Germline mutations in NUDT15 cause thiopurine intolerance during treatment of leukemia or autoimmune diseases. Previously, it has been shown that the mutations affect the enzymatic activity of the NUDT15 hydrolase due to decreased protein stability in vivo. Here we provide structural insights into protein destabilization in R139C and V18I mutants using thermolysin-based proteolysis and H/D exchange followed by mass spectrometry. Both mutants exhibited destabilization of the catalytic site, which was more pronounced at higher temperature. This structural perturbation is shared by the mutations despite their different positions within the protein structure. Reaction products of NUDT15 reverted these conformational abnormalities, demonstrating the importance of ligands for stabilization of a native state of the mutants. This study shows the action of pharmacogenetic variants in NUDT15 in a context of protein structure, which might open novel directions in personalized chemotherapy.
Hydrophobins are small proteins that play a role in a number of processes during the filamentous fungi growth and development. These proteins are characterized by the self-assembly of their molecules into an amphipathic membrane at hydrophilic-hydrophobic interfaces. Isolation and purification of hydrophobins generally present a challenge in their analysis. Hydrophobin SC3 from Schizophyllum commune was selected as a representative of class I hydrophobins in this work. A novel procedure for selective and effective isolation of hydrophobin SC3 based on solid-phase extraction with polytetrafluoroethylene microparticles loaded in a small self-made microcolumn is reported. The tailored binding of hydrophobins to polytetrafluoroethylene followed by harsh elution conditions resulted in a highly specific isolation of hydrophobin SC3 from the model mixture of ten proteins. The presented isolation protocol can have a positive impact on the analysis and utilization of these proteins including all class I hydrophobins. Hydrophobin SC3 was further subjected to reduction of its highly stable disulfide bonds and to chymotryptic digestion followed by mass spectrometric analysis. The isolation and digestion protocols presented in this work make the analysis of these highly hydrophobic and compact proteins possible.
- MeSH
- albuminy chemie MeSH
- ananasovník chemie MeSH
- bromelainy chemie MeSH
- Canavalia chemie MeSH
- chymotrypsin chemie MeSH
- cytochromy c chemie MeSH
- disulfidy chemie MeSH
- erytrocyty enzymologie MeSH
- extrakce na pevné fázi metody MeSH
- hmotnostní spektrometrie metody MeSH
- karboanhydrasy chemie MeSH
- kaseiny chemie MeSH
- koně MeSH
- konkanavalin A chemie MeSH
- kur domácí MeSH
- lidé MeSH
- mikrosféry * MeSH
- mléko enzymologie MeSH
- myokard metabolismus MeSH
- polytetrafluoroethylen chemie MeSH
- proteomika metody MeSH
- Schizophyllum chemie MeSH
- skot MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- tandemová hmotnostní spektrometrie MeSH
- thermolysin chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein misfolding has been proposed to be a common pathogenic mechanism in many inborn errors of metabolism including cystathionine β-synthase (CBS) deficiency. In this work, we describe the structural properties of nine CBS mutants that represent a common molecular pathology in the CBS gene. Using thermolysin in two proteolytic techniques, we examined conformation of these mutants directly in crude cell extracts after expression in E. coli. Proteolysis with thermolysin under native conditions appeared to be a useful technique even for very unstable mutant proteins, whereas pulse proteolysis in a urea gradient had limited values for the study of the majority of CBS mutants due to their instability. Mutants in the active core had either slightly increased unfolding (p.A114V, p.E302K and p.G307S) or extensive unfolding with decreased stability (p.H65R, p.T191M, p.I278T and p.R369C). The extent of the unfolding inversely correlated with the previously determined degree of tetrameric assembly and with the catalytic activity. In contrast, mutants bearing aminoacid substitutions in the C-terminal regulatory domain (p.R439Q and p.D444N) had increased global stability with decreased flexibility. This study shows that proteolytic techniques can reveal conformational abnormalities even for CBS mutants that have activity and/or a degree of assembly similar to the wild-type enzyme. We present here a methodological strategy that may be used in cell lysates to evaluate properties of proteins that tend to misfold and aggregate and that may be important for conformational studies of disease-causing mutations in the field of inborn errors of metabolism.
- MeSH
- časové faktory MeSH
- cystathionin-beta-synthasa genetika MeSH
- denaturace proteinů MeSH
- dimerizace MeSH
- Escherichia coli metabolismus MeSH
- kinetika MeSH
- konformace proteinů MeSH
- lidé MeSH
- močovina chemie MeSH
- mutace MeSH
- rozpouštědla MeSH
- sbalování proteinů MeSH
- terciární struktura proteinů MeSH
- thermolysin chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- bakteriální proteiny diagnostické užití MeSH
- cystathionin-beta-synthasa genetika izolace a purifikace MeSH
- denaturace proteinů MeSH
- konformace proteinů MeSH
- kultivační média MeSH
- močovina analýza MeSH
- mutace genetika MeSH
- průtoková injekční analýza metody využití MeSH
- stabilita enzymů genetika MeSH
- stabilita proteinů MeSH
- thermolysin chemie MeSH
- vrozené poruchy metabolismu enzymologie genetika MeSH
