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The Journal of Biological Chemistry Jan 2024The yeast vacuole membrane can phase separate into ordered and disordered domains, a phenomenon that is required for micro-lipophagy under nutrient limitation. Despite...
The yeast vacuole membrane can phase separate into ordered and disordered domains, a phenomenon that is required for micro-lipophagy under nutrient limitation. Despite its importance as a biophysical model and physiological significance, it is not yet resolved if specific lipidome changes drive vacuole phase separation. Here we report that the metabolism of sphingolipids (SLs) and their sorting into the vacuole membrane can control this process. We first developed a vacuole isolation method to identify lipidome changes during the onset of phase separation in early stationary stage cells. We found that early stationary stage vacuoles are defined by an increased abundance of putative raft components, including 40% higher ergosterol content and a nearly 3-fold enrichment in complex SLs (CSLs). These changes were not found in the corresponding whole cell lipidomes, indicating that lipid sorting is associated with domain formation. Several facets of SL composition-headgroup stoichiometry, longer chain lengths, and increased hydroxylations-were also markers of phase-separated vacuole lipidomes. To test SL function in vacuole phase separation, we carried out a systematic genetic dissection of their biosynthetic pathway. The abundance of CSLs controlled the extent of domain formation and associated micro-lipophagy processes, while their headgroup composition altered domain morphology. These results suggest that lipid trafficking can drive membrane phase separation in vivo and identify SLs as key mediators of this process in yeast.
Topics: Membranes; Phase Separation; Saccharomyces cerevisiae; Sphingolipids; Vacuoles; Lipidomics; Microscopy, Fluorescence
PubMed: 38013088
DOI: 10.1016/j.jbc.2023.105496 -
EMBO Reports Dec 2023Lysosomes are degradative organelles and signaling hubs that maintain cell and tissue homeostasis, and lysosomal dysfunction is implicated in aging and reduced...
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.
Topics: Animals; Humans; Microautophagy; Caenorhabditis elegans; Lysosomes; Intracellular Membranes; Endosomal Sorting Complexes Required for Transport; Autophagy; Microtubule-Associated Proteins; Apoptosis Regulatory Proteins; Protein Serine-Threonine Kinases
PubMed: 37987447
DOI: 10.15252/embr.202357300 -
Neuroscience Bulletin Mar 2024Autophagy involves the sequestration and delivery of cytoplasmic materials to lysosomes, where proteins, lipids, and organelles are degraded and recycled. According to... (Review)
Review
Autophagy involves the sequestration and delivery of cytoplasmic materials to lysosomes, where proteins, lipids, and organelles are degraded and recycled. According to the way the cytoplasmic components are engulfed, autophagy can be divided into macroautophagy, microautophagy, and chaperone-mediated autophagy. Recently, many studies have found that autophagy plays an important role in neurological diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, neuronal excitotoxicity, and cerebral ischemia. Autophagy maintains cell homeostasis in the nervous system via degradation of misfolded proteins, elimination of damaged organelles, and regulation of apoptosis and inflammation. AMPK-mTOR, Beclin 1, TP53, endoplasmic reticulum stress, and other signal pathways are involved in the regulation of autophagy and can be used as potential therapeutic targets for neurological diseases. Here, we discuss the role, functions, and signal pathways of autophagy in neurological diseases, which will shed light on the pathogenic mechanisms of neurological diseases and suggest novel targets for therapies.
Topics: Humans; Autophagy; Nervous System Diseases; Parkinson Disease; Alzheimer Disease; Huntington Disease
PubMed: 37856037
DOI: 10.1007/s12264-023-01120-y -
International Journal of Molecular... Oct 2023Autophagy is a lysosomal degradation process known as autophagic flux, involving the engulfment of damaged proteins and organelles by double-membrane autophagosomes. It... (Review)
Review
Autophagy is a lysosomal degradation process known as autophagic flux, involving the engulfment of damaged proteins and organelles by double-membrane autophagosomes. It comprises microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. Macroautophagy consists of three stages: induction, autophagosome formation, and autolysosome formation. Atg8-family proteins are valuable for tracking autophagic structures and have been widely utilized for monitoring autophagy. The conversion of LC3 to its lipidated form, LC3-II, served as an indicator of autophagy. Autophagy is implicated in human pathophysiology, such as neurodegeneration, cancer, and immune disorders. Moreover, autophagy impacts urological diseases, such as interstitial cystitis /bladder pain syndrome (IC/BPS), ketamine-induced ulcerative cystitis (KIC), chemotherapy-induced cystitis (CIC), radiation cystitis (RC), erectile dysfunction (ED), bladder outlet obstruction (BOO), prostate cancer, bladder cancer, renal cancer, testicular cancer, and penile cancer. Autophagy plays a dual role in the management of urologic diseases, and the identification of potential biomarkers associated with autophagy is a crucial step towards a deeper understanding of its role in these diseases. Methods for monitoring autophagy include TEM, Western blot, immunofluorescence, flow cytometry, and genetic tools. Autophagosome and autolysosome structures are discerned via TEM. Western blot, immunofluorescence, northern blot, and RT-PCR assess protein/mRNA levels. Luciferase assay tracks flux; GFP-LC3 transgenic mice aid study. Knockdown methods (miRNA and RNAi) offer insights. This article extensively examines autophagy's molecular mechanism, pharmacological regulation, and therapeutic application involvement in urological diseases.
Topics: Animals; Male; Mice; Humans; Testicular Neoplasms; Autophagy; Autophagosomes; Autophagy-Related Protein 8 Family; Mice, Transgenic; Cystitis; Microtubule-Associated Proteins; Lysosomes
PubMed: 37834333
DOI: 10.3390/ijms241914887 -
Aging Oct 2023Aging is inevitable and affects all cell types, thus yeast cells are often used as a model in aging studies. There are two approaches to studying aging in yeast:...
Aging is inevitable and affects all cell types, thus yeast cells are often used as a model in aging studies. There are two approaches to studying aging in yeast: replicative aging, which describes the proliferative potential of cells, and chronological aging, which is used for studying post-mitotic cells. While analyzing the chronological lifespan (CLS) of diploid cells, we discovered a remarkable phenomenon: ploidy reduction during aging progression. To uncover the mechanism behind this unusual process we used yeast strains undergoing a CLS assay, looking for various aging parameters. Cell mortality, regrowth ability, autophagy induction and cellular DNA content measurements indicated that during the CLS assay, dying cells lost their DNA, and only diploids survived. We demonstrated that autophagy was responsible for the gradual loss of DNA. The nucleophagy marker activation at the start of the CLS experiment correlated with the significant drop in cell viability. The activation of piecemeal microautophagy of nucleus (PMN) markers appeared to accompany the chronological aging process until the end. Our findings emphasize the significance of maintaining at least one intact copy of the genome for the survival of post-mitotic diploid cells. During chronological aging, cellular components, including DNA, are exposed to increasing stress, leading to DNA damage and fragmentation in aging cells. We propose that PMN-dependent clearance of damaged DNA from the nucleus helps prevent genome rearrangements. However, as long as one copy of the genome can be rebuilt, cells can still survive.
Topics: Saccharomyces cerevisiae; Diploidy; DNA; Autophagy
PubMed: 37815879
DOI: 10.18632/aging.205102 -
International Journal of Molecular... Sep 2023The heat shock factor 1 (HSF1)-mediated stress response pathway and autophagy processes play important roles in the maintenance of proteostasis. Autophagy processes are... (Review)
Review
The heat shock factor 1 (HSF1)-mediated stress response pathway and autophagy processes play important roles in the maintenance of proteostasis. Autophagy processes are subdivided into three subtypes: macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy. Recently, molecular chaperones and co-factors were shown to be involved in the selective degradation of substrates by these three autophagy processes. This evidence suggests that autophagy processes are regulated in a coordinated manner by the HSF1-mediated stress response pathway. Recently, various studies have demonstrated that proteostasis pathways including HSF1 and autophagy are implicated in longevity. Furthermore, they serve as therapeutic targets for aging-related diseases such as cancer and neurodegenerative diseases. In the future, these studies will underpin the development of therapies against various diseases.
Topics: Autophagy; Macroautophagy; Chaperone-Mediated Autophagy; Microautophagy; Longevity
PubMed: 37762105
DOI: 10.3390/ijms241813804 -
Molecular Neurobiology Mar 2024Autophagy is a conservative self-degradation system, which includes the two major processes of enveloping abnormal proteins, organelles and other macromolecules, and... (Review)
Review
Autophagy is a conservative self-degradation system, which includes the two major processes of enveloping abnormal proteins, organelles and other macromolecules, and transferring them into lysosomes for the subsequent degradation. It holds the stability of the intracellular environment under stress. So far, three types of autophagy have been found: microautophagy, chaperone-mediated autophagy and macroautophagy. Many diseases have the pathological process of autophagy dysfunction, such as nervous system diseases. Pyroptosis is one kind of programmed cell death mediated by gasdermin (GSDM). In this process of pyroptosis, the activated caspase-3, caspase-4/5/11, or caspase-1 cleaves GSDM into the N-terminal pore-forming domain (PFD). The oligomer of PFD combines with the cell membrane to form membrane holes, thus leading to pyroptosis. Pyroptosis plays a key role in multiple tissues and organs. Many studies have revealed that autophagy and pyroptosis participate in the nervous system, but the mechanisms need to be fully clarified. Here, we focused on the recent articles on the role and mechanism of pyroptosis and autophagy in the pathological processes of the nervous system.
Topics: Pyroptosis; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Autophagy; Nervous System; Caspases
PubMed: 37697221
DOI: 10.1007/s12035-023-03614-2 -
BioRxiv : the Preprint Server For... Aug 2023The Ccr4-Not complex containing the Not4 ubiquitin ligase regulates gene transcription and mRNA decay, yet it also has poorly defined roles in translation, proteostasis,...
The Ccr4-Not complex containing the Not4 ubiquitin ligase regulates gene transcription and mRNA decay, yet it also has poorly defined roles in translation, proteostasis, and endolysosomal-dependent nutrient signaling. To define how Ccr4-Not mediated ubiquitin signaling regulates these additional processes, we performed quantitative proteomics in the yeast lacking the Not4 ubiquitin ligase, and also in cells overexpressing either wild-type or functionally inactive ligase. Herein, we provide evidence that both increased and decreased Ccr4-Not ubiquitin signaling disrupts ribosomal protein (RP) homeostasis independently of reduced RP mRNA changes or reductions in known Not4 ribosomal substrates. Surprisingly, we also find that both Not4-mediated ubiquitin signaling, and the Ccr4 subunit, actively inhibit 40S ribosomal autophagy. This 40S autophagy is independent of canonical Atg7-dependent macroautophagy, thus indicating microautophagy activation is responsible. Furthermore, the Not4 ligase genetically interacts with endolysosomal pathway effectors to control both RP expression and 40S autophagy efficiency. Overall, we demonstrate that balanced Ccr4-Not ligase activity maintains RP homeostasis, and that Ccr4-Not ubiquitin signaling interacts with the endolysosomal pathway to both regulate RP expression and inhibit 40S ribosomal autophagy.
PubMed: 37693548
DOI: 10.1101/2023.08.28.555095 -
Science Advances Sep 2023Vitamin B is a vital micronutrient across cell types and tissues, and dysregulated B levels contribute to human disease. Despite its importance, how B vitamer levels are...
Vitamin B is a vital micronutrient across cell types and tissues, and dysregulated B levels contribute to human disease. Despite its importance, how B vitamer levels are regulated is not well understood. Here, we provide evidence that B dynamics are rapidly tuned by precise compartmentation of pyridoxal kinase (PDXK), the rate-limiting B enzyme. We show that canonical Wnt rapidly led to the accumulation of inactive B by shunting cytosolic PDXK into lysosomes. PDXK was modified with methyl-arginine Degron (MrDegron), a protein tag for lysosomes, which enabled delivery via microautophagy. Hyperactive lysosomes resulted in the continuous degradation of PDXK and B deficiency that promoted proliferation in Wnt-driven colorectal cancer (CRC) cells. Pharmacological or genetic disruption of the coordinated MrDegron proteolytic pathway was sufficient to reduce CRC survival in cells and organoid models. In sum, this work contributes to the repertoire of micronutrient-regulated processes that enable cancer cell growth and provides insight into the functional impact of B deficiencies for survival.
Topics: Humans; Vitamin B 6; Proteolysis; Peptide Hydrolases; Micronutrients; Vitamins
PubMed: 37682999
DOI: 10.1126/sciadv.adi2232 -
MedComm Oct 2023Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway that eliminates substrate proteins through heat-shock cognate protein 70 recognition and... (Review)
Review
Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway that eliminates substrate proteins through heat-shock cognate protein 70 recognition and lysosome-associated membrane protein type 2A-assisted translocation. It is distinct from macroautophagy and microautophagy. In recent years, the regulatory mechanisms of CMA have been gradually enriched, including the newly discovered NRF2 and p38-TFEB signaling, as positive and negative regulatory pathways of CMA, respectively. Normal CMA activity is involved in the regulation of metabolism, aging, immunity, cell cycle, and other physiological processes, while CMA dysfunction may be involved in the occurrence of neurodegenerative disorders, tumors, intestinal disorders, atherosclerosis, and so on, which provides potential targets for the treatment and prediction of related diseases. This article describes the general process of CMA and its role in physiological activities and summarizes the connection between CMA and macroautophagy. In addition, human diseases that concern the dysfunction or protective role of CMA are discussed. Our review deepens the understanding of the mechanisms and physiological functions of CMA and provides a summary of past CMA research and a vision of future directions.
PubMed: 37655052
DOI: 10.1002/mco2.347