scholarly journals 25-Hydroxyl-cholesterol Binds and Enhance Anti-viral Effects of Zebrafish Monomeric C-reactive Proteins

2018 ◽  
Author(s):  
Melissa Bello-Perez ◽  
Alberto Falco ◽  
Jose Antonio Encinar ◽  
Beatriz Novoa ◽  
Luis Perez ◽  
...  
Keyword(s):  
In Silico ◽  
Viral Infections ◽  
Solid Phase ◽  
Atherosclerotic Plaques ◽  
Dependent Manner ◽  
Structural Studies ◽  
Binding Assays ◽  
Zebrafish Model ◽  
Solid Phase Binding ◽  
Methyl Betacyclodextrin

AbstractC-reactive proteins (CRP) are among the faster acute-phase inflammation-responses coded by one gene in humans (hcrp) and seven genes (crp1-7) in zebrafish (Danio rerio). In this study, preferential 25-hydroxycholesterol (25HOCh) binding to zebrafish CRP1-7 compared to other lipids were predicted by in silico docking and confirmed by solid-phase binding-assays. In addition, 25HOCh enhanced methyl-betacyclodextrin-sensitive (Cholesterol-dependent) CRP1-7 anti-viral effects in a fine-tunned isoform-dependent manner. In silico and structural studies suggested that the crosstalk between the anti-viral enhancements of both 25HOCh and CRP1-7 were dependent on protein monomers rather than oligomers but differred among isoforms. The presence of oxidized cholesterols in human atherosclerotic plaques amplifies the importance that similar interactions may have for vascular and/or neurodegenerative diseases during viral infections. In this context, the zebrafish model offers a genetic tool to further investigate how the expression and functions of different CRP isoforms and/or transcript variants may be controlled.

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

Ultrastructural Evidence for Proteoglycans Mediating an Attachment of Type VI Collagen to Banded Collagen Fibrils

1997 ◽  
Vol 3 (S2) ◽  
pp. 153-154
Author(s):  
Douglas R. Keene ◽  
Catherine C. Ridgway ◽  
Renato V. Iozzo
Keyword(s):  
Dermatan Sulfate ◽  
Core Protein ◽  
Solid Phase ◽  
Collagen Fibrils ◽  
Binding Assays ◽  
Connective Tissues ◽  
Type Vi Collagen ◽  
Dermatan Sulfate Proteoglycan ◽  
Individual Type ◽  
Solid Phase Binding

Immunolocalizaton studies of type VI collagen in skin have previously demonstrated that type VI collagen forms a flexible network that anchors large interstitial structures such as nerves, blood vessels, and collagen fibers into the surrounding connective tissues matrix. The purpose of this study is to determine if individual type VI collagen microfilaments might be connected to banded collagen fibrils, thereby stabilizing the network.Solid phase binding assays suggest a specific, high affinity interaction between the core protein of the dermatan sulfate proteoglycan decorin and type VI collagen, and immunocytochemical studies in fetal and neonate rabbit cornea suggest an association of decorin with type VI microfilaments. Other studies in skin and perichondrium have localized decorin to a region between the d and e bands of banded collagen fibrils. However, no direct documentation has demonstrated a specific structural interaction between type VI microfilaments and banded collagen fibrils. We, therefore, sought to determine if type VI microfilaments cross banded collagen fibrils between the “d” and “e” bands.

scholarly journals Fibulin-1 mediates platelet adhesion via a bridge of fibrinogen

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2569-2577 ◽  
Author(s):  
S Godyna ◽  
M Diaz-Ricart ◽  
WS Argraves
Keyword(s):  
Extracellular Matrix ◽  
Vascular Injury ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Solid Phase ◽  
Blood Perfusion ◽  
General Mechanism ◽  
Binding Assays ◽  
Solid Phase Binding ◽  
Coated Surfaces

Fibulin-1 is a component of the extracellular matrix that surrounds vascular smooth muscle. This observation, along with the recent finding that fibulin-1 can bind fibrinogen (J Biol Chem 270:19458, 1995), prompted investigation into the potential role of fibulin-1 as a thrombogenic agent. In perfusion chamber assays, platelets in whole blood under flow conditions attached and spread on surfaces coated with fibulin-1. This adhesion was completely blocked by fibulin-1 antibodies. Platelets free of plasma did not attach to fibulin-1 coated surfaces; however, with the addition of fibrinogen, platelet adhesion to fibulin-1 took place. When detergent extracts of platelets were subjected to fibulin-1-Sepharose affinity chromatography, the integrin alpha IIb beta 3 was selected. Solid phase binding assays using purified components showed that integrin alpha IIb beta 3 could not bind directly to fibulin-1 but in the presence of fibrinogen the integrin bound to fibulin-1-coated surfaces. Monoclonal alpha IIb beta 3 antibodies capable of blocking its interaction with fibrinogen completely blocked platelet adhesion to fibulin-1 in both whole blood perfusion and static adhesion assays. The results show that fibulin-1 can support platelet attachment via a bridge of fibrinogen to the platelet integrin alpha IIb beta 3. When fibroblast monolayers containing extracellular matrix-incorporated fibulin-1 were used as adhesion substrates, platelet adhesion in the presence of fibrinogen could be inhibited by 30% using antibodies to fibulin-1. Following vascular injury, fibulin-1 present in the extracellular matrix of the vessel wall may therefore interact with plasma fibrinogen and promote platelet adhesion, leading to the formation of a platelet plug. Thus, fibulin-1 joins the list of matrix proteins including collagens I and IV and fibronectin that mediate platelet adhesion via a plasma protein bridge. This bridging phenomenon may represent a general mechanism by which platelets interact with exposed subendothelial matrices following vascular injury.

Immobilized whole algal cells for solid-phase binding assays

1986 ◽  
Vol 86 (2) ◽  
pp. 171-177 ◽  
Author(s):  
Paul Bubrick ◽  
Leon Goldstein ◽  
Asher Frensdorff
Keyword(s):  
Solid Phase ◽  
Binding Assays ◽  
Solid Phase Binding

Production of transferrin receptors byHistophilus ovis: three of five strains require two signals

2001 ◽  
Vol 47 (5) ◽  
pp. 417-423 ◽  
Author(s):  
Andrew Ekins ◽  
Donald F Niven
Keyword(s):  
Solid Phase ◽  
Iron Acquisition ◽  
Isolation Procedure ◽  
Transferrin Receptors ◽  
Binding Assays ◽  
Surface Receptors ◽  
Iron Restriction ◽  
Affinity Isolation ◽  
Solid Phase Binding ◽  
Bovine Transferrin

Five strains of Histophilus ovis (9L, 642A, 714, 5688T, and 3384Y) were investigated with respect to iron acquisition. All strains used ovine, bovine, and goat transferrins (Tfs), but not porcine or human Tfs, as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine, and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine transferrin (Tf), total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine, and goat) were bound by the same receptor(s). Membranes from organisms grown under iron-replete conditions in the presence or absence of bovine Tf failed to bind any of the test Tfs. An affinity-isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. It is concluded that all strains tested acquire Tf-bound iron by means of siderophore-independent mechanisms involving surface receptors analogous to the Tf-binding proteins (TbpA and TbpB) found in comparable organisms; although iron restriction alone is sufficient to promote the expression of these proteins by strains 9L and 642A, their production by strains 714, 5688T, and 3384Y appears to require two signals, iron restriction and the presence of Tf.Key words: Histophilus ovis, iron acquisition, transferrins, receptors, regulation.

New insights into the structural elements involved in the skin haemorrhage induced by snake venom metalloproteinases

2010 ◽  
Vol 104 (09) ◽  
pp. 485-497 ◽  
Author(s):  
Ana Oliveira ◽  
Adriana Paes Leme ◽  
Amanda Asega ◽  
Antonio Camargo ◽  
Jay Fox ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Snake Venom ◽  
Solid Phase ◽  
Structural Elements ◽  
Binding Assays ◽  
Domain Organisation ◽  
Solid Phase Binding ◽  
A Minor ◽  
Von Willebrand

SummaryHaemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon resulting in capillary disruption and extravasation. This study analysed structural elements important for the interaction of four Bothrops jararaca SVMPs of different domain organisation and glycosylation levels with plasma and extracellular matrix proteins: HF3 (P-III class) is highly glycosylated and ~80 times more haemorrhagic than bothropasin (P-III class), which has a minor carbohydrate moiety; BJ-PI (P-I class) is not haemorrhagic and the DC protein is composed of disintegrin-like/cysteine-rich domains of bothropasin. HF3, bothropasin and BJ-PI showed different degradation profiles of fibrinogen, fibronectin, vitronectin, von Willebrand factor, collagens IV and VI, laminin and Matrigel™; however, only bothropasin degraded collagen I. In solid-phase binding assays HF3 and bothropasin interacted with fibrinogen, fibronectin, laminin, collagens I and VI; the DC protein bound only to collagens I and VI; however, no binding of BJ-PI to these proteins was detected. N-deglycosylation caused loss of structural stability of bothropasin and BJ-PI but HF3 remained intact, although its haemorrhagic and fibrinogenolytic activities were partially impaired. Nevertheless, N-deglycosylated HF3 bound with higher affinity to collagens I and VI, although its proteolytic activity upon these collagens was not enhanced. This study demonstrates that features of carbohydrate moieties of haemorrhagic SVMPs may play a role in their interaction with substrates of the extracellular matrix, and the ability of SVMPs to degrade proteins in vitro does not correlate to their ability to cause haemorrhage, suggesting that novel, systemic approaches are necessary for understanding the mechanism of haemorrhage generation by SVMPs.

scholarly journals A Myopathy-linked Desmin Mutation Perturbs Striated Muscle Actin Filament Architecture

2009 ◽  
Vol 20 (3) ◽  
pp. 834-845 ◽  
Author(s):  
Gloria M. Conover ◽  
Syerra N. Henderson ◽  
Carol C. Gregorio
Keyword(s):  
Actin Filament ◽  
Thin Filament ◽  
Striated Muscle ◽  
Solid Phase ◽  
Skeletal Muscle Atrophy ◽  
Binding Assays ◽  
Concomitant Increase ◽  
Solid Phase Binding ◽  
Intracellular Aggregates ◽  
Filament Network

Desmin interacts with nebulin establishing a direct link between the intermediate filament network and sarcomeres at the Z-discs. Here, we examined a desmin mutation, E245D, that is located within the coil IB (nebulin-binding) region of desmin and that has been reported to cause human cardiomyopathy and skeletal muscle atrophy. We show that the coil IB region of desmin binds to C-terminal nebulin (modules 160-164) with high affinity, whereas binding of this desmin region containing the E245D mutation appears to enhance its interaction with nebulin in solid-phase binding assays. Expression of the desmin-E245D mutant in myocytes displaces endogenous desmin and C-terminal nebulin from the Z-discs with a concomitant increase in the formation of intracellular aggregates, reminiscent of a major histological hallmark of desmin-related myopathies. Actin filament architecture was strikingly perturbed in myocytes expressing the desmin-E245D mutant because most sarcomeres contained elongated or shorter actin filaments. Our findings reveal a novel role for desmin intermediate filaments in modulating actin filament lengths and organization. Collectively, these data suggest that the desmin E245D mutation interferes with the ability of nebulin to precisely regulate thin filament lengths, providing new insights into the potential molecular consequences of expression of certain disease-associated desmin mutations.

Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization

2001 ◽  
Vol 114 (22) ◽  
pp. 4127-4136
Author(s):  
Elizabeth Howes ◽  
John C. Pascall ◽  
Wolfgang Engel ◽  
Roy Jones
Keyword(s):  
Zona Pellucida ◽  
Solid Phase ◽  
Binding Assays ◽  
Vitro Fertilization ◽  
Sperm Binding ◽  
Secondary Receptor ◽  
Specificity Of Binding ◽  
Solid Phase Binding ◽  
Complementary Binding

The mouse zona pellucida glycoprotein, mZP2, is thought to be the secondary receptor on eggs for retention of acrosome-reacted sperm during fertilization. Here, we present evidence that one of its complementary binding proteins on sperm is proacrosin/acrosin. mZP2 binds to proacrosin null sperm considerably less effectively than to wild-type sperm. Binding is mediated by a strong ionic interaction between polysulphate groups on mZP2 and basic residues on an internal proacrosin peptide. The stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a polysulphonated anticancer drug, compete with mZP2 for complementary binding sites on proacrosin/acrosin in solid-phase binding assays. The same competitors also displace attached sperm from the zona pellucida of eggs in an in vitro fertilization system. This combination of genetic, biochemical and functional data supports the hypothesis that mZP2-proacrosin interactions are important for retention of acrosome-reacted sperm on the egg surface during fertilization. Safe mimetics of suramin have potential as non-steroidal antifertility agents.

Tissue factor pathway inhibitor-2 (TFPI-2) recognizes the complement and kininogen binding protein gC1qR/p33 (gC1qR): implications for vascular inflammation

2004 ◽  
Vol 92 (10) ◽  
pp. 811-819 ◽  
Author(s):  
Ramona Petrovan ◽  
Berhane Ghebrehiwet ◽  
Wolfram Ruf ◽  
Ellinor Peerschke
Keyword(s):  
Solid Phase ◽  
Vascular Inflammation ◽  
Fluid Phase ◽  
Cellular Protein ◽  
Binding Assays ◽  
Binding Studies ◽  
Atherosclerotic Lesions ◽  
Tissue Factor Pathway ◽  
Solid Phase Binding ◽  
Immunohistochemical Studies

SummaryEvidence is accumulating to suggest that TFPI-2 is involved in regulating pericellular proteases implicated in a variety of physiologic and pathologic processes including cancer cell invasion, vascular inflammation, and atherosclerosis. Recent immunohistochemical studies of advanced atherosclerotic lesions, demonstrated a similar tissue distribution for TFPI-2, High Molecular Weight Kininogen (HK), and gC1qR/p33 (gC1qR), a ubiquitously expressed, multicompartmental cellular protein involved in modulating complement, coagulation, and kinin cascades. Further studies to evaluate TFPI-2 interactions with gC1qR demonstrated direct interactions between gC1qR and TFPI-2 using immunoprecipitation and solid phase binding studies. Specific and saturable binding between TFPI-2 and gC1qR (estimated Kd: ∼ 70 nM) was observed by ELISA and surface plasmon resonance (Biacore) binding assays. Binding was inhibited by antibodies to gC1qR, and was strongly dependent on the Kunitz-2 domain of TFPI-2, as deletion of this domain reduced gC1qR-TFPI-2 interactions by approximately 75%. Deletion of gC1qR amino acids 74-95, involved in C1q binding, had no effect on gC1qR binding to TFPI-2, although antibodies to this region and purified C1q both inhibited binding, most likely via allosteric effects. In contrast, HK did not affect TFPI-2 binding to gC1qR. Binding of TFPI-2 to gC1qR produced statistically significant but modest reductions in TFPI-2 inhibition of plasmin, but had no effect on kallikrein inhibition in fluid phase chromogenic assays. Taken together, these data suggest that gC1qR may participate in tissue remodeling and inflammation by localizing TFPI-2 to the pericellular environment to modulate local protease activity and regulate HK activation.

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