114 research outputs found
The bvr locus of Listeria monocytogenes mediates virulence gene repression by beta-glucosides
Advancing multimer analysis of von Willebrand factor by single-molecule AFM imaging
The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773', Cys2773-Cys2771', and Cys2811-Cys2811'. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p. Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p. Cys2771Arg and p. Cys2773Arg, we found the majority of monomers (87 +/- 5% and 73 +/- 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants' different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 +/- 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p. Cys2771Arg, p. Cys2773Arg, and p. Cys2811Ala we found 'even-numbered' non-native multimers, i.e. multimers with monomers attached on both termini;a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis
Mutual A domain interactions in the force sensing protein von Willebrand factor
The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets
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DNA binds to a specific site of the adhesive blood-protein von Willebrand factor guided by electrostatic interactions.
Neutrophils release their intracellular content, DNA included, into the bloodstream to form neutrophil extracellular traps (NETs) that confine and kill circulating pathogens. The mechanosensitive adhesive blood protein, von Willebrand Factor (vWF), interacts with the extracellular DNA of NETs to potentially immobilize them during inflammatory and coagulatory conditions. Here, we elucidate the previously unknown molecular mechanism governing the DNA-vWF interaction by integrating atomistic, coarse-grained, and Brownian dynamics simulations, with thermophoresis, gel electrophoresis, fluorescence correlation spectroscopy (FCS), and microfluidic experiments. We demonstrate that, independently of its nucleotide sequence, double-stranded DNA binds to a specific helix of the vWF A1 domain, via three arginines. This interaction is attenuated by increasing the ionic strength. Our FCS and microfluidic measurements also highlight the key role shear-stress has in enabling this interaction. Our simulations attribute the previously-observed platelet-recruitment reduction and heparin-size modulation, upon establishment of DNA-vWF interactions, to indirect steric hindrance and partial overlap of the binding sites, respectively. Overall, we suggest electrostatics-guiding DNA to a specific protein binding site-as the main driving force defining DNA-vWF recognition. The molecular picture of a key shear-mediated DNA-protein interaction is provided here and it constitutes the basis for understanding NETs-mediated immune and hemostatic responses
Size Evolution of Ordered SiGe Islands Grown by Surface Thermal Diffusion on Pit-Patterned Si(100) Surface
The ordered growth of self-assembled SiGe islands by surface thermal diffusion in ultra high vacuum from a lithographically etched Ge stripe on pit-patterned Si(100) surface has been experimentally investigated. The total surface coverage of Ge strongly depends on the distance from the source stripe, as quantitatively verified by Scanning Auger Microscopy. The size distribution of the islands as a function of the Ge coverage has been studied by coupling atomic force microscopy scans with Auger spectro-microscopy data. Our observations are consistent with a physical scenario where island positioning is essentially driven by energetic factors, which predominate with respect to the local kinetics of diffusion, and the growth evolution mainly depends on the local density of Ge atoms
Gain-of-Function Variant pPro2555Arg of von Willebrand Factor Increases Aggregate Size through Altering Stem Dynamics
The multimeric plasma glycoprotein (GP) von Willebrand factor (VWF) is best known for recruiting platelets to sites of injury during primary hemostasis. Generally, mutations in the VWF gene lead to loss of hemostatic activity and thus the bleeding disorder von Willebrand disease. By employing cone and platelet aggregometry and microfluidic assays, we uncovered a platelet GPIIb/IIIa-dependent prothrombotic gain of function (GOF) for variant p.Pro2555Arg, located in the C4 domain, leading to an increase in platelet aggregate size. We performed complementary biophysical and structural investigations using circular dichroism spectra, small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, molecular dynamics simulations on the single C4 domain, and dimeric wild-type and p.Pro2555Arg constructs. C4-p.Pro2555Arg retained the overall structural conformation with minor populations of alternative conformations exhibiting increased hinge flexibility and slow conformational exchange. The dimeric protein becomes disordered and more flexible. Our data suggest that the GOF does not affect the binding affinity of the C4 domain for GPIIb/IIIa. Instead, the increased VWF dimer flexibility enhances temporal accessibility of platelet-binding sites. Using an interdisciplinary approach, we revealed that p.Pro2555Arg is the first VWF variant, which increases platelet aggregate size and shows a shear-dependent function of the VWF stem region, which can become hyperactive through mutations. Prothrombotic GOF variants of VWF are a novel concept of a VWF-associated pathomechanism of thromboembolic events, which is of general interest to vascular health but not yet considered in diagnostics. Thus, awareness should be raised for the risk they pose. Furthermore, our data implicate the C4 domain as a novel antithrombotic drug target
Streblocerus superserricaudatus sp. nov. from Venezuela
A new species of the genus Streblocerus is described from Venezuela and compared with European, Newfoundland and Australian specimens. The geographical distribution of the genus is discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42889/1/10750_2004_Article_BF00015509.pd
Gene-Gene Interaction Affects Coronary Artery Disease Risk
Introdução: Existem vários estudos que
comparam doentes coronários e controlos, no
sentido de determinar quais os polimorfismos
que apresentam risco acrescido de doença das
artérias coronárias (DC). Os seus resultados
têm sido muitas vezes contraditórios, mas
apresentam uma limitação suplementar:
avaliam os polimorfismos um a um, quando
na natureza os polimorfismos não existem
isolados. Põe-se a questão se serão mais
importantes associações de polimorfismos
mutados no mesmo gene ou em genes
diferentes.
Objectivo: Com o presente trabalho
pretendemos avaliar o risco da associação de
polimorfismos em termos de aparecimento de
DC no mesmo gene ou em genes diferentes.
Metodologia: Estudámos em 298 doentes
coronários e 298 controlos sãos o risco
associado aos polimorfismos (genótipos
considerados de risco), DD da Enzima de
Converaão da Angiotensina (ECA) I/D; GG da
ECA 8, MM do Angiotensinogénio (AGT) 174;
TT do AGT 235; TT da Metiltetrahidrofolato
Reductase (MTHFR) 677; AA da MTHFR
1298;RR da Paraoxonase1 (PON1) 192 e MM
da PON1 55. Posteriormente avaliámos o risco
ligado às associações no mesmo gene (DD da
ECA + GG da ECA 8; MM do AGT174 + TT
do AGT 235; TT da MTHFR 677 + AA da
MTHFR 1298). Finalmente, nos polimorfismos
que isoladamente apresentavam significância,
avaliámos o risco das associações de
polimorfismos a níveis funcionais diferentes (ECA + AGT; ECA + MTHFR; ECA + PON1.
Finalmente através de um modelo de regressão
logística fomos determinar quais as variáveis
que se relacionavam de forma significativa e
independente com a DC.
Resultados: Os polimorfismos isolados como:
ECA DD [P<0.0001], ECA 8 GG [P=0,023],
e MTHFR 1298 AA [P=0,049]), apresentaram
uma frequência mais elevada nos casos,
associando-se de forma significativa ao grupo
com DC. A associação de polimorfismos no
mesmo gene não teve efeito sinergístico ou
aditivo e não aumentou o risco de DC. A
associação polimórfica em genes diferentes
aumentou o risco de DC quando comparada
com o risco do polimorfismo isolado. No caso
da associação da ECA DD ou ECA 8 GG
com a PON1 192 RR, o risco quadruplicou
(OR passou de 1,8 para 4,2). Após regressão
logística o hábito tabágico, a história familiar,
o fibrinogénio, diabetes, a associação ECA
DD ou ECA 8 GG com a MTHFR 1298 AA
e a interacção ECA DD ou ECA 8 GG com
a PON1 192 RR permaneceram na equação,
mostrando ser factores de risco independente
para DC.
Conclusões: A associação de polimorfismos
mutados no mesmo gene nunca aumentou o
risco do polimorfismo isolado. A associação
com interacção de polimorfismos mutados
em genes diferentes, pertencentes a sistemas
fisiopatológicos e enzimáticos diferentes,
esteve sempre associada a maior risco do
que cada polimorfismo por si. Este trabalho
levanta, pela primeira vez, a possibilidade
de tentativa de compreensão do risco
genético coronário em conjunto e não de cada
polimorfismo por si.Introduction: Various studies have compared
coronary artery disease (CAD) patients
with controls in order to determine which
polymorphisms are associated with a higher
risk of disease. The results have often been
contradictory. Moreover, these studies
evaluated polymorphisms in isolation and not
in association, which is the way they occur in
nature.
Objective: Our purpose was to evaluate the
risk of CAD in patients with associated
polymorphisms in the same gene or in different
genes.
Methods: We evaluated the risk associated
with ACE DD, ACE 8 GG, AGT 174MM, AGT
235TT, MTHFR 677TT, MTHFR 1298AA,
PON1 192RR and PON1 55MM in 298
CAD patients and 298 healthy individuals.
We then evaluated the risk of associated
polymorphisms in the same gene (ACE DD
+ ACE 8 GG; AGT 174MM + AGT 235TT;
MTHFR 677TT + MTHFR 1298AA).
Finally, for the isolated polymorphisms
which were significant, we evaluated the risk
of polymorphism associations at different
functional levels (ACE + AGT; ACE +
MTHFR; ACE + PON1). Multiple logistic
regression was used to identify independent
risk factors for CAD.
Results: Isolated polymorphisms including
ACE DD (p<0.0001), ACE 8 GG (p=0.023), and MTHFR 1298AA (p=0.049) presented
with a significantly higher frequency in the
CAD group. An association of polymorphisms
in the same gene did not have an additive
or synergistic effect, nor did it increase the
risk of CAD. Polymorphic associations in
different genes increased the risk of CAD,
compared with the isolated polymorphisms.
The association of ACE DD or ACE 8 GG
with PON1 192RR increased the risk of CAD
fourfold (1.8 to 4.2). After logistic regression
analysis, current smoking, family history,
fibrinogen, diabetes, and the ACE DD or
ACE 8 GG + MTHFR 1298AA and ACE DD
or ACE 8 GG + PON1 192RR associations
remained in the model and proved to be
independent predictors of CAD.
Conclusions: The association of
polymorphisms in the same gene did not
increase the risk of the isolated polymorphism.
The association of polymorphisms in genes
belonging to different enzyme systems was
always linked to increased risk compared to
the isolated polymorphisms.
This study may contribute to a better
understanding of overall genetic risk for
CAD rather than that associated with each
polymorphism in isolation.info:eu-repo/semantics/publishedVersio
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