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Antiviral Research Nov 2022Naturally evolved immune-escape PreS2 mutant is an oncogenic caveat of liver cirrhosis and hepatocellular carcinoma (HCC) during chronic hepatitis B virus (HBV)...
Naturally evolved immune-escape PreS2 mutant is an oncogenic caveat of liver cirrhosis and hepatocellular carcinoma (HCC) during chronic hepatitis B virus (HBV) infection. PreS2 mutant is prevalent in above 50% of patients with HCC. In addition, intrahepatic expression of PreS2 mutant large surface antigen (PreS2-LHBS) induces endoplasmic reticulum stress, mitochondria dysfunction, cytokinesis failure, and subsequent chromosome hyperploidy. As PreS2-LHBS has no enzymatic activity, the development of PreS2-specific inhibitors can be challenging. In this study, we aim to identify inhibitors of PreS2-LHBS via the induction of protein-specific degradation. We set up a large-scale protein stability reporter platform and applied an FDA-approved drug library for the screening. We identified ABT199 as a negative modulator of PreS2-LHBS, which induced the degradation of PreS2-LHBS without affecting the general cell viability in both hepatoma and immortalized hepatocytes. Next, by affinity purification screening, we found that PreS2-LHBS interacted with HSC70, a microautophagy mediating chaperone. Simultaneously, inhibitions of lysosomal degradation or microautophagy restored the expression of PreS2-LHBS, suggesting microautophagy is involved in ABT199-induced PreS2-LHBS degradation. Notably, a 24-hr treatment of ABT199 was sufficient for the reduction of DNA damage and cytokinesis failure in PreS2-LHBS expressing hepatocytes. In addition, a persistent treatment of ABT199 for 3 weeks reversed chromosome hyperploidy in PreS2-LHBS cells and suppressed anchorage-independent growth of HBV-positive hepatoma cells. Together, this study identified ABT-199 as a negative modulator of PreS2-LHBS via mediating microautophagy. Our results indicate that long-term inhibition of PreS2-LHBS may serve as a novel strategy for the therapeutic prevention of HBV-mediated HCC.
Topics: Antigens, Surface; Carcinoma, Hepatocellular; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Humans; Liver Neoplasms; Microautophagy
PubMed: 36122619
DOI: 10.1016/j.antiviral.2022.105417 -
Aging Cell Oct 2022Autophagy is essential for protein quality control and regulation of the functional proteome. Failure of autophagy pathways with age contributes to loss of proteostasis...
Autophagy is essential for protein quality control and regulation of the functional proteome. Failure of autophagy pathways with age contributes to loss of proteostasis in aged organisms and accelerates the progression of age-related diseases. In this work, we show that activity of endosomal microautophagy (eMI), a selective type of autophagy occurring in late endosomes, declines with age and identify the sub-proteome affected by this loss of function. Proteomics of late endosomes from old mice revealed an aberrant glycation signature for Hsc70, the chaperone responsible for substrate targeting to eMI. Age-related Hsc70 glycation reduces its stability in late endosomes by favoring its organization into high molecular weight protein complexes and promoting its internalization/degradation inside late endosomes. Reduction of eMI with age associates with an increase in protein secretion, as late endosomes can release protein-loaded exosomes upon plasma membrane fusion. Our search for molecular mediators of the eMI/secretion switch identified the exocyst-RalA complex, known for its role in exocytosis, as a novel physiological eMI inhibitor that interacts with Hsc70 and acts directly at the late endosome membrane. This inhibitory function along with the higher exocyst-RalA complex levels detected in late endosomes from old mice could explain, at least in part, reduced eMI activity with age. Interaction of Hsc70 with components of the exocyst-RalA complex places this chaperone in the switch from eMI to secretion. Reduced intracellular degradation in favor of extracellular release of undegraded material with age may be relevant to the spreading of proteotoxicity associated with aging and progression of proteinopathies.
Topics: Aging; Animals; Autophagy; Endosomes; Lysosomes; Mice; Microautophagy; Protein Transport; Proteome
PubMed: 36116133
DOI: 10.1111/acel.13713 -
The Journal of Cell Biology Oct 2022Liquid-liquid phase separation (LLPS) triages protein cargoes for autophagic degradation. In this issue, Ohshima et al. (2022. J. Cell...
Liquid-liquid phase separation (LLPS) triages protein cargoes for autophagic degradation. In this issue, Ohshima et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202203102) demonstrate that the autophagy receptor NCOA4 interacts with ferritin particles to form liquid-like condensates via LLPS. The NCOA4-ferritin condensates are delivered to lysosomes for degradation via either canonical macroautophagy or endosomal microautophagy to maintain intracellular iron homeostasis.
Topics: Autophagy; Ferritins; Iron; Lysosomes; Nuclear Receptor Coactivators
PubMed: 36112419
DOI: 10.1083/jcb.202209004 -
The Journal of Cell Biology Oct 2022A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to...
A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to lysosomes to release iron ions. Two transport pathways have been reported: macroautophagy and ESCRT-dependent endosomal microautophagy. Although the membrane dynamics of these pathways differ, both require NCOA4, which is thought to be an autophagy receptor for ferritin. However, it is unclear whether NCOA4 only acts as an autophagy receptor in ferritin degradation. Here, we found that ferritin particles form liquid-like condensates in a NCOA4-dependent manner. Homodimerization of NCOA4 and interaction between FTH1 and NCOA4 (i.e., multivalent interactions between ferritin particles and NCOA4) were required for the formation of ferritin condensates. Disruption of these interactions impaired ferritin degradation. Time-lapse imaging and three-dimensional correlative light and electron microscopy revealed that these ferritin-NCOA4 condensates were directly engulfed by autophagosomes and endosomes. In contrast, TAX1BP1 was not required for the formation of ferritin-NCOA4 condensates but was required for their incorporation into autophagosomes and endosomes. These results suggest that NCOA4 acts not only as a canonical autophagy receptor but also as a driver to form ferritin condensates to facilitate the degradation of these condensates by macroautophagy (i.e., macroferritinophagy) and endosomal microautophagy (i.e., microferritinophagy).
Topics: Autophagy; Endosomes; Ferritins; Iron; Lysosomes; Nuclear Receptor Coactivators; Phagosomes; Transcription Factors
PubMed: 36066504
DOI: 10.1083/jcb.202203102 -
Antioxidants & Redox Signaling Mar 2023Autophagy is critical to cellular homeostasis. Emergence of the concept of regulated necrosis, such as necroptosis, ferroptosis, pyroptosis, and mitochondrial... (Review)
Review
Autophagy is critical to cellular homeostasis. Emergence of the concept of regulated necrosis, such as necroptosis, ferroptosis, pyroptosis, and mitochondrial membrane-permeability transition (MPT)-derived necrosis, has revolutionized the research into necrosis. Both altered autophagy and regulated necrosis contribute to major human diseases. Recent studies reveal an intricate interplay between autophagy and regulated necrosis. Understanding the interplay at the molecular level will provide new insights into the pathophysiology of related diseases. Among the three forms of autophagy, macroautophagy is better studied for its crosstalk with regulated necrosis. Macroautophagy seemingly can either antagonize or promote regulated necrosis, depending upon the form of regulated necrosis, the type of cells or stimuli, and other cellular contexts. This review will critically analyze recent advances in the molecular mechanisms governing the intricate dialogues between macroautophagy and main forms of regulated necrosis. The dual roles of autophagy, either pro-survival or pro-death characteristics, intricate the mechanistic relationship between autophagy and regulated necrosis at molecular level in various pathological conditions. Meanwhile, key components of regulated necrosis are also involved in the regulation of autophagy, which further complicates the interrelationship. Resolving the controversies over causation between altered autophagy and a specific form of regulated necrosis requires approaches that are more definitive, where rigorous evaluation of autophagic flux and the development of more reliable and specific methods to quantify each form of necrosis will be essential. The relationship between chaperone-mediated autophagy or microautophagy and regulated necrosis remains largely unstudied. 38, 550-580.
Topics: Humans; Apoptosis; Necrosis; Pyroptosis; Ferroptosis; Autophagy
PubMed: 36053716
DOI: 10.1089/ars.2022.0110 -
Molecular Biology of the Cell Nov 2022Huntington's disease is characterized by accumulation of the aggregation-prone mutant Huntingtin (mHTT) protein. Here, we show that expression of exon 1 of mHTT in mouse...
Huntington's disease is characterized by accumulation of the aggregation-prone mutant Huntingtin (mHTT) protein. Here, we show that expression of exon 1 of mHTT in mouse cultured cells activates IRE1, the transmembrane sensor of stress in the endoplasmic reticulum, leading to degradation of the mRNA and repositioning of lysosomes and late endosomes toward the microtubule organizing center. Overriding degradation results in excessive accumulation of mHTT aggregates in both cultured cells and primary neurons. Although mHTT is degraded by macroautophagy when highly expressed, we show that before the formation of large aggregates, mHTT is degraded via an ESCRT-dependent, macroautophagy-independent pathway consistent with endosomal microautophagy. This pathway is enhanced by degradation and appears to protect cells from a toxic, less aggregated form of mHTT.
Topics: Animals; Endoribonucleases; Endosomal Sorting Complexes Required for Transport; Huntingtin Protein; Mice; Protein Aggregates; Protein Serine-Threonine Kinases; RNA, Messenger
PubMed: 36044348
DOI: 10.1091/mbc.E22-07-0281 -
Autophagy Sep 2022Exosomes are a subtype of extracellular vesicles (EVs), released by all cell types, that originate from the invagination of the endosomal limiting membrane. These EVs...
Exosomes are a subtype of extracellular vesicles (EVs), released by all cell types, that originate from the invagination of the endosomal limiting membrane. These EVs can transport biological information in the form of proteins and RNA and have been the focus of intensive research over the last decade. It is becoming apparent that EVs can have important roles in health and disease. EVs are also promising noninvasive biomarkers of disease (liquid biopsies) and valuable vectors for innovative therapies. However, little is known about the mechanisms that regulate the loading of cytosolic proteins into exosomes. We recently showed that soluble proteins containing amino acid sequences biochemically related to the KFERQ motif are loaded into nascent exosomes at the endosomal limiting membrane, in a process mediated by LAMP2A. Because of the subcellular localization and machinery involved, this mechanism has many similarities with chaperone-mediated autophagy (CMA) and endosomal microautophagy (e-Mi), but also some important differences. In this punctum we will focus on the mechanistic details of xosomal AMP2A oading f argo (e-LLoC) as well as on its implications for intercellular and interorgan communication.
Topics: Autophagy; Cytosol; Endosomes; Exosomes; Proteins
PubMed: 35761480
DOI: 10.1080/15548627.2022.2092315 -
Cells Jun 2022Autophagy is a pleiotropic and evolutionarily conserved process in eukaryotes that encompasses different types of mechanisms by which cells deliver cytoplasmic... (Review)
Review
Autophagy is a pleiotropic and evolutionarily conserved process in eukaryotes that encompasses different types of mechanisms by which cells deliver cytoplasmic constituents to the lysosome for degradation. Interestingly, in mammals, two different and specialized autophagic pathways, (i) the chaperone-mediated autophagy (CMA) and (ii) the endosomal microautophagy (eMI), both rely on the use of the same cytosolic chaperone HSPA8 (also known as HSC70) for targeting specific substrates to the lysosome. However, this is not true for all organisms, and differences exist between species with respect to the coexistence of these two autophagic routes. In this paper, we present an in-depth analysis of the evolutionary history of the main components of CMA and eMI and discuss how the observed discrepancies between species may contribute to improving our knowledge of these two functions and their interplays.
Topics: Animals; Autophagy; Chaperone-Mediated Autophagy; Lysosomes; Macroautophagy; Mammals; Microautophagy
PubMed: 35741074
DOI: 10.3390/cells11121945 -
International Journal of General... 2022Proteostasis, also known as protein homeostasis, is critical for cell survival. Autophagy is a cellular process that degrades and recycles damaged or long-lived... (Review)
Review
Proteostasis, also known as protein homeostasis, is critical for cell survival. Autophagy is a cellular process that degrades and recycles damaged or long-lived proteins, misfolded proteins, and damaged or abnormal organelles in order to preserve homeostasis. Among the three forms of autophagy, chaperone-mediated autophagy (CMA) is distinct from macroautophagy and microautophagy; it does not require the formation of vacuoles and only degrades selected individual proteins. CMA helps to maintain cellular homeostasis by regulating protein quality, bioenergetics, and substrate-associated cellular processes at the right moment. This pathway's dysfunction has been linked to several diseases and disorders. Neurodegenerative diseases and cancer have received the most attention. In various neurodegenerative disorders, especially in their later stages, CMA activity declines. CMA has been shown to act as a tumor suppressor in cancer by destroying specific tumor promoters. Once a tumor has grown, it also helps tumor survival and the metastatic cascade. The presence of changes in CMA in these diseases disorders raises the idea of targeting CMA to restore cellular homeostasis as a potential therapeutic method. Manipulation of CMA activity may be effective therapeutic strategies for treating these diseases. Therefore, in this paper; we introduce the basic processes, regulatory mechanisms, and physiological functions of CMA; evidences supporting the role of impaired CMA function in neurodegeneration and cancer; and the potential of how targeting CMA could be a promising therapeutic method for the two diseases.
PubMed: 35734200
DOI: 10.2147/IJGM.S368364 -
Plant Signaling & Behavior Dec 2022Photosynthesis is an essential process that plants must regulate to survive in dynamic environments. Thus, chloroplasts (the sites of photosynthesis in plant and algae...
Photosynthesis is an essential process that plants must regulate to survive in dynamic environments. Thus, chloroplasts (the sites of photosynthesis in plant and algae cells) use multiple signaling mechanisms to report their health to the cell. Such signals are poorly understood but often involve reactive oxygen species (ROS) produced from the photosynthetic light reactions. One ROS, singlet oxygen (O), can signal to initiate chloroplast degradation, but the cellular machinery involved in identifying and degrading damaged chloroplasts (., chloroplast quality control pathways) is unknown. To provide mechanistic insight into these pathways, two recent studies have investigated degrading chloroplasts in the O over-producing () mutant. First, a structural analysis of degrading chloroplasts was performed with electron microscopy, which demonstrated that damaged chloroplasts can protrude into the central vacuole compartment with structures reminiscent of fission-type microautophagy. O-stressed chloroplasts swelled before these interactions, which may be a mechanism for their selective degradation. Second, the roles of autophagosomes and canonical autophagy (macroautophagy) were shown to be dispensable for O-initiated chloroplast degradation. Instead, putative fission-type microautophagy genes were induced by chloroplast O. Here, we discuss how these studies implicate this poorly understood cellular degradation pathway in the dismantling of O-damaged chloroplasts.
Topics: Arabidopsis; Arabidopsis Proteins; Chloroplasts; Reactive Oxygen Species; Singlet Oxygen
PubMed: 35676885
DOI: 10.1080/15592324.2022.2084955