STIP1 Homology And U-box Containing Protein 1 Research Articles

RACK1 degrades MAVS to promote bovine ephemeral fever virus replication via upregulating E3 ubiquitin ligase STUB1

Receptors for activated C kinase 1 (RACK1) could competitively combine with mitochondrial antiviral signaling protein (MAVS) to inhibit the type I interferon (IFN) signaling pathway during viral infection in vitro. However, whether RACK1 can degrade MAVS to enhance viral replication is still unknown. In this study, we found that bovine epidemic fever virus (BEFV) infection triggered the expression of RACK1. Overexpression of RACK1 promoted BEFV replication, while knockdown of RACK1 inhibited the replication of BEFV. Further research showed that RACK1 inhibited the type I IFN signaling pathway during BEFV infection by degrading MAVS, and RACK1 degraded MAVS via the ubiquitin-proteasome system. Mechanistically, RACK1 up-regulated the expression of E3 ubiquitin ligase STIP1 homology and U-box containing protein 1 (STUB1), thereby promoting the ubiquitination and degradation of MAVS. In addition, RACK1 degraded MAVS by enhancing the interaction between STUB1 and MAVS but not via its interaction with STUB1. Overall, our study reveals a novel mechanism by which RACK1 inhibits the type I IFN signaling pathway to BEFV infection through degradation of MAVS, thereby promoting viral infection. These findings provide a new perspective for the MAVS degradation regulated by RACK1.

Xbp1s-FoxO1 axis governs lipid accumulation and contractile performance in heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure and one for which no efficacious therapies exist. Obesity and lipid mishandling greatly contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are largely unknown. Here, we report that cardiomyocyte steatosis in HFpEF is coupled with increases in the activity of the transcription factor FoxO1 (Forkhead box protein O1). FoxO1 depletion, as well as over-expression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) in cardiomyocytes each ameliorates the HFpEF phenotype in mice and reduces myocardial lipid accumulation. Mechanistically, forced expression of Xbp1s in cardiomyocytes triggers ubiquitination and proteasomal degradation of FoxO1 which occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-box-containing protein 1) a novel and direct transcriptional target of Xbp1s. Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of cardiometabolic HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes.

Abstract 13192: Xbp1s - Foxo1 Axis Governs Lipid Accumulation and Cardiac Performance in Heart Failure With Preserved Ejection Fraction

Introduction: Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure (HF). Limited insight into underlying mechanisms has culminated in the longstanding absence of evidence-based therapies capable of mitigating the substantial morbidity and mortality associated with the syndrome. Existing clinical and epidemiological evidence suggests that excessive body fat and lipid mishandling contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are unknown. We recently developed a novel, clinically relevant, murine model of HFpEF, uncovering suppression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) signaling pathway as a critical driver of HFpEF pathogenesis. Objectives: To define and manipulate mechanisms downstream of Xbp1s in HFpEF and decipher its cardioprotective actions. Methods and Results: In the myocardium of experimental HFpEF, we detected cardiomyocyte steatosis coupled with increases in the abundance and activity of FoxO1 (Forkhead box protein O1), a conserved transcription factor involved in cell metabolism. FoxO1 depletion, as well as Xbp1s over-expression, in cardiomyocytes each ameliorated the HFpEF phenotype and reduced myocardial lipid accumulation. Strikingly, forced expression of Xbp1s in cardiomyocytes triggered proteasomal degradation of FoxO1. Furthermore, we discovered that FoxO1 is ubiquitinated upon Xbp1s over-expression, and Xbp1s-induced proteasomal degradation of FoxO1 occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-Box-containing protein 1), a protein we identified as a novel and direct transcriptional target of Xbp1s. Conclusions: Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes.

Iron regulatory protein 2 is required for artemether -mediated anti-hepatic fibrosis through ferroptosis pathway

BackgroundCurrently, the existing treatments have not cured the liver fibrosis thoroughly. Ferroptosis is a newly discovered way of cell death, which is closely related to many diseases. Previous studies have shown that ferroptosis plays an important role in the occurrence and development of liver fibrosis, but the further mechanism remains to be discovered. MethodsLX-2 cells were used as the research object, fibrosis activation index was detected by Western blot, PCR and Immunofluorescence, ferroptosis was detected by kits, the binding and interaction between IRP2 (iron regulatory protein 2) and STUB1 (STIP1 homology and U-box containing protein 1) were detected by Immunoprecipitation and ubiquitin test, and IRP2 knockdown mice were constructed by interfering plasmid to verify the results of in vitro experiment. ResultOur research showed that ART (artemether) had a good anti-fibrosis effect in vivo and in vitro, and ferroptosis played an important role in this process. Further studies have found that ART could lead to the accumulation of IRP 2 a in hepatic stellate cell by inhibiting the ubiquitination of it, thus inducing the increase of iron in HSC (hepatic stellate cell), which could product a large number of ROS (reactive oxide species), resulting the occurrence of ferroptosis in cells. Our findings provided an experimental basis for ART to become a drug for the treatment of liver fibrosis. ConclusionOur results show that IRP2-Iron-ROS axis is necessary for ART to induce ferroptosis in HSC and play an anti-fibrotic effect.

A reciprocal feedback of Myc and lncRNA MTSS1-AS contributes to extracellular acidity-promoted metastasis of pancreatic cancer.

Rationale: Previous studies have reported on the role of extracellular acidity in the metastasis of numerous cancers. However, the involvement of long noncoding RNA (lncRNA) in the extracellular acidity-induced cancer metastasis of pancreatic cancer (PC) remains unclear.Methods: Different expression levels of lncRNAs in PC cells under normal and acidic conditions were compared by RNA sequencing (RNA-seq). The effects of antisense lncRNA of metastasis suppressor 1 (MTSS1-AS) on acidic PC cells were assessed by gain- and loss-of-function experiments. Fluorescence in situ hybridization, RNA immunoprecipitation, RNA pull-down, Western blot, luciferase reporter, and Chromatin immunoprecipitation assays were employed to determine the regulatory mechanisms of MTSS1-AS in the acidity-induced metastasis of PC cells. The expression of MTSS1-AS and associated pathways were compared in PC samples and peritumoral normal tissues.Results: RNA-seq demonstrated that MTSS1-AS was significantly downregulated in pancreatic cells cultured with the acidic medium. The overexpression of MTSS1-AS remarkably inhibited the acidity-promoted metastasis of PC cells by upregulating the expression of its sense gene metastasis suppressor 1 (MTSS1). Mechanistically, MTSS1-AS scaffolded the interaction between E3 ubiquitin-protein ligase STIP1 homology and U-box containing protein 1 (STUB1) and transcription regulator myeloid zinc finger 1 (MZF1), leading to ubiquitination-mediated degradation of MZF1. Further, MZF1 inhibited the expression of MTSS1 by binding to the MTSS1 promoter. Thus, the acidity-reduced MTSS1-AS facilitated the stability of MZF1 and its inhibitory effect on MTSS1 transcription, thereby promoting the metastasis of PC cells under acidic conditions. Moreover, MTSS1-AS was transcriptionally repressed by the binding of MYC proto-oncogene (Myc) with initiator (Inr) elements of the MTSS1-AS promoter. Meanwhile, MTSS1-AS mutually repressed the expression of Myc by impairing the MZF1-mediated transcription activation of Myc, thereby forming a negative feedback loop between MTSS1-AS and Myc in acidic PC cells. In accordance with the experimental results, MTSS1-AS and MTSS1 were downregulated in PC and correlated with poor overall survival.Conclusions: The results implicated that a reciprocal feedback loop between Myc and MTSS1-AS contributed to the extracellular acidity-promoted metastasis of PC, and indicated that MTSS1-AS was a valuable biomarker and therapeutic target for PC.

Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4

ABSTRACT Accumulation of profibrotic myofibroblasts is involved in the process of fibrosis development during idiopathic pulmonary fibrosis (IPF) pathogenesis. TGFB (transforming growth factor β) is one of the major profibrotic cytokines for myofibroblast differentiation and NOX4 (NADPH oxidase 4) has an essential role in TGFB-mediated cell signaling. Azithromycin (AZM), a second-generation antibacterial macrolide, has a pleiotropic effect on cellular processes including proteostasis. Hence, we hypothesized that AZM may regulate NOX4 levels by modulating proteostasis machineries, resulting in inhibition of TGFB-associated lung fibrosis development. Human lung fibroblasts (LF) were used to evaluate TGFB-induced myofibroblast differentiation. With respect to NOX4 regulation via proteostasis, assays for macroautophagy/autophagy, the unfolded protein response (UPR), and proteasome activity were performed. The potential anti-fibrotic property of AZM was examined by using bleomycin (BLM)-induced lung fibrosis mouse models. TGFB-induced NOX4 and myofibroblast differentiation were clearly inhibited by AZM treatment in LF. AZM-mediated NOX4 reduction was restored by treatment with MG132, a proteasome inhibitor. AZM inhibited autophagy and enhanced the UPR. Autophagy inhibition by AZM was linked to ubiquitination of NOX4 via increased protein levels of STUB1 (STIP1 homology and U-box containing protein 1), an E3 ubiquitin ligase. An increased UPR by AZM was associated with enhanced proteasome activity. AZM suppressed lung fibrosis development induced by BLM with concomitantly reduced NOX4 protein levels and enhanced proteasome activation. These results suggest that AZM suppresses NOX4 by promoting proteasomal degradation, resulting in inhibition of TGFB-induced myofibroblast differentiation and lung fibrosis development. AZM may be a candidate for the treatment of the fibrotic lung disease IPF.

In vitro characterization of six STUB1 variants in spinocerebellar ataxia 16 reveals altered structural properties for the encoded CHIP proteins.

Spinocerebellar ataxia, autosomal recessive 16 (SCAR16) is caused by biallelic mutations in the STIP1 homology and U-box containing protein 1 (STUB1) gene encoding the ubiquitin E3 ligase and dimeric co-chaperone C-terminus of Hsc70-interacting protein (CHIP). It has been proposed that the disease mechanism is related to CHIP’s impaired E3 ubiquitin ligase properties and/or interaction with its chaperones. However, there is limited knowledge on how these mutations affect the stability, folding, and protein structure of CHIP itself. To gain further insight, six previously reported pathogenic STUB1 variants (E28K, N65S, K145Q, M211I, S236T, and T246M) were expressed as recombinant proteins and studied using limited proteolysis, size-exclusion chromatography (SEC), and circular dichroism (CD). Our results reveal that N65S shows increased CHIP dimerization, higher levels of α-helical content, and decreased degradation rate compared with wild-type (WT) CHIP. By contrast, T246M demonstrates a strong tendency for aggregation, a more flexible protein structure, decreased levels of α-helical structures, and increased degradation rate compared with WT CHIP. E28K, K145Q, M211I, and S236T also show defects on structural properties compared with WT CHIP, although less profound than what observed for N65S and T246M. In conclusion, our results illustrate that some STUB1 mutations known to cause recessive SCAR16 have a profound impact on the protein structure, stability, and ability of CHIP to dimerize in vitro. These results add to the growing understanding on the mechanisms behind the disorder.

Transcription factor RFX1 is ubiquitinated by E3 ligase STUB1 in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease caused by complex interactions between genes and the environment. The expression level of transcription factor regulatory factor X 1 (RFX1) is reduced in T cells from SLE patients. RFX1 can regulate epigenetic modifications of CD70 and CD11a and plays an important role in the development of SLE. However, the mechanisms that mediate reduction of RFX1 in SLE are unclear. Here, we demonstrate that RFX1 protein expression can be tightly regulated by polyubiquitination-mediated proteosomal degradation via STIP1 homology and U-box containing protein 1 (STUB1). The E3 ligase STUB1 is upregulated in CD4+T cells of SLE patients compared to healthy subjects. Overexpression of STUB1 in CD4+T cells leads to upregulation of levels of CD70 and CD11a in T cells. The modulation of STUB1 activity may provide a novel therapeutic approach for SLE.

Changes and significance of ubiquitination of Foxp3 protein during the acute phase of Kawasaki dis-ease

Objective To investigate the changes and significance of ubiquitination of Foxp3 protein during the acute phage of Kawasaki disease (KD). Methods Forty-eight children with KD and twenty-eight age-matched healthy children were recruited in this study. Co-immunoprecipitation and Western blot assays were performed to determine the poly-ubiquitination status of Foxp3 in CD4+ T cells. The percentages of CD4+ CD25highFoxp3+ regulatory T cells (Treg) and the levels of IL-10, transforming growth factor-β (TGF-β), cytotoxic T lymphocyte-associated protein-4 (CTLA4), STIP1 homology and U-Box containing protein 1 (STUB1), heat shock protein 70 (HSP70), ubiquitin-specific-processing protease 7 (USP7) and pSTAT3 were analyzed by flow cytometry analysis. Quantitative real-time PCR was used to evaluate the transcription levels of TLR1-10, IL-1R1, IL-1RAP, IL-6Rα, gp130, TNFR1, MyD88 and RIP1 in CD4+ T cells. Plasma concentrations of IL-1β, IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. Results (1) The percentages of Treg cells and the levels of IL-10, TGF-β, CTLA4 and forkhead box P3 (Foxp3) in patients with acute KD were lower than those of healthy subjects (P 0.05). Conclusion Hyper-ubiquitination of Foxp3 protein might be involved in the immune dysfunction during Kawasaki disease. Key words: Kawasaki disease; Foxp3; Ubiquitination; Regulatory T cells; Immune regulation

The Unfolding Tail of CHIP Mutation in Gordon Holmes Syndrome

Gordon Holmes Syndrome (GHS) is a rare neurodegenerative disorder characterized by cerebellar ataxia with hypogonadism. The pathophysiological mechanisms and genetic causes of GHS are largely unknown and therapies limited. We identified a homozygous mutation (c.737C→T, p.Thr246Met) in STIP1 homology and U‐box containing protein 1 (STUB1), the gene that encodes for C‐terminus of Hsp70 Interacting Protein (CHIP) in siblings that presented with GHS. CHIP plays a central role in regulating protein quality control as both an E3 ligase and molecular chaperone. Human mutations of CHIP are increasingly linked to ataxias, but the molecular mechanisms underlying this disease pathology remain undefined. Biophysical and cell culture studies reveal T246M CHIP has a disorganized Ubox domain and exists mostly as aggregates. Consequently, T246M CHIP exhibits loss of E3 ligase activity, but unexpectedly, the amount of T246M CHIP interacting with substrates, E2 enzymes and molecular chaperones is higher than wild‐type CHIP. We hypothesize that the sequestering of CHIP interacting proteins drives disease pathology. Interestingly, T246M CHIP maintains some chaperone activity as measured by its in vitro potentiation of AMPK and translocation/activation of HSF1 in cells. Thus the T246M mutation disrupts CHIP structure and function resulting in a dominant negative protein impairing ubiquitin‐dependent protein degradation. We developed a T246M CHIP mouse model of GHS and present preliminary data consistent with our hypothesis that T246M CHIP functions as a dominant negative protein causing GHS.

Regulation of IL-4 Receptor Signaling by STUB1 in Lung Inflammation

IL-4Rα, the common receptor component for IL-4 and IL-13, plays a critical role in IL-4- and IL-13-mediated signaling pathways that regulate airway inflammation and remodeling. However, the regulatory mechanisms underlying IL-4Rα turnover and its signal termination remain elusive. To evaluate the role of STUB1 (STIP1 homology and U-Box containing protein 1) in regulating IL-4R signaling in airway inflammation. The roles of STUB1 in IL-4Rα degradation and its signaling were investigated by immunoblot, immunoprecipitation, and flow cytometry. The involvement of STUB1 in airway inflammation was determined in vivo by measuring lung inflammatory cells infiltration, mucus production, serum lgE levels, and alveolar macrophage M2 activation in STUB1(-/-) mice. STUB1 expression was evaluated in airway epithelium of patients with asthma and lung tissues of subjects with chronic obstructive pulmonary disease. STUB1 interacted with IL-4Rα and targeted it for ubiquitination-mediated proteasomal degradation, terminating IL-4 or IL-13 signaling. STUB1 knockout cells showed increased levels of IL-4Rα and sustained STAT6 activation, whereas STUB1 overexpression reduced IL-4Rα levels. Mice deficient in STUB1 had spontaneous airway inflammation, alternative M2 activation of alveolar macrophage, and increased serum IgE. STUB1 levels were increased in airways of subjects with asthma or chronic obstructive pulmonary disease, suggesting that up-regulation of STUB1 might be an important feedback mechanism to dampen IL-4R signaling in airway inflammation. Our study identified a previously uncharacterized role for STUB1 in regulating IL-4R signaling, which might provide a new strategy for attenuating airway inflammation.