scholarly journals LIM domain-wide comprehensive mutagenesis reveals the role of leucine in CSRP3 protein stability

2021 ◽  
Author(s):  
Pankaj Kumar Chauhan ◽  
Ramanathan Sowdhamini
Keyword(s):  
Protein Structures ◽  
Screening Method ◽  
Md Simulations ◽  
Specific Protein ◽  
Lim Domain ◽  
Lim Domains ◽  
Screening Protocol ◽  
The Stability ◽  
Glycine Rich Protein

Cardiomyopathies are a severe and chronic cardiovascular burden worldwide, affecting a large cohort in the general population. Cysteine and glycine-rich protein 3 (CSRP3) is one of key proteins implicated in dominant dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). In this study, we device a rapid in-silico screening protocol that creates a mutational landscape map for all possible allowed and disallowed substitutions in the protein of interest. This map provides the structural and functional insights on the stability of LIM domains of CSRP3. Further, the sequence analysis delineates the eukaryotic CSRP3 protein orthologs which complements the mutational map. Next, we also evaluated the effect of HCM/DCM mutations on these domains. One of highly destabilising mutations - L44P (also disease causing) and a neutral mutation - L44M were further subjected to molecular dynamics (MD) simulations. The results establish that L44P substitution affects the LIM domain structure. The present study provides a useful perspective to our understanding of the role of mutations in the CSRP3 LIM domains and their evolution. Experimentally verifying every reported mutation can become challenging both in time and resources used. This study provides a novel screening method for quick identification of key mutation sites for specific protein structures that can reduce the burden on experimental research.

scholarly journals LIM Domain-Wide Comprehensive Virtual Mutagenesis Provides Structural Rationale for Cardiomyopathy Mutations in CSRP3

2021 ◽  
Author(s):  
Pankaj Kumar Chauhan ◽  
R. Sowdhamini
Keyword(s):  
Protein Structures ◽  
Screening Method ◽  
Md Simulations ◽  
Specific Protein ◽  
Lim Domain ◽  
Lim Domains ◽  
Screening Protocol ◽  
The Stability ◽  
Cardiomyopathy Mutations

Abstract Cardiomyopathies are a severe and chronic cardiovascular burden worldwide, affecting a large cohort in the general population. Cysteine and glycine-rich protein 3 (CSRP3) is one of key proteins implicated in dominant dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). In this study, we device a rapid in-silico screening protocol that creates a mutational landscape map for all possible allowed and disallowed substitutions in the protein of interest. This map provides the structural and functional insights on the stability of LIM domains of CSRP3. Further, the sequence analysis delineates the eukaryotic CSRP3 protein orthologs which complements the mutational map. Next, we also evaluated the effect of HCM/DCM mutations on these domains. One of highly destabilising mutations - L44P (also disease causing) and a neutral mutation - L44M were further subjected to molecular dynamics (MD) simulations. The results establish that L44P substitution affects the LIM domain structure. The present study provides a useful perspective to our understanding of the role of mutations in the CSRP3 LIM domains and their evolution. Experimentally verifying every reported mutation can become challenging both in time and resources used. This study provides a novel screening method for quick identification of key mutation sites for specific protein structures that can reduce the burden on experimental research.

scholarly journals Serine and Threonine Phosphorylation of the Paxillin LIM Domains Regulates Paxillin Focal Adhesion Localization and Cell Adhesion to Fibronectin

1998 ◽  
Vol 9 (7) ◽  
pp. 1803-1816 ◽  
Author(s):  
Michael C. Brown ◽  
Joseph A. Perrotta ◽  
Christopher E. Turner
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Binding Sites ◽  
Protein Localization ◽  
Model System ◽  
Lim Domain ◽  
Amino Terminal ◽  
Lim Domains ◽  
Inside Out

We have previously shown that the LIM domains of paxillin operate as the focal adhesion (FA)-targeting motif of this protein. In the current study, we have identified the capacity of paxillin LIM2 and LIM3 to serve as binding sites for, and substrates of serine/threonine kinases. The activities of the LIM2- and LIM3-associated kinases were stimulated after adhesion of CHO.K1 cells to fibronectin; consequently, a role for LIM domain phosphorylation in regulating the subcellular localization of paxillin after adhesion to fibronectin was investigated. An avian paxillin-CHO.K1 model system was used to explore the role of paxillin phosphorylation in paxillin localization to FAs. We found that mutations of paxillin that mimicked LIM domain phosphorylation accelerated fibronectin-induced localization of paxillin to focal contacts. Further, blocking phosphorylation of the LIM domains reduced cell adhesion to fibronectin, whereas constitutive LIM domain phosphorylation significantly increased the capacity of cells to adhere to fibronectin. The potentiation of FA targeting and cell adhesion to fibronectin was specific to LIM domain phosphorylation as mutation of the amino-terminal tyrosine and serine residues of paxillin that are phosphorylated in response to fibronectin adhesion had no effect on the rate of FA localization or cell adhesion. This represents the first demonstration of the regulation of protein localization through LIM domain phosphorylation and suggests a novel mechanism of regulating LIM domain function. Additionally, these results provide the first evidence that paxillin contributes to “inside-out” integrin-mediated signal transduction.

Factors affecting the amplitude of the τ angle in proteins: a revisitation

2017 ◽  
Vol 73 (7) ◽  
pp. 618-625 ◽  
Author(s):  
Nicole Balasco ◽  
Luciana Esposito ◽  
Luigi Vitagliano
Keyword(s):  
Protein Structures ◽  
Factors Affecting ◽  
X Ray ◽  
Backbone Conformation ◽  
Potential Factors ◽  
The Stability ◽  
Major Factors ◽  
The Impact ◽  
Open Issues

The protein folded state is the result of the fine balance of a variety of different forces. Even minor structural perturbations may have a significant impact on the stability of these macromolecules. Studies carried out in recent decades have led to the convergent view that proteins are endowed with a flexible spine. One of the open issues related to protein local backbone geometry is the identification of the factors that influence the amplitude of the τ (N—Cα—C) angle. Here, statistical analyses performed on an updated ensemble of X-ray protein structures by dissecting the contribution of the major factors that can potentially influence the local backbone geometry of proteins are reported. The data clearly indicate that the local backbone conformation has a prominent impact on the modulation of the τ angle. Therefore, a proper assessment of the impact of the other potential factors can only be appropriately evaluated when small (φ, ψ) regions are considered. Here, it is shown that when the contribution of the backbone conformation is removed by considering small (φ, ψ) areas, an impact of secondary structure, as defined byDSSP, and/or the residue type on τ is still detectable, although to a limited extent. Indeed, distinct τ-value distributions are detected for Pro/Gly and β-branched (Ile/Val) residues. The key role of the local backbone conformation highlighted here supports the use of variable local backbone geometry in protein refinement protocols.

Development of a rapid screening protocol for selection of strains resistant to spray drying and storage in dry powder

2010 ◽  
Vol 1 (2) ◽  
pp. 165-174 ◽  
Author(s):  
S. Reimann ◽  
F. Grattepanche ◽  
C. Baggenstos ◽  
E. Rezzonico ◽  
B. Berger ◽  
...  
Keyword(s):  
Spray Drying ◽  
Bifidobacterium Longum ◽  
Survival Rates ◽  
Screening Method ◽  
Rapid Screening ◽  
Fast Screening ◽  
Screening Protocol ◽  
The Stability ◽  
And Storage ◽  
Selection Of

An efficient screening method for selection of Bifidobacterium longum strains resistant to spray drying and storage was developed based on randomly amplified polymorphic DNA (RAPD) for identification of the best survivors in mixed strains bacterial preparations. Three different primers were used to generate RAPD profiles of 22 B. longum strains. All strains were distinguished according to their RAPD profiles except for the strain NCC2705 and its H2O2 resistant derivative variant. The 22 strains were grouped in 3 batches of 7, 7 and 8 strains and subjected to spray drying and storage at 30 and 37 °C under anaerobic conditions. Batch survival rates after spray drying reached 17.1±4.4%. Strains showing the highest prevalence and/or resistance to storage at 37 °C were selected from individual batches for subsequent spray drying and storage testing. After 67 days of storage, NCC572 was identified as the dominant strain in powder. The stability of strain NCC572 was confirmed by performing single spray drying and storage tests. Out of 22 B. longum strains, a robust strain was identified by combining RAPD with a simultaneous screening test for survival under spray drying and storage. The method allowed a fast screening of B. longum strains in mixture for resistance to spray drying and storage compared to traditional screening procedures carried out with individual strains, in the same conditions. This approach could be applied to other stress conditions.

scholarly journals Forever Young: Structural Stability of Telomeric Guanine-Quadruplexes in Presence of Oxidative DNA Lesions

2020 ◽  
Author(s):  
Tom Miclot ◽  
Camille Corbier ◽  
Alessio Terenzi ◽  
Cécilia Hognon ◽  
Stéphanie Grandemange ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Molecular Dynamics ◽  
Structural Stability ◽  
Md Simulations ◽  
Dna Lesions ◽  
Computational Investigation ◽  
Telomeric Dna ◽  
G Quadruplex ◽  
The Stability

AbstractHuman telomeric DNA (h-Telo), in G-quadruplex (G4) conformation, is characterized by a remarkable structural stability that confers it the capacity to resist to oxidative stress producing one or even clustered 8-oxoguanine lesions. We present a combined experimental/computational investigation, by using circular dichroism in aqueous solutions, cellular immunofluorescence assays and molecular dynamics (MD) simulations, that identifies the crucial role of the stability of G4s to oxidative lesions, related also to their biological role as inhibitors of telomerase, an enzyme overexpressed in most cancers associated to oxidative stress.

scholarly journals LIM domains of cysteine-rich protein 1 (CRP1) are essential for its zyxin-binding function

1998 ◽  
Vol 331 (3) ◽  
pp. 885-892 ◽  
Author(s):  
Karen L. SCHMEICHEL ◽  
Mary C. BECKERLE
Keyword(s):  
Binding Site ◽  
Protein Interactions ◽  
Specific Protein ◽  
Binding Activity ◽  
Structural Motif ◽  
Lim Domain ◽  
Lim Domains ◽  
Cysteine Rich Protein ◽  
Binding Function ◽  
Domain Mapping

Previous studies have demonstrated that the adhesion-plaque protein, zyxin, interacts specifically with a 23 kDa protein, called the cysteine-rich protein 1 (CRP1), which has been implicated in myogenesis. Primary sequence analyses have revealed that both zyxin and CRP1 exhibit multiple copies of a structural motif called the LIM domain. LIM domains, which are defined by the consensus CX2CX16–23HX2CX2CX2CX16–23CX2–3(C,H,D), are found in a variety of proteins that are involved in cell growth and differentiation. Recent studies have established that LIM domains are zinc-binding structures that mediate specific protein–protein interactions. For example, in the case of the zyxin–CRP1 interaction, one of zyxin's three LIM domains is necessary and sufficient for binding to CRP1. Because the CRP1 molecule is comprised primarily of two LIM domains, we were interested in the possibility that the binding site for zyxin on CRP1 might also be contained within a single LIM domain. Consistent with the hypothesis that the LIM domains of CRP1 are critical for the protein's zyxin-binding function, zinc-depleted CRP1 displays a reduced zyxin-binding activity. However, domain mapping analyses have revealed that neither of the two individual LIM domains of CRP1 can support a wild-type interaction with zyxin. Collectively, our results suggest that the binding site for zyxin on CRP1 is not contained within a single contiguous sequence of amino acids. Instead, the interaction appears to rely on the co-ordinate action of a number of residues that are displayed in both of CRP1's LIM domains.

Dual role of friction in granular flows: attenuation versus enhancement of instabilities

2013 ◽  
Vol 729 ◽  
pp. 484-495 ◽  
Author(s):  
Peter P. Mitrano ◽  
Steven R. Dahl ◽  
Andrew M. Hilger ◽  
Christopher J. Ewasko ◽  
Christine M. Hrenya
Keyword(s):  
Cooling System ◽  
Translational Motion ◽  
Granular Flows ◽  
Md Simulations ◽  
Particle Interactions ◽  
Frictional Dissipation ◽  
Inelastic Dissipation ◽  
The Stability ◽  
The Impact

AbstractFlow instabilities driven by the dissipative nature of particle–particle interactions have been well documented in granular flows. The bulk of previous studies on such instabilities have considered the impact of inelastic dissipation only and shown that instabilities are enhanced with increased dissipation. The impact of frictional dissipation on the stability of grains in a homogeneous cooling system is studied in this work using molecular dynamics (MD) simulations and kinetic-theory-based predictions. Surprisingly, both MD simulations and theory indicate that high levels of friction actually attenuate instabilities relative to the frictionless case, whereas moderate levels enhance instabilities compared to frictionless systems, as expected. The mechanism responsible for this behaviour is identified as the coupling between rotational and translational motion. These results have implications not only for granular materials, but also more generally to flows with dissipative interactions between constituent particles – cohesive systems with agglomeration, multiphase flows with viscous dissipation, etc.

An integrated screening method for evaluating the pulmonary toxicity of inhaled particulates: Role of microscopic techniques in assessing pulmonary macrophage clearance functions

1990 ◽  
Vol 48 (3) ◽  
pp. 826-827
Author(s):  
David B. Warheit ◽  
Lena Achinko ◽  
Mark A. Hartsky
Keyword(s):  
Bronchoalveolar Lavage ◽  
Pulmonary Toxicity ◽  
Screening Method ◽  
Pulmonary Response ◽  
Inhaled Particles ◽  
Cytochemical Analysis ◽  
Pulmonary Macrophages ◽  
Integrated Screening

There is a great need for the development of a rapid and reliable bioassay to evaluate the pulmonary toxicity of inhaled particles. A number of methods have been proposed, including lung clearance studies, bronchoalveolar lavage analysis, and in vitro cytotoxicity tests. These methods are often limited in scope inasmuch as they measure only one dimension of the pulmonary response to inhaled, instilled or incubated dusts. Accordingly, a comprehensive approach to lung toxicity studies has been developed.To validate the method, rats were exposed for 6 hours or 3 days to various concentrations of either aerosolized alpha quartz silica (Si) or carbonyl iron (CI) particles. Cells and fluids from groups of sham and dust-exposed animals were recovered by bronchoalveolar lavage (BAL). Alkaline phosphatase, LDH and protein values were measured in BAL fluids at several time points postexposure. Cells were counted and evaluated for viability, as well as differential and cytochemical analysis. In addition, pulmonary macrophages (PM) were cultured and studied for morphology, chemotaxis, and phagocytosis by scanning electron microscopy.

Role of the LIM domain binding protein LDB1 and the LIM only factor LMO4 in SHH subgroup medulloblastoma

2014 ◽  
Vol 226 (03) ◽  
Author(s):  
S Hutter ◽  
PA Northcott ◽  
M Kool ◽  
SM Pfister ◽  
D Kawauchi ◽  
...  
Keyword(s):  
Binding Protein ◽  
Lim Domain

The Diagnosis of Lupus Anticoagulants by the Activated Partial Thromboplastin Time - The Central Role of Phosphatidyl Serine

1984 ◽  
Vol 52 (02) ◽  
pp. 172-175 ◽  
Author(s):  
P R Kelsey ◽  
K J Stevenson ◽  
L Poller
Keyword(s):  
Screening Method ◽  
Coagulation Factors ◽  
Phosphatidyl Serine ◽  
Test System ◽  
Specific Factor ◽  
Marginal Effect ◽  
Constant Composition ◽  
Lupus Anticoagulants ◽  
Different Types

SummaryLiposomes of pure phospholipids were used in a modified APTT test system and the role of phosphatidyl serine (PS) in determining the sensitivity of the test system to the presence of lupus anticoagulants was assessed. Six consecutive patients with lupus anticoagulants and seven haemophiliacs with anticoagulants directed at specific coagulation factors, were studied. Increasing the concentration of phospholipid in the test system markedly reduced the sensitivity to lupus anticoagulants but had marginal effect on the specific factor inhibitors. The same effect was achieved when the content of PS alone was increased in a vehicle liposome of constant composition.The results suggest that the lupus anticoagulants can best be detected by a screening method using an APTT test with a reagent of low PS content. The use of a reagent rich in PS will largely abolish the lupus anticoagulant’s effect on the APTT. An approach using the two different types of reagent may facilitate differentiation of lupus inhibitors from other types of anticoagulant.

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