scholarly journals Multiomic analysis on human cell model of wolfram syndrome reveals changes in mitochondrial morphology and function

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Agnieszka Zmyslowska ◽  
Miljan Kuljanin ◽  
Beata Malachowska ◽  
Marcin Stanczak ◽  
Dominika Michalek ◽  
...  
Keyword(s):  
Er Stress ◽  
Human Cell ◽  
Proteomic Analysis ◽  
Protein Import ◽  
Cell Model ◽  
Mitochondrial Protein ◽  
Wolfram Syndrome ◽  
Mitochondrial Morphology ◽  
Pathway Enrichment Analysis ◽  
Signal Pathways

Abstract Background Wolfram syndrome (WFS) is a rare autosomal recessive syndrome in which diabetes mellitus and neurodegenerative disorders occur as a result of Wolframin deficiency and increased ER stress. In addition, WFS1 deficiency leads to calcium homeostasis disturbances and can change mitochondrial dynamics. The aim of this study was to evaluate protein levels and changes in gene transcription on human WFS cell model under experimental ER stress. Methods We performed transcriptomic and proteomic analysis on WFS human cell model—skin fibroblasts reprogrammed into induced pluripotent stem (iPS) cells and then into neural stem cells (NSC) with subsequent ER stress induction using tunicamycin (TM). Results were cross-referenced with publicly available RNA sequencing data in hippocampi and hypothalami of mice with WFS1 deficiency. Results Proteomic analysis identified specific signal pathways that differ in NSC WFS cells from healthy ones. Next, detailed analysis of the proteins involved in the mitochondrial function showed the down-regulation of subunits of the respiratory chain complexes in NSC WFS cells, as well as the up-regulation of proteins involved in Krebs cycle and glycolysis when compared to the control cells. Based on pathway enrichment analysis we concluded that in samples from mice hippocampi the mitochondrial protein import machinery and OXPHOS were significantly down-regulated. Conclusions Our results show the functional and morphological secondary mitochondrial damage in patients with WFS. Graphical Abstract

scholarly journals Multiomic Analysis on Human Cell Model of Wolfram Syndrome Reveals Changes In Mitochondrial Morphology And Function

2021 ◽  
Author(s):  
Agnieszka Zmyslowska ◽  
Miljan Kuljanin ◽  
Beata Malachowska ◽  
Marcin Stanczak ◽  
Dominika Michalek ◽  
...  
Keyword(s):  
Er Stress ◽  
Human Cell ◽  
Proteomic Analysis ◽  
Protein Import ◽  
Cell Model ◽  
Mitochondrial Protein ◽  
Wolfram Syndrome ◽  
Mitochondrial Morphology ◽  
Pathway Enrichment Analysis ◽  
Signal Pathways

Abstract Background: Wolfram syndrome (WFS) is a rare autosomal recessive syndrome in which diabetes mellitus and neurodegenerative disorders occur as a result of wolframin deficiency and increased ER stress. In addition, WFS1 deficiency leads to calcium homeostasis disturbances and can change mitochondrial dynamics. The aim of this study was to evaluate protein levels and changes in gene transcription on human WFS cell model under experimental ER stress. Methods: We performed transcriptomic and proteomic analysis on WFS human cell model - skin fibroblasts reprogrammed into induced pluripotent stem (iPS) cells and then into neural stem cells (NSC) with subsequent ER stress induction using tunicamycin (TM). Results were cross-referenced with publicly available RNA sequencing data in hippocampi and hypothalami of mice with WFS1 deficiency. Results: Proteomic analysis identified specific signal pathways that differ in NSC WFS cells from healthy ones. Next, detailed analysis of the proteins involved in the mitochondrial function showed the down-regulation of subunits of the respiratory chain complexes in NSC WFS cells, as well as the up-regulation of proteins involved in Krebs cycle and glycolysis when compared to the control cells. Based on pathway enrichment analysis we concluded that in samples from mice hippocampi the mitochondrial protein import machinery and OXPHOS were significantly down-regulated. Conclusions: Our results show the functional and morphological secondary mitochondrial damage in patients with WFS.

scholarly journals Shot-Gun Proteomic Analysis on Roots of Arabidopsis pldα1 Mutants Suggesting New Roles of PLDα1 in Mitochondrial Protein Import, Vesicular Trafficking and Glucosinolate Biosynthesis

2018 ◽  
Author(s):  
Tomáš Takáč ◽  
Olga Šamajová ◽  
Pavol Vadovič ◽  
Tibor Pechan ◽  
Jozef Šamaj
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Responses ◽  
Proteomic Analysis ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Import ◽  
Mitochondrial Import ◽  
Enzymatic Production ◽  
Glucosinolate Biosynthesis ◽  
Vacuolar Protein

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. We present here a shot-gun differential proteomic analysis on roots of two pldα1 mutants compared to the Col-0 wild type. Our data suggest new roles of PLDα1 in endomembrane transport, mitochondrial protein import and protein quality control and glucosinolate biosynthesis. Thus, we identified proteins involved in endocytosis, endoplasmic reticulum-Golgi transport and attachment sites of endoplasmic reticulum and plasma membrane (V-type proton ATPases, protein transport protein SEC13 homolog A, vesicle-associated protein 1-2, vacuolar protein sorting-associated protein 29, syntaxin-32, all upregulated in the mutants), mitochondrial import and electron transport chain (mitochondrial import inner membrane translocase subunits TIM23-2 and TIM13, mitochondrial NADH dehydrogenases, ATP synthases, cytochrome c oxidase subunit 6b-1, ADP,ATP carrier protein 2, downregulated in the mutants) and glucosinolate biosynthesis (3-isopropylmalate dehydrogenases 1, 2 and 3, methylthioalkylmalate synthase 1, cytochrome P450 83B1, Glutathione S-transferase F9, indole glucosinolate O-methyltransferase 1, adenylyl-sulfate kinase 1, all upregulated in mutants). Our results suggest broader biological roles of PLDα1 as anticipated so far.

scholarly journals Accessing Mitochondrial Protein Import in Living Cells by Protein Microinjection

2021 ◽  
Vol 9 ◽  
Author(s):  
Andrey Bogorodskiy ◽  
Ivan Okhrimenko ◽  
Ivan Maslov ◽  
Nina Maliar ◽  
Dmitrii Burkatovskii ◽  
...  
Keyword(s):  
Real Time ◽  
Protein Import ◽  
Expression System ◽  
Mitochondrial Protein ◽  
Living Cells ◽  
Mitochondrial Morphology ◽  
Mitochondrial Protein Import ◽  
Intact Cells ◽  
Endogenous Expression ◽  
Cellular Environment

Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected pre-protein. This suggests that a substantial amount of mitochondrial translocase complexes was involved in co-translational protein import of endogenously expressed pre-proteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.

Loss of the mitochondrial Hsp70 functions causes aggregation of mitochondria in yeast cells

2001 ◽  
Vol 114 (19) ◽  
pp. 3565-3574 ◽  
Author(s):  
Akemi Kawai ◽  
Shuh-ichi Nishikawa ◽  
Aiko Hirata ◽  
Toshiya Endo
Keyword(s):  
Protein Synthesis ◽  
Elevated Temperature ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Yeast Cells ◽  
Mitochondrial Morphology ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitochondrial Matrix ◽  
Mitochondrial Hsp70

Ssc1p, a member of the Hsp70 family in the mitochondrial matrix of budding yeast, mediates protein import into mitochondria and prevents irreversible aggregation of proteins in the mitochondrial matrix during folding/assembly or at elevated temperature. Here, we show that functional inactivation of the mitochondrial Hsp70 system causes aggregation of mitochondria. When temperature-sensitive mitochondrial Hsp70 mutant cells were incubated at restrictive temperature, a tubular network of mitochondria was collapsed to form aggregates. Inhibition of protein synthesis in the cytosol did not suppress the mitochondrial aggregation and functional impairment of Tim23, a subunit of mitochondrial protein translocator in the inner membrane, did not cause mitochondrial aggregation. Therefore defects of the Hsp70 function in protein import into mitochondria or resulting accumulation of precursor forms of mitochondrial proteins outside the mitochondria are not the causal reason for the aberrant mitochondrial morphology. By contrast, deletion of Mdj1p, a functional partner for mitochondrial Hsp70 in prevention of irreversible protein aggregation in the matrix, but not in protein import into mitochondria, caused aggregation of mitochondria, which was enhanced at elevated temperature (37°C). The aggregation of mitochondria at 37°C was reversed when the temperature was lowered to 23°C unless protein synthesis was blocked. On the basis of these results, we propose that the mitochondrial matrix contains a protein that is responsible for the maintenance of mitochondrial morphology and requires mitochondrial Hsp70 for its function.

Altered mitochondrial morphology and defective protein import reveal novel roles for Bax and/or Bak in skeletal muscle

2013 ◽  
Vol 305 (5) ◽  
pp. C502-C511 ◽  
Author(s):  
Yuan Zhang ◽  
Sobia Iqbal ◽  
Michael F. N. O'Leary ◽  
Keir J. Menzies ◽  
Ayesha Saleem ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Mitochondrial Function ◽  
Protein Import ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Protein ◽  
Heat Shock Protein 90 ◽  
Mitochondrial Morphology ◽  
Double Knockout ◽  
Mitochondrial Protein Import ◽  
Protein Components

The function Bax and/or Bak in constituting a gateway for mitochondrial apoptosis in response to apoptotic stimuli has been unequivocally demonstrated. However, recent work has suggested that Bax/Bak may have unrecognized nonapoptotic functions related to mitochondrial function in nonstressful environments. Wild-type (WT) and Bax/Bak double knockout (DKO) mice were used to determine alternative roles for Bax and Bak in mitochondrial morphology and protein import in skeletal muscle. The absence of Bax and/or Bak altered mitochondrial dynamics by regulating protein components of the organelle fission and fusion machinery. Moreover, DKO mice exhibited defective mitochondrial protein import, both into the matrix and outer membrane compartments, which was consistent with our observations of impaired membrane potential and attenuated expression of protein import machinery (PIM) components in intermyofibrillar mitochondria. Furthermore, the cytosolic chaperones heat-shock protein 90 (Hsp90) and binding immunoglobulin protein (BiP) were markedly increased with the deletion of Bax/Bak, indicating that the cytosolic environment related to protein folding may be changed in DKO mice. Interestingly, endurance training fully restored the deficiency of protein import in DKO mice, likely via the upregulation of PIM components and through improved cytosolic chaperone protein expression. Thus our results emphasize novel roles for Bax and/or Bak in mitochondrial function and provide evidence, for the first time, of a curative function of exercise training in ameliorating a condition of defective mitochondrial protein import.

scholarly journals Accessing mitochondrial protein import in living cells by protein microinjection

2020 ◽  
Author(s):  
Andrey Bogorodskiy ◽  
Ivan Okhrimenko ◽  
Ivan Maslov ◽  
Nina Maliar ◽  
Dmitry Burkatovskiy ◽  
...  
Keyword(s):  
Real Time ◽  
Protein Import ◽  
Expression System ◽  
Mitochondrial Protein ◽  
Living Cells ◽  
Mitochondrial Morphology ◽  
Mitochondrial Protein Import ◽  
Intact Cells ◽  
Endogenous Expression ◽  
Cellular Environment

AbstractMitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected preprotein. This suggests that a substantial amount of mitochondrial translocase complexes were involved in co-translational protein import of endogenously expressed preproteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.

scholarly journals Role of Essential Genes in Mitochondrial Morphogenesis inSaccharomyces cerevisiae

2005 ◽  
Vol 16 (11) ◽  
pp. 5410-5417 ◽  
Author(s):  
Katrin Altmann ◽  
Benedikt Westermann
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Transport Processes ◽  
Essential Genes ◽  
26S Proteasome ◽  
Organelle Transport ◽  
Mitochondrial Morphology ◽  
Strain Collection ◽  
Mitochondrial Morphogenesis ◽  
Yeast Genes

Mitochondria are essential organelles of eukaryotic cells. Inheritance and maintenance of mitochondrial structure depend on cytoskeleton-mediated organelle transport and continuous membrane fusion and fission events. However, in Saccharomyces cerevisiae most of the known components involved in these processes are encoded by genes that are not essential for viability. Here we asked which essential genes are required for mitochondrial distribution and morphology. To address this question, we performed a systematic screen of a yeast strain collection harboring essential genes under control of a regulatable promoter. This library contains 768 yeast mutants and covers approximately two thirds of all essential yeast genes. A total of 119 essential genes were found to be required for maintenance of mitochondrial morphology. Among these, genes were highly enriched that encode proteins involved in ergosterol biosynthesis, mitochondrial protein import, actin-dependent transport processes, vesicular trafficking, and ubiquitin/26S proteasome-dependent protein degradation. We conclude that these cellular pathways play an important role in mitochondrial morphogenesis and inheritance.

scholarly journals Effect of p53 on mitochondrial morphology, import, and assembly in skeletal muscle

2015 ◽  
Vol 308 (4) ◽  
pp. C319-C329 ◽  
Author(s):  
Ayesha Saleem ◽  
Sobia Iqbal ◽  
Yuan Zhang ◽  
David A. Hood
Keyword(s):  
Skeletal Muscle ◽  
Mitochondrial Function ◽  
Protein Import ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Protein ◽  
Mitochondrial Morphology ◽  
Functional Impairments ◽  
Native Page ◽  
Complex Iv ◽  
Related Proteins

The purpose of this study was to investigate whether p53 regulates mitochondrial function via changes in mitochondrial protein import, complex IV (COX) assembly, or the expression of key proteins involved in mitochondrial dynamics and degradation. Mitochondria from p53 KO mice displayed ultra-structural alterations and were more punctate in appearance. This was accompanied by protein-specific alterations in fission, fusion, and mitophagy-related proteins. However, matrix-destined protein import into subsarcolemmal or intermyofibrillar mitochondria was unaffected in the absence of p53, despite mitochondrial subfraction-specific reductions in Tom20, Tim23, mtHsp70, and mtHsp60 in the knockout (KO) mitochondria. Complex IV activity in isolated mitochondria was also unchanged in KO mice, but two-dimensional blue native-PAGE revealed a reduction in the assembly of complex IV within the IMF fractions from KO mice in tandem with lower levels of the assembly protein Surf1. This observed defect in complex IV assembly may facilitate the previously documented impairment in mitochondrial function in p53 KO mice. We suspect that these morphological and functional impairments in mitochondria drive a decreased reliance on mitochondrial respiration as a means of energy production in skeletal muscle in the absence of p53.

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