Use of a small molecule microarray screen to identify inhibitors of the catalytic RNA subunit of Methanobrevibacter smithii RNase P.

Despite interest in developing therapeutics that leverage binding pockets in structured RNAs-whose dysregulation leads to diseases-such drug discovery efforts are limited. Here, we have used a small molecule microarray (SMM) screen to find inhibitors of a large ribozyme: the Methanobrevibacter smithii RNase P RNA (Msm RPR, ∼300 nt). The ribonucleoprotein form of RNase P, which catalyzes the 5'-maturation of precursor tRNAs, is a suitable drug target as it is essential, structurally diverse across life domains, and present in low copy.

Mass Spectral Feature Analysis of Ubiquitylated Peptides Provides Insights into Probing the Dark Ubiquitylome.

Ubiquitylation is a structurally and functionally diverse post-translational modification that involves the covalent attachment of the small protein ubiquitin to other protein substrates. Trypsin-based proteomics is the most common approach for globally identifying ubiquitylation sites. However, we estimate that such methods are unable to detect ∼40% of ubiquitylation sites in the human proteome, , "the dark ubiquitylome", including many important for human health and disease.

Mass spectral feature analysis of ubiquitylated peptides provides insights into probing the dark ubiquitylome.

Ubiquitylation is a structurally and functionally diverse post translational modification that involves the covalent attachment of the small protein ubiquitin to other protein substrates. Trypsin-based proteomics is the most common approach for globally identifying ubiquitylation sites. However, we estimate that such methods are unable to detect ~40% of ubiquitylation sites in the human proteome - i.

Presence of sodefrin precursor-like factor pheromone candidates in mental and dorsal tail base glands in the plethodontid salamander, Karsenia koreana.

Plethodontid salamanders are well known for their distinct courtship rituals and the associated pheromonal signaling. However, little is known about pheromones produced in the lone Asian plethodontid species Karsenia koreana. Here, we examined the localization patterns of proteins of the sodefrin precursor-like factor (SPF) pheromone system in K.

Deep learning from harmonized peptide libraries enables retention time prediction of diverse post translational modifications.

In proteomics experiments, peptide retention time (RT) is an orthogonal property to fragmentation when assessing detection confidence. Advances in deep learning enable accurate RT prediction for any peptide from sequence alone, including those yet to be experimentally observed. Here we present Chronologer, an open-source software tool for rapid and accurate peptide RT prediction.

BRCA1/BARD1 intrinsically disordered regions facilitate chromatin recruitment and ubiquitylation.

BRCA1/BARD1 is a tumor suppressor E3 ubiquitin (Ub) ligase with roles in DNA damage repair and in transcriptional regulation. BRCA1/BARD1 RING domains interact with nucleosomes to facilitate mono-ubiquitylation of distinct residues on the C-terminal tail of histone H2A. These enzymatic domains constitute a small fraction of the heterodimer, raising the possibility of functional chromatin interactions involving other regions such as the BARD1 C-terminal domains that bind nucleosomes containing the DNA damage signal H2A K15-Ub and H4 K20me0, or portions of the expansive intrinsically disordered regions found in both subunits.

Scribe: Next Generation Library Searching for DDA Experiments.

Spectrum library searching is a powerful alternative to database searching for data dependent acquisition experiments, but has been historically limited to identifying previously observed peptides in libraries. Here we present Scribe, a new library search engine designed to leverage deep learning fragmentation prediction software such as Prosit. Rather than relying on highly curated DDA libraries, this approach predicts fragmentation and retention times for every peptide in a FASTA database.

Domain Expansion and Functional Diversification in Vertebrate Reproductive Proteins.

The rapid evolution of fertilization proteins has generated remarkable diversity in molecular structure and function. Glycoproteins of vertebrate egg coats contain multiple zona pellucida (ZP)-N domains (1-6 copies) that facilitate multiple reproductive functions, including species-specific sperm recognition. In this report, we integrate phylogenetics and machine learning to investigate how ZP-N domains diversify in structure and function.

Recurrent Co-Option and Recombination of Cytokine and Three Finger Proteins in Multiple Reproductive Tissues Throughout Salamander Evolution.

Reproductive proteins evolve at unparalleled rates, resulting in tremendous diversity of both molecular composition and biochemical function between gametes of different taxonomic clades. To date, the proteomic composition of amphibian gametes is largely a molecular mystery, particularly for Urodeles (salamanders and newts) for which few genomic-scale resources exist. In this study, we provide the first detailed molecular characterization of gametes from two salamander species ( and ) that are models of reproductive behavior.

Data-Independent Acquisition Protease-Multiplexing Enables Increased Proteome Sequence Coverage Across Multiple Fragmentation Modes.

The use of multiple proteases has been shown to increase protein sequence coverage in proteomics experiments; however, due to the additional analysis time required, it has not been widely adopted in routine data-dependent acquisition (DDA) proteomic workflows. Alternatively, data-independent acquisition (DIA) has the potential to analyze multiplexed samples from different protease digests, but has been primarily optimized for fragmenting tryptic peptides. Here we evaluate a DIA multiplexing approach that combines three proteolytic digests (Trypsin, AspN, and GluC) into a single sample.

Proteomics support the threespine stickleback egg coat as a protective oocyte envelope.

Ancestrally marine threespine stickleback fish (Gasterosteus aculeatus) have undergone an adaptive radiation into freshwater environments throughout the Northern Hemisphere, creating an excellent model system for studying molecular adaptation and speciation. Ecological and behavioral factors have been suggested to underlie stickleback reproductive isolation and incipient speciation, but reproductive proteins mediating gamete recognition during fertilization have so far remained unexplored. To begin to investigate the contribution of reproductive proteins to stickleback reproductive isolation, we have characterized the stickleback egg coat proteome.

Mediator subunit Med15 dictates the conserved "fuzzy" binding mechanism of yeast transcription activators Gal4 and Gcn4.

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a "fuzzy" protein interface.

CIDer: A Statistical Framework for Interpreting Differences in CID and HCD Fragmentation.

Library searching is a powerful technique for detecting peptides using either data independent or data dependent acquisition. While both large-scale spectrum library curators and deep learning prediction approaches have focused on beam-type CID fragmentation (HCD), resonance CID fragmentation remains a popular technique. Here we demonstrate an approach to model the differences between HCD and CID spectra, and present a software tool, CIDer, for converting libraries between the two fragmentation methods.

T Cells Specific for a Mycobacterial Glycolipid Expand after Intravenous Bacillus Calmette-Guérin Vaccination.

Intradermal vaccination with bacillus Calmette-Guérin (BCG) protects infants from disseminated tuberculosis, and i.v. BCG protects nonhuman primates (NHP) against pulmonary and extrapulmonary tuberculosis.

Indirect sexual selection drives rapid sperm protein evolution in abalone.

Sexual selection can explain the rapid evolution of fertilization proteins, yet sperm proteins evolve rapidly even if not directly involved in fertilization. In the marine mollusk abalone, sperm secrete enormous quantities of two rapidly evolving proteins, lysin and sp18, that are stored at nearly molar concentrations. We demonstrate that this extraordinary packaging is achieved by associating into Fuzzy Interacting Transient Zwitterion (FITZ) complexes upon binding the intrinsically disordered FITZ Anionic Partner (FITZAP).

Conservation of molecular and cellular phenotypes of invariant NKT cells between humans and non-human primates.

Invariant NKT (iNKT) cells in both humans and non-human primates are activated by the glycolipid antigen, α-galactosylceramide (α-GalCer). However, the extent to which the molecular mechanisms of antigen recognition and in vivo phenotypes of iNKT cells are conserved among primate species has not been determined. Using an evolutionary genetic approach, we found a lack of diversifying selection in CD1 genes over 45 million years of evolution, which stands in stark contrast to the history of the MHC system for presenting peptide antigens to T cells.

An annual cycle of gene regulation in the red-legged salamander mental gland: from hypertrophy to expression of rapidly evolving pheromones.

Background: Cell differentiation is mediated by synchronized waves of coordinated expression for hundreds to thousands of genes, and must be regulated to produce complex tissues and phenotypes. For many animal species, sexual selection has driven the development of elaborate male ornaments, requiring sex-specific differentiation pathways. One such male ornament is the pheromone-producing mental gland of the red-legged salamander (Plethodon shermani).

A Diverse Lipid Antigen-Specific TCR Repertoire Is Clonally Expanded during Active Tuberculosis.

Human T cells that recognize lipid Ags presented by highly conserved CD1 proteins often express semi-invariant TCRs, but the true diversity of lipid Ag-specific TCRs remains unknown. We use CD1b tetramers and high-throughput immunosequencing to analyze thousands of TCRs from ex vivo-sorted or in vitro-expanded T cells specific for the mycobacterial lipid Ag, glucose monomycolate. Our results reveal a surprisingly diverse repertoire resulting from editing of germline-encoded gene rearrangements analogous to MHC-restricted TCRs.

Skin glands of an aquatic salamander vary in size and distribution and release antimicrobial secretions effective against chytrid fungal pathogens.

Amphibian skin is unique among vertebrate classes, containing a large number of multicellular exocrine glands that vary among species and have diverse functions. The secretions of skin glands contain a rich array of bioactive compounds including antimicrobial peptides (AMPs). Such compounds are important for amphibian innate immune responses and may protect some species from chytridiomycosis, a lethal skin disease caused by the fungal pathogens (Bd) and (Bsal).

Solution structure of sperm lysin yields novel insights into molecular dynamics of rapid protein evolution.

Protein evolution is driven by the sum of different physiochemical and genetic processes that usually results in strong purifying selection to maintain biochemical functions. However, proteins that are part of systems under arms race dynamics often evolve at unparalleled rates that can produce atypical biochemical properties. In the marine mollusk abalone, lysin and vitelline envelope receptor for lysin (VERL) are a pair of rapidly coevolving proteins that are essential for species-specific interactions between sperm and egg.

Evolution of microRNA in primates.

MicroRNA play an important role in post-transcriptional regulation of most transcripts in the human genome, but their evolution across the primate lineage is largely uncharacterized. A particular miRNA can have one to thousands of messenger RNA targets, establishing the potential for a small change in sequence or overall miRNA structure to have profound phenotypic effects. However, the majority of non-human primate miRNA is predicted solely by homology to the human genome and lacks experimental validation.

Olfactory effects of a hypervariable multicomponent pheromone in the red-legged salamander, Plethodon shermani.

Chemical communication via chemosensory signaling is an essential process for promoting and modifying reproductive behavior in many species. During courtship in plethodontid salamanders, males deliver a mixture of non-volatile proteinaceous pheromones that activate chemosensory neurons in the vomeronasal epithelium (VNE) and increase female receptivity. One component of this mixture, Plethodontid Modulating Factor (PMF), is a hypervariable pheromone expressed as more than 30 unique isoforms that differ between individual males-likely driven by co-evolution with female receptors to promote gene duplication and positive selection of the PMF gene complex.

Co-option and evolution of non-olfactory proteinaceous pheromones in a terrestrial lungless salamander.

Gene co-option is a major force in the evolution of novel biological functions. In plethodontid salamanders, males deliver proteinaceous courtship pheromones to the female olfactory system or transdermally to the bloodstream. Molecular studies identified three families of highly duplicated, rapidly evolving pheromones (PRF, PMF, and SPF).

From molecules to mating: Rapid evolution and biochemical studies of reproductive proteins.

Unlabelled: Sexual reproduction and the exchange of genetic information are essential biological processes for species across all branches of the tree of life. Over the last four decades, biochemists have continued to identify many of the factors that facilitate reproduction, but the molecular mechanisms that mediate this process continue to elude us. However, a recurring observation in this research has been the rapid evolution of reproductive proteins.