scholarly journals Proteomic Analysis of Mitochondrial Protein Turnover: Identification of Novel Substrate Proteins of the Matrix Protease Pim1

2006 ◽  
Vol 26 (3) ◽  
pp. 762-776 ◽  
Author(s):  
Tamara Major ◽  
Birgit von Janowsky ◽  
Thomas Ruppert ◽  
Axel Mogk ◽  
Wolfgang Voos
Keyword(s):  
Protein Turnover ◽  
Protein Quality ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Differential Susceptibility ◽  
Cellular Protein ◽  
Major Influence ◽  
Mitochondrial Functions ◽  
Substrate Proteins

ABSTRACT ATP-dependent oligomeric proteases are major components of cellular protein quality control systems. To investigate the role of proteolytic processes in the maintenance of mitochondrial functions, we analyzed the dynamic behavior of the mitochondrial proteome of Saccharomyces cerevisiae by two-dimensional (2D) polyacrylamide gel electrophoresis. By a characterization of the influence of temperature on protein turnover in isolated mitochondria, we were able to define four groups of proteins showing a differential susceptibility to proteolysis. The protein Pim1/LON has been shown to be the main protease in the mitochondrial matrix responsible for the removal of damaged or nonnative proteins. To assess the substrate range of Pim1 under in vivo conditions, we performed a quantitative comparison of the 2D protein spot patterns between wild-type and pim1Δ mitochondria. We were able to identify a novel subset of mitochondrial proteins that are putative endogenous substrates of Pim1. Using an in organello degradation assay, we confirmed the Pim1-specific, ATP-dependent proteolysis of the newly identified substrate proteins. We could demonstrate that the functional integrity of the Pim1 substrate proteins, in particular, the presence of intact prosthetic groups, had a major influence on the susceptibility to proteolysis.

scholarly journals Truncation-Driven Lateral Association of α-Synuclein Hinders Amyloid Clearance by the Hsp70-based Disaggregase

2021 ◽  
Vol 22 (23) ◽  
pp. 12983
Author(s):  
Aitor Franco ◽  
Jorge Cuéllar ◽  
José Ángel Fernández-Higuero ◽  
Igor de la Arada ◽  
Natalia Orozco ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Protein Quality ◽  
Mechanical Action ◽  
Cellular Protein ◽  
Type B ◽  
Post Translational Modifications ◽  
Lateral Association ◽  
N Terminus ◽  
Protein Quality Control System

The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2. We find that, in contrast to aggregates of the protein lacking the N-terminus, which can be solubilized as efficiently as those of the WT protein, the deletion of the C-terminal domain, either in a recombinant context or as a consequence of calpain treatment, impaired Hsc70-mediated amyloid disassembly. Progressive removal of the negative charges at the C-terminal region induces lateral association of fibrils and type B* oligomers, precluding chaperone action. We propose that truncation-driven aggregate clumping impairs the mechanical action of chaperones, which includes fast protofilament unzipping coupled to depolymerization. Inhibition of the chaperone-mediated clearance of C-truncated species could explain their exacerbated toxicity and higher propensity to deposit found in vivo.

scholarly journals Role of membrane-bound and free polyribosomes in the synthesis of cytochrome c in rat liver

1974 ◽  
Vol 140 (2) ◽  
pp. 157-167 ◽  
Author(s):  
Néstor F. González-Cadavid ◽  
Carmen Sáez De Córdova
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Cell Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sodium Dodecyl ◽  
Membrane Bound

The functional distinction of membrane-bound and free polyribosomes for the synthesis of exportable and non-exportable proteins respectively is not so strict as was initially thought, and it was therefore decided to investigate their relative contribution to the elaboration of an internal protein integrated into a cell structure. Cytochrome c was chosen as an example of a soluble mitochondrial protein, and the incorporation of [14C]leucine and δ-amino[14C]laevulinate into the molecule was studied by using different ribosomal preparations from regenerating rat liver. A new procedure was devised for the purification of cytochrome c, based on ion-exchange chromatography combined with sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. In spite of cytochrome c being a non-exportable protein, the membrane-bound polyribosomes were at least as active as the free ribosomes in the synthesis in vitro of the apoprotein and the haem moiety. The detergent-treated ribosomes could also effect the synthesis of cytochrome c, although at a lower rate. Since in liver more than two-thirds of the ribosomes are bound to the endoplasmic-reticulum membranes, it is considered that in vivo they are responsible for the synthesis of most of the cytochrome c content of the cell. This suggests that in secretory tissues the endoplasmic reticulum plays a predominant role in mitochondrial biogenesis, although free ribosomes may participate in the partial turnover of some parts of the organelle. The hypothesis on the functional specialization of the different kinds of ribosomes was therefore modified to account for their parallel intervention in the synthesis of proteins associated with membranous structures.

scholarly journals A disease-associated G5703A mutation in human mitochondrial DNA causes a conformational change and a marked decrease in steady-state levels of mitochondrial tRNA(Asn).

1997 ◽  
Vol 17 (12) ◽  
pp. 6831-6837 ◽  
Author(s):  
H Hao ◽  
C T Moraes
Keyword(s):  
Mitochondrial Dna ◽  
Steady State ◽  
Oxidative Phosphorylation ◽  
Conformational Change ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Human Osteosarcoma Cells ◽  
Steady State Levels

We introduced mitochondrial DNA (mtDNA) from a patient with a mitochondrial myopathy into established mtDNA-less human osteosarcoma cells. The resulting transmitochondrial cybrid lines, containing either exclusively wild-type or mutated (G5703A transition in the tRNA[Asn] gene) mtDNA, were characterized and analyzed for oxidative phosphorylation function and steady-state levels of different RNA species. Functional studies showed that the G5703A mutation severely impairs oxidative phosphorylation function and mitochondrial protein synthesis. We detected a marked reduction in tRNA(Asn) steady-state levels which was not associated with an accumulation of intermediate transcripts containing tRNA(Asn) sequences or decreased transcription. Native polyacrylamide gel electrophoresis showed that the residual tRNA(Asn) fraction in mutant cybrids had an altered conformation, suggesting that the mutation destabilized the tRNA(Asn) secondary or tertiary structure. Our results suggest that the G5703 mutation causes a conformational change in the tRNA(Asn) which may impair aminoacylation. This alteration leads to a severe reduction in the functional tRNA(Asn) pool by increasing its in vivo degradation by mitochondrial RNases.

scholarly journals Effect of Tim23 knockdown in vivo on mitochondrial protein import and retrograde signaling to the UPRmt in muscle

2018 ◽  
Vol 315 (4) ◽  
pp. C516-C526 ◽  
Author(s):  
Ashley N. Oliveira ◽  
David A. Hood
Keyword(s):  
Protein Import ◽  
Protein Quality ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
Inhibitory Effect ◽  
Feedback Mechanism ◽  
Retrograde Signaling ◽  
Inner Membrane ◽  
The Face

The mitochondrial unfolded protein response (UPRmt) is a protein quality control mechanism that strives to achieve proteostasis in the face of misfolded proteins. Because of the reliance of mitochondria on both the nuclear and mitochondrial genomes, a perturbation of the coordination of these genomes results in a mitonuclear imbalance in which holoenzymes are unable to assume mature stoichiometry and thereby activates the UPRmt. Thus, we sought to perturb this genomic coordination by using a systemic antisense oligonucleotide (in vivo morpholino) targeted to translocase of the inner membrane channel subunit 23 (Tim23), the major channel of the inner membrane. This resulted in a 40% reduction in Tim23 protein content, a 32% decrease in matrix-destined protein import, and a trend to elevate reactive oxygen species (ROS) emission under maximal respiration conditions. This import defect activated the C/EBP homologous protein (CHOP) branch of the UPRmt, as evident from increases in caseinolytic mitochondrial matrix peptidase proteolytic subunit (ClpP) and chaperonin 10 (cpn10) but not the activating transcription factor 5 (ATF5) arm. Thus, in the face of proteotoxic stress, CHOP and ATF5 could be activated independently to regain proteostasis. Our second aim was to investigate the role of proteolytically derived peptides in mediating retrograde signaling. Peptides released from the mitochondrion following basal proteolysis were isolated and incubated with import reactions. Dose- and time-dependent effect of peptides on protein import was observed. Our data suggest that mitochondrial proteolytic byproducts exert an inhibitory effect on protein import, possibly to reduce excessive protein import as a potential negative feedback mechanism. The inhibition of import into the organelle also serves a retrograde function, possibly via ROS emission, to modify nuclear gene expression and ultimately improve folding capacity.

Abstract 119: Tempol Pre-treatment Restores Isoflurane Preconditioning in the Aged Cardiomyocytes: Involvement of Pink1/Parkin-mediated Mitophagy

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Li Ma ◽  
Qun Gao ◽  
Mario J Rebecchi ◽  
Lixin Liu
Keyword(s):  
Mitochondrial Protein ◽  
Autophagic Flux ◽  
Fischer 344 ◽  
Protein Ubiquitination ◽  
Chronic Oxidative Stress ◽  
Mitochondrial Functions ◽  
Pre Treatment ◽  
Lc3 Puncta ◽  
Cardiac Preconditioning

Background: Aging is associated with chronic oxidative stress and impaired cardiac preconditioning. Four-week pre-treatment with TEMPOL, an antioxidant, improves mitochondrial functions and restores isoflurane (ISO) preconditioning in the aging rat heart. Because mitophagy is implicated in cardiac preconditioning and declines with age, this study was designed to investigate how age influence mitophagy in response to preconditioning and whether TEMPOL pre-treatment improves it. Methods: Male Fischer 344 old (22-24 months) rats treated with or without 4-week TEMPOL (2 mM in drinking water) and young (4-6 months) untreated rats were used. Cardiomyocytes isolated from these rats were preconditioned with ISO and then collected for Western blot analyses of mitochondrial PINK1, Parkin and ubiquitination, or fixed for IF staining of co-localization of PINK1/Parkin with mitochondria. Autophagic flux was determined by IF staining of LC3 puncta, membrane-associated LC3-II and p62 in the presence and absences of bafilomycin A1, an autophagy inhibitor. ISO preconditioning was assessed by LDH release in a simulated hypoxia/reoxygenation model. Results: ISO protected young but not old cardiomyocytes. Four-week pre-treatment with TEMPOL in vivo restored ISO preconditioning in the old cardiomyocytes. In addition, ISO increased mitochondrial PINK1 and Parkin levels and promoted mitochondrial protein ubiquitination in the young, but not the old cardiomyocytes. TEMPOL pre-treatment improved these responses in the old cardiomyocytes. Aging impaired both baseline and ISO-induced autophagic flux, which was restored by TEMPOL pre-treatment. Inhibition of autophagy by bafilomycin abolished ISO preconditioning in the cardiomyocytes from the young and TEMPOL-pre-treated old rats. Conclusion: Aging reduces PINK1/Parkin-mediated mitophagy in response to ISO and impairs baseline as well as preconditioning-induced autophagic flux. A 4-week pre-treatment of TEMPOL reverses these changes and restores ISO preconditioning in the old cardiomyocytes. Our results may explain why cardiac preconditioning has failed in some clinical trials, because most patients were with pathologies, such as diabetes and advanced age, in which mitophagy is disrupted.

scholarly journals Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease

2011 ◽  
Vol 22 (5) ◽  
pp. 541-554 ◽  
Author(s):  
Tom Bender ◽  
Ilka Lewrenz ◽  
Sebastian Franken ◽  
Catherina Baitzel ◽  
Wolfgang Voos
Keyword(s):  
Quality Control ◽  
Protein Aggregation ◽  
Elevated Temperatures ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Cellular Protein ◽  
Control Measures ◽  
Mitochondrial Enzymes ◽  
Induced Aggregation

Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria.

scholarly journals Novel proteostasis reporter mouse reveals different effects of cytoplasmic and nuclear aggregates on protein quality control in neurons

2020 ◽  
Author(s):  
Sonja Blumenstock ◽  
Elena Katharina Schulz-Trieglaff ◽  
Anna-Lena Bolender ◽  
Kerstin Voelkl ◽  
Paul Lapios ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Cellular Protein ◽  
Aging Brain ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Age Related ◽  
Reporter Mice

AbstractThe cellular protein quality control machinery is important for preventing protein misfolding and aggregation, and decline in protein homeostasis (proteostasis) is believed to play a crucial role in age-related neurodegenerative disorders. However, how proteostasis capacity of neurons changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP-fused firefly luciferase (Fluc), a conformationally unstable protein that requires chaperones for proper folding and sensitively reacts to proteotoxic stress by formation of intracellular Fluc-EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked impairment in proteostasis in tauopathy mice, but not in Huntington’s disease mice. Mechanistic investigations in primary neuronal cultures demonstrate that cytoplasmic, but not nuclear, aggregates cause defects of cellular protein quality control. Thus, the Fluc-EGFP reporter mice enable new insights into proteostasis alterations in different diseases.

scholarly journals Mitochondrial c-Src regulates cell survival through phosphorylation of respiratory chain components

2012 ◽  
Vol 447 (2) ◽  
pp. 281-289 ◽  
Author(s):  
Masato Ogura ◽  
Junko Yamaki ◽  
Miwako K. Homma ◽  
Yoshimi Homma
Keyword(s):  
Oxidative Phosphorylation ◽  
Nadh Dehydrogenase ◽  
Kinase Inhibitor ◽  
Mitochondrial Protein ◽  
Phosphorylation Site ◽  
Respiration Activity ◽  
Site Analysis ◽  
Mitochondrial Functions ◽  
Respiratory Complex I

Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr193 of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr215 of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.

Differentiation between mitochondrial and cytoplasmic protein subunits of blowfly flight muscle mitochondria synthesized in vivo

1981 ◽  
Vol 50 (1) ◽  
pp. 377-390
Author(s):  
M.B. Ashour ◽  
M. Tribe
Keyword(s):  
Gel Electrophoresis ◽  
Flight Muscle ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Cytoplasmic Protein ◽  
Protein Subunits ◽  
Molecular Weights ◽  
And Function ◽  
Made In

Total adult blowfly flight-muscle mitochondrial protein was labelled in vivo with [14C]leucine. The labelled proteins were enumerated and characterized by their electrophoretic mobility using sodium dodcecyl sulphate/polyacrylamide gel electrophoresis and autoradiography. When different gel concentrations were used to resolve the maximum number of bands and their molecular weights, it was possible to observe at least 35 electrophoretic bands after staining and scanning; their molecular weights ranged between 11 000 and 130 000. When mitochondria were labelled in the presence of cycloheximide, only 6–8 bands could be identified on gradient gels after electrophoresis and autoradiography. By comparison, controls (where cycloheximide was absent), which were run alongside the drug-treated mitochondria, revealed 17–20 radioactively labelled bands from densitometric tracings. Whilst the molecular weights of these bands could be estimated, it was difficult to identify the precise nature and function of the proteins made in these mitochondria.

scholarly journals Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle

1976 ◽  
Vol 158 (2) ◽  
pp. 271-282 ◽  
Author(s):  
S J Higgins ◽  
J M Burchell ◽  
W I P Mainwaring
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Distinct Species ◽  
Specific Protein ◽  
Cellular Protein ◽  
Secretory Proteins ◽  
Receptor System ◽  
Basic Proteins ◽  
Synthetic Capacity

1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug.

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