Dihydroorotate dehydrogenase (DHODH) is an enzyme of the de novo pyrimidine synthesis pathway that provides nucleotides for RNA/DNA synthesis essential for proliferation. In mammalian cells, DHODH is localized in mitochondria, linked to the respiratory chain via the coenzyme Q pool. Here we discuss the role of DHODH in the oxidative phosphorylation system and in the initiation and progression of cancer. We summarize recent findings on DHODH biology, the progress made in the development of new, specific inhibitors of DHODH intended for cancer therapy, and the mechanistic insights into the consequences of DHODH inhibition.

• Klíčová slova
• Bioenergetics, Cancer, De novo pyrimidine synthesis, Dehydroorotate dehydrogenase, Oxidative phosphorylation,
• MeSH
• dihydroorotátdehydrogenasa MeSH
• inhibitory enzymů terapeutické užití   MeSH
• lidé MeSH
• mitochondrie genetika   metabolismus   MeSH
• nádory genetika   patologie   MeSH
• oxidativní fosforylace * MeSH
• oxidoreduktasy působící na CH-CH vazby antagonisté a inhibitory   genetika   MeSH
• proliferace buněk účinky léků   MeSH
• transport elektronů genetika   MeSH
• ubichinon analogy a deriváty   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• coenzyme Q10 MeSH Prohlížeč
• dihydroorotátdehydrogenasa MeSH
• inhibitory enzymů MeSH
• oxidoreduktasy působící na CH-CH vazby MeSH
• ubichinon MeSH

Annexin A1 (ANXA1) is the first characterized member of the annexins superfamily. It binds the cellular membrane phospholipids in Ca(2+) regulated manner. Annexin A1 has been found in several tissues and many physiological roles as hormones secretion, vesiculation, inflammatory response, apoptosis and differentiation have been shown. Its subcellular localization and binding with many partner proteins are altered accordingly with its physiological role. The Annexin A1 membrane localization is crucial for binding to receptors, suggesting a paracrine and juxtacrine extracellular action. Annexin A1 is subjected to several post-translational modifications. In particular the protein is phosphorylated on several residues both on the N-terminal functional domain and on the C-terminus core. Different kinases have been identified as responsible for the phosphorylation status of selective residues. The specific change in the phosphorylation status on the different sites alters ANXA1 localization, binding properties and functions. This review shows the physiological relevance of the ANXA1 phosphorylation leading to the conclusion that numerous and different roles of Annexin A1 could be associated with different phosphorylations to alter not only intracellular localization and bindings to its partners but also the extracellular receptor interactions.

• Klíčová slova
• ANXA1, Annexin A1, EGFR, PKA, PKC, Phosphorylation,
• MeSH
• aminokyseliny metabolismus   MeSH
• annexin A1 metabolismus   MeSH
• biologické modely MeSH
• buněčná membrána metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• aminokyseliny MeSH
• annexin A1 MeSH

The evolutionarily conserved Swi5-Sfr1 complex plays an important role in homologous recombination, a process crucial for the maintenance of genomic integrity. Here, we purified Schizosaccharomyces pombe Swi5-Sfr1 complex from meiotic cells and analyzed it by mass spectrometry. Our analysis revealed new phosphorylation sites on Swi5 and Sfr1. We found that mutations that prevent phosphorylation of Swi5 and Sfr1 do not impair their function but swi5 and sfr1 mutants encoding phosphomimetic aspartate at the identified phosphorylation sites are only partially functional. We concluded that during meiosis, Swi5 associates with Sfr1 and both Swi5 and Sfr1 proteins are phosphorylated. However, the functional relevance of Swi5 and Sfr1 phosphorylation remains to be determined.

• Klíčová slova
• DNA repair, Schizosaccharomyces pombe, Sfr1, Swi5, meiosis, phosphorylation, recombination,
• MeSH
• fosforylace MeSH
• homologní rekombinace * MeSH
• meióza MeSH
• oprava DNA * MeSH
• poškození DNA * MeSH
• Schizosaccharomyces pombe - proteiny genetika   metabolismus   MeSH
• Schizosaccharomyces genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Schizosaccharomyces pombe - proteiny MeSH
• Sfr1 protein, S pombe MeSH Prohlížeč
• Swi5 protein, S pombe MeSH Prohlížeč

Phosphorylated peptides are instrumental in studying protein phosphorylation events. In the present study, Raman optical activity (ROA) is employed to elucidate the structure of a dipeptide, L-alanyl-L-glutamine (L-Ala-L-Gln) and its two differently alkylated N-phosphorylated derivatives. Theoretical simulations were conducted to aid the interpretation of peptide conformation variations upon phosphorylation, and of the measured Raman and ROA spectra. Induced circularly polarized luminescence (CPL) was also recorded in solution, in the presence of a simple europium aqua ion. As the spectra are peptide specific, this type of stereochemical analysis is expected to aid identification of the phosphorylation sites also in other peptides and possibly proteins.

• Klíčová slova
• Biomolecular spectroscopy, Circularly polarized luminescence, Molecular dynamics, Peptide phosphorylation, Raman optical activity,
• MeSH
• dipeptidy * chemie   MeSH
• fosforylace MeSH
• molekulární modely MeSH
• Ramanova spektroskopie * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alanylglutamine MeSH Prohlížeč
• dipeptidy * MeSH

Analogs of carbonyl cyanide phenylhydrazone providing no reaction with nucleophilic groups and lacking acidobasic properties, respectively, were synthesized for study of mechanism of uncoupling effect on oxidative phosphorylation. Their retention, influence on proton transport, abilities to SH--groups modify and to stimulate respiration in rat liver mitochondria, together with their physico-chemical properties, namely lipophilicity, acidobasicity and reactivity were characterized. The substitution of acidic hydrogen of the imino group resulted in the loss of both acidobasicity and uncoupling effect on oxidative phosphorylation. A decreased reactivity resulted from the substitutions of nitrile groups with the uncoupling activity remaining preserved.

• MeSH
• jaterní mitochondrie účinky léků   metabolismus   MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• krysa rodu Rattus MeSH
• nitrily farmakologie   MeSH
• oxidativní fosforylace účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• nitrily MeSH

Cancer cell bioenergetics, maintaining mixed aerobic glycolysis (Warburg phenotype) and oxidative phosphorylation (OXPHOS), is not fully elucidated. Hypoxia-dependent OXPHOS suppression determines aerobic glycolysis. To elucidate further details, we studied hypoxic adaptation (up to 72 h at 5 % oxygen) of hepatocellular carcinoma HepG2 cells. The key regulatory component, hypoxia-inducible factor (HIF)-1α (HIF-1α) was stabilized at 5 h in 5 % oxygen for all three studied regimens, i.e. in glycolytic cells at 5 mM or 25 mM glucose, or in aglycemic (OXPHOS) cells when glucose was replaced by galactose. However, the conventional HIF-mediated suppression of respiration was prevented at aglycemia, which correlated with a high proportion of unphosphorylated pyruvate dehydrogenase (PDH) at 5 % oxygen. Such a modified HIF response in OXPHOS cells, termed as a non-canonical one, contrasted to conventional respiration suppression down to 45 % or 43 %, observed in hypoxia-adapted glycolytic cells at 5 mM or 25 mM glucose, respectively. These hypoxic glycolytic cells had normally highly phosphorylated PDH and most likely utilized pyruvate by aminotransferase reaction of glutaminolysis to feed at least suppressed respiration. Also, glycolytic cells were rather resistant towards the staurosporine-induced apoptosis, whereas aglycemic (OXPHOS) HepG2 cells exhibited much higher susceptibility. We conclude that aglycemia modulates the hypoxic HIF signaling toward a non-canonical response that is unable to carry out complete PDH phosphorylation, allowing a high pyruvate input for OXPHOS from the elevated glycolysis, which together with ongoing glutaminolysis maintain a virtually unchanged respiration. Similar OXPHOS revival may explain distinct tumor sensitivity to chemotherapy and other pharmacological interventions.

• Klíčová slova
• Cancer mitochondria, Glutaminolysis, HepG2 cells, Hypoxia-inducible factor, Non-canonical response to hypoxia,
• MeSH
• buněčné kultury MeSH
• buňky Hep G2 MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa metabolismus   MeSH
• hypoxie buňky MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• oxidativní fosforylace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktor 1 indukovatelný hypoxií - podjednotka alfa MeSH

The effects of phloretin and carbonyl-cyanide-m-chlorophenylhydrazone (CCCP), both inhibitors of oxidative phosphorylation, on renal urea excretion in Wistar rats were investigated. Phloretin and CCCP infusions did not influence plasma urea concentration (P(urea)), compared with controls (0.15 M NaCl and Tris solution in 0.15 M NaCl-a solvent for phloretin and CCCP). The fractional urea excretion (FEurea) was not altered by phloretin infusion. It decreased significantly only when compared with 0.15 M NaCl infusion (P < 0.05). CCCP infusion had no effect on FEurea. The total amount of urea excreted by urine (UureaV) was not altered by phloretin compared with controls. CCCP significantly enhanced Uurea V only when compared with 0.15 M NaCl (P < 0.001), not when compared with Tris. Glomerular Filtration rate (GFR) increased significantly during phloretin infusion (P < 0.001), CCCP (P < 0.001) and also after Tris in 0.15 M NaCl (P < 0.001), in comparison with 0.15 M NaCl alone. Our results showed that phloretin and CCCP had no effect on urea excretion in rats. The increase in GFR is attributed to Tris, not to phloretin or CCCP. It is concluded that inhibition of oxidative phosphorylation in kidney has no effect on urea excretion.

• MeSH
• floretin farmakologie   MeSH
• hodnoty glomerulární filtrace MeSH
• karbonylkyanid-m-chlorfenylhydrazon farmakologie   MeSH
• krysa rodu Rattus MeSH
• ledviny účinky léků   fyziologie   MeSH
• močovina krev   moč   MeSH
• oxidativní fosforylace účinky léků   MeSH
• potkani Wistar fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• floretin MeSH
• karbonylkyanid-m-chlorfenylhydrazon MeSH
• močovina MeSH

Phosphorylation, or dephosphorylation, is one of the most frequent post-translational modifications regulating protein-protein activity in eukaryotic cells. Whereas mature spermatozoa (as specialized cells) are transcriptionally inactive and do not synthesize new proteins, phosphorylation of sperm proteins is very important for the regulation of the sperm function. Although the post-testicular maturation of spermatozoa is a process common to all mammals, comparative studies showed significant differences in sperm surface proteins and the mechanisms of protein modification during the epididymal maturation. In our study, the evaluation of tyrosine phosphorylation, represented by the fluorescent patterns of used anti-phosphotyrosine antibodies (P-Tyr-01 and 4G10), in spermatozoa isolated from different regions of the epididymis - caput, corpus and cauda - was performed. Although in general both antibodies detected almost the same reaction patterns, we observed some dissimilarity associated with the binding specificity of the antibodies and also the segment-dependent manner of phosphorylated protein localization. These data were filled up by immunohistochemical analysis of testes and epididymides cryosections. Additionally, our phosphoproteomic study focused on evaluation of the changes in the pattern of tyrosine-phosphorylated proteins during the post-testicular maturation of bull spermatozoa (PY20 antibody). To summarize the results, an increasing trend of tyrosine phosphorylation of proteins during the maturation of bull sperm in the epididymis was consistently observed in all the methods/experiments.

• Klíčová slova
• Anti-phosphotyrosine antibodies, Bull sperm, Epididymis, Protein phosphorylation, Sperm maturation,
• MeSH
• epididymis cytologie   MeSH
• fluorescence MeSH
• fosforylace MeSH
• fosfotyrosin metabolismus   MeSH
• proteiny metabolismus   MeSH
• skot MeSH
• spermie cytologie   metabolismus   MeSH
• testis cytologie   MeSH
• zrání spermie * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfotyrosin MeSH
• proteiny MeSH

The enzymes involved in Euglena oxidative phosphorylation (OXPHOS) were characterized in this study. We have demonstrated that Euglena gracilis strain Z and its stable bleached non-photosynthetic mutant strain WgmZOflL both possess fully functional OXPHOS apparatus as well as pathways requiring terminal alternative oxidase(s) and alternative mitochondrial NADH-dehydrogenase(s). Light (or dark) and plastid (non)functionality seem to have little effect on oxygen consumption, the activities of the enzymes involved in OXPHOS and the action of respiration inhibitors in Euglena. This study also demonstrates biochemical properties of complex III (cytochrome c reductase) in Euglena.

• Klíčová slova
• Bleaching, Euglenozoa, Mitochondria, Respiratory chain, Trypanosomatids,
• MeSH
• elektronový transportní řetězec chemie   metabolismus   MeSH
• Euglena gracilis enzymologie   genetika   MeSH
• mitochondrie enzymologie   MeSH
• mutace MeSH
• oxidativní fosforylace * MeSH
• protozoální proteiny chemie   metabolismus   MeSH
• spotřeba kyslíku MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• elektronový transportní řetězec MeSH
• protozoální proteiny MeSH

Protein kinase N3 (PKN3) is a serine/threonine kinase implicated in tumor progression of multiple cancer types, however, its substrates and effector proteins still remain largely understudied. In the present work we aimed to identify novel PKN3 substrates in a phosphoproteomic screen using analog sensitive PKN3. Among the identified putative substrates we selected ARHGAP18, a protein from RhoGAP family, for validation of the screen and further study. We confirmed that PKN3 can phosphorylate ARHGAP18 in vitro and we also characterized the interaction of the two proteins, which is mediated via the N-terminal part of ARHGAP18. We present strong evidence that PKN3-ARHGAP18 interaction is increased upon ARHGAP18 phosphorylation and that the phosphorylation of ARHGAP18 by PKN3 enhances its GAP domain activity and contributes to negative regulation of active RhoA. Taken together, we identified new set of potential PKN3 substrates and revealed a new negative feedback regulatory mechanism of Rho signaling mediated by PKN3-induced ARHGAP18 activation.

• Klíčová slova
• ARHGAP18, PKN3, Rho-GTP, phosphoproteomic screen, phosphorylation,
• MeSH
• fosforylace MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• proteinkinasa C genetika   metabolismus   MeSH
• proteiny aktivující GTPasu metabolismus   MeSH
• proteomika metody   MeSH
• sekvence aminokyselin MeSH
• signální transdukce MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• zpětná vazba fyziologická MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ARHGAP18 protein, human MeSH Prohlížeč
• protein kinase N MeSH Prohlížeč
• proteinkinasa C MeSH
• proteiny aktivující GTPasu MeSH