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American Journal of Physiology. Cell... Jan 2018Cigarette-smoke (CS) exposure and aging are the leading causes of chronic obstructive pulmonary disease (COPD)-emphysema development, although the molecular mechanism...
Cigarette-smoke (CS) exposure and aging are the leading causes of chronic obstructive pulmonary disease (COPD)-emphysema development, although the molecular mechanism that mediates disease pathogenesis remains poorly understood. Our objective was to investigate the impact of CS exposure and aging on autophagy and the pathophysiological changes associated with lung aging (senescence) and emphysema progression. Beas2b cells, C57BL/6 mice, and human (GOLD 0-IV) lung tissues were used to determine the central mechanism involved in CS/age-related COPD-emphysema pathogenesis. Beas2b cells and murine lungs exposed to cigarette smoke extract (CSE)/CS showed a significant ( P < 0.05) accumulation of poly-ubiquitinated proteins and impaired autophagy marker, p62, in aggresome bodies. Moreover, treatment with the autophagy-inducing antioxidant drug cysteamine significantly ( P < 0.001) decreased CSE/CS-induced aggresome bodies. We also found a significant ( P < 0.001) increase in levels of aggresome bodies in the lungs of smokers and COPD subjects in comparison to nonsmoker controls. Furthermore, the presence and levels of aggresome bodies statistically correlated with severity of emphysema and alveolar senescence. In addition to CS exposure, lungs from old mice also showed accumulation of aggresome bodies, suggesting this as a common mechanism to initiate cellular senescence and emphysema. Additionally, Beas2b cells and murine lungs exposed to CSE/CS showed cellular apoptosis and senescence, which were both controlled by cysteamine treatment. In parallel, we evaluated the impact of CS on pulmonary exacerbation, using mice exposed to CS and/or infected with Pseudomonas aeruginosa ( Pa), and confirmed cysteamine's potential as an autophagy-inducing antibacterial drug, based on its ability to control CS-induced pulmonary exacerbation ( Pa-bacterial counts) and resulting inflammation. CS induced autophagy impairment accelerates lung aging and COPD-emphysema exacerbations and pathogenesis.
Topics: Animals; Autophagy; Case-Control Studies; Cell Line; Cellular Senescence; Cigarette Smoking; Cysteamine; Cytokines; Disease Models, Animal; Disease Progression; Epithelial Cells; Humans; Inclusion Bodies; Inflammation Mediators; Lung; Mice, Inbred C57BL; Pulmonary Disease, Chronic Obstructive; Pulmonary Emphysema; Sequestosome-1 Protein; Severity of Illness Index; Smoke; Ubiquitination
PubMed: 27413169
DOI: 10.1152/ajpcell.00110.2016 -
Molecular Cell Jan 2019Cell dormancy is a widespread mechanism used by bacteria to evade environmental threats, including antibiotics. Here we monitored bacterial antibiotic tolerance and...
Cell dormancy is a widespread mechanism used by bacteria to evade environmental threats, including antibiotics. Here we monitored bacterial antibiotic tolerance and regrowth at the single-cell level and found that each individual survival cell shows different "dormancy depth," which in return regulates the lag time for cell resuscitation after removal of antibiotic. We further established that protein aggresome-a collection of endogenous protein aggregates-is an important indicator of bacterial dormancy depth, whose formation is promoted by decreased cellular ATP level. For cells to leave the dormant state and resuscitate, clearance of protein aggresome and recovery of proteostasis are required. We revealed that the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the lag time for bacterial regrowth. Better understanding of the key factors regulating bacterial regrowth after surviving antibiotic attack could lead to new therapeutic strategies for combating bacterial antibiotic tolerance.
Topics: Adenosine Triphosphate; Anti-Bacterial Agents; Drug Resistance, Bacterial; Endopeptidase Clp; Energy Metabolism; Escherichia coli; Escherichia coli Proteins; HSP70 Heat-Shock Proteins; Heat-Shock Proteins; Hydrogen-Ion Concentration; Microbial Viability; Protein Aggregates; Single-Cell Analysis; Time Factors
PubMed: 30472191
DOI: 10.1016/j.molcel.2018.10.022 -
Cellular & Molecular Biology Letters 2016The ubiquitin-proteasome system (UPS) and autophagy are two distinct and interacting proteolytic systems. They play critical roles in cell survival under normal... (Review)
Review
The ubiquitin-proteasome system (UPS) and autophagy are two distinct and interacting proteolytic systems. They play critical roles in cell survival under normal conditions and during stress. An increasing body of evidence indicates that ubiquitinated cargoes are important markers of degradation. p62, a classical receptor of autophagy, is a multifunctional protein located throughout the cell and involved in many signal transduction pathways, including the Keap1-Nrf2 pathway. It is involved in the proteasomal degradation of ubiquitinated proteins. When the cellular p62 level is manipulated, the quantity and location pattern of ubiquitinated proteins change with a considerable impact on cell survival. Altered p62 levels can even lead to some diseases. The proteotoxic stress imposed by proteasome inhibition can activate autophagy through p62 phosphorylation. A deficiency in autophagy may compromise the ubiquitin-proteasome system, since overabundant p62 delays delivery of the proteasomal substrate to the proteasome despite proteasomal catalytic activity being unchanged. In addition, p62 and the proteasome can modulate the activity of HDAC6 deacetylase, thus influencing the autophagic degradation.
Topics: Animals; Autophagy; Humans; Proteasome Endopeptidase Complex; Sequestosome-1 Protein; Signal Transduction; Ubiquitinated Proteins
PubMed: 28536631
DOI: 10.1186/s11658-016-0031-z -
The Journal of Biological Chemistry Aug 2007Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative...
Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related gamma-aminobutyrate receptor-associated protein and gamma-aminobutyrate receptor-associated-like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62- and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62- and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 microm diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.
Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Autophagy; Cytosol; Fluorescent Dyes; HeLa Cells; Humans; Microtubule-Associated Proteins; Multiprotein Complexes; Protein Binding; RNA, Small Interfering; Sequestosome-1 Protein; Transfection; Ubiquitin
PubMed: 17580304
DOI: 10.1074/jbc.M702824200 -
Molecular Neurodegeneration Mar 2021Emerging evidence indicates that impaired mitophagy-mediated clearance of defective mitochondria is a critical event in Alzheimer's disease (AD) pathogenesis....
BACKGROUND
Emerging evidence indicates that impaired mitophagy-mediated clearance of defective mitochondria is a critical event in Alzheimer's disease (AD) pathogenesis. Amyloid-beta (Aβ) metabolism and the microtubule-associated protein tau have been reported to regulate key components of the mitophagy machinery. However, the mechanisms that lead to mitophagy dysfunction in AD are not fully deciphered. We have previously shown that intraneuronal cholesterol accumulation can disrupt the autophagy flux, resulting in low Aβ clearance. In this study, we examine the impact of neuronal cholesterol changes on mitochondrial removal by autophagy.
METHODS
Regulation of PINK1-parkin-mediated mitophagy was investigated in conditions of acute (in vitro) and chronic (in vivo) high cholesterol loading using cholesterol-enriched SH-SY5Y cells, cultured primary neurons from transgenic mice overexpressing active SREBF2 (sterol regulatory element binding factor 2), and mice of increasing age that express the amyloid precursor protein with the familial Alzheimer Swedish mutation (Mo/HuAPP695swe) and mutant presenilin 1 (PS1-dE9) together with active SREBF2.
RESULTS
In cholesterol-enriched SH-SY5Y cells and cultured primary neurons, high intracellular cholesterol levels stimulated mitochondrial PINK1 accumulation and mitophagosomes formation triggered by Aβ while impairing lysosomal-mediated clearance. Antioxidant recovery of cholesterol-induced mitochondrial glutathione (GSH) depletion prevented mitophagosomes formation indicating mitochondrial ROS involvement. Interestingly, when brain cholesterol accumulated chronically in aged APP-PSEN1-SREBF2 mice the mitophagy flux was affected at the early steps of the pathway, with defective recruitment of the key autophagy receptor optineurin (OPTN). Sustained cholesterol-induced alterations in APP-PSEN1-SREBF2 mice promoted an age-dependent accumulation of OPTN into HDAC6-positive aggresomes, which disappeared after in vivo treatment with GSH ethyl ester (GSHee). The analyses in post-mortem brain tissues from individuals with AD confirmed these findings, showing OPTN in aggresome-like structures that correlated with high mitochondrial cholesterol levels in late AD stages.
CONCLUSIONS
Our data demonstrate that accumulation of intracellular cholesterol reduces the clearance of defective mitochondria and suggest recovery of the cholesterol homeostasis and the mitochondrial scavenging of ROS as potential therapeutic targets for AD.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Autophagy; Cholesterol; Lysosomes; Mice, Transgenic; Mitochondria; Neurons; Ubiquitin-Protein Ligases
PubMed: 33685483
DOI: 10.1186/s13024-021-00435-6 -
Nature Cell Biology Apr 2022Protein degradation is critical to maintaining cellular homeostasis, and perturbation of the ubiquitin proteasome system leads to the accumulation of protein aggregates....
Protein degradation is critical to maintaining cellular homeostasis, and perturbation of the ubiquitin proteasome system leads to the accumulation of protein aggregates. These aggregates are either directed towards autophagy for destruction or sequestered into an inclusion, termed the aggresome, at the centrosome. Utilizing high-resolution quantitative analysis, here, we define aggresome assembly at the centrosome in human cells. Centriolar satellites are proteinaceous granules implicated in the trafficking of proteins to the centrosome. During aggresome assembly, satellites were required for the growth of the aggresomal structure from an initial ring of phosphorylated HSP27 deposited around the centrioles. The seeding of this phosphorylated HSP27 ring depended on the centrosomal proteins CP110, CEP97 and CEP290. Owing to limiting amounts of CP110, senescent cells, which are characterized by the accumulation of protein aggregates, were defective in aggresome formation. Furthermore, satellites and CP110-CEP97-CEP290 were required for the aggregation of mutant huntingtin. Together, these data reveal roles for CP110-CEP97-CEP290 and satellites in the control of cellular proteostasis and the aggregation of disease-relevant proteins.
Topics: Antigens, Neoplasm; Cell Cycle Proteins; Centrioles; Centrosome; Cilia; Cytoskeletal Proteins; HSP27 Heat-Shock Proteins; Humans; Microtubule-Associated Proteins; Protein Aggregates
PubMed: 35411088
DOI: 10.1038/s41556-022-00869-0 -
Cell Stem Cell Apr 2023Hematopoietic stem cells (HSCs) regenerate blood cells throughout life. To preserve their fitness, HSCs are particularly dependent on maintaining protein homeostasis...
Hematopoietic stem cells (HSCs) regenerate blood cells throughout life. To preserve their fitness, HSCs are particularly dependent on maintaining protein homeostasis (proteostasis). However, how HSCs purge misfolded proteins is unknown. Here, we show that in contrast to most cells that primarily utilize the proteasome to degrade misfolded proteins, HSCs preferentially traffic misfolded proteins to aggresomes in a Bag3-dependent manner and depend on aggrephagy, a selective form of autophagy, to maintain proteostasis in vivo. When autophagy is disabled, HSCs compensate by increasing proteasome activity, but proteostasis is ultimately disrupted as protein aggregates accumulate and HSC function is impaired. Bag3-deficiency blunts aggresome formation in HSCs, resulting in protein aggregate accumulation, myeloid-biased differentiation, and diminished self-renewal activity. Furthermore, HSC aging is associated with a severe loss of aggresomes and reduced autophagic flux. Protein degradation pathways are thus specifically configured in young adult HSCs to preserve proteostasis and fitness but become dysregulated during aging.
Topics: Proteostasis; Macroautophagy; Proteasome Endopeptidase Complex; Autophagy; Transcription Factors; Hematopoietic Stem Cells
PubMed: 36948186
DOI: 10.1016/j.stem.2023.02.010 -
Scientific Reports Sep 2022The aggresome is a protein turnover system in which proteins are trafficked along microtubules to the centrosome for degradation. Despite extensive focus on aggresomes...
The aggresome is a protein turnover system in which proteins are trafficked along microtubules to the centrosome for degradation. Despite extensive focus on aggresomes in immortalized cell lines, it remains unclear if the aggresome is conserved in all primary cells and all cell-states. Here we examined the aggresome in primary adult mouse dermal fibroblasts shifted into four distinct cell-states. We found that in response to proteasome inhibition, quiescent and immortalized fibroblasts formed aggresomes, whereas proliferating and senescent fibroblasts did not. Using this model, we generated a resource to provide a characterization of the proteostasis networks in which the aggresome is used and transcriptomic features associated with the presence or absence of aggresome formation. Using this resource, we validate a previously reported role for p38 MAPK signaling in aggresome formation and identify TAK1 as a novel driver of aggresome formation upstream of p38 MAPKs. Together, our data demonstrate that the aggresome is a non-universal protein degradation system which can be used cell-state specifically and provide a resource for studying aggresome formation and function.
Topics: Animals; Centrosome; Fibroblasts; Inclusion Bodies; Mice; Microtubules; Proteasome Endopeptidase Complex; Proteins
PubMed: 36056070
DOI: 10.1038/s41598-022-19055-1 -
Translational Neurodegeneration Dec 2020Parkinson's disease (PD) is diagnosed when patients exhibit bradykinesia with tremor and/or rigidity, and when these symptoms respond to dopaminergic medications. Yet in... (Review)
Review
Parkinson's disease (PD) is diagnosed when patients exhibit bradykinesia with tremor and/or rigidity, and when these symptoms respond to dopaminergic medications. Yet in the last years there was a greater recognition of additional aspects of the disease including non-motor symptoms and prodromal states with associated pathology in various regions of the nervous system. In this review we discuss current concepts of two major alterations found during the course of the disease: cytoplasmic aggregates of the protein α-synuclein and the degeneration of dopaminergic neurons. We provide an overview of new approaches in this field based on current concepts and latest literature. In many areas, translational research on PD has advanced the understanding of the disease but there is still a need for more effective therapeutic options based on the insights into the basic biological phenomena.
Topics: Dopamine; Dopaminergic Neurons; Humans; Parkinson Disease; Prodromal Symptoms; Translational Research, Biomedical; alpha-Synuclein
PubMed: 33256849
DOI: 10.1186/s40035-020-00223-0 -
International Journal of Molecular... May 2023A number of muscular disorders are hallmarked by the aggregation of misfolded proteins within muscle fibers. A specialized form of macroautophagy, termed aggrephagy, is... (Review)
Review
A number of muscular disorders are hallmarked by the aggregation of misfolded proteins within muscle fibers. A specialized form of macroautophagy, termed aggrephagy, is designated to remove and degrade protein aggregates. This review aims to summarize what has been studied so far about the direct involvement of aggrephagy and the activation of the key players, among others, p62, NBR1, Alfy, Tollip, Optineurin, TAX1BP1 and CCT2 in muscular diseases. In the first part of the review, we describe the aggrephagy pathway with the involved proteins; then, we illustrate the muscular disorder histologically characterized by protein aggregates, highlighting the role of aggrephagy pathway abnormalities in these muscular disorders.
Topics: Humans; Macroautophagy; Protein Aggregates; Autophagy; Apoptosis Regulatory Proteins; Muscular Diseases
PubMed: 37176163
DOI: 10.3390/ijms24098456