Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.
- MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- kyselina jantarová metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- posttranslační úpravy proteinů * MeSH
- proteasa La genetika metabolismus MeSH
- proteomika * MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The ends of eukaryotic chromosomes typically contain a 3' ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing-based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.
- MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA metabolismus MeSH
- G-kvadruplexy MeSH
- homeostáza telomer fyziologie MeSH
- jednovláknová DNA metabolismus MeSH
- kinetika MeSH
- konformace nukleové kyseliny MeSH
- oligonukleotidy genetika MeSH
- proteiny vázající telomery metabolismus MeSH
- retardační test MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- telomerasa genetika MeSH
- telomery metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast.
- MeSH
- DNA vazebné proteiny genetika metabolismus ultrastruktura MeSH
- elektronová mikroskopie MeSH
- fluorescenční polarizace MeSH
- fylogeneze MeSH
- genetická variace MeSH
- lidé MeSH
- molekulární evoluce MeSH
- oligonukleotidy genetika metabolismus MeSH
- proteiny vázající telomery klasifikace genetika metabolismus ultrastruktura MeSH
- retardační test MeSH
- Schizosaccharomyces pombe - proteiny genetika metabolismus ultrastruktura MeSH
- Schizosaccharomyces genetika MeSH
- sekvence nukleotidů MeSH
- telomery genetika metabolismus ultrastruktura MeSH
- transkripční faktory genetika metabolismus ultrastruktura MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Prezentujeme kazuistiku jinak zdravé 14leté dívky s jednotýdenní anamnézou kožní léze doprovázené jednostrannou epitrochleární a axilární lymfadenopatií. Pacientka neměla žádné systémové onemocnění a antibiotická léčba amoxicilinem a gentamicinem nevedla ke zlepšení. Vyšetření levé epitrochleární oblasti prokázalo zarudnutí kůže nad bolestivou fluktuující lymfatickou uzlinou velikosti 2 × 2 cm. Dívka přiznala častý kontakt s koťátkem a vzpomněla si, že mohla být asi týden před zjištěním rezistence poškrábaná na levé paži. Pacientka podstoupila chirurgickou drenáž po exstirpaci uzliny a léčbu doxycyklinem s klinickým zlepšením. Infekce Bartonella henselae byla potvrzena sérologickým vyšetřením.
We report the case of an otherwise healthy, 14-year-old girl having a one-week history of a skin lesion and concomitant unilateral epitrochlear and axillary lymphadenopathy. She had no systemic illness and noted no improvement with a course of amoxicillin and gentamycine. Examination of left epitrochlear area revealed a red, tender fluctuant lymf node measuring 2 by 2 cm. The child reported having regular contact with kitten and remembered being scratched by one week earlier, shortly before the mass appeared. She underwent drainage following lymph node extirpation and a course of doxycycline, with clinical improvement. Bartonella henselae infection was confirmed by serologic analysis.
- Klíčová slova
- epitrochleární hnisavá nekrotizující lymfadenitida,
- MeSH
- antibakteriální látky terapeutické užití MeSH
- axila MeSH
- Bartonella henselae imunologie izolace a purifikace patogenita MeSH
- diferenciální diagnóza MeSH
- granulom diagnóza chirurgie patologie MeSH
- lidé MeSH
- lymfatické uzliny chirurgie patologie MeSH
- mladiství MeSH
- nemoc z kočičího škrábnutí * diagnóza chirurgie terapie MeSH
- protilátky imunologie krev MeSH
- sérologické testy MeSH
- Check Tag
- lidé MeSH
- mladiství MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
