|
|
Mitochondria as a target of anticancer therapy.
Dvořák, Aleš ; Ježek, Petr (advisor) ; Poučková, Pavla (referee) ; Vecka, Marek (referee)
Mitochondrial isocitrate dehydrogenase 2 (IDH2) catalyzes reductive carboxylation (RC, reverse Krebs cycle pathway) and 2HG synthesis (2HG) - metabolite of which many scientists are interested. 2HG may be concurrently synthetized in cytosol by IDH1. RC is involved in anabolic reactions necessary for cell proliferation - produces citrate, fatty acid precursor - especially in hypoxia. IDH2 and IDH1 are not the only enzymes that are involved in 2HG synthesis. Recently, several enzymes, which participate in 2HG production, have been discovered. 2HG is useful in cancer diagnostics due to its overproduction by transformed cells. Moreover, 2HG may cause epigenetic changes via inhibition of 2-oxoglutarate dependent dioxygenase. In this work, the importance of RC and 2HG synthesis in cancer and healthy cells was investigated by gas chromatography with mass spectrometry detection as well as IDH2 influence. We found that IDH2 significantly participates in reverse RC and 2HG synthesis in breast cancer cell lines and uses glutaminolysis as a supplementary anaplerotic pathway. RC is increased by hypoxia, inhibition of respiration, and decreased by activation of respiration or hypocapnia. We confirmed 2HG synthesis and RC in healthy cells (fibroblasts, breast epithelial cells etc.) as well as in cancer cells....
|
|
|
Quantitative aspects of mitochondrial genome in cancer and diabetes
Alán, Lukáš ; Ježek, Petr (advisor) ; Teisinger, Jan (referee) ; Modrianský, Martin (referee)
Mitochondria are cellular powerhouses and physiological regulators with many features resembling their prokaryotic ancestors. They maintain their own mitochondrial DNA (mtDNA) encoding 13 inner mitochondrial membrane proteins of oxidative phosphorylation machinery, 22 transfer RNAs and two ribosomal RNAs. Healthy mitochondria in normal cells form a dynamic network consisting of highly interconnected tubules. The mitochondrial disorders are very heterogeneous and difficult to diagnose and cure. It is due to combining products of two genomes, the complexity of mitochondrial structure and due to insufficiency of current state of knowledge. To understand the mitochondrial nucleic acid species distribution, we have developed and established new techniques to visualize mitochondrial network, nucleoids and different RNA species together with qPCR techniques for monitoring the mitochondrial intactness. We determined nucleoid distribution and mtDNA amount following rotenone mediated respiratory inhibition or following degeneration of the electrical component of the protonmotive force by valinomycin treatment. Native mitochondria were mostly tubular with average nucleoid spacing 1.1 +/- 0.2µm which we termed as a nucleoid code. Subsequently induced fission resulted in mitochondrial network fragmentation and each...
|
|
| |
|
|
Mitochondrial RNA in mammalian cell
Balvín, Sebastian ; Ježek, Petr (advisor) ; Magner, Martin (referee)
Mitochondrion is an important organelle maintaining energy metabolism of the cell and participating in signalization, cell cycle and apoptosis. It's pathology causes several diseases. Replication, transcription and translation take place in mitochondria, similarly like in nucleus, though there are only 13 protein coding genes. However, these processes, as well as mitochondrial DNA and RNA, vary significantly from those present in nucleus. Mitochondrial DNA is circular and both strands are replicated separately. Mitochondria form polycistronic transcripts, which are subsequently processed by tRNA. Mitochondrial ribosome evolved from prokaryotic one, but contains only half as much rRNA. Missing rRNAs are replaced by ribosomal proteins. These ribosomes contain even more proteins compared to much larger cytoplasmatic eukaryotic ones. This work is focused on current topic of mitochondrial molecular genetics: mitochondrial rRNA and ribosome, especially ribosomal assembly. In this process mitochondrial rRNAs interact with nuclear encoded proteins. The whole process probably takes place on the inner mitochondrial membrane close to the nucleoid. Our understanding to whole mechanism can help us to find a way how to cure mitochondrial pathologies.
|
|
| |
|
| |
|
|
Mitochondrial uncoupling and mitochondrial morphology in relation to type-2 diabetes
Špaček, Tomáš ; Ježek, Petr (advisor) ; Kalous, Martin (referee) ; Vařecha, Miroslav (referee)
- 2 - 1 SUMMARY Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic -cells. Using high resolution respirometry and parallel mitochondrial membrane potential ( m) monitoring we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model for studies of insulin secretion in vitro. Upon glucose addition to glucose-depleted cells, we have demonstrated a simultaneous increase in respiration and m during GSIS and shown that the endogenous state 3/state 4 respiratory ratio hyperbolically increases with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the "toxic" effect of fatty acids on insulin secretion, GSIS has been studied after linoleic acid addition. The linoleic acid addition diminished the observed respiration increase, m jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose, which was caused by mitochondrial uncoupling. Energetic status of mitochondria, the rate of oxidative phosphorylation, is expressed not only by bioenergetic parameters measurable by classical biophysical methods (e.g. above mentioned respiration and...
|
|
|
Distribution of mitochondrial uncoupling proteins in selected tissues from mice and rat
Alán, Lukáš ; Flachs, Pavel (referee) ; Ježek, Petr (advisor)
Mitochondrial uncoupling proteins (UCPs) belong to the superfamily of mitochondrial anion-carriers. The longest known is UCP1, predominantly expressed in brown adipose tissue, where it takes part in nonshivering thermogenesis. In the late 1990s were discovered other sequence homologs of UCP1 with tissue specific distribution. The Function of these "new" uncoupling proteins is still uncertain. It is assumed that each of the isoforms has a specific function depending on the type of tissue. This thesis showed differences in tissue transcription pattern between rat and mice using RT-PCR absolute quantification. Significant differences in pattern were found in lungs, brain and muscle. In each case UCP expression was higher in mice tissues. Mice lungs express mainly UCP2. The difference in mice brain is caused by ucp4 and ucp5 genes transcription and finally in muscle is highest content of UCP3 mRNA. We investigated whether any of ucp transcript can complement ucp2 transcripton in spleen or lungs of ucp2 -/- mice. We did not find any difference which can explain, that in isolated lung mitochondria of fasted ucp2-/- mice were uncoupled in state 4. In the last project, we found relationship between ucp2 transcription in insulinoma INS-1E cells and oxygen levels of the cultivation atmosphere.
|
|
|
Non-canonical Bioenergetics of the Cell
Smolková, Katarína ; Ježek, Petr (advisor) ; Štukavec, Jan (referee) ; Lukeš, Julius (referee)
Cancer cells generally present abnormal bioenergetic properties including an elevated glucose uptake, a high glycolysis and a poorly efficient oxidative phosphorylation system. However, the determinants of cancer cells metabolic reprogramming remain unknown. The main question in this project was how environmental conditions in vivo can influence functioning of mitochondrial OXPHOS, because details of mitochondrial bioenergetics of cancer cells is poorly documented. We have combined two conditions, namely glucose and oxygen deprivation, to measure their potential interaction. We examined the impact of glucose deprivation and oxygen deprivation on cell survival, overall bioenergetics and OXPHOS protein expression. As a model, we have chosen a human breast carcinoma (HTB-126) and appropriate control (HTB-125) cultured cells, as large fraction of breast malignancies exhibit hypoxic tumor regions with low oxygen concentrations and poor glucose delivery. (...) Apoptosis is a natural, genetically controlled process of cell elimination. The mechanisms of its activation and regulation is a fundamental scientific question and growing body of evidence reveal further molecular pathways of apoptotic machinery. The well-known caspase activation cascade along with pro- and anti-apoptotic members of BCL family is the basic...
|
|
|
The role of CIDE proteins in apoptosis and mitochondrial network morphology under pathogenetic conditions
Šantorová, Jitka ; Ježek, Petr (advisor) ; Teisinger, Jan (referee) ; Kalous, Martin (referee)
Cď deďh-indrcing DFFIDNA fragmentationfactor]-likeeffector-a(CIDEa)' may initiďe 4optosis by disruptinga complex consisting of 40-kDa caspase-3-aďvďed nucleaseDFF40 md its 45.kDa inhibitor DFF45. We measured Íhe levels of mRNA CIDEa in rattus norvegicus tissues and deteďed high lwels of RNA in white aďpose tiszue. We have confirmedthepresenceof CIDEa in mitoohon&ia andimportanceof CIDE-C domain for this localization. We havealso performedimmrrnodetectionof subcellular&actionsofHela cells adapted for a tetracycline-inducible CIDEa expression. We have observed redistribution, enhancedupon treatuotrt with camptothecinor valinomycin, of CIDEa to nucleus. CIDEa contentincreasď in the nuclear fractionbut decreasedin oýosolio fraction in cells heatedto initiaÍe4optosis. We hypothesizethatCIDEa is sequesteredin mitochondriawhile transferof this pote,ntiallydangerousprotein from mitochondriainto nucleusintensifies or even initiates apoptosis. Mitochondria in nrrmerouscell types,especiďly in culturedcells, form a reticula network undergoing constant fusion and fission. The tbree dimensionď (3D) morphology of these networks however has not been stuďed in detail to our knowledge. We have investigated insďinoma INS-IE and hepatocellular oarcinoma HEP-G2 cells hansGoted \Yith mitoohondria-addressedGFP. Using 4Pi...
|
guest ::
RSS feed.