bims-lypmec 2024-01-14 papers

Issue of 2024‒01‒14
four papers selected by
Satoru Kobayashi, New York Institute of Technology

J Mol Med (Berl). 2024 Jan 06.

PRKAA2, MTOR, and TFEB in the regulation of lysosomal damage response and autophagy.

Mohd Shariq, Mohammad Firoz Khan, Reshmi Raj, Nuzhat Ahsan, Pramod Kumar.

Lysosomes function as critical signaling hubs that govern essential enzyme complexes. LGALS proteins (LGALS3, LGALS8, and LGALS9) are integral to the endomembrane damage response. If ESCRT fails to rectify damage, LGALS-mediated ubiquitination occurs, recruiting autophagy receptors (CALCOCO2, TRIM16, and SQSTM1) and VCP/p97 complex containing UBXN6, PLAA, and YOD1, initiating selective autophagy. Lysosome replenishment through biogenesis is regulated by TFEB. LGALS3 interacts with TFRC and TRIM16, aiding ESCRT-mediated repair and autophagy-mediated removal of damaged lysosomes. LGALS8 inhibits MTOR and activates TFEB for ATG and lysosomal gene transcription. LGALS9 inhibits USP9X, activates PRKAA2, MAP3K7, ubiquitination, and autophagy. Conjugation of ATG8 to single membranes (CASM) initiates damage repair mediated by ATP6V1A, ATG16L1, ATG12, ATG5, ATG3, and TECPR1. ATG8ylation or CASM activates the MERIT system (ESCRT-mediated repair, autophagy-mediated clearance, MCOLN1 activation, Ca2+ release, RRAG-GTPase regulation, MTOR modulation, TFEB activation, and activation of GTPase IRGM). Annexins ANAX1 and ANAX2 aid damage repair. Stress granules stabilize damaged membranes, recruiting FLCN-FNIP1/2, G3BP1, and NUFIP1 to inhibit MTOR and activate TFEB. Lysosomes coordinate the synergistic response to endomembrane damage and are vital for innate and adaptive immunity. Future research should unveil the collaborative actions of ATG proteins, LGALSs, TRIMs, autophagy receptors, and lysosomal proteins in lysosomal damage response.

Keywords: Autophagy receptors; LGALS; Lysosomal damage response; MTOR; PRKAA2; TFEB

DOI: https://doi.org/10.1007/s00109-023-02411-7

Curr Protoc. 2024 Jan;4(1): e950

Histodenz Separation of Lysosomal Subpopulations for Analysis of Chaperone-mediated Autophagy.

Calvin M Burns, Richard A Miller, S Joseph Endicott.

Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, in which proteins are individually selected for lysosomal degradation. CMA degradation targets bear a pentapeptide consensus motif that is recognized by the cytosolic chaperone HSPA8 (Hsc70), which participates in the trafficking of the target to the lysosomal surface. From there, it is translocated into the lysosomal lumen, independent of vesicle fusion, in a process dependent upon the lysosomal transmembrane protein LAMP2A. There are limited tools for studying CMA in whole cells and tissues, and many of the best techniques for studying CMA rely on the preparation of lysosome enriched fractions. Such experiments include (1) the in vitro evaluation of CMA substrate uptake activity, (2) the characterization of changes to lysosomal resident and CMA regulatory proteins, and (3) lysosomal targetomics, i.e., the use of quantitative proteomics to characterize lysosomal degradation targets. Previous studies using discontinuous metrizamide gradients have shown that a subpopulation of liver lysosomes is responsible for the majority of CMA activity ("CMA+ "). These CMA+ lysosomes are low density and have higher levels of MTORC2 relative to the "CMA- " lysosomes, which are high density and have higher levels of MTORC1. Because of safety concerns surrounding metrizamide, however, this compound is difficult to obtain, and it is impractically expensive. Here, we have provided protocols for isolation of lysosomal subpopulations for CMA-related analyses from mouse liver using Histodenz, a safe and affordable alternative to metrizamide. Supplementary protocols show how to perform CMA activity assays with appropriate statistical analysis, and how to analyze for lysosomal breakage/membrane integrity. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation of lysosomal subpopulations from mouse liver using discontinuous Histodenz gradients Alternate Protocol: Isolation of lysosomes from cultured cells using discontinuous Histodenz gradients Support Protocol 1: Verifying enrichment of lysosomal markers in lysosome-enriched fractions Support Protocol 2: Measuring in vitro uptake of CMA substrates Support Protocol 3: Measuring lysosomal membrane integrity by hexosaminidase assay.

Keywords: autophagy; density purification; lysosomes; organelle isolation

DOI: https://doi.org/10.1002/cpz1.950

J Biol Chem. 2024 Jan 09. pii: S0021-9258(24)00017-6. [Epub ahead of print] 105641

Disruption of lysosomal nutrient sensing scaffold hyperactivates mTORC1 contributing to pathogenesis of a fatal neurodegenerative disease.

Maria B Bagh, Abhilash P Appu, Tamal Sadhukhan, Avisek Mondal, Nisha Plavelil, Mahadevan Raghavankutty, Ajayan M Supran, Sriparna Sadhukhan, Aiyi Liu, Anil B Mukherjee.

The CLN1-disease, formerly called infantile neuronal ceroid lipofuscinosis (INCL), is a fatal hereditary neurodegenerative lysosomal storage disorder. This disease is caused by loss-of-function mutations in the CLN1 gene, encoding palmitoyl-protein thioesterase-1 (PPT1). PPT1 catalyzes depalmitoylation of S-palmitoylated proteins for degradation and clearance by lysosomal hydrolases. Numerous proteins, especially in the brain, require dynamic S-palmitoylation (palmitoylation-depalmitoylation cycles) for endosomal trafficking to their destination. While 23 palmitoyl-acyl transferases in the mammalian genome catalyze S-palmitoylation, depalmitoylation is catalyzed by thioesterases such as PPT1. Despite these discoveries, the pathogenic mechanism of CLN1-disease has remained elusive. Here we report that in the brain of Cln1-/- mice, which mimic CLN1-disease, the mechanistic target of rapamycin complex-1 (mTORC1)-kinase is hyperactivated. The activation of mTORC1 by nutrients requires its anchorage to lysosomal limiting membrane by Rag GTPases and Ragulator complex. These proteins form the lysosomal nutrient sensing scaffold to which mTORC1 must attach to activate. We found that in Cln1-/- mice, two constituent proteins of the Ragulator complex (vATPase and Lamtor1) require dynamic S-palmitoylation for endosomal trafficking to the lysosomal limiting membrane. Intriguingly, Ppt1-deficiency in Cln1-/- mice misrouted these proteins to the plasma membrane disrupting the lysosomal nutrient sensing scaffold. Despite this defect, mTORC1 was hyperactivated via the IGF1/PI3K/Akt-signaling pathway, which suppressed autophagy contributing to neuropathology. Importantly, pharmacological inhibition of PI3K/Akt suppressed mTORC1-activation, restored autophagy, and ameliorated neurodegeneration in Cln1-/- mice. Our findings reveal a previously unrecognized role of Cln1/Ppt1 in regulating mTORC1-activation and suggest that IGF1/PI3K/Akt may be a targetable pathway for CLN1 disease.

Keywords: CLN1-disease; IGF1-signaling; Lysosomal storage disease; Neurodegeneration; S-palmitoylation; mTORC1 activation

DOI: https://doi.org/10.1016/j.jbc.2024.105641

Sci China Life Sci. 2024 Jan 04.

Stay in touch with the endoplasmic reticulum.

Sha Sun, Gan Zhao, Mingkang Jia, Qing Jiang, Shulin Li, Haibin Wang, Wenjing Li, Yunyun Wang, Xin Bian, Yan G Zhao, Xun Huang, Ge Yang, Huaqing Cai, Jose C Pastor-Pareja, Liang Ge, Chuanmao Zhang, Junjie Hu.

The endoplasmic reticulum (ER), which is composed of a continuous network of tubules and sheets, forms the most widely distributed membrane system in eukaryotic cells. As a result, it engages a variety of organelles by establishing membrane contact sites (MCSs). These contacts regulate organelle positioning and remodeling, including fusion and fission, facilitate precise lipid exchange, and couple vital signaling events. Here, we systematically review recent advances and converging themes on ER-involved organellar contact. The molecular basis, cellular influence, and potential physiological functions for ER/nuclear envelope contacts with mitochondria, Golgi, endosomes, lysosomes, lipid droplets, autophagosomes, and plasma membrane are summarized.

Keywords: Golgi apparatus; autophagosome; endoplasmic reticulum; endosome; lipid droplets; lysosome; membrane contact site; mitochondria; nuclear envelope; plasma membrane

DOI: https://doi.org/10.1007/s11427-023-2443-9