BACKGROUND: Metastasis is the leading cause of cancer-related death in non-small cell lung cancer (NSCLC) patients. We previously showed that low HERC5 expression predicts early tumor dissemination and a dismal prognosis in NSCLC patients. Here, we performed functional studies to unravel the mechanism underlying the \"metastasis-suppressor\" effect of HERC5, with a focus on mitochondrial metabolism pathways.
METHODS: We assessed cell proliferation, colony formation potential, anchorage-independent growth, migration, and wound healing in NSCLC cell line models with HERC5 overexpression (OE) or knockout (KO). To study early tumor cell dissemination, we used these cell line models in zebrafish experiments and performed intracardial injections in nude mice. Mass spectrometry (MS) was used to analyze protein changes in whole-cell extracts. Furthermore, electron microscopy (EM) imaging, cellular respiration, glycolytic activity, and lactate production were used to investigate the relationships with mitochondrial energy metabolism pathways.
RESULTS: Using different in vitro NSCLC cell line models, we showed that NSCLC cells with low HERC5 expression had increased malignant and invasive properties. Furthermore, two different in vivo models in zebrafish and a xenograft mouse model showed increased dissemination and metastasis formation (in particular in the brain). Functional enrichment clustering of MS data revealed an increase in mitochondrial proteins in vitro when HERC5 levels were high. Loss of HERC5 leads to an increased Warburg effect, leading to improved adaptation and survival under prolonged inhibition of oxidative phosphorylation.
CONCLUSIONS: Taken together, these results indicate that low HERC5 expression increases the metastatic potential of NSCLC in vitro and in vivo. Furthermore, HERC5-induced proteomic changes influence mitochondrial pathways, ultimately leading to alterations in energy metabolism and demonstrating its role as a new potential metastasis suppressor gene.

Interferon-stimulated gene product 15 (ISG15) can be conjugated to substrates through ISGylation. Currently, the E3 ligase for porcine ISGylation remains unclear. Here, we identified porcine HERC5 and HERC6 (pHERC5/6) as ISGylation E3 ligases with pHERC6 acting as a major one by reconstitution of porcine ISGylation system in HEK-293 T cell via co-transfecting E1, E2 and porcine ISG15(pISG15) genes. Meanwhile, our data demonstrated that co-transfection of pISG15 and pHERC5/6 was sufficient to confer ISGylation, suggesting E1 and E2 of ISGylation are interchangeable between human and porcine. Using an immunoprecipitation based ISGylation analysis, our data revealed pHERC6 was a substrate for ISGylation and confirmed that K707 and K993 of pHERC6 were auto-ISGylation sites. Mutation of these sites reduced pHERC6 half-life and inhibited ISGylation, suggesting that auto-ISGylation of pHERC6 was required for effective ISGylation. Conversely, sustained ISGylation induced by overexpression of pISG15 and pHERC6 could be inhibited by a well-defined porcine ISGylation antagonist, the ovarian tumor (OTU) protease domain of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-nsp2 and PRRSV-nsp1β, further indicating such method could be used for identification of virus-encoded ISG15 antagonist. In conclusion, our study contributes new insights towards porcine ISGylation system and provides a novel tool for screening viral-encoded ISG15 antagonist.

The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) is essential to elicit type I interferon cascade response; thus, the activity of cGAS must be strictly regulated to boost the antiviral innate immunity. Here, we report that cGAS is responsible for the DNA-induced ISG15 conjugation system. The E3 HERC5 catalyzes the ISGylation of cytoplasmic cGAS at lysine 21, 187, 219, and 458, whereas Ubl carboxy-terminal hydrolase 18 removes the ISGylation of cGAS. The interaction of cGAS and HERC5 depends on the cGAS C-terminal domain and the RRC1-4 and RRC1-5 domains of HERC5. Mechanically, HERC5-catalyzed ISGylation promotes DNA-induced cGAS oligomerization and enhances cGAS enzymatic activity. Deficiency of ISGylation attenuates the downstream inflammatory gene expression induced by the cGAS-STING axis and the antiviral ability in mouse and human cells. Mice deficient in Isg15 or Herc6 are more vulnerable to herpes simplex virus 1 infection. Collectively, our study shows a positive feedback regulation of the cGAS-mediated innate immune pathway by ISGylation.

Osteoarthritis (OA) is a multifaceted chronic joint disease characterized by complex mechanisms. It has a detrimental impact on the quality of life for individuals in the middle-aged and elderly population while also imposing a significant socioeconomic burden. At present, there remains a lack of comprehensive understanding regarding the pathophysiology of OA. The objective of this study was to examine the genes, functional pathways, and immune infiltration characteristics associated with the development and advancement of OA.
The Gene Expression Omnibus (GEO) database was utilized to acquire gene expression profiles. The R software was employed to conduct the screening of differentially expressed genes (DEGs) and perform enrichment analysis on these genes. The OA-characteristic genes were identified using the Weighted Gene Co-expression Network Analysis (WGCNA) and the Lasso algorithm. In addition, the infiltration levels of immune cells in cartilage were assessed using single-sample gene set enrichment analysis (ssGSEA). Subsequently, a correlation analysis was conducted to examine the relationship between immune cells and the OA-characteristic genes.
A total of 80 DEGs were identified. As determined by functional enrichment, these DEGs were associated with chondrocyte metabolism, apoptosis, and inflammation. Three OA-characteristic genes were identified using WGCNA and the lasso algorithm, and their expression levels were then validated using the verification set. Finally, the analysis of immune cell infiltration revealed that T cells and B cells were primarily associated with OA. In addition, Tspan2, HtrA1 demonstrated a correlation with some of the infiltrating immune cells.
The findings of an extensive bioinformatics analysis revealed that OA is correlated with a variety of distinct genes, functional pathways, and processes involving immune cell infiltration. The present study has successfully identified characteristic genes and functional pathways that hold potential as biomarkers for guiding drug treatment and facilitating molecular-level research on OA.

African swine fever virus (ASFV) causes acute hemorrhagic infectious disease in pigs. The ASFV genome encodes various proteins that enable the virus to escape innate immunity; however, the underlying mechanisms are poorly understood. The present study found that ASFV MGF-360-10L significantly inhibits interferon (IFN)-β-triggered STAT1/2 promoter activation and the production of downstream IFN-stimulated genes (ISGs). ASFV MGF-360-10L deletion (ASFV-Δ10L) replication was impaired compared with the parental ASFV CN/GS/2018 strain, and more ISGs were induced by the ASFV-Δ10L in porcine alveolar macrophages in vitro. We found that MGF-360-10L mainly targets JAK1 and mediates its degradation in a dose-dependent manner. Meanwhile, MGF-360-10L also mediates the K48-linked ubiquitination of JAK1 at lysine residues 245 and 269 by recruiting the E3 ubiquitin ligase HERC5 (HECT and RLD domain-containing E3 ubiquitin protein ligase 5). The virulence of ASFV-Δ10L was significantly lower than that of the parental strain in vivo, which indicates that MGF-360-10L is a novel virulence factor of ASFV. Our findings elaborate the novel mechanism of MGF-360-10L on the STAT1/2 signaling pathway, expanding our understanding of the inhibition of host innate immunity by ASFV-encoded proteins and providing novel insights that could contribute to the development of African swine fever vaccines. IMPORTANCE African swine fever outbreaks remain a concern in some areas. There is no effective drug or commercial vaccine to prevent African swine fever virus (ASFV) infection. In the present study, we found that overexpression of MGF-360-10L strongly inhibited the interferon (IFN)-β-induced STAT1/2 signaling pathway and the production of IFN-stimulated genes (ISGs). Furthermore, we demonstrated that MGF-360-10L mediates the degradation and K48-linked ubiquitination of JAK1 by recruiting the E3 ubiquitin ligase HERC5. The virulence of ASFV with MGF-360-10L deletion was significantly less than parental ASFV CN/GS/2018. Our study identified a new virulence factor and revealed a novel mechanism by which MGF-360-10L inhibits the immune response, thus providing new insights into the vaccination strategies against ASFV.

The tumor suppressor p53 has been implicated in the pathogenesis of liver fibrosis. HERC5-mediated posttranslational ISG modification of the p53 protein is critical for controlling its activity. Here, we demonstrated that the expression of HERC5 and ISG15 is highly elevated, whereas p53 is downregulated, in fibrotic liver tissues of mice and transforming growth factor-β1 (TGF-β1)-induced LX2 cells. HERC5 siRNA clearly increased the protein expression of p53, but the mRNA expression of p53 was not obviously changed. The inhibition of lincRNA-ROR (ROR) downregulated HERC5 expression and elevated p53 expression in TGF-β1-stimulated LX-2 cells. Furthermore, the expression of p53 was almost unchanged after TGF-β1-stimulated LX-2 cells were co-transfected with a ROR-expressing plasmid and HERC5 siRNA. We further confirmed that miR-145 is a target gene of ROR. In addition, we also showed that ROR regulates the HERC5-mediated ISGylation of p53 through mir-145/ZEB2. Together, we propose that ROR/miR-145/ZEB2 might be involved in the course of liver fibrosis by regulating ISGylation of the p53 protein.

UNASSIGNED: The restoration of the Wnt/β-catenin pathway to alleviate alcoholic fatty liver disease (AFLD) progression is under study as a new strategy for alcoholic liver disease (ALD) treatment. Recent studies have indicated that interferon-stimulated gene 15 (ISG15) can covalently bind to β-catenin by HECT E3 ubiquitin ligase 5 (HERC5), leading to ISG degradation and downregulation of β-catenin levels. However, the relationship between β-catenin and the ISG15 system in AFLD remains unclear.
UNASSIGNED: Here, we explored the roles of the ISG15 system in β-catenin activation and in the pathogenesis of alcohol-induced liver injury and steatosis.
UNASSIGNED: In this study, HERC5 silencing upregulated β-catenin protein expression and inhibited lipid metabolism disorders and cell apoptosis. Reduced β-catenin protein expression, increased lipid metabolism disorders, and cell apoptosis were detected in cells induced with HERC5 overexpression, which was reversible with the reactive oxygen species (ROS) inhibitor. All the above results were statistically analyzed. Thus, these observations demonstrate that β-catenin ISGylation is a prominent regulator of ALD pathology, which works by regulating ROS to induce lipid metabolism disorders and cell apoptosis.
UNASSIGNED: Our findings provided the mechanism involved in the β-catenin ISGylation, allowing for future studies on the prevention or amelioration of liver injury in ALD.

Apoptosis-inducing factor (AIF) is a mitochondrion-localized flavoprotein with NADH oxidase activity. AIF normally acts as an oxidoreductase to catalyze the transfer of electrons between molecules, but it can also kill cells when exposed to certain stimuli. For example, intact AIF is cleaved upon exposure to DNA-damaging agents such as etoposide, and truncated AIF (tAIF) is released from the mitochondria to the cytoplasm and translocated to the nucleus where it induces apoptosis. Although the serial events during tAIF-mediated apoptosis and the transition of AIF function have been widely studied from various perspectives, their underlying regulatory mechanisms and the factors involved are not fully understood. Here, we demonstrated that tAIF is a target of the covalent conjugation of the ubiquitin-like moiety ISG15 (referred to as ISGylation), which is mediated by the ISG15 E3 ligase HERC5. In addition, ISGylation increases the stability of tAIF protein as well as its K6-linked polyubiquitination. Moreover, we found that ISGylation increases the nuclear translocation of tAIF upon cytotoxic etoposide treatment, subsequently causing apoptotic cell death in human lung A549 carcinoma cells. Collectively, these results suggest that HERC5-mediated ISG15 conjugation is a key factor in the positive regulation of tAIF-mediated apoptosis, highlighting a novel role of posttranslational ISG15 modification as a switch that allows cells to live or die under the stress that triggers tAIF release.

Ubiquitin-like proteins (Ubls) share some features with ubiquitin (Ub) such as their globular 3D structure and the ability to attach covalently to other proteins. Interferon Stimulated Gene 15 (ISG15) is an abundant Ubl that similar to Ub, marks many hundreds of cellular proteins, altering their fate. In contrast to Ub, , ISG15 requires interferon (IFN) induction to conjugate efficiently to other proteins. Moreover, despite the multitude of E3 ligases for Ub-modified targets, a single E3 ligase termed HERC5 (in humans) is responsible for the bulk of ISG15 conjugation. Targets include both viral and cellular proteins spanning an array of cellular compartments and metabolic pathways. So far, no common structural or biochemical feature has been attributed to these diverse substrates, raising questions about how and why they are selected. Conjugation of ISG15 mitigates some viral and bacterial infections and is linked to a lower viral load pointing to the role of ISG15 in the cellular immune response. In an apparent attempt to evade the immune response, some viruses try to interfere with the ISG15 pathway. For example, deconjugation of ISG15 appears to be an approach taken by coronaviruses to interfere with ISG15 conjugates. Specifically, coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2, encode papain-like proteases (PL1pro) that bear striking structural and catalytic similarities to the catalytic core domain of eukaryotic deubiquitinating enzymes of the Ubiquitin-Specific Protease (USP) sub-family. The cleavage specificity of these PLpro enzymes is for flexible polypeptides containing a consensus sequence (R/K)LXGG, enabling them to function on two seemingly unrelated categories of substrates: (i) the viral polyprotein 1 (PP1a, PP1ab) and (ii) Ub- or ISG15-conjugates. As a result, PLpro enzymes process the viral polyprotein 1 into an array of functional proteins for viral replication (termed non-structural proteins; NSPs), and it can remove Ub or ISG15 units from conjugates. However, by de-conjugating ISG15, the virus also creates free ISG15, which in turn may affect the immune response in two opposite pathways: free ISG15 negatively regulates IFN signaling in humans by binding non-catalytically to USP18, yet at the same time free ISG15 can be secreted from the cell and induce the IFN pathway of the neighboring cells. A deeper understanding of this protein-modification pathway and the mechanisms of the enzymes that counteract it will bring about effective clinical strategies related to viral and bacterial infections.

OBJECTIVE: This study aims to find differentially expressed ubiquitination-related gene(s) and elucidates their biological significance in breast cancer.
METHODS: Differentially expressed genes were profiled in MCF-7 and MDA-MB-231 cells by using PCR array method. Abnormal expression of HERC5 was studied in the cells and in breast cancer specimens via Quantitative Real-time PCR and western blot. Cell proliferation and cell migration abilities were evaluated by using cell counting kits, or through colony formation, wound healing and trans-well assays. HERC5 target proteins were investigated via proteomic, co-immunoprecipitation and western blot methods. Down-stream signaling pathways were investigated through gene expression/knockdown methods.
RESULTS: Huge increase of HERC5 expression was found in MCF-7 and MDA-MB-231 cells, knockdown of which repressed the cell proliferation and migration. HERC5 interacted with IFI16, mediated IFI16 ISGylation at K274 and facilitated IFI16 proteasomal degradation. IFI16 acted as a tumor suppressor and to some extent mediated the HERC5 function in the breast cancer (BC) cells. HERC5 was negatively correlated with IFI16 protein, while IFI16 was positively correlated to p53 expression at mRNA and protein levels, which indicates a novel signaling pathway - HERC5/IFI16/p53. HERC5 expression was increased in glucose-starved BC cells and in human breast cancer tissues, accompanied with the decrease of IFI16 and P53.
CONCLUSIONS: Our work reveals the abnormal expression of HERC5 and its carcinogenic role in breast cancer cells, which is probably mediated by an HERC5/IFI16/p53 signaling pathway. This work also provides potential diagnostic/therapeutic biomarkers for breast cancer diagnosis and treatment.