functions of its components, E3 ligases, deubiquitinating enzymes and proteasomes in cancer recurrence. We demonstrate how UPS components functionally or mechanistically interact with the pivotal proteins implicated in the recurrence program and reveal that modulators of the UPS hold promise to become an efficient adjuvant therapy for eradicating refractory tumor cells to impede tumor relapse.

Ring finger protein 138 inhibits transcription factor C/EBPα protein turnover leading to differentiation arrest in acute myeloid leukemia.

E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-trans-retinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited ß-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML.

Photobiomodulation therapy moderates cancer cachexia-associated muscle wasting through activating PI3K/AKT/FoxO3a pathway.

Cancer cachexia-associated muscle wasting as a multifactorial wasting syndrome, is an important factor affecting the long-term survival rate of tumor patients. Photobiomodulation therapy (PBMT) has emerged as a promising tool to cure and prevent many diseases. However, the effect of PBMT on skeletal muscle atrophy during cancer progression has not been fully demonstrated yet. Here, we found PBMT alleviated the atrophy of myotube diameter induced by cancer cells in vitro, and prevented cancer-associated muscle atrophy in mice bearing tumor. Mechanistically, the alleviation of muscle wasting by PBMT was found to be involved in inhibiting E3 ubiquitin ligases MAFbx and MuRF-1. In addition, transcriptomic analysis using RNA-seq and GSEA revealed that PI3K/AKT pathway might be involved in PBMT-prevented muscle cachexia. Next, we showed the protective effect of PBMT against muscle cachexia was totally blocked by AKT inhibitor in vitro and in vivo. Moreover, PBMT-activated AKT promoted FoxO3a phosphorylation and thus inhibiting the nucleus entry of FoxO3a. Lastly, in cisplatin-treated muscle cachexia model, PBMT had also been shown to ameliorate muscle atrophy through enhancing PI3K/AKT pathway to suppress MAFbx and MuRF-1 expression. These novel findings revealed that PBMT could be a promising therapeutic approach in treating muscle cachexia induced by cancer.

Contribution of γ-Glutamyl-Cysteine Ligases of Limosilactobacillus reuteri to the Formation of Kokumi-Active γ-Glutamyl Dipeptides in Sourdough Bread.

Kokumi-active γ-glutamyl dipeptides accumulate during sourdough fermentation. γ-Glutamylcysteine ligases (Gcls) of Limosilactobacillus reuteri synthesize γ-glutamyl dipeptides during growth in sourdough. This study aimed to evaluate the contribution of Gcls from strains of L. reuteri in the formation of kokumi-active γ-glutamyl dipeptides in sourdough bread. Among 12 acceptor amino acids, the three Gcls of L. reuteri were the most active to Cys. With the acceptor amino acids Ile, Leu, and Phe, Gcl1 was more active than Gcl2 and Gcl3. Accordingly, Gcl1 contributed to the γ-Glu-Ile synthesis in sourdough fermentation. Proofing and baking strongly influenced the concentration of γ-glutamyl dipeptides in bread. The addition of 10% sourdough increased the content of γ-Glu-Leu and γ-Glu-Phe but not of other γ-glutamyl dipeptides in bread. In conclusion, the accumulation of kokumi γ-glutamyl dipeptides in sourdoughs was attributed to the combined activity of cereal enzymes, γ-glutamyl-cysteine ligases, and other microbial enzymes.

Identification of an SCF Ubiquitin Ligase Complex that Contributes to Resistance Against Valsa Canker in Apple.

E3 ubiquitin ligases play a critical role in plant disease resistance. Among them, the Skp1-Cullin-F-box protein (SCF) ubiquitin ligase complex is the largest family and regulates the ubiquitination of a wide range of proteins. Apple Valsa canker (AVC) is a fungal disease of apple trees caused by the fungus Valsa mali, which can lead to significant economic losses. However, the function of the SCF complex in apple resistance to this disease is still largely unknown. In this study, we identified an SCF ubiquitin ligase complex that can enhance resistance to Valsa canker in apple. Disease evaluation experiments demonstrated that MdSkp1 increased apple resistance to AVC. Furthermore, MdSkp1 interacted with an F-box protein, MdSKIP14, and interacted with a cullin-1 protein, MdCUL1, to form an SCF ubiquitin ligase complex. Additionally, we revealed both MdSKIP14 and MdCUL1 as positive regulators of AVC resistance. In conclusion, our results identified an SCF complex capable of contributing to apple resistance against AVC, providing a theoretical basis for apple disease resistance and the sustainable development of the industry. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Regulation of lipid metabolism by E3 ubiquitin ligases in lipid-associated metabolic diseases.

Previous studies have progressively elucidated the involvement of E3 ubiquitin (Ub) ligases in regulating lipid metabolism. Ubiquitination, facilitated by E3 Ub ligases, modifies critical enzymes in lipid metabolism, enabling them to respond to specific signals. In this review, we aim to present a comprehensive analysis of the role of E3 Ub ligases in lipid metabolism, which includes lipid synthesis and lipolysis, and their influence on cellular lipid homeostasis through the modulation of lipid uptake and efflux. Furthermore, it explores how the ubiquitination process governs the degradation or activation of pivotal enzymes, thereby regulating lipid metabolism at the transcriptional level. Perturbations in lipid metabolism have been implicated in various diseases, including hepatic lipid metabolism disorders, atherosclerosis, diabetes, and cancer. Therefore, this review focuses on the association between E3 Ub ligases and lipid metabolism in lipid-related diseases, highlighting enzymes critically involved in lipid synthesis and catabolism, transcriptional regulators, lipid uptake translocators, and transporters. Overall, this review aims to identify gaps in current knowledge, highlight areas requiring further research, offer potential targeted therapeutic approaches, and provide a comprehensive outlook on clinical conditions associated with lipid metabolic diseases.

CDK-independent role of D-type cyclins in regulating DNA mismatch repair.

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.

CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells.

During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies. IMPORTANCE: HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.

Application of AlphaFold models in evaluating ligandable cysteines across E3 ligases.

Proteolysis Targeting Chimeras (PROTACs) are an emerging therapeutic modality and chemical biology tools for Targeted Protein Degradation (TPD). PROTACs contain a ligand targeting the protein of interest, a ligand recruiting an E3 ligase and a linker connecting these two ligands. There are over 600 E3 ligases known so far, but only a handful have been exploited for TPD applications. A key reason for this is the scarcity of ligands binding various E3 ligases and the paucity of structural data available, which complicates ligand design across the family. In this study, we aim to progress PROTAC discovery by proposing a shortlist of E3 ligases that can be prioritized for covalent targeting by performing systematic structural ligandability analysis on a chemoproteomic dataset of potentially reactive cysteines across hundreds of E3 ligases. One of the goals of this study is to apply AlphaFold (AF) models for ligandability evaluations, as for a vast majority of these ligases an experimental structure is not available in the protein data bank (PDB). Using a combination of pocket features, AF model quality and additional aspects, we propose a shortlist of E3 ligases and corresponding cysteines that can be prioritized to potentially discover covalent ligands and expand the PROTAC toolbox.