bims-lypmec 2023-11-26 papers

Issue of 2023‒11‒26
eight papers selected by
Satoru Kobayashi, New York Institute of Technology

EMBO Rep. 2023 Nov 21. e57300

Microautophagy regulated by STK38 and GABARAPs is essential to repair lysosomes and prevent aging.

Monami Ogura, Tatsuya Kaminishi, Takayuki Shima, Miku Torigata, Nao Bekku, Keisuke Tabata, Satoshi Minami, Kohei Nishino, Akiko Nezu, Maho Hamasaki, Hidetaka Kosako, Tamotsu Yoshimori, Shuhei Nakamura.

Lysosomes are degradative organelles and signaling hubs that maintain cell and tissue homeostasis, and lysosomal dysfunction is implicated in aging and reduced longevity. Lysosomes are frequently damaged, but their repair mechanisms remain unclear. Here, we demonstrate that damaged lysosomal membranes are repaired by microautophagy (a process termed "microlysophagy") and identify key regulators of the first and last steps. We reveal the AGC kinase STK38 as a novel microlysophagy regulator. Through phosphorylation of the scaffold protein DOK1, STK38 is specifically required for the lysosomal recruitment of the AAA+ ATPase VPS4, which terminates microlysophagy by promoting the disassembly of ESCRT components. By contrast, microlysophagy initiation involves non-canonical lipidation of ATG8s, especially the GABARAP subfamily, which is required for ESCRT assembly through interaction with ALIX. Depletion of STK38 and GABARAPs accelerates DNA damage-induced cellular senescence in human cells and curtails lifespan in C. elegans, respectively. Thus, microlysophagy is regulated by STK38 and GABARAPs and could be essential for maintaining lysosomal integrity and preventing aging.

Keywords: ESCRT; lysosome; microautophagy; non-canonical ATG8 lipidation

DOI: https://doi.org/10.15252/embr.202357300

Nat Rev Mol Cell Biol. 2023 Nov 24.

Lysosomes as coordinators of cellular catabolism, metabolic signalling and organ physiology.

Carmine Settembre, Rushika M Perera.

Every cell must satisfy basic requirements for nutrient sensing, utilization and recycling through macromolecular breakdown to coordinate programmes for growth, repair and stress adaptation. The lysosome orchestrates these key functions through the synchronised interplay between hydrolytic enzymes, nutrient transporters and signalling factors, which together enable metabolic coordination with other organelles and regulation of specific gene expression programmes. In this Review, we discuss recent findings on lysosome-dependent signalling pathways, focusing on how the lysosome senses nutrient availability through its physical and functional association with mechanistic target of rapamycin complex 1 (mTORC1) and how, in response, the microphthalmia/transcription factor E (MiT/TFE) transcription factors exert feedback regulation on lysosome biogenesis. We also highlight the emerging interactions of lysosomes with other organelles, which contribute to cellular homeostasis. Lastly, we discuss how lysosome dysfunction contributes to diverse disease pathologies and how inherited mutations that compromise lysosomal hydrolysis, transport or signalling components lead to multi-organ disorders with severe metabolic and neurological impact. A deeper comprehension of lysosomal composition and function, at both the cellular and organismal level, may uncover fundamental insights into human physiology and disease.

DOI: https://doi.org/10.1038/s41580-023-00676-x

J Biol Chem. 2023 Nov 16. pii: S0021-9258(23)02501-2. [Epub ahead of print] 105473

Human V-ATPase a-subunit isoforms bind specifically to distinct phosphoinositide phospholipids.

Connie Mitra, Samuel Winkley, Patricia M Kane.

V-ATPases are highly conserved multi-subunit enzymes that maintain the distinct pH of eukaryotic organelles. The integral membrane a-subunit is encoded by tissue- and organelle- specific isoforms, and its cytosolic N-terminal domain (aNT) modulates organelle specific regulation and targeting of V-ATPases. Organelle membranes have specific phosphatidylinositol phosphate (PIP) lipid enrichment linked to maintenance of organelle pH. In yeast, the aNT domains of the two a-subunit isoforms bind PIP lipids enriched in the organelle membranes where they reside; these interactions affect activity and regulatory properties of the V-ATPases containing each isoform. Humans have four a-subunit isoforms, and we hypothesize that the aNT domains of these isoforms will also bind to specific PIP lipids. The a1 and a2 isoforms of human V-ATPase a-subunits are localized to endolysosomes and Golgi, respectively. We determined that bacterially expressed Hua1NT and Hua2NT bind specifically to endolysosomal PIP lipids PI(3)P and PI(3,5)P2 and Golgi enriched PI(4)P, respectively. Despite the lack of canonical PIP binding sites, we identified potential binding sites in the HuaNT domains by sequence comparisons and existing subunit structures and models. We found that mutations at a similar location in the distal loops of both HuaNT isoforms compromise binding to their cognate PIP lipids, suggesting that these loops encode PIP specificity of the a-subunit isoforms. These data suggest a mechanism through which PIP lipid binding could stabilize and activate V-ATPases in distinct organelles.

Keywords: Golgi; V-ATPase; a-subunit isoforms; endosomes; liposomes; lysosomes; phosphoinositide

DOI: https://doi.org/10.1016/j.jbc.2023.105473

Cell Death Differ. 2023 Nov 23.

MLKL polymerization-induced lysosomal membrane permeabilization promotes necroptosis.

Shuzhen Liu, Preston Perez, Xue Sun, Ken Chen, Rojin Fatirkhorani, Jamila Mammadova, Zhigao Wang.

Mixed lineage kinase-like protein (MLKL) forms amyloid-like polymers to promote necroptosis; however, the mechanism through which these polymers trigger cell death is not clear. We have determined that activated MLKL translocates to the lysosomal membrane during necroptosis induction. The subsequent polymerization of MLKL induces lysosome clustering and fusion and eventual lysosomal membrane permeabilization (LMP). This LMP leads to the rapid release of lysosomal contents into the cytosol, resulting in a massive surge in cathepsin levels, with Cathepsin B (CTSB) as a significant contributor to the ensuing cell death as it cleaves many proteins essential for cell survival. Importantly, chemical inhibition or knockdown of CTSB protects cells from necroptosis. Furthermore, induced polymerization of the MLKL N-terminal domain (NTD) also triggers LMP, leading to CTSB release and subsequent cell death. These findings clearly establish the critical role of MLKL polymerization induced lysosomal membrane permeabilization (MPI-LMP) in the process of necroptosis.

DOI: https://doi.org/10.1038/s41418-023-01237-7

Cell Rep. 2023 Nov 22. pii: S2211-1247(23)01496-1. [Epub ahead of print]42(12): 113484

An atypical GABARAP binding module drives the pro-autophagic potential of the AML-associated NPM1c variant.

Hannah Mende, Anshu Khatri, Carolin Lange, Sergio Alejandro Poveda-Cuevas, Georg Tascher, Adriana Covarrubias-Pinto, Frank Löhr, Sebastian E Koschade, Ivan Dikic, Christian Münch, Anja Bremm, Lorenzo Brunetti, Christian H Brandts, Hannah Uckelmann, Volker Dötsch, Vladimir V Rogov, Ramachandra M Bhaskara, Stefan Müller.

The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options.

Keywords: AML; ATG8; Autophagy; CP: Cancer; CP: Molecular biology; GABARAP; LC3; NPM1; NPM1c; TFEB; atypical LIR; lysosome

DOI: https://doi.org/10.1016/j.celrep.2023.113484

Commun Biol. 2023 Nov 22. 6(1): 1147

Cellular senescence triggers intracellular acidification and lysosomal pH alkalinized via ATP6AP2 attenuation in breast cancer cells.

Wei Li, Kosuke Kawaguchi, Sunao Tanaka, Chenfeng He, Yurina Maeshima, Eiji Suzuki, Masakazu Toi.

Several chemotherapeutic drugs induce senescence in cancer cells; however, the mechanisms underlying intracellular pH dysregulation in senescent cells remain unclear. Adenosine triphosphatase H+ transporting accessory protein 2 (ATP6AP2) plays a critical role in maintaining pH homeostasis in cellular compartments. Herein, we report the regulatory role of ATP6AP2 in senescent breast cancer cells treated with doxorubicin (Doxo) and abemaciclib (Abe). A decline in ATP6AP2 triggers aberrant pH levels that impair lysosomal function and cause immune profile changes in senescent breast cancer cells. Doxo and Abe elicited a stable senescent phenotype and altered the expression of senescence-related genes. Additionally, senescent cells show altered inflammatory and immune transcriptional profiles due to reprogramming of the senescence-associated secretory phenotype. These findings elucidate ATP6AP2-mediated cellular pH regulation and suggest a potential link in immune profile alteration during therapy-induced senescence in breast cancer cells, providing insights into the mechanisms involved in the senescence response to anticancer therapy.

DOI: https://doi.org/10.1038/s42003-023-05433-6

Circulation. 2023 Nov 23.

USP28 Serves as a Key Suppressor of Mitochondrial Morphofunctional Defects and Cardiac Dysfunction in the Diabetic Heart.

Sai-Yang Xie, Shi-Qiang Liu, Tong Zhang, Wen-Ke Shi, Yun Xing, Wen-Xi Fang, Min Zhang, Meng-Ya Chen, Si-Chi Xu, Meng-Qi Fan, Lan-Lan Li, Heng Zhang, Nan Zhao, Zhao-Xiang Zeng, Si Chen, Xiao-Feng Zeng, Wei Deng, Qi-Zhu Tang.

BACKGROUND: The majority of people with diabetes are susceptible to cardiac dysfunction and heart failure, and conventional drug therapy cannot correct diabetic cardiomyopathy progression. Herein, we assessed the potential role and therapeutic value of USP28 (ubiquitin-specific protease 28) on the metabolic vulnerability of diabetic cardiomyopathy.METHODS: The type 2 diabetes mouse model was established using db/db leptin receptor-deficient mice and high-fat diet/streptozotocin-induced mice. Cardiac-specific knockout of USP28 in the db/db background mice was generated by crossbreeding db/m and Myh6-Cre+/USP28fl/fl mice. Recombinant adeno-associated virus serotype 9 carrying USP28 under cardiac troponin T promoter was injected into db/db mice. High glucose plus palmitic acid-incubated neonatal rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes were used to imitate diabetic cardiomyopathy in vitro. The molecular mechanism was explored through RNA sequencing, immunoprecipitation and mass spectrometry analysis, protein pull-down, chromatin immunoprecipitation sequencing, and chromatin immunoprecipitation assay.
RESULTS: Microarray profiling of the UPS (ubiquitin-proteasome system) on the basis of db/db mouse hearts and diabetic patients' hearts demonstrated that the diabetic ventricle presented a significant reduction in USP28 expression. Diabetic Myh6-Cre+/USP28fl/fl mice exhibited more severe progressive cardiac dysfunction, lipid accumulation, and mitochondrial disarrangement, compared with their controls. On the other hand, USP28 overexpression improved systolic and diastolic dysfunction and ameliorated cardiac hypertrophy and fibrosis in the diabetic heart. Adeno-associated virus serotype 9-USP28 diabetic mice also exhibited less lipid storage, reduced reactive oxygen species formation, and mitochondrial impairment in heart tissues than adeno-associated virus serotype 9-null diabetic mice. As a result, USP28 overexpression attenuated cardiac remodeling and dysfunction, lipid accumulation, and mitochondrial impairment in high-fat diet/streptozotocin-induced type 2 diabetes mice. These results were also confirmed in neonatal rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes. RNA sequencing, immunoprecipitation and mass spectrometry analysis, chromatin immunoprecipitation assays, chromatin immunoprecipitation sequencing, and protein pull-down assay mechanistically revealed that USP28 directly interacted with PPARα (peroxisome proliferator-activated receptor α), deubiquitinating and stabilizing PPARα (Lys152) to promote Mfn2 (mitofusin 2) transcription, thereby impeding mitochondrial morphofunctional defects. However, such cardioprotective benefits of USP28 were largely abrogated in db/db mice with PPARα deletion and conditional loss-of-function of Mfn2.
CONCLUSIONS: Our findings provide a USP28-modulated mitochondria homeostasis mechanism that involves the PPARα-Mfn2 axis in diabetic hearts, suggesting that USP28 activation or adeno-associated virus therapy targeting USP28 represents a potential therapeutic strategy for diabetic cardiomyopathy.

Keywords: PPAR alpha; USP28 protein, human; diabetic cardiomyopathies; heart failure; mitochondria

DOI: https://doi.org/10.1161/CIRCULATIONAHA.123.065603