Impact of inhibition of the autophagy-lysosomal pathway on biomolecules carbonylation and proteome regulation in rat cardiac cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Coliva G
PROVIDER: S-EPMC6859560 | biostudies-literature | 2019 May
REPOSITORIES: biostudies-literature
Publications
Impact of inhibition of the autophagy-lysosomal pathway on biomolecules carbonylation and proteome regulation in rat cardiac cells.
Coliva Giulia G Duarte Sofia S Pérez-Sala Dolores D Fedorova Maria M
Redox biology 20190124
Cells employ multiple defence mechanisms to sustain a wide range of stress conditions associated with accumulation of modified self-biomolecules leading to lipo- and proteotoxicity. One of such mechanisms involves activation of the autophagy-lysosomal pathway for removal and degradation of modified lipids, proteins and even organelles. Biomolecules carbonylation, an irreversible oxidative modification, occurs in a variety of pathological conditions and is generally viewed as a marker of oxidativ ...[more]
Similar Datasets
Project description:Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins-EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.
| S-EPMC7058366 | biostudies-literature
Project description:The autophagy lysosomal pathway (ALP) is a major mechanism for degrading intracellular macromolecules. The catabolic products can then be used by the cell for energy or as building blocks to make other macromolecules. Since its discovery, a variety of cellular pathways have emerged that target components with varying specificity for lysosomal degradation. Under some circumstances, lysosomes may release their contents into the extracellular space where they may serve signaling or pathogenic functions. The ALP is active in healthy cells, and the level of activity can be regulated by nutrient-sensing and metabolic signaling pathways. The ALP is the primary pathway by which lipids and damaged organelles are degraded and may be the only pathway capable of degrading aggregated proteins. As such, there has been intense interest in understanding the role of the ALP in the accumulation of aggregated misfolded proteins characteristic of many of the major adult-onset neurodegenerative diseases. This review focuses on recent advances in our understanding of the ALP and its potential relationship to the pathogenesis and treatment of neurodegenerative diseases.
| S-EPMC6773515 | biostudies-literature
Project description:The mechanisms of the control and activity of the autophagy-lysosomal protein degradation machinery are emerging as an important theme for neurodevelopment and neurodegeneration. However, the underlying regulatory and functional networks of known genes controlling autophagy and lysosomal function and their role in disease are relatively unexplored. We performed a systems biology-based integrative computational analysis to study the interactions between molecular components and to develop models for regulation and function of genes involved in autophagy and lysosomal function. Specifically, we analyzed transcriptional and microRNA-based post-transcriptional regulation of these genes and performed functional enrichment analyses to understand their involvement in nervous system-related diseases and phenotypes. Transcriptional regulatory network analysis showed that binding sites for transcription factors, SREBP1, USF, AP-1 and NFE2, are common among autophagy and lysosomal genes. MicroRNA enrichment analysis revealed miR-130, 98, 124, 204 and 142 as the putative post-transcriptional regulators of the autophagy-lysosomal pathway genes. Pathway enrichment analyses revealed that the mTOR and insulin signaling pathways are important in the regulation of genes involved in autophagy. In addition, we found that glycosaminoglycan and glycosphingolipid pathways also make a major contribution to lysosomal gene regulation. The analysis confirmed the known contribution of the autophagy-lysosomal genes to Alzheimer and Parkinson diseases and also revealed potential involvement in tuberous sclerosis, neuronal ceroidlipofuscinoses, sepsis and lung, liver and prostatic neoplasms. To further probe the impact of autophagy-lysosomal gene deficits on neurologically-linked phenotypes, we also mined the mouse knockout phenotype data for the autophagylysosomal genes and found them to be highly predictive of nervous system dysfunction. Overall this study demonstrates the utility of systems biology-based approaches for understanding the autophagy-lysosomal pathways and gaining additional insights into the potential impact of defects in these complex biological processes.
| S-EPMC3127210 | biostudies-literature
Project description:ATM (ataxia telangiectasia mutated) protein is found associated with multiple organelles including synaptic vesicles, endosomes and lysosomes, often in cooperation with ATR (ataxia telangiectasia and Rad3 related). Mutation of the ATM gene results in ataxia-telangiectasia (A-T), an autosomal recessive disorder with defects in multiple organs including the nervous system. Precisely how ATM deficiency leads to the complex phenotypes of A-T, however, remains elusive. Here, we reported that part of the connection may lie in autophagy and lysosomal abnormalities. We found that ATM was degraded through the autophagy pathway, while ATR was processed by the proteasome. Autophagy and lysosomal trafficking were both abnormal in atm-/- neurons and the deficits impacted cellular functions such as synapse maintenance, neuronal survival and glucose uptake. Upregulated autophagic flux was observed in atm-/- lysosomes, associated with a more acidic pH. Significantly, we found that the ATP6V1A (ATPase, H+ transporting, lysosomal V1 subunit A) proton pump was an ATM kinase target. In atm-/- neurons, lysosomes showed enhanced retrograde transport and accumulated in the perinuclear regions. We attributed this change to an unexpected physical interaction between ATM and the retrograde transport motor protein, dynein. As a consequence, SLC2A4/GLUT4 (solute carrier family 4 [facilitated glucose transporter], member 4) translocation to the plasma membrane was inhibited and trafficking to the lysosomes was increased, leading to impaired glucose uptake capacity. Together, these data underscored the involvement of ATM in a variety of neuronal vesicular trafficking processes, offering new and therapeutically useful insights into the pathogenesis of A-T.Abbreviations: 3-MA: 3-methyladenine; A-T: ataxia-telangiectasia; ALG2: asparagine-linked glycosylation 2 (alpha-1,3-mannosyltransferase); AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; ATG5: autophagy related 5; ATM: ataxia telangiectasia mutated; ATP6V1A: ATPase, H+ transporting, lysosomal V1 subunit A; ATR: ataxia-telangiectasia and Rad3 related; BFA1: bafilomycin A1; CC3: cleaved-CASP3; CGN: cerebellar granule neuron; CLQ: chloroquine; CN: neocortical neuron; CTSB: cathepsin B; CTSD: cathepsin D; DYNLL1: the light chain1 of dynein; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; Etop: etoposide; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HBS: HEPES-buffered saline; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HOMER1: homer protein homolog 1; KU: KU-60019; LAMP1: lysosomal-associated membrane protein 1; LC3B-II: LC3-phosphatidylethanolamine conjugate; Lyso: lysosome; LysopH-GFP: lysopHluorin-GFP; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule associated protein 2; MAPK14: mitogen-activated protein kinase 14; MAPK8/JNK1: mitogen-activated protein kinase 8; MCOLN1/TRPML1: mucolipin 1; OSBPL1A: oxysterol binding protein like 1A; PIKK: phosphatidylinositol 3 kinase related kinase; Rapa: rapamycin; RILP: rab interacting lysosomal protein; ROS: reactive oxygen species; SEM: standard error of mean; SLC2A4/GLUT4: solute carrier family 2 (facilitated glucose transporter), member 4; TSC2/tuberin: TSC complex subunit 2; ULK1: unc-51 like kinase 1; UPS: ubiquitin-proteasome system; VE: VE-822; WCL: whole-cell lysate; WT: wild type.
| S-EPMC8386711 | biostudies-literature
Project description:The etiology of preeclampsia (PE), a serious pregnancy complication, remains an enigma. We have demonstrated that proteinopathy, a pathologic feature of neurodegenerative diseases, is a key observation in the placenta and serum from PE patients. We hypothesize that the macroautophagy/autophagy machinery that mediates degradation of aggregated proteins and damaged organelles is impaired in PE. Here, we show that TFEB (transcription factor EB), a master transcriptional regulator of lysosomal biogenesis, and its regulated proteins, LAMP1, LAMP2, and CTSD (cathepsin D), were dysregulated in the placenta from early and late onset PE deliveries. Primary human trophoblasts and immortalized extravillous trophoblasts (EVTs) showed reduced TFEB expression and nuclear translocation as well as lysosomal protein content in response to hypoxia. Hypoxia-exposed trophoblasts also showed decreased PPP3/calcineurin phosphatase activity and increased XPO1/CRM1 (exportin 1), events that inhibit TFEB nuclear translocation. These proteins were also dysregulated in the PE placenta. These results are supported by observed lysosomal ultrastructural defects with decreased number of autolysosomes in hypoxia-treated primary human trophoblasts. Autophagy-deficient human EVTs exhibited poor TFEB nuclear translocation, reduced lysosomal protein expression and function, and increased MTORC1 activity. Sera from PE patients induced these features and protein aggregation in EVTs. Importantly, trophoblast-specific conditional atg7 knockout mice exhibited reduced TFEB expression with increased deposition of protein aggregates in the placenta. These results provide compelling evidence for a regulatory link between accumulation of protein aggregates and TFEB-mediated impaired lysosomal biogenesis and autophagy in the placenta of PE patients. Abbreviation: atg7: autophagy related 7; CTSD: cathepsin D; ER: endoplasmic reticulum; EVTs: extravillous trophoblasts; KRT7: keratin 7; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; mSt: mStrawberry; MTORC1: mechanistic target of rapamycin complex 1; NP: normal pregnancy; NPS: normal pregnancy serum; PE: preeclampsia; PES: preeclampsia serum; p-RPS6KB: phosphorylated ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; XPO1/CRM1: exportin 1.
| S-EPMC8386603 | biostudies-literature
Project description:Previous studies have demonstrated that autophagic vacuoles induced in hepatocytes of the perfused rat liver can be effectively separated from secondary lysosomes in self-generating gradients of colloidal silica/polyvinylpyrrolidone. Here we show that, as autophagy is increased by graded amino acid deprivation, the lysosomal marker N-acetyl-beta-D-glucosaminidase shifts progressively from dense to buoyant gradient fractions, an effect attributable to enhanced lysosome-autophagosome fusion and the formation of additional autolysosomes. Both the amount of the shift and the aggregate volume of autolysosomes were directly proportional to the increase in protein degradation above basal. Because the total quantity of protein sequestered in the vacuoles is also a direct function of deprivation-induced proteolysis, the relative specific activity of the marker enzyme in autolysosomes (enrichment) would be expected to remain constant over the full range of deprivation; numerical estimates of enrichment, in fact, did not differ from the mean (43.9) by more than 5%. The close correspondence between cytoplasmic sequestration and fusion was maintained, despite a greater than 3-fold increase in the average volume of individual vacuoles. This suggests the existence of an intrinsic regulatory mechanism which limits the number of fusions per vacuole, on the basis of information derived from vacuolar volume.
| S-EPMC1147726 | biostudies-other
Project description:Our previous study and others have demonstrated that autophagy is activated in ischemic astrocytes and contributes to astrocytic cell death. However, the mechanisms of ischemia-induced autophagy remain largely unknown. In this study, we established a rat's model of permanent middle cerebral artery occlusion (pMCAO) and an in vitro oxygen and glucose deprivation (OGD) model. Autophagy was inhibited by either pharmacological treatment with 3-methyladenine (3-MA) and wortmannin (Wort) or genetic treatment with knockdown of Atg5 in primary cultured astrocytes and knockout of Atg5 in mouse embryonic fibroblast (MEF) cells, respectively. We found that pharmacological or genetic inhibition of autophagy reversed pMCAO or OGD-induced increase in LC3-II, active cathepsin B and L, tBid, active caspase-3 and cytoplastic cytochrome c (Cyt-c), and suppressed the injury-induced reduction in mitochondrial Cyt-c in ischemic cortex, in injured astrocytes and MEF cells. Immunofluorescence analysis showed that 3-MA or Wort treatment reversed OGD-induced release of cathepsin B and L from the lysosome to the cytoplasm and activation of caspase-3 in the astrocytes. Furthermore, treatment of 3-MA or Wort decreased OGD-induced increase in lysosomal membrane permeability and enhanced OGD-induced upregulation of lysosomal heat shock protein 70.1B (Hsp70.1B) in astrocytes. Inhibition of autophagy by 3-MA or Wort reduced infarction volume in rats and protected OGD-induced astrocytic cell injury. A non-selective caspase inhibitor z-VAD-fmk or a specific caspase-3 inhibitor Q-DEVD-OPh also rescued OGD-induced astrocytic cell injury. In conclusion, our presenting data suggest that inhibition of autophagy blocks cathepsins-tBid-mitochondrial apoptotic signaling pathway via stabilization of lysosomal membranes, possibly due to upregulation of the lysosomal Hsp70.1B in ischemic astrocytes.
| S-EPMC5386481 | biostudies-literature
Project description:When pathological hypertrophy progresses to heart failure (HF), the prognosis is often very poor. Therefore, it is crucial to find new and effective intervention targets. Here, myocardium-specific Trim44 knockout rats were generated using CRISPR-Cas9 technology. Cardiac phenotypic observations revealed that Trim44 knockout affected cardiac morphology at baseline. Rats with Trim44 deficiency exhibited resistance to cardiac pathological changes in response to stimulation via isoproterenol (ISO) treatment, including improvement of cardiac remodeling and dysfunction by morphological and functional observations, reduced myocardial fibrosis and reduced expression of molecular markers of cardiac stress. Furthermore, signal transduction validation associated with growth and hypertrophy development in vivo and in vitro demonstrated that Trim44 deficiency inhibited the activation of signaling pathways involved in myocardial hypertrophy, especially response to pathological stress. In conclusion, the present study indicates that Trim44 knockout attenuates ISO-induced pathological cardiac remodeling through blocking the AKT/mTOR/GSK3β/P70S6K signaling pathway. This is the first study to demonstrate the function and importance of Trim44 in the heart at baseline and under pathological stress. Trim44 could be a novel therapeutic target for prevention of cardiac hypertrophy and HF.
| S-EPMC9441189 | biostudies-literature
Project description:Lysosomal autophagy inhibition (LAI) with hydroxychloroquine or DC661 can enhance cancer therapy, but tumor regrowth is common. To elucidate LAI resistance, proteomics and immunoblotting demonstrated that LAI induced lipid metabolism enzymes in multiple cancer cell lines. Lipidomics showed that LAI increased cholesterol, sphingolipids, and glycosphingolipids. These changes were associated with striking levels of GM1+ membrane microdomains (GMM) in plasma membranes and lysosomes. Inhibition of cholesterol/sphingolipid metabolism proteins enhanced LAI cytotoxicity. Targeting UDP-glucose ceramide glucosyltransferase (UGCG) synergistically augmented LAI cytotoxicity. While UGCG inhibition decreased LAI-induced GMM and augmented cell death, UGCG overexpression led to LAI resistance. Melanoma patients with high UGCG expression had significantly shorter disease-specific survival. The FDA-approved UGCG inhibitor eliglustat combined with LAI significantly inhibited tumor growth and improved survival in syngeneic tumors and a therapy-resistant patient-derived xenograft. These findings nominate UGCG as a new cancer target, and clinical trials testing UGCG inhibition in combination with LAI are warranted.
2022-03-08 | MSV000089020 | GNPS
Project description:Many neurodegenerative conditions are characterized by the deposition of protein aggregates (mainly amyloid-like) in the central nervous system (CNS). In post-mitotic CNS cells protein aggregation causes cytotoxicity by interfering with various cellular functions. Mutations in different genes may directly cause protein aggregation. However, genetic factors together with aging may contribute to the onset of protein aggregation also by affecting cellular degradative functions, in particular the autophagy-lysosomal pathway (ALP). Increasing body of evidence show that ALP dysfunction and protein aggregation are functionally interconnected and induce each other during neurodegenerative processes. We will summarize the findings supporting these concepts by focusing on lysosomal storage diseases (LSDs), a class of metabolic inherited conditions characterized by global lysosomal dysfunction and often associated to a severe neurodegenerative course. We propose a model by which the inherited lysosomal defects initiate aggregate-prone protein deposition, which, in turns, worsen ALP degradation function, thus generating a vicious cycle, which boost neurodegenerative cascades.
| S-EPMC7079699 | biostudies-literature