Abstract 1187: PDZ Domain Protein Arrays Define a Network of ABCA1 and SPLTC1 Protein-Protein Interactions that Regulate ABCA1 Trafficking and Efflux Activity

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Michael L Fitzgerald ◽  
Norimasa Tamehiro ◽  
Suiping Zhou ◽  
Keiichiro Okuhira
Keyword(s):  
Cardiovascular Disease ◽  
Protein Interactions ◽  
Cholesterol Efflux ◽  
Pdz Domain ◽  
Serine Palmitoyltransferase ◽  
Protein Protein Interactions ◽  
Data Set ◽  
Protein Arrays ◽  
Pdz Proteins ◽  
Increased Risk

Protein microarrays are an emerging high throughput proteomic tool that can be used to study protein-protein interactions. Here we use protein arrays that have 123 PDZ domains spotted in duplicate from 77 PDZ proteins testing over 700 potential interactions and mapping a network of protein interactions that encompasses the ABCA1 transporter and SPLTC1. SPTLC1, through its associated serine palmitoyltransferase activity, catalyzes the initial step in the synthesis of sphingomyelin whose plasma level has been associated with an increased risk of cardiovascular disease. Conversely, ABCA1 effluxes cellular cholesterol to maintain plasma HDL levels which prevents cardiovascular disease. Using hierarchical clustering and networking algorithms analysis of our data set shows PDZ proteins are able to discriminate between closely related PDZ binding motifs contained in the C-termini of ABCA1 and SPTLC1. Mutation of the SPTLC1 PDZ motif disrupted SPTLC1’s ability to bind PDZ proteins and destabilized SPTLC1 protein expression demonstrating the functional importance of the motif. Co-expression of SPTLC1 with ABCA1 in 293 cells inhibited cholesterol efflux activity of the transporter by up to 80% (p < 0.01) as we previously reported and here we show this is through a mechanism that blocks the exit of the transporter from the endoplasmic reticulum. In contrast, treatment of primary human fibroblasts or mouse macrophages with myriocin, a specific serine palmitoyltransferase inhibitor, increased efflux by nearly 60% (p < 0.05). Our results demonstrate the utility using protein arrays to map protein-protein interactions that we hypothesis form a regulatory network that integrates the cholesterol efflux and sphingolipid synthesis pathways. That SPTLC1 activity negatively regulates ABCA1 efflux further suggests inhibition of SPTLC1 activity may represent a new therapeutic target to prevent cardiovascular disease.

scholarly journals Exploring the Human-Nipah Virus Protein-Protein Interactome

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Luis Martinez-Gil ◽  
Natalia M. Vera-Velasco ◽  
Ismael Mingarro
Keyword(s):  
Protein Interactions ◽  
Affinity Purification ◽  
Nipah Virus ◽  
Productive Infection ◽  
Virus Protein ◽  
Protein Protein Interactions ◽  
Data Set ◽  
Wide Range ◽  
Host Virus Interactions ◽  
Virus Interactions

ABSTRACT Nipah virus is an emerging, highly pathogenic, zoonotic virus of the Paramyxoviridae family. Human transmission occurs by close contact with infected animals, the consumption of contaminated food, or, occasionally, via other infected individuals. Currently, we lack therapeutic or prophylactic treatments for Nipah virus. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. This aim led us to perform the present work, in which we identified 101 human-Nipah virus protein-protein interactions (PPIs), most of which (88) are novel. This data set provides a comprehensive view of the host complexes that are manipulated by viral proteins. Host targets include the PRP19 complex and the microRNA (miRNA) processing machinery. Furthermore, we explored the biologic consequences of the interaction with the PRP19 complex and found that the Nipah virus W protein is capable of altering p53 control and gene expression. We anticipate that these data will help in guiding the development of novel interventional strategies to counter this emerging viral threat. IMPORTANCE Nipah virus is a recently discovered virus that infects a wide range of mammals, including humans. Since its discovery there have been yearly outbreaks, and in some of them the mortality rate has reached 100% of the confirmed cases. However, the study of Nipah virus has been largely neglected, and currently we lack treatments for this infection. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. In the present work, we identified 101 human-Nipah virus protein-protein interactions using an affinity purification approach coupled with mass spectrometry. Additionally, we explored the cellular consequences of some of these interactions. Globally, this data set offers a comprehensive and detailed view of the host machinery's contribution to the Nipah virus's life cycle. Furthermore, our data present a large number of putative drug targets that could be exploited for the treatment of this infection.

scholarly journals Protein Arrays as Tools for Detection of Protein-Protein Interactions by Mass Spectrometry

2010 ◽  
pp. 725-727
Author(s):  
Christian F. W. Becker ◽  
Ron Wacker ◽  
Werner Bouschen ◽  
Ralf P. Seidel ◽  
Branko Kolaric ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Protein Arrays

The Yin and Yang of High-density Lipoprotein and Atherosclerotic Cardiovascular Disease: Focusing on Functionality and Cholesterol Efflux to Reframe the HDL Hypothesis

2021 ◽  
Vol 28 ◽  
Author(s):  
Shiva Ganjali ◽  
Gerald F. Watts ◽  
Maciej Banach ◽  
Željko Reiner ◽  
Petr Nachtigal ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
High Density Lipoprotein ◽  
Cholesterol Efflux ◽  
Association Studies ◽  
Plasma Concentrations ◽  
Density Lipoprotein ◽  
High Density ◽  
Atherosclerotic Cardiovascular Disease ◽  
Genome Wide Association Studies ◽  
Increased Risk

Abstract: The inverse relationship between low plasma high-density lipoprotein cholesterol (HDL-C) concentrations and increased risk of Atherosclerotic Cardiovascular Disease (ASCVD) is well-known. However, plasma HDL-C concentrations are highly variable in subjects with ASCVD. In clinical outcome trials, pharmacotherapies that increase HDL-C concentrations are not associated with a reduction in ASCVD events. A causal relationship between HDL-C and ASCVD has also been questioned by Mendelian randomization studies and genome-wide association studies of genetic variants associated with plasma HDL-C concentrations. The U-shaped association between plasma HDL-C concentrations and mortality observed in several epidemiological studies implicates both low and very high plasma HDL-C concentrations in the etiology of ASCVD and non-ASCVD mortality. These data do not collectively support a causal association between HDL-C and ASCVD risk. Therefore, the hypothesis concerning the association between HDL and ASCVD has shifted from focus on plasma concentrations to the concept of functionality, in particular cellular cholesterol efflux and HDL holoparticle transport. In this review, we focus on these new concepts and provide a new framework for understanding and testing the role of HDL in ASCVD.

scholarly journals Engineering selective competitors for the discrimination of highly conserved protein-protein interaction modules

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Charlotte Rimbault ◽  
Kashyap Maruthi ◽  
Christelle Breillat ◽  
Camille Genuer ◽  
Sara Crespillo ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Pdz Domain ◽  
Specific Inhibitor ◽  
Rational Approach ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Domain Specific ◽  
Postsynaptic Protein ◽  
Protein Interfaces ◽  
Direct Generation

Abstract Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by using phage display selection coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to contact the target interface. We apply this approach to specifically bind a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides a paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules.

scholarly journals Peptide Targeting of PDZ-Dependent Interactions as Pharmacological Intervention in Immune-Related Diseases

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6367
Author(s):  
Luis H. Gutiérrez-González ◽  
Selma Rivas-Fuentes ◽  
Silvia Guzmán-Beltrán ◽  
Angélica Flores-Flores ◽  
Jorge Rosas-García ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Interactions ◽  
Cell Types ◽  
Pharmacological Intervention ◽  
Protein Protein Interactions ◽  
Pdz Proteins ◽  
Immune Evasion Mechanism ◽  
Cellular Processes ◽  
Clinical Conditions ◽  
Peptide Targeting

PDZ (postsynaptic density (PSD95), discs large (Dlg), and zonula occludens (ZO-1)-dependent interactions are widely distributed within different cell types and regulate a variety of cellular processes. To date, some of these interactions have been identified as targets of small molecules or peptides, mainly related to central nervous system disorders and cancer. Recently, the knowledge of PDZ proteins and their interactions has been extended to various cell types of the immune system, suggesting that their targeting by viral pathogens may constitute an immune evasion mechanism that favors viral replication and dissemination. Thus, the pharmacological modulation of these interactions, either with small molecules or peptides, could help in the control of some immune-related diseases. Deeper structural and functional knowledge of this kind of protein–protein interactions, especially in immune cells, will uncover novel pharmacological targets for a diversity of clinical conditions.

scholarly journals In vivo crystals reveal critical features of the interaction between cystic fibrosis transmembrane conductance regulator (CFTR) and the PDZ2 domain of Na+/H+ exchange cofactor NHERF1

2020 ◽  
Vol 295 (14) ◽  
pp. 4464-4476
Author(s):  
Eleanor R. Martin ◽  
Alessandro Barbieri ◽  
Robert C. Ford ◽  
Robert C. Robinson
Keyword(s):  
Cystic Fibrosis ◽  
Protein Interactions ◽  
Pdz Domain ◽  
Binding Motif ◽  
Transmembrane Conductance Regulator ◽  
Protein Protein Interactions ◽  
Reporter System ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

Crystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under extremely nonphysiological protein, salt, and H+ concentrations. Here, we describe the development of a robust Inka1-Box (iBox)–PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semi-quantitative assay described here allows the measurement of in vivo protein-protein interactions using a novel GFP-linked reporter system that produces fluorescent readouts from protein crystals. We combined this assay with in vitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between the PDZ2 domain of Na+/H+ exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions −1 and −3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2-CFTR interaction. Our results suggest that this system could be utilized to screen additional protein-protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice.

scholarly journals Functional regulation of cystic fibrosis transmembrane conductance regulator-containing macromolecular complexes: a small-molecule inhibitor approach

2011 ◽  
Vol 435 (2) ◽  
pp. 451-462 ◽  
Author(s):  
Weiqiang Zhang ◽  
Himabindu Penmatsa ◽  
Aixia Ren ◽  
Chandanamali Punchihewa ◽  
Andrew Lemoff ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Small Molecule ◽  
Protein Interactions ◽  
Pdz Domain ◽  
Transmembrane Conductance Regulator ◽  
Protein Protein Interactions ◽  
Transmembrane Conductance ◽  
Macromolecular Complexes ◽  
Channel Function ◽  
Cystic Fibrosis Transmembrane

CFTR (cystic fibrosis transmembrane conductance regulator) has been shown to form multiple protein macromolecular complexes with its interacting partners at discrete subcellular microdomains to modulate trafficking, transport and signalling in cells. Targeting protein–protein interactions within these macromolecular complexes would affect the expression or function of the CFTR channel. We specifically targeted the PDZ domain-based LPA2 (type 2 lysophosphatidic acid receptor)–NHERF2 (Na+/H+ exchanger regulatory factor-2) interaction within the CFTR–NHERF2–LPA2-containing macromolecular complexes in airway epithelia and tested its regulatory role on CFTR channel function. We identified a cell-permeable small-molecule compound that preferentially inhibits the LPA2–NHERF2 interaction. We show that this compound can disrupt the LPA2–NHERF2 interaction in cells and thus compromises the integrity of macromolecular complexes. Functionally, it elevates cAMP levels in proximity to CFTR and upregulates its channel activity. The results of the present study demonstrate that CFTR Cl− channel function can be finely tuned by modulating PDZ domain-based protein–protein interactions within the CFTR-containing macromolecular complexes. The present study might help to identify novel therapeutic targets to treat diseases associated with dysfunctional CFTR Cl− channels.

The result of equilibrium-constant calculations strongly depends on the evaluation method used and on the type of experimental errors

2001 ◽  
Vol 359 (2) ◽  
pp. 411-418 ◽  
Author(s):  
Hendrik FUCHS ◽  
Reinhard GESSNER
Keyword(s):  
Protein Interactions ◽  
Evaluation Method ◽  
Equilibrium Constants ◽  
Scatchard Analysis ◽  
Protein Protein Interactions ◽  
Data Set ◽  
Slow Kinetics ◽  
Experimental Errors ◽  
Saturation Effects

The determination of equilibrium constants is a widespread tool both to understand and to characterize protein–protein interactions. A variety of different methods, among them Scatchard analysis, is used to calculate these constants. Although more than 1000 articles dealing with equilibrium constants are published every year, the effects of experimental errors on the results are often disregarded when interpreting the data. In the present study we theoretically analysed the effect of various types of experimental errors on equilibrium constants derived by three different methods. A computer simulation clearly showed that certain experimental errors, namely inaccurate background correction, inexact calibration, saturation effects, slow kinetics and simple scattering, can adversely affect the result. The analysis further revealed that, for a given type of error, the same data set can produce different results depending on the method used.

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