substrate recognition
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V posledním desetiletí v neurovědních oborech poměrně prudce vzrůstá zájem o pozoruhodnv fenomén miphcitm percepce (vnímání bez uvědomění). Jeho podstatou je schopnost jedince registrovat různé podněty ze svého okolí, aniž by si přitom jejich samotné existence byl vědom. Tyto podněty přesto mohou za určitých podmínek ovlivňovat jeho myšlení či chování a představovat tak více či méně významnou část jeho vnímání. „Slepé videm" (blindsight), skryté rozpoznání tváří u prozopagnózie či předmětů a tvarů u vizuální agnózie, stejně jako neuvědomované zpracování informací z opomíjené poloviny prostoru u neglect syndromu, představují nejčastější projevy organicky podmíněných disociací vizuální percepce a vědomí, tedy vnímání bez uvědomění u některých neurologických pacientů. U zcela zdravých jedinců se variantou takové disociace zdá být podprahové vnímání, jehož studium bylo V nedávné době zahájeno i na poli kognitivní neurofyziologie. S využitím metod ERP (kognitivní evokované potenciály), fMRI a PET byly získány první nadějné výsledky, které významně doplňuji klinická pozorování a společně konkretizují neuroanatomický a neurofyziologický substrát vnímání bez uvědomění. Bylo prokázáno, že mechanismus zpracování neuvědomovaných zrakových podnětů v lidském mozku se principiálně neliší od zpracování na úrovni vědomé. Na druhé straně typickým neurofyziologickým rysem vnímání bez uvědomění se v současnosti zdá být inkompletní aktivace stávajících neurokognitivních sítí pro zpracování externích podnětů. Nález významně rychlejšího zpracování neuvědomované informace představuje další charakteristický rys vizuální implicitní perčepce. Na základě srovnání literámích i vlastních údajů klinicko-patologických a experimentálních je autorem navržena koncepce existujícího neuronálního systému pro zajištění vědomé zkušenosti perčepce, rozsáhle distribuovaného mezi dorzálními paríetálními asociačními oblastmi a prefrontálním kortexem s dominantním postavením pravé hemisféry. Tento systém může být nahlížen jako oboustranná „aktivní brána" vědomí, regulující přístup vědomí k informacím, respektive nevpouštějící informaci příliš slabou nebo pro daného jedince v daný okamžik nevýznamnou.
During the last decade increasing attention to a peculiar phenomenon of implicit perception (perception without awareness) has been paid in neurosciences. In addition to consciously perceived stimuli. there is a great amount of signals from without and within the subject which are perceived and processed without any reportable awareness (i.e. unconsciously). These events can nevertheless influence our experience, thought, and action under some conditions. Blindsight, covert recognition of faces in prosopagnosia or object and shapes in visual agnosia as well as unconscious perception in neglect and extinction represent rare neurological visual disorders where perception and consciousness are dissociated as a result of brain damage. Some analogy of such dissociation in healthy subjects could be subliminal perception; its study was recently started in the field of cognitive neurophysiology. Results of sporadic ERP,fMRI and PET studies complement clinical observations and together particularíze neuroanatomical and neurophysiological substrate of implicit perception. So far it was proved that mechanism of unaware processing of visual stimuli in the human brain does not differ substantially from the aware processing at least from the electrophysiological viewpoint. The proof of significantly faster unaware information processing represents another distinctive feature of the visual perception without awareness. Based on the author's own experimental and clinical findings and comparing them with the results of the previously published studies, the crucial role of a large scale neural system for conscious experience of perception is suggested, distributed extensively among dorsal posterior association areas and the prefi*ontal cortex with the dominant role of the right hemisphere. This system can be considered as an interactive „gate" of consciousness regulating the access of consciousness to information as well as blocking transmission of too weak information into the awareness.
We have a continuing interest in applying the current knowledge of P450cam substrate recognition to engineer the enzyme for the biotransformation of unnatural substrates with the long-term aim of applications in the synthesis of fine chemicals and bioremediation of environmental contaminants. Comparison of the structure of target substrates with camphor, the natural substrate, lead to the design of active site mutations that had greatly enhanced activity for the oxidation of chlorinated benzenes and selectivity of (+)-?-pinene oxidation. The crystal structure of the F87W/Y96F/V247L mutant with 1,3,5-trichlorobenzene and (+)-?-pinene bound revealed the enzyme substrate contacts and provided insights into the activity and selectivity patterns. The structures also provided a novel basis for further engineering of P450cam for increased activity and selectivity for the oxidation of related compounds.
Nitric oxide (NO) produced by mammalian nitric oxide synthases (mNOSs) is an important mediator in a variety of physiological functions. Crystal structures of mNOSs have shown strong conservation of the active-site residue Val567 (numbering for rat neuronal NOS, nNOS). NOS-like proteins have been identified in several bacterial pathogens, and these display striking sequence identity to the oxygenase domain of mNOS (NOSoxy), with the exception of a Val to Ile mutation at the active site. Preliminary studies have highlighted the importance of this Val residue in NO-binding, substrate recognition, and oxidation in mNOSs. To further elucidate the role of this valine in substrate and substrate analogue recognition, we generated five Val567 mutants of the oxygenase domain of the neuronal NOS (nNOSoxy) and used UV-visible and EPR spectroscopy to investigate the effects of these mutations on the heme distal environment, the stability of the heme-FeII-CO complexes, and the binding of a series of substrate analogues. Our results are consistent with Val567 playing an important role in preserving the integrity of the active site for substrate binding, stability of heme-bound gaseous ligands, and potential NO production.
- Publikační typ
- časopisecké články MeSH
Molecular dynamics simulations of complexes between Norwalk virus RNA dependent RNA polymerase and its natural CTP and 2dCTP (both containing the O5'-C5'-C4'-O4' sequence of atoms bridging the triphosphate and sugar moiety) or modified coCTP (C5'-O5'-C4'-O4'), cocCTP (C5'-O5'-C4'-C4'') substrates were produced by means of CUDA programmable graphical processing units and the ACEMD software package. It enabled us to gain microsecond MD trajectories clearly showing that similar nucleoside triphosphates can bind surprisingly differently into the active site of the Norwalk virus RNA dependent RNA polymerase. It corresponds to their different modes of action (CTP-substrate, 2dCTP-poor substrate, coCTP-chain terminator, cocCTP-inhibitor). Moreover, extremely rare events-as repetitive pervasion of Arg182 into a potentially reaction promoting arrangement-were captured.
- MeSH
- cytidintrifosfát analogy a deriváty metabolismus MeSH
- infekce viry z čeledi Caliciviridae virologie MeSH
- lidé MeSH
- Norovirus enzymologie metabolismus MeSH
- RNA-dependentní RNA-polymerasa metabolismus MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Class IIa histone deacetylases (HDACs) play critical roles in vertebrate development and physiology, yet direct evidence of their intrinsic deacetylase activity and on substrate specificity regarding the peptide sequence is still missing. In this study, we designed and synthesized a combinatorial peptide library allowing us to profile class IIa HDACs sequence specificity at positions +3 through -3 from the central lysine modified by the well-accepted trifluoroacetyl function. Our data revealed a strong preference for bulky aromatic acids directly flanking the central trifluoroacetyllysine, while all class IIa HDACs disfavor positively charged residues and proline at the +1/-1 positions. The chemical nature of amino acid residues N-terminally to the central trifluoroacetyllysine has a more profound effect on substrate recognition as compared to residues located C-terminally. These findings were validated by designing selected favored and disfavored peptide sequences, with the favored ones are accepted with catalytic efficacy of 75 000 and 525 000 M-1 s-1 for HDAC7 and HDAC5, respectively. Results reported here could help in developing class IIa HDACs inhibitors and also in the search for new natural class IIa HDACs substrates.
Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases.
- MeSH
- cyklin-dependentní kinasy chemie metabolismus MeSH
- cykliny chemie metabolismus MeSH
- ELISA MeSH
- HeLa buňky MeSH
- hmotnostní spektrometrie MeSH
- imunoprecipitace MeSH
- konformace proteinů MeSH
- krystalizace MeSH
- lidé MeSH
- molekulární modely * MeSH
- multiproteinové komplexy chemie metabolismus MeSH
- substrátová specifita MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The R2TP complex is a HSP90 co-chaperone, which consists of four subunits: PIH1D1, RPAP3, RUVBL1, and RUVBL2. It is involved in the assembly of large protein or protein-RNA complexes such as RNA polymerase, small nucleolar ribonucleoproteins (snoRNPs), phosphatidylinositol 3 kinase-related kinases (PIKKs), and their complexes. While RPAP3 has a HSP90 binding domain and the RUVBLs comprise ATPase activities important for R2TP functions, PIH1D1 contains a PIH-N domain that specifically recognizes phosphorylated substrates of the R2TP complex. In this review we provide an overview of the current knowledge of the R2TP complex with the focus on the recently identified structural and mechanistic features of the R2TP complex functions. We also discuss the way R2TP regulates cellular response to stress caused by low levels of nutrients or by DNA damage and its possible exploitation as a target for anti-cancer therapy.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Fibroblast activation protein (FAP) is a non-classical serine protease expressed predominantly in conditions accompanied by tissue remodeling, particularly cancer. Due to its plasma membrane localization, FAP represents a promising molecular target for tumor imaging and treatment. The unique enzymatic activity of FAP facilitates development of diagnostic and therapeutic tools based on molecular recognition of FAP by substrates and small-molecule inhibitors, in addition to conventional antibody-based strategies. In this review, we provide background on the pathophysiological role of FAP and discuss its potential for diagnostic and therapeutic applications. Furthermore, we present a detailed analysis of the structural patterns crucial for substrate and inhibitor recognition by the FAP active site and determinants of selectivity over the related proteases dipeptidyl peptidase IV and prolyl endopeptidase. We also review published data on targeting of the tumor microenvironment with FAP antibodies, FAP-targeted prodrugs, activity-based probes and small-molecule inhibitors. We describe use of a recently developed, selective FAP inhibitor with low-nanomolar potency in inhibitor-based targeting strategies including synthetic antibody mimetics based on hydrophilic polymers and inhibitor conjugates for PET imaging. In conclusion, recent advances in understanding of the molecular structure and function of FAP have significantly contributed to the development of several tools with potential for translation into clinical practice.
- MeSH
- dipeptidylpeptidasa 4 metabolismus MeSH
- fibroblasty metabolismus MeSH
- katalytická doména MeSH
- lidé MeSH
- membránové proteiny chemie účinky léků metabolismus MeSH
- molekulární struktura MeSH
- nádorové mikroprostředí MeSH
- nádory diagnóza metabolismus terapie MeSH
- prekurzory léčiv MeSH
- serinové endopeptidasy chemie účinky léků metabolismus MeSH
- substrátová specifita MeSH
- želatinasy chemie účinky léků metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring nonpolar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance of nonpolar interactions governing GCPII affinity toward novel substrates as well as formerly unnoticed plasticity of the S1' specificity pocket. On the basis of those data, we designed, synthesized, and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.
- MeSH
- antigeny povrchové genetika MeSH
- dipeptidy chemická syntéza chemie farmakologie MeSH
- glutamátkarboxypeptidasa II antagonisté a inhibitory genetika MeSH
- kinetika MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- kvantová teorie MeSH
- lidé MeSH
- ligandy MeSH
- molekulární modely MeSH
- mutageneze cílená MeSH
- substrátová specifita MeSH
- termodynamika MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, N.I.H., Intramural MeSH
