Ying Zhao’s research while affiliated with Wenzhou Medical University and other places

Acute kidney injury (AKI), triggered by various stimuli including ischemia-reperfusion, nephrotoxic insult, and sepsis, is characterized by an abrupt deterioration in kidney function. Ubiquitination is a post-translational modification of proteins that plays a critical role in the pathogenesis and progression of AKI. In this study, we aimed to investigate the role and underlying mechanism of the deubiquitinating enzyme Josephin Domain-containing protein 2 (JOSD2) in AKI. We found that deficiency of JOSD2 exacerbated renal tubular injury and inflammation in AKI mice induced by cisplatin or ischemia-reperfusion injury. Conversely, the specific overexpression of JOSD2 in renal tubular epithelial cells effectively prevented renal tubular injury and inflammation induced in AKI mice. Mechanistically, we identified Sirtuin 7 (SIRT7) as a potential substrate of JOSD2 through mass spectrometry combined with co-immunoprecipitation analysis. JOSD2 removes the K63-linked ubiquitination of SIRT7 via its active site C24 and promotes P62-mediated autophagic degradation of SIRT7, which subsequently prevents the phosphorylation and nuclear translocation of P65 and reduces inflammatory responses in renal tubular epithelial cells. Our study reveals the role of the JOSD2-SIRT7 axis in regulating AKI-induced renal inflammation and highlights the potential of JOSD2 as a promising therapeutic target for AKI.

Recent studies have shown the crucial role of podocyte injury in the development of diabetic kidney disease (DKD). Deubiquitinating modification of proteins is widely involved in the occurrence and development of diseases. Here, we explore the role and regulating mechanism of a deubiquitinating enzyme, OTUD5, in podocyte injury and DKD. RNA-seq analysis indicates a significantly decreased expression of OTUD5 in HG/PA-stimulated podocytes. Podocyte-specific Otud5 knockout exacerbates podocyte injury and DKD in both type 1 and type 2 diabetic mice. Furthermore, AVV9-mediated OTUD5 overexpression in podocytes shows a therapeutic effect against DKD. Mass spectrometry and co-immunoprecipitation experiments reveal an inflammation-regulating protein, TAK1, as the substrate of OTUD5 in podocytes. Mechanistically, OTUD5 deubiquitinates K63-linked TAK1 at the K158 site through its active site C224, which subsequently prevents the phosphorylation of TAK1 and reduces downstream inflammatory responses in podocytes. Our findings show an OTUD5-TAK1 axis in podocyte inflammation and injury and highlight the potential of OTUD5 as a promising therapeutic target for DKD.

Kidney fibrosis is a prominent pathological feature of hypertensive kidney diseases (HKD). Recent studies have highlighted the role of ubiquitinating/deubiquitinating protein modification in kidney pathophysiology. Ovarian tumor Domain-Containing Protein 6A (OTUD6A) is a deubiquitinating enzyme involved in tumor progression. However, its role in kidney pathophysiology remains elusive. We aimed to investigate the role and underlying mechanism of OTUD6A during kidney fibrosis in HKD. The results revealed higher OTUD6A expression in kidney tissues of nephropathy patients and mice with chronic Ang II administration than that from the control ones. OTUD6A was mainly located in tubular epithelial cells. Moreover, OTUD6A deficiency significantly protected mice against Ang II-induced kidney dysfunction and fibrosis. Also, knocking OTUD6A down suppressed Ang II-induced fibrosis in cultured tubular epithelial cells, while overexpression of OTUD6A enhanced fibrogenic responses. Mechanistically, OTUD6A bounded to STAT3 and removed K63 linked- ubiquitin chains to promote STAT3 phosphorylation at tyrosine705 position and nuclear translocation, which then induced pro-fibrotic gene transcription in epithelial cells. These studies identified STAT3 as a direct substrate of OTUD6A and highlighted the pivotal role of OTUD6A in Ang II-induced kidney injury, indicating OTUD6A as a potential therapeutic target for HKD.

Background: CCN6 is a matricellular protein that critically regulates the tumourigenesis and progression of breast cancer. Although the tumour-suppressive function of CCN6 has been extensively studied, molecular mechanisms regulating protein levels of CCN6 remain largely unclear. This study aims to investigate the regulation of CCN6 by ubiquitination and deubiquitinating enzymes (DUBs) in breast cancer. Methods: A screening assay was performed to identify OTUB1 as the DUB for CCN6. Various biochemical methods were applied to elucidate the molecular mechanism of OTUB1 in the regulation of CCN6. The role of OTUB1-CCN6 interaction in breast cancer was studied with cell experiments and the allograft model. The correlation of OTUB1 and CCN6 in human breast cancer was determined by immunohistochemistry and Western blot. Results: We found that CCN6 protein levels were controlled by the ubiquitin-proteasome system. The K48 ubiquitination and degradation of CCN6 was inhibited by OTUB1, which directly interacted with CCN6 through its linker domain. Furthermore, OTUB1 inhibited the ubiquitination of CCN6 in a non-canonical manner. Deletion of OTUB1, concomitant with reduced CCN6 abundance, increased the migration, proliferation and viability of breast cancer cells. Supplementation of CCN6 abolished the effect of OTUB1 deletion on breast cancer. Importantly, OTUB1 expression was downregulated in human breast cancer and positively correlated with CCN6 levels. Conclusion: This study identified OTUB1 as a novel regulator of CCN6 in breast cancer.

Renal fibrosis is a crucial pathological feature of hypertensive renal disease (HRD). In-depth analysis of the pathogenesis of fibrosis is of great significance for the development of new drugs for the treatment of HRD. USP25 is a deubiquitinase that can regulate the progression of many diseases, but its function in the kidney remains unclear. We found that USP25 was significantly increased in human and mice HRD kidney tissues. In the HRD model induced by Ang II, USP25-/- mice showed significant aggravation of renal dysfunction and fibrosis compared with the control mice. Consistently, AAV9-mediated overexpression of USP25 significantly improved renal dysfunction and fibrosis. Mechanistically, USP25 inhibited the TGF-β pathway by reducing SMAD4 K63-linked polyubiquitination, thereby suppressing SMAD2 nuclear translocation. In conclusion, this study demonstrates for the first time that the deubiquitinase USP25 plays an important regulatory role in HRD.

Background: Pathological cardiac hypertrophy can lead to heart failure and is one of the leading causes of death globally. Understanding the molecular mechanism of pathological cardiac hypertrophy will contribute to the treatment of heart failure. DUBs (deubiquitinating enzymes) are essential to cardiac pathophysiology by precisely controlling protein function, localization, and degradation. This study set out to investigate the role and molecular mechanism of a DUB, USP25 (ubiquitin-specific peptidase 25), in pathological cardiac hypertrophy. Methods: The role of USP25 in myocardial hypertrophy was evaluated in murine cardiomyocytes in response to Ang II (angiotensin II) and transverse aortic constriction stimulation and in hypertrophic myocardium tissues of heart failure patients. Liquid chromotography with mass spectrometry/mass spectrometry analysis combined with Co-IP was used to identify SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2A), an antihypertrophy protein, as an interacting protein of USP25. To clarify the molecular mechanism of USP25 in the regulation of SERCA2a, we constructed a series of mutant plasmids of USP25. In addition, we overexpressed USP25 and SERCA2a in the heart with adenoassociated virus serotype 9 vectors to validate the biological function of USP25 and SERCA2a interaction. Results: We revealed increased protein level of USP25 in murine cardiomyocytes subject to Ang II and transverse aortic constriction stimulation and in hypertrophic myocardium tissues of patients with heart failure. USP25 deficiency aggravated cardiac hypertrophy and cardiac dysfunction under Ang II and transverse aortic constriction treatment. Mechanistically, USP25 bound to SERCA2a directly via its USP (ubiquitin-specific protease) domain and cysteine at position 178 of USP25 exerts deubiquitination to maintain the stability of the SERCA2a protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby maintaining calcium handling in cardiomyocytes. Moreover, restoration of USP25 expression via adenoassociated virus serotype 9 vectors in USP25-/- mice attenuated Ang II-induced cardiac hypertrophy and cardiac dysfunction, whereas myocardial overexpression of SERCA2a could mimic the effect of USP25. Conclusions: We confirmed that USP25 inhibited cardiac hypertrophy by deubiquitinating and stabilizing SERCA2a.

Renal fibrosis is a crucial pathological feature of hypertensive renal disease (HRD). In-depth analysis of the pathogenesis of fibrosis is of great significance for the development of new drugs for the treatment of HRD. USP25 is a deubiquitinase that can regulate the progression of many diseases, but its function in the kidney remains unclear. We found that USP25 was significantly increased in human and mouse HRD kidney tissues. In the HRD model induced by Ang II, USP25 −/− mice showed significant aggravation of renal dysfunction and fibrosis compared with the control mice. Consistently, AAV9-mediated overexpression of USP25 significantly improved renal dysfunction and fibrosis. Mechanistically, USP25 inhibited the TGF-β pathway by reducing SMAD4 k63-linked polyubiquitination, thereby suppressing SMAD2 nuclear translocation. In conclusion, this study demonstrates for the first time that the deubiquitinase USP25 plays an important regulatory role in HRD.

Acute lung injury (ALI), characterized by inflammatory damage, is a major clinical challenge. Developing specific treatment options for ALI requires the identification of novel targetable signaling pathways. Recent studies reported that endotoxin lipopolysaccharide (LPS) induced a TLR4-dependent activation of focal adhesion kinase (FAK) in colorectal adenocarcinoma cells, suggesting that FAK may be involved in LPS-induced inflammatory responses. Here, we investigated the involvement and mechanism of FAK in mediating LPS-induced inflammation and ALI. We show that LPS phosphorylates FAK in macrophages. Either FAK inhibitor, site-directly mutation, or siRNA knockdown of FAK significantly suppresses LPS-induced inflammatory cytokine production in macrophages. FAK inhibition also blocked LPS-induced activation of MAPKs and NFκB. Mechanistically, we demonstrate that activated FAK directly interacts with transforming growth factor-β-activated kinase-1 (TAK1), an upstream kinase of MAPKs and NFκB, and then phosphorylates TAK1 at Ser412. In a mouse model of LPS-induced ALI, pharmacological inhibition of FAK suppressed FAK/TAK activation and inflammatory response in lung tissues. These activities resulted in the preservation of lung tissues in LPS-challenged mice and increased survival during LPS-induced septic shock. Collectively, our results illustrate a novel FAK-TAK1-NFκB signaling axis in LPS-induced inflammation and ALI, and support FAK as a potential target for the treatment of ALI.

Giving the fact that the disorders of multiple receptor tyrosine kinases (RTKs) are characteristics of various cancers, we assumed that developing novel multi-target drugs might have an advantage in treating the complex cancers. Taking the multi-target c-Met inhibitor Foretinib as the leading compound, we discovered a novel series of 6,7-disubstituted-4-phenoxyquinoline derivatives bearing 1,8-naphthyridine-3-carboxamide moiety with the help of molecular docking. Among them, the most promising compound 33 showed a prominent activity against Hela (IC50 = 0.21 µM), A549 (IC50 = 0.39 µM), and MCF-7 (IC50 = 0.33 µM), which were 3.28-4.82 times more active than that of Foretinib. Additionally, compound 33 dose dependently induced apoptosis by arresting A549 cells at G1 phase. Enzymatic assays and docking analyses were further confirmed that compound 33 was a multi-target inhibitor with the strong potencies against c-Met (IC50 = 11.77 nM), MEK1 (IC50 = 10.71 nM), and Flt-3 (IC50 = 22.36 nM). In the A549 cells mediated xenograft mouse model, compound 33 inhibited the tumor growth (TGI = 64%) without obvious toxicity, establishing compound 33 as a promising candidate for cancer therapy.