Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains.
- MeSH
- adenosintrifosfát chemie MeSH
- aktivace enzymů MeSH
- analýza hlavních komponent MeSH
- DNA-helikasy chemie genetika metabolismus MeSH
- hydrolýza MeSH
- interakční proteinové domény a motivy * MeSH
- konformace proteinů MeSH
- multienzymové komplexy chemie MeSH
- mutace MeSH
- podjednotky proteinů chemie genetika metabolismus MeSH
- restrikční endonukleasy typu I chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
Purines are essential molecules for nucleic acid synthesis and are the most common carriers of chemical energy in all living organisms. The cellular pool of purines is maintained by the balance between their de novo synthesis (DNPS), recycling and degradation. DNPS includes ten reactions catalysed by six enzymes. To date, two genetically determined disorders of DNPS enzymes have been described, and the existence of other defects manifested by neurological symptoms and the accumulation of DNPS intermediates in bodily fluids is highly presumable. In the current study, we prepared specific recombinant DNPS enzymes and used them for the biochemical preparation of their commercially unavailable substrates. These compounds were used as standards for the development and validation of quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). To simulate manifestations of known and putative defects of DNPS we prepared CRISPR-Cas9 genome-edited HeLa cells deficient for the individual steps of DNPS (CR-cells), assessed the substrates accumulation in cell lysates and growth media and tested how the mutations affect assembly of the purinosome, the multi-enzyme complex of DNPS enzymes. In all model cell lines with the exception of one, an accumulation of the substrate(s) for the knocked out enzyme was identified. The ability to form the purinosome was reduced. We conclude that LC-MS/MS analysis of the dephosphorylated substrates of DNPS enzymes in bodily fluids is applicable in the selective screening of the known and putative DNPS disorders. This approach should be considered in affected individuals with neurological and neuromuscular manifestations of unknown aetiology. Prepared in vitro human model systems can serve in various studies that aim to provide a better characterization and understanding of physiology and pathology of DNPS, to study the role of each DNPS protein in the purinosome formation and represent an interesting way for the screening of potential therapeutic agents.
- MeSH
- chromatografie kapalinová MeSH
- CRISPR-Cas systémy * MeSH
- HeLa buňky MeSH
- lidé MeSH
- multienzymové komplexy chemie genetika metabolismus MeSH
- mutace MeSH
- puriny biosyntéza metabolismus MeSH
- substrátová specifita MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.
- MeSH
- endodeoxyribonukleasy chemie genetika metabolismus MeSH
- fosfáty metabolismus MeSH
- krystalizace MeSH
- molekulární sekvence - údaje MeSH
- multienzymové komplexy chemie genetika metabolismus MeSH
- rostlinné proteiny chemie genetika metabolismus MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- Solanum lycopersicum enzymologie genetika MeSH
- vazebná místa fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Many aspects of protein function regulation require specific protein-protein interactions to carry out the exact biochemical and cellular functions. The highly conserved members of the 14-3-3 protein family mediate such interactions and through binding to hundreds of other proteins provide multitude of regulatory functions, thus playing key roles in many cellular processes. The 14-3-3 protein binding can affect the function of the target protein in many ways including the modulation of its enzyme activity, its subcellular localization, its structure and stability, or its molecular interactions. In this minireview, we focus on mechanisms of the 14-3-3 protein-dependent regulation of three important 14-3-3 binding partners: yeast neutral trehalase Nth1, regulator of G-protein signaling 3 (RGS3), and phosducin.
- MeSH
- DNA-glykosylasy chemie ultrastruktura MeSH
- DNA-lyasa (apurinová nebo apyrimidinová) chemie ultrastruktura MeSH
- fosfoproteiny chemie ultrastruktura MeSH
- konformace proteinů MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- multienzymové komplexy chemie ultrastruktura MeSH
- oční proteiny chemie ultrastruktura MeSH
- proteiny 14-3-3 chemie ultrastruktura MeSH
- proteiny RGS chemie ultrastruktura MeSH
- proteiny vázající GTP - regulátory chemie ultrastruktura MeSH
- Schizosaccharomyces pombe - proteiny chemie ultrastruktura MeSH
- sekvence aminokyselin MeSH
- vazba proteinů MeSH
- vazebná místa 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
Type I plant nucleases play an important role in apoptotic processes and cell senescence. Recently, they have also been indicated to be potent anticancer agents in in vivo studies. The first structure of tomato nuclease I (TBN1) has been determined, its oligomerization and activity profiles have been analyzed and its unexpected activity towards phospholipids has been discovered, and conclusions are drawn regarding its catalytic mechanism. The structure-solution process required X-ray diffraction data from two crystal forms. The first form was used for phase determination; the second form was used for model building and refinement. TBN1 is mainly α-helical and is stabilized by four disulfide bridges. Three observed oligosaccharides are crucial for its stability and solubility. The active site is localized at the bottom of the positively charged groove and contains a zinc cluster that is essential for enzymatic activity. An equilibrium between monomers, dimers and higher oligomers of TBN1 was observed in solution. Principles of the reaction mechanism of the phosphodiesterase activity are suggested, with central roles for the zinc cluster, the nucleobase-binding pocket (Phe-site) and Asp70, Arg73 and Asn167. Based on the distribution of surface residues, possible binding sites for dsDNA and other nucleic acids with secondary structure were identified. The phospholipase activity of TBN1, which is reported for the first time for a nuclease, significantly broadens the substrate promiscuity of the enzyme, and the resulting release of diacylglycerol, which is an important second messenger, can be related to the role of TBN1 in apoptosis.
- MeSH
- deoxyribonukleasy chemie metabolismus MeSH
- fosfolipasy chemie metabolismus MeSH
- katalytická doména MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- multienzymové komplexy chemie metabolismus MeSH
- myši MeSH
- rostlinné proteiny chemie metabolismus MeSH
- Solanum lycopersicum enzymologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Adenylosuccinate lyase (ADSL) deficiency is neurometabolic disease characterized by accumulation of dephosphorylated enzyme substrates SAICA-riboside (SAICAr) and succinyladenosine (S-Ado) in body fluids of affected individuals. The phenotypic severity differs considerably among patients: neonatal fatal, severe childhood, and moderate phenotypic forms correlating with different values for the ratio between S-Ado and SAICAr concentrations in cerebrospinal fluid have been distinguished. To reveal the biochemical and structural basis for this phenotypic heterogeneity, we expressed and characterized 19 ADSL mutant proteins identified in 16 patients representing clinically distinct subgroups. Respecting compound heterozygosity and considering the homotetrameric structure of ADSL, we used intersubunit complementation and prepared and characterized genotype-specific heteromeric mutant ADSL complexes. We correlated clinical phenotypes with biochemical properties of the mutant proteins and predicted structural impacts of the mutations. We found that phenotypic severity in ADSL deficiency is correlated with residual enzymatic activity and structural stability of the corresponding mutant ADSL complexes and does not seem to result from genotype-specific disproportional catalytic activities toward one of the enzyme substrates. This suggests that the S-Ado/SAICAr ratio is probably not predictive of phenotype severity; rather, it may be secondary to the degree of the patient's development (i.e., to the age of the patient at the time of sample collection).
- MeSH
- adenylsukcinátlyasa chemie nedostatek genetika MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fenotyp MeSH
- genetická heterogenita MeSH
- lidé MeSH
- multienzymové komplexy chemie nedostatek genetika MeSH
- mutace genetika MeSH
- mutantní proteiny chemie genetika MeSH
- novorozenec MeSH
- sekundární struktura proteinů MeSH
- stabilita enzymů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
According to a general paradigm, proper DNA duplication from each replication origin is ensured by two protein complexes termed replisomes. In prokaryotes and in budding yeast Saccharomyces cerevisiae, these two replisomes seem to be associated with one another until DNA replication initiated from the origin has finished. This arrangement results in the formation of the loop of newly synthesized DNA. However, arrangement of replisomes in other eukaryotic organisms including vertebrate cells is largely unknown. Here, we used in vivo labeling of DNA segments in combination with the electron microscopy tomography to describe the organization of replisomes in human HeLa cells. The experiments were devised in order to distinguish between a model of independent replisomes and a model of replisome couples. The comparative analysis of short segments of replicons labeled in pulse-chase experiments of various length shows that replisomes in HeLa cells are organized into the couples during DNA replication. Moreover, our data enabled to suggest a new model of the organization of replicated DNA. According to this model, replisome couples produce loop with the associated arms in the form of four tightly associated 30nm fibers.
- MeSH
- bromodeoxyuridin metabolismus MeSH
- buněčné jádro metabolismus ultrastruktura MeSH
- chromatin fyziologie ultrastruktura MeSH
- deoxyuracilnukleotidy metabolismus MeSH
- DNA-dependentní DNA-polymerasy chemie metabolismus MeSH
- financování organizované MeSH
- HeLa buňky MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- modely genetické MeSH
- multienzymové komplexy chemie metabolismus MeSH
- počítačové zpracování obrazu MeSH
- replikace DNA fyziologie MeSH
- replikon genetika MeSH
- tomografie elektronová MeSH
- Check Tag
- lidé MeSH
Atomically flat mica surfaces were chemically modified with an alkyl trifluoromethyl ketone, a covalent inhibitor of esterase 2 from Alicyclobacillus acidocaldarius, which served as a tag for ligand-directed immobilization of esterase-linked proteins. Purified NADH oxidase from Thermus thermophilus and human exportin-t from cell lysates were anchored on the modified surfaces. The immobilization effectiveness of the proteins was studied by atomic force microscopy (AFM). It was shown that ligand-esterase interaction allowed specific attachment of exportin-t and resulted in high-resolution images and coverage patterns that were comparable with immobilized purified protein. Moreover, the biological functionality of immobilized human exportin-t in forming a quaternary complex with tRNA and the GTPase Ran-GTP, and the dimension changes before and after complex formation were also determined by AFM.
- MeSH
- bakteriální proteiny MeSH
- esterasy MeSH
- exprese genu MeSH
- financování organizované MeSH
- ketony farmakologie chemie MeSH
- lidé MeSH
- ligandy MeSH
- mikroskopie atomárních sil MeSH
- multienzymové komplexy genetika chemie MeSH
- NADH, NADPH oxidoreduktasy genetika chemie MeSH
- nukleocytoplazmatické transportní proteiny genetika chemie MeSH
- rekombinantní fúzní proteiny genetika chemie ultrastruktura MeSH
- silikáty hliníku chemie MeSH
- terciární struktura proteinů MeSH
- Thermus thermophilus enzymologie MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- MeSH
- aspartátaminotransferasy analýza krev MeSH
- elektroforéza v agarovém gelu metody využití MeSH
- imunochemie metody MeSH
- izoenzymy analýza krev MeSH
- lidé MeSH
- multienzymové komplexy analýza chemie krev MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
