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Despite osteoarthritis (OA) being recognised for over a century as a debilitating disease that affects millions, there are huge gaps in our understanding of the underlying pathophysiology that drives this disease. Present day studies that focussed on ubiquitination (Ub) and ubiquitylation-like (Ubl) modification related mechanisms have brought light into the possibility of attenuating OA development by targeting these specific proteins in chondrocytes. In the present review, we discuss recent advances in studies involving Ub ligases and deubiquitinating enzymes (DUBs) which are of importance in the development of OA, and may offer potential therapeutic strategies for OA. Such targets may involve attenuating proteases such as matrix metalloproteinases (MMP) 1, 8, 13, 4 and several A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) that are well known for their roles in cartilage breakdown. Ligases such as ubiquitin-conjugating enzymes (E2) and ubiquitin-ligating enzymes (E3) that are involved in extracellular matrix (ECM) degradation in OA and of their pathogenesis would be discussed. In addition to catabolic and degenerative downstream effects of Ub and DUBs in OA, inflammatory mechanisms most notably involving nuclear factor-kappa B (NF-κB) signalling pathways regulated through Ub and using various targeting molecules would also be highlighted. Challenges, gaps and insights from clinical trials will provide valuable guidance for future investigations on targeting ubiquitin-proteosome system (UPS) as a therapeutic option for OA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: With a decline of 17β-estradiol (E2) at menopause, E2 has been implicated in the accompanied loss of skeletal muscle mass and strength. We aimed at characterizing transcriptomic responses of skeletal muscle to E2 in female mice, testing the hypothesis that genes and pathways related to contraction and maintenance of mass are differentially expressed in ovariectomized mice with and without E2 treatment., Methods: Soleus and tibialis anterior (TA) muscles from C57BL/6 ovariectomized mice treated with placebo (OVX) or E2 (OVX + E2) for 60 days, or from skeletal muscle-specific ERα knockout (skmERαKO) mice and wild-type littermates (skmERαWT), were used for genome-wide expression profiling, quantitative real-time PCR and immunoblotting. Computational detection of estrogen response elements (EREs) was performed with EREFINDER., Results: We found 155 significantly regulated probe sets in response to E2 (p ≤ 0.001). Pathway analyses identified proteasome and ubiquitin-mediated proteolysis as two downregulated pathways in the E2 group. We confirmed downregulation (p ≤ 0.05) in levels of Fbxw7, Psmb6, Ube2h and Ubxn1, as well as pro-apoptotic Bnip3 and inflammatory factor Nfkbia. Computational analysis identified ERE in the promoter regions of Psmb6, Ube2h, Bnip3 and Nfkbia. The overall content of ubiquitinated proteins was modestly but significantly lower in TA muscles from OVX + E2 vs. OVX mice (p = 0.039). There were no differences between skmERαKO and skmERαWT mice or between skmERαKO/OVX and skmERαKO/OVX + E2 mice for any genes assessed, indicating that ERα is required for E2 regulation of those genes., Conclusions: These results suggest that a mechanism whereby E2 protects against losses of skeletal muscle mass and strength is regulation of ubiquitin-proteasomal mediators., (© 2025 The Author(s). Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)

Introduction: Chemotherapy-induced peripheral neuropathy (CIPN) is a common complication that affects an increasing number of cancer survivors. However, the current treatment options for CIPN are limited. Paclitaxel (PTX) is a widely used chemotherapeutic drug that induces senescence in cancer cells. While previous studies have demonstrated that Sonic hedgehog (Shh) can counteract cellular dysfunction during aging, its role in CIPN remains unknown., Objectives: Herein, the aim of this study was to investigate whether Shh activation could inhibits neuronal/glial senescence and alleviates CIPN., Methods: We treated ND7/23 neuronal cells and RSC96 Schwann cells with two selective Shh activators (purmorphamine [PUR] and smoothened agonist [SAG]) in the presence of PTX. Additionally, we utilized a CIPN mouse model induced by PTX injection. To assess cellular senescence, we performed a senescence-associated β-galactosidase (SA-β-gal) assay, measured reactive oxygen species (ROS) levels, and examined the expression of P16, P21, and γH2AX. To understand the underlying mechanisms, we conducted ubiquitin assays, LC-MS/MS, H&E staining, and assessed protein expression through Western blotting and immunofluorescence staining., Results: In vitro, we observed that Shh activation significantly alleviated the senescence-related decline in multiple functions included SA-β-gal activity, expression of P16 and P21, cell viability, and ROS accumulation in DRG sensory neurons and Schwann cells after PTX exposure. Furthermore, our in vivo experiments demonstrated that Shh activation significantly reduced axonal degeneration, demyelination, and improved nerve conduction. Mechanistically, we discovered that PTX reduced the protein level of SP1, which was ubiquitinated by the E3 ligase TRIM25 at the lysine 694 (K694), leading to increased CXCL13 expression, and we found that Shh activation inhibited PTX-induced neuronal/glial senescence and CIPN through the TRIM25-SP1-CXCL13 axis., Conclusion: These findings provide evidence for the role of PTX-induced senescence in DRG sensory neurons and Schwann cells, suggesting that Shh could be a potential therapeutic target for CIPN., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Macroautophagy/autophagy is a constitutively active catabolic lysosomal degradation pathway, often found dysregulated in human diseases. It is often considered to act in a cytoprotective manner and is commonly upregulated in cells undergoing stress. Its initiation is regulated at the protein level and does not require de novo protein synthesis. Historically, autophagy has been regarded as nonselective; however, it is now clear that different stimuli can lead to the selective degradation of cellular components via selective autophagy receptors (SARs). Due to its selective nature and the existence of multiple degradation pathways potentially acting in concert, monitoring of autophagy flux, i.e . selective autophagy-dependent protein degradation, should address this complexity. Here, we introduce a targeted proteomics approach monitoring abundance changes of 37 autophagy-related proteins covering process-relevant proteins such as the initiation complex and the Atg8-family protein lipidation machinery, as well as most known SARs. We show that proteins involved in autophagosome biogenesis are upregulated and spared from degradation under autophagy-inducing conditions in contrast to SARs, in a cell-line dependent manner. Classical bulk stimuli such as nutrient starvation mainly induce degradation of ubiquitin-dependent soluble SARs and not of ubiquitin-independent, membrane-bound SARs. In contrast, treatment with the iron chelator deferiprone leads to the degradation of ubiquitin-dependent and -independent SARs linked to mitophagy and reticulophagy/ER-phagy. Our approach is automatable and supports large-scale screening assays paving the way to (pre)clinical applications and monitoring of specific autophagy flux. Abbreviation: AMBRA1: autophagy and beclin 1 regulator 1; ATG: autophagy related; BafA1: bafilomycin A 1 ; BNIP1: BCL2 interacting protein 1; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3-like; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCPG1: cell cycle progression 1; CV: coefficients of variations; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DFP: deferiprone; ER: endoplasmic reticulum; FKBP8: FKBP prolyl isomerase 8; GABARAPL: GABA type A receptor associated protein like; LC: liquid chromatography; LOD: limit of detection; LOQ: limit of quantification; MAP1LC3: microtubule associated protein 1 light chain 3; MS: mass spectrometry; NCOA4: nuclear receptor coactivator 4; NBR1: NBR1 autophagy cargo receptor; NUFIP1: nuclear FMR1 interacting protein 1; OPTN: optineurin; PHB2: prohibitin 2; PNPLA2/ATGL: patatin like phospholipase domain containing 2; POI: protein of interest; PTM: posttranslational modification; PRM: parallel reaction monitoring; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RETREG1/FAM134B: reticulophagy regulator 1; RPS6KB1: ribosomal protein S6 kinase B1; RTN3: reticulon 3; SARs: selective autophagy receptors; SQSTM1/p62: sequestosome 1; STBD1: starch binding domain 1; TAX1BP1: Tax1 binding protein 1; TFEB: transcription factor EB; TNIP1: TNFAIP3 interacting protein 1; TOLLIP: toll interacting protein; ULK1: unc-51 like autophagy activating kinase 1; WBP2: WW domain binding protein 2; WDFY3/Alfy: WD repeat and FYVE domain containing 3; WIPI2: WD repeat domain, phosphoinositide interacting 2.

The host ESCRT-machinery repairs damaged endolysosomal membranes. If damage persists, selective macroautophagy/autophagy clears the damaged compartment. Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that damages the phagosomal membrane and targets ESCRT-mediated repair as part of its virulence program. The E3 ubiquitin ligases PRKN and SMURF1 promote autophagic capture of damaged, Mtb-containing phagosomes. Because ubiquitination is a reversible process, we anticipated that host deubiquitinases (DUBs) would also be involved. Here, we screened all predicted mouse DUBs for their role in ubiquitin targeting and control of intracellular Mtb. We show that USP8 (ubiquitin specific peptidase 8) colocalizes with intracellular Mtb, recognizes phagosomal membrane damage, and is required for ESCRT-dependent membrane repair. Furthermore, we show that USP8 regulates the NFE2L2/NRF2-dependent antioxidant signature. Taken together, our study demonstrates a central role of USP8 in promoting Mtb intracellular growth by promoting phagosomal membrane repair, limiting ubiquitin-driven selective autophagy, and reducing oxidative stress. Abbreviation: BMDMs: bone marrow-derived macrophages; CFUs: colony-forming units; DUB: deubiquitinase; ESCRT: endosomal sorting complexes required for transport; LLOMe: L-leucyl-L-leucine methyl ester; MFI: mean fluorescence intensity; MOI: multiplicity of infection; Mtb: Mycobacterium tuberculosis ; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; PMA: phorbol 12-myristate 13-acetate; ROS: reactive oxygen species; USP8: ubiquitin specific peptidase 8.

The 26S proteasome is involved in degrading and regulating the majority of proteins in eukaryotic cells, which requires a sophisticated balance of specificity and promiscuity. In this Review, we discuss the principles that underly substrate recognition and ATP-dependent degradation by the proteasome. We focus on recent insights into the mechanisms of conventional ubiquitin-dependent and ubiquitin-independent protein turnover, and discuss the plethora of modulators for proteasome function, including substrate-delivering cofactors, ubiquitin ligases and deubiquitinases that enable the targeting of a highly diverse substrate pool. Furthermore, we summarize recent progress in our understanding of substrate processing upstream of the 26S proteasome by the p97 protein unfoldase. The advances in our knowledge of proteasome structure, function and regulation also inform new strategies for specific inhibition or harnessing the degradation capabilities of the proteasome for the treatment of human diseases, for instance, by using proteolysis targeting chimera molecules or molecular glues., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. Springer Nature Limited.)

Individuals with genetic elimination of MLKL (mixed lineage kinase domain like pseudokinase) exhibit an increased susceptibility to neurodegenerative diseases like Alzheimer disease (AD). However, the mechanism is not yet fully understood. Here, we observed significant compromise in macroautophagy/autophagy in the brains of mlkl knockout (KO) mice, as evidenced by the downregulation of BECN1/Beclin1 and ULK1 (unc-51 like autophagy activating kinase 1). We identified UBA52 (ubiquitin A-52 residue ribosomal protein fusion product 1) as the binding partner of MLKL under physiological conditions. Loss of Mlkl induced a decrease in ubiquitin levels by preventing UBA52 cleavage. Furthermore, we demonstrated that the deubiquitinase (DUB) USP7 (ubiquitin specific peptidase 7) mediates the processing of UBA52, which is regulated by MLKL. Moreover, our results indicated that the reduction of BECN1 and ULK1 upon Mlkl loss is attributed to a decrease in their lysine 63 (K63)-linked polyubiquitination. Additionally, single-nucleus RNA sequencing revealed that the loss of Mlkl resulted in the disruption of multiple neurodegenerative disease-related pathways, including those associated with AD. These results were consistent with the observation of cognitive impairment in mlkl KO mice and exacerbation of AD pathologies in an AD mouse model with mlkl deletion. Taken together, our findings demonstrate that MLKL-USP7-UBA52 signaling is required for autophagy in brain through maintaining ubiquitin homeostasis, and highlight the contribution of Mlkl loss-induced ubiquitin deficits to the development of neurodegeneration. Thus, the maintenance of adequate levels of ubiquitin may provide a novel perspective to protect individuals from multiple neurodegenerative diseases through regulating autophagy. Abbreviations : 4HB: four-helix bundle; AAV: adeno-associated virus; AD: Alzheimer disease; AIF1: allograft inflammatory factor 1; APOE: apolipoprotein E; APP: amyloid beta precursor protein; Aβ: amyloid β; BECN1: beclin 1; co-IP: co-immunoprecipitation; DEGs: differentially expressed genes; DLG4: discs large MAGUK scaffold protein 4; DUB: deubiquitinase; EBSS: Earle's balanced salt solution; GFAP: glial fibrillary acidic protein; HRP: horseradish peroxidase; IL1B: interleukin 1 beta; IL6: interleukin 6; IPed: immunoprecipitated; KEGG: Kyoto Encyclopedia of Genes and Genomes; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MLKL: mixed lineage kinase domain like pseudokinase; NSA: necrosulfonamide; OPCs: oligodendrocyte precursor cells; PFA: paraformaldehyde; PsKD: pseudo-kinase domain; SYP: synaptophysin; UB: ubiquitin; UBA52: ubiquitin A-52 residue ribosomal protein fusion product 1; UCHL3: ubiquitin C-terminal hydrolase L3; ULK1: unc-51 like autophagy activating kinase 1; UMAP: uniform manifold approximation and projection; UPS: ubiquitin-proteasome system; USP7: ubiquitin specific peptidase 7; USP9X: ubiquitin specific peptidase 9 X-linked.

Distant metastasis is a primary factor contributing to the low survival rate of patients with nasopharyngeal carcinoma (NPC). Circular RNAs (circRNAs) are increasingly recognized for their roles in cancer initiation and progression. However, the mechanisms underlying the abnormal expression and biological function of circRNA in NPC remain unclear. In this study, we identified a new circRNA, circINADL, which was upregulated in NPC tissues and positively correlated with the clinical stage of NPC. We found that the FUS RNA binding protein (FUS) promoted the transcription of circINADL in NPC cells. Elevated circINADL levels were shown to enhance NPC cells metastasis. Mechanistically, circINADL attenuated the interaction between human antigen R (HuR) and the E3 ubiquitin ligase β-TrCP, thereby inhibited the ubiquitination and degradation of HuR. Consequently, CircINADL enhanced the stability of the HuR target gene Yes1-associated transcriptional regulator (YAP1), leading to the dysregulation of the Hippo signaling pathway. In conclusion, our study reveals the function of circINADL in promoting NPC metastasis and highlights its potential as a biomarker and therapeutic target for NPC treatment., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Due to various intracellular and external cues, cellular organelles are frequently stressed in both physiological and pathological conditions. Sensing these stresses initiates various signaling pathways which may lead to adaptation of the stressed cells or trigger its their death. At the unicellular level, this stress signaling involves a crosstalk between different organelles. At the multicellular level, such pathways can contribute to indicate the presence of a stressed cell to its neighboring cells. Here, we highlight the crucial and diverse roles played by Ubiquitin and Ubiquitin-like modification in organelle stress signaling., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

Autophagy is an essential cellular recycling process that maintains protein and organelle homeostasis. ATG9A vesicle recruitment is a critical early step in autophagy to initiate autophagosome biogenesis. The mechanisms of ATG9A vesicle recruitment are best understood in the context of starvation-induced nonselective autophagy, whereas less is known about the signals driving ATG9A vesicle recruitment to autophagy initiation sites in the absence of nutrient stress. Here we demonstrate that loss of ATG9A, or the lipid transfer protein ATG2, leads to the accumulation of phosphorylated p62 aggregates in nutrient replete conditions. Furthermore, we show that p62 degradation requires the lipid scramblase activity of ATG9A. Last, we present evidence that polyubiquitin is an essential signal that recruits ATG9A and mediates autophagy foci assembly in nutrient replete cells. Together, our data support a ubiquitin-driven model of ATG9A recruitment and autophagosome formation during basal autophagy., Competing Interests: Conflict of interest: The authors declare no financial conflicts of interest.

Targeted protein degradation (TPD) has emerged as a highly promising approach for eliminating disease-associated proteins in the field of drug discovery. Among the most advanced TPD technologies, PROteolysis TArgeting Chimera (PROTAC), functions by bringing a protein of interest (POI) into proximity with an E3 ubiquitin ligase, leading to ubiquitin (Ub)-dependent proteasomal degradation. However, the designs of most PROTACs are based on the utilization of a limited number of available E3 ligases, which significantly restricts their potential. Recent studies have shown that phytoplasmas, a group of bacterial plant pathogens, have developed several E3- and ubiquitin-independent proteasomal degradation (UbInPD) mechanisms for breaking down host targets. This suggests an alternative approach for substrate recruitment and TPD. Here, we present existing evidence that supports the feasibility of UbInPD in eukaryotic cells and propose candidate proteins that can serve as docking sites for the development of E3-independent PROTACs., (© 2024 The Author(s). BioEssays published by Wiley‐VCH GmbH.)

Stalled ribosomes cause collisions, impair protein synthesis, and generate potentially harmful truncated polypeptides. Eukaryotic cells utilize the ribosome-associated quality control (RQC) and no-go mRNA decay (NGD) pathways to resolve these problems. In yeast, the E3 ubiquitin ligase Hel2 recognizes and polyubiquitinates disomes and trisomes at the 40S ribosomal protein Rps20/uS10, thereby priming ribosomes for further steps in the RQC/NGD pathways. Recent studies have revealed high concentrations of disomes and trisomes in unstressed cells, raising the question of whether and how Hel2 selects long-term stalled disomes and trisomes. This study presents quantitative analysis of in vivo-formed Hel2•ribosome complexes and the dynamics of Hel2-dependent Rps20 ubiquitination and Ubp2/Ubp3-dependent deubiquitination. Our findings show that Hel2 occupancy progressively increases from translating monosomes to disomes and trisomes. We demonstrate that disomes and trisomes with mono- or di-ubiquitinated Rps20 resolve independently of the RQC component Slh1, while those with tri- and tetra-ubiquitinated Rps20 do not. Based on the results, we propose a model in which Hel2 translates the duration of ribosome stalling into polyubiquitin chain length. This mechanism allows for the distinction between transient and long-term stalling, providing the RQC machinery with a means to select fatally stalled ribosomes over transiently stalled ones., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Non-covalent protein-protein interactions are one of the most fundamental building blocks in cellular signalling pathways. Despite this, they have been historically hard to identify using conventional methods due to their often weak and transient nature. Using genetic code expansion and incorporation of commercially available unnatural amino acids, we have developed a highly accessible method whereby interactions between biotinylated ubiquitin-like protein (UBL) probes and their binding partners can be stabilised using ultraviolet (UV) light-induced crosslinks. The stabilised protein complexes can be purified using affinity purification and identified by mass spectrometry. The resultant covalent bonds can withstand even the harshest washing conditions, allowing for the removal of indirect binders whilst retaining and capturing weak and transient interactors that are commonly lost during wash steps. This technique is widely applicable and highly effective for identifying site-selective non-covalent interactors. Members of our team have previously demonstrated the benefit of this method using the small ubiquitin-like modifier (SUMO). Here, we provide further proof-of-principle validation of the method and highlight its generality by applying an optimised workflow to a lesser studied UBL, interferon stimulated gene 15 (ISG15). We show that this method is able to capture known ISG15 interactors from a complex protein mixture in a site-selective manner, only capturing proteins that specifically interact with the region of ISG15 where the unnatural amino acid was incorporated. This exquisite degree of sensitivity and specificity greatly improves upon previous screens aimed at identifying downstream non-covalent binders, or readers, of ISG15. Taken together, the approach opens the possibility of characterising previously undetected protein-protein interactions, with the potential of elucidating molecular mechanisms behind the most complex and poorly understood processes in the cell., Competing Interests: J.V.F. is an AstraZeneca employee and holds shares in the company. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare., (Copyright: © 2025 Sandy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Protein homeostasis is crucial for maintaining cardiomyocyte (CM) function. Disruption of proteostasis results in accumulation of protein aggregates causing cardiac pathologies such as hypertrophy, dilated cardiomyopathy (DCM), and heart failure. Here, we identify ubiquitin-specific peptidase 5 (USP5) as a critical determinant of protein quality control (PQC) in CM. CM-specific loss of mUsp5 leads to the accumulation of polyubiquitin chains and protein aggregates, cardiac remodeling, and eventually DCM. USP5 interacts with key components of the proteostasis machinery, including PSMD14, and the absence of USP5 increases activity of the ubiquitin-proteasome system and autophagic flux in CMs. Cardiac-specific hUSP5 overexpression reduces pathological remodeling in pressure-overloaded mouse hearts and attenuates protein aggregate formation in titinopathy and desminopathy models. Since CMs from humans with end-stage DCM show lower USP5 levels and display accumulation of ubiquitinated protein aggregates, we hypothesize that therapeutically increased USP5 activity may reduce protein aggregates during DCM. Our findings demonstrate that USP5 is essential for ubiquitin turnover and proteostasis in mature CMs.

Background: The sustained activation of androgen receptor splice variant-7 (AR-V7) is a key factor in the resistance of castration-resistant prostate cancer (CRPC) to second-generation anti-androgens such as enzalutamide (ENZ). The AR/AR-V7 protein is regulated by the E3 ubiquitin ligase STUB1 and a complex involving HSP70, but the precise mechanism remains unclear., Methods: High-throughput RNA sequencing was used to identify differentially expressed circular RNAs (circRNAs) in ENZ-resistant and control CRPC cells. The coding potential of circSRCAP was confirmed by polysome profiling and LC-MS. The function of circSRCAP was validated in vitro and in vivo using gain- and loss-of-function assays. Mechanistic insights were obtained through immunoprecipitation analyses., Results: A novel ENZ-resistant circRNA, circSRCAP, was identified and shown to be upregulated in ENZ-resistant C4-2B (ENZR-C4-2B) cells, correlating with increased AR-V7 protein levels. circSRCAP is generated via splicing by eIF4A3, forming a loop structure and is exported from the nucleus by the RNA helicase DDX39A. Mechanistically, circSRCAP encodes a 75-amino acid peptide (circSRCAP-75aa) that inhibits the ubiquitination of AR/AR-V7's co-chaperone protein HSP70 by disrupting the interaction with the E3 ligase STUB1. This process results in the upregulation of AR-V7 expression and promotes ENZ resistance in CRPC cells. Xenograft tumor models further confirmed the role of circSRCAP in CRPC progression and its potential as a therapeutic target for ENZ-resistant CRPC., Conclusions: circSRCAP provides an epigenetic mechanism influencing AR-V7 stability and offers a promising therapeutic target for treating ENZ-resistant CRPC., Competing Interests: Declarations. Ethics approval and consent to participate: The animal experimental procedures involving mice were carried out as prescribed by the National Guidelines for Animal Usage in Research (China) and were approved by the Institutional Animal Care and Use Committee of Xuzhou Medical University (202309T023). This study was conducted following approval by the Institutional Animal Care and Use Committee of Xuzhou Medical University (202309T023). All treatments were administered in accordance with the guidelines established by the committee. Consent for publication: All authors consent to publication. Competing interests: The authors have declared that no competing interest exists., (© 2025. The Author(s).)

Small molecule degraders such as PROteolysis TArgeting Chimeras (PROTACs) and molecular glues are new modalities for drug development and important tools for target validation. When appropriately optimized, both modalities lead to proteasomal degradation of the protein of interest (POI). Due to the complexity of the induced multistep degradation process, controls for degrader evaluation are critical and commonly used in the literature. However, comparative studies and evaluations of cellular potencies of these control compounds have not been published so far. Here, we investigated a diverse set of ubiquitin pathway inhibitors and evaluated their potency and utility within the CRBN and VHL-mediated degradation pathway. We used the HiBiT system to measure the level of target rescue after treatment with the control compounds. In addition, the cell health was assessed using a multiplexed high-content assay. These assays allowed us to determine nontoxic effective concentrations for control experiments and to perform rescue experiments in the absence of cellular toxicity.

The modification of proteins and other biomolecules with the small protein ubiquitin has enthralled scientists from many disciplines for decades, creating a broad research field. Ubiquitin research is particularly rich in molecular and mechanistic understanding due to a plethora of (poly)ubiquitin structures alone and in complex with ubiquitin machineries. Furthermore, due to its favorable properties, ubiquitin serves as a model system for many biophysical and computational techniques. Here, we review the current knowledge of ubiquitin signals through a ubiquitin-centric, structural biology lens. We amalgamate the information from 240 structures in the Protein Data Bank (PDB), combined with single-molecule, molecular dynamics, and nuclear magnetic resonance (NMR) studies, to provide a comprehensive picture of ubiquitin and polyubiquitin structures and dynamics. We close with a discussion of the latest frontiers in ubiquitin research, namely the modification of ubiquitin by other post-translational modifications (PTMs) and the notion that ubiquitin is attached to biomolecules beyond proteins., Competing Interests: Declaration of interests D.K. is a founder, shareholder, and SAB member of Entact Bio and Proxima Bio., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Porcine epidemic diarrhoea virus (PEDV) is an enteric pathogen that causes acute diarrhoea, dehydration and high mortality rates in suckling pigs. Tripartite motif 8 (TRIM8) has been shown to play multiple roles in the host's defence against viral infections. However, the functions of TRIM8 in regulating PEDV infection are still not well understood. In our study, we found a significant upregulation of TRIM8 following PEDV infection. We created TRIM8 knockout and overexpression cell lines and discovered that TRIM8 can inhibit PEDV replication within host cells. Co-immunoprecipitation assays revealed that TRIM8 directly interacts with the nucleocapsid protein (N) of PEDV, specifically within the coiled-coil structural domain of TRIM8. Furthermore, TRIM8 was shown to reduce the expression of the PEDV N protein in a dose-dependent manner. Mechanistically, TRIM8 inhibits the expression of PEDV N through K48-linked ubiquitin proteasome degradation. Transcriptomics analysis revealed that TRIM8 facilitates the expression of genes associated with several pathways, including the IL-17 signalling pathway, chemokine signalling pathway, and cytokine-cytokine receptor interaction. This suggests that TRIM8 plays a crucial role in boosting antiviral immune responses against PEDV infection. Our findings provide new insights into the functions and mechanisms of TRIM8 in regulating PEDV infection and highlight its potential as a molecular target for the prevention and control of this virus., Competing Interests: Declarations. Ethics approval and consent to participate: This article does not involve any studies involving human participants or animals conducted by any of the authors. Competing interests: The authors declare that they have no competing interests., (© 2025. The Author(s).)

The autophagy-related ubiquitin-like conjugation systems, the ATG8 and ATG12 systems, are universally conserved in eukaryotes. However, the covalent bond in the ATG12 system has recently been shown to be evolutionarily lost in Apicomplexa. Here, we show that all genes associated with the ATG12 system are absent in piroplasmida, a lineage within Apicomplexa. Comparative genomics of ATGs further shows that piroplasm ATG3 has lost the region necessary for ATG12 binding. However, our in vitro functional analysis using recombinant proteins demonstrated that ATG3 retained the ability to interact with ATG8 in Babesia bovis , a model species in piroplasmida. These findings provide evidence that the ATG8 system is functional, while the ATG12 system is completely lost in the common ancestor of piroplasmida and highlight the evolutionary flexibility of the ATG12 system in Apicomplexa.

The ubiquitin-proteasome system (UPS) is an important type of protein post-translational modification that affects the quantity and quality of various proteins and influences cellular processes such as the cell cycle, transcription, oxidative stress, and autophagy. Nanomaterials (NMs), which exhibit excellent physicochemical properties, can directly interact with the UPS and act as molecular-targeted drugs to induce changes in biological processes. This review provides an overview of the influence of NMs on the UPS of misfolded proteins and key proteins, which are related to cancer, neurodegenerative diseases and oxidative stress. This review also summarizes the role of modification processes involved in ubiquitination the biological effects of NMs and the mechanism of such effects of NMs through regulation of the UPS. This review deepens our understanding of the influence of NMs on the protein degradation process and provides new potential therapeutic targets for disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Uncontrollable cancer cell growth is characterized by the maintenance of cellular homeostasis through the continuous accumulation of misfolded proteins and damaged organelles. This review delineates the roles of two complementary and synergistic degradation systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system, in the degradation of misfolded proteins and damaged organelles for intracellular recycling. We emphasize the interconnected decision-making processes of degradation systems in maintaining cellular homeostasis, such as the biophysical state of substrates, receptor oligomerization potentials (e.g., p62), and compartmentalization capacities (e.g., membrane structures). Mitochondria, the cellular hubs for respiration and metabolism, are implicated in tumorigenesis. In the subsequent sections, we thoroughly examine the mechanisms of mitochondrial quality control (MQC) in preserving mitochondrial homeostasis in human cells. Notably, we explored the relationships between mitochondrial dynamics (fusion and fission) and various MQC processes-including the UPS, mitochondrial proteases, and mitophagy-in the context of mitochondrial repair and degradation pathways. Finally, we assessed the potential of targeting MQC (including UPS, mitochondrial molecular chaperones, mitochondrial proteases, mitochondrial dynamics, mitophagy and mitochondrial biogenesis) as cancer therapeutic strategies. Understanding the mechanisms underlying mitochondrial homeostasis may offer novel insights for future cancer therapies., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no competing interests. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable., (© 2025. The Author(s).)

Ubiquitination is cells' second most abundant posttranslational protein modification after phosphorylation. The ubiquitin-proteasome system (UPS) is critical in maintaining essential life processes such as cell cycle control, DNA damage repair, and apoptosis. Mutations in ubiquitination pathway genes are strongly linked to the development and spread of multiple cancers since several of the UPS family members possess oncogenic or tumor suppressor activities. This comprehensive review delves into understanding the ubiquitin code, shedding light on its role in cancer cell biology and immune evasion. Furthermore, we highlighted recent advances in the field for targeting the UPS pathway members for effective therapeutic intervention against human cancers. We also discussed the recent update on small-molecule inhibitors and PROTACs and their progress in preclinical and clinical trials.

Ubiquitin-proteasomal degradation of K + /Cl - cotransporter 2 (KCC2) in the ventral posteromedial nucleus (VPM) has been demonstrated to serve as a common mechanism by which the brain emerges from anesthesia and regains consciousness. Ubiquitin-proteasomal degradation of KCC2 during anesthesia is driven by E3 ligase Fbxl4. However, the mechanism by which ubiquitinated KCC2 is targeted to the proteasome has not been elucidated. We report in cultured neuro-2a cells that the valosin-containing protein (VCP) transported ubiquitinated KCC2 to the proteasome and in mice in vivo experiments that inhibition of VCP restored KCC2 expression in the VPM and enhanced the effects of anesthesia. In cultured neuro-2a cells, propofol-induced degradation of KCC2 was inhibited by VCP inhibitor DBeQ and VCP knockout plasmid sgRNA(VCP). Propofol-induced enhanced interaction between VCP and KCC2 was inhibited by knockout of Fbxl4 or Fas-associated factor 1 (FAF1). In in vivo studies, pharmacological or genetic inhibition of VCP in the VPM significantly prevented KCC2 degradation and enhanced propofol anesthesia; these effects were abrogated by a KCC2 antagonist VU0463271. These results demonstrate that the VCP controls ubiquitin-proteasomal degradation of KCC2 dependent on FAF1 recruitment and serves as a mechanism for the ubiquitin-proteasomal degradation of KCC2, which is responsible for the subsequent emergence from anesthesia., Competing Interests: Competing interests statement:The authors declare no competing interest.

Tsg101 is a highly conserved protein best known as an early-functioning component of cellular ESCRT machinery participating in recognition, sorting, and trafficking of cellular cargo to various intracellular destinations. It shares sequence and structural homology to canonical ubiquitin-conjugating (E2) enzymes and is linked to diverse events regulated by Ub signaling. How it might fulfill these roles is unclear. Here, we show that Tsg101 E2 mimicry permits interactions with diverse ubiquitin ligating (E3) enzymes and underlies its multifunctional capabilities. Coexpression of Tsg101 with the E3 ligase NNedd4-2s protected the enzyme from degradation and, remarkably, other widely divergent ligases as well. Structural alignment with UbcH5, a canonical E2 enzyme, revealed that recognition at the E2-E3 interface, a region broadly conserved despite sequence and structural differences in both E2 and E3 enzymes, was critical for protection. Nevertheless, UbcH5 failed to protect NNedd4-2s, indicating that the UEV chaperone function is unique to the variant. Studies using Cy5-Ub-VME showed that Tsg101-mediated protection reduced accessibility to Cys residues in the ligase. Access to Tsg101 Ub-binding sites was critical: Rabeprazole, which interferes with Tsg101 Ub-binding, diminished E3 ligase protection. Thus, E2 mimicry permitting control of E3 ligase ubiquitin signaling underlies Tsg101's broad ability to participate in multiple cellular functions. The study provides mechanistic insight into how Tsg101, by partnering with diverse E3 ligases, can contribute to a broad range of cellular activities., Competing Interests: Competing interests statement:The authors declare no competing interest.

Proteolysis-targeting chimera (PROTAC) is an emerging therapeutic technology that leverages the ubiquitin-proteasome system to target protein degradation. Due to its event-driven mechanistic characteristics, PROTAC has the potential to regulate traditionally non-druggable targets. Recently, AI-aided drug design has accelerated the development of PROTAC drugs. However, the rational design of PROTACs remains a considerable challenge. Here, we present an updated online database, PROTAC-DB 3.0. In this third version, we have expanded the database to include 6111 PROTACs (87% increase compared to the 2.0 version). Additionally, the database now contains 569 warheads (small molecules targeting the protein), 2753 linkers, and 107 E3 ligands (small molecules recruiting E3 ligases). The number of target-PROTAC-E3 ternary complex structures has also increased to 959. Recognizing the importance of druggability in PROTAC design, we have incorporated pharmacokinetic data to PROTAC-DB 3.0. To enhance user experience, we have added features for sorting based on molecular similarity and literature publication date. PROTAC-DB 3.0 is accessible at http://cadd.zju.edu.cn/protacdb/., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: RING finger protein 112 (RNF112) exerts a key role in human tumors. However, its biological function in colorectal cancer (CRC) has not been discussed. We aimed to explore the function and molecular mechanism of RNF112 in CRC., Results: In this study, RNF112 expression was notably decreased in CRC tissues and cells. Clinical analysis revealed a significant association between low RNF112 expression and tumor size, N classification and TNM stage. In vitro experiments demonstrated that overexpression of RNF112 repressed cell viability, promoted cell cycle arrest and apoptosis, while knocking down RNF112 had the opposite function. The tumor formation results in nude mice supported that RNF112 overexpression exerted anti-tumor effects by inhibiting cell growth and promoting cell apoptosis. Mechanistically, Krüppel-like factor 4 (KLF4) acted as an upstream regulator of RNF112 by mediating its transcription. Furthermore, we explored the downstream mechanism of RNF112 and discovered that it promoted ubiquitination and degradation of oncoprotein N-alpha-acetyltransferase 40 (NAA40) through ubiquitin ligase activity. In addition, overexpression of NAA40 eliminated the effect of RNF112 overexpression on CRC tumorigenesis., Conclusions: In summary, our findings confirm that RNF112, whose transcription is regulated by KLF4, inhibits CRC growth through promoting ubiquitin-dependent degradation of NAA40. We have unraveled the mechanism of KLF4-RNF112-NAA40 axis in CRC, which shed light on the therapeutic strategies for this disease., Competing Interests: Declarations. Ethical approval: Clinical study was approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University and conducted in accordance with the Declaration of Helsinki. Animal studies were approved by Medical Ethics Committee of Shengjing Hospital of China Medical University, and all procedures were conducted according to the National Research Council’s Guide for the Care and Use of Laboratory Animals. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Interferon regulatory factor 3 (IRF3) is a central hub transcription factor that controls host antiviral innate immunity. The expression and function of IRF3 are tightly regulated by the post-translational modifications. However, it is unknown whether unanchored ubiquitination and deubiquitination of IRF3 involve modulating antiviral innate immunity against RNA viruses. Here, we find that USP5, a deubiquitinase (DUB) regulating unanchored polyubiquitin, is downregulated during host anti-RNA viral innate immunity in a type I interferon (IFN-I) receptor (IFNAR)-dependent manner. USP5 is further identified to inhibit IRF3-triggered antiviral immune responses through its DUB enzyme activity. K48-linked unanchored ubiquitin promotes IRF3-driven transcription of IFN-β and induction of IFN-stimulated genes (ISGs) in a dose-dependent manner. USP5 simultaneously removes both K48-linked unanchored and K63-linked anchored polyubiquitin chains on IRF3. Our study not only provides evidence that unanchored ubiquitin regulates anti-RNA viral innate immunity but also proposes a novel mechanism for DUB-controlled IRF3 activation, suggesting that USP5 is a potential target for the treatment of RNA viral infectious diseases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Qiao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Muscle atrophy in pathological or diseased muscles arises from an imbalance between protein synthesis and degradation. Elevated levels of interleukin-6 (IL-6) are a hallmark of ischemic stroke and have been associated with muscle atrophy in certain pathological contexts. However, the mechanisms by which IL-6 induces muscle atrophy in the context of stroke remain unclear. To investigate these effects, we used a rat model of middle cerebral artery occlusion (MCAO) and an in vitro model with the C2C12 cell line to uncover potential molecular mechanisms underlying IL-6-induced muscle atrophy. Our findings revealed elevated protein and serum levels of IL-6, along with increased markers of muscle atrophy, in MCAO rats compared to sham controls. We also observed overactivation of protein ubiquitination pathways and downregulation of muscle regeneration markers in MCAO rats. Further analysis indicated that IL-6 contributes to increased muscle protein ubiquitination. Inhibition of IL-6 signaling led to a significant reduction in infarct size and improved neurological deficit scores. Targeting the IL-6/IL-6R signaling pathway presents a promising therapeutic approach to mitigate muscle atrophy in individuals affected by ischemic stroke., Competing Interests: Declarations. Competing Interest: The authors declare no competing interests. Consent for Publication: Not applicable. Ethical Approval: Animal studies were approved by the Affiliated Jiangning Hospital of Nanjing Medical University. This study was performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985)., (© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Conjugation of ubiquitin (Ub) and structurally related ubiquitin-like proteins (Ubls), essential for many cellular processes, employs multi-step reactions orchestrated by specific E1, E2 and E3 enzymes. The E1 enzyme activates the Ub/Ubl C-terminus in an ATP-dependent process that results in the formation of a thioester linkage with the E1 active site cysteine. The thioester-activated Ub/Ubl is transferred to the active site of an E2 enzyme which then interacts with an E3 enzyme to promote conjugation to the target substrate. The E1-E2-E3 enzymatic cascades utilize labile intermediates, extensive conformational changes, and vast combinatorial diversity of short-lived protein-protein complexes to conjugate Ub/Ubl to various substrates in a regulated manner. In this review, we discuss various chemical tools and methods used to study the consecutive steps of Ub/Ubl activation and conjugation, which are often too elusive for direct studies. We focus on methods developed to probe enzymatic activities and capture and characterize stable mimics of the transient intermediates and transition states, thereby providing insights into fundamental mechanisms in the Ub/Ubl conjugation pathways., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Intracellular proteins take part in almost every body function; thus, protein homeostasis is of utmost importance. The ubiquitin proteasome system (UPS) has a fundamental role in protein homeostasis. Its main role is to selectively eradicate impaired or misfolded proteins, thus halting any damage that could arise from the accumulation of these malfunctioning proteins. Proteasomes have a critical role in controlling protein homeostasis in all cell types, including stem cells. We will discuss the role of UPS enzymes as well as the 26S proteasome complex in stem cell biology from several angles. First, we shall overview common trends of proteasomal activity and gene expression of different proteasomal subunits and UPS enzymes upon passaging and differentiation of stem cells toward various cell lineages. Second, we shall explore the effect of modulating proteasomal activity in stem cells and navigate through the interrelation between proteasomes' activity and various proteasome-related transcription factors. Third, we will shed light on curated microRNAs and long non-coding RNAs using various bioinformatics tools that might have a possible role in regulating UPS in stem cells and possibly, upon manipulation, can enhance the differentiation process into different lineages and/or delay senescence upon cell passaging. This will help to decipher the role played by individual UPS enzymes and subunits as well as various interrelated molecular mediators in stem cells' maintenance and/or differentiation and open new avenues in stem cell research. This can ultimately provide a leap toward developing novel therapeutic interventions related to proteasome dysregulation., (© 2025 International Union of Biochemistry and Molecular Biology.)

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key regulator of mitochondrial biogenesis and antioxidative defenses, and it may play a critical role in Parkinson's disease (PD). F-box/WD repeat domain-containing protein (FBXW7), an E3 protein ligase, promotes the degradation of substrate proteins through the ubiquitin-proteasome system (UPS) and leads to the clearance of PGC-1α. Here, we elucidate a novel post-translational mechanism for regulating PGC-1α levels in neurons. We show that enhancing chaperone-mediated autophagy (CMA) activity promotes the CMA-mediated degradation of FBXW7 and consequently increases PGC-1α. We confirm the relevance of this pathway in vivo by showing decreased FBXW7 and increased PGC-1α as a result of boosting CMA selectively in dopaminergic (DA) neurons by overexpressing lysosomal-associated membrane protein 2A (LAMP2A) in TH-Cre-LAMP2-loxp conditional mice. We further demonstrate that these mice are protected against MPTP-induced oxidative stress and neurodegeneration. These results highlight a novel regulatory pathway for PGC-1α in DA neurons and suggest targeted increasing of CMA or decreasing FBXW7 in DA neurons as potential neuroprotective strategies in PD., (© 2024 Federation of European Biochemical Societies.)

Protein homeostasis (proteostasis) refers to the balance of the cellular protein environment, tightly regulated by pathways governing protein synthesis, folding, trafficking, and degradation. Growing evidence supports the interconnection of these pathways to ensure the robustness of the proteo-stasis network. A recent study by Park et al. showed that, in macroautophagy/autophagy-deficient cells, the loss of proteasome or nuclear pore components causes synthetic lethality, as cytoplasmic proteins that accumulate under impaired autophagy are transported to the nucleus and degraded by nuclear proteasomes. The authors illustrated the mechanistic basis for why cells with conditions such as Huntington disease, where both autophagy and cytoplasm-to-nuclear shuttling are compromised, are more vulnerable to proteostasis perturbation. Abbreviation : UPR: unfolded protein response; UPS: ubiquitin-proteasome system.

The ubiquitin-proteasome system (UPS) is responsible for degrading over 70-80% of cellular proteins. Consequently, proteolysis-targeting chimeras (PROTACs) are developed to induce the ubiquitination and subsequent degradation of proteins of interest (POIs) by the UPS. To amplify the therapeutic efficacy of PROTACs, energy metabolism regulation is first harnessed to boost UPS function in tumor cells. Proteomic and ubiquitinome analyzes reveal that total ubiquitinated proteins and proteasome activity are significantly increased in 143B and MDA-MB-231 tumor cells following fasting-mimicking diet (FMD) treatment. As a result, the degradation efficiency of PROTACs targeting focal adhesion kinase (FAK-P) or bromodomain-containing protein 4 (BRD4-P) is significantly enhanced in FMD-treated 143B and MDA-MB-231 tumor cells. Then, silica-coated iron oxide nanoparticles are developed modified with tumor cell membranes for targeted delivery of PROTACs. Magnetic resonance imaging (MRI) and fluorescence imaging confirm that nanocarriers significantly improve the delivery efficiency of PROTACs in FMD-treated 143B or MDA-MB-231 tumors. In vivo studies demonstrate that the antitumor efficacy of FAK-P and BRD4-P is greatly augmented when combined with targeted delivery and FMD treatment. Overall, this study presents a strategy to enhance the efficacy of PROTACs in cancer therapy., (© 2024 Wiley‐VCH GmbH.)

The pyroptosis of renal tubular epithelial cells leads to tubular loss and inflammation and then promotes renal fibrosis. The transcription factor Krüppel-like factor 4 (KLF4) can bidirectionally regulate the transcription of target genes. Our previous study revealed that sustained elevation of KLF4 is responsible for the transition of acute kidney injury (AKI) into chronic kidney disease (CKD) and renal fibrosis. In this study, we explored the upstream mechanisms of renal tubular epithelial cell pyroptosis from the perspective of posttranslational regulation and focused on the transcription factor KLF4. Mice were subjected to unilateral ureteral obstruction (UUO) surgery and euthanized on D7 or D14 for renal tissue harvesting. We showed that the pyroptosis of renal tubular epithelial cells mediated by both the Caspase-1/GSDMD and Caspase-3/GSDME pathways was time-dependently increased in UUO mouse kidneys. Furthermore, we found that the expression of the transcription factor KLF4 was also upregulated in a time-dependent manner in UUO mouse kidneys. Tubular epithelial cell-specific Klf4 knockout alleviated UUO-induced pyroptosis and renal fibrosis. In Ang II-treated mouse renal proximal tubular epithelial cells (MTECs), we demonstrated that KLF4 bound to the promoter regions of Caspase-3 and Caspase-1 and directly increased their transcription. In addition, we found that ubiquitin-specific protease 11 (USP11) was increased in UUO mouse kidneys. USP11 deubiquitinated KLF4. Knockout of Usp11 or pretreatment with the USP11 inhibitor mitoxantrone (3 mg/kg, i.p., twice a week for two weeks before UUO surgery) significantly alleviated the increases in KLF4 expression, pyroptosis and renal fibrosis. These results demonstrated that the increased expression of USP11 in renal tubular cells prevents the ubiquitin degradation of KLF4 and that elevated KLF4 promotes inflammation and renal fibrosis by initiating tubular cell pyroptosis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

By the ubiquitin-proteasomes, cellular proteins are structurally degraded and turnover. Many essential functions and regulations of cells are regulated and controlled by these proteins. Recent studies indicated that many cancer types have been associated with aberrations in the ubiquitination pathway, which involves three enzymatic steps. Dietary phytochemicals have been identified as having the potential to inhibit carcinogenesis recently. As part of this group of phytochemicals, curcumin can play a crucial role in suppressing carcinogenesis by changing many reactions affected by the ubiquitin-proteasome pathway. Due to its ability to change some biological processes such as NF-κB, inhibit some cyclins, and induce apoptosis, it can be used as a drug in cancer treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Ubiquitination is one of the most important post-translational modifications (PTMs) and involves the covalent attachment of ubiquitin to a lysine residue on a target protein. Despite ubiquitination playing a crucial role in regulating cellular processes, the ubiquitinated proteome has not been studied extensively in recombinant Chinese hamster ovary (CHO) cells. Moreover, ubiquitination modification in CHO cells is likely to have an impact on protein function related to the efficient productivity of biopharmaceuticals. In this chapter, we describe a comprehensive protocol for ubiquitin di-Glycine (diGly) peptide enrichment using an immunoprecipitation method from recombinant CHO cell proteins followed by Liquid chromatography-Mass spectrometry (LC-MS) analysis of the ubiquitinated proteome. The methods described are also applicable to differential ubiquitinated proteomic studies., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Ubiquitination regulates a myriad of eukaryotic signaling cascades by modifying substrate proteins, thereby determining their functions and fates. In this perspective, we discuss current challenges in investigating the ubiquitin system in the developing brain. We foster the concept that ubiquitination pathways are spatiotemporally regulated and tightly intertwined with molecular and cellular transitions during neurogenesis and neural circuit assembly. Focusing on the neurologically highly relevant class of homologous to E6AP C-terminus (HECT) ubiquitin ligases, we propose cross-disciplinary translational approaches bridging state-of-the-art cell biology, proteomics, biochemistry, structural biology and neuroscience to dissect ubiquitination in neurodevelopment and its specific perturbations in brain diseases. We highlight that a comprehensive understanding of ubiquitin signaling in the brain may reveal new horizons in basic neuroscience and clinical applications., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. Springer Nature America, Inc.)

Single cell sequencing and related databases have been widely used in the exploration of cancer occurrence and development, but there is still no in-depth explanation of specific and complicated cellular protein modification processes. Ubiquitin-Proteasome System (UPS), as a specific and precise protein modification and degradation process, plays an important role in the biological functions of cancer cell proliferation and apoptosis. Proteasomes, vital multi-catalytic proteinases in eukaryotic cells, play a crucial role in protein degradation and contribute to tumor regulation. The 26S proteasome, part of the ubiquitin-proteasome system. In this study, we have enrolled a common SITP process including analysis of single cell sequencing to elucidate a flow that can capture typical proteasome markers in the oncogenesis and progression of breast cancer. PSMD11, a key component of the 26S proteasome regulatory particle, has been identified as a critical survival factor in cancer cells. Results suggest that PSMD11's rapid degradation is linked to acute apoptosis in cancer cells, making it a potential target for cancer treatment. Our study explored the potential mechanisms of PSMD11 in breast cancer development. The findings revealed the feasibility of disclosing ubiquitinating biomarkers from public database, as well as presented new evidence supporting PSMD11 as a potential therapeutic biomarker for breast cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Mitochondrial dysfunction is a pivotal factor in the onset of podocyte damage, which is a central component in the pathogenesis of nephrotic syndrome (NS). However, the precise mechanisms underlying the changes in podocyte mitochondria remain elusive. Our study aims to clarify the potential mechanisms involved in the role of F-box protein 11 (Fbxo11) in NS, specifically concentrating on its impact on mitochondrial function. A mouse model was established by tail vein injection of adriamycin (ADR, 10 mg/kg) and was infected with lentivirus overexpressing Fbxo11 (lenti-Fbxo11-OE). Mouse podocytes (MPC-5) were infected with lenti-Fbxo11-OE, followed by treatment with 0.4 μg/mL of ADR. We identified the decreased expression of Fbxo11 in mouse renal tissues and MPC-5 cells induced by ADR. Lenti-Fbxo11-OE intervention relieved ADR-induced glomerular lesion, podocyte injury, and mitochondrial dysfunction. In vitro, overexpression of Fbxo11 in mouse podocytes improved mitochondrial function and reduced podocyte damage, thereby inhibiting podocyte apoptosis. Mechanistically, Fbxo11 decreased the protein expression of Fosl2 through ubiquitin-dependent proteasomal degradation. Rescue experiments revealed that overexpression of Fosl2 abolished the protective effects of Fbxo11 overexpression on mitochondrial damage and podocyte injury. Importantly, the regulatory effects of the Fbxo11/Fosl2 axis were reversed when treated with the mitochondrial fission inhibitor mdivi-1. Taken together, our results demonstrated that Fbxo11-mediated protein degradation of Fosl2 is critical for maintaining mitochondrial function and preventing podocyte injury during NS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

STING activation by cyclic dinucleotides induces IRF3- and NF-κB-mediated gene expression in mammals, as well as lipidation of LC3B at Golgi-related membranes. While mechanisms of the IRF3 response are well understood, the mechanisms of NF-κB activation via STING remain unclear. We report here that STING activation induces linear/M1-linked ubiquitin chain (M1-Ub) formation and recruitment of the LUBAC E3 ligase, HOIP, to LC3B-associated Golgi membranes where ubiquitin is also localized. Loss of HOIP prevents formation of M1-Ub chains and reduces STING-induced NF-κB and IRF3 signaling in human THP1 monocytes and mouse bone marrow-derived macrophages, without affecting STING activation. STING-induced LC3B lipidation is not required for M1-Ub chain formation or for immune-related gene expression, but the recently reported STING function in neutralizing Golgi pH may be involved. Thus, LUBAC synthesis of M1-linked ubiquitin chains mediates STING-induced innate immune signaling., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)