National Repository of Grey Literature 10 records found  Search took 0.00 seconds. 
Impact of downregulation of gene expression of the peptidase subunit ClpP of the mitochondrial protease ClpXP on structure and function of mitochondria in human cells
Kolařík, Daniel ; Stibůrek, Lukáš (advisor) ; Pecina, Petr (referee)
Mitochondria are some of the most complex organelles of eukaryotic cell. They have their own genome and transcriptional apparatus and maintain several key cellular functions. A substantial part of cellular energetic metabolism happens in the mitochondria, as well as formation of iron-sulfur complexes, synthesis of several key molecules and they are also the essential organelles for the apoptotic pathway. In order to maintain the quality of proteins in their oxidative environment, mitochondria have developed a complex system of proteases that reaches all the mitochondrial compartments that degrade damaged proteins and thus promote mitochondrial turnover. The aim of this work was to characterise function of ClpP subunit of ClpXP matrix protease, which role was not yet extensively investigated in human cells. Therefore, we used RNA-interference to silence expression of ClpP in HEK 293 cells and then we performed rescue experiment during which we reintroduced ClpP in cells. Our results show that the ClpP subunit does not actively participate in apoptotic pathway, nevertheless it is essential for correct assembly of all the respiratory complexes as well as the quality of mitochondria itself. We have also shown that the system of mitochondrial proteases is highly functional and that a lack of ClpP...
Functional characterization of LACE1 APTase and mitochondrial AAA proteases YME1L and AFG3L2 in mitochondrial protein homeostasis.
Tesařová, Jana ; Stibůrek, Lukáš (advisor) ; Kalous, Martin (referee) ; Pecina, Petr (referee)
Mitochondrial protein homeostasis is crucial for cellular function and integrity. It is ensured by many specific mitochondrial proteases with possible chaperone functions located across the various mitochondrial subcompartments. In the first part, we have focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 and YME1L of the mitochondrial inner membrane complexes m- and i-AAA in HEK293 cells. The double AFG3L2/YME1L knockdown cells showed severe alteration in OPA1 protein processing, marked elevation in OMA1 protease and severe reduction in SPG7. Our results reveal cooperative and partly redundant involvement of AFG3L2 and YME1L in the maintenance of mitochondrial protein homeostasis and further emphasize their importance for mitochondrial and cellular function and integrity. The aim of the second part was to characterize the cellular function of LACE1 (lactation elevated 1) in mitochondrial protein homeostasis. LACE1 protein is a human homologue of yeast Afg1 (ATPase family gene 1) ATPase. We show that LACE1 is a mitochondrial integral membrane protein that exists as a part of three complexes of approximately 140, 400 and 500 kDa. We demonstrate that LACE1 mediates degradation of nuclear-encoded complex IV subunits COX4, COX5A and COX6A. Using affinity...
The role of tissue specific isoforms of subunit 4 in assembly and function of cytochrome c oxidase
Čunátová, Kristýna ; Pecina, Petr (advisor) ; Stibůrek, Lukáš (referee)
Oxidative phosphorylation apparatus (OXPHOS) is responsible for production of majority of ATP in mammalian organisms. This process, occurring in the inner mitochondrial membrane, is partly regulated by nuclear-encoded subunits of cytochrome c oxidase (COX), the terminal enzyme of electron transport chain. Cox4 subunit, participating in OXPHOS regulation, is an early-assembly state subunit, which is necessary for incorporation of Cox2 catalytic subunit, thus for assembly of catalytically functional COX enzyme. Moreover, regulated expression of two isoforms (Cox4i1, Cox4i2) of Cox4 subunit is hypothesized to optimize respiratory chain function according to tissue oxygen supply. However, the functional impact of the isoform switch for mammalian tissues and cells is still only partly understood. In the present thesis, unique HEK293 cell line-based model with complete absence of subunit Cox4 (knock-out, KO) was prepared employing novel CRISPR CAS9-10A paired nickase technology and further characterized. Knock-out of both isoforms Cox4i1 and Cox4i2 (COX4i1/4i2 KO clones) showed general decrease of majority of Cox subunits resulting in total absence of fully assembled COX. Moreover, detected Complex I subunits as well as the content of assembled Complex I were decreased in COX4i1/4i2 KO clones. On the...
The function of p53 protein in mitochondria
Magdálková, Kateřina ; Stibůrek, Lukáš (advisor) ; Kalous, Martin (referee)
Protein p53 is known as a tumor supressor. In nucleus, p53 regulates the expression of its target genes, which are involved in cell cycle control, DNA repair and cell death. Protein p53 also has transcription-independent activities outside the nucleus. Certain amount of this protein can be found in mitochondria, where it is involved in mitochondrial genom integrity maintaining. Under stress conditions, p53 rapidly translocates to outer mitochondrial membrane, and takes a part in apoptotic signalling pathway. Keywords: p53, mitochondria, mtDNA, apoptosis
Regulation and Disorders of Mammalian Cytochrome c Oxidase
Kovářová, Nikola ; Houštěk, Josef (advisor) ; Stibůrek, Lukáš (referee) ; Kalous, Martin (referee)
Cytochrome c oxidase (COX) represents the terminal enzyme complex of respiratory chain metabolic pathway and it occurs as monomer, dimer or as a part of respiratory supercomplexes in the inner mitochondrial membrane. COX assembly process is complicated, highly regulated and depends on many ancillary proteins. Mutations in COX subunits, which are encoded by mitochondrial and nuclear DNA, or in genes encoding its assembly proteins are frequent cause of very severe mitochondrial disorders. SURF1 assembly protein participates in the first steps of COX assembly, but its exact function is not yet clarified. In humans, mutations of SURF1 gene lead to severe COX defect and fatal neurodegenerative disorder, Leigh syndrome. Knockout of SURF1 gene in mouse causes isolated COX defect as well, but less pronounced and without involvement of CNS. The aim of the thesis was detailed analysis of disturbed COX biogenesis in a condition of SURF1 gene mutations or SURF1 gene knockout, from assembly of COX monomer to interaction of COX into supercomplexes, and to the impact of isolated COX defect on other OXPHOS complexes. Mutations of SURF1 gene in patient's fibroblasts led to marked accumulation of COX assembly intermediates and to a defect in formation of functional COX monomer, which was preferentially built into an...
Mitochondrial proteases and their role in biogenesis of mitochondria
Krump, Ondřej ; Stibůrek, Lukáš (advisor) ; Pecina, Petr (referee)
Mitochondria are organelles of endosymbiotic origin responsible for many cellular functions, including bioenergetics, biosynthesis and apoptosis. Regulated protein turnover is crucial for proper mitochondrial function. It is controlled by cellular proteolytic system, especially by its mitochondrial part. This mitochondrial proteolytic system is comprised of severeal groups of proteases. The best characterized AAA+ proteases constitute hollow oligomeric complexes, in which the proteolytic domains are localized. Access to these domains is dependent on unfolding - an energy-consuming process driven by ATP hydrolysis mediated by ATPase domains of AAA+ protein. The main function of AAA proteases is proteolytic degradation of proteins, a part of quality control system of mitochondrial proteins. AAA proteases are localized freely in mitochondrial matrix (Lon and ClpXP), or anchorred in the inner membrane (i-AAA and m-AA). Processing peptidases cleave off the mitochondrial targetting sequences of nuclearly encoded mitochondrial proteins. Oligopeptidases cleave peptides produced by processing and proteolytical degradation to single amino acids. Incorrect function of various components of mitochondrial proteolytical system is implicated in several diseases, including certain forms of hereditary spastic paraplegia...
The role of human Sco1, Sco2, Surf1 and Oxa1l in the biogenesis of the oxidative phosphorylation system
Stibůrek, Lukáš
This thesis was prepared in the Laboratory for study of mitochondrial disorders at the Department of Pediatrics, First Faculty of Medicine, Charles University in Prague. The study of human mitochondrial biogenesis, particularly of the oxidative phosphorylation system (OXPHOS), and its pathologies has seen remarkable progress in past two decades. The knowledge of the complete sequence of the human genome, together with the results of elegant yeast studies aimed at the identification of respiratory important gene products, enabled us to identify and study the molecular and biochemical bases of numerous human mitochondrial pathologies. These studies not only continue to reveal the underlying disease mechanisms but shed completely new light on the various processes of mitochondrial biogenesis and function in humans. The work presented in this thesis was aimed mainly at the understanding of the various roles of nuclear-encoded mitochondrial inner membrane proteins Sco1, Sco2, Surf1 and Oxa1l in the assembly and/or maintenance of the five multimeric complexes of the human OXPHOS system. The presented data demonstrate that human Sco1 and Sco2 are involved, in a highly tissue-specific manner, in distinct posttranslational steps of maturation of the cytochrome c oxidase (CcO) subunit 2 (Cox2). Furthermore,...
The Role of Human Sco1, Sco2, Surf1 and Oxa1l in the Biogenesis of the Mitochondrial Oxidative Phosphorylation System
Stibůrek, Lukáš ; Zeman, Jiří (advisor) ; Entlicher, Gustav (referee) ; Pelouch, Václav (referee)
Disertační práce byla vypracována v Laboratoři pro studium mitochondriálních poruch, Kliniky dětského a dorostového lékařství a 1.LF UK v Praze. Studium mitochondriální biogeneze a jejích poruch, a to především s ohledem na biogenezi systému oxidativní fosforylace (OXPHOS), zaznamenalo v uplynulém desetiletí mimořádný pokrok. Znalost kompletní sekvence lidského genomu, spolu s výsledky elegantních kvasinkových studií zaměřených na identifikaci respiračně významných genových produktů, dovolila molekulární identifikaci a následné studium biochemické podstaty celé řady lidských mitochondriálních patologií. Tyto studie odhalují molekulární a biochemické mechanismy etiopatogeneze těchto chorob a současně přinášejí nové unikátní informace o lidské mitochondriální biogenezi jako takové. Výsledky a původní publikace prezentované v této disertační práci se týkají snahy o pochopení molekulárních rolí lidských asemblačních faktorů Sco1, Sco2, Surf1 a Oxa1l v biogenezi multimerních membránových komplexů lidského systému OXPHOS. Prezentovaná data ukazují, že lidské asemblační faktory Sco1 a Sco2 fungují vysoce tkáňově specifickým způsobem na úrovni odlišných posttranslačních kroků maturace podjednotky Cox2 cytochrom c oxidázy (CcO). Prezentovaná data dále naznačují, že oba výše zmíněné lidské SCO proteiny, a asemblační...

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