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1.
Blood ; 139(20): 3087-3098, 2022 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-35245360

RESUMO

Glycoprotein VI (GPVI) mediates collagen-induced platelet activation after vascular damage and is an important contributor to the onset of thrombosis, heart attack, and stroke. Animal models of thrombosis have identified GPVI as a promising target for antithrombotic therapy. Although for many years the crystal structure of GPVI has been known, the essential details of its interaction with collagen have remained elusive. Here, we present crystal structures of the GPVI ectodomain bound to triple-helical collagen peptides, which reveal a collagen-binding site across the ß-sheet of the D1 domain. Mutagenesis and binding studies confirm the observed binding site and identify Trp76, Arg38, and Glu40 as essential residues for binding to fibrillar collagens and collagen-related peptides (CRPs). GPVI binds a site on collagen comprising two collagen chains with the core formed by the sequence motif OGPOGP. Potent GPVI-binding peptides from Toolkit-III all contain OGPOGP; weaker binding peptides frequently contain a partial motif varying at either terminus. Alanine-scanning of peptide III-30 also identified two AGPOGP motifs that contribute to GPVI binding, but steric hindrance between GPVI molecules restricts the maximum binding capacity. We further show that no cooperative interactions could occur between two GPVI monomers binding to a stretch of (GPO)5 and that binding of ≥2 GPVI molecules to a fibril-embedded helix requires non-overlapping OGPOGP motifs. Our structure confirms the previously suggested similarity in collagen binding between GPVI and leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) but also indicates significant differences that may be exploited for the development of receptor-specific therapeutics.


Assuntos
Glicoproteínas da Membrana de Plaquetas , Trombose , Animais , Sítios de Ligação , Plaquetas/metabolismo , Colágeno/metabolismo , Peptídeos/química , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ligação Proteica , Trombose/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(41): 25759-25770, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32994342

RESUMO

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.


Assuntos
Coronavirus/fisiologia , Hemaglutininas Virais/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais de Fusão/genética , Vírion/metabolismo , Animais , Evolução Biológica , Linhagem Celular , Coronavirus/genética , Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Coronavirus Humano OC43/fisiologia , Coronavirus Bovino/genética , Coronavirus Bovino/metabolismo , Coronavirus Bovino/fisiologia , Hemaglutininas Virais/química , Hemaglutininas Virais/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Camundongos , Mutação , Ligação Proteica , Domínios Proteicos , Receptores Virais/metabolismo , Seleção Genética , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Vírion/genética , Ligação Viral , Liberação de Vírus
3.
Proc Natl Acad Sci U S A ; 116(7): 2681-2690, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30679277

RESUMO

Human betacoronaviruses OC43 and HKU1 are endemic respiratory pathogens and, while related, originated from independent zoonotic introductions. OC43 is in fact a host-range variant of the species Betacoronavirus-1, and more closely related to bovine coronavirus (BCoV)-its presumptive ancestor-and porcine hemagglutinating encephalomyelitis virus (PHEV). The ß1-coronaviruses (ß1CoVs) and HKU1 employ glycan-based receptors carrying 9-O-acetylated sialic acid (9-O-Ac-Sia). Receptor binding is mediated by spike protein S, the main determinant of coronavirus host specificity. For BCoV, a crystal structure for the receptor-binding domain S1A is available and for HKU1 a cryoelectron microscopy structure of the complete S ectodomain. However, the location of the receptor-binding site (RBS), arguably the single-most important piece of information, is unknown. Here we solved the 3.0-Å crystal structure of PHEV S1A We then took a comparative structural analysis approach to map the ß1CoV S RBS, using the general design of 9-O-Ac-Sia-binding sites as blueprint, backed-up by automated ligand docking, structure-guided mutagenesis of OC43, BCoV, and PHEV S1A, and infectivity assays with BCoV-S-pseudotyped vesicular stomatitis viruses. The RBS is not exclusive to OC43 and related animal viruses, but is apparently conserved and functional also in HKU1 S1A The binding affinity of the HKU1 S RBS toward short sialoglycans is significantly lower than that of OC43, which we attribute to differences in local architecture and accessibility, and which may be indicative for differences between the two viruses in receptor fine-specificity. Our findings challenge reports that would map the OC43 RBS elsewhere in S1A and that of HKU1 in domain S1B.


Assuntos
Coronavirus Humano OC43/fisiologia , Fusão de Membrana , Ácido N-Acetilneuramínico/metabolismo , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Acetilação , Animais , Sítios de Ligação , Humanos , Ratos , Receptores Virais/química
4.
J Biol Chem ; 294(12): 4634-4643, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30674550

RESUMO

Murine paired immunoglobulin receptor B (PirB) and its human ortholog leukocyte immunoglobulin-like receptor B2 (LILRB2) are widely expressed inhibitory receptors that interact with a diverse set of extracellular ligands and exert functions ranging from down-regulation of immune responses to inhibition of neuronal growth. However, structural information that could shed light on how PirB interacts with its ligands is lacking. Here, we report crystal structures of the PirB ectodomain; the first full ectodomain structure for a LILR family member, at 3.3-4.5 Å resolution. The structures reveal that PirB's six Ig-like domains are arranged at acute angles, similar to the structures of leukocyte immunoglobulin-like receptor (LILR) and killer-cell immunoglobulin-like receptor (KIR). We observe that this regular arrangement is followed throughout the ectodomain, resulting in an extended zigzag conformation. In two out of the five structures reported here, the repeating zigzag is broken by the first domain that can adopt two alternative orientations. Quantitative binding experiments revealed a 9 µm dissociation constant for PirB-myelin-associated glycoprotein (MAG) ectodomain interactions. Taken together, these structural findings and the observed PirB-MAG interactions are compatible with a model for intercellular signaling in which the PirB extracellular domains, which point away from the cell surface, enable interaction with ligands in trans.


Assuntos
Receptores Imunológicos/química , Animais , Cristalografia por Raios X , Ligantes , Camundongos , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Ressonância de Plasmônio de Superfície
5.
Vet Res ; 49(1): 115, 2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30486901

RESUMO

Staphylococcus aureus is a versatile opportunistic pathogen, causing disease in human and animal species. Its pathogenicity is linked to the ability of S. aureus to secrete immunomodulatory molecules. These evasion proteins bind to host receptors or their ligands, resulting in inhibitory effects through high affinity protein-protein interactions. Staphylococcal evasion molecules are often species-specific due to differences in host target proteins between species. We recently solved the crystal structure of murine TLR2 in complex with immunomodulatory molecule staphylococcal superantigen-like protein 3 (SSL3), which revealed the essential residues within SSL3 for TLR2 inhibition. In this study we aimed to investigate the molecular basis of the interaction on the TLR2 side. The SSL3 binding region on murine TLR2 was compared to that of other species through sequence alignment and homology modeling, which identified interspecies differences. To examine whether this resulted in altered SSL3 activity on the corresponding TLR2s, bovine, equine, human, and murine TLR2 were stably expressed in HEK293T cells and the ability of SSL3 to inhibit TLR2 was assessed. We found that SSL3 was unable to inhibit bovine TLR2. Subsequent loss and gain of function mutagenesis showed that the lack of inhibition is explained by the absence of two tyrosine residues in bovine TLR2 that play a prominent role in the SSL3-TLR2 interface. We found no evidence for the existence of allelic SSL3 variants that have adapted to the bovine host. Thus, within this paper we reveal the molecular determinants of the TLR2-SSL3 interaction which adds to our understanding of staphylococcal host specificity.


Assuntos
Proteínas de Bactérias/farmacologia , Superantígenos/farmacologia , Receptor 2 Toll-Like/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Bovinos , Clonagem Molecular , Simulação por Computador , Proteínas de Ligação a DNA , Células HEK293 , Cavalos , Humanos , Modelos Químicos , Modelos Moleculares , Conformação Proteica , Especificidade da Espécie , Staphylococcus aureus/fisiologia
6.
Cell Host Microbe ; 21(3): 356-366, 2017 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-28279346

RESUMO

Human beta1-coronavirus (ß1CoV) OC43 emerged relatively recently through a single zoonotic introduction. Like related animal ß1CoVs, OC43 uses 9-O-acetylated sialic acid as receptor determinant. ß1CoV receptor binding is typically controlled by attachment/fusion spike protein S and receptor-binding/receptor-destroying hemagglutinin-esterase protein HE. We show that following OC43's introduction into humans, HE-mediated receptor binding was selected against and ultimately lost through progressive accumulation of mutations in the HE lectin domain. Consequently, virion-associated receptor-destroying activity toward multivalent glycoconjugates was reduced and altered such that some clustered receptor populations are no longer cleaved. Loss of HE lectin function was also observed for another respiratory human coronavirus, HKU1. This thus appears to be an adaptation to the sialoglycome of the human respiratory tract and for replication in human airways. The findings suggest that the dynamics of virion-glycan interactions contribute to host tropism. Our observations are relevant also to other human respiratory viruses of zoonotic origin, particularly influenza A virus.


Assuntos
Adaptação Biológica , Coronavirus Humano OC43/genética , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Lectinas/genética , Lectinas/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Ligação Viral , Animais , Coronavirus Humano OC43/fisiologia , Humanos , Mutação , Ligação Proteica , Receptores Virais/metabolismo
7.
Blood ; 129(4): 520-524, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-28064200

RESUMO

The von Willebrand receptor complex, which is composed of the glycoproteins Ibα, Ibß, GPV, and GPIX, plays an essential role in the earliest steps in hemostasis. During the last 4 decades, it has become apparent that loss of function of any 1 of 3 of the genes encoding these glycoproteins (namely, GP1BA, GP1BB, and GP9) leads to autosomal recessive macrothrombocytopenia complicated by bleeding. A small number of variants in GP1BA have been reported to cause a milder and dominant form of macrothrombocytopenia, but only 2 tentative reports exist of such a variant in GP1BB By analyzing data from a collection of more than 1000 genome-sequenced patients with a rare bleeding and/or platelet disorder, we have identified a significant association between rare monoallelic variants in GP1BB and macrothrombocytopenia. To strengthen our findings, we sought further cases in 2 additional collections in the United Kingdom and Japan. Across 18 families exhibiting phenotypes consistent with autosomal dominant inheritance of macrothrombocytopenia, we report on 27 affected cases carrying 1 of 9 rare variants in GP1BB.


Assuntos
Plaquetas/metabolismo , Hemorragia/genética , Mutação , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Trombocitopenia/genética , Alelos , Plaquetas/patologia , Estudos de Casos e Controles , Feminino , Expressão Gênica , Genes Dominantes , Genoma Humano , Hemorragia/diagnóstico , Hemorragia/metabolismo , Hemorragia/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linhagem , Contagem de Plaquetas , Trombocitopenia/diagnóstico , Trombocitopenia/metabolismo , Trombocitopenia/patologia
8.
Proc Natl Acad Sci U S A ; 113(22): E3111-9, 2016 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-27185912

RESUMO

Hemagglutinin-esterases (HEs) are bimodular envelope proteins of orthomyxoviruses, toroviruses, and coronaviruses with a carbohydrate-binding "lectin" domain appended to a receptor-destroying sialate-O-acetylesterase ("esterase"). In concert, these domains facilitate dynamic virion attachment to cell-surface sialoglycans. Most HEs (type I) target 9-O-acetylated sialic acids (9-O-Ac-Sias), but one group of coronaviruses switched to using 4-O-Ac-Sias instead (type II). This specificity shift required quasisynchronous adaptations in the Sia-binding sites of both lectin and esterase domains. Previously, a partially disordered crystal structure of a type II HE revealed how the shift in lectin ligand specificity was achieved. How the switch in esterase substrate specificity was realized remained unresolved, however. Here, we present a complete structure of a type II HE with a receptor analog in the catalytic site and identify the mutations underlying the 9-O- to 4-O-Ac-Sia substrate switch. We show that (i) common principles pertaining to the stereochemistry of protein-carbohydrate interactions were at the core of the transition in lectin ligand and esterase substrate specificity; (ii) in consequence, the switch in O-Ac-Sia specificity could be readily accomplished via convergent intramolecular coevolution with only modest architectural changes in lectin and esterase domains; and (iii) a single, inconspicuous Ala-to-Ser substitution in the catalytic site was key to the emergence of the type II HEs. Our findings provide fundamental insights into how proteins "see" sugars and how this affects protein and virus evolution.


Assuntos
Coronavirus/enzimologia , Hemaglutininas Virais/metabolismo , Lectinas/metabolismo , Mutação/genética , Receptores Virais/metabolismo , Ácidos Siálicos/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Colo/metabolismo , Cristalografia por Raios X , Hemaglutininas Virais/química , Hemaglutininas Virais/genética , Humanos , Lectinas/química , Camundongos , Simulação de Acoplamento Molecular , Receptores de Coronavírus , Receptores Virais/química , Receptores Virais/genética , Ácidos Siálicos/química , Estereoisomerismo , Especificidade por Substrato , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética
9.
Structure ; 23(12): 2349-2357, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26585513

RESUMO

Inflammasomes are high molecular weight protein complexes that play a crucial role in innate immunity by activating caspase-1. Inflammasome formation is initiated when molecules originating from invading microorganisms activate nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) and induce NLR multimerization. Little is known about the conformational changes involved in NLR activation and the structural organization of NLR multimers. Here, we show by cryoelectron tomography that flagellin-induced NAIP5/NLRC4 multimers form right- and left-handed helical polymers with a diameter of 28 nm and a pitch of 6.5 nm. Subtomogram averaging produced an electron density map at 4 nm resolution, which was used for rigid body fitting of NLR subdomains derived from the crystal structure of dormant NLRC4. The resulting structural model of inflammasome-incorporated NLRC4 indicates that a prominent rotation of the LRR domain of NLRC4 is necessary for multimer formation, providing unprecedented insight into the conformational changes that accompany NLR activation.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas de Ligação ao Cálcio/química , Inflamassomos/química , Proteína Inibidora de Apoptose Neuronal/química , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Células HEK293 , Humanos , Inflamassomos/metabolismo , Camundongos , Proteína Inibidora de Apoptose Neuronal/metabolismo
10.
Proc Natl Acad Sci U S A ; 112(35): 11018-23, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26283364

RESUMO

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2-glycans with the conserved Lewis(X) binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2-Pam2CSK4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2-lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.


Assuntos
Endotoxinas/fisiologia , Staphylococcus aureus/fisiologia , Receptor 2 Toll-Like/antagonistas & inibidores , Dimerização , Endotoxinas/química , Endotoxinas/genética , Estrutura Molecular , Mutagênese , Ligação Proteica , Receptor 2 Toll-Like/química
11.
Cell Rep ; 11(12): 1966-78, 2015 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-26095364

RESUMO

Sialic acids (Sias), 9-carbon-backbone sugars, are among the most complex and versatile molecules of life. As terminal residues of glycans on proteins and lipids, Sias are key elements of glycotopes of both cellular and microbial lectins and thus act as important molecular tags in cell recognition and signaling events. Their functions in such interactions can be regulated by post-synthetic modifications, the most common of which is differential Sia-O-acetylation (O-Ac-Sias). The biology of O-Ac-Sias remains mostly unexplored, largely because of limitations associated with their specific in situ detection. Here, we show that dual-function hemagglutinin-esterase envelope proteins of nidoviruses distinguish between a variety of closely related O-Ac-Sias. By using soluble forms of hemagglutinin-esterases as lectins and sialate-O-acetylesterases, we demonstrate differential expression of distinct O-Ac-sialoglycan populations in an organ-, tissue- and cell-specific fashion. Our findings indicate that programmed Sia-O-acetylation/de-O-acetylation may be critical to key aspects of cell development, homeostasis, and/or function.


Assuntos
Acetilesterase/biossíntese , Hemaglutininas Virais/genética , Ácido N-Acetilneuramínico/genética , Ácidos Siálicos/genética , Proteínas Virais de Fusão/genética , Acetilação , Acetilesterase/genética , Animais , Regulação da Expressão Gênica , Genoma , Hemaglutininas Virais/química , Hemaglutininas Virais/metabolismo , Humanos , Lipídeos/química , Lipídeos/genética , Mamíferos , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Nidovirales/química , Proteínas/química , Proteínas/genética , Ácidos Siálicos/química , Especificidade da Espécie , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo
12.
Protein Expr Purif ; 99: 27-34, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24680733

RESUMO

Transient transfection of the human HEK293-EBNA1 cell line using polyethyleneimine is widely adopted for recombinant protein production. Whereas high expression of many targets is achieved, purification yields of some highly expressed proteins remain low due to aggregation. We hypothesized that for these proteins the expression rates achieved at standard transfection conditions are too high, causing an overload of the protein folding machinery. Here we present plasmid titration as an efficient method to vary expression rates for the optimization of soluble protein expression. In plasmid titration a dilution series of expression vector mixed with dummy plasmid is transfected in small scale cultures. Application to GFP shows that plasmid titration achieves a wide range of expression levels while maintaining high transfection efficiencies even at 500-fold plasmid dilution. Application of plasmid titration to selected Nod-like receptors (NLRs), which at standard conditions are highly expressed but poorly soluble, delays the onset of NLR aggregation and improves cell viability and the buildup of biomass. The amount of soluble protein depends on the combination of dilution factor and harvest day in a protein specific manner. For NOD1 50-fold plasmid dilution increases the amount of soluble protein approximately 5-fold. Due to its association with chaperones at all dilution factors tested we were unable to purify NOD1 to homogeneity. For NLRC4, which did not associate with chaperones, 10-fold plasmid dilution increased the purification yield 2-fold. This improvement, obtained with minimal effort due to the simplicity of the method, shows that reducing total expression may increase soluble protein yield.


Assuntos
Células HEK293/metabolismo , Plasmídeos/genética , Agregados Proteicos/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Transfecção/métodos , Proteínas Adaptadoras de Sinalização CARD/química , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/isolamento & purificação , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/isolamento & purificação , Morte Celular , Antígenos Nucleares do Vírus Epstein-Barr/genética , Proteínas de Fluorescência Verde/genética , Humanos , Polietilenoimina , Dobramento de Proteína/efeitos dos fármacos , Proteínas Recombinantes/genética , Solubilidade
13.
PLoS One ; 8(7): e67923, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23844130

RESUMO

BACKGROUND: RP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown. METHODS AND RESULTS: TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105(-/-) mice resulting in a higher proliferation rate. In RP105(-/-) mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982 ± 974 µm(2) vs.1947 ± 278 µm(2),p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105(-/-) mice this effect was more pronounced (10316±1243 µm(2) vs.4208 ± 555 µm(2),p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500 ± 573 vs.6581 ± 1894 µm(2),p<0.05), whereas expression of the single factors RP105 or MD1 had no effect. CONCLUSION: RP105 is a potent inhibitor of post-interventional neointima formation.


Assuntos
Antígenos CD/metabolismo , Vasos Sanguíneos/metabolismo , Neointima/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Antígenos CD/genética , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Artéria Femoral/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Imuno-Histoquímica , Lipopolissacarídeos/farmacologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Neointima/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Solubilidade , Receptor 4 Toll-Like/genética
14.
J Biol Chem ; 287(46): 38460-72, 2012 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-23012363

RESUMO

The NOD-like receptors NAIP5 and NLRC4 play an essential role in the innate immune response to the bacterial tail protein flagellin. Upon flagellin detection, NAIP5 and NLRC4 form a hetero-oligomeric inflammasome that induces caspase-1-dependent cell death. So far, both the mechanism of formation of the NAIP5-NLRC4 inflammasome and its structure are poorly understood. In this study we combine inflammasome reconstitution in HEK293 cells, purification of inflammasome components, and negative stain electron microscopy to address these issues. We find that a Salmonella typhimurium flagellin fragment comprising the D0 domain and the neighboring spoke region is able to co-precipitate NAIP5 and induce formation of the NAIP5-NLRC4 inflammasome. Comparison with smaller fragments indicates that flagellin recognition is mediated by its C-terminal residues as well as the spoke region. We reconstitute the inflammasome from purified flagellin, NAIP5, and NLRC4, thus proving that no other cellular components are required for its formation. Electron micrographs of the purified inflammasome provide unprecedented insight into its architecture, revealing disk-like complexes consisting of 11 or 12 protomers in which NAIP5 and NLRC4 appear to occupy equivalent positions. On the basis of our data, we propose a model for inflammasome formation wherein direct interaction of flagellin with a single NAIP5 induces the recruitment and progressive incorporation of NLRC4, resulting in the formation of a hetero-oligomeric inflammasome.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Adaptadoras de Sinalização CARD/química , Proteínas de Ligação ao Cálcio/química , Flagelina/metabolismo , Inflamassomos/metabolismo , Proteína Inibidora de Apoptose Neuronal/química , Salmonella typhimurium/metabolismo , Animais , Caspase 1/química , DNA Complementar/metabolismo , Células HEK293 , Humanos , Ligantes , Camundongos , Microscopia Eletrônica/métodos , Plasmídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína
15.
PLoS One ; 7(6): e37617, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22685543

RESUMO

We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames. Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles. As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs. Although we primarily designed this strategy to accelerate assembly of repetitive constructs for single-molecule force spectroscopy, we anticipate that this approach is equally applicable to the reconstitution and modification of complex modular sequences including structural and functional analysis of multi-domain proteins, synthetic biology or the modular construction of episomal vectors.


Assuntos
Clonagem Molecular/métodos , DNA/genética , Modelos Genéticos , Fases de Leitura Aberta/genética , Sequência de Aminoácidos , Sequência de Bases , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação ao Cálcio , Fibronectinas/química , Fibronectinas/genética , Fibronectinas/metabolismo , Células HEK293 , Humanos , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Temperatura de Transição
16.
Proc Natl Acad Sci U S A ; 109(14): 5253-8, 2012 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-22440751

RESUMO

Fibrillar collagens, the most abundant proteins in the vertebrate body, are involved in a plethora of biological interactions. Plasma protein von Willebrand factor (VWF) mediates adhesion of blood platelets to fibrillar collagen types I, II, and III, which is essential for normal haemostasis. High affinity VWF-binding sequences have been identified in the homotrimeric collagen types II and III, however, it is unclear how VWF recognizes the heterotrimeric collagen type I, the superstructure of which is unknown. Here we present the crystal structure of VWF domain A3 bound to a collagen type III-derived homotrimeric peptide. Our structure reveals that VWF-A3 interacts with all three collagen chains and binds through conformational selection to a sequence that is one triplet longer than was previously appreciated from platelet and VWF binding studies. The VWF-binding site overlaps those of SPARC (also known as osteonectin) and discodin domain receptor 2, but is more extended and shifted toward the collagen amino terminus. The observed collagen-binding mode of VWF-A3 provides direct structural constraints on collagen I chain registry. A VWF-binding site can be generated from the sequences RGQAGVMF, present in the two α1(I) chains, and RGEOGNIGF, in the unique α2(I) chain, provided that α2(I) is in the middle or trailing position. Combining these data with previous structural data on integrin binding to collagen yields strong support for the trailing position of the α2(I) chain, shedding light on the fundamental and long-standing question of the collagen I chain registry.


Assuntos
Colágeno Tipo I/química , Fator de von Willebrand/química , Sequência de Aminoácidos , Sítios de Ligação , Colágeno Tipo I/metabolismo , Humanos , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Fator de von Willebrand/metabolismo
17.
PLoS Pathog ; 8(1): e1002492, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22291594

RESUMO

The hemagglutinin-esterases (HEs), envelope glycoproteins of corona-, toro- and orthomyxoviruses, mediate reversible virion attachment to O-acetylated sialic acids (O-Ac-Sias). They do so through concerted action of distinct receptor-binding ("lectin") and receptor-destroying sialate O-acetylesterase ("esterase") domains. Most HEs target 9-O-acetylated Sias. In one lineage of murine coronaviruses, however, HE esterase substrate and lectin ligand specificity changed dramatically as these viruses evolved to use 4-O-acetylated Sias instead. Here we present the crystal structure of the lectin domain of mouse hepatitis virus (MHV) strain S HE, resolved both in its native state and in complex with a receptor analogue. The data show that the shift from 9-O- to 4-O-Ac-Sia receptor usage primarily entailed a change in ligand binding topology and, surprisingly, only modest changes in receptor-binding site architecture. Our findings illustrate the ease with which viruses can change receptor-binding specificity with potential consequences for host-, organ and/or cell tropism, and for pathogenesis.


Assuntos
Hemaglutininas Virais/química , Vírus da Hepatite Murina/química , Ácido N-Acetilneuramínico/química , Receptores Virais/química , Proteínas Virais de Fusão/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Hemaglutininas Virais/metabolismo , Humanos , Camundongos , Vírus da Hepatite Murina/fisiologia , Ácido N-Acetilneuramínico/metabolismo , Ratos , Ratos Wistar , Receptores Virais/metabolismo , Proteínas Virais de Fusão/metabolismo , Tropismo Viral/fisiologia
18.
Nat Commun ; 2: 385, 2011 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-21750539

RESUMO

von Willebrand factor (VWF) multimers mediate primary adhesion and aggregation of platelets. VWF potency critically depends on multimer size, which is regulated by a feedback mechanism involving shear-induced unfolding of the VWF-A2 domain and cleavage by the metalloprotease ADAMTS-13. Here we report crystallographic and single-molecule optical tweezers data on VWF-A2 providing mechanistic insight into calcium-mediated stabilization of the native conformation that protects A2 from cleavage by ADAMTS-13. Unfolding of A2 requires higher forces when calcium is present and primarily proceeds through a mechanically stable intermediate with non-native calcium coordination. Calcium further accelerates refolding markedly, in particular, under applied load. We propose that calcium improves force sensing by allowing reversible force switching under physiologically relevant hydrodynamic conditions. Our data show for the first time the relevance of metal coordination for mechanical properties of a protein involved in mechanosensing.


Assuntos
Cálcio/metabolismo , Modelos Moleculares , Conformação Proteica , Estabilidade Proteica , Fator de von Willebrand/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Linhagem Celular , Cromatografia de Afinidade , Cromatografia em Gel , Cristalografia , Humanos , Simulação de Dinâmica Molecular , Pinças Ópticas , Desnaturação Proteica , Fator de von Willebrand/isolamento & purificação
19.
Haematologica ; 95(5): 829-32, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20007141

RESUMO

The ERYTHROPOIETIN (EPO) gene is regulated by the transcription factor Hypoxia Inducible Factor-alpha (HIF-alpha). In this pathway, Prolyl Hydroxylase Domain protein 2 (PHD2) hydroxylates two prolyl residues in HIF-alpha, which in turn promotes HIF-alpha degradation by the von Hippel Lindau (VHL) protein. Evidence that HIF-2alpha is the important isoform for EPO regulation in humans comes from the recent observation that mutations in the HIF2A gene are associated with cases of erythrocytosis. We report here a new erythrocytosis-associated mutation, p.Asp539Glu, in the HIF2A gene. Similar to all reported cases, the affected residue is in close vicinity and C-terminal to the primary hydroxylation site in HIF-2alpha, Pro531. This mutation, however, is notable in producing a rather subtle amino acid substitution. Nonetheless, we find that this mutation compromises binding of HIF-2alpha to both PHD2 and VHL, and we propose that this mutation is the cause of erythrocytosis in this individual.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Mutação de Sentido Incorreto/genética , Policitemia/diagnóstico , Policitemia/genética , Adolescente , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Feminino , Humanos
20.
Blood ; 115(7): 1364-73, 2010 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-20007810

RESUMO

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), one of the most widely spread immune receptors, attenuates immune cell activation when bound to specific sites in collagen. The collagen-binding domain of LAIR-1 is homologous to that of glycoprotein VI (GPVI), a collagen receptor crucial for platelet activation. Because LAIR-1 and GPVI also display overlapping collagen-binding specificities, a common structural basis for collagen recognition would appear likely. Therefore, it is crucial to gain insight into the molecular interaction of both receptors with their ligand to prevent unwanted cross-reactions during therapeutic intervention. We determined the crystal structure of LAIR-1 and mapped its collagen-binding site by nuclear magnetic resonance (NMR) titrations and mutagenesis. Our data identify R59, E61, and W109 as key residues for collagen interaction. These residues are strictly conserved in LAIR-1 and GPVI alike; however, they are located outside the previously proposed GPVI collagen-binding site. Our data provide evidence for an unanticipated mechanism of collagen recognition common to LAIR-1 and GPVI. This fundamental insight will contribute to the exploration of specific means of intervention in collagen-induced signaling in immunity and hemostasis.


Assuntos
Colágeno/metabolismo , Glicoproteínas da Membrana de Plaquetas/química , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Sítios de Ligação/fisiologia , Cristalografia , Expressão Gênica , Humanos , Células K562 , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Ativação Plaquetária/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Imunológicos/genética , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade
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