bims-lypmec 2024-05-26 papers

Biophys Rep. 2024 Apr 30. 10(2): 111-120

Exploring lysosomal biology: current approaches and methods.

Lysosomes are the degradation centers and signaling hubs in the cell. Lysosomes undergo adaptation to maintain cell homeostasis in response to a wide variety of cues. Dysfunction of lysosomes leads to aging and severe diseases including lysosomal storage diseases (LSDs), neurodegenerative disorders, and cancer. To understand the complexity of lysosome biology, many research approaches and tools have been developed to investigate lysosomal functions and regulatory mechanisms in diverse experimental systems. This review summarizes the current approaches and tools adopted for studying lysosomes, and aims to provide a methodological overview of lysosomal research and related fields.

Keywords: C. elegans; Cultured cell; Lysosome; Method; Mice

DOI: https://doi.org/10.52601/bpr.2023.230028

Cell Mol Life Sci. 2024 May 22. 81(1): 227

Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis.

Lisa Gallwitz, Florian Bleibaum, Matthias Voss, Michaela Schweizer, Katharina Spengler, Dominic Winter, Frederic Zöphel, Stephan Müller, Stefan Lichtenthaler, Markus Damme, Paul Saftig.

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.

Keywords: Amyloid precursor protein; Autophagy; Bulk proteolysis; Cathepsins; Lysosome; Protease network; Proteolysis; Proteomics

DOI: https://doi.org/10.1007/s00018-024-05274-4

Front Immunol. 2024 ;15 1404846

The lysosomal trafficking regulator "LYST": an 80-year traffic jam.

Mackenzie E Turner, Jingru Che, Gabriel J M Mirhaidari, Catherine C Kennedy, Kevin M Blum, Sahana Rajesh, Jacob C Zbinden, Christopher K Breuer, Cameron A Best, Jenny C Barker.

Lysosomes and lysosome related organelles (LROs) are dynamic organelles at the intersection of various pathways involved in maintaining cellular hemostasis and regulating cellular functions. Vesicle trafficking of lysosomes and LROs are critical to maintain their functions. The lysosomal trafficking regulator (LYST) is an elusive protein important for the regulation of membrane dynamics and intracellular trafficking of lysosomes and LROs. Mutations to the LYST gene result in Chédiak-Higashi syndrome, an autosomal recessive immunodeficiency characterized by defective granule exocytosis, cytotoxicity, etc. Despite eight decades passing since its initial discovery, a comprehensive understanding of LYST's function in cellular biology remains unresolved. Accumulating evidence suggests that dysregulation of LYST function also manifests in other disease states. Here, we review the available literature to consolidate available scientific endeavors in relation to LYST and discuss its relevance for immunomodulatory therapies, regenerative medicine and cancer applications.

Keywords: Chédiak-Higashi syndrome; LYST; beige; cancer; immunotherapy; lysosomes; vesicle traffic; wound healing

DOI: https://doi.org/10.3389/fimmu.2024.1404846

Proc Natl Acad Sci U S A. 2024 May 28. 121(22): e2315690121

Tau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes.

Kevin Rose, Tyler Jepson, Sankalp Shukla, Alex Maya-Romero, Martin Kampmann, Ke Xu, James H Hurley.

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

Keywords: Alzheimer’s disease; ESCRT; STORM; astrocyte; lysosome

DOI: https://doi.org/10.1073/pnas.2315690121

Autophagy. 2024 May 18. 1-17

ROS-mediated lysosomal membrane permeabilization and autophagy inhibition regulate bleomycin-induced cellular senescence.

Zhangyang Qi, Weiqi Yang, Baibing Xue, Tingjun Chen, Xianjie Lu, Rong Zhang, Zhichao Li, Xiaoqing Zhao, Yang Zhang, Fabin Han, Xiaohong Kong, Ruikang Liu, Xue Yao, Rui Jia, Shiqing Feng.

Bleomycin exhibits effective chemotherapeutic activity against multiple types of tumors, and also induces various side effects, such as pulmonary fibrosis and neuronal defects, which limit the clinical application of this drug. Macroautophagy/autophagy has been recently reported to be involved in the functions of bleomycin, and yet the mechanisms of their crosstalk remain insufficiently understood. Here, we demonstrated that reactive oxygen species (ROS) produced during bleomycin activation hampered autophagy flux by inducing lysosomal membrane permeabilization (LMP) and obstructing lysosomal degradation. Exhaustion of ROS with N-acetylcysteine relieved LMP and autophagy defects. Notably, we observed that LMP and autophagy blockage preceded the emergence of cellular senescence during bleomycin treatment. In addition, promoting or inhibiting autophagy-lysosome degradation alleviated or exacerbated the phenotypes of senescence, respectively. This suggests the alternation of autophagy activity is more a regulatory mechanism than a consequence of bleomycin-induced cellular senescence. Taken together, we reveal a specific role of bleomycin-induced ROS in mediating defects of autophagic degradation and further regulating cellular senescence in vitro and in vivo. Our findings, conversely, indicate the autophagy-lysosome degradation pathway as a target for modulating the functions of bleomycin. These provide a new perspective for optimizing bleomycin as a clinically applicable chemotherapeutics devoid of severe side-effects.Abbreviations: AT2 cells: type II alveolar epithelial cells; ATG7: autophagy related 7; bEnd.3: mouse brain microvascular endothelial cells; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CCL2: C-C motif chemokine ligand 2; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; FTH1: ferritin heavy polypeptide 1; γ-H2AX: phosphorylated H2A.X variant histone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HUVEC: human umbilical vein endothelial cells; HT22: hippocampal neuronal cell lines; Il: interleukin; LAMP: lysosomal-associated membrane protein; LMP: lysosome membrane permeabilization; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NCOA4: nuclear receptor coactivator 4; PI3K: phosphoinositide 3-kinase; ROS: reactive oxygen species; RPS6KB/S6K: ribosomal protein S6 kinase; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; SAHF: senescence-associated heterochromatic foci; SASP: senescence-associated secretory phenotype; SEC62: SEC62 homolog, preprotein translocation; SEP: superecliptic pHluorin; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB.

Keywords: Autophagy; ROS; bleomycin; cellular senescence; lysosomal membrane permeabilization

DOI: https://doi.org/10.1080/15548627.2024.2353548

Biomolecules. 2024 May 13. pii: 573. [Epub ahead of print]14(5):

Failure of Autophagy in Pompe Disease.

Hung Do, Naresh K Meena, Nina Raben.

Autophagy is an evolutionarily conserved lysosome-dependent degradation of cytoplasmic constituents. The system operates as a critical cellular pro-survival mechanism in response to nutrient deprivation and a variety of stress conditions. On top of that, autophagy is involved in maintaining cellular homeostasis through selective elimination of worn-out or damaged proteins and organelles. The autophagic pathway is largely responsible for the delivery of cytosolic glycogen to the lysosome where it is degraded to glucose via acid α-glucosidase. Although the physiological role of lysosomal glycogenolysis is not fully understood, its significance is highlighted by the manifestations of Pompe disease, which is caused by a deficiency of this lysosomal enzyme. Pompe disease is a severe lysosomal glycogen storage disorder that affects skeletal and cardiac muscles most. In this review, we discuss the basics of autophagy and describe its involvement in the pathogenesis of muscle damage in Pompe disease. Finally, we outline how autophagic pathology in the diseased muscles can be used as a tool to fast track the efficacy of therapeutic interventions.

Keywords: Pompe disease; autophagy; enzyme replacement therapy; glycogen degradation; lysosome; muscle

DOI: https://doi.org/10.3390/biom14050573