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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...
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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...
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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...
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The role of c14orf2 protein in structure and function of mammalian ATP synthase
Ho, Dieu Hien ; Pecina, Petr (advisor) ; Panicucci Zíková, Alena (referee)
The F1Fo-ATP synthase (EC 3.6.3.14) is a key enzyme of the mitochondrial oxidative phosphorylation system (OXPHOS) - using the proton gradient generated by the respiratory chain it synthetizes approximately 90 % of cellular ATP. The subunit arrangement of its Fo domain has not been yet described in detail. At present, the research on ATP synthase research is focused mostly on revealing the structure of the proton channel a so that it is possible to precisely define the molecular mechanism of the ATP synthase rotation generation. The role of the supernumery subunits of Fo domain represents another unresolved issue. These proteins specific for eukaryotic ATP synthases are not essential for synthetic activity, instead they are putatively involved in assembly or stabilization of the enzyme complex. One of such subunits is the nuclear encoded MLQ protein (or also 6.8 kDa proteolipid or MP68), which is conserved only in vertebrates. The aim of this diploma thesis was to reveal the role of this subunit in the structure, assembly and function of the F1Fo-ATP synthase. For these purposes, cellular model of the HEK293 line with the deficiency of the MLQ protein was established employing the CRISPR/Cas9 method with paired nickases (the knock-out MLQ, MLQ KO) as part of the thesis. Three chosen MLQ KO lines...
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Mitochondrial beta-lactamase and its role in humans
Baudyšová, Alžběta ; Šácha, Pavel (advisor) ; Pecina, Petr (referee)
Cancer is one of the most frequent causes of death. Fortunately, human body has a number of various mechanisms that protect cells from tumorigenic transformation. One of those mechanisms are tumor suppressor genes. The latest described tumor suppressor gene encodes LACTB protein. LACTB is the mammalian homolog of bacterial beta-lactamases and penicillin binding proteins (PBPs). PBPs are involved in construction of bacterial cell walls (specifically in the synthesis of peptidoglycan) and they could be inhibited by penicillin antibiotics. Beta-lactamases are able to break the beta-lactam ring of penicillin and provide resistance to the antibiotics. The main topic of this work will be the LACTB protein. LACTB is localizated in the intermembrane space of mammalian mitochondria. Here it forms filaments whose physiological function still remains unknown. LACTB, apart from its connection with cancer, was also associated with obesity and penicillin allergy. Main focus of this work will be to gather all known information about the LACTB protein and put them into a wider context.
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Role of nuclear-encoded subunits of cytochrome c oxidase
Čunátová, Kristýna ; Pecina, Petr (advisor) ; Kalous, Martin (referee)
Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain and its main function is transfer of electrons to the terminal acceptor, oxygen. Moreover, COX contributes to the generation of proton gradient, which is indispensable for the production of vast majority of ATP molecules in the mammalian cells. The present work summarizes available data concerning the structure and function of nuclear-encoded subunits, which are specific for the eukaryotic form of COX. Particular emphasis is put on the role of subunit Cox4 and its regulatory properties within the enzyme. The study also describes other proteins associated with the enzyme, which are able to regulate the activity of COX, but are mainly involved in formation and stabilization of the respiratory supercomplexes. Based on the summarized data, three levels model of quaternary COX structure is postulated. They correspond to subunits of the catalytic center, nuclear-encoded subunits and substoichiometrically asociated proteins, which may give rise to several forms of COX with varying composition and differentially regulated function.
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New components and functions of mitochondrial ATP synthase.
Ho, Dieu Hien ; Pecina, Petr (advisor) ; Kalous, Martin (referee)
The system of oxidative phosphorylation, or respiratory chain in mitochondria gives the eukaryotic cell total majority of the energy it receives and uses in the form of ATP. F1Fo-ATP synthase, powered by the proton-motive force is directly responsible for the ATP synthesis. Diseases connected to the ATP synthesis can have even lethal consequences. There is therefore no doubt about the need for a detailed analysis of the structure of this enzyme. What is left is to reveal the structure of the transmembrane domains, which are not involved in the synthesis itself, but they can for example work as stabilisers or assembly factors. Outside the synthesis activity the dimers of F1Fo-ATP synthase are apparently taking part in the formation of the cristae of the inner membrane of a mitochondrion. Recently, the role of the enzyme is also considered in the creation of the mitochondrial permeability transition pore.
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Mitochondria as a target for anti-cancer therapy by vitamin E analogues.
Kľučková, Katarína ; Neužil, Jiří (advisor) ; Hyršlová Vaculová, Alena (referee) ; Pecina, Petr (referee)
(EN) Based on the promising results concerning the anti-cancer properties of redox-silent analogue vitamin E α-tocopheryl succinate (α-TOS), we prepared its mitochondrially targeted derivative MitoVES by attaching the positively charged triphenylphosphonium (TPP+ ) tag to α-TOS molecule. We tested the hypothesis that 'sending' the drug directly to its cellular site of action, mitochondria, should enhance its anti-cancer properties, which would result in more effective anti-cancer agent while making it possible to reduce the effective concentration. We provide evidence that, indeed, MitoVES is a highly effective anti-cancer compound, superior to untargeted α-TOS both in vitro and in vivo. We show that MitoVES exerts its anti-cancer effects by interfering with complex II (CII) activity specifically at the ubiquinone binding site (Qp), where it blocks further electron transfer resulting in increased reactive oxygen species (ROS) production, which then leads to apoptosis induction via the intrinsic mitochondrial pathway, preferentially engaging the pro-apoptotic Bak protein causing mitochondrial membrane permeabilisation. We further show that mitochondrial targeting on the basis of higher mitochondrial membrane potential (ΔΨ) is important for MitoVES pro-apoptotic activity. This feature endows the...
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