Cryo-EM structure of native human thyroglobulin

The thyroglobulin (TG) protein is essential to thyroid hormone synthesis, plays a vital role in the regulation of metabolism, development and growth and serves as intraglandular iodine storage. Its architecture is conserved among vertebrates. Synthesis of triiodothyronine (T3) and thyroxine (T4) hormones depends on the conformation, iodination and post-translational modification of TG. Although structural information is available on recombinant and deglycosylated endogenous human thyroglobulin (hTG) from patients with goiters, the structure of native, fully glycosylated hTG remained unknown. Here, we present the cryo-electron microscopy structure of native and fully glycosylated hTG from healthy thyroid glands to 3.2 Å resolution. The structure provides detailed information on hormonogenic and glycosylation sites. We employ liquid chromatography–mass spectrometry (LC-MS) to validate these findings as well as other post-translational modifications and proteolytic cleavage sites. Our results offer insights into thyroid hormonogenesis of native hTG and provide a fundamental understanding of clinically relevant mutations.

yHydra: Deep Learning enables an Ultra Fast Open Search by Jointly Embedding MS/MS Spectra and Peptides of Mass Spectrometry-based Proteomics

Mass spectrometry-based proteomics allows to study all proteins of a sample on a molecular level. The ever increasing complexity and amount of proteomics MS-data requires powerful and yet efficient computational and statistical analysis. In particular, most recent bottom-up MS-based proteomics studies consider either a diverse pool of post-translational modifications, employ large databases – as in metaproteomics or proteogenomics, contain multiple isoforms of proteins, include unspecific cleavage sites or even combinations thereof and thus face a computationally challenging situation regarding protein identification. In order to cope with resulting large search spaces, we present a deep learning approach that jointly embeds MS/MS spectra and peptides into the same vector space such that embeddings can be compared easily and interchangeable by using euclidean distances. In contrast to existing spectrum embedding techniques, ours are learned jointly with their respective peptides and thus remain meaningful. By visualizing the learned manifold of both spectrum and peptide embeddings in correspondence to their physicochemical properties our approach becomes easily interpretable. At the same time, our joint embeddings blur the lines between spectra and protein sequences, providing a powerful framework for peptide identification. In particular, we build an open search, which allows to search multiple ten-thousands of spectra against millions of peptides within seconds. yHydra achieves identification rates that are compatible with MSFragger. Due to the open search, delta masses are assigned to each identification which allows to unrestrictedly characterize post-translational modifications. Meaningful joint embeddings allow for faster open searches and generally make downstream analysis efficient and convenient for example for integration with other omics types.Availabilityupon [email protected]

MTSviewer: a database to visualize mitochondrial targeting sequences, cleavage sites, and mutations on protein structures

Mitochondrial dysfunction is implicated in a wide array of human diseases ranging from neurodegenerative disorders to cardiovascular defects. The coordinated localization and import of proteins into mitochondria is an essential process that ensures mitochondrial homeostasis and consequently cell survival. The localization and import of most mitochondrial proteins are driven by N-terminal mitochondrial targeting sequences (MTS), which interact with import machinery and are removed by the mitochondrial processing peptidase (MPP). The recent discovery of internal MTS's - those which are distributed throughout a protein and act as import regulators or secondary MPP cleavage sites - has expanded the role of both MTS's and MPP beyond conventional N-terminal regulatory pathways. Still, the global mutational landscape of MTS's remains poorly characterized, both from genetic and structural perspectives. To this end, we have integrated a variety of prediction tools into one harmonized R/Shiny database called MTSviewer, which combines MTS predictions, MPP cleavage sites, genetic variants, pathogenicity predictions, and N-terminomics data with structural visualization using AlphaFold models. Using this platform, we have generated a list of disease-linked variants in protein MTS's and their predicted consequences as a resource for their functional characterization. Overall, MTSviewer is a platform that can be used to interrogate MTS mutations and their potential effects on import and proteolysis across the mitochondrial proteome.

An Antibacterial Peptide with High Resistance to Trypsin Obtained by Substituting d-Amino Acids for Trypsin Cleavage Sites

The poor stability of antibacterial peptide to protease limits its clinical application. Among these limitations, trypsin mainly exists in digestive tract, which is an insurmountable obstacle to orally delivered peptides. OM19R is a random curly polyproline cationic antimicrobial peptide, which has high antibacterial activity against some gram-negative bacteria, but its stability against pancreatin is poor. According to the structure-activity relationship of OM19R, all cationic amino acid residues (l-arginine and l-lysine) at the trypsin cleavage sites were replaced with corresponding d-amino acid residues to obtain the designed peptide OM19D, which not only maintained its antibacterial activity but also enhanced the stability of trypsin. Proceeding high concentrations of trypsin and long-time (such as 10 mg/mL, 8 h) treatment, it still had high antibacterial activity (MIC = 16–32 µg/mL). In addition, OM19D also showed high stability to serum, plasma and other environmental factors. It is similar to its parent peptide in secondary structure and mechanism of action. Therefore, this strategy is beneficial to improve the protease stability of antibacterial peptides.

Homologous recombination repair creates mutations in the non-coding genome that alter Topoisomerase-1 cleavage sites & orchestrates irinotecan resistance

The selection of drug-resistant mammalian cell mutants requires multiple drug exposures. When cloned genetically identical cells are exposed to the drug, resistance is unlikely to result from the selection of pre-existent mutations. Therefore, adaptation must involve the generation of drug-resistant mutations de-novo. Understanding how adaptive mutations are generated and protect cells is important for our knowledge of cancer biology and evolution. Here, we studied the adaptation of cancer cells to topoisomerase (Top1) inhibitor irinotecan, which triggers DNA breaks, resulting in cytotoxicity. The resistance mechanism was based on the gradual accumulation of hundreds of thousands of recurrent mutations in non-coding DNA at sequence-specific Top1 cleavage sites. Repair of DSBs at these sites following initial irinotecan exposures created mutant sequences that were resistant to further Top1 cleavage. Therefore, by creating DNA breaks Top1 increases the rate of highly protective mutations specifically at such spots, thus explaining a puzzling need of dose escalation in resistance development.

Quantification of alternative 3′UTR isoforms from single cell RNA-seq data with scUTRquant

Although half of human genes use alternative polyadenylation (APA) to generate mRNA isoforms that encode the same protein but differ in their 3′UTRs, most single cell RNA-sequencing (scRNA-seq) pipelines only measure gene expression. Here, we describe an open-access pipeline, called scUTRquant (https://github.com/Mayrlab/scUTRquant), that measures gene and 3′UTR isoform expression from scRNA-seq data obtained from known cell types in any species. scUTRquant-derived gene and 3′UTR transcript counts were validated against standard methods which demonstrated their accuracy. 3′UTR isoform quantification was substantially more reproducible than previous methods. scUTRquant provides an atlas of high-confidence 3′ end cleavage sites at single-nucleotide resolution to allow APA comparison across mouse datasets. Analysis of 120 mouse cell types revealed that during differentiation genes either change their expression or they change their 3′UTR isoform usage. Therefore, we identified thousands of genes with 3′UTR isoform changes that have previously not been implicated in specific biological processes.

Pre-metazoan origin of neuropeptide signalling

Neuropeptides are a diverse class of signalling molecules in metazoans. They occur in all animals with a nervous system and also in neuron-less placozoans. However, their origin has remained unclear because no neuropeptide shows deep homology across lineages and none have been found in sponges. Here, we identify two neuropeptide precursors, phoenixin and nesfatin, with broad evolutionary conservation. By database searches, sequence alignments and gene-structure comparisons we show that both precursors are present in bilaterians, cnidarians, ctenophores and sponges. We also found phoenixin and a secreted nesfatin precursor homolog in the choanoflagellate Salpingoeca rosetta. Phoenixin in particular, is highly conserved, including its cleavage sites, suggesting that prohormone processing occurs also in choanoflagellates. In addition, based on phyletic patterns and negative pharmacological assays we question the originally proposed GPR-173 (SREB3) as a phoenixin receptor. Our findings indicate that signalling by secreted neuropeptide homologs has pre-metazoan origins and thus evolved before neurons.

HIV-1 Gag Non-Cleavage Site PI Resistance Mutations Stabilize Protease/Gag Substrate Complexes in Silico via a Substrate-Clamp

HIV-1 protease active site inhibitors are a key part of antiretroviral therapy, though resistance can evolve rendering therapy ineffective. Protease inhibitor resistance typically starts with primary mutations around the active site, which reduces inhibitor binding, protease affinity for substrate cleavage site residues P4-P4′, and viral replication. This is often followed by secondary mutations in the protease substrate-grooves which restore viral replication by increasing protease affinity for cleavage site residues P12-P5/P5′-P12′, while maintaining resistance. However, mutations in Gag alone can also result in resistance. The Gag resistance mutations can occur in cleavage sites (P12-P12′) to increase PR binding, as well as at non-cleavage sites. Here we show in silico that Gag non-cleavage site protease inhibitor resistance mutations can stabilize protease binding to Gag cleavage sites which contain structured subdomains on both sides: SP1/NC, SP2/p6, and MA/CA. The Gag non-cleavage site resistance mutations coordinated a network of H-bond interactions between the adjacent structured subdomains of the Gag substrates to form a substrate-clamp around the protease bound to cleavage site residues P12-P12′. The substrate-clamp likely slows protease disassociation from the substrate, restoring the cleavage rate in the presence of the inhibitor. Native Gag substrates can also form somewhat weaker substrate-clamps. This explains the 350-fold slower cleavage rate for the Gag CA/SP1 cleavage site in that the CA-SP1 substrate lacks structured subdomains on both sides of the cleavage site, and so cannot form a substrate-clamp around the PR.