catalytic activities
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The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis.
- MeSH
- inhibitory proteasomu farmakologie chemie MeSH
- katalytická doména * MeSH
- proteasomový endopeptidasový komplex * metabolismus chemie MeSH
- protozoální proteiny chemie metabolismus antagonisté a inhibitory genetika MeSH
- substrátová specifita MeSH
- Trichomonas vaginalis * enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
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.
The structures and functions of the components of ATP synthases, especially those subunits involved directly in the catalytic formation of ATP, are widely conserved in metazoans, fungi, eubacteria, and plant chloroplasts. On the basis of a map at 32.5-Å resolution determined in situ in the mitochondria of Trypanosoma brucei by electron cryotomography, it has been proposed that the ATP synthase in this species has a noncanonical structure and different catalytic sites in which the catalytically essential arginine finger is provided not by the α-subunit adjacent to the catalytic nucleotide-binding site as in all species investigated to date, but rather by a protein, p18, found only in the euglenozoa. A crystal structure at 3.2-Å resolution of the catalytic domain of the same enzyme demonstrates that this proposal is incorrect. In many respects, the structure is similar to the structures of F1-ATPases determined previously. The α3β3-spherical portion of the catalytic domain in which the three catalytic sites are found, plus the central stalk, are highly conserved, and the arginine finger is provided conventionally by the α-subunits adjacent to each of the three catalytic sites found in the β-subunits. Thus, the enzyme has a conventional catalytic mechanism. The structure differs from previous described structures by the presence of a p18 subunit, identified only in the euglenozoa, associated with the external surface of each of the three α-subunits, thereby elaborating the F1-domain. Subunit p18 is a pentatricopeptide repeat (PPR) protein with three PPRs and appears to have no function in the catalytic mechanism of the enzyme.
- MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- mitochondriální protonové ATPasy genetika metabolismus MeSH
- molekulární modely MeSH
- podjednotky proteinů MeSH
- protozoální proteiny genetika metabolismus MeSH
- regulace genové exprese enzymů MeSH
- Trypanosoma brucei brucei enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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
Acetohydroxy-acid synthases (AHAS) of two mutant strains Streptomyces cinnamonensis ACB-NLR-2 and BVR-18 were chosen for this study for their apparent activation by valine, which regularly acts as an allosteric inhibitor. Sequencing the ilvB genes coding for the AHAS catalytic subunit revealed two distant changes in the mutants, DeltaQ217 and E139A, respectively. Homology modeling was used to propose the structural changes caused by those mutations. In the mutant strain ACB-NLR-2 (resistant to 2-amino-3-chlorobutyrate and norleucine), deletion of Q217 affected a helix in ss-domain, distant from the active center. As no mutation was found in the regulatory subunit of this strain, DeltaQ217 in IlvB was supposed to be responsible for the observed valine activation, probably via changed properties on the proposed regulatory-catalytic subunit interface. In mutant strain BVR-18 (resistant to 2-oxobutyrate), substitution E139A occurred in a conservative loop near the active center. In vitro AHAS activity assay with the enzyme reconstituted from the wild-type regulatory and BVR-18 catalytic subunits proved that the substitution in the catalytic subunit led to the apparent activation of AHAS by valine. We suggest that the conservative loop participated in a conformational change transfer to the active center during the allosteric regulation.
- MeSH
- acetolaktátsynthasa genetika chemie metabolismus MeSH
- aktivace enzymů MeSH
- alosterická regulace imunologie MeSH
- bakteriální proteiny genetika chemie metabolismus MeSH
- bodová mutace MeSH
- katalytická doména imunologie MeSH
- konformace proteinů MeSH
- missense mutace MeSH
- molekulární modely MeSH
- rekombinantní fúzní proteiny chemie metabolismus MeSH
- sekvenční homologie aminokyselin MeSH
- Streptomyces enzymologie genetika MeSH
- substituce aminokyselin MeSH
- valin metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
Nijmegen breakage syndrome (NBS) is a rare human disease displaying chromosome instability, radiosensitivity, cancer predisposition, immunodeficiency, and other defects [1, 2]. NBS is complexed with MRE11 and RAD50 in a DNA repair complex [3-5] and is localized to telomere ends in association with TRF proteins [6, 7]. We show that blood cells from NBS patients have shortened telomere DNA ends. Likewise, cultured NBS fibroblasts that exhibit a premature growth cessation were observed with correspondingly shortened telomeres. Introduction of the catalytic subunit of telomerase, TERT, was alone sufficient to increase the proliferative capacity of NBS fibroblasts. However, NBS, but not TERT, restores the capacity of NBS cells to survive gamma irradiation damage. Strikingly, NBS promotes telomere elongation in conjunction with TERT in NBS fibroblasts. These results suggest that NBS is a required accessory protein for telomere extension. Since NBS patients have shortened telomeres, these defects may contribute to the chromosome instability and disease associated with NBS patients.
- MeSH
- chromozomální aberace MeSH
- chromozomální poruchy MeSH
- DNA vazebné proteiny MeSH
- fibroblasty fyziologie MeSH
- jaderné proteiny MeSH
- katalytická doména MeSH
- kultivované buňky MeSH
- lidé MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- syndrom MeSH
- telomerasa genetika metabolismus MeSH
- telomery genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- Research Support, U.S. Gov't, P.H.S. MeSH
Humoral immunity in mammals relies on the function of two developmentally and functionally distinct B-cell subsets-B1 and B2 cells. While B2 cells are responsible for the adaptive response to environmental antigens, B1 cells regulate the production of polyreactive and low-affinity antibodies for innate humoral immunity. The molecular mechanism of B-cell specification into different subsets is understudied. In this study, we identified lysine methyltransferase NSD2 (MMSET/WHSC1) as a critical regulator of B1 cell development. In contrast to its minor impact on B2 cells, deletion of the catalytic domain of NSD2 in primary B cells impairs the generation of B1 lineage. Thus, NSD2, a histone H3 K36 dimethylase, is the first-in-class epigenetic regulator of a B-cell lineage in mice.
- MeSH
- analýza přežití MeSH
- B-lymfocyty metabolismus MeSH
- histonlysin-N-methyltransferasa chemie metabolismus MeSH
- histony metabolismus MeSH
- humorální imunita MeSH
- katalytická doména * MeSH
- lysin metabolismus MeSH
- metylace MeSH
- myši inbrední C57BL MeSH
- novorozená zvířata MeSH
- přesmyk imunoglobulinových tříd MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zárodečné centrum lymfatické uzliny metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Východiska: Mnohočetný myelom je nevyléčitelné onemocnění. Jako standardní terapie je využívána vysokodávkovaná chemoterapie s autologní transplantací kmenových buněk či alogenní transplantací. Relaps onemocnění je však neodvratný, a proto jsou rozvíjeny i jiné směry léčby. Jedním z nich je buněčná imunoterapie, která využívá potenciálu cytotoxických T-lymfocytů. Jako nádorový antigen lze využít nádorově specifické proteiny. Jedním z nich je i katalytická podjednotka telomerázy hTERT a od ní odvozený nonapeptid vázající se na HLA-A2 systém molekul. Typ studie a soubor: Ve studii in vitro byla na souboru zdravých HLA-A2 pozitivních dárců testována možnost aktivace a identifikace myelom-specifických T-lymfocytů s využitím hTERT jako nádorového antigenu. Metody a výsledky: Z mononukleárních buněk periferní krve byly kultivovány T-lymfocyty a dendritické buňky. Dendritické buňky byly pulzovány nonapeptidem hTERT. Po opakované stimulaci T-lymfocytů takto pulzovanými dendritickými buňkami došlo k jejich aktivaci charakterizované produkcí interferonu gama. Závěry: Tato práce ukazuje možnost specifické aktivace a identifikace protinádorových T-lymfocytů, které lze využít při léčbě mnohočetného myelomu.
Backgrounds: Multiple myeloma is an incurable hematological disease. High-dose chemotherapy including autologous stem cell transplantation is recently considered a standard therapy for myeloma. Unfortunately, a relapse of the disease is inevitable. Therefore, new approaches such as immunotherapy have been considered recently. A specific activation of cytotoxic T cells can be reached using dendritic cells loaded with tumor-specific antigen. Catalytic subunit of telomerase hTERT and an HLA-A2-specific nonapeptide derived from hTERT can be used. Design and subjects: Activation and identification of myeloma-specific T cells from healthy HLA-A2 blood donors has been tested in an in vitro study using hTERT-derived nonapeptide as a tumor-specific antigen. Methods and results: T cells and dendritic cells were obtained from peripheral blood. T cells were repeatedly stimulated with hTERT nonapeptide- loaded dendritic cells. Activated myeloma-specific T cells produced interferon gamma and were evaluated by flow cytometry. Conclusion: This study demonstrates feasibility of an in vitro identification of tumor-specific T cells that can be used in myeloma therapy.
