scholarly journals A proteome-wide screen uncovers diverse roles for sequence context surrounding proline-rich motifs in Ena/VASP molecular recognition

2021 ◽  
Author(s):  
Theresa Hwang ◽  
Sara S. Parker ◽  
Samantha M. Hill ◽  
Meucci W. Ilunga ◽  
Robert A. Grant ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Structural Similarity ◽  
Low Complexity ◽  
Cellular Functions ◽  
Sequence Context ◽  
Proteomic Screen ◽  
Critical Elements ◽  
Intrinsically Disordered ◽  
Sequence Elements ◽  
Linear Motifs

Protein interactions between intrinsically disordered, short linear motifs (SLiMs) and modular recognition domains are critical elements in many signal transduction pathways. Yet for most interactions, it is still unclear how low-complexity SLiMs discriminate between highly conserved but biologically distinct SLiM-binding proteins. The Ena/VASP family of proteins pose one such specificity problem. Paralogs ENAH, VASP, and EVL bind to a 5-residue SLiM prevalent in the proteome and perform distinct cellular functions despite sharing high sequence and structural similarity. To interrogate how the sequence context of SLiMs impacts Ena/VASP interactions, we performed an unbiased proteomic screen against the ENAH EVH1 domain. We discovered unexpected ways in which local and distal sequence elements flanking native SLiMs modulate binding. Particularly notable is a peptide from PCARE that achieves paralog specificity by stabilizing a unique conformation adopted only by ENAH. A PCARE-derived peptide can selectively recruit ENAH and block its activity in cells, establishing it as a valuable reagent to disentangle the roles of Ena/VASP paralogs and a potential agent for modulating ENAH function in breast cancer. Guided by our analyses of native interactions, we designed the tightest known ENAH EVH1 binder with a dissociation constant of 50 nM and 400-600-fold selectivity over EVL and VASP. Our work demonstrates that sequence context plays a prominent role in dictating SLiM interactions and can inform the design of custom molecules that target SLiM-based interactions.

scholarly journals Peptide-based Interaction Proteomics

2020 ◽  
Vol 19 (7) ◽  
pp. 1070-1075 ◽  
Author(s):  
Katrina Meyer ◽  
Matthias Selbach
Keyword(s):  
Protein Interactions ◽  
Synthetic Peptides ◽  
Protein Protein Interactions ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Interaction Partners ◽  
Linear Motifs ◽  
Cellulose Membranes ◽  
Interaction Proteomics ◽  
Proteins Interactions

Protein-protein interactions are often mediated by short linear motifs (SLiMs) that are located in intrinsically disordered regions (IDRs) of proteins. Interactions mediated by SLiMs are notoriously difficult to study, and many functionally relevant interactions likely remain to be uncovered. Recently, pull-downs with synthetic peptides in combination with quantitative mass spectrometry emerged as a powerful screening approach to study protein-protein interactions mediated by SLiMs. Specifically, arrays of synthetic peptides immobilized on cellulose membranes provide a scalable means to identify the interaction partners of many peptides in parallel. In this minireview we briefly highlight the relevance of SLiMs for protein-protein interactions, outline existing screening technologies, discuss unique advantages of peptide-based interaction screens and provide practical suggestions for setting up such peptide-based screens.

Single-Molecule FRET of Intrinsically Disordered Proteins

2020 ◽  
Vol 71 (1) ◽  
pp. 391-414 ◽  
Author(s):  
Lauren Ann Metskas ◽  
Elizabeth Rhoades
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Intrinsically Disordered Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Intrinsically Disordered ◽  
Single Molecule Fret

Intrinsically disordered proteins (IDPs) are now widely recognized as playing critical roles in a broad range of cellular functions as well as being implicated in diverse diseases. Their lack of stable secondary structure and tertiary interactions, coupled with their sensitivity to measurement conditions, stymies many traditional structural biology approaches. Single-molecule Förster resonance energy transfer (smFRET) is now widely used to characterize the physicochemical properties of these proteins in isolation and is being increasingly applied to more complex assemblies and experimental environments. This review provides an overview of confocal diffusion-based smFRET as an experimental tool, including descriptions of instrumentation, data analysis, and protein labeling. Recent papers are discussed that illustrate the unique capability of smFRET to provide insight into aggregation-prone IDPs, protein–protein interactions involving IDPs, and IDPs in complex experimental milieus.

scholarly journals Prion Replication in the Mammalian Cytosol: Functional Regions within a Prion Domain Driving Induction, Propagation, and Inheritance

2018 ◽  
Vol 38 (15) ◽  
Author(s):  
Yvonne Duernberger ◽  
Shu Liu ◽  
Katrin Riemschoss ◽  
Lydia Paulsen ◽  
Romina Bester ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Low Complexity ◽  
Nucleation Site ◽  
Content Type ◽  
Amino Terminal ◽  
Intrinsically Disordered ◽  
Functional Regions ◽  
Sequence Elements ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal

ABSTRACTPrions of lower eukaryotes are transmissible protein particles that propagate by converting homotypic soluble proteins into growing protein assemblies. Prion activity is conferred by so-called prion domains, regions of low complexity that are often enriched in glutamines and asparagines (Q/N). The compositional similarity of fungal prion domains with intrinsically disordered domains found in many mammalian proteins raises the question of whether similar sequence elements can drive prion-like phenomena in mammals. Here, we define sequence features of the prototypeSaccharomyces cerevisiaeSup35 prion domain that govern prion activities in mammalian cells by testing the ability of deletion mutants to assemble into self-perpetuating particles. Interestingly, the amino-terminal Q/N-rich tract crucially important for prion induction in yeast was dispensable for the prion life cycle in mammalian cells. Spontaneous and template-assisted prion induction, growth, and maintenance were preferentially driven by the carboxy-terminal region of the prion domain that contains a putative soft amyloid stretch recently proposed to act as a nucleation site for prion assembly. Our data demonstrate that preferred prion nucleation domains can differ between lower and higher eukaryotes, resulting in the formation of prions with strikingly different amyloid cores.

Promiscuity as a functional trait: intrinsically disordered regions as central players of interactomes

2013 ◽  
Vol 454 (3) ◽  
pp. 361-369 ◽  
Author(s):  
Alexander Cumberworth ◽  
Guillaume Lamour ◽  
M. Madan Babu ◽  
Jörg Gsponer
Keyword(s):  
Low Complexity ◽  
Functional Trait ◽  
Protein Interaction Networks ◽  
Altered Expression ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Linear Motifs ◽  
Eukaryotic Genomes ◽  
Molecular Recognition Features ◽  
Disordered Regions

Because of their pervasiveness in eukaryotic genomes and their unique properties, understanding the role that ID (intrinsically disordered) regions in proteins play in the interactome is essential for gaining a better understanding of the network. Especially critical in determining this role is their ability to bind more than one partner using the same region. Studies have revealed that proteins containing ID regions tend to take a central role in protein interaction networks; specifically, they act as hubs, interacting with multiple different partners across time and space, allowing for the co-ordination of many cellular activities. There appear to be three different modules within ID regions responsible for their functionally promiscuous behaviour: MoRFs (molecular recognition features), SLiMs (small linear motifs) and LCRs (low complexity regions). These regions allow for functionality such as engaging in the formation of dynamic heteromeric structures which can serve to increase local activity of an enzyme or store a collection of functionally related molecules for later use. However, the use of promiscuity does not come without a cost: a number of diseases that have been associated with ID-containing proteins seem to be caused by undesirable interactions occurring upon altered expression of the ID-containing protein.

Mathematical and Computational Techniques for Drug Discovery: Promises and Developments

2019 ◽  
Vol 18 (32) ◽  
pp. 2774-2799 ◽  
Author(s):  
Krishnan Balasubramanian
Keyword(s):  
Drug Discovery ◽  
Protein Interactions ◽  
Quantum Chemical ◽  
Intrinsically Disordered Proteins ◽  
Hepatitis Virus ◽  
Disordered Proteins ◽  
Computational Techniques ◽  
Large Set ◽  
Structural Protein ◽  
Intrinsically Disordered

We review various mathematical and computational techniques for drug discovery exemplifying some recent works pertinent to group theory of nested structures of relevance to phylogeny, topological, computational and combinatorial methods for drug discovery for multiple viral infections. We have reviewed techniques from topology, combinatorics, graph theory and knot theory that facilitate topological and mathematical characterizations of protein-protein interactions, molecular-target interactions, proteomics, genomics and statistical data reduction procedures for a large set of starting chemicals in drug discovery. We have provided an overview of group theoretical techniques pertinent to phylogeny, protein dynamics especially in intrinsically disordered proteins, DNA base permutations and related algorithms. We consider computational techniques derived from high level quantum chemical computations such as QM/MM ONIOM methods, quantum chemical optimization of geometries complexes, and molecular dynamics methods for providing insights into protein-drug interactions. We have considered complexes pertinent to Hepatitis Virus C non-structural protein 5B polymerase receptor binding of C5-Arylidebne rhodanines, complexes of synthetic potential vaccine molecules with dengue virus (DENV) and HIV-1 virus as examples of various simulation studies that exemplify the utility of computational tools. It is demonstrated that these combinatorial and computational techniques in conjunction with experiments can provide promising new insights into drug discovery. These techniques also demonstrate the need to consider a new multiple site or allosteric binding approach to drug discovery, as these studies reveal the existence of multiple binding sites.

scholarly journals Short Linear Motifs Characterizing Snake Venom and Mammalian Phospholipases A2

Toxins ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 290
Author(s):  
Caterina Peggion ◽  
Fiorella Tonello
Keyword(s):  
Snake Venom ◽  
Protein Interactions ◽  
Biological Properties ◽  
Protein Protein Interactions ◽  
Sequence Alignments ◽  
Toxic Activity ◽  
Short Linear Motifs ◽  
Phospholipases A2 ◽  
Group I ◽  
Linear Motifs

Snake venom phospholipases A2 (PLA2s) have sequences and structures very similar to those of mammalian group I and II secretory PLA2s, but they possess many toxic properties, ranging from the inhibition of coagulation to the blockage of nerve transmission, and the induction of muscle necrosis. The biological properties of these proteins are not only due to their enzymatic activity, but also to protein–protein interactions which are still unidentified. Here, we compare sequence alignments of snake venom and mammalian PLA2s, grouped according to their structure and biological activity, looking for differences that can justify their different behavior. This bioinformatics analysis has evidenced three distinct regions, two central and one C-terminal, having amino acid compositions that distinguish the different categories of PLA2s. In these regions, we identified short linear motifs (SLiMs), peptide modules involved in protein–protein interactions, conserved in mammalian and not in snake venom PLA2s, or vice versa. The different content in the SLiMs of snake venom with respect to mammalian PLA2s may result in the formation of protein membrane complexes having a toxic activity, or in the formation of complexes whose activity cannot be blocked due to the lack of switches in the toxic PLA2s, as the motif recognized by the prolyl isomerase Pin1.

Current trends in mathematical modeling of tumor–microenvironment interactions: a survey of tools and applications

2010 ◽  
Vol 235 (4) ◽  
pp. 411-423 ◽  
Author(s):  
Katarzyna A Rejniak ◽  
Lisa J McCawley
Keyword(s):  
Mathematical Modeling ◽  
Protein Interactions ◽  
Computational Models ◽  
Chemical Species ◽  
Tumor Stroma ◽  
Cellular Functions ◽  
Complex Interactions ◽  
Expanding Population

In its simplest description, a tumor is comprised of an expanding population of transformed cells supported by a surrounding microenvironment termed the tumor stroma. The tumor microcroenvironment has a very complex composition, including multiple types of stromal cells, a dense network of various extracellular matrix (ECM) fibers interpenetrated by the interstitial fluid and gradients of several chemical species that either are dissolved in the fluid or are bound to the ECM structure. In order to study experimentally such complex interactions between multiple players, cancer is dissected and considered at different scales of complexity, such as protein interactions, biochemical pathways, cellular functions or whole organism studies. However, the integration of information acquired from these studies into a common description is as difficult as the disease itself. Computational models of cancer can provide cancer researchers with invaluable tools that are capable of integrating the complexity into organizing principles as well as suggesting testable hypotheses. We will focus in this Minireview on mathematical models in which the whole cell is a main modeling unit. We will present a current stage of such cell-focused mathematical modeling incorporating different stromal components and their interactions with growing tumors, and discuss what modeling approaches can be undertaken to complement the in vivo and in vitro experimentation.

scholarly journals PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

1996 ◽  
Vol 132 (3) ◽  
pp. 359-370 ◽  
Author(s):  
E F Smith ◽  
P A Lefebvre
Keyword(s):  
Protein Interactions ◽  
Insertional Mutagenesis ◽  
Flagellar Motility ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Central Pair ◽  
Central Apparatus ◽  
Immunofluorescence Labeling ◽  
Armadillo Repeats

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

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