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Autophagy Oct 2023Macroautophagy/autophagy is a regulated cellular degradation process essential as a pro-survival mechanism and integral to the regulation of diverse cellular processes...
Macroautophagy/autophagy is a regulated cellular degradation process essential as a pro-survival mechanism and integral to the regulation of diverse cellular processes in eukaryotes. During cellular stress and nutrient sensing, SQSTM1/p62 (sequestosome 1) functions as a key receptor for selective autophagy by shuttling ubiquitinated cargoes toward autophagic degradation making it a useful marker for monitoring autophagic flux. We present a straightforward and rapid flow cytometric assay for the quantitative measurement of intracellular SQSTM1 with improved sensitivity to conventional immunoblotting and with the benefit of higher throughput and reduced requirements for starting cellular materials for adequate analysis. We demonstrate that flow cytometry is able to detect similar trends in the measurement of intracellular SQSTM1 levels following serum starvation, genetic manipulations, and bafilomycin A/chloroquine treatments. The assays utilizes readily available reagents and equipment without the need for transfection and utilizes standard flow cytometry equipment. In the present studies, expression of reporter proteins was applied to a range of SQSTM1 expression levels generated by genetic and chemical manipulation in both mouse as well as human cells. In combination with appropriate controls and attention to cautionary issues, this assay offers the ability to assess an important measure of autophagic capacity and flux. ATG5: autophagy related 5 ATG7: autophagy related 7 BafA: bafilomycin A1 BMDM: bone marrow-derived macrophages CQ: chloroquine EBV: Epstein-Barr Virus EDTA: ethylenediaminetetraacetic acid FBS: fetal bovine serum gMFI: geometric mean fluorescent intensity HD: healthy donor MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3 MedianFI: median fluorescent intensity NTC: non-target control PBMC: peripheral blood mononuclear cells RPMI: Roswell Park Memorial Institution SQSTM1/p62: sequestosome 1 WT: wild type.
Topics: Humans; Mice; Animals; Sequestosome-1 Protein; Autophagy; Leukocytes, Mononuclear; Flow Cytometry; Epstein-Barr Virus Infections; Herpesvirus 4, Human; Transcription Factors
PubMed: 37335017
DOI: 10.1080/15548627.2023.2224074 -
Autophagy Aug 2023Macroautophagy/autophagy is an important process responsible for protein turnover and cell survival in amino acid-deprived conditions, especially for leucine (Leu). With...
Macroautophagy/autophagy is an important process responsible for protein turnover and cell survival in amino acid-deprived conditions, especially for leucine (Leu). With the dramatic advances in mass spectrometry, many new post-translational modifications (PTMs) have been identified. However, whether these PTMs regulate autophagy remains unclear. Here we found global lysine crotonylation levels are significantly upregulated during Leu deprivation-induced autophagy. A comprehensive crotonylome profiling showed that YWHA/14-3-3 proteins are significantly enriched in the Leu regulated-crotonylome. The inhibition of YWHAE/14-3-3ε crotonylation by mutating two crotonylated sites to arginine, K73R K78R, significantly attenuates autophagy induced by Leu deprivation. Molecular dynamics suggest that YWHAE K73 and K78 crotonylations decrease protein conformation and thermodynamic stability. Moreover, we found crotonylation of YWHAE releases PPM1B to dephosphorylate ULK1 and consequently activate autophagy. Decrotonylation of YWHAE is mediated by HDAC7 whose activity is inhibited significantly by Leu deprivation. Taken together, our finding reveals a critical role of YWHAE crotonylation in Leu deprivation-induced autophagy.
Topics: Leucine; 14-3-3 Proteins; Autophagy; Mass Spectrometry; Protein Processing, Post-Translational
PubMed: 36628438
DOI: 10.1080/15548627.2023.2166276 -
EMBO Reports Nov 2023Transcription factor EB (TFEB) is a basic helix-loop-helix leucine zipper transcription factor that acts as a master regulator of lysosomal biogenesis, lysosomal... (Review)
Review
Transcription factor EB (TFEB) is a basic helix-loop-helix leucine zipper transcription factor that acts as a master regulator of lysosomal biogenesis, lysosomal exocytosis, and macro-autophagy. TFEB contributes to a wide range of physiological functions, including mitochondrial biogenesis and innate and adaptive immunity. As such, TFEB is an essential component of cellular adaptation to stressors, ranging from nutrient deprivation to pathogenic invasion. The activity of TFEB depends on its subcellular localisation, turnover, and DNA-binding capacity, all of which are regulated at the post-translational level. Pathological states are characterised by a specific set of stressors, which elicit post-translational modifications that promote gain or loss of TFEB function in the affected tissue. In turn, the resulting increase or decrease in survival of the tissue in which TFEB is more or less active, respectively, may either benefit or harm the organism as a whole. In this way, the post-translational modifications of TFEB account for its otherwise paradoxical protective and deleterious effects on organismal fitness in diseases ranging from neurodegeneration to cancer. In this review, we describe how the intracellular environment characteristic of different diseases alters the post-translational modification profile of TFEB, enabling cellular adaptation to a particular pathological state.
Topics: Lysosomes; Protein Processing, Post-Translational; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
PubMed: 37728021
DOI: 10.15252/embr.202357574 -
Journal of Advanced Research May 2024Dysregulated alterations in organelle structure and function have a significant connection with cell death, as well as the occurrence and development of inflammatory... (Review)
Review
BACKGROUND
Dysregulated alterations in organelle structure and function have a significant connection with cell death, as well as the occurrence and development of inflammatory diseases. Maintaining cell viability and inhibiting the release of inflammatory cytokines are essential measures to treat inflammatory diseases. Recently, many studies have showed that autophagy selectively targets dysfunctional organelles, thereby sustaining the functional stability of organelles, alleviating the release of multiple cytokines, and maintaining organismal homeostasis. Organellophagy dysfunction is critically engaged in different kinds of cell death and inflammatory diseases.
AIM OF REVIEW
We summarized the current knowledge of organellophagy (e.g., mitophagy, reticulophagy, golgiphagy, lysophagy, pexophagy, nucleophagy, and ribophagy) and the underlying mechanisms by which organellophagy regulates cell death.
KEY SCIENTIFIC CONCEPTS OF REVIEW
We outlined the potential role of organellophagy in the modulation of cell fate during the inflammatory response to develop an intervention strategy for the organelle quality control in inflammatory diseases.
PubMed: 38740259
DOI: 10.1016/j.jare.2024.05.012 -
Cancers Nov 2023Rhabdomyosarcoma is a rare cancer arising in skeletal muscle that typically impacts children and young adults. It is a worldwide challenge in child health as treatment... (Review)
Review
Rhabdomyosarcoma is a rare cancer arising in skeletal muscle that typically impacts children and young adults. It is a worldwide challenge in child health as treatment outcomes for metastatic and recurrent disease still pose a major concern for both basic and clinical scientists. The treatment strategies for rhabdomyosarcoma include multi-agent chemotherapies after surgical resection with or without ionization radiotherapy. In this comprehensive review, we first provide a detailed clinical understanding of rhabdomyosarcoma including its classification and subtypes, diagnosis, and treatment strategies. Later, we focus on chemotherapy strategies for this childhood sarcoma and discuss the impact of three mechanisms that are involved in the chemotherapy response including apoptosis, macro-autophagy, and the unfolded protein response. Finally, we discuss in vivo mouse and zebrafish models and in vitro three-dimensional bioengineering models of rhabdomyosarcoma to screen future therapeutic approaches and promote muscle regeneration.
PubMed: 37958442
DOI: 10.3390/cancers15215269 -
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 -
Ageing Research Reviews Dec 2023Vascular ageing, characterized by structural and functional changes in blood vessels of which arterial stiffness and endothelial dysfunction are key components, is... (Review)
Review
Vascular ageing, characterized by structural and functional changes in blood vessels of which arterial stiffness and endothelial dysfunction are key components, is associated with increased risk of cardiovascular and other age-related diseases. As the global population continues to age, understanding the underlying mechanisms and developing effective therapeutic interventions to mitigate vascular ageing becomes crucial for improving cardiovascular health outcomes. Therefore, this review provides an overview of the current knowledge on pharmacological modulation of vascular ageing, highlighting key strategies and promising therapeutic targets. Several molecular pathways have been identified as central players in vascular ageing, including oxidative stress and inflammation, the renin-angiotensin-aldosterone system, cellular senescence, macroautophagy, extracellular matrix remodelling, calcification, and gasotransmitter-related signalling. Pharmacological and dietary interventions targeting these pathways have shown potential in ameliorating age-related vascular changes. Nevertheless, the development and application of drugs targeting vascular ageing is complicated by various inherent challenges and limitations, such as certain preclinical methodological considerations, interactions with exercise training and sex/gender-related differences, which should be taken into account. Overall, pharmacological modulation of endothelial dysfunction and arterial stiffness as hallmarks of vascular ageing, holds great promise for improving cardiovascular health in the ageing population. Nonetheless, further research is needed to fully elucidate the underlying mechanisms and optimize the efficacy and safety of these interventions for clinical translation.
Topics: Humans; Aging; Oxidative Stress; Cellular Senescence; Vascular Stiffness; Signal Transduction
PubMed: 37956927
DOI: 10.1016/j.arr.2023.102122 -
Nature Communications Aug 2023Organelles are shaped by curvature-generating proteins, which include the reticulons and REEPs that are involved in forming the endoplasmic reticulum (ER). A conserved...
Organelles are shaped by curvature-generating proteins, which include the reticulons and REEPs that are involved in forming the endoplasmic reticulum (ER). A conserved REEP subfamily differs from the ER-shaping REEPs in abundance and membrane topology and has unidentified functions. Here, we show that Rop1, the single member of this family in the fission yeast Schizosacharomyces pombe, is crucial for the macroautophagy of organelles and cytosolic proteins. Rop1 is needed for the formation of phagophores, cup-like structures consisting of two closely apposed membrane sheets that encapsulate cargo. It is recruited at early stages to phagophores and is required for their maturation into autophagosomes. Rop1 function relies on its ability to generate high membrane curvature and on its colocalization with the autophagy component Atg2 that is thought to reside at the phagophore rim. We propose that Rop1 facilitates the formation and growth of the double-membrane structure of the autophagosome.
Topics: Membrane Proteins; Autophagosomes; Schizosaccharomyces; Macroautophagy; Autophagy; Autophagy-Related Proteins
PubMed: 37553386
DOI: 10.1038/s41467-023-40530-4 -
Communications Biology Aug 2023Human WIPI β-propellers function as PI3P effectors in autophagy, with WIPI4 and WIPI3 being able to link autophagy control by AMPK and TORC1 to the formation of...
Human WIPI β-propellers function as PI3P effectors in autophagy, with WIPI4 and WIPI3 being able to link autophagy control by AMPK and TORC1 to the formation of autophagosomes. WIPI1, instead, assists WIPI2 in efficiently recruiting the ATG16L1 complex at the nascent autophagosome, which in turn promotes lipidation of LC3/GABARAP and autophagosome maturation. However, the specific role of WIPI1 and its regulation are unknown. Here, we discovered the ABL-ERK-MYC signalling axis controlling WIPI1. As a result of this signalling, MYC binds to the WIPI1 promoter and represses WIPI1 gene expression. When ABL-ERK-MYC signalling is counteracted, increased WIPI1 gene expression enhances the formation of autophagic membranes capable of migrating through tunnelling nanotubes to neighbouring cells with low autophagic activity. ABL-regulated WIPI1 function is relevant to lifespan control, as ABL deficiency in C. elegans increased gene expression of the WIPI1 orthologue ATG-18 and prolonged lifespan in a manner dependent on ATG-18. We propose that WIPI1 acts as an enhancer of autophagy that is physiologically relevant for regulating the level of autophagic activity over the lifespan.
Topics: Animals; Humans; Autophagosomes; Autophagy; Caenorhabditis elegans; Longevity; Macroautophagy; Proto-Oncogene Proteins c-abl
PubMed: 37620393
DOI: 10.1038/s42003-023-05236-9 -
Nature Communications Nov 2023Autophagosomes are double-membrane vesicles generated intracellularly to encapsulate substrates for lysosomal degradation during autophagy. Phase separated p62 body...
Autophagosomes are double-membrane vesicles generated intracellularly to encapsulate substrates for lysosomal degradation during autophagy. Phase separated p62 body plays pivotal roles during autophagosome formation, however, the underlying mechanisms are still not fully understood. Here we describe a spatial membrane gathering mode by which p62 body functions in autophagosome formation. Mass spectrometry-based proteomics reveals significant enrichment of vesicle trafficking components within p62 body. Combining cellular experiments and biochemical reconstitution assays, we confirm the gathering of ATG9 and ATG16L1-positive vesicles around p62 body, especially in Atg2ab DKO cells with blocked lipid transfer and vesicle fusion. Interestingly, p62 body also regulates ATG9 and ATG16L vesicle trafficking flux intracellularly. We further determine the lipid contents associated with p62 body via lipidomic profiling. Moreover, with in vitro kinase assay, we uncover the functions of p62 body as a platform to assemble ULK1 complex and invigorate PI3KC3-C1 kinase cascade for PI3P generation. Collectively, our study raises a membrane-based working model for multifaceted p62 body in controlling autophagosome biogenesis, and highlights the interplay between membraneless condensates and membrane vesicles in regulating cellular functions.
Topics: Autophagosomes; Autophagy; Macroautophagy; Phagosomes; Autophagy-Related Proteins; Lipids
PubMed: 37957156
DOI: 10.1038/s41467-023-42829-8