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Annual Review of Cell and Developmental... Oct 2023Every eukaryotic cell contains two distinct multisubunit protein kinase complexes that each contain a TOR (target of rapamycin) protein as the catalytic subunit. These... (Review)
Review
Every eukaryotic cell contains two distinct multisubunit protein kinase complexes that each contain a TOR (target of rapamycin) protein as the catalytic subunit. These ensembles, designated TORC1 and TORC2, serve as nutrient and stress sensors, signal integrators, and regulators of cell growth and homeostasis, but they differ in their composition, localization, and function. TORC1, activated on the cytosolic surface of the vacuole (or, in mammalian cells, on the cytosolic surface of the lysosome), promotes biosynthesis and suppresses autophagy. TORC2, located primarily at the plasma membrane (PM), maintains the proper levels and bilayer distribution of all PM components (sphingolipids, glycerophospholipids, sterols, and integral membrane proteins), which are needed for the membrane expansion that accompanies cell growth and division and for combating insults to PM integrity. This review summarizes our current understanding of the assembly, structural features, subcellular distribution, and function and regulation of TORC2, obtained largely through studies conducted with .
PubMed: 37339679
DOI: 10.1146/annurev-cellbio-011723-030346 -
Journal of Experimental Botany Oct 2023In response to changing environmental conditions, plants activate cellular responses to enable them to adapt. One such response is autophagy, in which cellular... (Review)
Review
In response to changing environmental conditions, plants activate cellular responses to enable them to adapt. One such response is autophagy, in which cellular components, for example proteins and organelles, are delivered to the vacuole for degradation. Autophagy is activated by a wide range of conditions, and the regulatory pathways controlling this activation are now being elucidated. However, key aspects of how these factors may function together to properly modulate autophagy in response to specific internal or external signals are yet to be discovered. In this review we discuss mechanisms for regulation of autophagy in response to environmental stress and disruptions in cell homeostasis. These pathways include post-translational modification of proteins required for autophagy activation and progression, control of protein stability of the autophagy machinery, and transcriptional regulation, resulting in changes in transcription of genes involved in autophagy. In particular, we highlight potential connections between the roles of key regulators and explore gaps in research, the filling of which can further our understanding of the autophagy regulatory network in plants.
Topics: Autophagy; Gene Expression Regulation; Protein Processing, Post-Translational; Homeostasis; Vacuoles
PubMed: 37358252
DOI: 10.1093/jxb/erad211 -
MBio Oct 2023Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. , the causative agent, has perfected many ways to replicate...
Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. , the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the / system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as .
Topics: Humans; Vacuoles; Dictyostelium; Endoplasmic Reticulum; Mycobacterium marinum; Mycobacterium tuberculosis; Tuberculosis
PubMed: 37676004
DOI: 10.1128/mbio.00943-23 -
Scientific Reports Oct 2023Vesicular transport driven by membrane trafficking systems conserved in eukaryotes is critical to cellular functionality and homeostasis. It is known that homotypic...
Vesicular transport driven by membrane trafficking systems conserved in eukaryotes is critical to cellular functionality and homeostasis. It is known that homotypic fusion and vacuole protein sorting (HOPS) and class C core endosomal vacuole tethering (CORVET) interact with Rab-GTPases and SNARE proteins to regulate vesicle transport, fusion, and maturation in autophagy and endocytosis pathways. In this study, we identified two novel "Hybrid" tethering complexes in mammalian cells in which one of the subunits of HOPS or CORVET is replaced with the subunit from the other. Substrates taken up by receptor-mediated endocytosis or pinocytosis were transported by distinctive pathways, and the newly identified hybrid complexes contributed to pinocytosis in the presence of HOPS, whereas receptor-mediated endocytosis was exclusively dependent on HOPS. Our study provides new insights into the molecular mechanisms of the endocytic pathway and the function of the vacuolar protein sorting-associated (VPS) protein family.
Topics: Animals; Vacuoles; Vesicular Transport Proteins; Endosomes; Endocytosis; SNARE Proteins; Membrane Fusion; Saccharomyces cerevisiae Proteins; Mammals
PubMed: 37907479
DOI: 10.1038/s41598-023-45418-3 -
Cancer Letters Nov 2023Rigosertib (RGS) is a benzyl styryl sulfone which exhibits impressive cytotoxicity in cancer cells. However, its modulating effect on tumor immune microenvironment...
Rigosertib (RGS) is a benzyl styryl sulfone which exhibits impressive cytotoxicity in cancer cells. However, its modulating effect on tumor immune microenvironment remains elusive. In our experiments, compared with immunodeficient mouse model, increased tumor growth arrest and robust anti-tumor immunity were observed in RGS-treated colorectal cancer (CRC) isograft tumors in immunocompetent mice. Intriguingly, RGS markedly down-regulated programmed cell death ligand 1 (PD-L1) expression in both vivo and in vitro. Meanwhile, RGS increased autophagic vacuole number in CRC cells as seen by transmission electron microscopy and immunofluorescence. Moreover, increased LC3-II level and tandem-mRFP- GFP- LC3 labeled vacuole accumulation demonstrated RGS-induced autophagic flux. Mechanistically, it is the activation of AMP-activated protein kinase-UNC-51-like kinase 1 (AMPK-ULK1) axis, rather than the canonical mTOR signaling pathway, that plays a pivotal role in RGS-induced autophagy. AMPK-ULK1 dependent autophagy inhibition, by either short interfering RNA or chemical inhibitors, blocked RGS-induced PD-L1 degradation. Finally, RGS exhibited synergistic anti-tumor activity with cytotoxic T-lymphocyte-associated protein 4 monoclonal antibody in the CRC isograft model. Furthermore, apart from the immunomodulatory effect, we also confirmed the direct cytotoxicity of RGS in inducing mitochondria-related apoptosis. Altogether, considering its PD-L1 inhibitory and cytotoxic effects, RGS could be a promising drug for CRC therapy.
Topics: Animals; Mice; AMP-Activated Protein Kinases; Autophagy; B7-H1 Antigen; Colorectal Neoplasms; Sulfones; Tumor Microenvironment
PubMed: 37805162
DOI: 10.1016/j.canlet.2023.216422 -
Acta Myologica : Myopathies and... 2023Massive parallel sequencing methods, such as exome, genome, and targeted DNA sequencing, have aided molecular diagnosis of genetic diseases in the last 20 years.... (Review)
Review
Massive parallel sequencing methods, such as exome, genome, and targeted DNA sequencing, have aided molecular diagnosis of genetic diseases in the last 20 years. However, short-read sequencing methods still have several limitations, such inaccurate genome assembly, the inability to detect large structural variants, and variants located in hard-to-sequence regions like highly repetitive areas. The recently emerged PacBio single-molecule real-time (SMRT) and Oxford nanopore technology (ONT) long-read sequencing (LRS) methods have been shown to overcome most of these technical issues, leading to an increase in diagnostic rate. LRS methods are contributing to the detection of repeat expansions in novel disease-causing genes (e.g., , and causing an Oculopharyngodistal myopathy or causing a Myopathy with rimmed ubiquitin-positive autophagic vacuolation), of structural variants (e.g., in ), and of single nucleotide variants in repetitive regions ( and ). Moreover, these methods have simplified the characterization of the D4Z4 repeats in , facilitating the diagnosis of Facioscapulohumeral muscular dystrophy (FSHD). We review recent studies that have used either ONT or PacBio SMRT sequencing methods and discuss different types of variants that have been detected using these approaches in individuals with neuromuscular disorders.
Topics: Humans; Sequence Analysis, DNA; Muscular Dystrophy, Facioscapulohumeral; Repetitive Sequences, Nucleic Acid; High-Throughput Nucleotide Sequencing
PubMed: 38406378
DOI: 10.36185/2532-1900-394 -
Trends in Cell Biology Dec 2023Autophagy is a self-catabolic process through which cellular components are delivered to lysosomes for degradation. There are three types of autophagy, i.e.,... (Review)
Review
Autophagy is a self-catabolic process through which cellular components are delivered to lysosomes for degradation. There are three types of autophagy, i.e., macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy. In macroautophagy, a portion of the cytoplasm is wrapped by the autophagosome, which then fuses with lysosomes and delivers the engulfed cytoplasm for degradation. In CMA, the translocation of cytosolic substrates to the lysosomal lumen is directly across the limiting membrane of lysosomes. In microautophagy, lytic organelles, including endosomes or lysosomes, take up a portion of the cytoplasm directly. Although macroautophagy has been investigated extensively, microautophagy has received much less attention. Nonetheless, it has become evident that microautophagy plays a variety of cellular roles from yeast to mammals. Here we review the very recent updates of microautophagy. In particular, we focus on the feature of the degradative substrates and the molecular machinery that mediates microautophagy.
PubMed: 38104013
DOI: 10.1016/j.tcb.2023.11.005 -
Nature Communications Oct 2023The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for autophagosome-lysosome fusion in mammals, yet...
The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for autophagosome-lysosome fusion in mammals, yet reconstituting the mammalian HOPS complex remains a challenge. Here we propose a "hook-up" model for mammalian HOPS complex assembly, which requires two HOPS sub-complexes docking on membranes via membrane-associated Rabs. We identify Rab39A as a key small GTPase that recruits HOPS onto autophagic vesicles. Proper pairing with Rab2 and Rab39A enables HOPS complex assembly between proteoliposomes for its tethering function, facilitating efficient membrane fusion. GTP loading of Rab39A is important for the recruitment of HOPS to autophagic membranes. Activation of Rab39A is catalyzed by C9orf72, a guanine exchange factor associated with amyotrophic lateral sclerosis and familial frontotemporal dementia. Constitutive activation of Rab39A can rescue autophagy defects caused by C9orf72 depletion. These results therefore reveal a crucial role for the C9orf72-Rab39A-HOPS axis in autophagosome-lysosome fusion.
Topics: Animals; Autophagy; C9orf72 Protein; Catalysis; Guanosine Triphosphate; Mammals; Membrane Fusion; Vacuoles
PubMed: 37821429
DOI: 10.1038/s41467-023-42003-0 -
Frontiers in Plant Science 2023The ras gene from rat brain (RAB) family of small GTPases is highly conserved among eukaryotes and regulates endomembrane trafficking pathways. RAB7, in particular, has... (Review)
Review
The ras gene from rat brain (RAB) family of small GTPases is highly conserved among eukaryotes and regulates endomembrane trafficking pathways. RAB7, in particular, has been linked to various processes involved in regulating endocytic and autophagic pathways. Plants have several copies of RAB7 proteins that reflect the intricacy of their endomembrane transport systems. RAB7 activity regulates different pathways of endomembrane trafficking in plants: (1) endocytic traffic to the vacuole; (2) biosynthetic traffic to the vacuole; and (3) recycling from the late endosome to the secretory pathway. During certain developmental and stress related processes another pathway becomes activated (4) autophagic trafficking towards the vacuole that is also regulated by RAB7. RAB7s carry out these functions by interacting with various effector proteins. Current research reveals many unexplored RAB7 functions in connection with stress responses. Thus, this review describes a comprehensive summary of current knowledge of plant RAB7's functions, discusses unresolved challenges, and recommends prospective future research directions.
PubMed: 37662169
DOI: 10.3389/fpls.2023.1240973 -
Nature Metabolism Oct 2023Amino acid homeostasis is critical for many cellular processes. It is well established that amino acids are compartmentalized using pH gradients generated between...
Amino acid homeostasis is critical for many cellular processes. It is well established that amino acids are compartmentalized using pH gradients generated between organelles and the cytoplasm; however, the dynamics of this partitioning has not been explored. Here we develop a highly sensitive pH reporter and find that the major amino acid storage compartment in Saccharomyces cerevisiae, the lysosome-like vacuole, alkalinizes before cell division and re-acidifies as cells divide. The vacuolar pH dynamics require the uptake of extracellular amino acids and activity of TORC1, the v-ATPase and the cycling of the vacuolar specific lipid phosphatidylinositol 3,5-bisphosphate, which is regulated by the cyclin-dependent kinase Pho85 (CDK5 in mammals). Vacuolar pH regulation enables amino acid sequestration and mobilization from the organelle, which is important for mitochondrial function, ribosome homeostasis and cell size control. Collectively, our data provide a new paradigm for the use of dynamic pH-dependent amino acid compartmentalization during cell growth/division.
Topics: Animals; Vacuoles; Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae; Homeostasis; Amino Acids; Cell Division; Cell Cycle; Hydrogen-Ion Concentration; Mammals
PubMed: 37640943
DOI: 10.1038/s42255-023-00872-1