ABSTRACT
Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of ß-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.
Subject(s)
Adipose Tissue, Brown/metabolism , Constitutive Androstane Receptor/metabolism , Lipolysis , Receptors, G-Protein-Coupled/metabolism , Thermogenesis , Adipocytes/metabolism , Animals , COS Cells , Cells, Cultured , Chlorocebus aethiops , Cold Temperature , Dietary Fats/pharmacology , Humans , Mice, Inbred C57BL , Phenotype , Receptors, G-Protein-Coupled/genetics , Signal Transduction , Sympathetic Nervous System/metabolism , Transcription, GeneticABSTRACT
The control over the extent and timing of G protein signaling is provided by the regulator of G protein signaling (RGS) proteins that deactivate G protein α subunits (Gα). Mammalian genomes encode 20 canonical RGS and 16 Gα genes with key roles in physiology and disease. To understand the principles governing the selectivity of Gα regulation by RGS, we examine the catalytic activity of all canonical human RGS proteins and their selectivity for a complete set of Gα substrates using real-time kinetic measurements in living cells. The data reveal rules governing RGS-Gα recognition, the structural basis of its selectivity, and provide principles for engineering RGS proteins with defined selectivity. The study also explores the evolution of RGS-Gα selectivity through ancestral reconstruction and demonstrates how naturally occurring non-synonymous variants in RGS alter signaling. These results provide a blueprint for decoding signaling selectivity and advance our understanding of molecular recognition principles.
Subject(s)
GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein alpha Subunits/physiology , RGS Proteins/genetics , Animals , Female , GTP-Binding Protein Regulators/metabolism , GTP-Binding Protein alpha Subunits/genetics , HEK293 Cells , Humans , Kinetics , Male , Mice , Mice, Inbred C57BL , Neurons/metabolism , Primary Cell Culture , Protein Binding , RGS Proteins/metabolism , RGS Proteins/physiology , Signal Transduction/geneticsABSTRACT
Although there is much focus on the impact of mutations on structured protein domains, less is known about their impact on unstructured regions. In this issue, Meyer et al. demonstrate that mutations resulting in the emergence of new short linear peptide motifs within intrinsically disordered protein regions can cause human genetic diseases by gain of function.
Subject(s)
Gain of Function Mutation , Peptides , Humans , MutationABSTRACT
Natural genetic variation in the human genome is a cause of individual differences in responses to medications and is an underappreciated burden on public health. Although 108 G-protein-coupled receptors (GPCRs) are the targets of 475 (â¼34%) Food and Drug Administration (FDA)-approved drugs and account for a global sales volume of over 180 billion US dollars annually, the prevalence of genetic variation among GPCRs targeted by drugs is unknown. By analyzing data from 68,496 individuals, we find that GPCRs targeted by drugs show genetic variation within functional regions such as drug- and effector-binding sites in the human population. We experimentally show that certain variants of µ-opioid and Cholecystokinin-A receptors could lead to altered or adverse drug response. By analyzing UK National Health Service drug prescription and sales data, we suggest that characterizing GPCR variants could increase prescription precision, improving patients' quality of life, and relieve the economic and societal burden due to variable drug responsiveness. VIDEO ABSTRACT.
Subject(s)
Pharmacogenetics/methods , Pharmacogenomic Variants , Receptors, G-Protein-Coupled/genetics , Software , Binding Sites , Drug Prescriptions/standards , HEK293 Cells , Humans , Protein Binding , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolismABSTRACT
In his commentary in this issue of Molecular Cell,1 Struhl reasons that the term "intrinsically disordered regions" represents a vague and confusing concept for protein function. However, the term "intrinsically disordered" highlights the important physicochemical characteristic of conformational heterogeneity. Thus, "intrinsically disordered" is the counterpart to the term "folded, " with neither term having specific functional implications.
Subject(s)
Intrinsically Disordered Proteins , Intrinsically Disordered Proteins/metabolism , Protein ConformationABSTRACT
G protein-coupled receptors (GPCRs) are the largest group of cell surface receptors in humans that signal in response to diverse inputs and regulate a plethora of cellular processes. Hence, they constitute one of the primary drug target classes. Progress in our understanding of GPCR dynamics, activation and signalling has opened new possibilities for selective drug development. A key advancement has been provided by the concept of biased agonism, which describes the ability of ligands acting at the same GPCR to elicit distinct cellular signalling profiles by preferentially stabilizing different active conformational states of the receptor. Application of this concept raises the prospect of 'designer' biased agonists as optimized therapeutics with improved efficacy and/or reduced side-effect profiles. However, this application will require a detailed understanding of the spectrum of drug actions and a structural understanding of the drug-receptor interactions that drive distinct pharmacologies. The recent revolution in GPCR structural biology provides unprecedented insights into ligand binding, conformational dynamics and the control of signalling outcomes. These insights, together with new approaches to multi-dimensional analysis of drug action, are allowing refined classification of drugs according to their pharmacodynamic profiles, which can be linked to receptor structure and predictions of preclinical drug efficacy.
Subject(s)
Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/physiology , Animals , Humans , Ligands , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
G-protein-coupled receptors (GPCRs) are membrane proteins that modulate physiology across human tissues in response to extracellular signals. GPCR-mediated signalling can differ because of changes in the sequence1,2 or expression3 of the receptors, leading to signalling bias when comparing diverse physiological systems4. An underexplored source of such bias is the generation of functionally diverse GPCR isoforms with different patterns of expression across different tissues. Here we integrate data from human tissue-level transcriptomes, GPCR sequences and structures, proteomics, single-cell transcriptomics, population-wide genetic association studies and pharmacological experiments. We show how a single GPCR gene can diversify into several isoforms with distinct signalling properties, and how unique isoform combinations expressed in different tissues can generate distinct signalling states. Depending on their structural changes and expression patterns, some of the detected isoforms may influence cellular responses to drugs and represent new targets for developing drugs with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling that considers how combinatorial expression of isoforms in a particular cell type, tissue or organism collectively influences receptor signalling and drug responses.
Subject(s)
Protein Isoforms/chemistry , Protein Isoforms/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/drug effects , Transcriptome , Databases, Factual , Gene Expression Profiling , HEK293 Cells , Humans , Molecular Targeted Therapy , Organ Specificity/drug effects , Protein Isoforms/genetics , Proteomics , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/genetics , Signal Transduction/genetics , Single-Cell AnalysisABSTRACT
In this Editorial, our Chief Editor and members of our Advisory Editorial Board discuss recent breakthroughs, current challenges, and emerging opportunities in single-cell biology and share their vision of "where the field is headed."
ABSTRACT
Gene fusions are common cancer-causing mutations, but the molecular principles by which fusion protein products affect interaction networks and cause disease are not well understood. Here, we perform an integrative analysis of the structural, interactomic, and regulatory properties of thousands of putative fusion proteins. We demonstrate that genes that form fusions (i.e., parent genes) tend to be highly connected hub genes, whose protein products are enriched in structured and disordered interaction-mediating features. Fusion often results in the loss of these parental features and the depletion of regulatory sites such as post-translational modifications. Fusion products disproportionately connect proteins that did not previously interact in the protein interaction network. In this manner, fusion products can escape cellular regulation and constitutively rewire protein interaction networks. We suggest that the deregulation of central, interaction-prone proteins may represent a widespread mechanism by which fusion proteins alter the topology of cellular signaling pathways and promote cancer.
Subject(s)
Gene Fusion , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Protein Interaction Maps , Computational Biology , Databases, Protein , Humans , Protein Interaction Mapping , Protein Processing, Post-Translational , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , UbiquitinationABSTRACT
The selective coupling of G-protein-coupled receptors (GPCRs) to specific G proteins is critical to trigger the appropriate physiological response. However, the determinants of selective binding have remained elusive. Here we reveal the existence of a selectivity barcode (that is, patterns of amino acids) on each of the 16 human G proteins that is recognized by distinct regions on the approximately 800 human receptors. Although universally conserved positions in the barcode allow the receptors to bind and activate G proteins in a similar manner, different receptors recognize the unique positions of the G-protein barcode through distinct residues, like multiple keys (receptors) opening the same lock (G protein) using non-identical cuts. Considering the evolutionary history of GPCRs allows the identification of these selectivity-determining residues. These findings lay the foundation for understanding the molecular basis of coupling selectivity within individual receptors and G proteins.
Subject(s)
Heterotrimeric GTP-Binding Proteins/chemistry , Heterotrimeric GTP-Binding Proteins/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Amino Acid Sequence , Binding Sites , Evolution, Molecular , Humans , Internet , Models, Molecular , Protein Binding , Protein Conformation , Substrate Specificity , User-Computer InterfaceABSTRACT
Excessive expansions of glutamine (Q)-rich repeats in various human proteins are known to result in severe neurodegenerative disorders such as Huntington's disease and several ataxias. However, the physiological role of these repeats and the consequences of more moderate repeat variation remain unknown. Here, we demonstrate that Q-rich domains are highly enriched in eukaryotic transcription factors where they act as functional modulators. Incremental changes in the number of repeats in the yeast transcriptional regulator Ssn6 (Cyc8) result in systematic, repeat-length-dependent variation in expression of target genes that result in direct phenotypic changes. The function of Ssn6 increases with its repeat number until a certain threshold where further expansion leads to aggregation. Quantitative proteomic analysis reveals that the Ssn6 repeats affect its solubility and interactions with Tup1 and other regulators. Thus, Q-rich repeats are dynamic functional domains that modulate a regulator's innate function, with the inherent risk of pathogenic repeat expansions.
Subject(s)
Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Base Sequence , DNA, Fungal/genetics , Gene Expression Regulation, Fungal , Glutamine/chemistry , Molecular Sequence Data , Protein Interaction Domains and Motifs , Protein Stability , Repetitive Sequences, Amino Acid , Repressor Proteins/chemistry , Repressor Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , SolubilityABSTRACT
Genes and gene products interact with each other to form signal transduction networks in the cell. The interactome networks are under intricate regulation in physiological conditions, but could go awry upon genome instability caused by genetic mutations. In the past decade with next-generation sequencing technologies, an increasing number of genomic mutations have been identified in a variety of disease patients and healthy individuals. As functional and systematic studies on these mutations leap forward, they begin to reveal insights into cellular homeostasis and disease mechanisms. In this review, we discuss recent advances in the field of network biology and signaling pathway perturbations upon genomic changes, and highlight the success of various omics datasets in unraveling genotype-to-phenotype relationships.
Subject(s)
Genotype , Phenotype , Signal Transduction/genetics , Animals , Gene Regulatory Networks , HumansABSTRACT
Alternative splicing can expand the diversity of proteomes. Homologous mutually exclusive exons (MXEs) originate from the same ancestral exon and result in polypeptides with similar structural properties but altered sequence. Why would some genes switch homologous exons and what are their biological impact? Here, we analyse the extent of sequence, structural and functional variability in MXEs and report the first large scale, structure-based analysis of the biological impact of MXE events from different genomes. MXE-specific residues tend to map to single domains, are highly enriched in surface exposed residues and cluster at or near protein functional sites. Thus, MXE events are likely to maintain the protein fold, but alter specificity and selectivity of protein function. This comprehensive resource of MXE events and their annotations is available at: http://gene3d.biochem.ucl.ac.uk/mxemod/. These findings highlight how small, but significant changes at critical positions on a protein surface are exploited in evolution to alter function.
Subject(s)
Alternative Splicing/genetics , Exons/genetics , Genome/genetics , Proteins , Animals , Evolution, Molecular , Genomics , Humans , Proteins/genetics , Proteins/physiologyABSTRACT
Class A G-protein-coupled receptors (GPCRs) are a large family of membrane proteins that mediate a wide variety of physiological functions, including vision, neurotransmission and immune responses. They are the targets of nearly one-third of all prescribed medicinal drugs such as beta blockers and antipsychotics. GPCR activation is facilitated by extracellular ligands and leads to the recruitment of intracellular G proteins. Structural rearrangements of residue contacts in the transmembrane domain serve as 'activation pathways' that connect the ligand-binding pocket to the G-protein-coupling region within the receptor. In order to investigate the similarities in activation pathways across class A GPCRs, we analysed 27 GPCRs from diverse subgroups for which structures of active, inactive or both states were available. Here we show that, despite the diversity in activation pathways between receptors, the pathways converge near the G-protein-coupling region. This convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices 3, 6 and 7 that releases G-protein-contacting residues. The convergence of activation pathways may explain how the activation steps initiated by diverse ligands enable GPCRs to bind a common repertoire of G proteins.
Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Binding Sites , Conserved Sequence , Humans , Ligands , Models, Molecular , Protein Structure, Secondary , Receptors, G-Protein-Coupled/classification , Receptors, G-Protein-Coupled/genetics , Receptors, Vasopressin/chemistry , Receptors, Vasopressin/genetics , Receptors, Vasopressin/metabolism , Signal Transduction , Structural Homology, ProteinABSTRACT
A molecular description of functional modules in the cell is the focus of many high-throughput studies in the postgenomic era. A large portion of biomolecular interactions in virtually all cellular processes is mediated by compact interaction modules, referred to as peptide motifs. Such motifs are typically less than ten residues in length, occur within intrinsically disordered regions, and are recognized and/or posttranslationally modified by structured domains of the interacting partner. In this review, we suggest that there might be over a million instances of peptide motifs in the human proteome. While this staggering number suggests that peptide motifs are numerous and the most understudied functional module in the cell, it also holds great opportunities for new discoveries.
Subject(s)
Proteins/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Humans , Molecular Biology , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs , Protein Processing, Post-TranslationalABSTRACT
We report a bioinformatic workflow and subsequent discovery of a new polyethylene terephthalate (PET) hydrolase, which we named MG8, from the human saliva metagenome. MG8 has robust PET plastic degradation activities under different temperature and salinity conditions, outperforming several naturally occurring and engineered hydrolases in degrading PET. Moreover, we genetically encoded 2,3-diaminopropionic acid (DAP) in place of the catalytic serine residue of MG8, thereby converting a PET hydrolase into a covalent binder for bio-functionalization of PET. We show that MG8(DAP), in conjunction with a split green fluorescent protein system, can be used to attach protein cargos to PET as well as other polyester plastics. The discovery of a highly active PET hydrolase from the human metagenome-currently an underexplored resource for industrial enzyme discovery-as well as the repurposing of such an enzyme into a plastic functionalization tool, should facilitate ongoing efforts to degrade and maximize reusability of PET.
Subject(s)
Hydrolases , Polyethylene Terephthalates , Genetic Code , Humans , Hydrolases/metabolism , Metagenome , Plastics/chemistry , Polyethylene Terephthalates/chemistry , Saliva/metabolismABSTRACT
Intrinsically disordered proteins (IDPs) can protect cells from diverse stresses by forming higher order assemblies such as reversible aggregates or granules. Recently, Boothby et al. show that IDPs protect tardigrades against desiccation by forming a glass-like amorphous matrix, highlighting that material properties of disordered proteins can confer adaptation during stress.
Subject(s)
Intrinsically Disordered Proteins/metabolism , Stress, PhysiologicalABSTRACT
G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are â¼800 human GPCRs and 16 different Gα genes, this raises the question of whether a universal allosteric mechanism governs Gα activation. Here we show that different GPCRs interact with and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR-Gα system diversified rapidly, while conserving the allosteric activation mechanism.
Subject(s)
Allosteric Regulation , Evolution, Molecular , GTP-Binding Protein alpha Subunits/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Binding Sites , Computational Biology , Conserved Sequence , Enzyme Activation , GTP-Binding Protein alpha Subunits/chemistry , GTP-Binding Protein alpha Subunits/genetics , Genetic Engineering , Guanosine Diphosphate/metabolism , Humans , Models, Molecular , Mutation , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, G-Protein-Coupled/chemistry , Signal Transduction , Substrate Specificity , ras Proteins/chemistry , ras Proteins/metabolismABSTRACT
Messenger RNA (mRNA) export from the nucleus is essential for eukaryotic gene expression. Here we identify a transcript-selective nuclear export mechanism affecting certain human transcripts, enriched for functions in genome duplication and repair, controlled by inositol polyphosphate multikinase (IPMK), an enzyme catalyzing inositol polyphosphate and phosphoinositide turnover. We studied transcripts encoding RAD51, a protein essential for DNA repair by homologous recombination (HR), to characterize the mechanism underlying IPMK-regulated mRNA export. IPMK depletion or catalytic inactivation selectively decreases RAD51 protein abundance and the nuclear export of RAD51 mRNA, thereby impairing HR. Recognition of a sequence motif in the untranslated region of RAD51 transcripts by the mRNA export factor ALY requires IPMK. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), an IPMK product, restores ALY recognition in IPMK-depleted cell extracts, suggesting a mechanism underlying transcript selection. Our findings implicate IPMK in a transcript-selective mRNA export pathway controlled by phosphoinositide turnover that preserves genome integrity in humans.