The Generation of An Aptamer Inhibitor of Murine Von Willebrand Factor (VWF) Mediated Platelet Aggregation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4312-4312 ◽  
Author(s):  
Melissa Woelfel ◽  
Simon De Meyer ◽  
Patricia Wagner ◽  
Kathleen E McGinness ◽  
Denisa D. Wagner ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Protein Interactions ◽  
In Vitro Selection ◽  
Health Concern ◽  
Single Digit ◽  
Vein Wall ◽  
High Affinity

Abstract Abstract 4312 Venous thromboembolism (VTE) is a national health concern, with an occurrence of over 900,000 cases per year and over 300,000 deaths per year. The total number of cases of VTE and the incidence of VTE-related deaths exceeds those related to both myocardial infarction and stroke. With an aging population, the incidence of VTE has also been increasing. Current treatment of venous thromboembolism with anti-coagulation is not optimal. There is a risk of bleeding, thrombus extension, pain and swelling as well as a recurrence rate of 3–9%. A significant inflammatory response occurs with venous thromboembolism. This inflammation can influence the extent of thrombosis, vein wall fibrosis and valve damage in the thrombosed vein. In a high percentage of VTE patients a condition of venous insufficiency known as post-thrombotic syndrome (PTS) can develop. PTS is associated with stasis ulceration, dermatitis and edema. Venous thrombogenesis is influenced by platelet (PLT) and leukocyte (WBC) adhesion as well as interactions between these cells. There is growing evidence to suggest that VWF interactions with PLT GPIbα can mediate some of these early events. This is evidenced by the reduction in PLT/WBC recruitment and reduced thrombus growth seen in either VWF or GPIbα deficient mice. These data point to a role for VWF in VTE. We sought to develop an aptamer to mouse VWF that would inhibit its interactions with platelet GPIbα. The availability of this tool would support investigations into the role of VWF in mouse models of VTE. Aptamers are oligonucleotides that fold into three-dimensional structures and specifically bind to ligands with high affinity. Aptamers bound to proteins can modify and/or inhibit protein-protein interactions. Using an in vitro selection method known as Systematic Evolution of Ligands by EXponential enrichment (SELEX), we generated aptamers that bind to murine VWF (mVWF) from a modified RNA pool. Nine of these aptamers bind to mVWF with single-digit or sub- nanomolar affinity. A subset of these aptamers also binds to human VWF (hVWF). The aptamers that bind to hVWF inhibit platelet adhesion/aggregation in human whole blood. Further in vitro characterization has demonstrated that five of these aptamers specifically inhibit the interaction between mVWF and recombinant human GPIbα, but do not interfere with the binding of mVWF to collagen. These five aptamers were also active in vivo in a FeCl3-induced thrombosis model in mice. Intravenous injection of the anti-mVWF aptamers prolonged time to occlusion from a baseline of 10–15 minutes to either 25–35 minutes or >40 minutes in this model, depending on the aptamer. These results demonstrate that we have identified high affinity aptamers to mVWF that specifically disrupt mVWF binding to platelets and have an antithrombotic effect in an in vivo mouse model of thrombosis. These aptamers will allow us to investigate the role of VWF in murine models of venous thrombosis. This project was supported by Award Number R01HL095091 from the National Heart, Lung, And Blood Institute. Disclosures: Woelfel: Archemix Corporation: Employment. Wagner:Archemix Corporation: Employment. McGinness:Archemix Corporation: Employment. Schaub:Archemix Corporation: Employment.

Temperature-dependence of the DnaA–DNA interaction and its effect on the autoregulation of dnaA expression

2012 ◽  
Vol 449 (2) ◽  
pp. 333-341 ◽  
Author(s):  
Chiara Saggioro ◽  
Anne Olliver ◽  
Bianca Sclavi
Keyword(s):  
Temperature Dependence ◽  
Binding Sites ◽  
Dna Interaction ◽  
Bacterial Dna ◽  
Dnaa Protein ◽  
High Affinity ◽  
Key Factor

The DnaA protein is a key factor for the regulation of the timing and synchrony of initiation of bacterial DNA replication. The transcription of the dnaA gene in Escherichia coli is regulated by two promoters, dnaAP1 and dnaAP2. The region between these two promoters contains several DnaA-binding sites that have been shown to play an important role in the negative auto-regulation of dnaA expression. The results obtained in the present study using an in vitro and in vivo quantitative analysis of the effect of mutations to the high-affinity DnaA sites reveal an additional effect of positive autoregulation. We investigated the role of transcription autoregulation in the change of dnaA expression as a function of temperature. While negative auto-regulation is lost at dnaAP1, the effects of both positive and negative autoregulation are maintained at the dnaAP2 promoter upon lowering the growth temperature. These observations can be explained by the results obtained in vitro showing a difference in the temperature-dependence of DnaA–ATP binding to its high- and low-affinity sites, resulting in a decrease in DnaA–ATP oligomerization at lower temperatures. The results of the present study underline the importance of the role for autoregulation of gene expression in the cellular adaptation to different growth temperatures.

scholarly journals A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

1995 ◽  
Vol 15 (10) ◽  
pp. 5214-5225 ◽  
Author(s):  
A D Catling ◽  
H J Schaeffer ◽  
C W Reuter ◽  
G R Reddy ◽  
M J Weber
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Serum Stimulation ◽  
And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

scholarly journals Air pollution and childhood obesity

2020 ◽  
Vol 63 (10) ◽  
pp. 382-388
Author(s):  
Moon Young Seo ◽  
Shin-Hye Kim ◽  
Mi Jung Park
Keyword(s):  
Air Pollution ◽  
Childhood Obesity ◽  
Physical Inactivity ◽  
Animal Studies ◽  
Epidemiologic Studies ◽  
Health Concern ◽  
Postnatal Exposure

Childhood obesity is a global health concern. Air pollution is also a crucial health threat, especially in developing countries. Over the past decade, a number of epidemiologic and animal studies have suggested a possible role of pre- or postnatal exposure to air pollutants on childhood obesity. Although no clear mechanism has been elucidated, physical inactivity, oxidative stress, and epigenetic modifications have been suggested as possible mechanisms by which obesity develops due to air pollution. In this review, we summarize and review previous epidemiologic studies linking air pollution and childhood obesity and discuss the possible mechanisms underlying air pollution-induced obesity based on in vivo and in vitro evidence.

Acetylation of GATA-1 Is Required for Chromatin Occupancy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1179-1179 ◽  
Author(s):  
Janine M. Lamonica ◽  
Christopher R. Vakoc ◽  
Gerd A. Blobel
Keyword(s):  
Protein Interactions ◽  
Dependent Manner ◽  
Protein Levels ◽  
Defective Mutant ◽  
Cellular Target ◽  
Stable Association ◽  
Peptide Affinity

Abstract All three hematopoietic GATA transcription factors GATA-1, GATA-2, and GATA-3 are acetylated, although the in vivo role of this modification remains unclear. It has been proposed that acetylation of GATA-1 increases its affinity for DNA in vitro, although this finding has not been observed by others. To study the role of GATA-1 acetylation, we examined the functions of an acetylation-defective mutant of GATA-1 in maturing erythroid cells. We found that removal of the acetylation sites in GATA-1 largely abrogates its biological activity but does not impair its nuclear localization, steady state protein levels, or its ability to bind naked GATA elements in vitro. However, chromatin immunoprecipitation (ChIP) experiments revealed that mutant GATA-1 was dramatically impaired in binding to its cellular target sites in vivo, including genes that are normally activated (α- and β-globin, EKLF, FOG-1, Band3, and AHSP) and repressed (GATA-2 and c-kit) by GATA-1. Together, these results suggest that acetylation is required for GATA-1 chromatin occupancy. These findings point to a novel function for transcription factor acetylation, perhaps by facilitating protein interactions required for stable association with chromatin templates in vivo. To identify proteins that interact with acetylated GATA-1, we performed peptide affinity chromatography using acetylated GATA-1 peptides. Using this technique coupled with mass spectrometry, several proteins that bind to GATA-1 peptides in an acetylation-dependent manner were identified. The identified proteins contain known acetyl-lysine binding modules (bromodomains) consistent with their binding properties. The in vivo role of these proteins with regard to GATA-1 function is being examined and will be discussed.

scholarly journals Mobilization of Chimeric oriT Plasmids by F and R100-1: Role of Relaxosome Formation in Defining Plasmid Specificity

2000 ◽  
Vol 182 (14) ◽  
pp. 4022-4027 ◽  
Author(s):  
Richard A. Fekete ◽  
Laura S. Frost
Keyword(s):  
Binding Sites ◽  
Integration Host Factor ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Negative Dominance ◽  
Transfer Apparatus

ABSTRACT Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage atnic in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene. The efficiency of cleavage atnic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT. High-affinity binding of TraY in cis to oriTallowed detection of cleavage at nic but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome).

scholarly journals MK2a inhibitor CMPD1 abrogates chikungunya virus infection by modulating actin remodeling pathway

2021 ◽  
Author(s):  
Soma Chattopadhyay ◽  
Prabhudutta Mamidi ◽  
Tapas Kumar Nayak ◽  
Abhishek Kumar ◽  
Sameer Kumar ◽  
...  
Keyword(s):  
Viral Infection ◽  
Gene Silencing ◽  
Chikungunya Virus ◽  
Late Phase ◽  
Health Concern ◽  
Chikv Infection ◽  
Viral Copy Number

Chikungunya virus (CHIKV) epidemics around the world have created public health concern with the unavailability of effective drugs and vaccines. This emphasizes the need for molecular understanding of host-virus interactions for developing effective targeted antivirals. Microarray analysis was carried out using CHIKV strain (Prototype and Indian) infected Vero cells and two host isozymes, MK2 and MK3 were selected for further analysis. Gene silencing and drug treatment were performed in vitro and in vivo to unravel the role of MK2/MK3 in CHIKV infection. Gene silencing of MK2 and MK3 abrogated around 58% CHIKV progeny release from the host cell and a MK2 activation (a) inhibitor (CMPD1) treatment demonstrated 68% inhibition of viral infection suggesting a major role of MAPKAPKs during the late phase of CHIKV infection in vitro. Further, it was observed that the inhibition in viral infection is primarily due to the abrogation of lamellipodium formation through modulation of factors involved in the actin cytoskeleton remodeling pathway that is responsible for releasing the virus from the infected cells.  Moreover, CHIKV-infected C57BL/6 mice demonstrated reduction in the viral copy number, lessened disease score and better survivability after CMPD1 treatment. In addition, reduction in expression of key pro-inflammatory mediators such as CXCL13, RAGE, FGF, MMP9 and increase in HGF (a CHIKV infection recovery marker) was observed indicating the effectiveness of this drug against CHIKV. Additionally, CMPD1 also inhibited HSV1 and SARS CoV2-19 infection in vitro. Taken together it can be proposed that MK2 and MK3 are crucial host factors for CHIKV infection and can be considered as key targets for developing effective anti-CHIKV strategies in future.

scholarly journals PDZ Motifs in PTP-BL and RIL Bind to Internal Protein Segments in the LIM Domain Protein RIL

1998 ◽  
Vol 9 (3) ◽  
pp. 671-683 ◽  
Author(s):  
Edwin Cuppen ◽  
Herlinde Gerrits ◽  
Barry Pepers ◽  
Bé Wieringa ◽  
Wiljan Hendriks
Keyword(s):  
Protein Interactions ◽  
Tyrosine Phosphatase ◽  
Specific Interaction ◽  
Protein Protein Interactions ◽  
Lim Domain ◽  
Binding Interface ◽  
Mouse Tissues

The specificity of protein–protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells.

scholarly journals A high-affinity antibody against the CSP N-terminal domain lacks Plasmodium falciparum inhibitory activity

2020 ◽  
Vol 217 (11) ◽  
Author(s):  
Elaine Thai ◽  
Giulia Costa ◽  
Anna Weyrich ◽  
Rajagopal Murugan ◽  
David Oyen ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Humoral Response ◽  
Health Concern ◽  
Functional Studies ◽  
High Affinity ◽  
Terminal Domain ◽  
C Terminus ◽  
Immunogen Design

Malaria is a global health concern, and research efforts are ongoing to develop a superior vaccine to RTS,S/AS01. To guide immunogen design, we seek a comprehensive understanding of the protective humoral response against Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP). In contrast to the well-studied responses to the repeat region and the C-terminus, the antibody response against the N-terminal domain of PfCSP (N-CSP) remains obscure. Here, we characterized the molecular recognition and functional efficacy of the N-CSP–specific monoclonal antibody 5D5. The crystal structure at 1.85-Å resolution revealed that 5D5 binds an α-helical epitope in N-CSP with high affinity through extensive shape and charge complementarity and the unusual utilization of an antibody N-linked glycan. Nevertheless, functional studies indicated low 5D5 binding to live Pf sporozoites and lack of sporozoite inhibition in vitro and in vivo. Overall, our data do not support the inclusion of the 5D5 N-CSP epitope into the next generation of CSP-based vaccines.

scholarly journals Role of an RNA polymerase interacting protein, MsRbpA, from Mycobacterium smegmatis in phenotypic tolerance to rifampicin

Microbiology ◽  
2010 ◽  
Vol 156 (3) ◽  
pp. 873-883 ◽  
Author(s):  
Abhinav Dey ◽  
Amit Kumar Verma ◽  
Dipankar Chatterji
Keyword(s):  
Rna Polymerase ◽  
Protein Interactions ◽  
Mycobacterium Smegmatis ◽  
Streptomyces Coelicolor ◽  
Polymerase Activity ◽  
Current Standard ◽  
Chemotherapeutic Regimen

Rifampicin and its derivatives are at the forefront of the current standard chemotherapeutic regimen for active tuberculosis; they act by inhibiting the transcription activity of prokaryotic RNA polymerase. Rifampicin is believed to interact with the β subunit of RNA polymerase. However, it has been observed that protein–protein interactions with RNA polymerase core enzyme lead to its reduced susceptibility to rifampicin. This mechanism became more diversified with the discovery of RbpA, a novel RNA polymerase-binding protein, in Streptomyces coelicolor that could mitigate the effect of rifampicin on RNA polymerase activity. MsRbpA is a homologue of RbpA in Mycobacterium smegmatis. On deciphering the role of MsRbpA in M. smegmatis we found that it interacts with RNA polymerase and increases the rifampicin tolerance levels, both in vitro and in vivo. It interacts with the β subunit of RNA polymerase. However, it was found to be incapable of rescuing rifampicin-resistant RNA polymerases in the presence of rifampicin at the respective IC50.

scholarly journals Light-responsive monobodies for dynamic control of customizable protein binding

2020 ◽  
Author(s):  
César Carrasco-López ◽  
Evan M. Zhao ◽  
Agnieszka A. Gil ◽  
Nathan Alam ◽  
Jared E. Toettcher ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Dynamic Control ◽  
Binding Activity ◽  
Sh2 Domain ◽  
Protein Protein Interactions ◽  
High Affinity ◽  
Protein Binders ◽  
Basic And Applied Research

ABSTRACTCustomizable, high affinity protein-protein interactions, such as those mediated by antibodies and antibody-like molecules, are invaluable to basic and applied research and have become pillars for modern therapeutics. The ability to reversibly control the binding activity of these proteins to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, a light-controlled monobody (OptoMB) that works in vitro and in vivo, whose affinity for its SH2-domain target exhibits a 300-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. This OptoMB belongs to a new class of light-sensitive protein binders we call OptoBinders (OptoBNDRs) which, by virtue of their ability to be designed to bind any protein of interest, have the potential to find new powerful applications as light-switchable binders of untagged proteins with high affinity and selectivity, and with the temporal and spatial precision afforded by light.

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