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1.
Haematologica ; 2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31558672

RESUMO

In the complex with Von Willebrand Factor, factor VIII (FVIII) is protected from rapid clearance from the circulation. Although it has been established that the FVIII binding site resides in the N-terminal D'-D3 domains of Von Willebrand Factor, detailed information about the amino acid regions that contribute to FVIII binding is still lacking. In the present study, Hydrogen- deuterium exchange mass spectrometry was employed to gain insight into the FVIII binding region on Von Willebrand Factor. To this end, time-dependent deuterium incorporation was assessed in D'-D3 and the FVIII - D'-D3 complex. Data showed reduced deuterium incorporation in D region Arg782-Cys799 in the FVIII - D'-D3 complex compared to D'- D3. This implies that this region interacts with FVIII. Site-directed mutagenesis of the six-individual charged amino acids in Arg782-Cys799 into alanine residues followed by surface plasmon resonance analysis and solid phase binding studies revealed that replacement of Asp796 affected FVIII binding. A marked decrease in FVIII binding was observed for the D'-D3 Glu787Ala variant. The same was observed for D'-D3 variants in which Asp796 and Glu787 were replaced by Asn796 and Gln787. Site-directed mutagenesis of Leu786, which together with Glu787 and Cys789 forms a short helical region in the crystal structure of D'-D3, also had a marked impact on FVIII binding. The combined results show that amino acid region Arg782-Cys799 is part of a FVIII binding surface. Our study provides new insight into FVIII - Von Willebrand Factor complex formation and defects therein that may be associated with bleedings caused by markedly reduced levels of FVIII.

2.
Biochem J ; 475(17): 2819-2830, 2018 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-30111575

RESUMO

The D'-D3 fragment of von Willebrand factor (VWF) can be divided into TIL'-E'-VWD3-C8_3-TIL3-E3 subdomains of which TIL'-E'-VWD3 comprises the main factor VIII (FVIII)-binding region. Yet, von Willebrand disease (VWD) Type 2 Normandy (2N) mutations, associated with impaired FVIII interaction, have been identified in C8_3-TIL3-E3. We now assessed the role of the VWF (sub)domains for FVIII binding using isolated D', D3 and monomeric C-terminal subdomain truncation variants of D'-D3. Competitive binding assays and surface plasmon resonance analysis revealed that D' requires the presence of D3 for effective interaction with FVIII. The isolated D3 domain, however, did not show any FVIII binding. Results indicated that the E3 subdomain is dispensable for FVIII binding. Subsequent deletion of the other subdomains from D3 resulted in a progressive decrease in FVIII-binding affinity. Chemical footprinting mass spectrometry suggested increased conformational changes at the N-terminal side of D3 upon subsequent subdomain deletions at the C-terminal side of the D3. A D'-D3 variant with a VWD type 2N mutation in VWD3 (D879N) or C8_3 (C1060R) also revealed conformational changes in D3, which were proportional to a decrease in FVIII-binding affinity. A D'-D3 variant with a putative VWD type 2N mutation in the E3 subdomain (C1225G) showed, however, normal binding. This implies that the designation VWD type 2N is incorrect for this variant. Results together imply that a structurally intact D3 in D'-D3 is indispensable for effective interaction between D' and FVIII explaining why specific mutations in D3 can impair FVIII binding.

3.
J Allergy Clin Immunol ; 142(4): 1285-1296, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29477724

RESUMO

BACKGROUND: The genetic cause of primary immunodeficiency disease (PID) carries prognostic information. OBJECTIVE: We conducted a whole-genome sequencing study assessing a large proportion of the NIHR BioResource-Rare Diseases cohort. METHODS: In the predominantly European study population of principally sporadic unrelated PID cases (n = 846), a novel Bayesian method identified nuclear factor κB subunit 1 (NFKB1) as one of the genes most strongly associated with PID, and the association was explained by 16 novel heterozygous truncating, missense, and gene deletion variants. This accounted for 4% of common variable immunodeficiency (CVID) cases (n = 390) in the cohort. Amino acid substitutions predicted to be pathogenic were assessed by means of analysis of structural protein data. Immunophenotyping, immunoblotting, and ex vivo stimulation of lymphocytes determined the functional effects of these variants. Detailed clinical and pedigree information was collected for genotype-phenotype cosegregation analyses. RESULTS: Both sporadic and familial cases demonstrated evidence of the noninfective complications of CVID, including massive lymphadenopathy (24%), unexplained splenomegaly (48%), and autoimmune disease (48%), features prior studies correlated with worse clinical prognosis. Although partial penetrance of clinical symptoms was noted in certain pedigrees, all carriers have a deficiency in B-lymphocyte differentiation. Detailed assessment of B-lymphocyte numbers, phenotype, and function identifies the presence of an increased CD21low B-cell population. Combined with identification of the disease-causing variant, this distinguishes between healthy subjects, asymptomatic carriers, and clinically affected cases. CONCLUSION: We show that heterozygous loss-of-function variants in NFKB1 are the most common known monogenic cause of CVID, which results in a temporally progressive defect in the formation of immunoglobulin-producing B cells.

4.
J Biol Chem ; 292(15): 6381-6388, 2017 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-28246168

RESUMO

HABP2 (hyaluronan-binding protein 2) is a Ca2+-dependent serine protease with putative roles in blood coagulation and fibrinolysis. A G221E substitution, known as the Marburg I polymorphism, reportedly affects HABP2 function and has been associated with increased risk for cardiovascular disease. However, the importance of Gly-221 for HABP2 activity is unclear. Here, we used G221E, G221A, and G221S mutants to assess the role of Gly-221 in HABP2 catalysis. The G221E variant failed to activate the single-chain urokinase-type plasminogen activator, and the G221A and G221S variants displayed moderately reduced single-chain urokinase-type plasminogen activator activation. Activity toward the peptide substrate S-2288 was markedly decreased in all HABP2 variants, with G221E being the most defective and G221A being the least defective. In the absence of Ca2+, S-2288 cleavage by wild-type HABP2 was Na+-dependent, with Km decreasing from 3.0 to 0.6 mm upon titration from 0 to 0.3 m Na+ In the presence of 5 mm Ca2+, Km was further reduced to 0.05 mm, but without an appreciable contribution of Na+ At physiological concentrations of Na+ and Ca2+, the three HABP2 variants, and particularly G221E, displayed a major Km increase for S-2288. Chemical footprinting revealed that Ile-16 is significantly less protected from chemical modification in G221E than in wild-type HABP2, suggesting impaired insertion of the N terminus into the G221E protease domain, with a concomitant impact on catalytic activity. Homology modeling suggested that the Glu-221 side chain could sterically hinder insertion of the N terminus into the HABP2 protease domain, helping to explain the detrimental effects of Glu-221 substitution on HABP2 activity.


Assuntos
Serina Endopeptidases/química , Substituição de Aminoácidos , Cálcio/química , Catálise , Glicina/química , Glicina/genética , Mutação de Sentido Incorreto , Domínios Proteicos , Serina Endopeptidases/genética , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/genética
5.
Haematologica ; 102(4): 686-694, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28057741

RESUMO

Factor VIII C-domains are believed to have specific functions in cofactor activity and in interactions with von Willebrand factor. We have previously shown that factor VIII is co-targeted with von Willebrand factor to the Weibel-Palade bodies in blood outgrowth endothelial cells, even when factor VIII carries mutations in the light chain that are associated with defective von Willebrand factor binding. In this study, we addressed the contribution of individual factor VIII C-domains in intracellular targeting, von Willebrand factor binding and cofactor activity by factor VIII/V C-domain swapping. Blood outgrowth endothelial cells were transduced with lentivirus encoding factor V, factor VIII or YFP-tagged C-domain chimeras, and examined by confocal microscopy. The same chimeras were produced in HEK293-cells for in vitro characterization and chemical foot-printing by mass spectrometry. In contrast to factor VIII, factor V did not target to Weibel-Palade bodies. The chimeras showed reduced Weibel-Palade body targeting, suggesting that this requires the factor VIII C1-C2 region. The factor VIII/V-C1 chimera did not bind von Willebrand factor and had reduced affinity for activated factor IX, whereas the factor VIII/V-C2 chimera showed a minor reduction in von Willebrand factor binding and normal interaction with activated factor IX. This suggests that mainly the C1-domain carries factor VIII-specific features in assembly with von Willebrand factor and activated factor IX. Foot-printing analysis of the chimeras revealed increased exposure of lysine residues in the A1/C2- and C1/C2-domain interface, suggesting increased C2-domain mobility and disruption of the natural C-domain tandem pair orientation. Apparently, this affects intracellular trafficking, but not extracellular function.


Assuntos
Fator VIII/metabolismo , Fator V/metabolismo , Domínios e Motivos de Interação entre Proteínas , Células Endoteliais/metabolismo , Fator V/química , Fator V/genética , Fator VIII/química , Fator VIII/genética , Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transporte Proteico , Relação Estrutura-Atividade , Fator de von Willebrand/metabolismo
6.
Biochem J ; 468(1): 65-72, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25728577

RESUMO

Tandem mass tags (TMTs) were utilized in a novel chemical footprinting approach to identify lysine residues that mediate the interaction of receptor-associated protein (RAP) with cluster II of LDL (low-density lipoprotein) receptor (LDLR)-related protein (LRP). The isolated RAP D3 domain was modified with TMT-126 and the D3 domain-cluster II complex with TMT-127. Nano-LC-MS analysis revealed reduced modification with TMT-127 of peptides including Lys(256), Lys(270) and Lys(305)-Lys(306) suggesting that these residues contribute to cluster II binding. This agrees with previous findings that Lys(256) and Lys(270) are critical for binding cluster II sub-domains [Fisher, Beglova and Blacklow (2006) Mol. Cell 22, 277-283]. Cluster II-binding studies utilizing D3 domain variants K(256)A, K(305)A and K(306)A now showed that Lys(306) contributes to cluster II binding as well. For full-length RAP, we observed that peptides including Lys(60), Lys(191), Lys(256), Lys(270) and Lys(305)-Lys(306) exhibited reduced modification with TMT in the RAP-cluster II complex. Notably, Lys(60) has previously been implicated to mediate D1 domain interaction with cluster II. Our results suggest that also Lys(191) of the D2 domain contributes to cluster II binding. Binding studies employing the RAP variants K(191)A, K(256)A, K(305)A and K(306)A, however, revealed a modest reduction in cluster II binding for the K(256)A variant only. This suggests that the other lysine residues can compensate for the absence of a single lysine residue for effective complex assembly. Collectively, novel insight has been obtained into the contribution of lysine residues of RAP to cluster II binding. In addition, we propose that TMTs can be utilized to identify lysine residues critical for protein complex formation.


Assuntos
Proteína Associada a Proteínas Relacionadas a Receptor de LDL/química , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Humanos , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Lisina/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Pegadas de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem
7.
ACS Chem Biol ; 8(10): 2133-9, 2013 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-23962156

RESUMO

Elucidation of subcellular signaling networks by multiparameter imaging is hindered by a lack of sensitive FRET pairs spectrally compatible with the classic CFP/YFP pair. Here, we present a generic strategy to enhance the traditionally poor sensitivity of red FRET sensors by developing self-associating variants of mOrange and mCherry that allow sensors to switch between well-defined on- and off states. Requiring just a single mutation of the mFruit domain, this new FRET pair improved the dynamic range of protease sensors up to 10-fold and was essential to generate functional red variants of CFP-YFP-based Zn(2+) sensors. The large dynamic range afforded by the new red FRET pair allowed simultaneous use of differently colored Zn(2+) FRET sensors to image Zn(2+) over a broad concentration range in the same cellular compartment.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Bibliotecas de Moléculas Pequenas , Zinco/química
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