Structural Insights into the Evolution of a Non-Biological Protein: Importance of Surface Residues in Protein Fold Optimization

Phylogenetic profiling of amino acid substitution patterns in proteins has led many to conclude that most structural information is carried by interior core residues that are solvent inaccessible. This conclusion is based on the observation that buried residues generally tolerate only conserved sequence changes, while surface residues allow more diverse chemical substitutions. This notion is now changing as it has become apparent that both core and surface residues play important roles in protein folding and stability. Unfortunately, the ability to identify specific mutations that will lead to enhanced stability remains a challenging problem. Here we discuss two mutations that emerged from an in vitro selection experiment designed to improve the folding stability of a non-biological ATP binding protein. These mutations alter two solvent accessible residues, and dramatically enhance the expression, solubility, thermal stability, and ligand binding affinity of the protein. The significance of both mutations was investigated individually and together, and the X-ray crystal structures of the parent sequence and double mutant protein were solved to a resolution limit of 2.8 and 1.65 Å, respectively. Comparative structural analysis of the evolved protein to proteins found in nature reveals that our non-biological protein evolved certain structural features shared by many thermophilic proteins. This experimental result suggests that protein fold optimization by in vitro selection offers a viable approach to generating stable variants of many naturally occurring proteins whose structures and functions are otherwise difficult to study

International Consensus Based Review and Recommendations for Minimum Reporting Standards in Research on Transcutaneous Vagus Nerve Stimulation (Version 2020)

Given its non-invasive nature, there is increasing interest in the use of transcutaneous vagus nerve stimulation (tVNS) across basic, translational and clinical research. Contemporaneously, tVNS can be achieved by stimulating either the auricular branch or the cervical bundle of the vagus nerve, referred to as transcutaneous auricular vagus nerve stimulation(VNS) and transcutaneous cervical VNS, respectively. In order to advance the field in a systematic manner, studies using these technologies need to adequately report sufficient methodological detail to enable comparison of results between studies, replication of studies, as well as enhancing study participant safety. We systematically reviewed the existing tVNS literature to evaluate current reporting practices. Based on this review, and consensus among participating authors, we propose a set of minimal reporting items to guide future tVNS studies. The suggested items address specific technical aspects of the device and stimulation parameters. We also cover general recommendations including inclusion and exclusion criteria for participants, outcome parameters and the detailed reporting of side effects. Furthermore, we review strategies used to identify the optimal stimulation parameters for a given research setting and summarize ongoing developments in animal research with potential implications for the application of tVNS in humans. Finally, we discuss the potential of tVNS in future research as well as the associated challenges across several disciplines in research and clinical practice

A consensus protocol for functional connectivity analysis in the rat brain

Task-free functional connectivity in animal models provides an experimental framework to examine connectivity phenomena under controlled conditions and allows for comparisons with data modalities collected under invasive or terminal procedures. Currently, animal acquisitions are performed with varying protocols and analyses that hamper result comparison and integration. Here we introduce StandardRat, a consensus rat functional magnetic resonance imaging acquisition protocol tested across 20 centers. To develop this protocol with optimized acquisition and processing parameters, we initially aggregated 65 functional imaging datasets acquired from rats across 46 centers. We developed a reproducible pipeline for analyzing rat data acquired with diverse protocols and determined experimental and processing parameters associated with the robust detection of functional connectivity across centers. We show that the standardized protocol enhances biologically plausible functional connectivity patterns relative to previous acquisitions. The protocol and processing pipeline described here is openly shared with the neuroimaging community to promote interoperability and cooperation toward tackling the most important challenges in neuroscience

Genome-wide identification and phenotypic characterization of seizure-associated copy number variations in 741,075 individuals

Copy number variants (CNV) are established risk factors for neurodevelopmental disorders with seizures or epilepsy. With the hypothesis that seizure disorders share genetic risk factors, we pooled CNV data from 10,590 individuals with seizure disorders, 16,109 individuals with clinically validated epilepsy, and 492,324 population controls and identified 25 genome-wide significant loci, 22 of which are novel for seizure disorders, such as deletions at 1p36.33, 1q44, 2p21-p16.3, 3q29, 8p23.3-p23.2, 9p24.3, 10q26.3, 15q11.2, 15q12-q13.1, 16p12.2, 17q21.31, duplications at 2q13, 9q34.3, 16p13.3, 17q12, 19p13.3, 20q13.33, and reciprocal CNVs at 16p11.2, and 22q11.21. Using genetic data from additional 248,751 individuals with 23 neuropsychiatric phenotypes, we explored the pleiotropy of these 25 loci. Finally, in a subset of individuals with epilepsy and detailed clinical data available, we performed phenome-wide association analyses between individual CNVs and clinical annotations categorized through the Human Phenotype Ontology (HPO). For six CNVs, we identified 19 significant associations with specific HPO terms and generated, for all CNVs, phenotype signatures across 17 clinical categories relevant for epileptologists. This is the most comprehensive investigation of CNVs in epilepsy and related seizure disorders, with potential implications for clinical practice

Identification and characterization of the zebra finch (Taeniopygia guttata) sperm proteome

Spermatozoa exhibit remarkable variability in size, shape, and performance. Our understanding of the molecular basis of this variation, however, is limited, especially in avian taxa. The zebra finch (Taeniopygia guttata) is a model organism in the study of avian sperm biology and sperm competition. Using LC-MS based proteomics, we identify and describe 494 proteins of the zebra finch sperm proteome (ZfSP). Gene ontology and associated bioinformatics analyses revealed a rich repertoire of proteins essential to sperm structure and function, including proteins linked to metabolism and energetics, as well as tubulin binding and microtubule related functions. The ZfSP also contained a number of immunity and defense proteins and proteins linked to sperm motility and sperm-egg interactions. Additionally, while most proteins in the ZfSP appear to be evolutionarily constrained, a small subset of proteins are evolving rapidly. Finally, in a comparison with the sperm proteome of the domestic chicken, we found an enrichment of proteins linked to catalytic activity and cytoskeleton related processes. As the first described passerine sperm proteome, and one of only two characterized avian sperm proteomes, the ZfSP provides a significant step towards a platform for studies of the molecular basis of sperm function and evolution in birds

The Old and the New: Discovery Proteomics Identifies Putative Novel Seminal Fluid Proteins in Drosophila

Karr TL, Southern H, Rosenow MA, Goßmann T, Snook RR. The Old and the New: Discovery Proteomics Identifies Putative Novel Seminal Fluid Proteins in Drosophila. Molecular & Cellular Proteomics. 2019;18(Suppl. 1):S23-S33

Sperm Proteome Maturation in the Mouse Epididymis

<div><p>In mammals, transit through the epididymis, which involves the acquisition, loss and modification of proteins, is required to confer motility and fertilization competency to sperm. The overall dynamics of maturation is poorly understood, and a systems level understanding of the complex maturation process will provide valuable new information about changes occurring during epididymal transport. We report the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. This study identified 765 proteins that are present in sperm obtained from all three segments. We identified 1766 proteins that are potentially added (732) or removed (1034) from sperm during epididymal transit. Phenotypic analyses of the caput, corpus and cauda sperm proteomes identified 60 proteins that have known sperm phenotypes when mutated, or absent from sperm. Our analysis indicates that as much as one-third of proteins with known sperm phenotypes are added to sperm during epididymal transit. GO analyses revealed that cauda sperm are enriched for specific functions including sperm-egg recognition and motility, consistent with the observation that sperm acquire motility and fertilization competency during transit through the epididymis. In addition, GO analyses revealed that the immunity protein profile of sperm changes during sperm maturation. Finally, we identified components of the 26S proteasome, the immunoproteasome, and a proteasome activator in mature sperm.</p></div

Enriched immune system process GO categories for proteins in the caput and cauda sperm proteomes.

<p>Network immune system process diagram depicting GO categories enriched in the caput (blue) and cauda (magenta) sperm proteomes. The darker the node color, the stronger the enrichment while white nodes indicate an equal abundance of that functional category in the list of added and removed proteins.</p