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Of the seven in absentia homologue (SIAH) family, three members have been identified in the human genome. In contrast to the E3 ubiquitin ligase encoding SIAH1 and SIAH2, little is known on the regulation and function of SIAH3 in tumorigenesis. In this study, we reveal that SIAH3 is frequently epigenetically silenced in different cancer entities, including cutaneous melanoma, lung adenocarcinoma and head and neck cancer. Low SIAH3 levels correlate with an impaired survival of cancer patients. Additionally, induced expression of SIAH3 reduces cell proliferation and induces cell death. Functionally, SIAH3 negatively affects cellular metabolism by shifting cells form aerobic oxidative phosphorylation to glycolysis. SIAH3 is localized in the mitochondrion and interacts with proteins involved in mitochondrial ribosome biogenesis and translation. We also report that SIAH3 interacts with ubiquitin ligases, including SIAH1 or SIAH2, and is degraded by them. These results suggest that SIAH3 acts as an epigenetically controlled tumor suppressor by regulating cellular metabolism through the inhibition of oxidative phosphorylation., (© 2024 The Author(s). International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

AIMP2-DX2 (hereafter DX2) is an oncogenic variant of aminoacyl-tRNA synthetase-interacting multifunctional protein 2 (AIMP2) that mediates tumorigenic interactions with various factors involved in cancer. Reducing the levels of DX2 can effectively inhibit tumorigenesis. We previously reported that DX2 can be degraded through Siah1-mediated ubiquitination. In this study, we identified a compound, SDL01, which enhanced the interaction between DX2 and Siah1, thereby facilitating the ubiquitin-dependent degradation of DX2. SDL01 was found to bind to the pocket surrounding the N-terminal flexible region and GST domain of DX2, causing a conformational change that stabilized its interaction with Siah1. Our findings demonstrate that protein-protein interactions (PPIs) can be modulated through chemically induced conformational changes., Competing Interests: Declaration of interests This work has been patented in 2020 under patent number (WO2020204548A1, worldwide applications)., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mitophagy eliminates dysfunctional mitochondria, and defects in this cellular housekeeping mechanism are implicated in various age-related diseases. Here, we found that mitophagy suppression by the protein Siah3 promoted developmental axonal remodeling in mice. Siah3-deficient mice displayed increased peripheral sensory innervation. Cultured Siah3-deficient sensory neurons exhibited delays in both axonal degeneration and caspase-3 activation in response to withdrawal of nerve growth factor. Mechanistically, Siah3 was transcriptionally induced by the loss of trophic support and formed a complex with the cytosolic E3 ubiquitin ligase parkin, a core component of mitophagy, in transfected cells. Axons of Siah3-deficient neurons mounted profound mitophagy upon initiation of degeneration but not under basal conditions. Neurons lacking both Siah3 and parkin did not exhibit the delay in trophic deprivation-induced axonal degeneration or the induction of axonal mitophagy that was seen in Siah3-deficient neurons. Our findings reveal that mitophagy regulation acts as a gatekeeper of a physiological axon elimination program.

Developmental axon pruning is controlled by a careful balance of pro- and anti-apoptotic signals, which are activated in response to external cues to sculpt mature neuronal circuitry. In this issue of Science Signaling , Abraham et al. define a safeguard against apoptotic axon pruning and illustrate that Siah3 represses Parkin-mediated mitophagy to control the availability of axonal mitochondria that activate the pruning process.

Casein kinase 1ε (CK1ε) and axis inhibitor 1 (AXIN1) are crucial components of the β-catenin destruction complex in canonical Wnt signaling. CK1ε has been shown to interact with AXIN1, but its physiological function and role in tumorigenesis remain unknown. In this study, we found that CK1δ/ε inhibitors significantly enhanced AXIN1 protein level in colorectal cancer (CRC) cells through targeting CK1ε. Mechanistically, CK1ε promoted AXIN1 degradation by the ubiquitin-proteasome pathway by promoting the interaction of E3 ubiquitin-protein ligase SIAH1 with AXIN1. Genetic or pharmacological inhibition of CK1ε and knockdown of SIAH1 downregulated the expression of Wnt/β-catenin-dependent genes, suppressed the viability of CRC cells, and restrained tumorigenesis and progression of CRC in vitro and in vivo. In summary, our results demonstrate that CK1ε exerted its oncogenic role in CRC occurrence and progression by regulating the stability of AXIN1. These findings reveal a novel mechanism by which CK1ε regulates the Wnt/β-catenin signaling pathway and highlight the therapeutic potential of targeting the CK1ε/SIAH1 axis in CRC., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

It has been shown that the formation of filopodia is a key step in tumor cell metastasis, but there is limited research regarding its mechanism. In this study, we demonstrated that fatty acid synthase (FASN) promoted filopodia formation in liver cancer cells by regulating fascin actin-bundling protein 1 (FSCN1), a marker protein for filopodia. Mechanistically, on the one hand, the accumulation of FASN is caused by the enhanced deubiquitination of FASN mediated by UCHL5 (ubiquitin c-terminal hydrolase L5). In this pathway, low expression of SIAH1 (Seven in absentia homolog 1) can decrease the ubiquitination and degradation of ADRM1 (adhesion regulating molecule 1) thereby increasing its protein level, which will recruit and activate the deubiquitination enzyme UCHL5, leading to FASN undergo deubiquitination and escape from proteasomal degradation. On the other hand, the accumulation of FASN is related to its weakened ubiquitination, where SIAH1 directly acts as a ubiquitin ligase toward FASN, and low expression of SIAH1 reduces the ubiquitination and degradation of FASN. Both the two pathways are involved in the regulation of FASN in liver cancer. Our results reveal a novel mechanism for FASN accumulation due to the low expression of SIAH1 in human liver cancer and suggest an important role of FASN in filopodia formation in liver cancer cells., (© 2024. The Author(s).)

Nucleoprotein (N) is well known for its function in the encapsidation of the genomic RNAs of negative-strand RNA viruses, which leads to the formation of ribonucleoproteins that serve as templates for viral transcription and replication. However, the function of the N protein in other aspects during viral infection is far from clear. In this study, the N protein of snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus, was proved to be ubiquitinated mainly via K63-linked ubiquitination. We identified nine host E3 ubiquitin ligases that interacted with SHVV N, among which seven E3 ubiquitin ligases facilitated ubiquitination of the N protein. Further investigation revealed that only two E3 ubiquitin ligases, Siah E3 ubiquitin protein ligase 2 (Siah2) and leucine-rich repeat and sterile alpha motif containing 1 (LRSAM1), mediated K63-linked ubiquitination of the N protein. SHVV infection upregulated the expression of Siah2 and LRSAM1, which maintained the stability of SHVV N. Besides, overexpression of Siah2 or LRSAM1 promoted SHVV replication, while knockdown of Siah2 or LRSAM1 inhibited SHVV replication. Deletion of the ligase domain of Siah2 or LRSAM1 did not affect their interactions with SHVV N but reduced the K63-linked ubiquitination of SHVV N and SHVV replication. In summary, Siah2 and LRSAM1 mediate K63-linked ubiquitination of SHVV N to facilitate SHVV replication, which provides novel insights into the role of the N proteins of negative-strand RNA viruses., Importance: Ubiquitination of viral protein plays an important role in viral replication. However, the ubiquitination of the nucleoprotein (N) of negative-strand RNA viruses has rarely been investigated. This study aimed at investigating the ubiquitination of the N protein of a fish rhabdovirus SHVV (snakehead vesiculovirus), identifying the related host E3 ubiquitin ligases, and determining the role of SHVV N ubiquitination and host E3 ubiquitin ligases in viral replication. We found that SHVV N was ubiquitinated mainly via K63-linked ubiquitination, which was mediated by host E3 ubiquitin ligases Siah2 (Siah E3 ubiquitin protein ligase 2) and LRSAM1 (leucine-rich repeat and sterile alpha motif containing 1). The data suggested that Siah2 and LRSAM1 were hijacked by SHVV to ubiquitinate the N protein for viral replication, which exhibited novel anti-SHVV targets for drug design., Competing Interests: The authors declare no conflict of interest.

Hepatitis B virus (HBV) infects approximately 300 million people worldwide, causing chronic infections. The HBV X protein (HBx) is crucial for viral replication and induces reactive oxygen species (ROS), leading to cellular damage. This study explores the relationship between HBx-induced ROS, p53 activation, and HBV replication. Using HepG2 and Hep3B cell lines that express the HBV receptor NTCP, we compared ROS generation and HBV replication relative to p53 status. Results indicated that HBV infection significantly increased ROS levels in p53-positive HepG2-NTCP cells compared to p53-deficient Hep3B-NTCP cells. Knockdown of p53 reduced ROS levels and enhanced HBV replication in HepG2-NTCP cells, whereas p53 overexpression increased ROS and inhibited HBV replication in Hep3B-NTCP cells. The ROS scavenger N-acetyl-L-cysteine (NAC) reversed these effects. The study also found that ROS-induced degradation of the HBx is mediated by the E3 ligase Siah-1, which is activated by p53. Mutations in p53 or inhibition of its transcriptional activity prevented ROS-mediated HBx degradation and HBV inhibition. These findings reveal a p53-dependent negative feedback loop where HBx-induced ROS increases p53 levels, leading to Siah-1-mediated HBx degradation and HBV replication inhibition. This study offers insights into the molecular mechanisms of HBV replication and identifies potential therapeutic targets involving ROS and p53 pathways.

As an important protein encoded by hepatitis B virus (HBV), HBV X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). It has been shown that seven in absentia homologue 1 (SIAH1) could regulates the degradation of HBx through the ubiquitin-proteasome pathway. However, as a member of SIAH family, the regulatory effects of SIAH2 on HBx remain unclear. In this study, we first confirmed that SIAH2 could reduce the protein levels of HBx depending on its E3 ligase activity. Moreover, SIAH2 interacted with HBx and induced its K48-linked polyubiquitination and proteasomal degradation. Furthermore, we provided evidence that SIAH2 inhibits HBx-associated HCC cells proliferation by regulating HBx. In conclusion, our study identified a novel role for SIAH2 in promoting HBx degradation and SIAH2 exerts an inhibitory effect in the proliferation of HBx-associated HCC through inducing the degradation of HBx. Our study provides a new idea for the targeted degradation of HBx and may have great huge significance into providing novel evidence for the targeted therapy of HBV-infected HCC., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Triple-negative breast cancer (TNBC) is the most lethal and aggressive subtype of breast cancer, and chemoresistance is the major determinant of TNBC treatment failure. This study explores the molecular mechanism of TNBC chemoresistance. The Cancer Genome Atlas, breast cancer integrative platform, and GEPIA databases were used to analyze the expression and correlation of YTHDF1 and seven in absentia homology 2 (SIAH2) in breast cancer. Knockdown of YTHDF1 and SIAH2, or overexpression of SIAH2 in vitro and in vivo, was conducted to evaluate the impact of changes in YTHDF1 and SIAH2 expression on TNBC cell proliferation, apoptosis, stemness, drug resistance, and Hippo pathway gene expression. YTHDF1 and SIAH2 were highly expressed in breast cancer patients and TNBC cells. Knockdown of YTHDF1 and SIAH2 significantly inhibited proliferation and stemness and promoted apoptosis and chemosensitivity of TNBC cells. Mechanistically, the knockdown of YTHDF1 inhibited the expression of SIAH2, thereby downregulating the Hippo pathway, which inhibited proliferation and stemness and promoted apoptosis and chemosensitivity of TNBC cells. The current findings revealed the regulatory mechanism of YTHDF1 in TNBC and clarified the role of the YTHDF1/SIAH2 axis in TNBC drug resistance and stemness. This could provide new insights into the vital role of targeting YTHDF1/SIAH2 to suppress drug resistance and stemness in TNBC cells., (© 2023 The Authors. Molecular Carcinogenesis published by Wiley Periodicals LLC.)

Inflammation-mediated dysfunction of cardiomyocytes is the main cause of diabetic cardiomyopathy (DCM). The present study aimed to investigate the roles of siah E3 ubiquitin protein ligase 1 (SIAH1) in DCM. The online dataset GSE4172 was used to analyze the differentially expressed genes in myocardial inflammation of DCM patients. RT-qPCR was conducted to detect mRNA levels. Enzyme-Linked Immunosorbent Assay (ELISA) was performed to detect cytokine release. Western blot was used to detect protein expression. Lactate dehydrogenase (LDH) assay was used to determine cytotoxicity. In vitro ubiquitination assay was applied to determine the ubiquitination of nuclear factor kappa B inhibitor alpha (1κВ-α). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect the death of cardiomyocytes. Flow cytometry was applied for determining cardiomyocyte pyroptosis. The results showed that SIAH1 was overexpressed in human inflammatory cardiomyopathy. High expression of SIAH1 was associated with inflammatory response. SIAH1 was also overexpressed lipopolysaccharide (LPS)-induced inflammatory cardiomyopathy model in vitro. However, SIAH1 knockdown suppressed the inflammatory-related pyroptosis of cardiomyocytes. SIAH1 promoted the ubiquitination of 1κВ-α and activated nuclear factor kappa В (NF-κВ) signaling, which promoted the pyroptosis of cardiomyocytes. In conclusion, SIAH1 exacerbated the progression of human inflammatory cardiomyopathy via inducing the ubiquitination of 1κВ-α and activation of NF-κВ signaling. Therefore, SIAHI/IκB-α/NF-κB signaling may be a potential target for human inflammatory cardiomyopathy.

SIAH2 function as an oncogene in various cancer. However, the roles of SIAH2 in hepatocellular carcinoma (HCC) are still unknown. This study aimed to investigate the roles of SIAH2 in HCC. Immunohistochemistry was used determine SIAH2 and ACSL4 expression in clinical samples. RT-qPCR was used to determine mRNA expression. Western blot assay was applied for determining protein expression. Ubiquitination assay was conducted for determining ubiquitination of ACSL4. Xenograft experiment was applied for determining tumor growth. Flow cytometry was applied to determine the functions of CD4+ and CD8+ T cells. SIAH2 expression was overexpressed in HCC tumors. High levels of SIAH2 predicted poor outcomes. However, SIAH2 knockdown promoted the proliferation of CD8+ T cells as well as promoted the ferroptosis of tumor cells, inhibiting tumor growth in HCC. ACSL4 is required for CD8+ T cell-mediated ferroptosis of HCC cells. However, SIAH2 induced ubiquitination of ACSL4 and inhibited its expression. SIAH2 specific inhibitor menadione promoted the immune checkpoint blockade. Taken together, SIAH2-mediated inactivation of CD8+ T cells inhibits the ferroptosis of HCC via mediating ubiquitination of ACSL4. Therefore, targeting SIAH2 may be a promising strategy for HCC.

The hepatitis B virus (HBV) is constantly exposed to significant oxidative stress characterized by elevated levels of reactive oxygen species (ROS), such as H 2 O 2 , during infection in hepatocytes of patients. In this study, we demonstrated that H 2 O 2 inhibits HBV replication in a p53-dependent fashion in human hepatoma cell lines expressing sodium taurocholate cotransporting polypeptide. Interestingly, H 2 O 2 failed to inhibit the replication of an HBV X protein (HBx)-null HBV mutant, but this defect was successfully complemented by ectopic expression of HBx. Additionally, H 2 O 2 upregulated p53 levels, leading to increased expression of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induced the ubiquitination-dependent proteasomal degradation of HBx. The inhibitory effect of H 2 O 2 was nearly abolished not only by treatment with a representative antioxidant, N-acetyl-L-cysteine but also by knockdown of either p53 or Siah-1 using specific short hairpin RNA, confirming the role of p53 and Siah-1 in the inhibition of HBV replication by H 2 O 2 . The present study provides insights into the mechanism that regulates HBV replication under conditions of oxidative stress in patients.

Interleukin-1β (IL-1β) has been described to exert important effect on synapses in the brain. Here, we explored if the synapses in the hippocampus would be adversely affected following intracerebral IL-1β injection and, if so, to clarify the underlying molecular mechanisms. Adult male Sprague-Dawley rats were divided into control, IL-1β, IL-1β + PD98059, and IL-1β + MG132 groups and then sacrificed for detection of synaptophysin (syn) protein level, synaptosome glutamate release, and synapse ultrastructure by western blotting, glutamate kit and electron microscopy, respectively. These rats were tested by Morris water maze for learning and memory ability. It was determined by western blotting whether IL-1β exerted the effect of on syn and siah1 expression in primary neurons via extracellular regulated protein kinases (ERK) signaling pathway. Intrahippocampal injection of IL-1β in male rats and sacrificed at 8d resulted in a significant decrease in syn protein, damage of synapse structure, and abnormal release of neurotransmitters glutamate. ERK inhibitor and proteosome inhibitor treatment reversed the above changes induced by IL-1β both in vivo and in vitro. In primary cultured neurons incubated with IL-1β, the expression level of synaptophysin was significantly downregulated coupled with abnormal glutamate release. Furthermore, use of PD98059 had confirmed that ERK signaling pathway was implicated in synaptic disorders caused by IL-1β treatment. The present results suggest that exogenous IL-1β can suppress syn protein level and glutamate release. A possible mechanism for this is that IL-1β induces syn degradation that is regulated by the E3 ligase siah1 via the ERK signaling pathway., (© 2023 Wiley Periodicals LLC.)

Background: We previously report that yes-associated protein (YAP), the core downstream effector of Hippo pathway, promotes the malignant progression of glioblastoma (GBM). However, although classical regulatory mechanisms of YAP are well explored, how YAP is modulated by the Hippo-independent manner remains poorly understood. Meanwhile, the nonreceptor tyrosine kinase Fyn-related kinase (FRK), which exhibits low expression and possesses tumor suppressor effects in GBM, is reported to be involved in regulation of protein phosphorylation. Here, we examined whether FRK could impede tumor progression by modulating YAP activities., Methods: Human GBM cells and intracranial GBM model were used to assess the effects of FRK and YAP on the malignant biological behaviors of GBM. Immunoblotting and immunohistochemistry were used to detect the expression of core proteins in GBM tissues. Co-immunoprecipitation, proximity ligation assay, luciferase assay and ubiquitination assay were utilized to determine the protein-protein interactions and related molecular mechanisms., Results: The expression levels of FRK and YAP were inversely correlated with each other in glioma tissues. In addition, FRK promoted the ubiquitination and degradation of YAP, leading to tumor suppression in vitro and in vivo. Mechanistically, FRK interacted with and phosphorylated YAP on Tyr391/407/444, which recruited the classical E3 ubiquitin ligase Siah1 to catalyze ubiquitination and eventually degradation of YAP. Siah1 is required for YAP destabilization initiated by FRK., Conclusions: We identify a novel mechanism by which FRK orchestrates tumor-suppression effect through phosphorylating YAP and inducing its ubiquitination by Siah1. FRK-Siah1-YAP signaling axis may serve as a potential therapeutic target for GBM treatment., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Human CtIP maintains genomic integrity primarily by promoting 5' DNA end resection, an initial step of the homologous recombination (HR). A few mechanisms have been suggested as to how CtIP recruitment to damage sites is controlled, but it is likely that we do not yet have full understanding of the process. Here, we provide evidence that CtIP recruitment and functioning are controlled by the SIAH2 E3 ubiquitin ligase. We found that SIAH2 interacts and ubiquitinates CtIP at its N-terminal lysine residues. Mutating the key CtIP lysine residues impaired CtIP recruitment to DSBs and stalled replication forks, DSB end resection, overall HR repair capacity of cells, and recovery of stalled replication forks, suggesting that the SIAH2-induced ubiquitination is important for relocating CtIP to sites of damage. Depleting SIAH2 consistently phenocopied these results. Overall, our work suggests that SIAH2 is a new regulator of CtIP and HR repair, and emphasizes that SIAH2-mediated recruitment of the CtIP is an important step for CtIP's function during HR repair., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Seven in absentia homolog 1 (Siah1) has been shown plays important roles in the pathogenesis and development of multiple cancers. However, the functions and mechanisms of Siah1 in non-small cell lung cancer (NSCLC) remain unclear. In our study, we found that knock down of Siah1 could inhibit the proliferation of NSCLC cells, while over-expression of Siah1 had the opposite effects. Molecularly, the bioinformatics analysis determined that notch receptor 1 (Notch1) might be the potential target of Siah1. Subsequently, we identified that Siah1 acted as an E3 ligase to promote the ubiquitination and stabilization of Notch1 through the proteasome pathway. Furthermore, the results showed that the Siah1 expression was directly correlated with CTR9 in human NSCLC tissues. Finally, Siah1 could promote Akt phosphorylation through regulating Notch1, thus promoting the proliferation of NSCLC cells. In conclusion, our study demonstrated that Siah1 acts as an oncogene, can ubiquitinate and stabilize Notch1 by proteasome pathway, which promotes Akt phosphorylation and ultimately leads to NSCLC cell proliferation., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The E3 ligase SIAH1 is deregulated in human cancers and correlated with poor prognosis, but its contributions to chemoresistance in epithelial ovarian cancer (EOC) are not evident. Herein we found that SIAH1 was decreased in EOC tumour tissues and cell lines and negatively correlated with the RPS3 levels. SIAH1 overexpression suppressed tumour cell growth, colony formation, invasion, metastasis, and cisplatin resistance in vivo and in vitro . SIAH1 promoted RPS3 ubiquitination and degradation using the RING-finger domain, and these steps were required for RPS3 localization to the cytoplasm, which led to subsequent NF-κB inactivation and thereby conferred chemosensitivity. Moreover, ectopic expression of RPS3 or depletion of RPS3 ubiquitination mediated by SIAH1 via the K214R mutant significantly impaired cisplatin-induced tumour suppression in cells stably expressing SIAH1. Together, our findings reveal a tumour suppressor function of SIAH1 and provide evidence showing that the SIAH1-RPS3-NF-κB axis may act as an appealing strategy for tackling treatment resistance in EOC.

Background: Using clinical samples and database analysis, this study aimed to investigate the signaling pathways that mediated degeneration of nucleus pulposus cells (NPCs) in patients with intervertebral disc degeneration (IDD)., Methods: NPCs were extracted from enucleated intervertebral discs of IDD patients, and the senescence, apoptosis, and extracellular matrix (ECM) synthesis levels of cells were confirmed by β-galactosidase (SA-β-gal), Western blot, and measurement of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH). The microarray expression profile of GSE56081 was downloaded to screen differentially expressed mRNAs. CO-IP and ubiquitination assays were used to determine the targeted regulation of XIAP by SIAH1. Methylation of mRNA was verified by m6A RIP and actinomycin D assays., Results: NPCs extracted from the enucleated intervertebral discs of IDD patients exhibited marked senescence, apoptosis, elevated levels of inflammation, and decreased ECM synthesis. The expression of SIAH1 was significantly elevated in NPCs of IDD patients, and SIAH1 knockdown reversed senescence, apoptosis, elevated levels of inflammation, and decreased ECM synthesis in NPCs of IDD patients. CO-IP and ubiquitination assays indicated that SIAH1 can target and ubiquitinate XIAP. Besides, MeRIP-qPCR and actinomycin experiments showed that METTL3-mediated m6A can methylate SIAH1 mRNA., Conclusion: In IDD patients, SIAH1 can target and ubiquitinate XIAP, thereby mediating senescence, apoptosis, increased inflammation, and decreased ECM synthesis of NPCs, while METTL3-mediated m6A can methylate SIAH1 mRNA, producing harmful effects., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Reactive oxygen species (ROS) exposure triggers granulosa cells' (GCs) senescence, which is an important causal factor for premature ovarian failure (POF). However, underlying mechanism in this process remains unknown. In our study, we observed increased ROS levels in POF ovarian tissues, POF patient follicular GCs and cyclophosphamide (CTX) pretreated GCs. Correspondingly, increased SIAH1, reduced TRF2 and GC senescence were also found in these cases. Silencing of SIAH1 rescued ROS-induced TRF2 reduction and cell senescence in GCs. Moreover, SIAH1 co-localized with TRF2 in the cytoplasm, facilitating its ubiquitination degradation, further leading to telomere abnormalities in GCs. In conclusion, our findings indicate that ROS induces telomere abnormalities by augmenting SIAH1-mediated TRF2 degradation, leading to cell senescence in GCs in POF processing., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Well-differentiated and dedifferentiated liposarcomas are rare soft tissue tumors originating in adipose tissue that share genetic abnormalities but have significantly different metastatic potential. Dedifferentiated liposarcoma (DDLPS) is highly aggressive and has an overall 5-year survival rate of 30% as compared to 90% for well-differentiated liposarcoma (WDLPS). This discrepancy may be connected to their potential to form adipocytes, where WDLPS is adipogenic but DDLPS is adipogenic-impaired. Normal adipogenesis requires Zinc Finger Protein 423 (ZFP423), a transcriptional coregulator of Perixosome Proliferator Activated Receptor gamma (PPARG2) mRNA expression that defines committed preadipocytes. Expression of ZFP423 in preadipocytes is promoted by Seven-In-Absentia Homolog 2 (SIAH2)-mediated degradation of Zinc Finger Protein 521 (ZFP521). This study investigated the potential role of ZFP423, SIAH2 and ZFP521 in the adipogenic potential of WDLPS and DDLPS., Methods: Human WDLPS and DDLPS fresh and paraffin-embedded tissues were used to assess the gene and protein expression of proadipogenic regulators. In parallel, normal adipose tissue stromal cells along with WDLPS and DDLPS cell lines were cultured, genetically modified, and induced to undergo adipogenesis in vitro., Results: Impaired adipogenic potential in DDLPS was associated with reduced ZFP423 protein levels in parallel with reduced PPARG2 expression, potentially involving regulation of ZFP521. SIAH2 protein levels did not define a clear distinction related to adipogenesis in these liposarcomas. However, in primary tumor specimens, SIAH2 mRNA was consistently upregulated in DDLPS compared to WDLPS when assayed by fluorescence in situ hybridization or real-time PCR., Conclusions: These data provide novel insights into ZFP423 expression in adipogenic regulation between WDLPS and DDLPS adipocytic tumor development. The data also introduces SIAH2 mRNA levels as a possible molecular marker to distinguish between WDLPS and DDLPS., (© 2022. The Author(s).)

The dysregulation of the ubiquitin‑proteasome system will result in the abnormal accumulation and dysfunction of proteins, thus leading to severe diseases. Seven in absentia homolog 1 (Siah1), an E3 ubiquitin ligase, has attracted wide attention due to its varied functions in physiological and pathological conditions, and the numerous newly discovered Siah1 substrates. In cancer and nervous system diseases, the functions of Siah1 as a promoter or a suppressor of diseases are related to the change in cellular microenvironment and subcellular localization. At the same time, complex upstream regulations make Siah1 different from other E3 ubiquitin ligases. Understanding the molecular mechanism of Siah1 will help the study of various signaling pathways and benefit the therapeutic strategy of human diseases (e.g., cancer and nervous system diseases). In the present review, the functions and regulations of Siah1 are described. Moreover, novel substrates of Siah1 discovered in recent studies will be highlighted in cancer and nervous system diseases, providing ideas for future research and clinical targeted therapies using Siah1.

Seven in absentia homolog 2 (Siah2), an RING E3 ubiquitin ligases, has been characterized to play the vital role in tumorigenesis and cancer progression. Numerous studies have determined that Siah2 promotes tumorigenesis in a variety of human malignancies such as prostate, lung, gastric, and liver cancers. However, several studies revealed that Siah2 exhibited tumor suppressor function by promoting the proteasome-mediated degradation of several oncoproteins, suggesting that Siah2 could exert its biological function according to different stages of tumor development. Moreover, Siah2 is subject to complex regulation, especially the phosphorylation of Siah2 by a variety of protein kinases to regulate its stability and activity. In this review, we describe the structure and regulation of Siah2 in human cancer. Moreover, we highlight the critical role of Siah2 in tumorigenesis. Furthermore, we note that the potential clinical applications of targeting Siah2 in cancer therapy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

N6-methyladenosine (m6A) has been reported as an important mechanism of post-transcriptional regulation. Programmed death ligand 1 (PD-L1) is a primary immune inhibitory molecule expressed on tumor cells that promotes immune evasion. In addition, seven in absentia homolog 2 (Siah2), a RING E3 ubiquitin ligase, has been involved in tumorigenesis and cancer progression. However, the role of m6A-METTL14-Siah2-PD-L1 axis in immunotherapy remains to be elucidated. In this study, we showed that METTL14, a component of the m 6 A methyltransferase complex, induced Siah2 expression in cholangiocarcinoma (CCA). METTL14 was shown to enrich m 6 A modifications in the 3'UTR region of the Siah2 mRNA, thereby promoting its degradation in an YTHDF2-dependent manner. Furthermore, co-immunoprecipitation experiments demonstrated that Siah2 interacted with PD-L1 by promoting its K63-linked ubiquitination. We also observed that in vitro and in vivo Siah2 knockdown inhibited T cells expansion and cytotoxicity by sustaining tumor cell PD-L1 expression. The METTL14-Siah2-PD-L1-regulating axis was further confirmed in human CCA specimens. Analysis of specimens from patients receiving anti-PD1 immunotherapy suggested that tumors with low Siah2 levels were more sensitive to anti-PD1 immunotherapy. Taken together, our results evidenced a new regulatory mechanism of Siah2 by METTL14-induced mRNA epigenetic modification and the potential role of Siah2 in cancer immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The editor CH declared a shared parent affiliation with the authors HZ, LO, GJ at the time of the review., (Copyright © 2022 Zheng, Zheng, Wang, Tao, Huang, Yang, Ouyang, Duan, Zhang, Chen, Xiang, Jin, Fang, Zhou and Liang.)

Most clinical and experimental studies have suggested that hepatitis C virus (HCV) is dominant over hepatitis B virus (HBV) during coinfection, although the mechanism remains unclear. Here, we found that HCV core protein inhibits HBV replication by downregulating HBx levels during coinfection in human hepatoma cells. For this effect, HCV core protein increased reactive oxygen species levels in the mitochondria and activated the ataxia telangiectasia mutated-checkpoint kinase two pathway in the nucleus, resulting in an upregulation of p53 levels. Accordingly, HCV core protein induced p53-dependent activation of seven in absentia homolog one expression, an E3 ligase of HBx, resulting in the ubiquitination and proteasomal degradation of HBx. The effect of the HCV core protein on HBx levels was accurately reproduced in both a 1.2-mer HBV replicon and in vitro HBV infection systems, providing evidence for the inhibition of HBV replication by HCV core protein. The present study may provide insights into the mechanism of HCV dominance in HBV- and HCV-coinfected patients.

Proteasomal activator 28 gamma (PA28γ) upregulates the levels of HBx, a regulatory protein of hepatitis B virus (HBV) to stimulate HBV replication; however, the detailed mechanism remains unknown. Here, we found that PA28γ impaired the ability of seven in absentia homolog 1 (Siah-1) as an E3 ubiquitin ligase of HBx. PA28γ competitively inhibited the binding of Siah-1 to HBx in human hepatoma cells. Accordingly, PA28γ increased the stability of HBx and decreased HBx ubiquitination, abolishing the potential of Siah-1 to downregulate HBx levels. PA28γ also executed its role as an antagonist of Siah-1 during HBV replication, as demonstrated by an in vitro HBV replication system. The present study may provide insights into the mechanisms underlying the regulation of HBV replication., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This review focuses on the consequences of GAPDH S -nitrosylation at the catalytic cysteine residue. The widespread hypothesis according to which S -nitrosylation causes a change in GAPDH structure and its subsequent binding to the Siah1 protein is considered in detail. It is assumed that the GAPDH complex with Siah1 is transported to the nucleus by carrier proteins, interacts with nuclear proteins, and induces apoptosis. However, there are several conflicting and unproven elements in this hypothesis. In particular, there is no direct confirmation of the interaction between the tetrameric GAPDH and Siah1 caused by S -nitrosylation of GAPDH. The question remains as to whether the translocation of GAPDH into the nucleus is caused by S -nitrosylation or by some other modification of the catalytic cysteine residue. The hypothesis of the induction of apoptosis by oxidation of GAPDH is considered. This oxidation leads to a release of the coenzyme NAD + from the active center of GAPDH, followed by the dissociation of the tetramer into subunits, which move to the nucleus due to passive transport and induce apoptosis. In conclusion, the main tasks are summarized, the solutions to which will make it possible to more definitively establish the role of nitric oxide in the induction of apoptosis.

Aberrant expression of microRNAs (miRNAs) has been linked with ischemic brain injury (IBI), but the mechanistic actions behind the associated miRNAs remain to be determined. Of note, miR-129-5p was revealed to be downregulated in the serum of patients with IBI. In silico prediction identified a putative target gene, siah E3 ubiquitin protein ligase 1 (SIAH1), of miR-129-5p. Accordingly, this study plans to clarify the functional relevance of the interplay of miR-129-5p and SIAH1 in IBI. IBI was modeled by exposing human hippocampal neuronal cells to oxygen-glucose deprivation (OGD) in vitro and by occluding the middle cerebral artery (MCAO) in a mouse model in vivo. Apoptosis of hippocampal neuronal cells was assessed by annexin V-FITC/PI staining and TUNEL staining. The area of cerebral infarction was measured using TTC staining, along with neurological scoring on modeled mice. Loss of hippocampal neuronal cells in the peri-infarct area was monitored using Nissl staining. Downregulated miR-129-5p expression was found in OGD-induced hippocampal neuronal cells and MCAO-treated mice. Mechanistically, miR-129-5p was validated to target and inhibit SIAH1 through the application of dual-luciferase reporter assay. Additionally, enforced miR-129-5p inhibited the apoptosis of OGD-induced cells and decreased the cerebral infarct area, neurological scores and apoptosis of hippocampal neuronal cells by downregulating SIAH1 and activating the mTOR signaling pathway. Taken together, the results of this study reveal the important role and underlying mechanism of miR-129-5p in IBI, providing a promising biomarker for preventive and therapeutic strategies., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The DNA damage-responsive tumor suppressors p53 and HIPK2 are well established regulators of cell fate decision-making and regulate the cellular sensitivity to DNA-damaging drugs. Here, we identify Deleted in Azoospermia-associated protein 2 (DAZAP2), a small adaptor protein, as a novel regulator of HIPK2 and specifier of the DNA damage-induced p53 response. Knock-down or genetic deletion of DAZAP2 strongly potentiates cancer cell chemosensitivity both in cells and in vivo using a mouse tumour xenograft model. In unstressed cells, DAZAP2 stimulates HIPK2 polyubiquitination and degradation through interplay with the ubiquitin ligase SIAH1. Upon DNA damage, HIPK2 site-specifically phosphorylates DAZAP2, which terminates its HIPK2-degrading function and triggers its re-localization to the cell nucleus. Interestingly, nuclear DAZAP2 interacts with p53 and specifies target gene expression through modulating a defined subset of p53 target genes. Furthermore, our results suggest that DAZAP2 co-occupies p53 response elements to specify target gene expression. Collectively, our findings propose DAZAP2 as novel regulator of the DNA damage-induced p53 response that controls cancer cell chemosensitivity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Ubiquitination has a central role in numerous biological processes, including cell development, stress responses and ageing. Perturbed ubiquitination has been implicated in human diseases ranging from cancer to neurodegenerative diseases. SIAH1 encodes a RING-type E3 ubiquitin ligase involved in protein ubiquitination. Among numerous other roles, SIAH1 regulates metabotropic glutamate receptor signalling and affects neural cell fate. Moreover, SIAH1 positively regulates Wnt signalling through ubiquitin-mediated degradation of Axin and accumulation of β-catenin., Methods: Trio exome sequencing followed by Sanger validation was undertaken in five individuals with syndromic developmental delay. Three-dimensional structural modelling was used to predict pathogenicity of affected residues. Wnt stimulatory activity was measured by luciferase reporter assays and Axin degradation assays in HEK293 cells transfected with wild-type and mutant SIAH1 expression plasmids., Results: We report five unrelated individuals with shared features of developmental delay, infantile hypotonia, dysmorphic features and laryngomalacia, in whom exome sequencing identified de novo monoallelic variants in SIAH1 . In silico protein modelling suggested alteration of conserved functional sites. In vitro experiments demonstrated loss of Wnt stimulatory activity with the SIAH1 mutants, suggesting variant pathogenicity., Conclusion: Our results lend support to SIAH1 as a candidate Mendelian disease gene for a recognisable syndrome, further strengthening the connection between SIAH1 and neurodevelopmental disorders. Furthermore, the results suggest that dysregulation of the Wnt/β-catenin pathway may be involved in the pathogenesis., Competing Interests: Competing interests: TBP is an employee of GeneDx., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Developmental regulation of the vertebrate visual system has been a focus of investigation for generations as understanding this critical time period has direct implications on our understanding of congenital blinding disease. The majority of studies to date have focused on transcriptional regulation mediated by morphogen gradients and signaling pathways. However, recent studies of post translational regulation during ocular development have shed light on the role of the ubiquitin proteasome system (UPS). This rather ubiquitous yet highly diverse system is well known for regulating protein function and localization as well as stability via targeting for degradation by the 26S proteasome. Work from many model organisms has recently identified UPS activity during various milestones of ocular development including retinal morphogenesis, retinal ganglion cell function as well as photoreceptor homeostasis. In particular work from flies and zebrafish has highlighted the role of the E3 ligase enzyme family, Seven in Absentia Homologue (Siah) during these events. In this review, we summarize the current understanding of UPS activity during Drosophila and vertebrate ocular development, with a major focus on recent findings correlating Siah E3 ligase activity with two major developmental stages of vertebrate ocular development, retinal morphogenesis and photoreceptor specification and survival., (© 2021 The Author(s).)

Background: Helicobacter pylori-mediated gastric carcinogenesis is initiated by a plethora of signaling events in the infected gastric epithelial cells (GECs). The E3 ubiquitin ligase seven in absentia homolog 2 (Siah2) is induced in GECs in response to H. pylori infection. Posttranslational modifications of Siah2 orchestrate its function as well as stability. The aim of this study was to evaluate Siah2 phosphorylation status under the influence of H. pylori infection and its impact in gastric cancer progression., Methods: H. pylori-infected various GECs, gastric tissues from H. pylori-infected GC patients and H. felis-infected C57BL/6 mice were evaluated for Siah2 phosphorylation by western blotting or immunofluorescence microscopy. Coimmunoprecipitation assay followed by mass spectrometry were performed to identify the kinases interacting with Siah2. Phosphorylation sites of Siah2 were identified by using various plasmid constructs generated by site-directed mutagenesis. Proteasome inhibitor MG132 was used to investigate proteasome degradation events. The importance of Siah2 phosphorylation on tumorigenicity of infected cells were detected by using phosphorylation-null mutant and wild type Siah2 stably-transfected cells followed by clonogenicity assay, cell proliferation assay, anchorage-independent growth and transwell invasion assay., Results: Siah2 was phosphorylated in H. pylori-infected GECs as well as in metastatic GC tissues at residues serine 6  (Ser 6 ) and threonine 279  (Thr 279 ). Phosphorylation of Siah2 was mediated by MRCKβ, a Ser/Thr protein kinase. MRCKβ was consistently expressed in uninfected GECs and noncancer gastric tissues but its level decreased in infected GECs as well as in metastatic tissues which had enhanced Siah2 expression. Infected murine gastric tissues showed similar results. MRCKβ could phosphorylate Siah2 but itself got ubiquitinated from this interaction leading to the proteasomal degradation of MRCKβ and use of proteasomal inhibitor MG132 could rescue MRCKβ from Siah2-mediated degradation. Ser 6 and Thr 279 phosphorylated-Siah2 was more stable and tumorigenic than its non-phosphorylated counterpart as revealed by the proliferation, invasion, migration abilities and anchorage-independent growth of stable-transfected cells., Conclusions: Increased level of Ser 6 and Thr 279 -phosphorylated-Siah2 and downregulated MRCKβ were prominent histological characteristics of Helicobacter-infected gastric epithelium and metastatic human GC. MRCKβ-dependent Siah2 phosphorylation stabilized Siah2 which promoted anchorage-independent survival and proliferative potential of GECs. Phospho-null mutants of Siah2 (S6A and T279A) showed abated tumorigenicity.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets 1-3 , but a substantial subset of proteins are resistant to targeted degradation using existing approaches 4,5 . Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL6 6 . We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.

Evidence is lacking as to how developing neurons integrate mitogenic signals with microenvironment cues to control proliferation and differentiation. We determine that the Siah2 E3 ubiquitin ligase functions in a coincidence detection circuit linking responses to the Shh mitogen and the extracellular matrix to control cerebellar granule neurons (CGN) GZ occupancy. We show that Shh signaling maintains Siah2 expression in CGN progenitors (GNPs) in a Ras/Mapk-dependent manner. Siah2 supports ciliogenesis in a feed-forward fashion by restraining cilium disassembly. Efforts to identify sources of the Ras/Mapk signaling led us to discover that GNPs respond to laminin, but not vitronectin, in the GZ microenvironment via integrin β1 receptors, which engages the Ras/Mapk cascade with Shh, and that this niche interaction is essential for promoting GNP ciliogenesis. As GNPs leave the GZ, differentiation is driven by changing extracellular cues that diminish Siah2-activity leading to primary cilia shortening and attenuation of the mitogenic response.

This study aimed to reveal the mechanism of miR-146b-5p in the differentiation of bone marrow mesenchymal stem cells (BMSCs) derived from children with aplastic anemia (AA). Here, we found that miR-146b-5p was highly expressed in BMSCs from children with AA, and the BMSCs surface markers expressions in BMSCs derived from children with AA and the healthy controls exerted no significant differences. Besides, the overexpression of miR-146b-5p in normal human-derived BMSCs promoted the adipogenic differentiation of BMSCs. Furthermore, miR-146b-5p negatively regulated SIAH2 luciferase activity, and the interference with miR-146b-5p reduced the stability of PPARγ protein and inhibited SIAH2-mediated ubiquitination of PPARγ protein. Besides, the interference with miR-146b-5p was beneficial for ameliorating AA in a mouse model of AA. Overall, our results found that miR-146b-5p was highly expressed in BMSCs from children with AA, and our further studies indicated that miR-146b-5p improved AA via promoting SIAH2-mediated ubiquitination of PPARγ protein.

Purpose: TRAF4 plays an important role in the development and progression of breast cancer, but its impact on chemotherapy resistance is as yet, however, poorly understood., Methods: Western blotting, immunoprecipitation, and immunofluorescence staining were used to identify and verify that TRAF4 was a novel substrate of SIAH1 and prevented SIAH1-mediated β-catenin degradation. Cell proliferation analysis and Flow cytometry analysis were utilized to detect TRAF4's function on the growth-inhibitory effect of etoposide. Immunohistochemistry was used to detect the expression of TRAF4, SIAH1, and β-catenin. Statistical analysis was used to analyze the relationships between them with clinical parameters and curative effect of chemotherapy pathologically., Results: Our results suggested that TRAF4 prevents SIAH1-mediated β-catenin degradation. TRAF4 was a novel substrate of SIAH1 and the TRAF domain of TRAF4 was critical for binding to SIAH1. TRAF4 reduced the growth-inhibitory effect of etoposide via reducing the number of S-phase cells and suppressing cell apoptosis. Concordantly, we found that breast cancer patients with a low-TRAF4 expression benefited most from chemotherapy, who had higher tumor volume reduction rate and better pathological response, while, the high-TRAF4 expression group had lower tumor volume reduction rate and poor pathological response., Conclusions: TRAF4 was a novel substrate of SIAH1 and prevented SIAH1-mediated β-catenin degradation, which explains the protective effect of TRAF4 on β-catenin during cell stress and links TRAF4 to chemotherapy resistance in tumors. These findings implicated a novel pathway for the oncogenic function of TRAF4.

The Siah1 and Siah2 ubiquitin ligases are implicated in diverse biological processes ranging from cellular stress responses, signaling to transcriptional regulation. A key substrate of Siah1 is ELL2, which undergoes proteolysis upon polyubiquitination. ELL2 stimulates transcriptional elongation and is a subunit of the Super Elongation Complex (SEC) essential for HIV-1 transactivation. Previously, multiple transcriptional and post-translational mechanisms are reported to control Siah's expression and activity. Here we show that the activity of Siah1/2 can also be suppressed by host cell factor 1 (HCF1), and the hitherto poorly characterized HCF2, which themselves are not degraded but can bind and block the substrate-binding domain (SBD) of Siah1/2 to prevent their autoubiquitination and trans-ubiquitination of downstream targets including ELL2. This effect stabilizes ELL2 and enhances the ELL2-SEC formation for robust HIV-1 transactivation. Thus, our study not only identifies HCF1/2 as novel activators of HIV-1 transcription through inhibiting Siah1 to stabilize ELL2, but also reveals the SBD of Siah1/2 as a previously unrecognized new target for HCF1/2 to exert this inhibition., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Among all of the Super Elongation Complex (SEC) components, ELL1 (also known as ELL) is the only bona fide elongation factor that directly stimulates transcription elongation by RNA polymerase II. However, the mechanism(s) of functional regulation of ELL1 (referred to as ELL hereafter), through its stabilization, is completely unknown. Here, we report a function of human DBC1 in regulating ELL stability involving HDAC3, p300, and Siah1. Mechanistically, we show that p300-mediated site-specific acetylation increases, whereas HDAC3-mediated deacetylation decreases, ELL stability through polyubiquitylation by the E3 ubiquitin ligase Siah1. DBC1 competes with HDAC3 for the same binding sites on ELL and thus increases its acetylation and stability. Knockdown of DBC1 reduces ELL levels and expression of a significant number of genes, including those involved in glucose metabolism. Consistently, Type 2 diabetes patient-derived peripheral blood mononuclear cells show reduced expression of DBC1 and ELL and associated key target genes required for glucose homeostasis. Thus, we describe a pathway of regulating stability and functions of key elongation factor ELL for expression of diverse sets of genes, including ones that are linked to Type 2 diabetes pathogenesis., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The intracellular levels of the cytoprotective enzyme heme oxygenase-1 (HO-1) are tightly controlled. Here, we reveal a novel mechanism preventing the exaggerated expression of HO-1. The analysis of mice with a knock-out in the ubiquitin E3 ligase seven in absentia homolog 2 (SIAH2) showed elevated HO-1 protein levels in specific organs such as heart, kidney and skeletal muscle. Increased HO-1 protein amounts were also seen in human cells deleted for the SIAH2 gene. The higher HO-1 levels are not only due to an increased protein stability but also to elevated expression of the HO-1 encoding HMOX1 gene, which depends on the transcription factor nuclear factor E2-related factor 2 (NRF2), a known SIAH2 target. Dependent on its RING (really interesting new gene) domain, expression of SIAH2 mediates proteasome-dependent degradation of its interaction partner HO-1. Additionally SIAH2-deficient cells are also characterized by reduced expression levels of glutathione peroxidase 4 (GPX4), rendering the knock-out cells more sensitive to ferroptosis.

Rationale : Androgen receptor splice variant 7 (AR-V7) is a leading cause of the development of castration-resistant prostate cancer (CRPC). However, the regulation and function of AR-V7 at levels of post-translational modifications in prostate cancer therapy remain poorly understood. Here, we conducted a library screen of natural products to identify potential small molecules responsible for AR-V7 protein degradation in human prostate cancer cell lines. Methods : A natural product library was used to screen the inhibitor of AR-V7. Co-IP and biomass spectrum assays were used to identify the AR-V7-interacting proteins, whereas western blot, confocal microscopy, RNA interfering, and gene transfection were used to validate these interactions. Cell viability, EDU staining, and colony formation assays were employed to detect cell growth and proliferation. Flowcytometry assays were used to detect the distribution of cell cycle. Mouse xenograft models were used to study the anti-CRPC effects in vivo . Results : This screen identified rutaecarpine, one of the major components of the Chinese medicine Evodia rutaecarpa , as a novel chemical that selectively induces AR-V7 protein degradation via K48-linked ubiquitination. Mechanically, this effect relies on rutaecarpine inducing the formation of a GRP78-AR-V7 protein complex, which further recruits the E3 ligase SIAH2 to directly promote the ubiquitination of AR-V7. Consequently, the genetic and pharmacological activation of the GRP78-dependent AR-V7 protein degradation restores the sensitivity of castration-resistant prostate cancer to anti-androgen therapy in cell culture and animal models. Conclusions : These findings not only provide a new approach for overcoming castration-resistance in prostate cancer therapy, but also increase our understanding about the interplay between molecular chaperones and ubiquitin ligase in shaping protein stability., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Aims: It has been shown that up-regulation of E3 ubiquitin ligase seven-in-absentia-homolog 2 (Siah2) and activation of Hippo signaling pathway effector yes-associated protein (YAP) are involved in the development of pulmonary arterial hypertension (PAH). However, it is still unclear whether Siah2 activates YAP in monocrotaline (MCT)-induced PAH rat models., Main Methods: Intraperitoneal injection of MCT was used to induce PAH rat models. The right ventricular systolic pressure (RVSP), right ventricle hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-SMA, Ki-67 and TUNEL staining were performed to evaluate the development of PAH. Protein levels of Siah2, Lats1/2, YAP phosphorylation and total YAP, and the subcellular localization of YAP were examined using immunoblotting. Proteasome activity was measured by an assay kit., Key Findings: The protein level of Siah2 was significantly increased in MCT-induced PAH rats, this was accompanied with the proteasome-dependent degradation of Lats1/2 and subsequent up-regulation and dephosphorylation of YAP and its nuclear localization. Administration of PAH rats with Siah2 inhibitor Vitamin K3 or proteasome inhibitor MG-132 dramatically suppressed MCT-induced down-regulation of Lats1/2 and activation of YAP, finally reduced RVSP, RVHI, %MT, pulmonary arterial muscularization, pulmonary arterial smooth muscle cells (PASMCs) proliferation and enhanced PASMCs apoptosis in PAH rats., Significance: Siah2 contributes to the development of MCT-induced PAH by destabilizing Lats1/2 and subsequently stimulating YAP activation. Inhibition of Siah2 or proteasome alleviates pulmonary arterial remodeling through inactivation of YAP, indicating Siah2 ubiquitin ligase as a novel target might have potential value in the management of PAH., (Copyright © 2019. Published by Elsevier Inc.)

Understanding the mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here, we report that growth of BRAF-mutant melanoma cells is inhibited, up to complete rejection, in Siah2 -/- mice. Growth-inhibited tumors exhibit increased numbers of intra-tumoral activated T cells and decreased expression of Ccl17, Ccl22, and Foxp3. Marked reduction in Treg proliferation and tumor infiltration coincide with G1 arrest in tumor infiltrated Siah2 -/- Tregs in vivo or following T cell stimulation in culture, attributed to elevated expression of the cyclin-dependent kinase inhibitor p27, a Siah2 substrate. Growth of anti-PD-1 therapy resistant melanoma is effectively inhibited in Siah2 -/- mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and Siah2 loss. Low SIAH2 and FOXP3 expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy.

Ventricular enlargement occurs in several neurodegenerative and psychiatric diseases. A large genome-wide association study (GWAS) has identified seven loci associated with ventricular volume. The rate of ventricular enlargement increased in the progression of disease from normal cognition to dementia. Here, we aimed to use the rate of ventricular enlargement as an endophenotype for the development and progression of neurodegenerative diseases to discover more common genetic variants. We performed a GWAS of the rate of ventricular enlargement using 507 nondemented non-Hispanic white participants from the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. Linear regression model was used to identify the association of the rate of ventricular enlargement with single nucleotide polymorphisms (SNPs) in PLINK software. The associations of genome-wide significant SNPs with other four phenotypes were further discussed. Two SNPs (rs11620312, P = 4.04×10 -8 ; rs79174114, P = 4.28×10 -8 ) within SIAH3 gene in linkage disequilibrium (LD) reached genome-wide significance for association with increased rate of ventricular enlargement. Some intergenic SNPs and SNPs within NKAIN2, TBC1D2, GALNT18, ABCC1 and SRCIN1 genes were identified as potential candidates. SIAH3 rs11620312-C carriers were associated with poor cognition and brain hypometabolism longitudinally. Our findings indicated that SIAH3 gene may have potential influence on the pathogenesis of neurodegenerative diseases.

Elevated plasma homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is an independent risk factor for neurodegenerative diseases. Hcy, even at a low concentration, can promote free radical formation and increase oxidative stress, leading to neuronal death, which may be an important mechanism underlying the pathogenesis of neurodegenerative diseases. Although several reports have indicated that the nuclear translocation of glyceraldehyde 3‑phosphate dehydrogenase (GAPDH) may be involved in Hcy‑induced apoptosis, the exact mechanism remains to be fully elucidated. The siah E3 ubiquitin protein ligase 1 (siah‑1) gene was found to be critical for the translocation of GAPDH from the cytoplasm to the nucleus. In the present study, the role of siah‑1 was investigated in the nuclear translocation of GAPDH in rat C6 astroglioma cells treated with Hcy. C6 cells were treated with various concentrations of Hcy for 48 h and the expression level of siah‑1 was examined using reverse transcription‑quantitative polymerase chain reaction and western blotting analysis. In addition, the subcellular localization of siah‑1 and GAPDH and the interaction between these two factors were investigated by immunofluorescence staining and co‑immunoprecipitation assay, respectively. The results showed that Hcy at a high concentration increased the expression of siah‑1 and induced nuclear translocation of siah‑1 and GAPDH. In addition, siah‑1 knockdown by siah‑1 small interfering RNA significantly decreased the Hcy‑induced nuclear accumulation of GAPDH and inhibited the impairment of C6 cells. These findings suggest that siah‑1 is involved in Hcy‑induced cell damage by promoting the nuclear translocation of GAPDH.

During early meiotic prophase, homologous chromosomes are connected along their entire lengths by a proteinaceous tripartite structure known as the synaptonemal complex (SC). Although the components that comprise the SC are predominantly studied in this canonical ribbon-like structure, they can also polymerize into repeated structures known as polycomplexes. We find that in Drosophila oocytes, the ability of SC components to assemble into canonical tripartite SC requires the E3 ubiquitin ligase Seven in absentia (Sina). In sina mutant oocytes, SC components assemble into large rod-like polycomplexes instead of proper SC. Thus, the wild-type Sina protein inhibits the polymerization of SC components, including those of the lateral element, into polycomplexes. These polycomplexes persist into meiotic stages when canonical SC has been disassembled, indicating that Sina also plays a role in controlling SC disassembly. Polycomplexes induced by loss-of-function sina mutations associate with centromeres, sites of double-strand breaks, and cohesins. Perhaps as a consequence of these associations, centromere clustering is defective and crossing over is reduced. These results suggest that while features of the polycomplexes can be recognized as SC by other components of the meiotic nucleus, polycomplexes nonetheless fail to execute core functions of canonical SC., Competing Interests: The authors have declared that no competing interests exist.

Inhibition of ubiquitin ligases with small molecule remains a very challenging task, given the lack of catalytic activity of the target and the requirement of disruption of its interactions with other proteins. Siah1/2, which are E3 ubiquitin ligases, are implicated in melanoma and prostate cancer and represent high-value drug targets. We utilized three independent screening approaches in our efforts to identify small-molecule Siah1/2 inhibitors: Affinity Selection-Mass Spectrometry, a protein thermal shift-based assay and an in silico based screen. Inhibitors were assessed for their effect on viability of melanoma and prostate cancer cultures, colony formation, prolyl-hydroxylase-HIF1α signaling, expression of selected Siah2-related transcripts, and Siah2 ubiquitin ligase activity. Several analogs were further characterized, demonstrating improved efficacy. Combination of the top hits identified in the different assays demonstrated an additive effect, pointing to complementing mechanisms that underlie each of these Siah1/2 inhibitors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Patients suffering from glioblastoma have a dismal prognosis, indicating the need for new therapeutic targets. Here we provide evidence that the DNA damage kinase HIPK2 and its negative regulatory E3-ubiquitin ligase SIAH1 are critical factors controlling temozolomide-induced cell death. We show that HIPK2 downregulation (HIPK2kd) significantly reduces the level of apoptosis. This was not the case in glioblastoma cells expressing the repair protein MGMT, suggesting that the primary DNA lesion responsible for triggering HIPK2-mediated apoptosis is O 6 -methylguanine. Upon temozolomide treatment, p53 becomes phosphorylated whereby HIPK2kd had impact exclusively on ser46, but not ser15. Searching for the transcriptional target of p-p53ser46, we identified the death receptor FAS (CD95, APO-1) being involved. Thus, the expression of FAS was attenuated following HIPK2kd, supporting the conclusion that HIPK2 regulates temozolomide-induced apoptosis via p-p53ser46-driven FAS expression. This was substantiated in chromatin-immunoprecipitation experiments, in which p-p53ser46 binding to the Fas promotor was regulated by HIPK2. Other pro-apoptotic proteins such as PUMA, NOXA, BAX, and PTEN were not affected in HIPK2kd, and also double-strand breaks following temozolomide remained unaffected. We further show that downregulation of the HIPK2 inactivator SIAH1 significantly ameliorates temozolomide-induced apoptosis, suggesting that the ATM/ATR target SIAH1 together with HIPK2 plays a proapoptotic role in glioma cells exhibiting p53wt status. A database analysis revealed that SIAH1, but not SIAH2, is significantly overexpressed in glioblastomas. IMPLICATIONS: The identification of a novel apoptotic pathway triggered by the temozolomide-induced DNA damage O 6 -methylguanine supports the role of p53 in the decision between survival and death and suggests SIAH1 and HIPK2 as new therapeutic targets., (©2019 American Association for Cancer Research.)

Background: Long noncoding RNAs (lncRNAs) have emerged as critical players in cancer progression, but their functions in colorectal cancer (CRC) metastasis have not been systematically clarified., Methods: lncRNA expression profiles in matched normal and CRC tissue were checked using microarray analysis. The biological roles of a novel lncRNA, namely RP11-138 J23.1 (RP11), in development of CRC were checked both in vitro and in vivo. Its association with clinical progression of CRC was further analyzed., Results: RP11 was highly expressed in CRC tissues, and its expression increased with CRC stage in patients. RP11 positively regulated the migration, invasion and epithelial mesenchymal transition (EMT) of CRC cells in vitro and enhanced liver metastasis in vivo. Post-translational upregulation of Zeb1, an EMT-related transcription factor, was essential for RP11-induced cell dissemination. Mechanistically, the RP11/hnRNPA2B1/mRNA complex accelerated the mRNA degradation of two E3 ligases, Siah1 and Fbxo45, and subsequently prevented the proteasomal degradation of Zeb1. m 6 A methylation was involved in the upregulation of RP11 by increasing its nuclear accumulation. Clinical analysis showed that m 6 A can regulate the expression of RP11, further, RP11 regulated Siah1-Fbxo45/Zeb1 was involved in the development of CRC., Conclusions: m 6 A-induced lncRNA RP11 can trigger the dissemination of CRC cells via post-translational upregulation of Zeb1. Considering the high and specific levels of RP11 in CRC tissues, our present study paves the way for further investigations of RP11 as a predictive biomarker or therapeutic target for CRC.

Osteosarcoma (OS) patients often exhibit pulmonary metastasis, which results in high patient mortality. Our present study established the doxorubicin (Dox) resistant human OS MG-63 and HOS cells and named them MG-63/Dox and HOS/Dox, respectively. The Dox resistant OS cells had greater invasion ability than that of parental cells. The expression of ZEB1, while not FOXM1, Snail, HIF-1α, or Sp1, was significantly increased in Dox resistant OS cells. Silencing of ZEB1 can attenuate the metastasis and increase Dox sensitivity of MG-63/Dox and HOS/Dox cells. The upregulation of ZEB1 can increase of the expression of interlukin-6 (IL-6). Anti-IL-6 inhibited the invasion and increase the Dox sensitivity of MG-63/Dox and HOS/Dox cells. There was no significant difference of ZEB1 mRNA between Dox resistant and control cells. The upregulation of ZEB1 in Dox resistant OS cells can be attributed to the increase of protein half-life. This was confirmed by results that the inhibitor of proteasomal degradation can increase ZEB1 in Dox resistant OS cells. Over expression of SIAH1 can inhibit the expression of ZEB1 and increase the Dox sensitivity of MG-63/Dox and HOS/Dox cells. Collectively, we confirmed that SIAH1 induced ZEB1 is involved in the Dox resistance of OS cells.