Complex II (CII) activity controls phenomena that require crosstalk between metabolism and signaling, including neurodegeneration, cancer metabolism, immune activation, and ischemia-reperfusion injury. CII activity can be regulated at the level of assembly, a process that leverages metastable assembly intermediates. The nature of these intermediates and how CII subunits transfer between metastable complexes remains unclear. In this work, we identify metastable species containing the SDHA subunit and its assembly factors, and we assign a preferred temporal sequence of appearance of these species during CII assembly. Structures of two species show that the assembly factors undergo disordered-to-ordered transitions without the appearance of significant secondary structure. The findings identify that intrinsically disordered regions are critical in regulating CII assembly, an observation that has implications for the control of assembly in other biomolecular complexes.
- MeSH
- katalytická doména * MeSH
- sekundární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Pheochromocytoma and paraganglioma (PPGL) are neuroendocrine tumors with frequent mutations in genes linked to the tricarboxylic acid cycle. However, no pathogenic variant has been found to date in succinyl-CoA ligase (SUCL), an enzyme that provides substrate for succinate dehydrogenase (SDH; mitochondrial complex II [CII]), a known tumor suppressor in PPGL. METHODS: A cohort of 352 patients with apparently sporadic PPGL underwent genetic testing using a panel of 54 genes developed at the National Institutes of Health, including the SUCLG2 subunit of SUCL. Gene deletion, succinate levels, and protein levels were assessed in tumors where possible. To confirm the possible mechanism, we used a progenitor cell line, hPheo1, derived from a human pheochromocytoma, and ablated and re-expressed SUCLG2. RESULTS: We describe 8 germline variants in the guanosine triphosphate-binding domain of SUCLG2 in 15 patients (15 of 352, 4.3%) with apparently sporadic PPGL. Analysis of SUCLG2-mutated tumors and SUCLG2-deficient hPheo1 cells revealed absence of SUCLG2 protein, decrease in the level of the SDHB subunit of SDH, and faulty assembly of the complex II, resulting in aberrant respiration and elevated succinate accumulation. CONCLUSIONS: Our study suggests SUCLG2 as a novel candidate gene in the genetic landscape of PPGL. Large-scale sequencing may uncover additional cases harboring SUCLG2 variants and provide more detailed information about their prevalence and penetrance.
- MeSH
- feochromocytom * genetika patologie MeSH
- lidé MeSH
- nádory nadledvin * genetika patologie MeSH
- paragangliom * genetika patologie MeSH
- sukcinátdehydrogenasa genetika metabolismus MeSH
- zárodečné mutace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
Pancreatic cancer is one of the deadliest forms of cancer, which is attributed to lack of effective treatment options and drug resistance. Mitochondrial inhibitors have emerged as a promising class of anticancer drugs, and several inhibitors of the electron transport chain (ETC) are being clinically evaluated. We hypothesized that resistance to ETC inhibitors from the biguanide class could be induced by inactivation of SMAD4, an important tumor suppressor involved in transforming growth factor β (TGFβ) signaling, and associated with altered mitochondrial activity. Here we show that, paradoxically, both TGFβ-treatment and the loss of SMAD4, a downstream member of TGFβ signaling cascade, induce resistance to biguanides, decrease mitochondrial respiration, and fragment the mitochondrial network. Mechanistically, the resistance of SMAD4-deficient cells is mediated by increased mitophagic flux driven by MAPK/ERK signaling, whereas TGFβ-induced resistance is autophagy-independent and linked to epithelial-to-mesenchymal transition (EMT). Interestingly, mitochondria-targeted tamoxifen, a complex I inhibitor under clinical trial, overcomes resistance mediated by SMAD4-deficiency or TGFβ signaling. Our data point to differential mechanisms underlying the resistance to treatment in PDAC arising from TGFβ signaling and SMAD4 loss, respectively. The findings will help the development of mitochondria-targeted therapy for pancreatic cancer patients with SMAD4 as a plausible predictive marker.
- MeSH
- lidé MeSH
- mitofagie MeSH
- nádory slinivky břišní genetika metabolismus patologie MeSH
- protein Smad4 metabolismus MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Pancreatic cancer is recognized as one of the most fatal tumors due to its aggressiveness and resistance to therapy. Statins were previously shown to inhibit the proliferation of cancer cells via various signaling pathways. In healthy tissues, statins activate the heme oxygenase pathway, nevertheless the role of heme oxygenase in pancreatic cancer is still controversial. The aim of this study was to evaluate, whether anti-proliferative effects of statins in pancreatic cancer cells are mediated via the heme oxygenase pathway. METHODS: In vitro effects of various statins and hemin, a heme oxygenase inducer, on cell proliferation were evaluated in PA-TU-8902, MiaPaCa-2 and BxPC-3 human pancreatic cancer cell lines. The effect of statins on heme oxygenase activity was assessed and heme oxygenase-silenced cells were used for pancreatic cancer cell proliferation studies. Cell death rate and reactive oxygen species production were measured in PA-TU-8902 cells, followed by evaluation of the effect of cerivastatin on GFP-K-Ras trafficking and expression of markers of invasiveness, osteopontin (SPP1) and SOX2. RESULTS: While simvastatin and cerivastatin displayed major anti-proliferative properties in all cell lines tested, pravastatin did not affect the cell growth at all. Strong anti-proliferative effect was observed also for hemin. Co-treatment of cerivastatin and hemin increased anti-proliferative potential of these agents, via increased production of reactive oxygen species and cell death compared to individual treatment. Heme oxygenase silencing did not prevent pancreatic cancer cells from the tumor-suppressive effect of cerivastatin or hemin. Cerivastatin, but not pravastatin, protected Ras protein from trafficking to the cell membrane and significantly reduced expressions of SPP1 (p < 0.05) and SOX2 (p < 0.01). CONCLUSIONS: Anti-proliferative effects of statins and hemin on human pancreatic cancer cell lines do not seem to be related to the heme oxygenase pathway. While hemin triggers reactive oxygen species-induced cell death, cerivastatin targets Ras protein trafficking and affects markers of invasiveness.
- MeSH
- apoptóza účinky léků MeSH
- hemoxygenasa-1 genetika metabolismus MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- nádorové buňky kultivované MeSH
- nádory slinivky břišní farmakoterapie metabolismus patologie MeSH
- pankreas metabolismus MeSH
- pohyb buněk účinky léků MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proliferace buněk účinky léků MeSH
- ras proteiny genetika metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- signální transdukce MeSH
- statiny farmakologie MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
Gain-of-function (GOF) mutations in ion channels are rare events, which lead to increased agonist sensitivity or altered gating properties, and may render the channel constitutively active. Uncovering and following characterization of such mutants contribute substantially to the understanding of the molecular basis of ion channel functioning. Here we give an overview of some GOF mutants in polymodal ion channels specifically involved in transduction of painful stimuli--TRPV1 and TRPA1, which are scrutinized by scientists due to their important role in development of some pathological pain states. Remarkably, a substitution of single amino acid in the S4-S5 region of TRPA1 (N855S) has been recently associated with familial episodic pain syndrome. This mutation increases chemical sensitivity of TRPA1, but leaves the voltage sensitivity unchanged. On the other hand, mutations in the analogous region of TRPV1 (R557K and G563S) severely affect all aspects of channel activation and lead to spontaneous activity. Comparison of the effects induced by mutations in homologous positions in different TRP receptors (or more generally in other distantly related ion channels) may elucidate the gating mechanisms conserved during evolution.
- MeSH
- bolest patofyziologie MeSH
- kationtové kanály TRP chemie genetika metabolismus MeSH
- kationtové kanály TRPV chemie genetika metabolismus MeSH
- lidé MeSH
- mícha patofyziologie MeSH
- mutace genetika MeSH
- percepce bolesti MeSH
- proteiny nervové tkáně chemie genetika metabolismus MeSH
- vápníkové kanály chemie genetika metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat, or highly depolarizing voltages, and also by 2-aminoethoxydiphenyl borate (2-APB), a common activator of the related thermally gated vanilloid TRP channels TRPV1, TRPV2, and TRPV3. In these channels, the conserved charged residues in the intracellular S4-S5 region have been proposed to constitute part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. The molecular basis of this gating event is poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and identified four potential voltage-sensing residues (Arg(557), Glu(570), Asp(576), and Arg(579)) that, when specifically mutated, altered the functionality of the channel with respect to voltage, capsaicin, heat, 2-APB, and/or their interactions in different ways. The nonfunctional charge-reversing mutations R557E and R579E were partially rescued by the charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2-APB, revealing the specific role of Lys(571) in chemical sensitivity. Surprisingly, substitutions at homologous residues in TRPV2 or TRPV3 had no effect on temperature- and 2-APB-induced activity. Thus, the charged residues in S4 and the S4-S5 linker contribute to voltage sensing in TRPV1 and, despite their highly conserved nature, regulate the temperature and chemical gating in the various TRPV channels in different ways.
- MeSH
- elektrofyziologie metody MeSH
- iontové kanály chemie MeSH
- kationtové kanály TRP metabolismus MeSH
- kationtové kanály TRPV metabolismus MeSH
- lidé MeSH
- membránové proteiny chemie MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- mutageneze cílená MeSH
- sekvence aminokyselin MeSH
- sekvenční analýza DNA MeSH
- sekvenční homologie aminokyselin MeSH
- terciární struktura proteinů MeSH
- vysoká teplota MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- bolest MeSH
- buňky zadních rohů míšních fyziologie MeSH
- financování organizované MeSH
- hyperalgezie chemicky indukované MeSH
- kapsaicin farmakokinetika farmakologie MeSH
- kationtové kanály TRP fyziologie MeSH
- kationtové kanály TRPV fyziologie MeSH
- nociceptory MeSH
- termoreceptory MeSH
- změny tělesné teploty MeSH
