Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Int J Mol Sci ; 21(6)2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32244989

RESUMO

The Center of Biomedical Research Excellence in Matrix Biology strives to improve our understanding of extracellular matrix at molecular, cellular, tissue, and organismal levels to generate new knowledge about pathophysiology, normal development, and regenerative medicine. The primary goals of the Center are to i) support junior investigators, ii) enhance the productivity of established scientists, iii) facilitate collaboration between both junior and established researchers, and iv) build biomedical research infrastructure that will support research relevant to cell-matrix interactions in disease progression, tissue repair and regeneration, and v) provide access to instrumentation and technical support. A Pilot Project program provides funding to investigators who propose applying their expertise to matrix biology questions. Support from the National Institute of General Medical Sciences at the National Institutes of Health that established the Center of Biomedical Research Excellence in Matrix Biology has significantly enhanced the infrastructure and the capabilities of researchers at Boise State University, leading to new approaches that address disease diagnosis, prevention, and treatment. New multidisciplinary collaborations have been formed with investigators who may not have previously considered how their biomedical research programs addressed fundamental and applied questions involving the extracellular matrix. Collaborations with the broader matrix biology community are encouraged.

2.
Mar Drugs ; 17(12)2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31795126

RESUMO

KTM is a 16 amino acid peptide with the sequence WCCSYPGCYWSSSKWC. Here, we present the nuclear magnetic resonance (NMR) structure and bioactivity of this rationally designed α-conotoxin (α-CTx) that demonstrates potent inhibition of rat α3ß2-nicotinic acetylcholine receptors (rα3ß2-nAChRs). Two bioassays were used to test the efficacy of KTM. First, a qualitative PC12 cell-based assay confirmed that KTM acts as a nAChR antagonist. Second, bioactivity evaluation by two-electrode voltage clamp electrophysiology was used to measure the inhibition of rα3ß2-nAChRs by KTM (IC50 = 0.19 ± 0.02 nM), and inhibition of the same nAChR isoform by α-CTx MII (IC50 = 0.35 ± 0.8 nM). The three-dimensional structure of KTM was determined by NMR spectroscopy, and the final set of 20 structures derived from 32 distance restraints, four dihedral angle constraints, and two disulfide bond constraints overlapped with a mean global backbone root-mean-square deviation (RMSD) of 1.7 ± 0.5 Å. The structure of KTM did not adopt the disulfide fold of α-CTx MII for which it was designed, but instead adopted a flexible ribbon backbone and disulfide connectivity of C2-C16 and C3-C8 with an estimated 12.5% α-helical content. In contrast, α-CTx MII, which has a native fold of C2-C8 and C3-C16, has an estimated 38.1% α-helical secondary structure. KTM is the first reported instance of a Framework I (CC-C-C) α-CTx with ribbon connectivity to display sub-nanomolar inhibitory potency of rα3ß2-nAChR subtypes.

3.
Structure ; 25(1): 94-106, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-27989620

RESUMO

The ß-barrel assembly machine (BAM) mediates the biogenesis of outer membrane proteins (OMPs) in Gram-negative bacteria. BamA, the central BAM subunit composed of a transmembrane ß-barrel domain linked to five polypeptide transport-associated (POTRA) periplasmic domains, is thought to bind nascent OMPs and undergo conformational cycling to catalyze OMP folding and insertion. One model is that conformational flexibility between POTRA domains is part of this conformational cycling. Nuclear magnetic resonance (NMR) spectroscopy was used here to study the flexibility of the POTRA domains 1-5 in solution. NMR relaxation studies defined effective rotational correlational times and together with residual dipolar coupling data showed that POTRA1-2 is flexibly linked to POTRA3-5. Mutants of BamA that restrict flexibility between POTRA2 and POTRA3 by disulfide crosslinking displayed impaired function in vivo. Together these data strongly support a model in which conformational cycling of hinge motions between POTRA2 and POTRA3 in BamA is required for biological function.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Periplasma , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de Proteína
4.
Proc Natl Acad Sci U S A ; 113(46): E7169-E7175, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27799531

RESUMO

An essential early step in the assembly of human spliceosomes onto pre-mRNA involves the recognition of regulatory RNA cis elements in the 3' splice site by the U2 auxiliary factor (U2AF). The large (U2AF65) and small (U2AF35) subunits of the U2AF heterodimer contact the polypyrimidine tract (Py-tract) and the AG-dinucleotide, respectively. The tandem RNA recognition motif domains (RRM1,2) of U2AF65 adopt closed/inactive and open/active conformations in the free form and when bound to bona fide Py-tract RNA ligands. To investigate the molecular mechanism and dynamics of 3' splice site recognition by U2AF65 and the role of U2AF35 in the U2AF heterodimer, we have combined single-pair FRET and NMR experiments. In the absence of RNA, the RRM1,2 domain arrangement is highly dynamic on a submillisecond time scale, switching between closed and open conformations. The addition of Py-tract RNA ligands with increasing binding affinity (strength) gradually shifts the equilibrium toward an open conformation. Notably, the protein-RNA complex is rigid in the presence of a strong Py-tract but exhibits internal motion with weak Py-tracts. Surprisingly, the presence of U2AF35, whose UHM domain interacts with U2AF65 RRM1, increases the population of the open arrangement of U2AF65 RRM1,2 in the absence and presence of a weak Py-tract. These data indicate that the U2AF heterodimer promotes spliceosome assembly by a dynamic population shift toward the open conformation of U2AF65 to facilitate the recognition of weak Py-tracts at the 3' splice site. The structure and RNA binding of the heterodimer was unaffected by cancer-linked myelodysplastic syndrome mutants.


Assuntos
Sítios de Splice de RNA , RNA/metabolismo , Fator de Processamento U2AF/metabolismo , Dimerização , Humanos , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , RNA/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spliceossomos/metabolismo , Fator de Processamento U2AF/química , Fator de Processamento U2AF/genética
5.
Biochemistry ; 54(28): 4307-19, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26132046

RESUMO

Resonance assignments are the first step in most NMR studies of protein structure, function, and dynamics. Standard protein assignment methods employ through-bond backbone experiments on uniformly (13)C/(15)N-labeled proteins. For larger proteins, this through-bond assignment procedure often breaks down due to rapid relaxation and spectral overlap. The challenges involved in studies of larger proteins led to efficient methods for (13)C labeling of side chain methyl groups, which have favorable relaxation properties and high signal-to-noise. These methyls are often still assigned by linking them to the previously assigned backbone, thus limiting the applications for larger proteins. Here, a structure-based procedure is described for assignment of (13)C(1)H3-labeled methyls by comparing distance information obtained from three-dimensional methyl-methyl nuclear Overhauser effect (NOE) spectroscopy with the X-ray structure. The Ile, Leu, or Val (ILV) methyl type is determined by through-bond experiments, and the methyl-methyl NOE data are analyzed in combination with the known structure. A hierarchical approach was employed that maps the largest observed "NOE-methyl cluster" onto the structure. The combination of identification of ILV methyl type with mapping of the NOE-methyl clusters greatly simplifies the assignment process. This method was applied to the inactive and active forms of the 42-kDa ILV (13)C(1)H3-methyl labeled extracellular signal-regulated kinase 2 (ERK2), leading to assignment of 60% of the methyls, including 90% of Ile residues. A series of ILV to Ala mutants were analyzed, which helped confirm the assignments. These assignments were used to probe the local and long-range effects of ligand binding to inactive and active ERK2.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Domínio Catalítico , Cristalografia por Raios X , Isoleucina/análise , Leucina/análise , Metilação , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Nucleotídeos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Ratos , Valina/análise
6.
Methods Enzymol ; 558: 333-362, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26068746

RESUMO

Biological activity in the cell is predominantly mediated by large multiprotein and protein-nucleic acid complexes that act together to ensure functional fidelity. Nuclear magnetic resonance (NMR) spectroscopy is the only method that can provide information for high-resolution three-dimensional structures and the conformational dynamics of these complexes in solution. Mapping of binding interfaces and molecular interactions along with the characterization of conformational dynamics is possible for very large protein complexes. In contrast, de novo structure determination by NMR becomes very time consuming and difficult for protein complexes larger than 30 kDa as data are noisy and sparse. Fortunately, high-resolution structures are often available for individual domains or subunits of a protein complex and thus sparse data can be used to define their arrangement and dynamics within the assembled complex. In these cases, NMR can therefore be efficiently combined with complementary solution techniques, such as small-angle X-ray or neutron scattering, to provide a comprehensive description of the structure and dynamics of protein complexes in solution. Particularly useful are NMR-derived paramagnetic relaxation enhancements (PREs), which provide long-range distance restraints (ca. 20Å) for structural analysis of large complexes and also report on conformational dynamics in solution. Here, we describe the use of PREs from sample production to structure calculation, focusing on protein-RNA complexes. On the basis of recent examples from our own research, we demonstrate the utility, present protocols, and discuss potential pitfalls when using PREs for studying the structure and dynamic features of protein-RNA complexes.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Drosophila/química , Proteínas Nucleares/química , Proteínas com Motivo de Reconhecimento de RNA/química , Proteínas de Ligação a RNA/química , RNA/química , Ribonucleoproteínas/química , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Espectroscopia de Ressonância de Spin Eletrônica/instrumentação , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Humanos , Espectroscopia de Ressonância Magnética/instrumentação , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Soluções , Marcadores de Spin , Fator de Processamento U2AF , Termodinâmica
7.
J Am Chem Soc ; 136(19): 7068-76, 2014 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-24734879

RESUMO

Multidomain proteins containing intrinsically disordered linkers exhibit large-scale dynamic modes that play key roles in a multitude of molecular recognition and signaling processes. Here, we determine the conformational space sampled by the multidomain splicing factor U2AF65 using complementary nuclear magnetic resonance spectroscopy and small-angle scattering data. Available degrees of conformational freedom are initially stochastically sampled and experimental data then used to delineate the potential energy landscape in terms of statistical probability. The spatial distribution of U2AF65 conformations is found to be highly anisotropic, comprising significantly populated interdomain contacts that appear to be electrostatic in origin. This hypothesis is supported by the reduction of signature PREs reporting on expected interfaces with increasing salt concentration. The described spatial distribution reveals the complete spectrum of the unbound forms of U2AF65 that coexist with the small percentage of a preformed RNA-bound domain arrangement required for polypyrimidine-tract recognition by conformational selection. More generally, the proposed approach to describing conformational equilibria of multidomain proteins can be further combined with other experimental data that are sensitive to domain dynamics.


Assuntos
Proteínas Nucleares/química , Ribonucleoproteínas/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Espalhamento a Baixo Ângulo , Fator de Processamento U2AF , Eletricidade Estática , Difração de Raios X
8.
Angew Chem Int Ed Engl ; 53(17): 4475-9, 2014 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-24623579

RESUMO

Ultra-high-field NMR spectroscopy requires an increased bandwidth for heteronuclear decoupling, especially in biomolecular NMR applications. Composite pulse decoupling cannot provide sufficient bandwidth at practical power levels, and adiabatic pulse decoupling with sufficient bandwidth is compromised by sideband artifacts. A novel low-power, broadband heteronuclear decoupling pulse is presented that generates minimal, ultra-low sidebands. The pulse was derived using optimal control theory and represents a new generation of decoupling pulses free from the constraints of periodic and cyclic sequences. In comparison to currently available state-of-the-art methods this novel pulse provides greatly improved decoupling performance that satisfies the demands of high-field biomolecular NMR spectroscopy.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Simulação por Computador , Humanos
9.
J Mol Biol ; 411(1): 83-95, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21624375

RESUMO

The CS-RDC-NOE Rosetta program was used to generate the solution structure of a 27-kDa fragment of the Escherichia coli BamC protein from a limited set of NMR data. The BamC protein is a component of the essential five-protein ß-barrel assembly machine in E. coli. The first 100 residues in BamC were disordered in solution. The Rosetta calculations showed that BamC101₋344 forms two well-defined domains connected by an ~18-residue linker, where the relative orientation of the domains was not defined. Both domains adopt a helix-grip fold previously observed in the Bet v 1 superfamily. ¹5N relaxation data indicated a high degree of conformational flexibility for the linker connecting the N-terminal domain and the C-terminal domain in BamC. The results here show that CS-RDC-NOE Rosetta is robust and has a high tolerance for misassigned nuclear Overhauser effect restraints, greatly simplifying NMR structure determinations.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas Ligadas a Lipídeos/química , Biologia Computacional/métodos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dobramento de Proteína , Software
10.
GSTF Int J Bioinforma Biotechnol ; 1(1): 43-48, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25309886

RESUMO

The amino propeptide of collagen α1(XI) (NPP) has been shown to bind glycosaminoglycans and to form a dimer. While these are independent biochemical events, it is likely that dimerization facilitates the interaction with glycosaminoglycans or alternatively, that glycosaminoglycan interaction facilitates the formation of an NPP:NPP dimer. The computer program MODELLER was used to generate a homology model of the collagen α1(XI) NPP monomer using the crystal structure of the closely related noncollagenous-4 (NC4) domain of collagen α1(IX) (PDB:2UUR) as the template. Additionally, a dimer model of collagen α1(XI) NPP domain was created based upon the thrombospondin dimer template (PDB:1Z78). The structure of the dimer created in MODELLER was validated by comparison to a dimer model generated by docking two monomers of PDB:2UUR using ClusPro. Calculations of relative binding energy for the interaction between each collagen α1(XI) NPP model and glycosaminoglycans as ligands was performed using AutoDock4. Computational results support a higher affinity between heparan sulfate and a dimer compared to a monomer. These findings are supported by affinity chromatography experiments in which distinct monomer and dimer peaks were observed. Sequential point mutation studies of the putative binding site (147-KKKITK-152) indicated the importance of the basic lysine residue for binding to heparan sulfate. Two orders of magnitude change in binding affinity was predicted when comparing wild type to the mutation K152A. Experimental data supports the predicted change in affinity.

11.
J Hum Evol ; 60(2): 205-212, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21190724

RESUMO

While the hominid fossil record clearly shows that brain size has rapidly expanded over the last ~2.5 M.yr. the forces driving this change remain unclear. One popular hypothesis proposes that metabolic adaptations in response to dietary shifts supported greater encephalization in humans. An increase in meat consumption distinguishes the human diet from that of other great apes. Creatine, an essential metabolite for energy homeostasis in muscle and brain tissue, is abundant in meat and was likely ingested in higher quantities during human origins. Five phosphocreatine circuit proteins help regulate creatine utilization within energy demanding cells. We compared the expression of all five phosphocreatine circuit genes in cerebral cortex, cerebellum, and skeletal muscle tissue for humans, chimpanzees, and rhesus macaques. Strikingly, SLC6A8 and CKB transcript levels are higher in the human brain, which should increase energy availability and turnover compared to non-human primates. Combined with other well-documented differences between humans and non-human primates, this allocation of energy to the cerebral cortex and cerebellum may be important in supporting the increased metabolic demands of the human brain.


Assuntos
Evolução Biológica , Encéfalo/metabolismo , Fosfocreatina/metabolismo , Primatas/genética , Animais , Creatina Quinase Forma BB/genética , Creatina Quinase Forma MM/genética , Creatina Quinase Mitocondrial/genética , Humanos , Macaca mulatta , Proteínas de Membrana Transportadoras/genética , Músculo Esquelético/metabolismo , Pan troglodytes
12.
Proc Biol Sci ; 278(1708): 961-9, 2011 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-21177690

RESUMO

There are numerous anthropological analyses concerning the importance of diet during human evolution. Diet is thought to have had a profound influence on the human phenotype, and dietary differences have been hypothesized to contribute to the dramatic morphological changes seen in modern humans as compared with non-human primates. Here, we attempt to integrate the results of new genomic studies within this well-developed anthropological context. We then review the current evidence for adaptation related to diet, both at the level of sequence changes and gene expression. Finally, we propose some ways in which new technologies can help identify specific genomic adaptations that have resulted in metabolic and morphological differences between humans and non-human primates.


Assuntos
Evolução Biológica , Hominidae/genética , Hominidae/metabolismo , Adaptação Fisiológica , Animais , Sequência de Bases , Dieta , Expressão Gênica , Genômica , Hominidae/anatomia & histologia , Hominidae/fisiologia , Humanos , Primatas/genética , Primatas/metabolismo , Primatas/fisiologia
13.
PLoS One ; 5(9)2010 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-20824057

RESUMO

Because many species-specific phenotypic differences are assumed to be caused by differential regulation of gene expression, many recent investigations have focused on measuring transcript abundance. Despite the availability of high-throughput platforms, quantitative real-time polymerase chain reaction (RT-QPCR) is often the method of choice because of its low cost and wider dynamic range. However, the accuracy of this technique heavily relies on the use of multiple valid control genes for normalization. We created a pipeline for choosing genes potentially useful as RT-QPCR control genes for measuring expression between human and chimpanzee samples across multiple tissues, using published microarrays and a measure of tissue-specificity. We identified 13 genes from the pipeline and from commonly used control genes: ACTB, USP49, ARGHGEF2, GSK3A, TBP, SDHA, EIF2B2, GPDH, YWHAZ, HPTR1, RPL13A, HMBS, and EEF2. We then tested these candidate genes and validated their expression stability across species. We established the rank order of the most preferable set of genes for single and combined tissues. Our results suggest that for at least three tissues (cerebral cortex, liver, and skeletal muscle), EIF2B2, EEF2, HMBS, and SDHA are useful genes for normalizing human and chimpanzee expression using RT-QPCR. Interestingly, other commonly used control genes, including TBP, GAPDH, and, especially ACTB do not perform as well. This pipeline could be easily adapted to other species for which expression data exist, providing taxonomically appropriate control genes for comparisons of gene expression among species.


Assuntos
Evolução Molecular , Expressão Gênica , Reação em Cadeia da Polimerase/normas , Primatas/genética , Animais , Córtex Cerebral/metabolismo , Humanos , Fígado/metabolismo , Músculo Esquelético/metabolismo , Especificidade de Órgãos , Pan troglodytes/genética , Pan troglodytes/metabolismo , Primatas/metabolismo , Padrões de Referência , Especificidade da Espécie
14.
Brain Res ; 1288: 1-8, 2009 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-19591812

RESUMO

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, is known to contain naturally occurring genetic variation in it's promoter region that associates with a number of neuropsychological disorders. As such, examining non-coding regions is important for understanding tyrosine hydroxylase function in human health and disease. We examined approximately 2 kb upstream of the translation start site within humans and non-human primates to obtain a fine resolution map of evolutionarily and functionally relevant cis-regulatory differences. Our study investigated Macaca mulatta, Pan troglodytes, Gorilla gorilla, and Homo sapiens haplotypes using transient dual-luciferase transfection in three neuroblastoma cell lines to assay the impact of naturally occurring sequence variation on expression level. In addition to trans effects between cell lines, there are several significant expression differences between primate species, but the most striking difference was seen between human haplotypes in one cell line. Underlying this variation are numerous sequence polymorphisms, two of which influence expression within humans in a non-additive and cell line-specific manner. This study highlights functional consequences of tyrosine hydroxylase genetic variation in primates. Additionally, the results emphasize the importance of examining more than one cell line, the existence of multiple functional variants in a given promoter region and the presence of non-additive cis-interactions.


Assuntos
Regulação Enzimológica da Expressão Gênica/genética , Variação Genética/genética , Tirosina 3-Mono-Oxigenase/genética , Análise de Variância , Animais , Linhagem Celular Tumoral , Células Cultivadas , Clonagem Molecular , Gorilla gorilla , Haplótipos , Humanos , Macaca mulatta , Neurônios/fisiologia , Pan troglodytes , Polimorfismo Genético/genética , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
15.
J Biomed Mater Res A ; 90(1): 247-55, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18496861

RESUMO

Collagen is a major component of the newly synthesized pericellular microenvironment of chondrocytes. Collagen types II, IX, and XI are synthesized and assembled into higher ordered complexes by a mechanism in which type XI collagen plays a role in nucleation of new fibrils, and in limiting fibril diameter. This study utilizes a cell line derived from the Swarm rat chondrosarcoma that allows the accumulation and assembly of pericellular matrix. Immunofluorescence and atomic force microscopy were used to assess early intermediates of fibril formation. Results indicate that this cell line synthesizes and secretes chondrocyte-specific pericellular matrix molecules including types II, IX, and XI collagen and is suitable for the study of newly synthesized collagen matrix under the experimental conditions used. AFM data indicate that small fibrils or assemblies of microfibrils are detectable and may represent precursors of the approximately 20 nm thin fibrils reported in cartilage. Treatment with hyaluronidase indicates that the dimensions of the small fibrils may be dependent upon the presence of hyaluronan within the matrix. This study provides information on the composition and organization of the newly synthesized extracellular matrix that plays a role in establishing the material properties and performance of biological materials such as cartilage.


Assuntos
Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Colágeno Tipo IX/metabolismo , Colágeno Tipo XI/metabolismo , Matriz Extracelular/metabolismo , Animais , Linhagem Celular Tumoral , Condrócitos/citologia , Colágeno Tipo II/ultraestrutura , Colágeno Tipo IX/ultraestrutura , Colágeno Tipo XI/ultraestrutura , Matriz Extracelular/química , Hialuronoglucosaminidase/metabolismo , Microfibrilas/metabolismo , Microfibrilas/ultraestrutura , Microscopia de Força Atômica , Fenótipo , Ratos
16.
Am J Pathol ; 170(5): 1725-38, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17456777

RESUMO

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are both characterized pathologically by the presence of neuronal inclusions termed Lewy bodies (LBs). A common feature found in LBs are aggregates of alpha-synuclein (alpha-Syn), and although it is now recognized that alpha-Syn is the major building block for these toxic filaments, the mechanism of how this occurs remains unknown. In the present study, we demonstrate that proteolytic processing of alpha-Syn by the protease calpain I leads to the formation of aggregated high-molecular weight species and adoption of a beta-sheet structure. To determine whether calpain-cleavage of alpha-Syn occurs in PD and DLB, we designed site-directed calpain-cleavage antibodies to alpha-Syn and tested their utility in several animal model systems. Detection of calpain-cleaved alpha-Syn was evident in mouse models of cerebral ischemia and PD and in a Drosophila model of PD. In the human PD and DLB brain, calpain-cleaved alpha-Syn antibodies immunolabeled LBs and neurites in the substantia nigra. Moreover, calpain-cleaved alpha-Syn fragments identified within LBs colocalized with activated calpain in neurons of the PD and DLB brains. These findings suggest that calpain I may participate in the disease-linked aggregation of alpha-Syn in various alpha-synucleinopathies.


Assuntos
Encéfalo/metabolismo , Calpaína/metabolismo , Doença por Corpos de Lewy/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Animais , Área Sob a Curva , Western Blotting , Linhagem Celular Tumoral , Drosophila , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunoprecipitação , Doença por Corpos de Lewy/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurônios/metabolismo , Doença de Parkinson/patologia , Estrutura Secundária de Proteína , alfa-Sinucleína/química
17.
Protein Expr Purif ; 52(2): 403-9, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17166742

RESUMO

The amino terminal domain of collagen type XI alpha1 chain is a noncollagenous structure that is essential for the regulation of fibrillogenesis in developing cartilage. The amino terminal domain is alternatively spliced at the mRNA level, resulting in proteins expressed as splice variants. These splice variants, or isoforms, have unique distribution in growing tissues, alluding to distinct roles in development. We report here a rapid and straightforward method for expression, purification and in vitro folding of recombinant collagen XI isoforms alpha1(XI) NTD[p7] and alpha1(XI) NTD[p6b+7]. The recombinant isoforms were expressed in Escherichia coli as bacterial inclusion bodies. Unfolded carboxy terminal polyhistidine tagged proteins were purified via nickel affinity chromatography and refolded with specific protocols optimized for each isoform. Purity was assessed by SDS-PAGE and correct secondary structure by a comparison of circular dichroism data with that obtained for Npp. Protein expression and purification of the recombinant collagen XI splice variants will allow further studies to elucidate the structure and molecular interactions with components of the extracellular matrix. This research will clarify the mechanism of collagen XI mediated regulation of collagen fibrillogenesis.


Assuntos
Processamento Alternativo/fisiologia , Colágeno Tipo XI/metabolismo , Expressão Gênica , Dobramento de Proteína , Animais , Sequência de Bases , Dicroísmo Circular , Colágeno Tipo XI/química , Colágeno Tipo XI/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/fisiologia , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
J Biol Chem ; 281(51): 39507-16, 2006 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-17062562

RESUMO

Collagen type XI is a constituent of the pericellular matrix of chondrocytes and plays a role in the regulation of fibrillogenesis. The amino-terminal domain of collagen type XI alpha1 chain is a noncollagenous structure that has been identified on the surface of cartilage collagen fibrils. The biochemical composition of the amino-terminal domain varies due to alternative splicing of the primary transcript. Recombinantly expressed alpha1(XI) aminoterminal domain isoforms were used in this study to investigate potential interactions. Purified products were analyzed for heparan sulfate binding properties. The results demonstrated that two additional binding sites exist within the alpha1(XI) aminoterminal domain, one within the amino propeptide and one within the variable region of the amino-terminal domain. Analysis of relative affinities indicated that the site located within the amino propeptide (site 1) was of similar affinity to sites that exist within the major triple helix of collagen type XI. Substitution of amino acid residues 147 to 152 within the amino propeptide by site-directed mutagenesis resulted in altered affinity for heparan sulfate. The binding site located within the variable region (site 2) demonstrated significantly higher affinity than other sites within the molecule. Displacement of collagen type XI within the pericellular matrix was observed in cell culture in the presence of excess heparan sulfate and by treatment with heparinase. These studies suggest two additional binding sites located within the noncollagenous amino-terminal domain that may play a role in the function of collagen type XI. The localization of collagen type XI within the pericellular matrix may be dependent upon interactions with heparan sulfate proteoglycans, and these are likely to take place in an isoform-specific manner.


Assuntos
Colágeno Tipo XI/química , Heparitina Sulfato/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Colágeno/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Dobramento de Proteína , Isoformas de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos
19.
Protein Sci ; 14(6): 1526-37, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15930001

RESUMO

Fibrillar collagens are the principal structural molecules of connective tissues. The assembly of collagen fibrils is regulated by quantitatively minor fibrillar collagens, types V and XI. A unique amino-terminal propeptide domain of these collagens has been attributed this regulatory role. The structure of the amino terminal propeptide has yet to be determined. Low sequence similarity necessitated a secondary structure-based method to carry out homology modeling based upon the determined structure of LNS family members, named for a common structure in the laminin LG5 domain, the neurexin 1B domain and the sex hormone binding globulin. Distribution of amino acids within the model suggested glycosaminoglycan interaction and calcium binding. These activities were tested experimentally. Sequence analyses of existing genes for collagens indicate that 16 known collagen alpha chains may contain an LNS domain. A similar approach may prove useful for structure/function studies of similar domains in other collagens with similar domains. This will provide mechanistic details of the organization and assembly of the extracellular matrix and the underlying basis of structural integrity in connective tissues. The absolute requirement for collagen XI in skeletal growth is indicated by collagen XI deficiencies such as chondrodystrophies found in the cho/cho mouse and in humans with Stickler syndrome.


Assuntos
Colágeno Tipo XI/química , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA