Protein AMPylation by an Evolutionarily Conserved Pseudokinase.

Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities.

The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein.

Sexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa-animals, plants, and protists (including important human pathogens like Plasmodium)-suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life.

A Single Kinase Generates the Majority of the Secreted Phosphoproteome.

The existence of extracellular phosphoproteins has been acknowledged for over a century. However, research in this area has been undeveloped largely because the kinases that phosphorylate secreted proteins have escaped identification. Fam20C is a kinase that phosphorylates S-x-E/pS motifs on proteins in milk and in the extracellular matrix of bones and teeth. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing, mass spectrometry, and biochemistry, we identify more than 100 secreted phosphoproteins as genuine Fam20C substrates. Further, we show that Fam20C exhibits broader substrate specificity than previously appreciated. Functional annotations of Fam20C substrates suggest roles for the kinase beyond biomineralization, including lipid homeostasis, wound healing, and cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.

The WAVE regulatory complex links diverse receptors to the actin cytoskeleton.

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.

EGFR-mediated Beclin 1 phosphorylation in autophagy suppression, tumor progression, and tumor chemoresistance.

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.

Beclin 2 functions in autophagy, degradation of G protein-coupled receptors, and metabolism.

The molecular mechanism of autophagy and its relationship to other lysosomal degradation pathways remain incompletely understood. Here, we identified a previously uncharacterized mammalian-specific protein, Beclin 2, which, like Beclin 1, functions in autophagy and interacts with class III PI3K complex components and Bcl-2. However, Beclin 2, but not Beclin 1, functions in an additional lysosomal degradation pathway. Beclin 2 is required for ligand-induced endolysosomal degradation of several G protein-coupled receptors (GPCRs) through its interaction with GASP1. Beclin 2 homozygous knockout mice have decreased embryonic viability, and heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, and obesity and insulin resistance. Our findings identify Beclin 2 as a converging regulator of autophagy and GPCR turnover and highlight the functional and mechanistic diversity of Beclin family members in autophagy, endolysosomal trafficking, and metabolism.

Membrane protein structure predictions for exploration.

A daring experiment is performed. Using sequence alignments to predict contacts between residues in protein spatial structures, Hopf et al. are publishing untested de novo structure models for 11 transmembrane protein families. Will their models stand the test of time and hold up to experimentation? The prospects are excellent.

Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.

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.

What one genus of showy moths can say about migration, adaptation, and wing pattern.

The Ornate Moth, Utetheisa ornatrix, has served as a model species in chemical ecology studies for decades. Like in the widely publicized stories of the Monarch and other milkweed butterflies, the Ornate Moth and its relatives are tropical insects colonizing whole continents assisted by their chemical defenses. With the recent advances in genomic techniques and evo-devo research, it is becoming a model for studies in other areas, from wing pattern development to phylogeography, from toxicology to epigenetics. We used a genomic approach to learn about Utetheisa's evolution, detoxification, dispersal abilities, and wing pattern diversity. We present an evolutionary genomic analysis of the worldwide genus Utetheisa, then focusing on U. ornatrix. Our reference genome of U. ornatrix reveals gene duplications in the regions possibly associated with detoxification abilities, which allows them to feed on toxic food plants. Finally, comparative genomic analysis of over 100 U. ornatrix specimens from the museum with apparent differences in wing patterns suggest the potential roles of cortex and lim3 genes in wing pattern formation of Lepidoptera and the utility of museum-preserved collection specimens for wing pattern research.

Classification of domains in predicted structures of the human proteome.

Recent advances in protein structure prediction have generated accurate structures of previously uncharacterized human proteins. Identifying domains in these predicted structures and classifying them into an evolutionary hierarchy can reveal biological insights. Here, we describe the detection and classification of domains from the human proteome. Our classification indicates that only 62% of residues are located in globular domains. We further classify these globular domains and observe that the majority (65%) can be classified among known folds by sequence, with a smaller fraction (33%) requiring structural data to refine the domain boundaries and/or to support their homology. A relatively small number (966 domains) cannot be confidently assigned using our automatic pipelines, thus demanding manual inspection. We classify 47,576 domains, of which only 23% have been included in experimental structures. A portion (6.3%) of these classified globular domains lack sequence-based annotation in InterPro. A quarter (23%) have not been structurally modeled by homology, and they contain 2,540 known disease-causing single amino acid variations whose pathogenesis can now be inferred using AF models. A comparison of classified domains from a series of model organisms revealed expansions of several immune response-related domains in humans and a depletion of olfactory receptors. Finally, we use this classification to expand well-known protein families of biological significance. These classifications are presented on the ECOD website (http://prodata.swmed.edu/ecod/index_human.php).

ECOD domain classification of 48 whole proteomes from AlphaFold Structure Database using DPAM2.

Protein structure prediction has now been deployed widely across several different large protein sets. Large-scale domain annotation of these predictions can aid in the development of biological insights. Using our Evolutionary Classification of Protein Domains (ECOD) from experimental structures as a basis for classification, we describe the detection and cataloging of domains from 48 whole proteomes deposited in the AlphaFold Database. On average, we can provide positive classification (either of domains or other identifiable non-domain regions) for 90% of residues in all proteomes. We classified 746,349 domains from 536,808 proteins comprised of over 226,424,000 amino acid residues. We examine the varying populations of homologous groups in both eukaryotes and bacteria. In addition to containing a higher fraction of disordered regions and unassigned domains, eukaryotes show a higher proportion of repeated proteins, both globular and small repeats. We enumerate those highly populated domains that are shared in both eukaryotes and bacteria, such as the Rossmann domains, TIM barrels, and P-loop domains. Additionally, we compare the sampling of homologous groups from this whole proteome set against our stable ECOD reference and discuss groups that have been enriched by structure predictions. Finally, we discuss the implication of these results for protein target selection for future classification strategies for very large protein sets.

A switch to feeding on cycads generates parallel accelerated evolution of toxin tolerance in two clades of Eumaeus caterpillars (Lepidoptera: Lycaenidae).

We assembled a complete reference genome of Eumaeus atala, an aposematic cycad-eating hairstreak butterfly that suffered near extinction in the United States in the last century. Based on an analysis of genomic sequences of Eumaeus and 19 representative genera, the closest relatives of Eumaeus are Theorema and Mithras We report natural history information for Eumaeus, Theorema, and Mithras Using genomic sequences for each species of Eumaeus, Theorema, and Mithras (and three outgroups), we trace the evolution of cycad feeding, coloration, gregarious behavior, and other traits. The switch to feeding on cycads and to conspicuous coloration was accompanied by little genomic change. Soon after its origin, Eumaeus split into two fast evolving lineages, instead of forming a clump of close relatives in the phylogenetic tree. Significant overlap of the fast evolving proteins in both clades indicates parallel evolution. The functions of the fast evolving proteins suggest that the caterpillars developed tolerance to cycad toxins with a range of mechanisms including autophagy of damaged cells, removal of cell debris by macrophages, and more active cell proliferation.

DisEnrich: database of enriched regions in human dark proteome.

MOTIVATION: Intrinsically disordered proteins (IDPs) are involved in numerous processes crucial for living organisms. Bias in amino acid composition of these proteins determines their unique biophysical and functional features. Distinct intrinsically disordered regions (IDRs) with compositional bias play different important roles in various biological processes. IDRs enriched in particular amino acids in human proteome have not been described consistently. RESULTS: We developed DisEnrich-the database of human proteome IDRs that are significantly enriched in particular amino acids. Each human protein is described using Gene Ontology (GO) function terms, disorder prediction for the full-length sequence using three methods, enriched IDR composition and ranks of human proteins with similar enriched IDRs. Distribution analysis of enriched IDRs among broad functional categories revealed significant overrepresentation of R- and Y-enriched IDRs in metabolic and enzymatic activities and F-enriched IDRs in transport. About 75% of functional categories contain IDPs with IDRs significantly enriched in hydrophobic residues that are important for protein-protein interactions. AVAILABILITY AND IMPLEMENTATION: The database is available at http://prodata.swmed.edu/DisEnrichDB/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics Advances online.

Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone.

Ghrelin is a 28 amino acid, appetite-stimulating peptide hormone secreted by the food-deprived stomach. Serine-3 of ghrelin is acylated with an eight-carbon fatty acid, octanoate, which is required for its endocrine actions. Here, we identify GOAT (Ghrelin O-Acyltransferase), a polytopic membrane-bound enzyme that attaches octanoate to serine-3 of ghrelin. Analysis of the mouse genome revealed that GOAT belongs to a family of 16 hydrophobic membrane-bound acyltransferases that includes Porcupine, which attaches long-chain fatty acids to Wnt proteins. GOAT is the only member of this family that octanoylates ghrelin when coexpressed in cultured endocrine cell lines with prepro-ghrelin. GOAT activity requires catalytic asparagine and histidine residues that are conserved in this family. Consistent with its function, GOAT mRNA is largely restricted to stomach and intestine, the major ghrelin-secreting tissues. Identification of GOAT will facilitate the search for inhibitors that reduce appetite and diminish obesity in humans.

Genomics Reveals the Origins of Historical Specimens.

Centuries of zoological studies have amassed billions of specimens in collections worldwide. Genomics of these specimens promises to reinvigorate biodiversity research. However, because DNA degrades with age in historical specimens, it is a challenge to obtain genomic data for them and analyze degraded genomes. We developed experimental and computational protocols to overcome these challenges and applied our methods to resolve a series of long-standing controversies involving a group of butterflies. We deduced the geographical origins of several historical specimens of uncertain provenance that are at the heart of these debates. Here, genomics tackles one of the greatest problems in zoology: countless old specimens that serve as irreplaceable embodiments of species concepts cannot be confidently assigned to extant species or population due to the lack of diagnostic morphological features and clear documentation of the collection locality. The ability to determine where they were collected will resolve many on-going disputes. More broadly, we show the utility of applying genomics to historical museum specimens to delineate the boundaries of species and populations, and to hypothesize about genotypic determinants of phenotypic traits.

Gauging ages of tiger swallowtail butterflies using alternate SNP analyses.

Divergence times underpin diverse evolutionary hypotheses, but conflicting age estimates across studies diminish the validity of such hypotheses. These conflicts have continued to grow as large genomics datasets become commonplace and analytical approaches proliferate. To provide more stable temporal intervals, age estimations should be interpreted in the context of both the type of data and analysis being used. Here, we use multispecies coalescent (MSC), concatenation-based, and categorical data transformation approaches on genome-wide SNP data to infer divergence ages within the Papilio glaucus group of tiger swallowtail butterflies in North America. While the SNP data supported previously recognized relationships within the group (P. multicaudata, ((P. eurymedon, P. rutulus), (P. appalachiensis, P. canadensis, P. glaucus))), estimated ages of divergence between the major lineages varied substantially among analyses. MSC produced wide credibility intervals particularly for deeper nodes, reflecting uncertainty in the coalescence times as a possible result of conflicting signal across gene trees. Concatenation, in contrast, gave narrower and more well-defined posterior distributions for the node ages; however, the higher precision of these time estimates is a likely artefact due to more simplistic underlying assumptions of this approach that do not account for conflict among gene trees. Transformed categorical data analysis gave the least precise and the most variable results, with its simple substitution model coupled with a relaxed clock tending to produce spurious results from large genome-wide datasets. While median node ages differed considerably between analyses (∼2 Mya between MSC and concatenation-based results), their corresponding credibility intervals nonetheless highlight common temporal patterns for deeper divergences in the group as well as finer-scale phylogeography. Age distributions across analyses support an origin of the group during the warm period of the early to mid-Pliocene. Late Pliocene climate aridification and cooling drove divergence between eastern and western groups that further diversified during the period of repeated Pleistocene glaciations. Our results provide a structured comparative assessment of divergence time estimates and evolutionary relationships in a well-studied group of butterflies, and support better understanding of analytical biases in divergence time estimation.

A lytic polysaccharide monooxygenase-like protein functions in fungal copper import and meningitis.

Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While C. neoformans Cu+ import and virulence are dependent on the Ctr1 and Ctr4 proteins, little is known concerning extracellular Cu ligands that participate in this process. We identified a C. neoformans gene, BIM1, that is strongly induced during Cu limitation and which encodes a protein related to lytic polysaccharide monooxygenases (LPMOs). Surprisingly, bim1 mutants are Cu deficient, and Bim1 function in Cu accumulation depends on Cu2+ coordination and cell-surface association via a glycophosphatidyl inositol anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse infection models. These studies demonstrate a role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis.

Genomes of skipper butterflies reveal extensive convergence of wing patterns.

For centuries, biologists have used phenotypes to infer evolution. For decades, a handful of gene markers have given us a glimpse of the genotype to combine with phenotypic traits. Today, we can sequence entire genomes from hundreds of species and gain yet closer scrutiny. To illustrate the power of genomics, we have chosen skipper butterflies (Hesperiidae). The genomes of 250 representative species of skippers reveal rampant inconsistencies between their current classification and a genome-based phylogeny. We use a dated genomic tree to define tribes (six new) and subtribes (six new), to overhaul genera (nine new) and subgenera (three new), and to display convergence in wing patterns that fooled researchers for decades. We find that many skippers with similar appearance are distantly related, and several skippers with distinct morphology are close relatives. These conclusions are strongly supported by different genomic regions and are consistent with some morphological traits. Our work is a forerunner to genomic biology shaping biodiversity research.

Gypsy moth genome provides insights into flight capability and virus-host interactions.

Since its accidental introduction to Massachusetts in the late 1800s, the European gypsy moth (EGM; Lymantria dispar dispar) has become a major defoliator in North American forests. However, in part because females are flightless, the spread of the EGM across the United States and Canada has been relatively slow over the past 150 years. In contrast, females of the Asian gypsy moth (AGM; Lymantria dispar asiatica) subspecies have fully developed wings and can fly, thereby posing a serious economic threat if populations are established in North America. To explore the genetic determinants of these phenotypic differences, we sequenced and annotated a draft genome of L. dispar and used it to identify genetic variation between EGM and AGM populations. The 865-Mb gypsy moth genome is the largest Lepidoptera genome sequenced to date and encodes ∼13,300 proteins. Gene ontology analyses of EGM and AGM samples revealed divergence between these populations in genes enriched for several gene ontology categories related to muscle adaptation, chemosensory communication, detoxification of food plant foliage, and immunity. These genetic differences likely contribute to variations in flight ability, chemical sensing, and pathogen interactions among EGM and AGM populations. Finally, we use our new genomic and transcriptomic tools to provide insights into genome-wide gene-expression changes of the gypsy moth after viral infection. Characterizing the immunological response of gypsy moths to virus infection may aid in the improvement of virus-based bioinsecticides currently used to control larval populations.