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1.
Hum Mol Genet ; 25(24): 5311-5320, 2016 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-27798099

RESUMEN

Retinoschisin, an octameric retinal-specific protein, is essential for retinal architecture with mutations causing X-linked retinoschisis (XLRS), a monogenic form of macular degeneration. Most XLRS-associated mutations cause intracellular retention, however a subset are secreted as octamers and the cause of their pathology is ill-defined. Therefore, here we investigated the solution structure of the retinoschisin monomer and the impact of two XLRS-causing mutants using a combinatorial approach of biophysics and cryo-EM. The retinoschisin monomer has an elongated structure which persists in the octameric assembly. Retinoschisin forms a dimer of octamers with each octameric ring adopting a planar propeller structure. Comparison of the octamer with the hexadecamer structure indicated little conformational change in the retinoschisin octamer upon dimerization, suggesting that the octamer provides a stable interface for the construction of the hexadecamer. The H207Q XLRS-associated mutation was found in the interface between octamers and destabilized both monomeric and octameric retinoschisin. Octamer dimerization is consistent with the adhesive function of retinoschisin supporting interactions between retinal cell layers, so disassembly would prevent structural coupling between opposing membranes. In contrast, cryo-EM structural analysis of the R141H mutation at ∼4.2Šresolution was found to only cause a subtle conformational change in the propeller tips, potentially perturbing an interaction site. Together, these findings support distinct mechanisms of pathology for two classes of XLRS-associated mutations in the retinoschisin assembly.


Asunto(s)
Proteínas del Ojo/química , Proteínas del Ojo/genética , Retinosquisis/genética , Relación Estructura-Actividad , Animales , Células COS , Chlorocebus aethiops , Microscopía por Crioelectrón , Proteínas del Ojo/ultraestructura , Humanos , Mutación/genética , Conformación Proteica , Multimerización de Proteína , Retina/química , Retina/patología , Retinosquisis/patología
2.
J Biol Chem ; 289(18): 12842-51, 2014 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-24627488

RESUMEN

The adjacent fibrinogen (Fg)- and fibronectin (Fn)-binding sites on Fn-binding protein A (FnBPA), a cell surface protein from Staphylococcus aureus, are implicated in the initiation and persistence of infection. FnBPA contains a single Fg-binding site (that also binds elastin) and multiple Fn-binding sites. Here, we solved the structure of the N2N3 domains containing the Fg-binding site of FnBPA in the apo form and in complex with a Fg peptide. The Fg binding mechanism is similar to that of homologous bacterial proteins but without the requirement for "latch" strand residues. We show that the Fg-binding sites and the most N-terminal Fn-binding sites are nonoverlapping but in close proximity. Although Fg and a subdomain of Fn can form a ternary complex on an FnBPA protein construct containing a Fg-binding site and single Fn-binding site, binding of intact Fn appears to inhibit Fg binding, suggesting steric regulation. Given the concentrations of Fn and Fg in the plasma, this mechanism might result in targeting of S. aureus to fibrin-rich thrombi or elastin-rich tissues.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Fibrinógeno/metabolismo , Fibronectinas/metabolismo , Staphylococcus aureus/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Fibrinógeno/química , Fibronectinas/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Staphylococcus aureus/genética , Resonancia por Plasmón de Superficie
3.
J Biol Chem ; 286(44): 38311-38320, 2011 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-21840989

RESUMEN

Bacterial fibronectin-binding proteins (FnBPs) contain a large intrinsically disordered region (IDR) that mediates adhesion of bacteria to host tissues, and invasion of host cells, through binding to fibronectin (Fn). These FnBP IDRs consist of Fn-binding repeats (FnBRs) that form a highly extended tandem ß-zipper interaction on binding to the N-terminal domain of Fn. Several FnBR residues are highly conserved across bacterial species, and here we investigate their contribution to the interaction. Mutation of these residues to alanine in SfbI-5 (a disordered FnBR from the human pathogen Streptococcus pyogenes) reduced binding, but for each residue the change in free energy of binding was <2 kcal/mol. The structure of an SfbI-5 peptide in complex with the second and third F1 modules from Fn confirms that the conserved FnBR residues play equivalent functional roles across bacterial species. Thus, in SfbI-5, the binding energy for the tandem ß-zipper interaction with Fn is distributed across the interface rather than concentrated in a small number of "hot spot" residues that are frequently observed in the interactions of folded proteins. We propose that this might be a common feature of the interactions of IDRs and is likely to pose a challenge for the development of small molecule inhibitors of FnBP-mediated adhesion to and invasion of host cells.


Asunto(s)
Adhesinas Bacterianas/química , Fibronectinas/química , Streptococcus pyogenes/metabolismo , Adhesinas Bacterianas/metabolismo , Calorimetría , Cristalografía por Rayos X/métodos , Humanos , Cinética , Espectroscopía de Resonancia Magnética/métodos , Cadenas de Markov , Conformación Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Resonancia por Plasmón de Superficie , Termodinámica
4.
Mol Membr Biol ; 27(4-6): 147-59, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20446876

RESUMEN

To operate as a rotary motor, the ATP-hydrolyzing domain of the vacuolar H(+)-ATPase must be connected to a fixed structure in its membrane-bound proton pump domain by a mechanical stator. Although low-resolution structural data and spectroscopic analysis indicate that a filament-like subunit E/subunit G heterodimer performs this role, more detailed information about the relative arrangement of these subunits is limited. We have used a site-directed cross-linking approach to show that, in both bacterial and yeast V-type ATPases, the N-terminal alpha-helical segments of the G and E subunits are closely aligned over a distance of up to 40 A. Furthermore, cross-linking coupled to mass spectrometry shows that the C-terminal end of G is anchored at the C-terminal globular domain of subunit E. These data are consistent with a stator model comprising two approximately 150 A long parallel alpha-helices linked to each other at both ends, stabilized by a coiled-coil arrangement and capped by the globular C-terminal domain of E that connects the cytoplasmic end of the helical structure to the V-ATPase catalytic domain.


Asunto(s)
ATPasas de Translocación de Protón Vacuolares/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dicroismo Circular , Reactivos de Enlaces Cruzados/química , Disulfuros/química , Enterococcus/enzimología , Enterococcus/genética , Immunoblotting , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo
5.
J Biol Chem ; 284(38): 25938-43, 2009 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-19617354

RESUMEN

Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Missense mutations in fibulin 5 cause the elastin disorder cutis laxa and have been associated with age-related macular degeneration, a leading cause of blindness. We investigated the structure, hydrodynamics, and oligomerization of fibulin 5 using small angle x-ray scattering, EM, light scattering, circular dichroism, and sedimentation. Compact structures for the monomer were determined by small angle x-ray scattering and EM, and are supported by close agreement between the theoretical sedimentation of the structures and the experimental sedimentation of the monomer in solution. EM showed that monomers associate around a central cavity to form a dimer. Light scattering and equilibrium sedimentation demonstrated that the equilibrium between the monomer and the dimer is dependent upon NaCl and Ca2+ concentrations and that the dimer is dominant under physiological conditions. The dimerization of fragments containing just the cbEGF domains suggests that intermolecular interactions between cbEGFs cause dimerization of fibulin 5. It is possible that fibulin 5 functions as a dimer during elastinogenesis or that dimerization may provide a method for limiting interactions with binding partners such as tropoelastin.


Asunto(s)
Proteínas de la Matriz Extracelular/química , Multimerización de Proteína/fisiología , Cloruro de Sodio/química , Calcio/química , Calcio/metabolismo , Dicroismo Circular , Cutis Laxo/genética , Cutis Laxo/metabolismo , Tejido Elástico/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Degeneración Macular/genética , Degeneración Macular/metabolismo , Mutación Missense , Unión Proteica/fisiología , Estructura Cuaternaria de Proteína/fisiología , Dispersión de Radiación , Cloruro de Sodio/metabolismo , Tropoelastina/química , Tropoelastina/genética , Tropoelastina/metabolismo , Rayos X
6.
Hum Mutat ; 27(6): 568-74, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16652333

RESUMEN

Age-related macular degeneration (ARMD) is the leading cause of irreversible visual loss in the Western world, affecting approximately 25 million people worldwide. The pathogenesis is complex and missense mutations in FBLN5 have been reported in association with ARMD. We have investigated the role of fibulin 5 in ARMD by completing the first European study of the gene FBLN5 in ARMD (using 2 European cohorts of 805 ARMD patients and 279 controls) and by determining the functional effects of the missense mutations on fibulin 5 expression. We also correlated the FBLN5 genotype with the ARMD phenotype. We found two novel sequence changes in ARMD patients that were absent in controls and expressed these and the other nine reported FBLN5 mutations associated with ARMD and two associated with the autosomal recessive disease cutis laxa. Fibulin 5 secretion was significantly reduced (P<0.001) for four ARMD (p.G412E, p.G267S, p.I169 T, and p.Q124P) and two cutis laxa (p.S227P, p.C217R) mutations. These results suggest that some missense mutations associated with ARMD lead to decreased fibulin 5 secretion with a possible corresponding reduction in elastinogenesis. This study confirms the previous work identifying an association between FBLN5 mutations and ARMD and for the first time suggests a functional mechanism by which these mutations can lead to ARMD. It further demonstrates that FBLN5 mutations can be associated with different phenotypes of ARMD (not limited to the previously described cuticular drusen type). Such knowledge may ultimately lead to the development of novel therapies for this common disease.


Asunto(s)
Cutis Laxo/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Degeneración Macular/genética , Factores de Edad , Animales , Células COS , Chlorocebus aethiops , Estudios de Cohortes , Cutis Laxo/metabolismo , Análisis Mutacional de ADN , Proteínas de la Matriz Extracelular/fisiología , Genotipo , Humanos , Degeneración Macular/diagnóstico , Degeneración Macular/metabolismo , Mutación Missense , Fenotipo , Pliegue de Proteína , Radiografía , Retina/diagnóstico por imagen , Retina/patología
7.
PLoS One ; 5(6): e10985, 2010 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-20539757

RESUMEN

BACKGROUND: To identify molecular mechanisms underlying SCN5A-related sick sinus syndrome (SSS), a rare type of SSS, in parallel experiments we elucidated the electrophysiological properties and the cell surface localization of thirteen human Na(v)1.5 (hNa(v)1.5) mutant channels previously linked to this disease. METHODOLOGY/PRINCIPAL FINDINGS: Mutant hNa(v)1.5 channels expressed by HEK293 cells and Xenopus oocytes were investigated by whole-cell patch clamp and two-microelectrode voltage clamp, respectively. HEK293 cell surface biotinylation experiments quantified the fraction of correctly targeted channel proteins. Our data suggested three distinct mutant channel subtypes: Group 1 mutants (L212P, P1298L, DelF1617, R1632H) gave peak current densities and cell surface targeting indistinguishable from wild-type hNa(v)1.5. Loss-of-function of these mutants resulted from altered channel kinetics, including a negative shift of steady-state inactivation and a reduced voltage dependency of open-state inactivation. Group 2 mutants (E161K, T220I, D1275N) gave significantly reduced whole-cell currents due to impaired cell surface localization (D1275N), altered channel properties at unchanged cell surface localization (T220I), or a combination of both (E161K). Group 3 mutant channels were non-functional, due to an almost complete lack of protein at the plasma membrane (T187I, W1421X, K1578fs/52, R1623X) or a probable gating/permeation defect with normal surface localisation (R878C, G1408R). CONCLUSIONS/SIGNIFICANCE: This study indicates that multiple molecular mechanisms, including gating abnormalities, trafficking defects, or a combination of both, are responsible for SCN5A-related familial SSS.


Asunto(s)
Proteínas Musculares/genética , Síndrome del Seno Enfermo/genética , Canales de Sodio/genética , Anciano , Biotina/metabolismo , Línea Celular , Tamización de Portadores Genéticos , Humanos , Microelectrodos , Proteínas Musculares/fisiología , Mutación , Canal de Sodio Activado por Voltaje NAV1.5 , Técnicas de Placa-Clamp , Canales de Sodio/fisiología
8.
Invest Ophthalmol Vis Sci ; 51(5): 2356-62, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20007835

RESUMEN

PURPOSE: AMD has a complex etiology with environmental and genetic risk factors. Ten fibulin 5 sequence variants have been associated with AMD and two other fibulin 5 mutations cause autosomal-recessive cutis laxa. Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Biophysical techniques were used to detect structural changes in the fibulin 5 mutants and to determine whether changes are predictive of pathogenicity. METHODS: Native PAGE, nonreduced SDS-PAGE, size-exclusion column multiangle laser light scattering, sedimentation velocity, and circular dichroism (CD) were used to investigate the mobility, hydrodynamic radii, folding, and oligomeric states of the fibulin 5 mutants in the absence and presence of Ca(2+). RESULTS: CD showed that all mutants are folded, although perturbations to secondary structure contents were detected. Both cutis laxa mutants increased dimerization. Most other mutants slightly increased self-association in the absence of Ca(2+) but this was also demonstrated by G202R, a polymorphism detected in a control individual. The AMD-associated mutant G412E showed lower-than-expected mobility during native-PAGE, the largest hydrodynamic radius for the monomer form and the highest levels of aggregation in both the absence and presence of Ca(2+). CONCLUSIONS: The results identified structural differences for the disease-causing cutis laxa mutants and for one AMD variant (G412E), suggesting that this may also be pathogenic. Although the other AMD-associated mutants showed no gross structural differences, they cannot be excluded as pathogenic by differences outside the scope of this study-for example, disruption of heterointeractions.


Asunto(s)
Cutis Laxo/genética , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Degeneración Macular/genética , Mutación Missense , Calcio/farmacología , Cromatografía en Gel , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Humanos , Estructura Molecular , Mutagénesis Sitio-Dirigida , Pliegue de Proteína
9.
Biochemistry ; 44(10): 3933-41, 2005 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-15751969

RESUMEN

Vacuolar H(+)-ATPases (V-ATPases) are multi-subunit membrane proteins that couple ATP hydrolysis to the extrusion of protons from the cytoplasm. Although they share a common macromolecular architecture and rotational mechanism with the F(1)F(0)-ATPases, the organization of many of the specialized V-ATPase subunits within this rotary molecular motor remains uncertain. In this study, we have identified sequence segments involved in linking putative stator subunits in the Saccharomyces V-ATPase. Precipitation assays revealed that subunits Vma5p (subunit C) and Vma10p (subunit G), expressed as glutathione-S-transferase fusion proteins in E. coli, are both able to interact strongly with Vma4p (subunit E) expressed in a cell-free system. GST-Vma10p also associated with Vma2p and Vma1p, the core subunits of the ATP-hydrolyzing domain, and was able to self-associate to form a dimer. Mutations within the first 19-residue region of Vma4p, which disrupted interaction with Vma5p in vitro, also prevented the Vma4p polypeptide from restoring V-ATPase function in a complementation assay in vivo. These mutations did not prevent assembly of Vma5p (subunit C) and Vma2p (subunit B) into an inactive complex at the vacuolar membrane, indicating that Vma5p must make multiple interactions involving other V-ATPase subunits. A second, highly conserved region of Vma4p between residues 19 and 38 is involved in binding Vma10p. This region is highly enriched in charged residues, suggesting a role for electrostatic effects in Vma4p-Vma10p interaction. These protein interaction studies show that the N-terminal region of Vma4p is a key factor not only in the stator structure of the V-ATPase rotary molecular motor, but also in mediating interactions with putative regulatory subunits.


Asunto(s)
Proteínas Motoras Moleculares/metabolismo , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Secuencia de Aminoácidos , Animales , Sistema Libre de Células/enzimología , Escherichia coli/enzimología , Escherichia coli/genética , Prueba de Complementación Genética , Humanos , Proteínas Motoras Moleculares/biosíntesis , Proteínas Motoras Moleculares/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional/genética , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , ATPasas de Translocación de Protón Vacuolares/biosíntesis , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/genética
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