Haloalkane dehalogenases (HLDs) are a family of α/β-hydrolase fold enzymes that employ SN2 nucleophilic substitution to cleave the carbon-halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Atomic-level knowledge of both the inner organization and supramolecular complexation of HLDs is thus crucial to understand their catalytic and noncatalytic functions. Here, crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85 Å resolution. The structures show a canonical αβα-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate-histidine-aspartate) and uncommon active-site pocket reveal the molecular specificities of a catalytic apparatus that exhibits a rare (S)-enantiopreference. Additionally, the structures reveal a previously unobserved mode of symmetric homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering. Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its uncommon enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution.

• Klíčová slova
• DmmarA, Mycobacterium marinum, SAXS, X-ray crystallography, enantioselectivity, haloalkane dehalogenases, homodimerization, surface loops,
• MeSH
• hydrolasy chemie   MeSH
• kyselina asparagová MeSH
• Mycobacterium marinum * metabolismus   MeSH
• stereoizomerie MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• kyselina asparagová MeSH

RNase L, a key enzyme in the host defense system, is activated by the binding of 2'-5'-linked oligoadenylates (2-5A) to the N-terminal ankyrin repeat domain, which causes the inactive monomer to form a catalytically active homodimer. We focused on the structural changes of human RNase L as a result of interactions with four different activators: natural 2-5 pA(4) and three tetramers with 3'-end AMP units replaced with ribo-, arabino- and xylo-configured phosphonate analogs of AMP (pA(3)X). The extent of the RNase L dimerization and its cleavage activity upon binding of all these activators were similar. A drop-coating deposition Raman (DCDR) spectroscopy possessed uniform spectral changes upon binding of all of the tetramers, which verified the same binding mechanism. The estimated secondary structural composition of monomeric RNase L is 44% α-helix, 28% β-sheet, 17% β-turns and 11% of unordered structures, whereas dimerization causes a slight decrease in α-helix and increase in β-sheet (ca. 2%) content. The dimerization affects at least three Tyr, five Phe and two Trp residues. The α-β structural switch may fix domain positions in the hinge region (residues ca. 336-363) during homodimer formation.

• MeSH
• adeninnukleotidy chemie   metabolismus   MeSH
• ankyrinová repetice MeSH
• endoribonukleasy chemie   metabolismus   MeSH
• konformace proteinů MeSH
• lidé MeSH
• multimerizace proteinu MeSH
• Ramanova spektroskopie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2-5A-dependent ribonuclease MeSH Prohlížeč
• adeninnukleotidy MeSH
• endoribonukleasy MeSH

Little data on the role played in vivo by chloroplast protein AtDeg2 as a chaperone is available. Therefore, we sought for chloroplast proteins protected from high irradiance-induced interprotein aggregation via disulphide bridges by AtDeg2 acting as a holdase. To reach this goal, we performed analyses which involved comparative diagonal electrophoreses of lysates of chloroplasts isolated from wild type (WT) plants and transgenic plants 35S:AtDEG2ΔPDZ1-GFP which expressed AtDeg2 lacking its chaperone activity but retaining the protease activity. The results of the analyses indicate that AtDeg2 acting as a holdase prevents a single chloroplast protein, i.e., the γ1 subunit of ATP synthase from long-term high irradiance-induced homodimerization mediated by disuplhide bridges and this allows us to better understand a complexity of physiological significance of AtDeg2 - the chloroplast protein of dual protease/chaperone activity.

• Klíčová slova
• Deg2, chaperone, elevated irradiance, homodimerization, protease,
• Publikační typ
• časopisecké články MeSH

In current work, we used recombinant OspC protein derived from B. afzelii strain BRZ31 in the native homodimeric fold for mice immunization and following selection process to produce three mouse monoclonal antibodies able to bind to variable parts of up to five different OspC proteins. Applying the combination of mass spectrometry assisted epitope mapping and affinity based theoretical prediction we have localized regions responsible for antigen-antibody interactions and approximate epitopes' amino acid composition. Two mAbs (3F4 and 2A9) binds to linear epitopes located in previously described immunogenic regions in the exposed part of OspC protein. The third mAb (2D1) recognises highly conserved discontinuous epitope close to the ligand binding domain 1.

• Klíčová slova
• Borrelia burgdorferi, OspC, epitope mapping, immunoprecipitation, monoclonal antibody, outer surface protein C, protein alignment,
• MeSH
• antigeny bakteriální chemie   genetika   imunologie   MeSH
• Borrelia burgdorferi chemie   genetika   imunologie   MeSH
• mapování epitopu * MeSH
• multimerizace proteinu * MeSH
• myši inbrední BALB C MeSH
• myší monoklonální protilátky chemie   imunologie   MeSH
• myši MeSH
• proteiny vnější bakteriální membrány chemie   genetika   imunologie   MeSH
• sbalování proteinů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny bakteriální MeSH
• myší monoklonální protilátky MeSH
• OspC protein MeSH Prohlížeč
• proteiny vnější bakteriální membrány MeSH

Following the detection of individual members of the family of galectins it is an obvious challenge to define the extent of functional overlap/divergence among these proteins. As a step to address this issue a comparative profiling has been started in the mouse as a model organism, combining sequence analysis, expression patterns and structural features in the cases of the homodimeric galectins-1, -2 and -7. Close relationship was apparent at the level of global gene organization. Scrutiny of the proximal promoter regions for putative transcription-factor-binding sites by two search algorithms uncovered qualitative and quantitative differences with potential to influence the combinatorial functionality of regulatory sequences. RT-PCR mapping with samples from an array of 17 organs revealed significant differences, separating rather ubiquitous gene expression of galectin-1 from the more restricted individual patterns of galectins-2 and -7. Using specific antisera obtained by affinity depletion including stringent controls to ascertain lack of cross-reactivity these results were corroborated at the level of galectin localization in fixed tissue sections. Nuclear presence was seen in the case of galectin-1. In addition to nonidentical expression profiles the mapping of the carbohydrate recognition domains of galectins-1 and -7 by homology modelling and docking of naturally occurring complex tetra- and pentasaccharides disclosed a series of sequence deviations which may underlie disparate affinities for cell surface glycans/glycomimetic peptides. In view of applicability the presented data can serve as useful reference to delineate changes with respect to disease and in genetically engineered models. To enable more general conclusions on the galectin network it is warranted to further pursue this combined approach within this lectin family.

• MeSH
• databáze nukleových kyselin * MeSH
• dimerizace MeSH
• galektin 1 chemie   genetika   metabolismus   MeSH
• galektin 2 chemie   genetika   metabolismus   MeSH
• galektiny chemie   genetika   metabolismus   MeSH
• imunohistochemie MeSH
• konformace sacharidů MeSH
• ligandy MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• orgánová specificita MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• promotorové oblasti (genetika) genetika   MeSH
• regulace genové exprese MeSH
• sacharidy chemie   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• stanovení celkové genové exprese * MeSH
• transkripční faktory metabolismus   MeSH
• vazebná místa MeSH
• výpočetní biologie * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• galektin 1 MeSH
• galektin 2 MeSH
• galektiny MeSH
• Lgals7 protein, mouse MeSH Prohlížeč
• ligandy MeSH
• messenger RNA MeSH
• sacharidy MeSH
• transkripční faktory MeSH

Hydrogen/deuterium (H/D) exchange or chemical cross-linking by soluble carbodiimide (EDC) was employed in combination with high-resolution mass spectrometry (MS) to extend our knowledge about contact surface regions involved in the well-characterized model of interaction between two molecules of human 14-3-3ζ regulatory protein. The H/D exchange experiment provided low resolution mapping of interaction in the homodimeric 14-3-3ζ complex. A lower level of deuteration, suggesting structural protection, of two sequential segments has been demonstrated for dimeric 14-3-3ζ wild type relative to the monomeric mutant 14-3-3ζ S58D. The N-terminal sequence (the first 27 residues) from one subunit interacts with region αC'and αD'-helices (residues 45-98) of the other molecule across the dimer interface. To identify interacting amino acid residues within the studied complex, a chemical cross-linking reaction was carried out to produce the covalent homodimer, which was detected by SDS-PAGE. The MS analysis (following tryptic in-gel digestion) employing both high resolution and tandem mass spectrometry revealed cross-linked amino acid residues. Two alternative salt bridges between Glu81 and either Lys9 or the N-terminal amino group have been found to participate in transient interactions of the 14-3-3ζ isotype homodimerization. The data obtained, which have never previously been reported, were used to modify the published 14-3-3 crystal structure using molecular modeling. Based on our findings, utilization of this combination of experimental approaches, which preserve protein native structures, is suitable for mapping the contact between two proteins and also allows for the description of transient interactions or of regions with flexible structure in the studied protein complexes.

• MeSH
• hmotnostní spektrometrie metody   MeSH
• karbodiimidy chemie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• mapování interakce mezi proteiny MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• mutace MeSH
• proteiny 14-3-3 chemie   genetika   izolace a purifikace   metabolismus   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• vodík-deuteriová výměna metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• karbodiimidy MeSH
• proteiny 14-3-3 MeSH
• reagencia zkříženě vázaná MeSH
• rekombinantní proteiny MeSH
• YWHAB protein, human MeSH Prohlížeč

BACKGROUND: Since the demonstration that the protease of the human immunodeficiency virus (HIV Pr) is essential in the viral life cycle, this enzyme has become one of the primary targets for antiviral drug design. The murine monoclonal antibody 1696 (mAb1696), produced by immunization with the HIV-1 protease, inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides that include the N terminus of the enzyme, a region that is highly conserved in sequence among different viral strains and that, furthermore, is crucial for homodimerization to the active enzymatic form. RESULTS: We report here the crystal structure at 2.7 A resolution of a recombinant single-chain Fv fragment of mAb1696 as a complex with a cross-reactive peptide of the HIV-1 protease. The antibody-antigen interactions observed in this complex provide a structural basis for understanding the origin of the broad reactivity of mAb-1696 for the HIV-1 and HIV-2 proteases and their respective N-terminal peptides. CONCLUSION: A possible mechanism of HIV-protease inhibition by mAb1696 is proposed that could help the design of inhibitors aimed at binding inactive monomeric species.

• MeSH
• aspartátové endopeptidasy chemie   imunologie   metabolismus   MeSH
• chemické modely MeSH
• HIV-proteasa chemie   imunologie   metabolismus   MeSH
• imunoglobuliny - Fab fragmenty chemie   metabolismus   MeSH
• inhibitory HIV-proteasy chemie   farmakologie   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• monoklonální protilátky chemie   farmakologie   MeSH
• peptidové fragmenty imunologie   metabolismus   MeSH
• protilátky virové chemie   metabolismus   MeSH
• vazebná místa protilátek MeSH
• zkřížené reakce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• aspartátové endopeptidasy MeSH
• HIV-proteasa MeSH
• imunoglobuliny - Fab fragmenty MeSH
• inhibitory HIV-proteasy MeSH
• monoklonální protilátky MeSH
• p16 protease, Human immunodeficiency virus 2 MeSH Prohlížeč
• peptidové fragmenty MeSH
• protilátky virové MeSH

Truncating genetic variants of SORL1, encoding the endosome recycling receptor SORLA, have been accepted as causal of Alzheimer's disease (AD). However, most genetic variants observed in SORL1 are missense variants, for which it is complicated to determine the pathogenicity level because carriers come from pedigrees too small to be informative for penetrance estimations. Here, we describe three unrelated families in which the SORL1 coding missense variant rs772677709, that leads to a p.Y1816C substitution, segregates with Alzheimer's disease. Further, we investigate the effect of SORLA p.Y1816C on receptor maturation, cellular localization, and trafficking in cell-based assays. Under physiological circumstances, SORLA dimerizes within the endosome, allowing retromer-dependent trafficking from the endosome to the cell surface, where the luminal part is shed into the extracellular space (sSORLA). Our results showed that the p.Y1816C mutant impairs SORLA homodimerization in the endosome, leading to decreased trafficking to the cell surface and less sSORLA shedding. These trafficking defects of the mutant receptor can be rescued by the expression of the SORLA 3Fn-minireceptor. Finally, we find that iPSC-derived neurons with the engineered p.Y1816C mutation have enlarged endosomes, a defining cytopathology of AD. Our studies provide genetic as well as functional evidence that the SORL1 p.Y1816C variant is causal for AD. The partial penetrance of the mutation suggests this mutation should be considered in clinical genetic screening of multiplex early-onset AD families.

• Klíčová slova
• 3Fn-domain, SORL1-associated Alzheimer’s disease, SORLA, dimerization, retromer,
• MeSH
• Alzheimerova nemoc * genetika   metabolismus   patologie   MeSH
• endozomy * metabolismus   MeSH
• HEK293 buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• membránové transportní proteiny * genetika   metabolismus   MeSH
• missense mutace MeSH
• multimerizace proteinu MeSH
• proteiny související s LDL-receptory * genetika   metabolismus   MeSH
• rodokmen * MeSH
• senioři MeSH
• transport proteinů MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• membránové transportní proteiny * MeSH
• proteiny související s LDL-receptory * MeSH
• SORL1 protein, human MeSH Prohlížeč

Hydrolysis of ATP by the mitochondrial F-ATPase is inhibited by a protein called IF1 . In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1 , is expressed exclusively in the procyclic stage, where the F-ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F-ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1 -ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C-terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1 , the inhibitory capacity of the C-terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1 . This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C-terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1 -ATPase, and this difference in the mechanism of regulation of the F-ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases.

• Klíčová slova
• IF 1, Trypanosoma brucei, ATP synthase, F-ATPase, F1Fo-ATPase, TbIF1, enzyme inhibitor, trypanosome,
• MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• mutace MeSH
• protein inhibující ATPasu MeSH
• proteiny chemie   genetika   farmakologie   MeSH
• protonové ATPasy antagonisté a inhibitory   MeSH
• regulace genové exprese * MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• skot MeSH
• Trypanosoma brucei brucei enzymologie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibitory enzymů MeSH
• proteiny MeSH
• protonové ATPasy MeSH

Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group. The structure of monomeric Δ16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural in vitro TBEVC homodimerization, the dimeric interfaces were identified by hydrogen deuterium exchange mass spectrometry (MS). Although the assembly of flaviviruses occurs in endoplasmic reticulum-derived vesicles, we observed that TBEVC protein also accumulated in the nuclei and nucleoli of infected cells. In addition, the predicted bipartite nuclear localization sequence in the TBEVC C-terminal part was confirmed experimentally, and we described the interface between TBEVC bipartite nuclear localization sequence and import adapter protein importin-alpha using X-ray crystallography. Furthermore, our coimmunoprecipitation coupled with MS identification revealed 214 interaction partners of TBEVC, including viral envelope and nonstructural NS5 proteins and a wide variety of host proteins involved mainly in rRNA processing and translation initiation. Metabolic labeling experiments further confirmed that TBEVC and other flaviviral capsid proteins are able to induce translational shutoff and decrease of 18S rRNA. These findings may substantially help to design a targeted therapy against TBEV.

• Klíčová slova
• capsid, nucleolus, nucleus, protein structure, tick-borne flaviviruses, translational shutoff,
• MeSH
• kapsida metabolismus   MeSH
• RNA virová metabolismus   MeSH
• virové nestrukturální proteiny metabolismus   MeSH
• virové plášťové proteiny genetika   metabolismus   MeSH
• viry klíšťové encefalitidy * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA virová MeSH
• virové nestrukturální proteiny MeSH
• virové plášťové proteiny MeSH