Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

AbstractThe BCL-2 family is a challenging set of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are desirable as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL, BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective and potent recognition of their target BCL-2 protein. For anti-apoptotic targets, competitive inhibition of their canonical protein-protein interactions is demonstrated. Co-crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug bound like conformation. These results indicate Affimers can be used as alternative templates to inspire design of selective BCL-2 family modulators, and provide proof-of-concept for the elaboration of selective non-antibody binding reagents for use in cell-biology applications.

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Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.m109.001743 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16369-16376 ◽

Cited By ~ 23

Author(s):

Xuebo Hu ◽

Sungkwon Kang ◽

Xiaoyue Chen ◽

Charles B. Shoemaker ◽

Moonsoo M. Jin

Keyword(s):

Protein Interactions ◽

Secretory Pathway ◽

Coiled Coil ◽

Affinity Maturation ◽

Antibody Binding ◽

Soluble Form ◽

Protein Protein Interactions ◽

Yeast Surface ◽

Two Hybrid

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.

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Insulin signalling: putting the G- in protein-protein interactions

Biochemical Journal ◽

10.1042/bj20040619 ◽

2004 ◽

Vol 380 (1) ◽

pp. e11-e12 ◽

Cited By ~ 10

Author(s):

Craig C. MALBON

Keyword(s):

Insulin Receptor ◽

Cross Talk ◽

Protein Interactions ◽

Tyrosine Kinases ◽

Cell Biology ◽

Receptor Tyrosine Kinases ◽

Insulin Signalling ◽

Cell Signalling ◽

Protein Protein Interactions ◽

Proximal Point

Cell signalling via receptor tyrosine kinases, such as the insulin receptor, and via heterotrimeric G-proteins, such as Gαi, Gαs and Gαq family members, constitute two of most avidly studied paradigms in cell biology. That elements of these two populous signalling pathways must cross-talk to achieve proper signalling in the regulation of cell proliferation, differentiation and metabolism has been anticipated, but the evolution of our thinking and the analysis of such cross-talk have lagged behind the ever-expanding troupe of players and the recognition of multivalency as the rule, rather than the exception, in signalling biology. New insights have been provided by Kreuzer et al. in this issue of the Biochemical Journal, in which insulin is shown to provoke recruitment of Gαi-proteins to insulin-receptor-based complexes that can regulate the gain of insulin-receptor-catalysed autophosphorylation, a proximal point in the insulin-sensitive cascade of signalling. Understanding the convergence and cross-talk of signals from the receptor tyrosine kinases and G-protein-coupled receptor pathways in physical, spatial and temporal contexts will remain a major challenge of cell biology.

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The Disordered Cellular Multi-Tasker WIP and Its Protein–Protein Interactions: A Structural View

Biomolecules ◽

10.3390/biom10071084 ◽

2020 ◽

Vol 10 (7) ◽

pp. 1084 ◽

Cited By ~ 2

Author(s):

Chana G. Sokolik ◽

Nasrin Qassem ◽

Jordan H. Chill

Keyword(s):

Protein Interactions ◽

Cell Biology ◽

Intrinsically Disordered Proteins ◽

Disordered Proteins ◽

Actin Binding ◽

Structural Description ◽

Protein Protein Interactions ◽

Intrinsically Disordered ◽

Terminal Domain ◽

Binding Partners

WASp-interacting protein (WIP), a regulator of actin cytoskeleton assembly and remodeling, is a cellular multi-tasker and a key member of a network of protein–protein interactions, with significant impact on health and disease. Here, we attempt to complement the well-established understanding of WIP function from cell biology studies, summarized in several reviews, with a structural description of WIP interactions, highlighting works that present a molecular view of WIP’s protein–protein interactions. This provides a deeper understanding of the mechanisms by which WIP mediates its biological functions. The fully disordered WIP also serves as an intriguing example of how intrinsically disordered proteins (IDPs) exert their function. WIP consists of consecutive small functional domains and motifs that interact with a host of cellular partners, with a striking preponderance of proline-rich motif capable of interactions with several well-recognized binding partners; indeed, over 30% of the WIP primary structure are proline residues. We focus on the binding motifs and binding interfaces of three important WIP segments, the actin-binding N-terminal domain, the central domain that binds SH3 domains of various interaction partners, and the WASp-binding C-terminal domain. Beyond the obvious importance of a more fundamental understanding of the biology of this central cellular player, this approach carries an immediate and highly beneficial effect on drug-design efforts targeting WIP and its binding partners. These factors make the value of such structural studies, challenging as they are, readily apparent.

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Hypothesis: huntingtin may function in membrane association and vesicular traffickingThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

Biochemistry and Cell Biology ◽

10.1139/o06-181 ◽

2006 ◽

Vol 84 (6) ◽

pp. 912-917 ◽

Cited By ~ 34

Author(s):

Ray Truant ◽

Randy Atwal ◽

Anjee Burtnik

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Protein Interactions ◽

Cell Biology ◽

Genetic Disorder ◽

Scaffolding Protein ◽

Membrane Association ◽

Protein Protein Interactions ◽

Polyglutamine Expansion ◽

Exact Function

Huntington’s disease is a progressive neurodegenerative genetic disorder that is caused by a CAG triplet-repeat expansion in the first exon of the IT15 gene. This CAG expansion results in polyglutamine expansion in the 350 kDa huntingtin protein. The exact function of huntingtin is unknown. Understanding the pathological triggers of mutant huntingtin, and distinguishing the cause of disease from downstream effects, is critical to designing therapeutic strategies and defining long- and short-term goals of therapy. Many studies that have sought to determine the functions of huntingtin by determining huntingtin’s protein–protein interactions have been published. Through these studies, huntingtin has been seen to interact with a large number of proteins, and is likely a scaffolding protein for protein–protein interactions. Recently, using imaging, integrative proteomics, and cell biology, huntingtin has been defined as a membrane-associated protein, with activities related to axonal trafficking of vesicles and mitochondria. These functions have also been attributed to some huntingtin-interacting proteins. Additionally, discoveries of a membrane association domain and a palmitoylation site in huntingtin reinforce the fact that huntingtin is membrane associated. In Huntington’s disease mouse and fly models, axonal vesicle trafficking is inhibited, and lack of proper uptake of neurotrophic factors may be an important pathological trigger leading to striatal cell death in Huntington’s disease. Here we discuss recent advances from many independent groups and methodologies that are starting to resolve the elusive function of huntingtin in vesicle transport, and evidence that suggests that huntingtin may be directly involved in membrane interactions.

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Modulating the Bcl-2 Family of Apoptosis Suppressors for Potential Therapeutic Benefit in Cancer

Hematology ◽

10.1182/asheducation-2005.1.226 ◽

2005 ◽

Vol 2005 (1) ◽

pp. 226-230 ◽

Cited By ~ 46

Author(s):

Gordon C. Shore ◽

Jean Viallet

Keyword(s):

Protein Interactions ◽

Therapeutic Benefit ◽

Protein Protein Interactions ◽

Cancer Treatments ◽

The Family ◽

Challenges And Opportunities ◽

Apoptosis Pathways ◽

Current Therapies ◽

Cellular Oncogenes ◽

Potential Therapeutic Benefit

Abstract Members of the BCL-2 family of proteins regulate and execute many cell intrinsic apoptosis pathways, including those arising from dysregulated expression of cellular oncogenes. Since pro-survival members of the family are often strongly elevated in diverse cancers, with the potential to confer resistance to both endogenous cell death stimuli and many cancer treatments, there has been intense interest to develop strategies to therapeutically modulate their activity. Although encouraging genetic and pharmacological preclinical proof of concept has been obtained, the challenge for clinical development will be to devise strategies that address the fact that multiple pro-survival members are typically up-regulated in a given cancer and the family operates primarily through protein-protein interactions. Moreover, since several current therapies themselves are known to stimulate the levels of one or more family members, there will be additional challenges (and opportunities) in exploiting this target in the clinic. In this review, we describe the rationale for targeting the BCL-2 family of apoptosis suppressors in cancer and the progress that has been made in modulating the family by small molecule antagonists.

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The use of 4,4,4-trifluorothreonine to stabilize extended peptide structures and mimic β-strands

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.13.276 ◽

2017 ◽

Vol 13 ◽

pp. 2842-2853 ◽

Cited By ~ 1

Author(s):

Yaochun Xu ◽

Isabelle Correia ◽

Tap Ha-Duong ◽

Nadjib Kihal ◽

Jean-Louis Soulier ◽

...

Keyword(s):

Protein Interactions ◽

Amyloid Peptide ◽

Central Position ◽

Aggregation Process ◽

Proof Of Concept ◽

Protein Protein Interactions ◽

Conformational Studies ◽

Dynamics Simulations ◽

Β Sheet ◽

Central Residue

Pentapeptides having the sequence R-HN-Ala-Val-X-Val-Leu-OMe, where the central residue X is L-serine, L-threonine, (2S,3R)-L-CF3-threonine and (2S,3S)-L-CF3-threonine were prepared. The capacity of (2S,3S)- and (2S,3R)-CF3-threonine analogues to stabilize an extended structure when introduced in the central position of pentapeptides is demonstrated by NMR conformational studies and molecular dynamics simulations. CF3-threonine containing pentapeptides are more prone to mimic β-strands than their natural Ser and Thr pentapeptide analogues. The proof of concept that these fluorinated β-strand mimics are able to disrupt protein–protein interactions involving β-sheet structures is provided. The CF3-threonine containing pentapeptides interact with the amyloid peptide Aβ1-42 in order to reduce the protein–protein interactions mediating its aggregation process.

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Engineered illumination devices for optogenetic control of cellular signaling dynamics

10.1101/675892 ◽

2019 ◽

Cited By ~ 1

Author(s):

Nicole A. Repina ◽

Thomas McClave ◽

Xiaoping Bao ◽

Ravi S. Kane ◽

David V. Schaffer

Keyword(s):

Cell Fate ◽

High Throughput ◽

Protein Interactions ◽

Cell Biology ◽

Low Cost ◽

Optical Design ◽

Embryonic Stem ◽

Protein Protein Interactions ◽

Optogenetic Control

ABSTRACTSpatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, currentin vitromethods typically do not allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics impact cell behavior. Here we show that optogenetic Wnt/β-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using novel engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). The optical design of LAVA devices was optimized for uniform illumination of multi-well cell culture plates to enable high-throughput, spatiotemporal optogenetic activation of signaling pathways and protein-protein interactions. Using the LAVA devices, variation in light intensity induced a dose-dependent response in optoWnt activation and downstream Brachyury expression in human embryonic stem cells (hESCs). Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models embryonic presentation of Wnt signalsin vitro. The engineered LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology.

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Finding functional associations between prokaryotic virus orthologous groups: a proof of concept

BMC Bioinformatics ◽

10.1186/s12859-021-04343-w ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Nikolaos Pappas ◽

Bas E. Dutilh

Keyword(s):

Protein Interactions ◽

Systematic Evaluation ◽

Proof Of Concept ◽

Protein Protein Interactions ◽

Viral Genomes ◽

False Discovery ◽

Machine Learning Approach ◽

Recent Developments ◽

Significant Step ◽

Functional Context

Abstract Background The field of viromics has greatly benefited from recent developments in metagenomics, with significant efforts focusing on viral discovery. However, functional annotation of the increasing number of viral genomes is lagging behind. This is highlighted by the degree of annotation of the protein clusters in the prokaryotic Virus Orthologous Groups (pVOGs) database, with 83% of its current 9518 pVOGs having an unknown function. Results In this study we describe a machine learning approach to explore potential functional associations between pVOGs. We measure seven genomic features and use them as input to a Random Forest classifier to predict protein–protein interactions between pairs of pVOGs. After systematic evaluation of the model’s performance on 10 different datasets, we obtained a predictor with a mean accuracy of 0.77 and Area Under Receiving Operation Characteristic (AUROC) score of 0.83. Its application to a set of 2,133,027 pVOG-pVOG interactions allowed us to predict 267,265 putative interactions with a reported probability greater than 0.65. At an expected false discovery rate of 0.27, we placed 95.6% of the previously unannotated pVOGs in a functional context, by predicting their interaction with a pVOG that is functionally annotated. Conclusions We believe that this proof-of-concept methodology, wrapped in a reproducible and automated workflow, can represent a significant step towards obtaining a more complete picture of bacteriophage biology.

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Optothermophoretic flipping method for biomolecule interaction enhancement in biosensing

10.21203/rs.3.rs-253405/v1 ◽

2021 ◽

Author(s):

Jiajie Chen ◽

Youjun Zeng ◽

Jie Zhou ◽

Xueliang Wang ◽

Ruibiao Miyan ◽

...

Keyword(s):

Single Molecule ◽

Protein Interactions ◽

Concentration Level ◽

Brownian Diffusion ◽

Proof Of Concept ◽

Protein Protein Interactions ◽

Effective Contact ◽

All Optical ◽

Single Molecule Analysis ◽

Biomolecule Sensing

Abstract The widely used surface-based biomolecule sensing scheme has greatly facilitated the investigation of protein-protein interactions in lab-on-a-chip microfluidic systems. However, in most biosensing schemes, the interactions are driven in a passive way: The overall sensing time and sensitivity are totally dependent on the Brownian diffusion process, which has greatly hindered their efficiency, especially at low concentration level or single-molecule analysis. To break this limitation, we developed an all-optical active method termed optothermophoretic flipping (OTF). The biomolecules were first enriched to aggregation and then pushed to their counterparts for effective contact via a flipped thermophoresis. As a proof-of-concept experiment, we tested its performance via antibody-antigen binding on a surface plasmon resonance imaging (SPRi) platform. We achieved 36.9-fold sensitivity enhancement in this first temporal modulated approach that manipulates biomolecules for interaction enhancement. This method promises to be widely adopted in various biosensing platforms that require ultrasensitivity in colloidal sciences and biochemical studies.

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