bims-lypmec 2024-03-17 papers

Issue of 2024‒03‒17
five papers selected by
Satoru Kobayashi, New York Institute of Technology

Int J Biol Sci. 2024 ;20(5): 1905-1926

Impaired TFEB-mediated autophagy-lysosome fusion promotes tubular cell cycle G2/M arrest and renal fibrosis by suppressing ATP6V0C expression and interacting with SNAREs.

Xiang Ren, Jing Wang, Huizhi Wei, Xing Li, Yiqun Tian, Zhixian Wang, Yisheng Yin, Zihao Guo, Zhenliang Qin, Minglong Wu, Xiaoyong Zeng.

Increasing evidence suggests that autophagy plays a major role during renal fibrosis. Transcription factor EB (TFEB) is a critical regulator of autophagy- and lysosome-related gene transcription. However, the pathophysiological roles of TFEB in renal fibrosis and fine-tuned mechanisms by which TFEB regulates fibrosis remain largely unknown. Here, we found that TFEB was downregulated in unilateral ureteral obstruction (UUO)-induced human and mouse fibrotic kidneys, and kidney-specific TFEB overexpression using recombinant AAV serotype 9 (rAAV9)-TFEB in UUO mice alleviated renal fibrosis pathogenesis. Mechanically, we found that TFEB's prevention of extracellular matrix (ECM) deposition depended on autophagic flux integrity and its subsequent blockade of G2/M arrest in tubular cells, rather than the autophagosome synthesis. In addition, we together RNA-seq with CUT&Tag analysis to determine the TFEB targeted gene ATP6V0C, and revealed that TFEB was directly bound to the ATP6V0C promoter only at specific site to promote its expression through CUT&Run-qPCR and luciferase reporter assay. Interestingly, TFEB induced autophagic flux integrity, mainly dependent on scaffold protein ATP6V0C-mediated autophagosome-lysosome fusion by bridging with STX17 and VAMP8 (major SNARE complex) by co-immunoprecipitation analysis, rather than its mediated lysosomal acidification and degradation function. Moreover, we further investigated the underlying mechanism behind the low expression of TEFB in UUO-induced renal fibrosis, and clearly revealed that TFEB suppression in fibrotic kidney was due to DNMT3a-associated TFEB promoter hypermethylation by utilizing methylation specific PCR (MSP) and bisulfite-sequencing PCR (BSP), which could be effectively recovered by 5-Aza-2'-deoxycytidine (5A-za) to alleviate renal fibrosis pathogenesis. These findings reveal for the first time that impaired TFEB-mediated autophagosome-lysosome fusion disorder, tubular cell G2/M arrest and renal fibrosis appear to be sequentially linked in UUO-induced renal fibrosis and suggest that DNMT3a/TFEB/ATP6V0C may serve as potential therapeutic targets to prevent renal fibrosis.

Keywords: Renal fibrosis; V-ATPase; autophagy; cell cycle; methylation; transcription factor EB

DOI: https://doi.org/10.7150/ijbs.91480

FASEB Bioadv. 2024 Mar;6(3): 85-102

ROCK1 deficiency preserves caveolar compartmentalization of signaling molecules and cell membrane integrity.

Jianjian Shi, Lei Wei.

In this study, we investigated the roles of ROCK1 in regulating structural and functional features of caveolae located at the cell membrane of cardiomyocytes, adipocytes, and mouse embryonic fibroblasts (MEFs) as well as related physiopathological effects. Caveolae are small bulb-shaped cell membrane invaginations, and their roles have been associated with disease conditions. One of the unique features of caveolae is that they are physically linked to the actin cytoskeleton that is well known to be regulated by RhoA/ROCKs pathway. In cardiomyocytes, we observed that ROCK1 deficiency is coincident with an increased caveolar density, clusters, and caveolar proteins including caveolin-1 and -3. In the mouse cardiomyopathy model with transgenic overexpressing Gαq in myocardium, we demonstrated the reduced caveolar density at cell membrane and reduced caveolar protein contents. Interestingly, coexisting ROCK1 deficiency in cardiomyocytes can rescue these defects and preserve caveolar compartmentalization of β-adrenergic signaling molecules including β1-adrenergic receptor and type V/VI adenylyl cyclase. In cardiomyocytes and adipocytes, we detected that ROCK1 deficiency increased insulin signaling with increased insulin receptor activation in caveolae. In MEFs, we identified that ROCK1 deficiency increased caveolar and total levels of caveolin-1 and cell membrane repair ability after mechanical or chemical disruptions. Together, these results demonstrate that ROCK1 can regulate caveolae plasticity and multiple functions including compartmentalization of signaling molecules and cell membrane repair following membrane disruption by mechanical force and oxidative damage. These findings provide possible molecular insights into the beneficial effects of ROCK1 deletion/inhibition in cardiomyocytes, adipocytes, and MEFs under certain diseased conditions.

Keywords: ROCK1; cardiomyocyte hypertrophy; caveolae; compartmentalization; insulin signaling; mechanical injury; plasma membrane repair; β‐adrenergic signaling

DOI: https://doi.org/10.1096/fba.2024-00015

Int J Mol Sci. 2024 Feb 29. pii: 2843. [Epub ahead of print]25(5):

UBA6 Inhibition Accelerates Lysosomal TRPML1 Depletion and Exosomal Secretion in Lung Cancer Cells.

Dongun Lee, Peter Chang-Whan Lee, Jeong Hee Hong.

Ubiquitin-like modifier-activating enzyme 6 (UBA6) is a member of the E1 enzyme family, which initiates the ubiquitin-proteasome system (UPS). The UPS plays critical roles not only in protein degradation but also in various cellular functions, including neuronal signaling, myocardial remodeling, immune cell differentiation, and cancer development. However, the specific role of UBA6 in cellular functions is not fully elucidated in comparison with the roles of the UPS. It has been known that the E1 enzyme is associated with the motility of cancer cells. In this study, we verified the physiological roles of UBA6 in lung cancer cells through gene-silencing siRNA targeting UBA6 (siUBA6). The siUBA6 treatment attenuated the migration of H1975 cells, along with a decrease in lysosomal Ca2+ release. While autophagosomal proteins remained unchanged, lysosomal proteins, including TRPML1 and TPC2, were decreased in siUBA6-transfected cells. Moreover, siUBA6 induced the production of multivesicular bodies (MVBs), accompanied by an increase in MVB markers in siUBA6-transfected H1975 cells. Additionally, the expression of the exosomal marker CD63 and extracellular vesicles was increased by siUBA6 treatment. Our findings suggest that knock-down of UBA6 induces lysosomal TRPML1 depletion and inhibits endosomal trafficking to lysosome, and subsequently, leads to the accumulation of MVBs and enhanced exosomal secretion in lung cancer cells.

Keywords: UBA6; endosomal trafficking; exosome; lung cancer; multivesicular body

DOI: https://doi.org/10.3390/ijms25052843

J Mol Cell Cardiol. 2024 Mar 13. pii: S0022-2828(24)00034-8. [Epub ahead of print]189 83-89

Myocardial glycophagy flux dysregulation and glycogen accumulation characterize diabetic cardiomyopathy.

Kimberley M Mellor, Upasna Varma, Parisa Koutsifeli, Lorna J Daniels, Victoria L Benson, Marco Annandale, Xun Li, Yohanes Nursalim, Johannes V Janssens, Kate L Weeks, Kim L Powell, Terence J O'Brien, Rajesh Katare, Rebecca H Ritchie, James R Bell, Roberta A Gottlieb, Lea M D Delbridge.

Diabetic heart disease morbidity and mortality is escalating. No specific therapeutics exist and mechanistic understanding of diabetic cardiomyopathy etiology is lacking. While lipid accumulation is a recognized cardiomyocyte phenotype of diabetes, less is known about glycolytic fuel handling and storage. Based on in vitro studies, we postulated the operation of an autophagy pathway in the myocardium specific for glycogen homeostasis - glycophagy. Here we visualize occurrence of cardiac glycophagy and show that the diabetic myocardium is characterized by marked glycogen elevation and altered cardiomyocyte glycogen localization. We establish that cardiac glycophagy flux is disturbed in diabetes. Glycophagy may represent a potential therapeutic target for alleviating the myocardial impacts of metabolic disruption in diabetic heart disease.

Keywords: Autophagy; Cardiac glycogen; Cardiac metabolism; Diabetic heart disease; Glycophagy

DOI: https://doi.org/10.1016/j.yjmcc.2024.02.009

Chemistry. 2024 Mar 12. e202400111

pH-Responsive Aminobenzocoumarins as Fluorescent Probes for Biological Acidity.

Karin Schniererová, Hana Janeková, Jakub Joniak, Martin Putala, Peter Stacko, Henrieta Stankovičová.

Regulation of pH plays an essential role in orchestrating the delicate cellular machinery responsible for life as we know it. Its abnormal values are indicative of aberrant cellular behavior and associated with pathologies including cancer progression or solid tumors. Here, we report a series of bent and linear aminobenzocoumarins decorated with different substituents. We investigate their photophysical properties and demonstrate that the probes display strong pH-responsive fluorescence "turn on" behavior in highly acidic environments, with enhancement up to 300-fold. In combination with their low cytotoxicity, this behavior enabled their application in bioimaging of acidic lysosomes in live human cells. We believe that these molecules serve as attractive lead structures for future rational design of novel biocompatible fluorescent pH probes.

Keywords: benzocoumarin; coumarin; electron transfer; fluorescence; pH-probe

DOI: https://doi.org/10.1002/chem.202400111