A rational blueprint for the design of chemically-controlled protein switches

AbstractSmall-molecule responsive protein switches are crucial components to control synthetic cellular activities. However, the repertoire of small-molecule protein switches is insufficient for many applications, including those in the translational spaces, where properties such as safety, immunogenicity, drug half-life, and drug side-effects are critical. Here, we present a computational protein design strategy to repurpose drug-inhibited protein-protein interactions as OFF- and ON-switches. The designed binders and drug-receptors form chemically-disruptable heterodimers (CDH) which dissociate in the presence of small molecules. To design ON-switches, we converted the CDHs into a multi-domain architecture which we refer to as activation by inhibitor release switches (AIR) that incorporate a rationally designed drug-insensitive receptor protein. CDHs and AIRs showed excellent performance as drug responsive switches to control combinations of synthetic circuits in mammalian cells. This approach effectively expands the chemical space and logic responses in living cells and provides a blueprint to develop new ON- and OFF-switches for basic and translational applications.

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Targeting epigenetic protein–protein interactions with small-molecule inhibitors

Future Medicinal Chemistry ◽

10.4155/fmc-2020-0082 ◽

2020 ◽

Vol 12 (14) ◽

pp. 1305-1326 ◽

Cited By ~ 1

Author(s):

Brian M Linhares ◽

Jolanta Grembecka ◽

Tomasz Cierpicki

Keyword(s):

Small Molecules ◽

Small Molecule ◽

Protein Interactions ◽

Small Molecule Inhibitors ◽

Chemical Space ◽

Scaffolding Proteins ◽

Protein Protein Interactions ◽

Post Translational Modifications ◽

Inhibitor Development ◽

Interdisciplinary Approaches

Epigenetic protein–protein interactions (PPIs) play essential roles in regulating gene expression, and their dysregulations have been implicated in many diseases. These PPIs are comprised of reader domains recognizing post-translational modifications on histone proteins, and of scaffolding proteins that maintain integrities of epigenetic complexes. Targeting PPIs have become focuses for development of small-molecule inhibitors and anticancer therapeutics. Here we summarize efforts to develop small-molecule inhibitors targeting common epigenetic PPI domains. Potent small molecules have been reported for many domains, yet small domains that recognize methylated lysine side chains on histones are challenging in inhibitor development. We posit that the development of potent inhibitors for difficult-to-prosecute epigenetic PPIs may be achieved by interdisciplinary approaches and extensive explorations of chemical space.

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Faculty Opinions recommendation of Protein-protein interactions monitored in mammalian cells via complementation of beta -lactamase enzyme fragments.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1005525.65954 ◽

2002 ◽

Author(s):

Marilyn Parsons

Keyword(s):

Protein Interactions ◽

Mammalian Cells ◽

Beta Lactamase ◽

Protein Protein Interactions

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Faculty Opinions recommendation of Importance of Rigidity in Designing Small Molecule Drugs To Tackle Protein-Protein Interactions (PPIs) through Stabilization of Desired Conformers.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732129727.793539183 ◽

2017 ◽

Author(s):

John Lowe

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Protein Protein Interactions ◽

Small Molecule Drugs

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IL-14 MAPPIT: a versatile METHOD to study protein-protein interactions in mammalian cells

Pigment Cell Research ◽

10.1034/j.1600-0749.2003.08323.x ◽

2003 ◽

Vol 16 (5) ◽

pp. 577-577

Author(s):

J. Tavernier ◽

S. Eyckerman ◽

I. Lemmens ◽

S. Lievens ◽

J. Vandekerckhove ◽

...

Keyword(s):

Protein Interactions ◽

Mammalian Cells ◽

Protein Protein Interactions

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Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

Nuclear Receptor Signaling ◽

10.1621/nrs.04021 ◽

2006 ◽

Vol 4 (1) ◽

pp. nrs.04021 ◽

Cited By ~ 14

Author(s):

Kristen L. Koterba ◽

Brian G. Rowan

Keyword(s):

Energy Transfer ◽

Protein Interactions ◽

Fusion Proteins ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Renilla Luciferase ◽

Receptor Protein ◽

Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

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Elucidating Protein-protein Interactions Through Computational Approaches and Designing Small Molecule Inhibitors Against them for Various Diseases

Current Topics in Medicinal Chemistry ◽

10.2174/1568026618666181025114903 ◽

2018 ◽

Vol 18 (20) ◽

pp. 1719-1736 ◽

Cited By ~ 3

Author(s):

Sharanya Sarkar ◽

Khushboo Gulati ◽

Manikyaprabhu Kairamkonda ◽

Amit Mishra ◽

Krishna Mohan Poluri

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Small Molecule Inhibitors ◽

Computational Techniques ◽

Protein Protein Interactions ◽

Computational Tools ◽

Computational Approaches ◽

Protein Protein Interaction ◽

Wide Range ◽

Therapeutic Measures

Background: To carry out wide range of cellular functionalities, proteins often associate with one or more proteins in a phenomenon known as Protein-Protein Interaction (PPI). Experimental and computational approaches were applied on PPIs in order to determine the interacting partners, and also to understand how an abnormality in such interactions can become the principle cause of a disease. Objective: This review aims to elucidate the case studies where PPIs involved in various human diseases have been proven or validated with computational techniques, and also to elucidate how small molecule inhibitors of PPIs have been designed computationally to act as effective therapeutic measures against certain diseases. Results: Computational techniques to predict PPIs are emerging rapidly in the modern day. They not only help in predicting new PPIs, but also generate outputs that substantiate the experimentally determined results. Moreover, computation has aided in the designing of novel inhibitor molecules disrupting the PPIs. Some of them are already being tested in the clinical trials. Conclusion: This review delineated the classification of computational tools that are essential to investigate PPIs. Furthermore, the review shed light on how indispensable computational tools have become in the field of medicine to analyze the interaction networks and to design novel inhibitors efficiently against dreadful diseases in a shorter time span.

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β-Catenin is a pH sensor with decreased stability at higher intracellular pH

The Journal of Cell Biology ◽

10.1083/jcb.201712041 ◽

2018 ◽

Vol 217 (11) ◽

pp. 3965-3976 ◽

Cited By ~ 17

Author(s):

Katharine A. White ◽

Bree K. Grillo-Hill ◽

Mario Esquivel ◽

Jobelle Peralta ◽

Vivian N. Bui ◽

...

Keyword(s):

Intracellular Ph ◽

Protein Interactions ◽

Mammalian Cells ◽

Ph Sensor ◽

Adherens Junction ◽

Protein Protein Interactions ◽

Adherens Junction Protein ◽

Junction Protein ◽

Evolutionarily Conserved ◽

Ph Dependent

β-Catenin functions as an adherens junction protein for cell–cell adhesion and as a signaling protein. β-catenin function is dependent on its stability, which is regulated by protein–protein interactions that stabilize β-catenin or target it for proteasome-mediated degradation. In this study, we show that β-catenin stability is regulated by intracellular pH (pHi) dynamics, with decreased stability at higher pHi in both mammalian cells and Drosophila melanogaster. β-Catenin degradation requires phosphorylation of N-terminal residues for recognition by the E3 ligase β-TrCP. While β-catenin phosphorylation was pH independent, higher pHi induced increased β-TrCP binding and decreased β-catenin stability. An evolutionarily conserved histidine in β-catenin (found in the β-TrCP DSGIHS destruction motif) is required for pH-dependent binding to β-TrCP. Expressing a cancer-associated H36R–β-catenin mutant in the Drosophila eye was sufficient to induce Wnt signaling and produced pronounced tumors not seen with other oncogenic β-catenin alleles. We identify pHi dynamics as a previously unrecognized regulator of β-catenin stability, functioning in coincidence with phosphorylation.

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TULP1 and TUB Are Required for Specific Localization of PRCD to Photoreceptor Outer Segments

International Journal of Molecular Sciences ◽

10.3390/ijms21228677 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8677

Author(s):

Lital Remez ◽

Ben Cohen ◽

Mariela J. Nevet ◽

Leah Rizel ◽

Tamar Ben-Yosef

Keyword(s):

Protein Interactions ◽

Mammalian Cells ◽

Outer Segment ◽

Protein Protein Interactions ◽

Photoreceptor Outer Segment ◽

Photoreceptor Outer Segments ◽

Outer Segments ◽

Yeast Two Hybrid System ◽

Specific Localization ◽

Recruitment System

Photoreceptor disc component (PRCD) is a small protein which is exclusively localized to photoreceptor outer segments, and is involved in the formation of photoreceptor outer segment discs. Mutations in PRCD are associated with retinal degeneration in humans, mice, and dogs. The purpose of this work was to identify PRCD-binding proteins in the retina. PRCD protein-protein interactions were identified when implementing the Ras recruitment system (RRS), a cytoplasmic-based yeast two-hybrid system, on a bovine retina cDNA library. An interaction between PRCD and tubby-like protein 1 (TULP1) was identified. Co-immunoprecipitation in transfected mammalian cells confirmed that PRCD interacts with TULP1, as well as with its homolog, TUB. These interactions were mediated by TULP1 and TUB highly conserved C-terminal tubby domain. PRCD localization was altered in the retinas of TULP1- and TUB-deficient mice. These results show that TULP1 and TUB, which are involved in the vesicular trafficking of several photoreceptor proteins from the inner segment to the outer segment, are also required for PRCD exclusive localization to photoreceptor outer segment discs.

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