Building synthetic cellular organization

AbstractThe second messenger cyclic di-GMP regulates a variety of processes in bacteria, many of which are centered around the decision whether to adopt a sessile or a motile life style. Regulatory circuits include pathogenicity, biofilm formation, and motility in a wide variety of bacteria, and play a key role in cell cycle progression in Caulobacter crescentus. Interestingly, multiple, seemingly independent c-di-GMP pathways have been found in several species, where deletions of individual c-di-GMP synthetases (DGCs) or hydrolases (PDEs) have resulted in distinct phenotypes that would not be expected based on a freely diffusible second messenger. Several recent studies have shown that individual signaling nodes exist, and additionally, that protein/protein interactions between DGCs, PDEs and c-di-GMP receptors play an important role in signaling specificity. Additionally, subcellular clustering has been shown to be employed by bacteria to likely generate local signaling of second messenger, and/or to increase signaling specificity. This review highlights recent findings that reveal how bacteria employ spatial cues to increase the versatility of second messenger signaling.

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

10.1101/651364 ◽

2019 ◽

Cited By ~ 1

Author(s):

Jennifer A. Miles ◽

Fruzsina Hobor ◽

James Taylor ◽

Christian Tiede ◽

Philip R. Rowell ◽

...

Keyword(s):

Protein Interactions ◽

Cell Biology ◽

Competitive Inhibition ◽

Antibody Binding ◽

Proof Of Concept ◽

Protein Protein Interactions ◽

The Family ◽

Selective Ligands ◽

Binding Cleft ◽

Characterisation Methods

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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Development of a Split Esterase 1 for Protein-Protein 2 Interaction Dependent Small Molecule Activation

10.26434/chemrxiv.8258633.v1 ◽

2019 ◽

Author(s):

Krysten A. Jones ◽

Kaitlin Kentala ◽

Michael Beck ◽

Weiwei An ◽

Alexander Lippert ◽

...

Keyword(s):

Synthetic Biology ◽

Small Molecule ◽

Protein Interactions ◽

Simultaneous Detection ◽

Cell Killing ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Biological Studies ◽

Cytotoxic Molecule ◽

Measured Input

Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small molecule-based imaging agents are widely used in biological studies, and small molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure a protein-protein interactions and protein-protein interaction inhibitor engagement. We demonstrate the esterase-based reporter system is compatible with other commonly-used split reporter imaging systems for the simultaneous detection of multiple protein-protein interactions. Finally, we develop a system for selective small molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications.

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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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Development of a Split Esterase for Protein-Protein Interaction Dependent Small Molecule Activation

10.26434/chemrxiv.8258633 ◽

2019 ◽

Cited By ~ 1

Author(s):

Krysten A. Jones ◽

Kaitlin Kentala ◽

Michael Beck ◽

Weiwei An ◽

Alexander Lippert ◽

...

Keyword(s):

Synthetic Biology ◽

Small Molecule ◽

Protein Interaction ◽

Protein Interactions ◽

Cell Killing ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Protein Protein Interaction ◽

Cytotoxic Molecule ◽

Measured Input

Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small molecule-based imaging agents are widely used in biological studies, and small molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure a protein-protein interactions and protein-protein interaction inhibitor engagement. We demonstrate the esterase-based reporter system is compatible with other commonly-used split reporter imaging systems for the simultaneous detection of multiple protein-protein interactions. Finally, we develop a system for selective small molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications.

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Discovery of lipid-mediated protein-protein interactions in living cells using metabolic labeling with photoactivatable clickable probes

10.1101/2021.12.15.472799 ◽

2021 ◽

Author(s):

Roman O Fedoryshchak ◽

Andrii Gorelik ◽

Mengjie Shen ◽

Maria M Shchepinova ◽

Inmaculada Perez-Dorado ◽

...

Keyword(s):

Protein Interactions ◽

Cell Biology ◽

Regulatory Elements ◽

Metabolic Labeling ◽

Protein Protein Interactions ◽

Post Translational Modifications ◽

X Ray ◽

Design And Synthesis ◽

Human Proteins

Protein-protein interactions (PPIs) are essential and pervasive regulatory elements in cell biology. Despite development of a range of techniques to probe PPIs in living systems, there is a dearth of approaches to capture interactions driven by specific post-translational modifications (PTMs). Myristoylation is a lipid PTM added to more than 200 human proteins, where it may regulate membrane localization, stability or activity. Here we report design and synthesis of a panel of novel photocrosslinkable and clickable myristic acid analog probes, and their characterization as efficient substrates for human N myristoyltransferases NMT1 and NMT2, both biochemically and through X-ray co-crystallography. We demonstrate metabolic incorporation of probes to label NMT substrates in cell culture and in situ intracellular photoactivation to form a covalent crosslink between modified proteins and their interactors, capturing a snapshot of interactions driven by the presence of the lipid PTM. Proteomic analyses revealed both known and multiple novel interactors of a series of myristoylated proteins, including ferroptosis suppressor protein FSP1 and spliceosome-associated RNA helicase DDX46. The concept exemplified by these probes offers an efficient approach for exploring the PTM-specific interactome, which may prove broadly applicable to other PTMs.

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Computational Methods for Prediction of Protein-Protein Interactions

Materials Science and Engineering ◽

10.4018/978-1-5225-1798-6.ch012 ◽

2017 ◽

pp. 309-340

Author(s):

Sneha Rai ◽

Sonika Bhatnagar

Keyword(s):

Network Topology ◽

Protein Interactions ◽

Biological Significance ◽

Experimental Methods ◽

Ease Of Use ◽

Protein Protein Interactions ◽

Cellular Processes ◽

Large Numbers ◽

Transient Interactions ◽

Ppi Prediction

The key signaling pathways in cellular processes involve protein-protein interactions (PPIs). A perturbation in the balance of PPIs occurs in various pathophysiological processes. There are a large numbers of experimental methods for detection of PPIs. However, experimental PPI determination is time consuming, expensive, error prone and does not effectively cover transient interactions. Therefore, overlaying and integration of predictive methods with experimental results provides statistical robustness and biological significance to the PPI data. In this chapter, the authors describe PPIs in terms of types, importance, and biological consequences. This chapter also provides a comprehensive description on various computational approaches for PPI prediction. Prediction of PPI can be done through: 1) Genomic information based methods 2) Structure based methods 3) Network topology based methods: 4) Literature and data mining based methods 5) Machine learning methods. For ease of use and convenience, a summary of various databases and software for PPI prediction has been provided.

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