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1. In silico analysis of crustacean hyperglycemic hormone family G protein-coupled receptor candidates

   https://doi.org/10.3389/fendo.2023.1322800  

   Kozma, Mihika T.; Pérez-Moreno, Jorge L.; Gandhi, Neha S.; Hernandez Jeppesen, Luisanna; Durica, David S.; Ventura, Tomer; Mykles, Donald L. (January 2024, Frontiers in Endocrinology)

   Vrecl, M. (Ed.)

   Ecdysteroid molting hormone synthesis is directed by a pair of molting glands or Y-organs (YOs), and this synthesis is inhibited by molt-inhibiting hormone (MIH). MIH is a member of the crustacean hyperglycemic hormone (CHH) neuropeptide superfamily, which includes CHH and insect ion transport peptide (ITP). It is hypothesized that the MIH receptor is a Class A (Rhodopsin-like) G protein-coupled receptor (GPCR). The YO of the blackback land crab,Gecarcinus lateralis, expresses 49 Class A GPCRs, three of which (Gl-CHHR-A9, -A10, and -A12) were provisionally assigned as CHH-like receptors. CrusTome, a transcriptome database assembled from 189 crustaceans and 12 ecdysozoan outgroups, was used to deorphanize candidate MIH/CHH GPCRs, relying on sequence homology to three functionally characterized ITP receptors (BNGR-A2, BNGR-A24, and BNGR-A34) in the silk moth,Bombyx mori. Phylogenetic analysis and multiple sequence alignments across major taxonomic groups revealed extensive expansion and diversification of crustacean A2, A24, and A34 receptors, designatedCHHFamilyReceptorCandidates (CFRCs). The A2 clade was divided into three subclades; A24 clade was divided into five subclades; and A34 was divided into six subclades. The subclades were distinguished by conserved motifs in extracellular loop (ECL) 2 and ECL3 in the ligand-binding region. Eleven of the 14 subclades occurred in decapod crustaceans. InG. lateralis, seven CFRC sequences, designated Gl-CFRC-A2α1, -A24α, -A24β1, -A24β2, -A34α2, -A34β1, and -A34β2, were identified; the three A34 sequences corresponded to Gl-GPCR-A12, -A9, and A10, respectively. ECL2 in all the CFRC sequences had a two-stranded β-sheet structure similar to human Class A GPCRs, whereas the ECL2 of decapod CFRC-A34β1/β2 had an additional two-stranded β-sheet. We hypothesize that this second β-sheet on ECL2 plays a role in MIH/CHH binding and activation, which will be investigated further with functional assays. 

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2. A Machine Learning Approach for the Discovery of Ligand-Specific Functional Mechanisms of GPCRs

   https://doi.org/10.3390/molecules24112097  

   Plante, Ambrose; Shore, Derek M.; Morra, Giulia; Khelashvili, George; Weinstein, Harel (June 2019, Molecules)

   G protein-coupled receptors (GPCRs) play a key role in many cellular signaling mechanisms, and must select among multiple coupling possibilities in a ligand-specific manner in order to carry out a myriad of functions in diverse cellular contexts. Much has been learned about the molecular mechanisms of ligand-GPCR complexes from Molecular Dynamics (MD) simulations. However, to explore ligand-specific differences in the response of a GPCR to diverse ligands, as is required to understand ligand bias and functional selectivity, necessitates creating very large amounts of data from the needed large-scale simulations. This becomes a Big Data problem for the high dimensionality analysis of the accumulated trajectories. Here we describe a new machine learning (ML) approach to the problem that is based on transforming the analysis of GPCR function-related, ligand-specific differences encoded in the MD simulation trajectories into a representation recognizable by state-of-the-art deep learning object recognition technology. We illustrate this method by applying it to recognize the pharmacological classification of ligands bound to the 5-HT2A and D2 subtypes of class-A GPCRs from the serotonin and dopamine families. The ML-based approach is shown to perform the classification task with high accuracy, and we identify the molecular determinants of the classifications in the context of GPCR structure and function. This study builds a framework for the efficient computational analysis of MD Big Data collected for the purpose of understanding ligand-specific GPCR activity. 

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3. Homo-oligomerization of the human adenosine A2A receptor is driven by the intrinsically disordered C-terminus

   https://doi.org/10.7554/eLife.66662  

   Nguyen, Khanh Dinh; Vigers, Michael; Sefah, Eric; Seppälä, Susanna; Hoover, Jennifer Paige; Schonenbach, Nicole Star; Mertz, Blake; O'Malley, Michelle Ann; Han, Songi (July 2021, eLife)

   null (Ed.)

   G protein-coupled receptors (GPCRs) have long been shown to exist as oligomers with functional properties distinct from those of the monomeric counterparts, but the driving factors of oligomerization remain relatively unexplored. Herein, we focus on the human adenosine A 2A receptor (A 2A R), a model GPCR that forms oligomers both in vitro and in vivo. Combining experimental and computational approaches, we discover that the intrinsically disordered C-terminus of A 2A R drives receptor homo-oligomerization. The formation of A 2A R oligomers declines progressively with the shortening of the C-terminus. Multiple interaction types are responsible for A 2A R oligomerization, including disulfide linkages, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. These interactions are enhanced by depletion interactions, giving rise to a tunable network of bonds that allow A 2A R oligomers to adopt multiple interfaces. This study uncovers the disordered C-terminus as a prominent driving factor for the oligomerization of a GPCR, offering important insight into the effect of C-terminus modification on receptor oligomerization of A 2A R and other GPCRs reconstituted in vitro for biophysical studies. 

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4. Evaluating conserved domains and motifs of decapod gonadotropin-releasing hormone G protein-coupled receptor superfamily

   https://doi.org/10.3389/fendo.2024.1348465  

   Buckley, Sean J; Nguyen, Tuan Viet; Cummins, Scott F; Elizur, Abigail; Fitzgibbon, Quinn P; Smith, Gregory S; Mykles, Donald L; Ventura, Tomer (February 2024, Frontiers in Endocrinology)

   None (Ed.)

   G protein-coupled receptors (GPCRs) are an ancient family of signal transducers that are both abundant and consequential in metazoan endocrinology. The evolutionary history and function of the GPCRs of the decapod superfamilies of gonadotropin-releasing hormone (GnRH) are yet to be fully elucidated. As part of which, the use of traditional phylogenetics and the recycling of a diminutive set of mis-annotated databases has proven insufficient. To address this, we have collated and revised eight existing and three novel GPCR repertoires for GnRH of decapod species. We developed a novel bioinformatic workflow that included clustering analysis to capture likely GnRH receptor-like proteins, followed by phylogenetic analysis of the seven transmembrane-spanning domains. A high degree of conservation of the sequences and topology of the domains and motifs allowed the identification of species-specific variation (up to ~70%, especially in the extracellular loops) that is thought to be influential to ligand-binding and function. Given the key functional role of the DRY motif across GPCRs, the classification of receptors based on the variation of this motif can be universally applied to resolve cryptic GPCR families, as was achieved in this work. Our results contribute to the resolution of the evolutionary history of invertebrate GnRH receptors and inform the design of bioassays in their deorphanization and functional annotation. 

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5. Identifying transgene insertions in Caenorhabditis elegans genomes with Oxford Nanopore sequencing

   https://doi.org/10.7717/peerj.18100  

   Adams, Paula E; Thies, Jennifer L; Sutton, John M; Millwood, Joshua D; Caldwell, Guy A; Caldwell, Kim A; Fierst, Janna L (January 2024, PeerJ)

   Genetically modified organisms are commonly used in disease research and agriculture but the precise genomic alterations underlying transgenic mutations are often unknown. The position and characteristics of transgenes, including the number of independent insertions, influences the expression of both transgenic and wild-type sequences. We used long-read, Oxford Nanopore Technologies (ONT) to sequence and assemble two transgenic strains ofCaenorhabditis eleganscommonly used in the research of neurodegenerative diseases: BY250 (pPdat-1::GFP) and UA44 (GFP and humanα-synuclein), a model for Parkinson’s research. After scaffolding to the reference, the final assembled sequences were ∼102 Mb with N50s of 17.9 Mb and 18.0 Mb, respectively, and L90s of six contiguous sequences, representing chromosome-level assemblies. Each of the assembled sequences contained more than 99.2% of the Nematoda BUSCO genes found in theC. elegansreference and 99.5% of the annotatedC. elegansreference protein-coding genes. We identified the locations of the transgene insertions and confirmed that all transgene sequences were inserted in intergenic regions, leaving the organismal gene content intact. The transgenicC. elegansgenomes presented here will be a valuable resource for Parkinson’s research as well as other neurodegenerative diseases. Our work demonstrates that long-read sequencing is a fast, cost-effective way to assemble genome sequences and characterize mutant lines and strains. 

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