Cellular responses to stimuli underpin discoveries in drug development, synthetic biology, and general life sciences. We introduce a library comprising 6144 synthetic promoters, each shorter than 250 bp, designed as transcriptional readouts of cellular stimulus responses in massively parallel reporter assay format. This library facilitates precise detection and amplification of transcriptional activity from our promoters, enabling the systematic development of tunable reporters with dynamic ranges of 50-100 fold.
View Article and Find Full Text PDFG-Protein Pathway Suppressor 2 (GPS2) is an inhibitor of non-proteolytic K63 ubiquitination mediated by the E2 ubiquitin-conjugating enzyme Ubc13. Previous studies have associated GPS2-mediated restriction of ubiquitination with the regulation of insulin signaling, inflammatory responses and mitochondria-nuclear communication across different tissues and cell types. However, a detailed understanding of the targets of GPS2/Ubc13 activity is lacking.
View Article and Find Full Text PDFThree proton-sensing G protein-coupled receptors (GPCRs), GPR4, GPR65, and GPR68, respond to changes in extracellular pH to regulate diverse physiology and are implicated in a wide range of diseases. A central challenge in determining how protons activate these receptors is identifying the set of residues that bind protons. Here, we determine structures of each receptor to understand the spatial arrangement of putative proton sensing residues in the active state.
View Article and Find Full Text PDFIdentifying the drug-target interactome of small molecule therapeutics is essential for understanding the full pharmacological effects of a compound. These therapies often induce changes within the cellular proteome, leading to unexpected consequences such as changes in the targets complexation state or off-target interactions between the compound and additional proteins. Currently, unbiased target-ID approaches are being used to embark on this task.
View Article and Find Full Text PDFUnderstanding how small molecules bind to specific protein complexes in living cells is critical to understanding their mechanism-of-action. Unbiased chemical biology strategies for direct readout of protein interactome remodelling by small molecules would provide advantages over target-focused approaches, including the ability to detect previously unknown ligand targets and complexes. However, there are few current methods for unbiased profiling of small molecule interactomes.
View Article and Find Full Text PDFUnbiased chemical biology strategies for direct readout of protein interactome remodelling by small molecules provide advantages over target-focused approaches, including the ability to detect previously unknown targets, and the inclusion of chemical off-compete controls leading to high-confidence identifications. We describe the BioTAC system, a small-molecule guided proximity labelling platform, to rapidly identify both direct and complexed small molecule binding proteins. The BioTAC system overcomes a limitation of current approaches, and supports identification of both inhibitor bound and molecular glue bound complexes.
View Article and Find Full Text PDFCellular transcription enables cells to adapt to various stimuli and maintain homeostasis. Transcription factors bind to transcription response elements (TREs) in gene promoters, initiating transcription. Synthetic promoters, derived from natural TREs, can be engineered to control exogenous gene expression using endogenous transcription machinery.
View Article and Find Full Text PDFObjective: Dysfunctional, unhealthy expansion of white adipose tissue due to excess dietary intake is a process at the root of obesity and Type 2 Diabetes development. The objective of this study is to contribute to a better understanding of the underlying mechanism(s) regulating the early stages of adipose tissue expansion and adaptation to dietary stress due to an acute, high-fat diet (HFD) challenge, with a focus on the communication between adipocytes and other stromal cells.
Methods: We profiled the early response to high-fat diet exposure in wildtype and adipocyte-specific GPS2-KO (GPS2-AKO) mice at the cellular, tissue and organismal level.
Enzymatic and cellular signalling biosensors are used to decipher the activities of complex biological systems. Biosensors for monitoring G protein-coupled receptors (GPCRs), the most drugged class of proteins in the human body, are plentiful and vary in utility, form and function. Their applications have continually expanded our understanding of this important protein class.
View Article and Find Full Text PDFDeep mutational scanning provides new insights into how mutations alter the expression and activity of the potassium ion channel Kir2.1, which is associated with many diseases.
View Article and Find Full Text PDFADP-ribosylation is a reversible post-translational modification where an ADP-ribose moiety is covalently attached to target proteins by ADP-ribosyltransferases (ARTs). Although best known for its nuclear roles, ADP-ribosylation is increasingly recognized as a key regulatory strategy across cellular compartments. ADP-ribosylation of mitochondrial proteins has been widely reported, but the exact nature of mitochondrial ART enzymes is debated.
View Article and Find Full Text PDFThe MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions. MRGPRX2 couples to both G and G in mast cells.
View Article and Find Full Text PDFCellular homeostasis in eukaryotic cells requires synchronized coordination of multiple organelles. A key role in this stage is played by mitochondria, which have recently emerged as highly interconnected and multifunctional hubs that process and coordinate diverse cellular functions. Beyond producing ATP, mitochondria generate key metabolites and are central to apoptotic and metabolic signaling pathways.
View Article and Find Full Text PDFThe chemogenetic technology designer receptors exclusively activated by designer drugs (DREADDs) afford remotely reversible control of cellular signaling, neuronal activity and behavior. Although the combination of muscarinic-based DREADDs with clozapine-N-oxide (CNO) has been widely used, sluggish kinetics, metabolic liabilities and potential off-target effects of CNO represent areas for improvement. Here, we provide a new high-affinity and selective agonist deschloroclozapine (DCZ) for muscarinic-based DREADDs.
View Article and Find Full Text PDFG-protein-coupled receptors (GPCRs) remain major drug targets, despite our incomplete understanding of how they signal through 16 non-visual G-protein signal transducers (collectively named the transducerome) to exert their actions. To address this gap, we have developed an open-source suite of 14 optimized bioluminescence resonance energy transfer (BRET) Gαβγ biosensors (named TRUPATH) to interrogate the transducerome with single pathway resolution in cells. Generated through exhaustive protein engineering and empirical testing, the TRUPATH suite of Gαβγ biosensors includes the first Gα15 and GαGustducin probes.
View Article and Find Full Text PDFRecent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state.
View Article and Find Full Text PDFDirected evolution, artificial selection toward designed objectives, is routinely used to develop new molecular tools and therapeutics. Successful directed molecular evolution campaigns repeatedly test diverse sequences with a designed selective pressure. Unicellular organisms and their viral pathogens are exceptional for this purpose and have been used for decades.
View Article and Find Full Text PDFBackground: Psoriasis (PSO) is an immune-mediated inflammatory disease associated with metabolic and cardiovascular comorbidities. It is now known that resolution of inflammation is an active process locally controlled by specialized proresolving mediators (SPMs), named resolvins (Rvs), protectins, and maresins.
Objective: It is unknown whether these potent lipid mediators (LMs) are involved in PSO pathophysiology and if the skin and blood have disease-specific SPMs phenotype profiles.
Prostaglandins Leukot Essent Fatty Acids
February 2017
Specialized pro-resolving mediators (SPM), e.g. Resolvin D1, Protectin D1, Lipoxin A₄, and Resolvin E1 have each shown to be active in ocular models reducing inflammation.
View Article and Find Full Text PDFWith early life likely to have existed in a hot environment, enzymes had to cope with an inherent drop in catalytic speed caused by lowered temperature. Here we characterize the molecular mechanisms underlying thermoadaptation of enzyme catalysis in adenylate kinase using ancestral sequence reconstruction spanning 3 billion years of evolution. We show that evolution solved the enzyme's key kinetic obstacle-how to maintain catalytic speed on a cooler Earth-by exploiting transition-state heat capacity.
View Article and Find Full Text PDFNeurologic disorders are frequently a result of inappropriate electrical and/or chemical signaling of neurons and glia. Ultimate remediation would necessitate reprogramming these signals. Historically, correcting neuronal and glial signaling is accomplished via drug therapy/administration, although they frequently fail to effectively and fully treat the underlying disorder.
View Article and Find Full Text PDFLarge wildfires of increasing frequency and severity threaten local populations and natural resources and contribute carbon emissions into the earth-climate system. Although wildfires have been researched and modeled for decades, no verifiable physical theory of spread is available to form the basis for the precise predictions needed to manage fires more effectively and reduce their environmental, economic, ecological, and climate impacts. Here, we report new experiments conducted at multiple scales that appear to reveal how wildfire spread derives from the tight coupling between flame dynamics induced by buoyancy and fine-particle response to convection.
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