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Loss-of-function mutations in the human gene encoding the neuron-specific Ca2+ channel CaV2.1 are linked to the neurological disease episodic ataxia type 2 (EA2), as well as neurodevelopmental disorders such as developmental delay and developmental epileptic encephalopathy. Disease-associated CaV2.1 mutants may exhibit defective proteostasis and promote endoplasmic reticulum (ER)-associated degradation of their wild-type (WT) counterpart in a dominant-negative manner. The E3 ubiquitin ligase RNF138 was previously shown to mediate EA2-related aberrant degradation of CaV2.1 at the ER. Herein we aimed to elucidate the ER proteostasis mechanism of CaV2.1. The peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1) was identified as a novel neuronal CaV2.1 binding partner that promoted polyubiquitination and proteasomal degradation of CaV2.1. Suppression of endogenous Pin1 level with either shRNA knockdown or the Pin1 inhibitor all-trans retinoic acid (ATRA) enhanced endogenous CaV2.1 protein level in neurons, and attenuated ER-associated degradation of CaV2.1 WT and EA2-causing mutants. Detailed mutation analyses suggested that Pin1 interacted with specific phosphorylated serine/threonine-proline motifs in the intracellular II-III loop and the distal carboxy-terminal region of human CaV2.1. We further generated Pin1-insensitive CaV2.1 constructs and demonstrated that, during ER quality control, Pin1 served as an upstream regulator of CaV2.1 polyubiquitination and degradation by RNF138. Pin1 regulation was required for the dominant-negative effect of EA2 missense mutants, but not nonsense mutants, on CaV2.1 WT protein expression. Our data are consistent with the idea that CaV2.1 proteostasis at the ER, as well as dominant-negative suppression of disease-causing loss-of-function mutants on CaV2.1 WT, entail both Pin1/RNF138-dependent and -independent mechanisms. [ABSTRACT FROM AUTHOR]

Many proteins have been identified subject to ubiquitin (Ub)-independent proteasome degradation (UbIPD), a process mediated by Ub-like (UBL) proteins. UBL-conjugates can be processed by 19S proteasomes by mechanisms different from Ub-conjugates and by proteasome activator 28γ, which was previously thought to work as an activator of UbIPD. Ub and UBLs coordinate proteasome degradation to maintain protein homeostasis. UBL proteins, enzymes and modification substrates are tightly associated with proteostasis in muscle and neurodegenerative diseases. Although it is believed that ubiquitin (Ub) modification is required for protein degradation in the proteasome system (UPS), several proteins are subject to Ub-independent proteasome degradation, and in many cases ubiquitin-like (UBL) modifications, including neddylation, FAT10ylation, SUMOylation, ISGylation, and urmylation, are essential instead. In this Review, we focus on UBL-dependent proteasome degradation (UBLPD), on proteasome regulators especially shuttle factors and receptors, as well as potential competition and coordination with UPS. We propose that there is a distinct UBL–proteasome system (UBLPS) that might be underestimated in protein degradation. Finally, we investigate the association of UBLPD with muscle wasting and neurodegenerative diseases in which the proteasome is abnormally activated and impaired, respectively, and suggest strategies to modulate UBLPD for disease therapy. [ABSTRACT FROM AUTHOR]

Cervical cancer is predominantly driven by persistent infections with high‐risk human papillomavirus and the continuous activity of its E6 and E7 oncoproteins. This study explored the role of heat shock proteins 70 kDa (HSP70s) in enhancing the function of these oncoproteins and examined the impact of SHetA2, an investigational new drug, on this interaction. We found that HSP70 specifically binds to E7, but not E6, protein and that SHetA2 disrupts this binding. This disruption led to a significant reduction in E6 and E7 mRNA and E7 protein levels, while effects on E6 protein levels were minimal. SHetA2 treatment also resulted in altered levels of cell cycle regulatory proteins, reduced cell cycle progression, and decreased metabolic viability in cervical cancer cell lines and xenograft models. These findings support the potential of SHetA2 to impair cervical cancer progression by targeting HSP70/E7 interactions, highlighting its promise as a therapeutic strategy for treating cervical cancer. [ABSTRACT FROM AUTHOR]

The Herpesviridae include the Epstein–Barr Virus (EBV) and the Kaposi Sarcoma-associated Herpesvirus (KSHV), both of which are oncogenic gamma-herpesviruses. These viruses manipulate host cellular mechanisms, including through ubiquitin-mediated pathways, to promote viral replication and oncogenesis. Ubiquitin, a regulatory protein which tags substrates for degradation or alters their function, is manipulated by both EBV and KSHV to facilitate viral persistence and cancer development. EBV infects approximately 90% of the global population and is implicated in malignancies including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), post-transplant lymphoproliferative disorder (PTLD), and nasopharyngeal carcinoma. EBV latency proteins, notably LMP1 and EBNA3C, use ubiquitin-mediated mechanisms to inhibit apoptosis, promote cell proliferation, and interfere with DNA repair, contributing to tumorigenesis. EBV's lytic proteins, including BZLF1 and BPLF1, further disrupt cellular processes to favor oncogenesis. Similarly, KSHV, a causative agent of Kaposi's Sarcoma and lymphoproliferative disorders, has a latency-associated nuclear antigen (LANA) and other latency proteins that manipulate ubiquitin pathways to degrade tumor suppressors, stabilize oncogenic proteins, and evade immune responses. KSHV's lytic cycle proteins, such as RTA and Orf64, also use ubiquitin-mediated strategies to impair immune functions and promote oncogenesis. This review explores the ubiquitin-mediated interactions of EBV and KSHV proteins, elucidating their roles in viral oncogenesis. Understanding these mechanisms offers insights into the similarities between the viruses, as well as provoking thought about potential therapeutic targets for herpesvirus-associated cancers. [ABSTRACT FROM AUTHOR]

Abstract Loss-of-function mutations in the human gene encoding the neuron-specific Ca2+ channel CaV2.1 are linked to the neurological disease episodic ataxia type 2 (EA2), as well as neurodevelopmental disorders such as developmental delay and developmental epileptic encephalopathy. Disease-associated CaV2.1 mutants may exhibit defective proteostasis and promote endoplasmic reticulum (ER)-associated degradation of their wild-type (WT) counterpart in a dominant-negative manner. The E3 ubiquitin ligase RNF138 was previously shown to mediate EA2-related aberrant degradation of CaV2.1 at the ER. Herein we aimed to elucidate the ER proteostasis mechanism of CaV2.1. The peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1) was identified as a novel neuronal CaV2.1 binding partner that promoted polyubiquitination and proteasomal degradation of CaV2.1. Suppression of endogenous Pin1 level with either shRNA knockdown or the Pin1 inhibitor all-trans retinoic acid (ATRA) enhanced endogenous CaV2.1 protein level in neurons, and attenuated ER-associated degradation of CaV2.1 WT and EA2-causing mutants. Detailed mutation analyses suggested that Pin1 interacted with specific phosphorylated serine/threonine-proline motifs in the intracellular II-III loop and the distal carboxy-terminal region of human CaV2.1. We further generated Pin1-insensitive CaV2.1 constructs and demonstrated that, during ER quality control, Pin1 served as an upstream regulator of CaV2.1 polyubiquitination and degradation by RNF138. Pin1 regulation was required for the dominant-negative effect of EA2 missense mutants, but not nonsense mutants, on CaV2.1 WT protein expression. Our data are consistent with the idea that CaV2.1 proteostasis at the ER, as well as dominant-negative suppression of disease-causing loss-of-function mutants on CaV2.1 WT, entail both Pin1/RNF138-dependent and -independent mechanisms.

The PDZ-LIM domain-containing protein PDLIM2 is a common tumor suppressor and a key immune modulator. One main function of PDLIM2 is to promote the ubiquitination and proteasomal degradation of nuclear activated NF-κB RelA, a physiologically indispensable transcription factor whose persistent activation has been linked to almost all cancer types and inflammation-associated diseases. However, it remains unknown how PDLIM2 exerts this physiologically and pathogenically important function. Here, we show that PDLIM2 acts as a ubiquitin ligase enhancer, termed E5. It stabilizes ROC1, an essential component of SKP1/Cullin/F-box protein (SCF) ubiquitin ligases, and chaperones the ROC1-SCFβ-TrCP ubiquitin ligase to ubiquitinate nuclear RelA for proteasomal degradation in the nucleus. Consistently, silencing of ROC1, Cullin 1 or the F-box protein β-TrCP blocks RelA ubiquitination and degradation by PDLIM2. These data provide new mechanistic insights into how PDLIM2 promotes nuclear RelA ubiquitination and degradation, thereby serving as a critical tumor suppressor and a vital immune regulator. They also improve our understanding of the complex cascade of the ubiquitination and NF-κB pathways, particularly given the well-known role of the ROC1-SCFβ-TrCP ubiquitin ligase in initiating NF-κB activation by directly binding to and ubiquitinating NF-κB inhibitors for the proteasomal degradation in the cytoplasm. [ABSTRACT FROM AUTHOR]

Abstract Background Gastric cancer (GC) ranks fifth in global cancer incidence and third in mortality rate among all cancer types. Circular RNAs (circRNAs) have been extensively demonstrated to regulate multiple malignant biological behaviors in GC. Emerging evidence suggests that several circRNAs derived from FNDC3B play pivotal roles in cancer. However, the role of circFNDC3B in GC remains elusive. Methods We initially screened circFNDC3B with translation potential via bioinformatics algorithm prediction. Subsequently, Sanger sequencing, qRT-PCR, RNase R, RNA-FISH and nuclear-cytoplasmic fractionation assays were explored to assess the identification and localization of circ0003692, a circRNA derived from FNDC3B. qRT-PCR and ISH were performed to quantify expression of circ0003692 in human GC tissues and adjacent normal tissues. The protein-encoding ability of circ0003692 was investigated through dual-luciferase reporter assay and LC/MS. The biological behavior of circ0003692 in GC was confirmed via in vivo and in vitro experiments. Additionally, Co-IP and rescue experiments were performed to elucidate the interaction between the encoded protein and c-Myc. Results We found that circ0003692 was significantly downregulated in GC tissues. Circ0003692 had the potential to encode a novel protein FNDC3B-267aa, which was downregulated in GC cells. We verified that FNDC3B-267aa, rather than circ0003692, inhibited GC migration in vitro and in vivo. Mechanistically, FNDC3B-267aa directly interacted with c-Myc and promoted proteasomal degradation of c-Myc, resulting in the downregulation of c-Myc-Snail/Slug axis. Conclusions Our study revealed that the novel protein FNDC3B-267aa encoded by circ0003692 suppressed GC metastasis through binding to c-Myc and enhancing proteasome-mediated degradation of c-Myc. The study offers the potential applications of circ0003692 or FNDC3B-267aa as therapeutic targets for GC. Graphical abstract The mechanism of circ0003692 in suppressing metastasis of GC. FNDC3B-267aa encoded by circ0003692 interacted with c-Myc and promoted the proteasomal degradation of c-Myc, thereby down-regulated c-Myc-Snail/Slug axis and EMT pathway.

APOBEC3G (A3G) restricts HIV-1 replication primarily by reducing viral cDNA and inducing G-to-A hypermutations in viral cDNA. HIV-1 encodes virion infectivity factor (Vif) to counteract A3G primarily by excluding A3G viral encapsidation. Even though the Vif-induced exclusion is robust, studies suggest that A3G is still detectable in the virion. The impact of encapsidated A3G in the HIV-1 replication is unclear. Using a highly sensitive next-generation sequencing (NGS)-based G-to-A hypermutation detecting assay, we found that wild-type HIV-1 produced from A3G-expressing T-cells induced higher G-to-A hypermutation frequency in viral cDNA than HIV-1 from non-A3G-expressing T-cells. Interestingly, although the virus produced from A3G-expressing T-cells induced higher hypermutation frequency, there was no significant difference in viral infectivity, revealing a disassociation of cDNA G-to-A hypermutation to viral infectivity. We also measured G-to-A hypermutation in the viral RNA genome. Surprisingly, our data showed that hypermutation frequency in the viral RNA genome was significantly lower than in the integrated DNA, suggesting a mechanism exists to preferentially select intact genomic RNA for viral packing. This study revealed a new insight into the mechanism of HIV-1 counteracting A3G antiviral function and might lay a foundation for new antiviral strategies. [ABSTRACT FROM AUTHOR]

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children and adolescents. Respecting the age of the patients and the tumor aggressiveness, investigation of the molecular mechanisms of RMS tumorigenesis is directed toward the identification of novel therapeutic targets. To contribute to a better understanding of the molecular pathology of RMS, we investigated ankyrin repeat domain 1 (ANKRD1), designated as a potential marker for differential diagnostics. In this study, we used three RMS cell lines (SJRH30, RD, and HS-729) to assess its expression profile, intracellular localization, and turnover. They express wild-type ANKRD1, as judged by the sequencing of the open reading frame. Each cell line expressed a different amount of ANKRD1 protein, although the transcript level was similar. According to western blot analysis, ANKRD1 protein was expressed at detectable levels in the SJRH30 and RD cells (SJRH30 > RD), but not in the HS-729, even after immunoprecipitation. Immunocytochemistry revealed nuclear and cytoplasmic localization of ANKRD1 in all examined cell lines. Moreover, the punctate pattern of ANKRD1 staining in the nuclei of RD and HS-729 cells overlapped with coilin, indicating its association with Cajal bodies. We have shown that RMS cells are not able to overexpress ANKRD1 protein, which can be attributed to its proteasomal degradation. The unsuccessful attempt to overexpress ANKRD1 in RMS cells indicates the possibility that its overexpression may have detrimental effects for RMS cells and opens a window for further research into its role in RMS pathogenesis and for potential therapeutic targeting. [ABSTRACT FROM AUTHOR]

The ribosome is a molecular machine essential for protein synthesis, which is composed of approximately 80 different ribosomal proteins (Rps). Studies in yeast and cell culture systems have revealed that the intracellular level of Rps is finely regulated by negative feedback mechanisms or ubiquitin-proteasome system, which prevents over- or under-abundance of Rps in the cell. However, in vivo evidence for the homeostatic regulation of intracellular Rp levels has been poor. Here, using Drosophila genetics, we show that intracellular Rp levels are regulated by proteasomal degradation of excess Rps that are not incorporated into the ribosome. By establishing an EGFP-fused Rp gene system that can monitor endogenously expressed Rp levels, we found that endogenously expressed EGFP-RpS20 or -RpL5 is eliminated from the cell when RpS20 or RpL5 is exogenously expressed. Notably, the level of endogenously expressed Hsp83, a housekeeping gene, was not affected by exogenous expression of Hsp83, suggesting that the strict negative regulation of excess protein is specific for intracellular Rps. Further analyses revealed that the maintenance of cellular Rp levels is not regulated at the transcriptional level but by proteasomal degradation of excess free Rps as a protein quality control mechanism. Our observations provide not only the in vivo evidence for the homeostatic regulation of Rp levels but also a novel genetic strategy to study in vivo regulation of intracellular Rp levels and its role in tissue homeostasis via cell competition. Key words: ribosomal protein, proteasomal degradation, Drosophila

Activating cGAS‐STING pathway has great potential to achieve effective antitumor immunotherapy. However, mutant p53 (mutp53), a commonly observed genetic alteration in over 50% of human cancer, will impede the therapeutic performance of the cGAS‐STING pathway. Herein, multifunctional ZIF‐8@MnO2 nanoparticles are constructed to degrade mutp53 and facilitate the cGAS‐STING pathway. The synthesized ZIF‐8@MnO2 can release Zn2+ and Mn2+ in cancer cells to induce oxidative stress and cytoplasmic leakage of fragmented mitochondrial double‐stranded DNAs (dsDNAs). Importantly, the released Zn2+ induces variable degradation of multifarious p53 mutants through proteasome ubiquitination, which can alleviate the inhibitory effects of mutp53 on the cGAS‐STING pathway. In addition, the released Mn2+ further increases the sensitivity of cGAS to dsDNAs as immunostimulatory signals. Both in vitro and in vivo results demonstrate that ZIF‐8@MnO2 effectively promotes the cGAS‐STING pathway and synergizes with PD‐L1 checkpoint blockades, leading to remarkable regression of local tumors as well as distant metastases of breast cancer. This study proposes an inorganic metal ion‐based nanoplatform to enhance the cGAS‐STING‐mediated antitumor immunotherapy, especially to those tumors with mutp53 expression. [ABSTRACT FROM AUTHOR]

Activation of hepatic stellate cells (HSCs) plays a critical role in liver fibrosis. However, the molecular basis for HSC activation remains poorly understood. Herein, we demonstrate that primary cilia are present on quiescent HSCs but exhibit a significant loss upon HSC activation which correlates with decreased levels of the ciliary protein intraflagellar transport 88 (IFT88). Ift88-knockout mice are more susceptible to chronic carbon tetrachloride-induced liver fibrosis. Mechanistic studies show that the X-linked inhibitor of apoptosis (XIAP) functions as an E3 ubiquitin ligase for IFT88. Transforming growth factor-β (TGF-β), a profibrotic factor, enhances XIAP-mediated ubiquitination of IFT88, promoting its proteasomal degradation. Blocking XIAP-mediated IFT88 degradation ablates TGF-β-induced HSC activation and liver fibrosis. These findings reveal a previously unrecognized role for ciliary homeostasis in regulating HSC activation and identify the XIAP–IFT88 axis as a potential therapeutic target for liver fibrosis. Synopsis: Profibrotic stimuli disrupt primary cilia on quiescent hepatic stellate cells (HSCs) by inducing XIAP-mediated ubiquitination and proteasomal degradation of IFT88. This contributes to HSC activation and liver fibrosis. Primary cilia exist on multiple cell types in the liver, but only cilia present on HSCs disassemble during liver fibrosis. TGF-β enhances XIAP-mediated ubiquitination of IFT88, promoting its proteasomal degradation. Blocking XIAP-mediated IFT88 degradation ablates TGF-β-induced HSC activation. Profibrotic stimuli disrupt primary cilia on quiescent hepatic stellate cells (HSCs) by inducing XIAP-mediated ubiquitination and proteasomal degradation of IFT88. This contributes to HSC activation and liver fibrosis. [ABSTRACT FROM AUTHOR]

The gaseous hormone ethylene plays pivotal roles in plant growth and development. The rate-limiting enzyme of ethylene biosynthesis in seed plants is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). ACS proteins are encoded by a multigene family and the expression of ACS genes is highly regulated, especially at a post-translational level. AtACS7, the only type III ACS in Arabidopsis, is degraded in a 26S proteasome-dependent pathway. Here, by using liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) analysis, two lysine residues of AtACS7, lys285 (K285) and lys366 (K366), were revealed to be ubiquitin-modified in young, light-grown Arabidopsis seedlings but not in etiolated seedlings. Deubiquitylation-mimicking mutations of these residues significantly increased the stability of the AtACS7K285RK366R mutant protein in cell-free degradation assays. All results suggest that K285 and K366 are the major ubiquitination sites on AtACS7, providing deeper insights into the post-translational regulation of AtACS7 in Arabidopsis. [ABSTRACT FROM AUTHOR]

The dual leucine zipper kinase (DLK) alias mitogen-activated protein 3 kinase 12 (MAP3K12) has gained much attention in recent years. DLK belongs to the mixed lineage kinases, characterized by homology to serine/threonine and tyrosine kinase, but exerts serine/threonine kinase activity. DLK has been implicated in many diseases, including several neurodegenerative diseases, glaucoma, and diabetes mellitus. As a MAP3K, it is generally assumed that DLK becomes phosphorylated and activated by upstream signals and phosphorylates and activates itself, the downstream serine/threonine MAP2K, and, ultimately, MAPK. In addition, other mechanisms such as protein–protein interactions, proteasomal degradation, dephosphorylation by various phosphatases, palmitoylation, and subcellular localization have been shown to be involved in the regulation of DLK activity or its fine-tuning. In the present review, the diverse mechanisms regulating DLK activity will be summarized to provide better insights into DLK action and, possibly, new targets to modulate DLK function. [ABSTRACT FROM AUTHOR]

Background: Tumor metastasis and recurrence are major causes of mortality in patients with hepatocellular carcinoma (HCC) that is still lack of effective therapeutic targets and drugs. Previous reports implied that ras homolog family member C (RhoC) plays a toxic role on metastasis and proliferation of cancer. Methods: In this research, the correlation between RhoC and metastasis ability was confirmed by in vitro experiments and TCGA database. We explored whether quercetin could inhibit cell migration or invasion by transwell assay. Real‐time PCR, overexpression and ubiquitination assay, etc. were applied in mechanism study. Primary HCC cells and animal models including patient‐derived xenografts (PDXs) were employed to evaluate the anti‐metastasis effects of quercetin. Results: Clinical relevance and in vitro experiments further confirmed the level of RhoC was positively correlated with invasion and metastasis ability of HCC. Then we uncovered that quercetin could attenuate invasion and metastasis of HCC by downregulating RhoC's level in vitro, in vivo and PDXs. Furthermore, mechanistic investigations displayed quercetin hindered the E3 ligase expression of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) leading to enhancement of RhoC's ubiquitination and proteasomal degradation. Conclusions: Our research has revealed the novel mechanisms quercetin regulates degradation of RhoC level by targeting SMURF2 and identified quercetin may be a potential compound for HCC therapy. [ABSTRACT FROM AUTHOR]

Human sapoviruses (HuSaVs) and noroviruses are considered the leading cause of acute gastroenteritis worldwide. While extensive research has focused on noroviruses, our understanding of sapoviruses (SaVs) and their interactions with the host's immune response remains limited. HuSaVs have been challenging to propagate in vitro, making the porcine sapovirus (PSaV) Cowden strain a valuable model for studying SaV pathogenesis. In this study we show, for the first time, that PSaV Cowden strain has mechanisms to evade the host's innate immune response. The virus 3C-like protease (NS6) inhibits type I IFN production by targeting TBK1. Catalytically active NS6, both during ectopic expression and during PSaV infection, targets TBK1 which is then led for rapid degradation by the proteasome. Moreover, deletion of TBK1 from porcine cells led to an increase in PSaV titres, emphasizing its role in regulating PSaV infection. Additionally, we successfully established PSaV infection in IPEC-J2 cells, an enterocytic cell line originating from the jejunum of a neonatal piglet. Overall, this study provides novel insights into PSaV evasion strategies, opening the way for future investigations into SaV–host interactions, and enabling the use of a new cell line model for PSaV research. [ABSTRACT FROM AUTHOR]

Heart development is a spatiotemporally regulated process that extends from the embryonic phase to postnatal stages. Disruption of this highly orchestrated process can lead to congenital heart disease or predispose the heart to cardiomyopathy or heart failure. Consequently, gaining an in-depth understanding of the molecular mechanisms governing cardiac development holds considerable promise for the development of innovative therapies for various cardiac ailments. While significant progress in uncovering novel transcriptional and epigenetic regulators of heart development has been made, the exploration of post-translational mechanisms that influence this process has lagged. Culling-RING E3 ubiquitin ligases (CRLs), the largest family of ubiquitin ligases, control the ubiquitination and degradation of ~20% of intracellular proteins. Emerging evidence has uncovered the critical roles of CRLs in the regulation of a wide range of cellular, physiological, and pathological processes. In this review, we summarize current findings on the versatile regulation of cardiac morphogenesis and maturation by CRLs and present future perspectives to advance our comprehensive understanding of how CRLs govern cardiac developmental processes. [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome system is one of the most important protein stability regulation systems. It can precisely regulate host immune responses by targeting signaling proteins. TRAF6 is a crucial E3 ubiquitin ligase in host antiviral signaling pathway. Here, we discovered that EF-hand domain-containing protein D2 (EFHD2) collaborated with the E3 ubiquitin ligase Smurf1 to potentiate the degradation of TRAF6, hence facilitating RNA virus Siniperca chuatsi rhabdovirus infection. The mechanism analysis revealed that EFHD2 interacted with Smurf1 and enhanced its protein stability by impairing K48-linked polyubiquitination of Smurf1, thereby promoting Smurf1-catalyzed degradation of TRAF6. This study initially demonstrated a novel mechanism by which viruses utilize host EFHD2 to achieve immune escape and provided a new perspective on the exploration of mammalian innate immunity. IMPORTANCE Viruses induce host cells to activate several antiviral signaling pathways. TNF receptor-associated factor 6 (TRAF6) plays an essential role in these pathways. Numerous studies have been done on the mechanisms of TRAF6-mediated resistance to viral invasion. However, little is known about the strategies that viruses employ to antagonize TRAF6-mediated antiviral signaling pathway. Here, we discovered that EFHD2 functions as a host factor to promote viral replication. Mechanistically, EFHD2 potentiates Smurf1 to catalyze the ubiquitin-proteasomal degradation of TRAF6 by promoting the deubiquitination and stability of Smurf1, which in turn inhibits the production of proinflammatory cytokines and interferons. Our study also provides a new perspective on mammalian resistance to viral invasion. [ABSTRACT FROM AUTHOR]

ABSTRACTGcn4p belongs to conserved AP-1 transcription factors involved in many cellular processes, including cell proliferation, stress response, and nutrient availability in yeast and mammals. AP-1 activities are regulated at different levels, such as translational activation or protein degradation, which increases the variability of regulation under different conditions. Gcn4p activity in unstructured yeast liquid cultures increases upon amino acid deficiency and is rapidly eliminated upon amino acid excess. Gcn2p kinase is the major described regulator of Gcn4p that enables GCN4 mRNA translation via the uORFs mechanism. Here, we show that Gcn4p is specifically active in U cells in the upper regions and inactive in L cells in the lower regions of differentiated colonies. Using in situ microscopy in combination with analysis of mutants and strains with GFP at different positions in the translational regulatory region of Gcn4p, we show that cell-specific Gcn4p activity is independent of Gcn2p or other translational or transcriptional regulation. Genetically, biochemically, and microscopically, we identified cell-specific proteasomal degradation as a key mechanism that diversifies Gcn4p function between U and L cells. The identified regulation leading to active Gcn4p in U cells with amino acids and efficient degradation in starved L cells differs from known regulations of Gcn4p in yeast but shows similarities to the activity of AP-1 ATF4 in mammals during insulin signaling. These findings may open new avenues for understanding the parallel activities of Gcn4p/ATF4 and reveal a novel biological role for cell type-specific regulation of proteasome-dependent degradation.IMPORTANCEIn nature, microbes usually live in spatially structured communities and differentiate into precisely localized, functionally specialized cells. The coordinated interplay of cells and their response to environmental changes, such as starvation, followed by metabolic adaptation, is critical for the survival of the entire community. Transcription factor Gcn4p is responsible for yeast adaptation under amino acid starvation in liquid cultures, and its activity is regulated mainly at the level of translation involving Gcn2p kinase. Whether Gcn4p functions in structured communities was unknown. We show that translational regulation of Gcn4p plays no role in the development of colony subpopulations; the main regulation occurs at the level of stabilization of the Gcn4p molecule in the cells of one subpopulation and its proteasomal degradation in the other. This regulation ensures specific spatiotemporal activity of Gcn4p in the colony. Our work highlights differences in regulatory networks in unorganized populations and organized structures of yeast, which in many respects resemble multicellular organisms.

Pregnane X receptor (PXR) is a ligand-activated nuclear receptor that transcriptionally upregulates drug-metabolizing enzymes [e.g., cytochrome P450 3A4 (CYP3A4)] and transporters. Although the regulation of PXR target genes is well-characterized, less is known about the regulation of PXR protein level. By screening an RNAi library, we identified the F-box-only protein 44 (FBXO44) as a novel E3 ligase for PXR. PXR abundance increases upon knockdown of FBXO44, and, inversely, decreases upon overexpression of FBXO44. Further analysis revealed that FBXO44 interacts with PXR, leading to its ubiquitination and proteasomal degradation, and we determined that the F-box associated domain of FBXO44 and the ligand binding domain of PXR are required for the functional interaction. In summary, FBXO44 regulates PXR protein abundance, which has downstream consequences for CYP3A4 levels and drug–drug interactions. The results of this study provide new insight into the molecular mechanisms that regulate PXR protein level and activity and suggest the importance of considering how modulating E3 ubiquitin ligase activities will affect PXR-mediated drug metabolism.

Background/purpose: Proliferation-associated protein 2G4 (PA2G4) has alternative transcriptional and translational initiation. One dominant transcript ENST00000303305 could be translated into two protein isoforms (PA2G4-P42 and PA2G4-P48). In this study, we aimed to explore the effects of PA2G4-P42 and PA2G4-P48 on the proliferation of head and neck squamous cell carcinoma (HNSCC) and the mechanisms regulating PA2G4-P48 stability. Materials and methods: HNSCC cell lines HSC2 and SCC25 with relatively low PA2G4 expression were used for in-vitro cell studies. PA2G4-P42 and PA2G4-P48 overexpression lentiviruses were generated. In vitro cell proliferation was assessed by CCK-8 and colony formation. In vivo tumor cell proliferation was assessed by HSC2 cell-derived xenograft tumors. Liquid chromatography-mass spectrometry (LC-MS)/MS and co-immunoprecipitation (co-IP) assays were applied to check PA2G4-P48 interacting partners. Cycloheximide (CHX) chase and ubiquitin-based co-IP assays were also performed. Results: PA2G4-P48 was the dominant isoform, with substantially higher expression than PA2G4-P42 in HNSCC. PA2G4-P48 overexpression enhanced HNSCC cell proliferation, but PA2G4-P42 overexpression slowed the proliferation. MCTS1 interacted with PA2G4-P48, but not PA2G4-P42. PA2G4 protein but not its mRNA expression was decreased in cells with MCTS1 knockdown. MG132 treatment abrogated this alteration. MCTS1 overexpression significantly elevated the half-life of PA2G4-P48, while its knockdown drastically reduced the half-life compared with the control cells. In addition, MCTS1 overexpression significantly decreased the polyubiquitination of exogenous flag-tagged PA2G4-P48. MCTS1 overexpression-induced cell proliferation was hampered by knocking down of PA2G4-P48. Conclusion: PA2G4-P42 and PA2G4-P48 exert growth-suppressive and growth-promoting effects in HNSCC, respectively. MCTS1 can interact with PA2G4-P48 and prolong its half-life by reducing its poly-ubiquitination.

Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.

Abstract Activating cGAS‐STING pathway has great potential to achieve effective antitumor immunotherapy. However, mutant p53 (mutp53), a commonly observed genetic alteration in over 50% of human cancer, will impede the therapeutic performance of the cGAS‐STING pathway. Herein, multifunctional ZIF‐8@MnO2 nanoparticles are constructed to degrade mutp53 and facilitate the cGAS‐STING pathway. The synthesized ZIF‐8@MnO2 can release Zn2+ and Mn2+ in cancer cells to induce oxidative stress and cytoplasmic leakage of fragmented mitochondrial double‐stranded DNAs (dsDNAs). Importantly, the released Zn2+ induces variable degradation of multifarious p53 mutants through proteasome ubiquitination, which can alleviate the inhibitory effects of mutp53 on the cGAS‐STING pathway. In addition, the released Mn2+ further increases the sensitivity of cGAS to dsDNAs as immunostimulatory signals. Both in vitro and in vivo results demonstrate that ZIF‐8@MnO2 effectively promotes the cGAS‐STING pathway and synergizes with PD‐L1 checkpoint blockades, leading to remarkable regression of local tumors as well as distant metastases of breast cancer. This study proposes an inorganic metal ion‐based nanoplatform to enhance the cGAS‐STING‐mediated antitumor immunotherapy, especially to those tumors with mutp53 expression.

Abstract Background Tumor metastasis and recurrence are major causes of mortality in patients with hepatocellular carcinoma (HCC) that is still lack of effective therapeutic targets and drugs. Previous reports implied that ras homolog family member C (RhoC) plays a toxic role on metastasis and proliferation of cancer. Methods In this research, the correlation between RhoC and metastasis ability was confirmed by in vitro experiments and TCGA database. We explored whether quercetin could inhibit cell migration or invasion by transwell assay. Real‐time PCR, overexpression and ubiquitination assay, etc. were applied in mechanism study. Primary HCC cells and animal models including patient‐derived xenografts (PDXs) were employed to evaluate the anti‐metastasis effects of quercetin. Results Clinical relevance and in vitro experiments further confirmed the level of RhoC was positively correlated with invasion and metastasis ability of HCC. Then we uncovered that quercetin could attenuate invasion and metastasis of HCC by downregulating RhoC's level in vitro, in vivo and PDXs. Furthermore, mechanistic investigations displayed quercetin hindered the E3 ligase expression of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) leading to enhancement of RhoC's ubiquitination and proteasomal degradation. Conclusions Our research has revealed the novel mechanisms quercetin regulates degradation of RhoC level by targeting SMURF2 and identified quercetin may be a potential compound for HCC therapy.

ABSTRACT The accessory protein ORF6 of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key interferon (IFN) antagonist that strongly suppresses the production of primary IFN as well as the expression of IFN-stimulated genes. However, how host cells respond to ORF6 remains largely unknown. Our research of ORF6-binding proteins by pulldown revealed that E3 ligase components such as Cullin 4B (CUL4B), DDB1, and RBX1 are potential ORF6-interacting proteins. Further study found that the substrate recognition receptor PRPF19 interacts with CUL4B, DDB1, and RBX1 to form a CRL4B-based E3 ligase, which catalyzes ORF6 ubiquitination and subsequent degradation. Overexpression of PRPF19 promotes ORF6 degradation, releasing ORF6-mediated IFN inhibition, which inhibits SARS-CoV-2 replication. Moreover, we found that activation of CUL4B by the neddylation inducer etoposide alleviates lung lesions in a SARS-CoV-2 mouse infection model. Therefore, targeting ORF6 for degradation may be an effective therapeutic strategy against SARS-CoV-2 infection.IMPORTANCEThe cellular biological function of the ubiquitin-proteasome pathway as an important modulator for the regulation of many fundamental cellular processes has been greatly appreciated. The critical role of the ubiquitin-proteasome pathway in viral pathogenesis has become increasingly apparent. It is a powerful tool that host cells use to defend against viral infection. Some cellular proteins can function as restriction factors to limit viral infection by ubiquitin-dependent degradation. In this research, we identificated of CUL4B-DDB1-PRPF19 E3 Ubiquitin Ligase Complex can mediate proteasomal degradation of ORF6, leading to inhibition of viral replication. Moreover, the CUL4B activator etoposide alleviates disease development in a mouse infection model, suggesting that this agent or its derivatives may be used to treat infections caused by SARS-CoV-2. We believe that these results will be extremely useful for the scientific and clinic communities in their search for cues and preventive measures to combat the COVID-19 pandemic.

Non-alcoholic fatty liver disease (NAFLD), and its progressive form steatohepatitis (NASH), represent a genetically and phenotypically diverse entity for which there is no approved therapy, making it imperative to define the spectrum of pathways contributing to its pathogenesis. Rare variants in genes encoding nuclear envelope proteins cause lipodystrophy with early-onset NAFLD/NASH; we hypothesized that common variants in nuclear envelope-related genes might also contribute to hepatic steatosis and NAFLD. Using hepatic steatosis as the outcome of interest, we performed an association meta-analysis of nuclear envelope-related coding variants in three large discovery cohorts (N >120,000 participants), followed by phenotype association studies in large validation cohorts (N >600,000) and functional testing of the top steatosis-associated variant in cell culture. A common protein-coding variant, rs6461378 (SUN1 H118Y), was the top steatosis-associated variant in our association meta-analysis (p <0.001). In ancestrally distinct validation cohorts, rs6461378 associated with histologic NAFLD and with NAFLD-related metabolic traits including increased serum fatty acids, type 2 diabetes, hypertension, cardiovascular disease, and decreased HDL. SUN1 H118Y was subject to increased proteasomal degradation relative to wild-type SUN1 in cells, and SUN1 H118Y-expressing cells exhibited insulin resistance and increased lipid accumulation. Collectively, these data support a potential causal role for the common SUN1 variant rs6461378 in NAFLD and metabolic disease. Non-alcoholic fatty liver disease (NAFLD), with an estimated global prevalence of nearly 30%, is a growing cause of morbidity and mortality for which there is no approved pharmacologic therapy. Our data provide a rationale for broadening current concepts of NAFLD genetics and pathophysiology to include the nuclear envelope, and particularly Sad1 and UNC84 domain containing 1 (SUN1), as novel contributors to this common liver disease. Furthermore, if future studies confirm causality of the common SUN1 H118Y variant, it has the potential to become a broadly relevant therapeutic target in NAFLD and metabolic disease. [Display omitted] • Association meta-analysis identified a strong positive association between a coding variant in SUN1 (rs6461378-T) and hepatic steatosis. • SUN1 variant positively associated with histologic NAFLD and NAFLD-related metabolic traits in separate validation cohorts. • This variant increased SUN1 degradation compared to the wild-type protein under nutrient-limited conditions (serum starvation). • The SUN1 variant increased lipid droplet accumulation and blunted insulin signaling in human hepatoma cell lines. [ABSTRACT FROM AUTHOR]

Pregnane X receptor (PXR) is a ligand-activated nuclear receptor that transcriptionally upregulates drug-metabolizing enzymes [ e. g., cytochrome P450 3A4 (CYP3A4)] and transporters. Although the regulation of PXR target genes is well-characterized, less is known about the regulation of PXR protein level. By screening an RNAi library, we identified the F-box-only protein 44 (FBXO44) as a novel E3 ligase for PXR. PXR abundance increases upon knockdown of FBXO44, and, inversely, decreases upon overexpression of FBXO44. Further analysis revealed that FBXO44 interacts with PXR, leading to its ubiquitination and proteasomal degradation, and we determined that the F-box associated domain of FBXO44 and the ligand binding domain of PXR are required for the functional interaction. In summary, FBXO44 regulates PXR protein abundance, which has downstream consequences for CYP3A4 levels and drug–drug interactions. The results of this study provide new insight into the molecular mechanisms that regulate PXR protein level and activity and suggest the importance of considering how modulating E3 ubiquitin ligase activities will affect PXR-mediated drug metabolism. PXR is a xenobiotic receptor that controls the expression of drug-metabolizing enzymes and transporters. FBXO44 was identified as an E3 ligase that regulates PXR protein level by targeting PXR for ubiquitination and proteasomal degradation. [Display omitted] [ABSTRACT FROM AUTHOR]

Thalidomide has a dark history as a teratogen, but in recent years, its derivates have been shown to function as potent chemotherapeutic agents. These drugs bind cereblon (CRBN), the substrate receptor of an E3 ubiquitin ligase complex, and modify its degradation targets. Despite these insights, remarkably little is known about the normal function of cereblon in development. Here, we employ Ciona, a simple invertebrate chordate, to identify endogenous Crbn targets. In Ciona, Crbn is specifically expressed in developing muscles during tail elongation before they acquire contractile activity. Crbn expression is activated by Mrf, the ortholog of MYOD1, a transcription factor important for muscle differentiation. CRISPR/Cas9-mediated mutations of Crbn lead to precocious onset of muscle contractions. By contrast, overexpression of Crbn delays contractions and is associated with decreased expression of contractile protein genes such as troponin. This reduction is possibly due to reduced Mrf protein levels without altering Mrf mRNA levels. Our findings suggest that Mrf and Crbn form a negative feedback loop to control the precision of muscle differentiation during tail elongation. [ABSTRACT FROM AUTHOR]

Proliferation-associated protein 2G4 (PA2G4) has alternative transcriptional and translational initiation. One dominant transcript ENST00000303305 could be translated into two protein isoforms (PA2G4-P42 and PA2G4-P48). In this study, we aimed to explore the effects of PA2G4-P42 and PA2G4-P48 on the proliferation of head and neck squamous cell carcinoma (HNSCC) and the mechanisms regulating PA2G4-P48 stability. HNSCC cell lines HSC2 and SCC25 with relatively low PA2G4 expression were used for in-vitro cell studies. PA2G4-P42 and PA2G4-P48 overexpression lentiviruses were generated. In vitro cell proliferation was assessed by CCK-8 and colony formation. In vivo tumor cell proliferation was assessed by HSC2 cell-derived xenograft tumors. Liquid chromatography-mass spectrometry (LC-MS)/MS and co-immunoprecipitation (co-IP) assays were applied to check PA2G4-P48 interacting partners. Cycloheximide (CHX) chase and ubiquitin-based co-IP assays were also performed. PA2G4-P48 was the dominant isoform, with substantially higher expression than PA2G4-P42 in HNSCC. PA2G4-P48 overexpression enhanced HNSCC cell proliferation, but PA2G4-P42 overexpression slowed the proliferation. MCTS1 interacted with PA2G4-P48, but not PA2G4-P42. PA2G4 protein but not its mRNA expression was decreased in cells with MCTS1 knockdown. MG132 treatment abrogated this alteration. MCTS1 overexpression significantly elevated the half-life of PA2G4-P48, while its knockdown drastically reduced the half-life compared with the control cells. In addition, MCTS1 overexpression significantly decreased the polyubiquitination of exogenous flag-tagged PA2G4-P48. MCTS1 overexpression-induced cell proliferation was hampered by knocking down of PA2G4-P48. PA2G4-P42 and PA2G4-P48 exert growth-suppressive and growth-promoting effects in HNSCC, respectively. MCTS1 can interact with PA2G4-P48 and prolong its half-life by reducing its poly-ubiquitination. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis, is one of the commonest causes of liver dysfunction. Adipose triglyceride lipase (ATGL) is closely related to lipid turnover and hepatic steatosis as the speed-limited triacylglycerol lipase in liver lipolysis. However, the expression and regulation of ATGL in NAFLD remain unclear. Herein, our results showed that ATGL protein levels were decreased in the liver tissues of high-fat diet (HFD)-fed mice, naturally obese mice, and cholangioma/hepatic carcinoma patients with hepatic steatosis, as well as in the oleic acid-induced hepatic steatosis cell model, while ATGL mRNA levels were not changed. ATGL protein was mainly degraded through the proteasome pathway in hepatocytes. Beta-transducin repeat containing (BTRC) was upregulated and negatively correlated with the decreased ATGL level in these hepatic steatosis models. Consequently, BTRC was identified as the E3 ligase for ATGL through predominant ubiquitination at the lysine 135 residue. Moreover, adenovirus-mediated knockdown of BTRC ameliorated steatosis in HFD-fed mouse livers and oleic acid-treated liver cells via upregulating the ATGL level. Taken together, BTRC plays a crucial role in hepatic steatosis as a new ATGL E3 ligase and may serve as a potential therapeutic target for treating NAFLD. [ABSTRACT FROM AUTHOR]

APOBEC3G (A3G) restricts HIV-1 replication primarily by reducing viral cDNA and inducing G-to-A hypermutations in viral cDNA. HIV-1 encodes virion infectivity factor (Vif) to counteract A3G primarily by excluding A3G viral encapsidation. Even though the Vif-induced exclusion is robust, studies suggest that A3G is still detectable in the virion. The impact of encapsidated A3G in the HIV-1 replication is unclear. Using a highly sensitive next-generation sequencing (NGS)-based G-to-A hypermutation detecting assay, we found that wild-type HIV-1 produced from A3G-expressing T-cells induced higher G-to-A hypermutation frequency in viral cDNA than HIV-1 from non-A3G-expressing T-cells. Interestingly, although the virus produced from A3G-expressing T-cells induced higher hypermutation frequency, there was no significant difference in viral infectivity, revealing a disassociation of cDNA G-to-A hypermutation to viral infectivity. We also measured G-to-A hypermutation in the viral RNA genome. Surprisingly, our data showed that hypermutation frequency in the viral RNA genome was significantly lower than in the integrated DNA, suggesting a mechanism exists to preferentially select intact genomic RNA for viral packing. This study revealed a new insight into the mechanism of HIV-1 counteracting A3G antiviral function and might lay a foundation for new antiviral strategies.

ABSTRACT: More and more studies have demonstrated that proteasomal degradation occurs in the development of various diseases, including ventricular remodeling, which is a cardiac pathological change and seriously makes patient outcomes worse. Our preliminary results showed that Guanxin V, an effective and safe complementary and alternative medicine for ventricular remodeling, reverses ventricular hypertrophy by transforming growth factor-beta 1 (TGF-β1), but the specific mechanism needs to be explored. The left anterior descending coronary artery was ligated to build a ventricular remodeling model. Cardiac function and histopathology were measured. Fibrosis-related indicators were detected. Moreover, cardiomyocytes were exposed to hydrogen peroxide to construct an in vitro model of ventricular remodeling. The stability of the Vimentin protein was assessed with cycloheximide and MG132. Endogenous and exogenous TGF-β1-Vimentin interactions were detected by co-immunoprecipitation. Guanxin V significantly eased heart function and improved fibrosis in ventricular remodeling. Mechanistically, Guanxin V promoted TGF-β1-mediated proteasomal degradation of Vimentin and reduced the TGF-β1-Vimentin interaction. Here, we reported a completely new mechanism, Guanxin V alleviates ventricular remodeling by promoting and targeting TGF-β1-mediated proteasomal degradation of Vimentin, which provides a new target for the management of ventricular remodeling and lays the foundation for the further clinical promotion of Guanxin V.

In ALK-positive anaplastic large cell lymphoma (ALK+ALCL), a small subset of cancer stem-like (or RR) cells characterized by high Myc expression have been identified. We hypothesize that NPM-ALK contributes to their high Myc expression. While transfection of NPM-ALK into HEK293 cells effectively increased Myc by inhibiting its proteosomal degradation (PD-Myc), this effect was dramatically attenuated when the full-length NPM1 (FL-NPM1) was downregulated using shRNA, highlighting the importance of the NPM-ALK:FL-ALK heterodimers in this context. Consistent with this concept, immunoprecipitation experiments showed that the heterodimers are abundant only in RR cells, in which the half-life of Myc is substantially longer than the bulk cells. Fbw7γ, a key player in PD-Myc, is sequestered by the heterodimers in RR cells, and this finding correlates with a Myc phosphorylation pattern indicative of ineffective PD-Myc. Using confocal microscopy and immunofluorescence staining, we found that the fusion signal between ALK and FL-NPM1, characteristic of the heterodimers, correlates with the Myc level in ALK+ALCL cells from cell lines and patient samples. To conclude, our findings have revealed a novel oncogenic function of NPM-ALK in the nucleus. Specifically, the NPM-ALK:FL-NPM1 heterodimers increase cancer stemness by blocking PD-Myc and promoting Myc accumulation in the cancer stem-like cell subset. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy plays a protective role in sepsis-induced liver injury. As a member of class B scavenger receptors, CD36 plays important roles in various disorders, such as atherosclerosis and fatty liver disease. Here we found that the expression of CD36 in hepatocytes was increased in patients and a mouse model with sepsis, accompanied by impaired autophagy flux. Furthermore, hepatocyte cd36 knockout (cd36-HKO) markedly improved liver injury and the impairment of autophagosome-lysosome fusion in lipopolysaccharide (LPS)-induced septic mice. Ubqln1 (ubiquilin 1) overexpression (OE) in hepatocyte blocked the protective effect of cd36-HKO on LPS-induced liver injury in mice. Mechanistically, with LPS stimulation, CD36 on the plasma membrane was depalmitoylated and distributed to the lysosome, where CD36 acted as a bridge molecule linking UBQLN1 to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and hence promoting the proteasomal degradation of SNARE proteins, resulting in fusion impairment. Overall, our data reveal that CD36 is essential for modulating the proteasomal degradation of autophagic SNARE proteins in a UBQLN1-dependent manner. Targeting CD36 in hepatocytes is effective for improving autophagic flux in sepsis and therefore represents a promising therapeutic strategy for clinical treatment of septic liver injury. Abbreviations: AAV8: adeno-associated virus 8; AOSC: acute obstructive suppurative cholangitis; ATP1A1: ATPase, Na+/K+ transporting, alpha 1 polypeptide; CASP3: caspase 3; CASP8: caspase 8; CCL2: chemokine (C-C motif) ligand 2; cd36-HKO: hepatocyte-specific cd36 knockout; Co-IP: co-immunoprecipitation; CQ: chloroquine; Cys: cysteine; GOT1: glutamic-oxaloacetic transaminase 1, soluble; GPT: glutamic–pyruvic transaminase, soluble; IL1B: interleukin 1 beta; IL6: interleukin 6; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LDH, lactate dehydrogenase; LPS: lipopolysaccharide; LYPLA1: lysophospholipase 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OE: overexpression; qPCR: quantitative polymerase chain reaction; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TNF: tumor necrosis factor; TRIM: tripartite motif-containing; UBA: ubiquitin-associated; UBL: ubiquitin-like; UBQLN: ubiquilin; VAMP8: vesicle associated membrane protein 8; WT: wild-type. [ABSTRACT FROM AUTHOR]

Ribosomes are the workplace for protein biosynthesis. Protein production required for normal cell function is tightly linked to ribosome abundance. It is well known that ribosomal genes are actively transcribed and ribosomal messenger RNAs (mRNAs) are rapidly translated, and yet ribosomal proteins have relatively long half-lives. These observations raise questions as to how homeostasis of ribosomal proteins is controlled. Here, we show that ribosomal proteins, while posttranslationally stable, are subject to high-level cotranslational protein degradation (CTPD) except for those synthesized as ubiquitin (Ub) fusion precursors. The N-terminal Ub moiety protects fused ribosomal proteins from CTPD. We further demonstrate that cotranslational folding efficiency and expression level are two critical factors determining CTPD of ribosomal proteins. Different from canonical posttranslational degradation, we found that CTPD of all the ribosomal proteins tested in this study does not require prior ubiquitylation. This work provides insights into the regulation of ribosomal protein homeostasis and furthers our understanding of the mechanism and biological significance of CTPD. [ABSTRACT FROM AUTHOR]

Misfolded proteins retained in the endoplasmic reticulum cause many human diseases. ER-associated degradation (ERAD) is one of the protein quality and quantity control system located at ER, which is responsible for translocating the misfolded proteins or properly folded but excess proteins out of the ER for proteasomal degradation. Recent studies have revealed that mice with ERAD deficiency in specific cell types exhibit impaired metabolism homeostasis and metabolic diseases. Here, we highlight the ERAD physiological functions in metabolic disorders in a substrate-dependent and cell type-specific manner. [ABSTRACT FROM AUTHOR]

Ataxin-7 maintains the integrity of Spt-Ada-Gcn5-Acetyltransferase (SAGA), an evolutionarily conserved coactivator in stimulating preinitiation complex (PIC) formation for transcription initiation, and thus, its upregulation or downregulation is associated with various diseases. However, it remains unknown how ataxin-7 is regulated that could provide new insights into disease pathogenesis and therapeutic interventions. Here, we show that ataxin-7’s yeast homologue, Sgf73, undergoes ubiquitylation and proteasomal degradation. Impairment of such regulation increases Sgf73’s abundance, which enhances recruitment of TATA box-binding protein (TBP) (that nucleates PIC formation) to the promoter but impairs transcription elongation. Further, decreased Sgf73 level reduces PIC formation and transcription. Thus, Sgf73 is fine-tuned by ubiquitin–proteasome system (UPS) in orchestrating transcription. Likewise, ataxin-7 undergoes ubiquitylation and proteasomal degradation, alteration of which changes ataxin-7’s abundance that is associated with altered transcription and cellular pathologies/diseases. Collectively, our results unveil a novel UPS regulation of Sgf73/ataxin-7 for normal cellular health and implicate alteration of such regulation in diseases. [ABSTRACT FROM AUTHOR]

Voltage-gated ClC-2 channels are essential for chloride homeostasis. Complete knockout of mouse ClC-2 leads to testicular degeneration and neuronal myelin vacuolation. Gain-of-function and loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the genetic diseases aldosteronism and leukodystrophy, respectively. The protein homeostasis (proteostasis) mechanism of ClC-2 is currently unclear. Here, we aimed to identify the molecular mechanism of endoplasmic reticulum-associated degradation of ClC-2, and to explore the pathophysiological significance of disease-associated anomalous ClC-2 proteostasis. In both heterologous expression system and native neuronal and testicular cells, ClC-2 is subject to significant regulation by cullin-RING E3 ligase-mediated polyubiquitination and proteasomal degradation. The cullin 4 (CUL4)-damage-specific DNA binding protein 1 (DDB1)-cereblon (CRBN) E3 ubiquitin ligase co-exists in the same complex with and promotes the degradation of ClC-2 channels. The CRBN-targeting immunomodulatory drug lenalidomide and the cullin E3 ligase inhibitor MLN4924 promotes and attenuates, respectively, proteasomal degradation of ClC-2. Analyses of disease-related ClC-2 mutants reveal that aldosteronism and leukodystrophy are associated with opposite alterations in ClC-2 proteostasis. Modifying CUL4 E3 ligase activity with lenalidomide and MLN4924 ameliorates disease-associated ClC-2 proteostasis abnormality. Our results highlight the significant role and therapeutic potential of CUL4 E3 ubiquitin ligase in regulating ClC-2 proteostasis.

Abstract Background Mutations in the human gene encoding the neuron-specific Eag1 (KV10.1; KCNH1) potassium channel are linked to congenital neurodevelopmental diseases. Disease-causing mutant Eag1 channels manifest aberrant gating function and defective protein homeostasis. Both the E3 ubiquitin ligase cullin 7 (Cul7) and the small acid protein 14-3-3 serve as binding partners of Eag1. Cul7 mediates proteasomal and lysosomal degradation of Eag1 protein, whereas over-expression of 14-3-3 notably reduces Eag1 channel activity. It remains unclear whether 14-3-3 may also contribute to Eag1 protein homeostasis. Results In human cell line and native rat neurons, disruptions of endogenous 14-3-3 function with the peptide inhibitor difopein or specific RNA interference up-regulated Eag1 protein level in a transcription-independent manner. Difopein hindered Eag1 protein ubiquitination at the endoplasmic reticulum and the plasma membrane, effectively promoting the stability of both immature and mature Eag1 proteins. Suppression of endogenous 14-3-3 function also reduced excitotoxicity-associated Eag1 degradation in neurons. Difopein diminished Cul7-mediated Eag1 degradation, and Cul7 knock-down abolished the effect of difopein on Eag1. Inhibition of endogenous 14-3-3 function substantially perturbed the interaction of Eag1 with Cul7. Further structural analyses suggested that the intracellular Per-Arnt-Sim (PAS) domain and cyclic nucleotide-binding homology domain (CNBHD) of Eag1 are essential for the regulatory effect of 14-3-3 proteins. Significantly, suppression of endogenous 14-3-3 function reduced Cul7-mediated degradation of disease-associated Eag1 mutant proteins. Conclusion Overall these results highlight a chaperone-like role of endogenous 14-3-3 proteins in regulating Eag1 protein homeostasis, as well as a therapeutic potential of 14-3-3 modulators in correcting defective protein expression of disease-causing Eag1 mutants.

Respiratory syncytial virus, or RSV, is a leading cause of viral pneumonia and bronchiolitis in children and other susceptible populations. RSV infection dysregulates the immune response leading to exaggerated inflammation in the airway. Among other responses, RSV induces macroautophagy/autophagy, a key process that regulates immune response during infection. We investigated the molecular mechanisms underlying RSV-induced autophagy and showed that the RSV nonstructural NS2 protein promotes autophagy using a dual mechanism. First, NS2 interacts with and stabilizes the autophagy regulator BECN1 (beclin 1), augmenting its intracellular availability for autophagy induction. Second, NS2 interferes with BECN1 ISGylation, thus restricting the intracellular pool of the anti-autophagy ISGylated form of BECN1. Thus, the viral protein (i.e., NS2)-autophagy-ISGylation axis represents a yet unknown regulatory network for viruses. As many viruses induce autophagy that shapes virus-associated immune responses including inflammation, exploring viral protein-autophagy-ISGylation regulatory networks can aid in developing interventions to curb exaggerated immune responses such as inflammation for treating virus-associated inflammatory diseases.

Colorectal cancer ranks among the third most common human malignant diseases and is one of the leading causes of cancer-related deaths globally. Colon cancer cells are hypoxic and display disturbed protein homeostasis. Ubiquitin-ligase-initiated proteasomal degradation as well as its prevention by deubiquitinases (DUBs) are supposed to contribute to the above-mentioned disturbances. However, not much is known about the involvement of ubiquitinating and deubiquitinating enzymes in colon cancer and their effect on the hypoxia response. Here, we identify the DUB ubiquitin-specific protease 10 (USP10) as an important player in the control of colon cancer progression and a new modifier of the hypoxia response. Mechanistically, we show that knockout of USP10 in different colon cancer cells causes an elevation in HIF-1α but not HIF-2α protein levels under both normoxic and hypoxic conditions. In addition, the lack of USP10 increased cellular migration, reduced cell adhesion, and switched the energy phenotype towards increased glycolysis and enhanced extracellular acidification. These changes were at least partially caused by HIF-1α, as the knockdown of HIF-1α rescued the cellular phenotype caused by USP10 deficiency. Interestingly, the USP10-dependent increase in HIF-1 α was neither caused by enhanced transcription nor prolonged half-life but via mTOR/S6K mediated HIF-1α protein synthesis. Together, the current findings indicate that USP10 is able to participate in colon carcinogenesis by modulating the hypoxia response and may therefore represent a new therapeutic target. [ABSTRACT FROM AUTHOR]

Lysine residues are one of the main sites for posttranslational modifications of proteins, and lysine ubiquitination of the Machado-Joseph disease protein ataxin-3 is implicated in its cellular function and polyglutamine expansiondependent toxicity. Despite previously undertaken efforts, the individual roles of specific lysine residues of the ataxin-3 sequence are not entirely understood and demand further analysis. By retaining single lysine residues of otherwise lysinefree wild-type and polyglutamine-expanded ataxin-3, we assessed the effects of a site-limited modifiability on ataxin-3 protein levels, aggregation propensity, localization, and stability. We confirmed earlier findings that levels of lysine-free ataxin-3 are reduced due to its decreased stability, which led to a diminished load of SDS-insoluble species of its polyglutamine-expanded form. The isolated presence of several single lysine residues within the N-terminus of polyglutamineexpanded ataxin-3 significantly restored its aggregate levels, with highest fold changes induced by the presence of lysine 8 or lysine 85, respectively. Ataxin-3 lacking all lysine residues presented a slightly increased nuclear localization, which was counteracted by the reintroduction of lysine 85, whereas presence of either lysine 8 or lysine 85 led to a significantly higher ataxin-3 stability. Moreover, lysine-free ataxin-3 showed increased toxicity and binding to K48-linked polyubiquitin chains, whereas the reintroduction of lysine 85, located between the ubiquitin-binding sites 1 and 2 of ataxin-3, normalized its binding affinity. Overall, our data highlight the relevance of lysine residues 8 and 85 of ataxin-3 and encourage further analyses, to evaluate the potential of modulating posttranslational modifications of these sites for influencing pathophysiological characteristics of the Machado-Joseph disease protein. [ABSTRACT FROM AUTHOR]

Tankyrase (PARP5)-mediated PARsylation is a post-translational protein modification that creates a recognition site for the E3-ubiquitin ligase ring finger protein 146 (RNF146), leading to ubiquitylation and proteasomal degradation of its substrates, such as the adaptor protein SH3-domain binding protein 2 (3BP2). Missense mutations in SH3BP2 , which are associated with the autosomal dominant disorder Cherubism, are clustered in the binding motif for tankyrase and uncouple 3BP2 from tankyrase-mediated negative regulation. Stabilization of 3BP2 due to Cherubism mutations results in the activation of SRC and SYK kinases in osteoclasts, leading to craniofacial dysmorphic features in patients with Cherubism. Other tankyrase substrates, including the tumor suppressor genes AXIN , AMOT , PTEN , and LKB1 , suggest that tankyrase inhibitors have potential therapeutic benefit in selected tumors. However, recent in vivo studies show that mice lacking tankyrase in the myeloid-monocytic lineage develop severe systemic inflammation, demonstrating potential toxicities associated with tankyrase inhibition. Modification of proteins by ADP-ribose (PARsylation) is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes exemplified by PARP1, which controls chromatin organization and DNA repair. Additionally, PARsylation induces ubiquitylation and proteasomal degradation of its substrates because PARsylation creates a recognition site for E3-ubiquitin ligase. The steady-state levels of the adaptor protein SH3-domain binding protein 2 (3BP2) is negatively regulated by tankyrase (PARP5), which coordinates ubiquitylation of 3BP2 by the E3-ligase ring finger protein 146 (RNF146). 3BP2 missense mutations uncouple 3BP2 from tankyrase-mediated negative regulation and cause Cherubism, an autosomal dominant autoinflammatory disorder associated with craniofacial dysmorphia. In this review, we summarize the diverse biological processes, including bone dynamics, metabolism, and Toll-like receptor (TLR) signaling controlled by tankyrase-mediated PARsylation of 3BP2, and highlight the therapeutic potential of this pathway. [ABSTRACT FROM AUTHOR]

Appropriate treatment options are lacking for hepatitis E virus (HEV)-infected pregnant women and immunocompromised individuals. Thus, we aimed to identify efficient anti-HEV drugs through high-throughput screening, validate them in vitro and in vivo (in a preclinical animal study), and elucidate their underlying antiviral mechanism of action. Using appropriate cellular and rodent HEV infection models, we studied a critical pathway for host-HEV interactions and performed a preclinical study of the corresponding antivirals, which target proteostasis of the HEV replicase. We found 17 inhibitors that target HEV-HSP90 interactions by unbiased compound library screening on human hepatocytes harboring an HEV replicon. Inhibitors of HSP90 (iHSP90) markedly suppressed HEV replication with efficacy exceeding that of conventional antivirals (IFNα and ribavirin) in vitro. Mechanistically, iHSP90 treatment released the viral replicase ORF1 protein from the ORF1-HSP90 complex and triggered rapid ubiquitin/proteasome-mediated degradation of ORF1, resulting in abrogated HEV replication. Furthermore, a preclinical trial in a Mongolian gerbil HEV infection model showed this novel anti-HEV strategy to be safe, efficient, and able to prevent HEV-induced liver damage. In this study, we uncover a proteostatic pathway that is critical for host-HEV interactions and we provide a foundation from which to translate this new understanding of the HEV life cycle into clinically promising antivirals. Appropriate treatment options for hepatitis E virus (HEV)-infected pregnant women and immunocompromised patients are lacking; hence, there is an urgent need for safe and effective HEV-specific therapies. This study identified new antivirals (inhibitors of HSP90) that significantly limit HEV infection by targeting the viral replicase for degradation. Moreover, these anti-HEV drugs were validated in an HEV rodent model and were found to be safe and efficient for prevention of HEV-induced liver injury in preclinical experiments. Our findings substantially promote the understanding of HEV pathobiology and pave the way for antiviral development. [Display omitted] • We identified 17 HSP90 inhibitors via high-throughput screening of anti-HEV compounds using replicon models. • Inhibiting the HEV replicase-HSP90 interaction releases ORF1 for K48-linked ubiquitination and proteasomal degradation. • Cytosolic ORF1-HSP90 aggregates are critical for HEV replication. • Preclinical studies demonstrate that targeting HSP90 prevents sustained HEV infection and liver injury in rodents. [ABSTRACT FROM AUTHOR]

TGF-β signaling is a vital regulator for maintaining articular cartilage homeostasis. Runx transcription factors, downstream targets of TGF-β signaling, have been studied in the context of osteoarthritis (OA). Although Runx partner core binding factor β (Cbfβ) is known to play a pivotal role in chondrocyte and osteoblast differentiation, the role of Cbfβ in maintaining articular cartilage integrity remains obscure. This study investigated Cbfβ as a novel anabolic modulator of TGF-β signaling and determined its role in articular cartilage homeostasis. Cbfβ significantly decreased in aged mouse articular cartilage and human OA cartilage. Articular chondrocyte-specific Cbfb-deficient mice (Cbfb△ac/△ac) exhibited early cartilage degeneration at 20 weeks of age and developed OA at 12 months. Cbfb△ac/△ac mice showed enhanced OA progression under the surgically induced OA model in mice. Mechanistically, forced expression of Cbfβ rescued Type II collagen (Col2α1) and Runx1 expression in Cbfβ-deficient chondrocytes. TGF-β1-mediated Col2α1 expression failed despite the p-Smad3 activation under TGF-β1 treatment in Cbfβ-deficient chondrocytes. Cbfβ protected Runx1 from proteasomal degradation through Cbfβ/Runx1 complex formation. These results indicate that Cbfβ is a novel anabolic regulator for cartilage homeostasis, suggesting that Cbfβ could protect OA development by maintaining the integrity of the TGF-β signaling pathway in articular cartilage. [ABSTRACT FROM AUTHOR]

The dual leucine zipper kinase (DLK) alias mitogen-activated protein 3 kinase 12 (MAP3K12) has gained much attention in recent years. DLK belongs to the mixed lineage kinases, characterized by homology to serine/threonine and tyrosine kinase, but exerts serine/threonine kinase activity. DLK has been implicated in many diseases, including several neurodegenerative diseases, glaucoma, and diabetes mellitus. As a MAP3K, it is generally assumed that DLK becomes phosphorylated and activated by upstream signals and phosphorylates and activates itself, the downstream serine/threonine MAP2K, and, ultimately, MAPK. In addition, other mechanisms such as protein–protein interactions, proteasomal degradation, dephosphorylation by various phosphatases, palmitoylation, and subcellular localization have been shown to be involved in the regulation of DLK activity or its fine-tuning. In the present review, the diverse mechanisms regulating DLK activity will be summarized to provide better insights into DLK action and, possibly, new targets to modulate DLK function.

Heart development is a spatiotemporally regulated process that extends from the embryonic phase to postnatal stages. Disruption of this highly orchestrated process can lead to congenital heart disease or predispose the heart to cardiomyopathy or heart failure. Consequently, gaining an in-depth understanding of the molecular mechanisms governing cardiac development holds considerable promise for the development of innovative therapies for various cardiac ailments. While significant progress in uncovering novel transcriptional and epigenetic regulators of heart development has been made, the exploration of post-translational mechanisms that influence this process has lagged. Culling-RING E3 ubiquitin ligases (CRLs), the largest family of ubiquitin ligases, control the ubiquitination and degradation of ~20% of intracellular proteins. Emerging evidence has uncovered the critical roles of CRLs in the regulation of a wide range of cellular, physiological, and pathological processes. In this review, we summarize current findings on the versatile regulation of cardiac morphogenesis and maturation by CRLs and present future perspectives to advance our comprehensive understanding of how CRLs govern cardiac developmental processes.

A20 haploinsufficiency (HA20) is an autoinflammatory disease caused by heterozygous loss-of-function variations in TNFAIP3, the gene encoding the A20 protein. Diagnosis of HA20 is challenging due to its heterogeneous clinical presentation and the lack of pathognomonic symptoms. While the pathogenic effect of TNFAIP3 truncating variations is clearly established, that of missense variations is difficult to determine. Herein, we identified a novel TNFAIP3 variation, p.(Leu236Pro), located in the A20 ovarian tumor (OTU) domain and demonstrated its pathogenicity. In the patients’ primary cells, we observed reduced A20 levels. Protein destabilization was predicted in silico for A20_Leu236Pro and enhanced proteasomal degradation was confirmed in vitro through a flow cytometry-based functional assay. By applying this approach to the study of another missense variant, A20_Leu275Pro, for which no functional characterization has been performed to date, we showed that this variant also undergoes enhanced proteasomal degradation. Moreover, we showed a disrupted ability of A20_Leu236Pro to inhibit the NF-κB pathway and to deubiquitinate its substrate TRAF6. Structural modeling revealed that two residues involved in OTU pathogenic missense variations (i.e. Glu192Lys and Cys243Tyr) establish common interactions with Leu236. Interpretation of newly identified missense variations is challenging, requiring, as illustrated here, functional demonstration of their pathogenicity. Together with functional studies, in silico structure analysis is a valuable approach that allowed us (i) to provide a mechanistic explanation for the haploinsufficiency resulting from missense variations and (ii) to unveil a region within the OTU domain critical for A20 function.