Copper, an indispensable trace element in living organisms, plays a pivotal role in human physiological processes. Wilson's disease (WD), an inherited disorder of copper metabolism, is caused by mutations in the ATP7B gene. This genetic malfunction disrupts the dynamics of copper transport and metabolism, thereby impairing ceruloplasmin synthesis and copper excretion. The resultant accumulation of copper in various tissues and organs precipitates a cascade of cellular demise and functional impairment. Notably, cuproptosis, a recently discovered copper‑dependent regulated cell death mechanism, distinctly deviates from conventional cell death paradigms. This novel mode of cell death involves the interaction of copper with lipoacylated proteins within the tricarboxylic acid cycle, leading to proteinotoxic stress and culminating in cell death. In the realm of pathophysiology, cuproptosis has emerged as a pivotal player in a spectrum of diseases, with WD standing as a paradigm closely intertwined with the dysregulation of copper metabolism. This study aimed to encapsulate the pivotal molecular underpinnings of cuproptosis and delve into its crucial involvement in the etiopathogenesis of WD. By elucidating these mechanisms, the present analysis contributes significantly to the nuanced understanding of the pathological underpinnings of WD, thereby providing fresh insights and evidence that may direct innovative therapeutic strategies for this condition.

Plant immunity involves complex regulatory mechanisms that mediate the activation of defense responses against pathogens. Protein degradation via ubiquitination plays a crucial role in modulating these defenses, with E3 ubiquitin ligases functioning as central regulators. This study investigates the role of SlATL2, an ARABIDOPSIS TÓXICOS EN LEVADURA (ATL)-type E3 ubiquitin ligase localized in the plasma membrane, in the immune response of tomato plants against Pseudomonas syringae pv. tomato (Pst) DC3000. Our findings demonstrate that SlATL2 expression is induced upon Pst DC3000 infection and treatment with defense hormones salicylic acid and jasmonic acid. Functionally, SlATL2 negatively regulates immune responses, impairing resistance to Pst DC3000 and suppressing flg22-triggered immunity. In addition, SlATL2 limits pathogen-induced reactive oxygen species and callose accumulation by targeting the COP9 signalosome subunit 5a (SlCSN5a), a key positive regulator of tomato defense responses against Pst DC3000. This interaction, which occurs via the N-terminal residue of SlATL2, results in the ubiquitination and 26S proteasomal degradation of SlCSN5a, thereby suppressing SA-dependent expression of defense response genes associated and limiting reactive oxygen species production. This work sheds light on the molecular mechanism through which the E3 ubiquitin ligase SlATL2 attenuates tomato immune responses by targeting a COP9 signalosome subunit for degradation. These discoveries deepen our insights into the post-translational mechanisms governing plant immune responses and provide fresh opportunities to bolster crop resistance against bacterial pathogens.

Bladder cancer (BC) is a common urological malignancy for which effective treatments are lacking. In recent years, valosin-containing protein (VCP) has emerged as a potential target for the treatment of cancers. CB-5083 is a VCP inhibitor that has been evaluated in phase I clinical trials. However, drug resistance and severe side effects hamper the application of CB-5083. Mounting evidence suggests that combined treatment is a useful strategy to improve anticancer efficiency with lower toxicity. The aim of this study was to evaluate the combined effects of CB-5083 and luteolin (Lut), a natural flavonoid, on BC cells.

Cellular viability was measured via MTT assays. The cell cycle distribution, degree of cell death and mitochondrial membrane potential were assayed via flow cytometry. mRNA levels were assayed via qRT-PCR. Protein levels were measured via western blotting. RNA interference was applied to knockdown genes. Xenograft experiments were conducted to evaluate the toxicity in vivo.

Cotreatment with CB-5083 and luteolin synergistically reduced the viability of BC cells. In addition, cotreatment with CB-5083 and Lut synergistically induced cell cycle arrest at the G1 phase and apoptosis in BC cells. Mechanistically, CB-5083/Lut cooperatively reduced the expression of Bcl-xl and Mcl-1 in BC cells. Moreover, CB-5083 and Lut synergistically induced endoplasmic reticulum (ER) stress in BC cells. The genetic or pharmacological inhibition of ER stress markedly reduced the degree of apoptosis induced by CB-5083, Lut or their combination in BC cells. In addition, combined treatment with CB-5083 and Lut synergistically repressed the growth of BC cells in vivo.

Our data suggest that combined treatment with CB-5083 and Lut might be applied to treat BC.

Penaeus vannamei hemocyanin (PvHMC) exhibits multifunctional roles in immunity, often mediated by various post-translational modifications. While linear ubiquitination catalyzed by LUBAC in mammals regulates immune signaling, its role in crustacean immunity remains unclear. Here, we investigated the regulatory mechanism of PvHMC linear ubiquitination mediated by an E3 ligase PvLUBEL (a HOIP homolog), with emphasis on its role in shrimp immunity defending against diverse pathogens. We detected linear ubiquitination of PvHMC in multiple tissues, including hemocytes and the hepatopancreas. During Vibrio parahaemolyticus infection, the expression of PvLUBEL and the level of PvHMC linear ubiquitination were suppressed, whereas infection by white spot syndrome virus (WSSV) led to their upregulation. Structural analyses revealed that PvLUBEL, which shares a conserved RING-IBR-RING (RBR) domain with mammalian HOIP, serves as the catalytic subunit. Notably, inhibition of PvLUBEL via HOIPIN-1 (a covalent inhibitor) or RNA interference (RNAi) significantly reduced PvHMC linear ubiquitination, thereby increasing pathogen proliferation and decreasing host survival. These findings unveil a novel post-translational regulatory mechanism in which PvLUBEL-mediated linear ubiquitination of PvHMC underpins the shrimp immune response against aquaculture pathogens.

PROTACs that degrade target proteins to treat diseases represent a highly promising strategy in drug design. However, the degradation of target proteins in nondisease tissues may lead to systemic toxicity. Herein, capitalizing on the characteristic overexpression of PSMA in prostate cancer, we devised PSMA-guided PROTACs-specific targeting to prostate cancer. By conjugation of AR degraders and BET degraders separately with PSMA ligands via cleavable linkers, two classes of PSMA-guided PROTACs were obtained. In vitro experiments demonstrated that PSMA-guided PROTACs selectively degraded target proteins in PSMA-overexpressing prostate cancer cells without affecting target proteins in other cells. In vivo studies revealed that compared to conventional PROTACs, PSMA-guided PROTACs enhanced drug exposure in prostate cancer tissues and prolonged half-life and consequently achieved stronger and more sustained therapeutic effects. This work provides a novel avenue for disease tissue-specific PROTAC research, holding significant implications for targeted therapy in prostate cancer.

The efficient induction of antigen-specific CD8+ T cell activation is crucial in the development of mRNA tumor vaccines. Endogenous antigens are primarily degraded through the ubiquitin-proteasome system, followed by antigen presentation via major histocompatibility complex class I (MHC-I) molecules, leading to the activation of CD8+ T cells. Therefore, in this study, a novel mRNA vaccine was developed by fusing the mRNA sequence encoding the antigen with a proteasome-targeting peptide (PTP), aiming to enhance proteasomal targeting of the antigen and facilitate its degradation through the ubiquitin-proteasome system, thereby inducing a stronger CD8+ T cell immune response. This study confirmed a significant increase in antigen expression of the antigen-PTP fused mRNA vaccine upon treatment with a VHL inhibitor, as well as notable upregulation of genes associated with the MHC-I antigen-presenting pathway following treatment with the antigen-PTP fused mRNA vaccine. The intramuscular administration of the antigen-PTP fused mRNA vaccine significantly promoted the activation of dendritic cells, macrophages, and T cells in draining lymph nodes and spleens. Additionally, in TC-1 tumor-bearing mice, it markedly suppressed tumor growth, facilitated infiltration of intratumoral antigen-specific CD8+ T cells, and induced immune memory.

The Speckle-type POZ protein (SPOP), a substrate adaptor of the cullin-RING E3 ligase complex, mediates both the degradation and non-degradative ubiquitination of substrates, which are crucial for regulating various biological functions and cellular processes. Dysregulation of SPOP-mediated ubiquitination has been implicated in several cancers. Emerging evidence suggests that SPOP functions as a double-edged sword: acting as a tumor suppressor in prostate cancer (PCa), hepatocellular carcinoma (HCC), and colorectal cancer (CRC), while potentially serving as an oncoprotein in kidney cancer (KC). Therefore, SPOP's role in tumorigenesis appears to be tissue- or context-dependent. Numerous downstream substrates of SPOP have been identified across various cancers, where they regulate carcinogenesis, metabolic reprogramming, cell death, immune evasion, therapy resistance, and tumor microenvironment (TME) remodeling. However, the definitive role of SPOP in these cancers requires further investigation. A comprehensive understanding of the molecular mechanisms of SPOP in different cancer types will provide new insights into its function in oncogenesis, potentially advancing anti-cancer drug development. Here, we summarize the latest findings on SPOP's functions and structural features, its regulatory mechanisms, the roles of its substrates in various cancers, and SPOP-targeting strategies.

E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) are pivotal regulators of bone homeostasis, orchestrating osteoblast differentiation, proliferation, and osteoclast activity by controlling protein degradation and stability. This review delineates the roles of key E3 ligases (e.g., Smurf1, Smurf2, TRIM family) and DUBs (e.g., USP family) in bone formation and resorption. E3 ligases such as Smurf1/2 inhibit osteogenesis by degrading BMP/Smad signaling components, while TRIM proteins and HERC ligases promote osteoblast differentiation. Conversely, DUBs like USP2 and USP34 stabilize β-catenin and Smad1/RUNX2, enhancing osteogenic pathways, whereas USP10 and USP12 suppress differentiation. Dysregulation of these enzymes contributes to osteoporosis, fracture non-union, and other bone disorders. The interplay between ubiquitination and deubiquitination, alongside the regulatory role of miRNA and environmental factors, underscores their therapeutic potential. Future research should focus on developing therapies targeting E3 ubiquitin ligases, deubiquitinases, miRNA regulators, and small-molecule inhibitors to restore bone homeostasis in osteoporosis and fracture healing disorders.

TIP60 is a crucial lysine acetyltransferase protein that catalyzes the acetylation of histone and non-histone proteins. This enzyme plays a crucial role in maintaining genomic integrity, by participating in DNA damage repair, ensuring accurate chromosomal segregation, and regulating a myriad of cellular processes such as apoptosis, autophagy, and wound-induced cell migration. One of the primary mechanisms through which TIP60 executes these diverse cellular functions is via post-translational modifications (PTMs). Over the years, extensive studies have demonstrated the importance of PTMs in controlling protein functions. This review aims to summarize the findings on PTMs occurring on the TIP60 protein and their functional implications. We also discuss previously uncharacterized PTM sites identified on TIP60 and examine their relationship with cancer-associated mutations, with a particular focus on residues potentially modified by various PTMs, to understand the cause of deregulation of TIP60 in various cancers.

Ubiquitination plays vital roles in modulating pathogen-host cell interactions. RNF213, a E3 ligase, can catalyze the ubiquitination of lipopolysaccharide (LPS) and is crucial for antibacterial immunity in mammals. Shigella flexneri, an LPS-containing pathogenic bacterium, has developed mechanisms to evade host antibacterial defenses during infection. However, the precise strategies by which S. flexneri circumvents RNF213-mediated antibacterial immunity remain poorly understood. Here, through comprehensive biochemical, structural and cellular analyses, we reveal that the E3 effector IpaH1.4 of S. flexneri can directly target human RNF213 via a specific interaction between the IpaH1.4 LRR domain and the RING domain of RNF213, and mediate the ubiquitination and proteasomal degradation of RNF213 in cells. Furthermore, we determine the cryo-EM structure of human RNF213 and the crystal structure of the IpaH1.4 LRR/RNF213 RING complex, elucidating the molecular mechanism underlying the specific recognition of RNF213 by IpaH1.4. Finally, our cell based functional assays demonstrate that the targeting of host RNF213 by IpaH1.4 promotes S. flexneri proliferation within infected cells. In summary, our work uncovers an unprecedented strategy employed by S. flexneri to subvert the key host immune factor RNF213, thereby facilitating bacterial proliferation during invasion.

Motile cilia are organelles found on many eukaryotic cells that play critical roles in development and fertility. Human CFAP298 has been implicated in the transport/assembly of ciliary dyneins, and defects in this protein cause primary ciliary dyskinesia. However, neither the exact function nor the structure of CFAP298 have been elucidated. Here, we took advantage of Chlamydomonas, a ciliated alga, to study the structure and function of FBB18, an ortholog of CFAP298. Multiple ciliary dyneins were greatly reduced in cilia of Chlamydomonas fbb18 mutants. In addition, we found that both the stability of ciliary dynein heavy chains (HCs) and the association between HCs and intermediate/light chains (IC/LCs) are greatly reduced in fbb18 cytoplasm, strongly suggesting that FBB18 functions in the cytoplasmic assembly (the so-called "preassembly") of dynein complexes from HC/IC/LCs. Furthermore, X-ray crystallography revealed that FBB18 forms a bilobed structure with globular domains at both ends of the molecule, connected by an α-helical bundle. Unexpectedly, one globular domain shows high similarity to ubiquitin, a small protein critical for the modification of a variety of protein complexes, and this ubiquitin-like domain is indispensable for the molecular function of FBB18. Our results demonstrate that FBB18, a specialized member of the ubiquitin-like protein family, plays a critical role in dynein preassembly, most likely by mediating diverse interactions between dynein HCs, molecular chaperone(s), and other preassembly factor(s) using the ubiquitin-like domain as well as other regions, and by facilitating the proper folding of dynein HCs.

Endoreplication is characterized by multiple rounds of DNA replication without cell division and determines the growth and final size of endoreplicating cells and tissues in eukaryotes. The cyclic ubiquitination and degradation of several cell cycle regulators are required for endoreplication progression. However, the deubiquitinase that deubiquitinates and stabilizes key factors to modulate endoreplication remains unknown. Here, we found in the endoreplicating Drosophila salivary gland and Bombyx silk gland that the depletion of ubiquitin-specific peptidase 8 (USP8) led to endoreplication arrest and a decrease in gland size. Mechanistically, we showed that USP8 interacted with the Fizzy-related (Fzr) protein, a conserved master regulator of endoreplication, thereby deubiquitinating and stabilizing Fzr to modulate endoreplication. Moreover, the molecular chaperone heat shock protein 70 (Hsp70) mediated proper folding of Fzr and increased the interaction between Fzr and USP8, thereby promoting the deubiquitination and stabilization of Fzr. Together, our study demonstrates that USP8 and Hsp70 regulate endoreplication by synergistically maintaining Fzr stability though deubiquitination.

Proteasomes, essential protease complexes in protein homeostasis, adapt to metabolic changes through intracellular movements. As the executive arm of the ubiquitin-proteasome system, they selectively degrade poly-ubiquitinated proteins in an ATP-dependent process. The primary proteasome configuration involved in this degradation is the 26S proteasome, which is composed of a proteolytically active core particle flanked by two regulatory particles. In metabolically active cells, such as proliferating yeast and mammalian cancer cells, 26S proteasomes are predominantly nuclear and actively engaged in protein degradation. However, during nutrient deprivation or stress-induced quiescence, proteasome localization changes. In quiescent yeast, proteasomes initially accumulate at the nuclear envelope. During prolonged quiescence with decreased ATP levels, proteasomes exit the nucleus and are sequestered into cytoplasmic membraneless organelles, so-called proteasome storage granules (PSGs). In mammalian cells, starvation and stress trigger formation of membraneless organelles containing proteasomes and poly-ubiquitinated substrates. The proteasome condensates are motile, reversible, and contribute to stress resistance and improved fitness during aging. Proteasome condensation may involve liquid-liquid phase separation, a mechanism underlying the assembly of membraneless organelles.

The E2/E3 hybrid enzyme UBE2O plays important roles in key biological events, but its autoubiquitination mechanism remains largely unclear. In this study, we determined the crystal structures of full-length (FL) UBE2O from Trametes pubescens (tp) and its ubiquitin-conjugating (UBC) domain. The dimeric FL-tpUBE2O structure revealed interdomain interactions between the conserved regions (CR1-CR2) and UBC. The dimeric intermolecular and canonical ubiquitin/UBC interactions are mechanistically important for UBE2O functions in catalyzing the formation of free polyubiquitin chains and substrate ubiquitination. Beyond dimerization, autoubiquitination within the CR1-CR2 domain also regulates tpUBE2O activity. Additionally, we show that tpUBE2O catalyzes the formation of all seven types of polyubiquitin chains in vitro. The CR1-CR2/UBC and canonical ubiquitin/UBC interactions are important for the polyubiquitination of AMP-activated protein kinase α2 (AMPKα2) by human UBE2O (hUBE2O), which leads to tumorigenesis. These structural insights lay the groundwork for understanding UBE2O's mechanisms and developing structure-based therapeutics targeting UBE2O.

Abscisic acid insensitive 5 (ABI5) is a pivotal transcription factor in abscisic acid (ABA) signaling, playing an essential role in plant growth and responses to abiotic stress. This key regulator is subject to multifaceted regulation, especially on post-translational mechanisms. Recent research has shed light on the post-translational regulation of ABI5, encompassing both post-translational modifications (PTMs) and the modulation of its transcriptional activity. In this review, we provide a comprehensive overview of the current knowledge surrounding the post-translational regulation of ABI5, along with the influence of various cofactors on its transcriptional activity and protein stability. The potential biological roles of PTMs of ABI5 in the context of ABA signaling and plant stress responses are also explored. As ABI5 is one of the most extensively studied proteins in the context of plant ABA signaling and environmental stress responses, a sophisticated and precise understanding of the regulatory mechanisms that govern ABI5 is not only beneficial for its application in genetic engineering but also helpful for our exploration in the fundamental principles of post-translational regulation.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of cellular homeostasis, overseeing the expression of a wide array of genes involved in cytoprotective processes such as antioxidant and proteostasis control, mitochondrial function, inflammation, and the metabolism of lipids and glucose. The accumulation of misfolded proteins triggers the release, stabilization, and nuclear translocation of NRF2, which in turn enhances the expression of critical components of both the proteasomal and lysosomal degradation pathways. This process facilitates the clearance of toxic protein aggregates, thereby actively maintaining cellular proteostasis. As we age, the efficiency of the NRF2 pathway declines due to several factors including increased activity of its repressors, impaired NRF2-mediated antioxidant and cytoprotective gene expression, and potential epigenetic changes, though the precise mechanisms remain unclear. This leads to diminished antioxidant defenses, increased oxidative damage, and exacerbated metabolic dysregulation and inflammation-key contributors to age-related diseases. Given NRF2's role in mitigating proteotoxic stress, the pharmacological modulation of NRF2 has emerged as a promising therapeutic strategy, even in aged preclinical models. By inducing NRF2, it is possible to mitigate the damaging effects of oxidative stress, metabolic dysfunction, and inflammation, thus reducing protein misfolding. The review highlights NRF2's therapeutic implications for neurodegenerative diseases and cardiovascular conditions, emphasizing its role in improving proteostasis and redox homeostasis Additionally, it summarizes current research into NRF2 as a therapeutic target, offering hope for innovative treatments to counteract the effects of aging and associated diseases.

Hypoxia Inducible transcription Factors (HIFs) are central to the metazoan oxygen-sensing response. Under low oxygen conditions (hypoxia), HIFs are stabilised and govern an adaptive transcriptional programme to cope with prolonged oxygen starvation. However, when oxygen is present, HIFs are continuously degraded by the proteasome in a process involving prolyl hydroxylation and subsequent ubiquitination by the Von Hippel Lindau (VHL) E3 ligase. The essential nature of VHL in the HIF response is well established but the role of other enzymes involved in ubiquitination is less clear. Deubiquitinating enzymes (DUBs) counteract ubiquitination and provide an important regulatory aspect to many signalling pathways involving ubiquitination. In this review, we look at the complex network of ubiquitination and deubiquitination in controlling HIF signalling in normal and low oxygen tensions. We discuss the relative importance of DUBs in opposing VHL, and explore roles of DUBs more broadly in hypoxia, in both VHL and HIF independent contexts. We also consider the catalytic and non-catalytic roles of DUBs, and elaborate on the potential benefits and challenges of inhibiting these enzymes for therapeutic use.

The co-evolution of plants and pathogens has enabled them to 'outsmart' each other by promoting their own defence responses and suppressing those of the other. While plants are reliant on their sophisticated immune signalling pathways, pathogens make use of effector proteins to achieve the objective. This entails rapid regulation of underlying molecular mechanisms for prompt induction of associated signalling events in both plants as well as pathogens. The past decade has witnessed the emergence of post-translational modification (PTM) of proteins as a key a factor in modulating cellular responses. The ability of PTMs to expand the functional diversity of the proteome and induce rapid changes at the appropriate time enables them to play crucial roles in the regulation of plant-pathogen interactions. Therefore, this review will delve into the intricate interplay of five major PTMs involved in plant defence and pathogen countermeasures. We discuss how plants employ PTMs to fortify their immune networks, and how pathogen effectors utilize/target host modification systems to gain entry into plants and cause disease. We also emphasize the need for identification of novel PTMs and propose the use of PTM pathways as potential targets for genome editing approaches.

This study aimed to elucidate the role of USP25 in a mouse model of anti-glomerular basement membrane glomerulonephritis (anti-GBM GN).

USP25-deficient anti-GBM GN mice were generated, and their nephritis progression was monitored. Naïve CD4+ T cells were isolated from spleen lymphocytes and stimulated to differentiate into Th1, Th2, and Th17 cells. This approach was used to investigate the impact of USP25 on CD4+ T lymphocyte differentiation in vitro. Furthermore, changes in USP25 expression were monitored during Th17 differentiation, both in vivo and in vitro.

USP25-/- mice with anti-GBM GN exhibited accelerated renal function deterioration, increased infiltration of Th1 and Th17 cells, and elevated RORγt transcription. In vitro experiments demonstrated that USP25-/- CD4+ T lymphocytes had a higher proportion for Th17 cell differentiation and exhibited higher RORγt levels upon stimulation. Wild-type mice with anti-GBM GN showed higher USP25 levels compared to healthy mice, and a positive correlation was observed between USP25 levels and Th17 cell counts. Similar trends were observed in vitro.

USP25 plays a crucial role in mitigating renal histopathological and functional damage during anti-GBM GN in mice. This protective effect is primarily attributed to USP25's ability to inhibit the differentiation of naïve CD4+ T cells into Th17 cells. The underlying mechanism may involve the downregulation of RORγt. Additionally, during increased inflammatory responses or Th17 cell differentiation, USP25 expression is activated, forming a negative feedback regulatory loop that attenuates immune activation.

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

Ubiquitin C-terminal hydrolase 3 (UCHL3) is a cysteine protease that plays a crucial role in cell cycle regulation, DNA repair, and apoptosis by carrying out deubiquitination and deneddylation activities. It has emerged as a promising therapeutic target for certain cancers due to its ability to stabilize oncoproteins. The dysregulation of UCHL3 also has been associated with neurodegenerative diseases, underscoring its significance in maintaining protein homeostasis within cells. Research on UCHL3, including studies on Uchl3 knockout mice, has revealed its involvement in learning deficits, cellular stress responses, and retinal degeneration. This review delves into the cellular processes controlled by UCHL3 and its role in health and disease progression, as well as the development of UCHL3 inhibitors. Further investigation into the molecular mechanisms and physiological functions of UCHL3 is crucial for a comprehensive understanding of its impact on health and disease.

Hepatitis B virus is one of the leading causes behind the neoplastic transformation of liver tissue and associated mortality. Despite the availability of many therapies and vaccines, the pathogenic landscape of the virus remains elusive; urging the development of novel strategies based on the fundamental infectious and transformative modalities of the virus-host interactome. Ubiquitination is a widely observed post-translational modification of several proteins, which either regulates the proteins' turnover or impacts their functionalities. In recent years, ample amount of literature has accumulated regarding the ubiquitination dynamics of the HBV proteins as well as the host proteins during HBV infection and carcinogenesis; with direct and detailed characterization of the involvement of HBV in these processes. Interestingly, while many of these ubiquitination events restrict HBV life cycle and carcinogenesis, several others promote the emergence of hepatocarcinoma by putting the virus in an advantageous position. This review sums up the snowballing literature on ubiquitination-mediated regulation of the host-HBV crosstalk, with special emphasis on its influence on the establishment and progression of hepatocellular carcinoma on a molecular level. With the advent of cutting-edge ubiquitination-targeted therapeutic approaches, the findings emanating from this review may potentiate the identification of novel anti-HBV targets for the formulation of novel anticancer strategies to control the HBV-induced hepato-carcinogenic process on a global scale.

Ubiquitin carboxyl-terminal hydrolase 19 (USP19) is a unique deubiquitinase, characterized by multiple variants generated by alternative splicing. Several variants bear a C-terminal transmembrane domain that anchors them to the endoplasmic reticulum. Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from endoplasmic reticulum-associated protein degradation in a catalytic-independent manner, promote autophagy, and address proteins to lysosomal degradation via endosomal microautophagy. USP19 has recently emerged as the protein responsible for the unconventional secretion of misfolded proteins including Parkinson's disease-associated protein α-synuclein. Despite mounting evidence that USP19 plays crucial roles in several biological processes, the underlying mechanisms are unclear due to lack of information on the physiological substrates of USP19. Herein, we used high-resolution quantitative proteomics to analyze changes in the secretome and cell proteome induced by the loss of USP19 to identify proteins whose secretion or turnover is regulated by USP19. We found that ablation of USP19 induced significant proteomic alterations both in and out of the cell. Loss of USP19 impaired the release of several lysosomal proteins, including legumain (LGMN) and several cathepsins. In order to understand the underlaying mechanism, we dissected the USP19-regulated secretion of LGMN in several cell types. We found that LGMN was not a deubiquitinase substrate of USP19 and that its USP19-dependent release did not require their direct interaction. LGMN secretion occurred by a mechanism that involved the Golgi apparatus, autophagosome formation, and lysosome function. This mechanism resembled the recently described "lysosomal exocytosis," by which lysosomal hydrolases are secreted, when ubiquitination of p62 is increased in cells lacking deubiquitinases such as USP15 and USP17. In conclusion, our proteomic characterization of USP19 has identified a collection of proteins in the secretome and within the cell that are regulated by USP19, which link USP19 to the secretion of lysosomal proteins, including LGMN.

Many reported proteasome inhibitors, including the three clinically approved inhibitors, bortezomib, carfilzomib, and ixazomib, have peptidic structures. In this study, using a hybrid and versatile strategy for covalent virtual screening by combining warhead screening and preprocessing with GOLD and CovDock software that were applied to the ZINC virtual library, we identified multiple proteasome inhibitors with new nonpeptidic structural scaffolds. Proteasome inhibition assays confirmed the inhibitory activities of these new compounds. These results demonstrate the effectiveness of our computational strategy for large-scale covalent virtual screening. Furthermore, these identified proteasome inhibitors may serve as starting points for the development of a new class of nonpeptidic therapeutic agents.

Ubiquitination is widely involved in the progression of hepatocellular carcinoma (HCC) by regulating various cellular processes. However, systematic strategies for screening core ubiquitin-related genes, clarifying their functions and mechanisms, and ultimately developing potential therapeutics for patients with HCC are still lacking.

Cox and LASSO regression analyses were performed to construct a ubiquitin-related gene prediction model for HCC. Loss- and gain-of-function studies, transcriptomic and metabolomics analysis were used to explore the function and mechanism of UBE2S on HCC cell glycolysis and growth.

Based on 1,423 ubiquitin-related genes, a four-gene signature was successfully constructed to evaluate the prognosis of patients with HCC. UBE2S was identified in this signature with the potential to predict the survival of patients with HCC. E2F2 transcriptionally upregulated UBE2S expression by directly binding to its promoter. UBE2S positively regulated glycolysis in a HIF-1α-dependent manner, thus promoting the proliferation of HCC cells. Mechanistically, UBE2S enhanced K11-linkage polyubiquitination at lysine residues 171 and 196 of VHL independent of E3 ligase, thereby indirectly stabilizing HIF-1α protein levels by mediating the degradation of VHL by the proteasome. In particular, the combination of cephalomannine, a small molecule compound that inhibits the expression of UBE2S, and PX-478, an inhibitor of HIF-1α, significantly improved the anti-tumor efficacy.

UBE2S is identified as a key biomarker in HCC among the thousands of ubiquitin-related genes and promotes glycolysis by E3 enzyme-independent ubiquitination, thus serving as a therapeutic target for the treatment of HCC.

The Nab-paclitaxel combined with gemcitabine (AG) regimen is the main chemotherapy regimen for pancreatic cancer, but drug resistance often occurs. Currently, the ability to promote sensitization in drug-resistant cases is an important clinical issue, and the strategy of repurposing conventional drugs is a promising strategy. This study aimed to identify a classic drug that targets chemotherapy resistance's core signaling pathways and combine it with the AG regimen to enhance chemosensitivity. We also aimed to find reliable predictive biomarkers of drug combination sensitivity. Using RNA sequencing, we found that abnormal PI3K/Akt pathway activation plays a central role in mediating resistance to the AG regimen. Subsequently, through internal and external verification of randomly selected AG-resistant patient-derived organoid (PDO) and PDO xenograft models, we discovered for the first time that the classic anti-inflammatory drug sulindac K-80003, an inhibitor of the PI3K/Akt pathway that we focused on, promoted sensitization in half (14/28) of AG-resistant pancreatic ductal adenocarcinoma cases. Through RNA-sequencing, multiplex immunofluorescent staining, and immunohistochemistry experiments, we identified cFAM124A as a novel biomarker through which sulindac K-80003 promotes AG sensitization. Its role as a sensitization marker is explained via the following mechanism: cFAM124A enhances both the mRNA expression of cathepsin L and the activity of the cathepsin L enzyme. This dual effect stimulates the cleavage of RXRα, leading to large amounts of truncated RXRα, which serves as a direct target of K-80003. Consequently, this process results in the pathological activation of the PI3K/Akt pathway. In summary, our study provides a new treatment strategy and novel biological target for patients with drug-resistant pancreatic cancer.

Based on the recent evidence of IL-1 inhibition in patients with rheumatoid arthritis (RA) and concomitant type 2 diabetes (T2D), we evaluated the synovial tissue expression of IL-1 related genes in relationship to the ubiquitin-proteasome system and the effects of insulin on ubiquitinated proteins in fibroblast-like synoviocytes (FLSs).

The synovial expression of IL-1 pathway genes was compared in early (< 1 year) treatment-naïve RA patients with T2D (RA/T2D n = 16) and age- and sex-matched RA patients without T2D (n = 16), enrolled in the Pathobiology of Early Arthritis Cohort (PEAC). The synovial expression of ubiquitin in macrophages and synovial lining fibroblasts was also assessed by Immunohistochemistry/immunofluorescence and correlated with synovial pathotypes. Finally, FLSs from RA patients (n = 5) were isolated and treated with human insulin (200 and 500 nM) and ubiquitinated proteins were assessed by western blot.

Synovial tissues of RA/T2D patients were characterised by a consistent reduced expression of ubiquitin-proteasome genes. More specifically, ubiquitin genes (UBB, UBC, and UBA52) and genes codifying proteasome subunits (PSMA2, PSMA6, PSMA7, PSMB1, PSMB3, PSMB4, PSMB6, PSMB8, PSMB9, PSMB10, PSMC1, PSMD9, PSME1, and PSME2) were significantly lower in RA/T2D patients. On the contrary, genes regulating fibroblast functions (FGF7, FGF10, FRS2, FGFR3, and SOS1), and genes linked to IL-1 pathway hyper-activity (APP, IRAK2, and OSMR) were upregulated in RA/T2D. Immunohistochemistry showed a significant reduction of the percentage of ubiquitin-positive cells in synovial tissues of RA/T2D patients. Ubiquitin-positive cells were also increased in patients with a lympho-myeloid pathotype compared to diffuse myeloid or pauci-immune-fibroid. Finally, in vitro experiments showed a reduction of ubiquitinated proteins in RA-FLSs treated with a high concentration of insulin (500 nM).

A different IL-1 pathway gene expression was observed in the synovial tissues of early treatment-naïve RA/T2D patients, linked to decreased expression of the ubiquitin-proteasome system. These findings may provide a mechanistic explanation of the observed clinical benefits of IL-1 inhibition in patients with RA and concomitant T2D.

Rad6 is a canonical ubiquitin-conjugating enzyme known for its role in regulating chromosome-related cellular processes in yeast and has been proven to have multiple functions in Beauveria bassiana, including insect-pathogenic lifestyle, UV damage repair, and conidiation. However, previous studies have only reported the key role of Rad6 in regulating conidial production in a nutrient-rich medium, without any deep mechanism analyses. In this study, we found that the disruption of Rad6 leads to a profound reduction in conidial production, irrespective of whether the fungus is cultivated in nutrient-rich or nutrient-poor environments. The absence of rad6 exerts a suppressive effect on the transcription of essential genes in the central developmental pathway, namely, brlA, abaA, and wetA, resulting in a direct downregulation of conidiation capacity. Additionally, mutant strains exhibited a more pronounced decline in both conidial generation and hyphal development when cultured in nutrient-rich conditions. This observation correlates with the downregulation of the central developmental pathway (CDP) downstream gene vosA and the upregulation of flaA in nutrient-rich cultures. Moreover, single-transcriptomics analyses indicated that irregularities in biotin metabolism, DNA repair, and tryptophan metabolism are the underlying factors contributing to the reduced conidial production. Comprehensive dual transcriptomics analyses pinpointed abnormal biotin metabolism as the primary cause of conidial production decline. Subsequently, we successfully restored conidial production in the Rad6 mutant strain through the supplementation of biotin, further confirming the transcriptomic evidence. Altogether, our findings underscore the pivotal role of Rad6 in influencing biotin metabolism, subsequently impacting the expression of CDP genes and ultimately shaping the asexual life cycle of B. bassiana.

The unfolding dynamics of ubiquitin were studied using a combination of x-ray solution scattering (XSS) and molecular dynamics (MD) simulations. The kinetic analysis of the XSS ubiquitin signals showed that the protein unfolds through a two-state process, independent of the presence of destabilizing salts. In order to characterize the ensemble of unfolded states in atomic detail, the experimental XSS results were used as a constraint in the MD simulations through the incorporation of x-ray scattering derived potential to drive the folded ubiquitin structure toward sampling unfolded states consistent with the XSS signals. We detail how biased MD simulations provide insight into unfolded states that are otherwise difficult to resolve and underscore how experimental XSS data can be combined with MD to efficiently sample structures away from the native state. Our results indicate that ubiquitin samples unfolded in states with a high degree of loss in secondary structure yet without a collapse to a molten globule or fully solvated extended chain. Finally, we propose how using biased-MD can significantly decrease the computational time and resources required to sample experimentally relevant nonequilibrium states.

Radiation-induced pneumonitis (RIP) seriously limits the application of radiation therapy in the treatment of thoracic tumors, and its etiology and pathogenesis remain elusive. This study aimed to elucidate the role of ubiquitin-specific peptidase 11 (USP11) in the progression of RIP and the associated underlying mechanisms.

Changes in cytokines and infiltrated immune cells were detected by enzyme-linked immunosorbent assays and immunohistochemistry after exposure to 20 Gy x-ray with whole-thorax irradiation. The effects of USP11 expression on endothelial cell proliferation and apoptosis were analyzed by costaining of CD31/Ki67 and CD31/caspase-3 in vivo, and the production of cytokines and reactive oxygen species was confirmed by reverse-transcription polymerase chain reaction and flow cytometry in vitro. Comprehensive proteome and ubiquitinome analyses were used for USP11 substrate screening after radiation. Results were verified by Western blotting and coimmunoprecipitation experiments. Recombinant adeno-associated virus lung vectors expressing OTUD5 were used for localized overexpression of OTUD5 in mouse pulmonary tissue, and immunohistochemistry was conducted to analyze cytokine expression.

The progression of RIP was significantly alleviated by reduced expression of proinflammatory cytokines in both Usp11-knockout (Usp11-/-) mice and in mice treated with the USP11 inhibitor mitoxantrone. Likewise, the absence of USP11 resulted in decreased permeability of pulmonary vessels and neutrophils and macrophage infiltration. The proliferation rates of endothelial cells were prominently increased in the Usp11-/- lung, whereas apoptosis in Usp11-/- lungs decreased after irradiation compared with that observed in Usp11+/+ lungs. Conversely, USP11 overexpression increased proinflammatory cytokine expression and reactive oxygen species production in endothelial cells after radiation. Comprehensive proteome and ubiquitinome analyses indicated that USP11 overexpression upregulates the expression of several deubiquitinating enzymes, including USP22, USP33, and OTUD5. We demonstrate that USP11 deubiquitinates OTUD5 and implicates the OTUD5-STING signaling pathway in the progression of the inflammatory response in endothelial cells.

USP11 exacerbates RIP by triggering an inflammatory response in endothelial cells both in vitro and in vivo, and the OTUD5-STING pathway is involved in the USP11-dependent promotion of RIP. This study provides experimental support for the development of precision intervention strategies targeting USP11 to mitigate RIP.

Gliomas are highly malignant and invasive tumors lacking clear boundaries. Previous bioinformatics and experimental analyses have indicated that F-box and leucine-rich repeat protein 6 (FBXL6), a protein crucial for the cell cycle and tumorigenesis, is highly expressed in certain types of tumors. The high expression level of FBXL6 is reported to promote tumor growth and adversely affect patient survival. However, the molecular mechanism, prognostic value and drug sensitivity of FBXL6 in glioma remain unclear. To address this, the present study analyzed FBXL6 expression in gliomas, utilizing data from The Cancer Genome Atlas and Chinese Glioma Genome Atlas databases. Analysis of FBXL6 mRNA expression levels, combined with patient factors such as age, sex and tumor grade using Kaplan-Meier plots and nomograms, demonstrated a strong correlation between FBXL6 expression and glioma progression. Co-expression networks provided further insights into the biological function of FBXL6. Additionally, using CIBERSORT and TISDB tools, the correlation between FBXL6 expression correlation tumor-infiltrating immune cells and immune genes was demonstrated to be statistically significant. These findings were validated by examining FBXL6 mRNA and protein levels in glioma tissues using various techniques, including western blot, reverse transcription-quantitative PCR and immunohistochemistry. These assays demonstrated the role of FBXL6 in glioma progression. Furthermore, drug sensitivity analysis demonstrated a strong correlation between FBXL6 expression and various drugs, which indicated that FBXL6 may potentially act as a future promising therapeutic target in glioma treatment. Therefore, the present study identified FBXL6 as a diagnostic and prognostic marker in patients with gliomas and highlighted its potential role in glioma progression.

Histone H1 is involved in chromatin compaction and dynamics. In human cells, the H1 complement is formed by different amounts of somatic H1 subtypes, H1.0-H1.5 and H1X. The amount of each variant depends on the cell type, the cell cycle phase, and the time of development and can be altered in disease. However, the mechanisms regulating H1 protein levels have not been described. We have analyzed the contribution of the proteasome to the degradation of H1 subtypes in human cells using two different inhibitors: MG132 and bortezomib. H1 subtypes accumulate upon treatment with both drugs, indicating that the proteasome is involved in the regulation of H1 protein levels. Proteasome inhibition caused a global increase in cytoplasmatic H1, with slight changes in the composition of H1 bound to chromatin and chromatin accessibility and no alterations in the nucleosome repeat length. The analysis of the proteasome degradation pathway showed that H1 degradation is ubiquitin-independent. The whole protein and its C-terminal domain can be degraded directly by the 20S proteasome in vitro. Partial depletion of PA28γ revealed that this regulatory subunit contributes to H1 degradation within the cell. Our study shows that histone H1 protein levels are under tight regulation to prevent its accumulation in the nucleus. We revealed a new regulatory mechanism for histone H1 degradation, where the C-terminal disordered domain is responsible for its targeting and degradation by the 20S proteasome, a process enhanced by the regulatory subunit PA28γ.

Protein ubiquitination is a critical and widely distributed post-translational modification (PTM) involved in the regulation of almost every cellular process and pathway in cells, such as proteostasis, DNA repair, trafficking, and immunity. Mass spectrometry (MS)-based proteomics is a robust tool to decode the complexity of ubiquitin networks by disclosing the proteome-wide ubiquitination sites, the length, linkage and topology of ubiquitin chains, the chemical modification of ubiquitin chains, and the crosstalk between ubiquitination and other PTMs. In this chapter, we discuss the application of MS in the interpretation of the ubiquitin code.

The increasing incidence of metabolic diseases, including obesity, type 2 diabetes mellitus (T2DM), and non-alcoholic fatty liver disease (NAFLD), in the past decade is a serious concern worldwide. Disruption of cellular protein homeostasis has been considered as a crucial contributor to the pathogenesis of metabolic diseases. To maintain protein homeostasis, cells have evolved multiple dynamic and self-regulating quality control processes to adapt new environmental conditions and prevent prolonged damage. Among them, the ubiquitin proteasome system (UPS), the primary proteolytic pathway for degradation of aberrant proteins via ubiquitination, has an essential role in maintaining cellular homeostasis in response to intracellular stress. Correspondingly, accumulating evidences have shown that dysregulation of ubiquitination can aggravate various metabolic derangements in many tissues, including the liver, skeletal muscle, pancreas, and adipose tissue, and is involved in the initiation and progression of diverse metabolic diseases. In this part, we will summarize the role of ubiquitination in the pathogenesis of metabolic diseases, including obesity, T2DM and NAFLD, and discuss its potential as a therapeutic target.

In previous studies, downregulation of USP9Y and DDX3Y in lung cancer (LC) tissues was identified, while their function in LC progression remains elusive. In our current work, we intended to elucidate the effect and mechanisms of USP9Y and DDX3Y in LC. Gene downregulation has been confirmed in our LC tissues and cells. The effect of USP9Y or DDX3Y on LC cell malignancies was analyzed by functional assay. Both USP9Y and DDX3Y overexpression showed suppressive impact on LC cell malignancies. USP9Y overexpression has also been demonstrated to inhibit tumorigenesis in vivo. Based on GEPIA database, it was found that there was a positive correlation between the levels of USP9Y and DDX3Y in LC tissues. The mRNA expression of DDX3Y was not affected by USP9Y overexpression, while its protein levels were significantly up-regulated in USP9Y overexpressed LC cells. Moreover, USP9Y interacted with DDX3Y and has been demonstrated to stabilize DDX3Y expression by preventing its degradation via deubiquitination. In conclusion, USP9Y and DDX3Y exerted antioncogenic effects on the cell proliferation potential, cell cycle process, apoptosis, and tumorigenesis of LC. USP9Y binds to DDX3Y to prevent DDX3Y degradation through deubiquitination.

RNA-dependent RNA polymerase (RdRp) is a good target of anti-RNA virus agents; not only it is pivotal for the RNA virus replication cycle and highly conserved among RNA viruses across different families, but also lacks human homolog. Recently, human single-chain antibody (HuscFv) that bound to thumb domain of hepatitis C virus (HCV) RNA-dependent RNA polymerase (functionalized NS5B protein) was produced and engineered into cell-penetrating antibody (super antibody) in the form of cell-penetrating peptide (penetratin, PEN)-linked HuscFv (PEN-HuscFv34). The super antibody was produced and purified from inclusion body (IB) of a pen-huscfv34-vector-transformed Escherichia coli. The super antibody inhibited replication of alpha- and beta- coronaviruses, flaviviruses, and picornaviruses that were tested (broadly effective); thus, it has high potential for developing further towards a pan-anti-RNA virus agent. However, production, purification, and refolding of the super antibody molecules from the bacterial IB are laborious and hurdles to large-scale production. Therefore, in this study, Sortase-self-cleave method and bacteria surface display system were combined and modified for the super antibody production.

BL21 (DE3) ΔA E. coli, a strain lacking predominant outer membrane protein (OmpA) and ion and OmpT proteases, that displayed a membrane-anchored fusion protein, i.e., chimeric lipoprotein (Lpp')-OmpA', SUMO, Sortase protease, Sortase cleavage site (LPET↓G) and PEN-HuscFv34-6× His was generated. The soluble PEN-HuscFv34-6× His with glycine at the N-terminus could be released from the E. coli surface, simply by incubating the bacterial cells in a Sortase-cleavage buffer. After centrifugation, the G-PEN-HuscFv34-6× His could be purified from the supernatant. The purified G-PEN-HuscFv34-6× retained original cell-penetrating ability (being super antibody) and the broadly effective anti-RNA virus activity of the original IB-derived-PEN-HuscFv34.

The functionalized super antibody to RNA virus RdRp was successfully produced by using combined Sortase self-cleave and bacterial surface display systems with modification. The display system is suitable for downstream processing in a large-scale production of the super antibody. It is applicable also for production of other recombinant proteins in soluble free-folding form.

Genome stability is critical for normal functioning of cells, it depends on accuracy of DNA replication, chromosome segregation, and DNA repair. Cellular defense mechanisms against DNA damage are important for preventing cancer development and aging. The E3 ubiquitin ligase RNF168 of the RING superfamily is an essential component of the complex responsible for ubiquitination of the H2A/H2A.X histones near DNA double-strand breaks, which is a key step in attracting repair factors to the damage site. In this study, we unequivocally showed that RNF168 does not have the ability to directly distinguish architecture of polyubiquitin chains, except for the tropism of its two ubiquitin-binding domains UDM1/2 to K63 ubiquitin chains. Analysis of intracellular chromatosomal environment of the full-length RNF168 and its domains using the ligand-induced bioluminescence resonance energy transfer (BRET) revealed that the C-terminal part of UDM1 is associated with the K63 ubiquitin chains; RING and the N-terminal part of UDM2 are sterically close to the K63- and K48-ubiquitin chains, while the C-terminal part of UDM1 is co-localized with all possible ubiquitin variants. Our observations together with the available structural data suggest that the C-terminal part of UDM1 binds the K63 polyubiquitin chains on the linker histone H1; RING and the N-terminal part of UDM2 are located in the central part of nucleosome and sterically close to H1 and K48-ubiquitinated alternative substrates of RNF168, such as JMJD2A/B demethylases, while the C-terminal part of UDM1 is in the region of activated ubiquitin residue associated with E2 ubiquitin ligase, engaged by RNF168.