Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of ...unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.
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► A biotinylated small molecule precipitates RNA granule proteins from cell lysates ► Low complexity sequences in these proteins form hydrogels ► Amyloid-like fibers within the gels can trap LCS domains from other proteins ► The cell-free in vitro reactions model RNA granule architecture and formation
RNA-binding proteins with regions of low complexity sequence can form hydrogels in vitro comprised of amyloid-like fibers either via nucleation by a small molecule or by self-organization. Unlike pathologic amyloids, the fibers are dynamic and can incorporate low complexity domains from different proteins, suggesting a basis for assembly of RNA granules within cells.
Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic processes, including autophagy, and aberrant activation of such receptors is a common ...feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated VPS34 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors and restores autophagy in non-small-cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance.
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•EGFR negatively regulates autophagy by binding to Beclin 1•Active EGFR phosphorylates Beclin 1 and alters its interactome•EGFR suppression of Beclin 1 may contribute to tumor progression in lung cancer•Lung cancer responses to EGFR inhibitors may involve activation of Beclin 1
The oncogenic receptor tyrosine kinase EGFR regulates the core autophagy machinery by phosphorylating the autophagy protein Beclin 1. This regulation may contribute to the progression and chemoresistance of non-small-cell lung carcinoma with active EGFR mutations.
Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information ...facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or "fold"). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies.
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•The DeepSAV predictor of SAV functional impact was updated and improved.•The diversity of multiple sequence alignment is an important factor in DeepSAV performance.•DBSAV provides ...DeepSAV scores for human SAVs and GTS scores for human genes.•DBSAV is a valuable resource for mechanistic interpretation of human SAVs.
Deleterious single amino acid variation (SAV) is one of the leading causes of human diseases. Evaluating the functional impact of SAVs is crucial for diagnosis of genetic disorders. We previously developed a deep convolutional neural network predictor, DeepSAV, to evaluate the deleterious effects of SAVs on protein function based on various sequence, structural, and functional properties. DeepSAV scores of rare SAVs observed in the human population are aggregated into a gene-level score called GTS (Gene Tolerance of rare SAVs) that reflects a gene's tolerance to deleterious missense mutations and serves as a useful tool to study gene-disease associations. In this study, we aim to enhance the performance of DeepSAV by using expanded datasets of pathogenic and benign variants, more features, and neural network optimization. We found that multiple sequence alignments built from vertebrate-level orthologs yield better prediction results compared to those built from mammalian-level orthologs. For multiple sequence alignments built from BLAST searches, optimal performance was achieved with a sequence identify cutoff of 50% to remove distant homologs. The new version of DeepSAV exhibits the best performance among standalone predictors of deleterious effects of SAVs. We developed the DBSAV database (http://prodata.swmed.edu/DBSAV) that reports GTS scores of human genes and DeepSAV scores of SAVs in the human proteome, including pathogenic and benign SAVs, population-level SAVs, and all possible SAVs by single nucleotide variations. This database serves as a useful resource for research of human SAVs and their relationships with protein functions and human diseases.
The WAVE regulatory complex (WRC) controls actin cytoskeletal dynamics throughout the cell by stimulating the actin-nucleating activity of the Arp2/3 complex at distinct membrane sites. However, the ...factors that recruit the WRC to specific locations remain poorly understood. Here, we have identified a large family of potential WRC ligands, consisting of ∼120 diverse membrane proteins, including protocadherins, ROBOs, netrin receptors, neuroligins, GPCRs, and channels. Structural, biochemical, and cellular studies reveal that a sequence motif that defines these ligands binds to a highly conserved interaction surface of the WRC formed by the Sra and Abi subunits. Mutating this binding surface in flies resulted in defects in actin cytoskeletal organization and egg morphology during oogenesis, leading to female sterility. Our findings directly link diverse membrane proteins to the WRC and actin cytoskeleton and have broad physiological and pathological ramifications in metazoans.
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•Many potential WRC ligands defined by a peptide motif (WIRS) were identified•Motif binds to a conserved WRC surface formed by Sra and Abi subunits•WIRS/WRC interaction regulates oogenesis in flies
A short peptide motif that binds to a conserved surface of the WAVE regulatory complex (WRC) has been identified in a large family of diverse membrane proteins. This interaction recruits the WRC to membranes, regulates the actin cytoskeleton, and is important during Drosophila development.
During virus infection, the adaptor proteins MAVS and STING transduce signals from the cytosolic nucleic acid sensors RIG-I and cGAS, respectively, to induce type I interferons (IFNs) and other ...antiviral molecules. Here we show that MAVS and STING harbor two conserved serine and threonine clusters that are phosphorylated by the kinases IKK and/or TBK1 in response to stimulation. Phosphorylated MAVS and STING then bind to a positively charged surface of interferon regulatory factor 3 (IRF3) and thereby recruit IRF3 for its phosphorylation and activation by TBK1. We further show that TRIF, an adaptor protein in Toll-like receptor signaling, activates IRF3 through a similar phosphorylation-dependent mechanism. These results reveal that phosphorylation of innate adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate the type I IFN pathway.
RIG-I and MDA5 detect viral RNA in the cytoplasm and activate signaling cascades leading to the production of type-I interferons. RIG-I is activated through sequential binding of viral RNA and ...unanchored lysine-63 (K63) polyubiquitin chains, but how polyubiquitin activates RIG-I and whether MDA5 is activated through a similar mechanism remain unresolved. Here, we showed that the CARD domains of MDA5 bound to K63 polyubiquitin and that this binding was essential for MDA5 to activate the transcription factor IRF3. Mutations of conserved residues in MDA5 and RIG-I that disrupt their ubiquitin binding also abrogated their ability to activate IRF3. Polyubiquitin binding induced the formation of a large complex consisting of four RIG-I and four ubiquitin chains. This hetero-tetrameric complex was highly potent in activating the antiviral signaling cascades. These results suggest a unified mechanism of RIG-I and MDA5 activation and reveal a unique mechanism by which ubiquitin regulates cell signaling and immune response.
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► Like RIG-I, MDA5 activates IRF3 in a cell-free system ► Both RIG-I and MDA5 CARD domains bind K63 polyubiquitin chains and activate IRF3 ► Polyubiquitin binding is required for the activation of RIG-I and MDA5 ► Polyubiquitin binding induces the formation of a highly active RIG-I tetramer
Rossmann folds are ancient, frequently diverged domains found in many biological reaction pathways where they have adapted for different functions. Consequently, discernment and classification of ...their homologous relations and function can be complicated. We define a minimal Rossmann-like structure motif (RLM) that corresponds for the common core of known Rossmann domains and use this motif to identify all RLM domains in the Protein Data Bank (PDB), thus finding they constitute about 20% of all known 3D structures. The Evolutionary Classification of protein structure Domains (ECOD) classifies RLM domains in a number of groups that lack evidence for homology (X-groups), which suggests that they could have evolved independently multiple times. Closely related, homologous RLM enzyme families can diverge to bind different ligands using similar binding sites and to catalyze different reactions. Conversely, non-homologous RLM domains can converge to catalyze the same reactions or to bind the same ligand with alternate binding modes. We discuss a special case of such convergent evolution that is relevant to the polypharmacology paradigm, wherein the same drug (methotrexate) binds to multiple non-homologous RLM drug targets with different topologies. Finally, assigning proteins with RLM domain to the Enzyme Commission classification suggest that RLM enzymes function mainly in metabolism (and comprise 38% of reference metabolic pathways) and are overrepresented in extant pathways that represent ancient biosynthetic routes such as nucleotide metabolism, energy metabolism, and metabolism of amino acids. In fact, RLM enzymes take part in five out of eight enzymatic reactions of the Wood-Ljungdahl metabolic pathway thought to be used by the last universal common ancestor (LUCA). The prevalence of RLM domains in this ancient metabolism might explain their wide distribution among enzymes.
De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we ...find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly smaller autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.
Fumarate hydratase (FH) is a tumor suppressor, but how it acts is unclear. Two reports in this issue of Cancer Cell reveal that FH deficiency leads to succination of Keap1, stabilization of Nrf2, and ...induction of stress-response genes including HMOX1, which is important for the survival of FH-deficient cells.
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