Proteasome Research Articles

The effective multiplicity of infection for HCMV depends on the activity of the cellular 20S proteasome.

Human cytomegalovirus (HCMV) is a betaherpesvirus capable of infecting numerous cell types and persisting throughout an infected individual's life. Disease usually occurs in individuals with compromised or underdeveloped immune systems. Several antivirals exist but have limitations relating to toxicity and resistance. HCMV replication involves upregulation of host proteasomal activities, which play important roles in the temporal stages of replication. Here, we defined the impact on replication kinetics of the proteasome inhibitor, bortezomib. We demonstrate that bortezomib significantly reduces levels of viral genomes and infectious virions produced from a population of cells. Inhibition reduced expression of viral proteins that are influenced by genome synthesis. When added prior to 24 hpi, we observe decreases in PCNA and Cdk1 while increases in p21 whose regulations contribute to efficient replication. This response synergized with an antiviral, maribavir. Since some replication occurred, we tested the hypothesis that a subset of infected cells might break through inhibition. Initially, we simulated bortezomib activities using a mechanistic computational model of late-lytic replication. Upon reducing multiplicity of infection (MOI) in silico, we observed near-identical simulated results compared to experimental data. Next, we analyzed replication using live-cell imaging. This revealed treated cultures do contain a population of cells with fully developed late-stage cytoplasmic assembly compartments but at significantly lower numbers. We refer to this as the effective MOI. Overall, our studies support a hypothesis in which 20S proteasome inhibition disrupts HCMV replication by reducing the MOI to an effective MOI, defined by a fraction of infected cells capable of progressing to fulminant infection.IMPORTANCEHuman cytomegalovirus (HCMV) infection and reactivation continues to contribute to morbidity and mortality around the world. Antiviral compounds are available but have limitations. Here, we have defined the impact of the proteasome inhibitor bortezomib on HCMV replication. Proteasomal activities play a critical role in temporal changes required for replication. We demonstrate that disrupting these activities inhibits viral replication while likely supporting increased antiviral activity of the anti-HCMV agent, maribavir. Using a combination of live-cell imaging and computational tools, we discover that a subset of infected cells progresses to fulminant infection, which we define as the effective multiplicity of infection, and this subset would otherwise be missed when analyzing the average of the population.

Deubiquitinase USP37 enhances the anti-HIV-2/SIV ability of the host restriction factor SAMHD1.

The Vpx protein encoded by HIV-2/simian immunodeficiency virus (SIV) can antagonize the restriction of the host intrinsic restriction factor, SAMHD1, in nondividing cells by promoting its polyubiquitination and subsequent degradation, thereby facilitating viral replication and immune evasion. However, the role of deubiquitinating enzymes (DUBs) in the dynamics of virus and host remains poorly understood. Here, we demonstrate that DUB USP37 significantly reverses the Vpx-mediated degradation of SAMHD1 in various HIV-2/SIV subtypes by interacting with SAMHD1 and removing its ubiquitin chains. Notably, USP37 deubiquitinates SAMHD1 by directly recognizing SAMHD1 rather than by targeting the E3 ubiquitin ligase. The deubiquitinase activity of USP37 and its ubiquitin interacting motifs are essential for the deubiquitination of SAMHD1, whereas the phosphorylation state of USP37 does not influence its activity. Additionally, USP37 enhances the suppression of the retrotransposition of LINE-1 elements by SAMHD1 via stabilizing SAMHD1. Our findings provide important evidence that enhancing the deubiquitinating activity of some DUBs results in the stability of the host restriction factor and might be a viable strategy against HIV/SIV infections.IMPORTANCESAMHD1 is a multifunctional protein, including restricting virus replication, maintaining genomic integrity through DNA repair, modulating the immune response by influencing the production of type I interferons and other cytokines, and affecting cancer cell proliferation and sensitivity to chemotherapy. However, HIV-2/simian immunodeficiency virus (SIV)-encoded Vpx and the host E3 ligase TRIM21 can induce the degradation of SAMHD1 via the ubiquitin-proteasome pathway. Therefore, it is necessary to find the strategy to stabilize SAMHD1. Our study demonstrates that the deubiquitinase USP37 reverses Vpx- and TRIM21-mediated degradation of SAMHD1, thereby inhibiting SIV replication and LINE-1 activity by stabilizing SAMHD1. Thus, we report a novel role of USP37, which represents a potentially useful target for the development of new drugs.

FolSas2 is a regulator of early effector gene expression during Fusarium oxysporum infection.

Fusarium oxysporum f. sp. lycopersici (Fol) that causes a globally devastating wilt disease on tomato relies on the secretion of numerous effectors to mount an infection, but how the pathogenic fungus precisely regulates expression of effector genes during plant invasion remains elusive. Here, using molecular and cellular approaches, we show that the histone H4K8 acetyltransferase FolSas2 is a transcriptional regulator of early effector gene expression in Fol. Autoacetylation of FolSas2 on K269 represses K335 ubiquitination, preventing its degradation by the 26S proteasome. During the early infection process, Fol elevates FolSas2 acetylation by differentially changing transcription of itself and the FolSir1 deacetylase, leading to specific accumulation of the enzyme at this stage. FolSas2 subsequently activates the expression of an array of effectors genes, and as a consequence, Fol invades tomato successfully. These findings reveal a regulatory mechanism of effector gene expression via autoacetylation of a histone modifier during plant fungal invasion.

Elucidation of the late steps in the glycan-dependent ERAD of soluble misfolded glycoproteins.

The endoplasmic reticulum (ER) utilizes ER-associated degradation (ERAD), a highly conserved eukaryotic pathway, to eliminate misfolded or unassembled proteins and maintain protein homeostasis in cells. The clearance of misfolded glycoproteins involves several distinct steps, including the recognition of a specific glycan signal, retrotranslocation to the cytosol, and subsequent degradation of the misfolded protein by the ubiquitin proteasome system. Confocal microscopy was used to track the fate of a well-characterized ERAD substrate via a self-complementing split fluorescent protein assay. The results demonstrate that a misfolded variant of the STRUBBELIG (SUB) extracellular protein domain (SUBEX-C57Y) is retrotranslocated to the cytosol when transiently expressed in Nicotiana benthamiana leaf epidermal cells. Retrotranslocation requires a protein domain with a lesion that is exposed in the lumen of the ER, N-glycan trimming by α-mannosidases, HRD1-mediated ubiquitination, and the ATPase function of CDC48. The retrotranslocated SUBEX-C57Y ERAD substrate undergoes deglycosylation, and proteasomal degradation is blocked by a catalytically inactive cytosolic peptide N-glycanase. These findings define distinct aspects of ERAD that have been elusive until now and may represent the default pathway for degrading misfolded glycoproteins in plants.

Elucidation of Ubiquitin-Related Functions via an Ubiquitin Overexpression Approach

To identify new ubiquitin-related functions using yeast, we searched for mutants conferring a temperature-sensitivity phenotype that could be rescued through ubiquitin overexpression. Screening of mutants using this overexpression strategy identified SPC2, which encodes a subunit of the endoplasmic reticulum (ER) signal peptidase complex (SPC). Ubiquitin overexpression rescued a high-temperature sensitivity of spc2 deletion mutant, suggesting that ubiquitin could compensate for Spc2 loss-of-function at high temperatures. The double mutant of Spc2 and Hrd1, an ER E3 ubiquitin ligase, showed a synergistic growth defect at higher temperatures. A weak genetic interaction was also observed between spc2Δ and cdc48-3 mutation. The results suggest a close functional relationship between SPC and the ubiquitin–proteasome system in yeast and further provide proof-of-principle for this ubiquitin overexpression approach to identify novel ubiquitin-related genes and associated cellular processes.

Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways

A hallmark of neurodegenerative diseases (NDs) is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.

The E3 ubiquitin ligase TaGW2 facilitates TaSnRK1γ and TaVPS24 degradation to enhance stripe rust susceptibility in wheat.

Wheat stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst), threatens global wheat production, and therefore discovering genes involved in stripe rust susceptibility is essential for balancing yield with disease resistance in sustainable breeding strategies. Although TaGW2 is well known to negatively regulate wheat kernel size and weight, its role in stress response remains unclear. Here, we found that TaGW2 transcription levels increased following inoculation with Pst or treatment with flg22 or chitin. TaGW2 knockdown lines showed enhanced resistance to multiple Pst races, while TaGW2 overexpression reduced host defence response, promoted Pst growth and development and increased wheat susceptibility to Pst. Additionally, TaGW2 could mediate the ubiquitination and degradation of both TaSnRK1γ and TaVPS24 via the 26S proteasome pathway. Silencing TaSnRK1γ or TaVPS24 in wheat increased sensitivity to Pst, whereas overexpressing either gene enhanced wheat defence response, indicating that TaSnRK1γ and TaVPS24 act as positive regulators of Pst resistance. This study reveals a previously unrecognized mechanism inhibiting plant immunity to Pst through TaGW2-mediated ubiquitination and degradation of TaSnRK1γ and TaVPS24. This work also provides crucial genetic resources for breeding high-yield, stripe rust-resistant wheat varieties.

Engineered platelets as targeted protein degraders and application to breast cancer models.

Clinical application of chimeric molecules for targeted protein degradation has been limited by unfavorable drug-like properties and biosafety concerns arising from nonspecific biodistribution after systemic administration. Here we develop a method to engineer platelets for degradation of either intracellular or extracellular proteins of interest (POIs) in vivo by covalently labeling heat shock protein 90 (HSP90) in platelets with a POI ligand. The degrader platelets (DePLTs) target wound areas and undergo activation. Depending on the tethered POI ligand and transport mechanism of the prelabeled HSP90, activated DePLTs can mediate targeted protein degradation in the target cell through the ubiquitin-proteasome machinery or the lysosome. HSP90 packaged into platelet-derived microparticles uses the ubiquitin-proteasome system to degrade intracellular POIs, whereas released free HSP90 redirects extracellular POIs to lysosomal degradation. In postsurgical breast cancer mouse models, DePLTs engineered with corresponding POI ligands effectively degrade intracellular bromodomain-containing protein 4 or extracellular programmed cell death ligand 1, thereby suppressing cancer recurrence or metastasis.

The co-chaperone DNAJA2 buffers proteasomal degradation of cytosolic proteins with missense mutations

Mutations can disrupt a protein's native function by causing misfolding, which is generally handled by an intricate protein quality control network. To better understand the triaging mechanisms of misfolded cytosolic proteins, we screened a human mutation library to identify a panel of unstable mutations. The degradation of these mutated cytosolic proteins is largely dependent on the ubiquitin proteasome system. Using BioID proximity labelling, we found that the co-chaperones DNAJA1 and DNAJA2 are key interactors with one of the mutated proteins. Notably, the absence of DNAJA2 increases the turnover of the mutant but not the wild-type protein. Our work indicates that specific missense mutations in cytosolic proteins can promote enhanced interactions with molecular chaperones. Assessment of the broader panel of cytosolic mutant proteins shows that the co-chaperone DNAJA2 exhibits two distinct behaviours: acting to stabilize a wide array of cytosolic proteins including wild type variants, and specifically “buffer” some mutant proteins to reduce their turnover. Our work illustrates how distinct elements of the protein homeostasis network are utilized in the presence of a cytosolic misfolded protein.

Ubiquitin Ligase U-Box51 Positively Regulates Drought Stress in Potato (Solanum tuberosum L.)

The ubiquitin-proteasome system (UPS) is a key protein degradation pathway in eukaryotes, in which E3 ubiquitin ligases mediate protein ubiquitination, directly or indirectly targeting substrate proteins to regulate various biological processes, including plant growth, hormone signaling, immune responses, and adaptation to abiotic stress. In this study, we identified plant U-box protein 51 in Solanum tuberosum (StPUB51) as an E3 ubiquitin ligase through transcriptomic analysis, and used it as a candidate gene for gene-function analysis. Quantitative real-time PCR (qRT-PCR) was used to examine StPUB51 expression across different tissues, and its expression patterns under simulated drought stress induced by polyethylene glycol (PEG 6000) were assessed. Transgenic plants overexpressing StPUB51 and plants with down-regulated StPUB51 expression were generated to evaluate drought tolerance. The activities of key antioxidant enzymes-superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) as well as malondialdehyde (MDA) content in transgenic plants’ leaves were measured under drought conditions. Protein–protein interactions involving StPUB51 were explored via yeast two-hybrid (Y2H) screening, with interaction verification by bimolecular fluorescence complementation (BiFC). StPUB51 was predominantly expressed in stems, with lower expression observed in tubers, and its expression was significantly upregulated in response to 20% PEG-6000 simulated drought. Subcellular localization assays revealed nuclear localization of the StPUB51 protein. Under drought stress, StPUB51-overexpressing plants exhibited enhanced SOD, POD, and CAT activities and reduced MDA levels, in contrast to plants with suppressed StPUB51 expression. Y2H and BiFC analyses identified two interacting proteins, StSKP2A and StGATA1, which may be functionally linked to StPUB51. Collectively, these findings suggest that StPUB51 plays a positive regulatory role in drought tolerance, enhancing resilience in potato growth and stress adaptation.

Ubiquitin proteasome system in cardiac fibrosis.

Cardiac fibrosis, including reactive fibrosis and replacement fibrosis, is a common pathological process in most cardiovascular diseases. The ubiquitin proteasome system (UPS) plays an important role in the development of fibrosis by mediating the degradation and synthesis of proteins involved in transforming growth factor-β (TGF-β)-dependent and TGF-β-independent fibrous pathways. This review aims to provide an overview of ubiquitinated and deubiquitinated molecules that participating in cardiac fibrosis, with the ultimate purpose to identify promising targets for therapeutic strategies. The UPS primarily impacts cardiac fibrosis through modulation of the TGF-β signaling pathway targeting key molecules involved, including the TGF-β receptors, Smad2/3/4 complexes, and inhibitory Smad7, thereby influencing fibrotic processes. In addition to its effect on TGF-β signaling, UPS also regulates pro-fibrotic pathways independent of TGF-β, including p53, AKT1-p38, and JNK1/2. Understanding these pathways is critical due to their involvement in diverse fibrotic mechanisms. The interplay between ubiquitination and deubiquitination of crucial pathways and molecules is pivotal in cardiac fibrosis and represents a promising area for identifying novel therapeutic targets. Different types of cardiac fibrosis involve distinct fibrotic pathways, leading to differential effects of E3 ligases and DUBs across various cardiac fibrotic diseases. Insights into UPS-mediated regulation of cardiac fibrosis provides potential anti-fibrotic therapeutic strategies, emphasizing the importance of targeting UPS components specific to the heart for effective therapy against cardiac fibrosis.

Ginkgo biloba extract alleviates deltamethrin-induced testicular injury by upregulating SKP2 and inhibiting Beclin1-independent autophagy

BackgroundMale infertility is a worldwide concern that is associated with a decline in sperm quality. Environmental pollutants such as deltamethrin (DM) have harmful effects on male reproductive organs. By maintaining intracellular redox homeostasis, ginkgo biloba extract (GBE) can alleviate male reproductive dysfunction. However, research on the mechanisms by which GBE alleviates reproductive toxicity induced by DM is limited. PurposeIn this study, we investigated whether GBE can alleviate DM-induced testicular and Sertoli cell reproductive toxicity by modulating SKP2 and Beclin1, thus providing a theoretical basis for the development of novel therapeutic approaches. Study DesignWe explored the role of GBE in mitigating DM-induced testicular damage, with a specific focus on the intricate involvement of ubiquitination and autophagy. MethodsAn experimental model was constructed using ICR male mice and the TM4 cell line. Tissue, cellular, and sperm morphological changes were observed through methods such as Hematoxylin and Eosin (H&E) staining, Periodate-Schiff (PAS) staining, ultrastructural observation, immunohistochemistry, and immunofluorescence. Enzyme and hormone levels were measured, and gene and protein levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting techniques. ResultsIn vivo experiments showed that DM exposure led to decreased sex hormone levels, increased seminiferous tubule diameter and impaired spermatogenesis. Meanwhile, DM exposure was found to decrease ubiquitination levels, leading to mitochondrial damage and further escalation of mitochondrial autophagy. Furthermore, in the DM-induced cell model, the upregulation of Beclin1 expression was associated with the inhibition of the ubiquitin‒proteasome system (UPS) and SKP2, thereby exacerbating autophagy. However, GBE has demonstrated notable efficacy in alleviating the reproductive toxicity induced by DM. ConclusionOur findings highlighted that SKP2 is a key regulator of Beclin1-independent autophagy and that GBE exerts therapeutic effects by upregulating SKP2 and inhibiting Beclin1 activation, which ameliorates autophagy and reduces DM-induced testicular damage.

Diosmin reduces the stability of Snail and Cyclin D1 by targeting FAK to inhibit NSCLC progression

BackgroundIn different tumours, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase, is upregulated and hence, it represents a promising target for cancer therapy. However, the development of FAK kinase inhibitors has faced a number of challenges. It is therefore imperative that new, effective FAK kinase inhibitors be identified promptly. MethodsSmall molecules that target FAK were identified through molecular docking and validated through surface plasmon resonance (SPR) and cell thermal shift analysis. We investigated the pharmacological effects of FAK kinase inhibitors using CCK-8, colony formation, EdU, and Transwell assays and cell cycle analysis. The molecular mechanism was determined via methods such as coimmunoprecipitation, RNA pull-down and RNA immunoprecipitation. ResultsHere, we confirmed that diosmin (Dio) is an inhibitor of FAK and demonstrated its anti-proliferative and anti-metastatic effects in lung adenocarcinoma. Mechanistically, Dio inhibited tumour proliferation and metastasis by impeding the catalytic activity of FAK. Dio activated the ubiquitin proteasome pathway to induce Cyclin D1 degradation, while inhibiting tumour proliferation and reversing the epithelial mesenchymal transition (EMT) process by reducing the mRNA stability of Snail, thereby inhibiting cancer metastasis. In addition, the inhibitory effect of Dio on lung adenocarcinoma was validated in a mouse xenograft model. ConclusionThese results support the tumour-promoting role of FAK in lung adenocarcinoma by stabilizing Cyclin D1 and Snail and suggest that Dio is a promising candidate for FAK inhibition.

Spatial transcriptomics in focal cortical dysplasia type IIb

Focal cortical dysplasia (FCD) type IIb (FCD IIb) is an epileptogenic malformation of the neocortex that is characterized by cortical dyslamination, dysmorphic neurons (DNs) and balloon cells (BCs). Approximately 30–60% of lesions are associated with brain somatic mutations in the mTOR pathway. Herein, we investigated the transcriptional changes around the DNs and BCs regions in freshly frozen brain samples from three patients with FCD IIb by using spatial transcriptomics. We demonstrated that the DNs region in a gene enrichment network enriched for the mTOR signalling pathway, autophagy and the ubiquitin‒proteasome system, additionally which are involved in regulating membrane potential, may contribute to epileptic discharge. Moreover, differential expression analysis further demonstrated stronger expression of components of the inflammatory response and complement activation in the BCs region. And the DNs and BCs regions exhibited common functional modules, including regulation of cell morphogenesis and developmental growth. Furthermore, the expression of representative proteins in the functional enrichment module mentioned above was increased in the lesions of FCD IIb, such as p62 in DNs and BCs, UCHL1 in DNs, and C3 and CLU in BCs, which was confirmed via immunohistochemistry. Collectively, we constructed a spatial map showing the potential effects and functions of the DNs and BCs regions at the transcriptomic level and generated publicly available data on human FCD IIb to facilitate future research on human epileptogenesis.

20S Proteasomes, Hsp27 and LC3B Content in Plasma Exosomes of Gastric Cancer Patients, Association with Metabolic Disorders

Введение. Рак желудка (РЖ) является одним из распространенных онкологических заболеваний как в России, так и в мире. Ранняя диагностика данной патологии представляет важную проблему. За последние несколько лет были проведены исследования по использованию неинвазивной жидкостной биопсии при опухолях различных локализаций, которая проста в использовании и позволяет изучить внеклеточные везикулы плазмы крови. В настоящее время данное направление является перспективным в современной молекулярной онкологии. Цель. Изучение содержания белков 20S протеасом, Hsp27 и LC3B в экзосомах плазмы крови при раке желудка, по сравнению с экзосомами, выделенными у больных с атрофическим гастритом с учетом метаболических нарушений и ожирения. Материалы и методы. В исследование вошли 18 больных раком желудка, проходивших лечение в клиниках НИИ онкологии Томского НИМЦ. Группу сравнения составили 6 больных с атрофическим гастритом. Больные были разделены на группы в зависимости от стадии опухолевого процесса. T1-2 стадия опухоли была отмечена у 12 пациентов, а T3-4 — у 6 пациентов. Экзосомы плазмы крови выделяли с помощью метода ультрацентрифугирования. Типирование проводили с помощью маркера CD9. Содержание белков 20S протеасом, Hsp27 и LC3B в экзосомах плазмы проводили методом Вестерн блоттинг. Результаты. В результате проведенного исследования был отмечен рост экзосом плазмы крови с содержанием 20S протеасом и LC3B в 3,8 и 1,4 раза, соответственно, у больных с раком желудка, по сравнению с пациентами без опухолевой патологии (атрофическим гастритом). При этом содержание Hsp27 в экзосомах, выделенных из плазмы крови больных, было снижено в 2,3 раза. При исследовании не была выявлена взаимосвязь стадии опухолевого процесса и содержания 20S протеасом в экзосомах крови. Однако рост содержания LC3B в экзосомах был сопряжен с ростом стадии опухолевого процесса. Выводы. Выявлен рост 20S протеасом и LC3B в экзосомах плазмы, выделенных у больных с раком желудка. При этом уровень белка Hsp27 в экзосомах был снижен у больных со злокачественными опухолями. Отмечена связь содержания белка LC3B и стадией опухолевого процесса, что требует дальнейшего изучения. В проведенном исследовании сформированы и обоснованы подходы для применения маркеров жидкостной биопсии при формировании групп риска и диагностики рака желудка. При этом особое значение придается метаболическими нарушениям как основным триггерам опухолевой прогрессии.

Targeting PRMT5 through PROTAC for the treatment of triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is currently the most aggressive subtype of breast cancer, characterized by high heterogeneity and strong invasiveness, and currently lacks effective therapies. PRMT5, a type II protein arginine methyltransferase, is upregulated in numerous cancers, including TNBC, and plays a critical role, marked it as an attractive therapeutic target. PROTAC (Proteolysis Targeting Chimeras) is an innovative drug development technology that utilizes the ubiquitin-proteasome system (UPS) to degrade target proteins, which is characterized by higher activity, enhanced safety, lower resistance, and reduced toxicity, offering significant value for clinical translation.MethodsThis study utilizes the PROTAC technology to develop potential degraders targeting PRMT5 in vitro and in vivo.ResultsThrough the design, synthesis and screening of a series of targeted compounds, we identified YZ-836P as an effective compound that exerted cytotoxic effects and reduced the protein levels of PRMT5 and its key downstream target protein KLF5 in TNBC after 48 h. Its efficacy was significantly superior to the PRMT5 PROTAC degraders that had been reported. YZ-836P induced G1 phase cell cycle arrest and significantly induced apoptosis in TNBC cells. Additionally, we demonstrated that YZ-836P promoted the ubiquitination and degradation of PRMT5 in a cereblon (CRBN)-dependent manner. Notably, YZ-836P exhibited pronounced efficacy in inhibiting the growth of TNBC patient-derived organoids and xenografts in nude mice.ConclusionsThese findings position YZ-836P as a promising candidate for advancing treatment modalities for TNBC.Trial registrationEthics Committee of Yunnan Cancer Hospital, KYCS2023-078. Registered 7 June 2023.

Resurrecting p53 by an Artificial Nano-Protein for Potent Photodynamic Retinoblastoma Therapy.

The treatment of retinoblastoma (RB), a formidable eye cancer that affects infants and children, is not only aimed at saving lives but at preserving ocular function, maintaining optimal visual acuity, and enhancing the overall quality of life. Photodynamic therapy has already been established as a secure and dependable therapeutic modality for the treatment of ocular diseases that effectively preserves ocular function; however, it fails to provide satisfactory outcomes against RB. To address this formidable challenge, groundbreaking advancement is aspired by delving into the genetic characteristics of RB, which initially involves the wild-type p53 pathway but is subsequently suppressed by MDM2 and MDMX. In this study, an artificial nanoprotein (ANP) is developed through the fusion of human serum albumin with a potent MDM2/MDMX degrading peptide and the porphyrin-photosensitizer verteporfin (VP). ANP effectively dismantles MDM2/MDMX proteins within the ubiquitin-proteasome degradation pathway and triggers VP-PDT-mediated cell death through mitochondrial impairment. Remarkably, ANP exhibited remarkable therapeutic efficacy in both subcutaneous and in situ RB mouse models, while maintaining a highly favorable biosafety profile. Collectively, ANP not only provides compelling evidence for sensitizing photodynamic RB therapy by reactivating p53, but also serves as an exceptionally potent photosensitizer with promising potential for clinical translation in RB treatment.

Quiescent cells maintain active degradation-mediated protein quality control requiring proteasome, autophagy and nucleus-vacuole junctions

Many cells spend a major part of their life in quiescence, a reversible state characterized by a distinct cellular organization and metabolism. In glucose-depleted quiescent yeast cells, there is a metabolic shift from glycolysis to mitochondrial respiration, and a large fraction of proteasomes are reorganized into cytoplasmic granules containing disassembled particles. Given these changes, the operation of protein quality control (PQC) in quiescent cells, in particular the reliance on degradation-mediated PQC and the specific pathways involved, remains unclear. By examining model misfolded proteins expressed in glucose-depleted quiescent yeast cells, we found that misfolded proteins are targeted for selective degradation requiring functional 26S proteasomes. This indicates that a significant pool of proteasomes remains active in degrading quality control substrates. Misfolded proteins were degraded in a manner dependent on the E3 ubiquitin ligases Ubr1 and San1, with Ubr1 playing a dominant role. In contrast to exponentially growing cells, the efficient clearance of certain misfolded proteins additionally required intact nucleus-vacuole junctions (NVJ) and Cue5-independent selective autophagy. Our findings suggest that proteasome activity, autophagy, and NVJ-dependent degradation operate in parallel. Together the data demonstrate that quiescent cells maintain active PQC that relies primarily on selective protein degradation. The necessity of multiple degradation pathways for the removal of misfolded proteins during quiescence underscores the importance of misfolded protein clearance in this cellular state.

Deciphering potential molecular mechanisms in clear cell renal cell carcinoma based on the ubiquitin-conjugating enzyme E2 related genes: Identifying UBE2C correlates to infiltration of regulatory T cells.

Renal clear cell carcinoma (ccRCC) is a highly aggressive and common form of kidney cancer, with limited treatment options for advanced stages. Recent studies have highlighted the importance of the ubiquitin-proteasome system in tumor progression, particularly the role of ubiquitin-conjugating enzyme E2 (UBE2) family members. However, the prognostic significance of UBE2-related genes (UBE2RGs) in ccRCC remains unclear. In this study, bulk RNA-sequencing and single-cell RNA-sequencing data from ccRCC patients were retrieved from the Cancer Genome Atlas and Gene Expression Omnibus databases. Differential expression analysis was performed to identify UBE2RGs associated with ccRCC. A combination of 10 machine learning methods was applied to develop an optimal prognostic model, and its predictive performance was evaluated using area under the curve (AUC) values for 1-, 3-, and 5-year overall survival (OS) in both training and validation cohorts. Functional enrichment analyses of gene ontology and Kyoto Encyclopedia of Genes and Genomes were conducted to explore the biological pathways involved. Correlation analysis was conducted to investigate the association between the risk score and tumor mutational burden (TMB) and immune cell infiltration. Immunotherapy and chemotherapy sensitivity were assessed by immunophenoscore and tumor immune, dysfunction, and exclusion scores to identify potential predictive significance. In vitro, knockdown of the key gene UBE2C in 786-O cells by specific small interfering RNA to validate its impact on apoptosis, migration, cell cycle, migration, invasion of tumor cells, and induction of regulatory T cells (Tregs). Analysis of sc-RNA revealed that UBE2 activity was significantly upregulated in malignant cells, suggesting its role in tumor progression. A three-gene prognostic model comprising UBE2C, UBE2D3, and UBE2T was constructed by Lasoo Cox regression and demonstrated robust predictive accuracy, with AUC values of 0.745, 0.766, and 0.771 for 1-, 3-, and 5-year survival, respectively. The model was validated as an independent prognostic factor in ccRCC. Patients in the high-risk group had a worse prognosis, higher TMB scores, and low responsiveness to immunotherapy. Additionally, immune infiltration and chemotherapy sensitivity analyses revealed that UBE2RGs are associated with various immune cells and drugs, suggesting that UBE2RGs could be a potential therapeutic target for ccRCC. In vitro experiments confirmed that the reduction of UBE2C led to an increase in apoptosis rate, as well as a decrease in tumor cell invasion and metastasis abilities. Additionally, si-UBE2C cells reduced the release of the cytokine Transforming Growth Factor-beta 1 (TGF-β1), leading to a decreased ratio of Tregs in the co-culture system. This study presents a novel three-gene prognostic model based on UBE2RGs that demonstrates significant predictive value for OS, immunotherapy, and chemotherapy in ccRCC patients. The findings underscore the potential of UBE2 family members as biomarkers and therapeutic targets in ccRCC, warranting further investigation in prospective clinical trials.

Mechanistic exploration of ubiquitination-mediated pathways in cerebral ischemic injury.

The ubiquitin-proteasome system (UPS) plays a pivotal role in regulating protein homeostasis and cellular processes, including protein degradation, trafficking, DNA repair, and cell signaling. During cerebral ischemia, ischemic conditions profoundly disrupt UPS activity, leading to proteasomal dysfunction and the accumulation of abnormal proteins. This imbalance contributes to neuronal injury and cell death observed in ischemic stroke. The UPS is intricately linked to various signaling pathways crucial for neuronal survival, inflammation, and cellular stress response, such as NF-κB, TRIM, TRIP, JAK-STAT, PI3K/Akt, and ERK1/2. Alterations in the ubiquitination process can significantly impact the activation and regulation of these pathways, exacerbating ischemic brain injury. Therapeutic approaches targeting the UPS in cerebral ischemia aim to rebalance protein levels, reduce proteotoxic stress, and mitigate neuronal injury. Strategies include proteasome inhibition, targeting specific ubiquitin ligases and deubiquitinating enzymes, and modulating ubiquitination-mediated regulation of key signaling pathways implicated in ischemia-induced pathophysiology. Therefore, the present review discusses the molecular mechanisms underlying UPS dysfunction in ischemic stroke is crucial for developing effective therapeutic interventions. Modulating ubiquitination-mediated pathways through therapeutic interventions targeting specific UPS components holds significant promise for mitigating ischemic brain injury and promoting neuroprotection and functional recovery in patients with cerebral ischemia.