Project description:Here, we sample small proteomes from the interior enamel of 10 fossils deposited at seven paleontological sites between 1.5 and 29 Ma in the Turkana Basin, a region of northern Kenya in the East African Rift System. We find enamel protein fragments in all fossil specimens including a 29 Ma Arsinoitherium from Topernawi, belonging to a now extinct mammalian order. Identified proteins include the classical structural enamel proteins amelogenin, enamelin, and ameloblastin, but also uncommon enamel proteins including collagens and proteases. Protein fragment abundance and average length decline in progressively older fossils, but we observe significant variability in Early Miocene preservation from site to site, with proboscidean fossils from 16 Ma Buluk preserving substantially more proteins than Rhinocerotidae and Anthracotheriidae fossils from the marginally older Locherangan (18 Ma) and Hippopotamidae from the younger site of Napudet (c. 5-11 Ma). Most specimens yield known clade-specific diagenetiforms that support field taxonomic identifications, with the notable exception of the Arsinoitherium that is without living relatives. Consensus phylogenetic trees suggest the potential for paleoproteomics in supporting taxonomic identifications and resolving evolutionary relationships of extinct taxa but should be approached with caution due to sometimes sparse fragment identification and the potential for sequence diagenesis. We identify numerous likely modifications that support the ancient age of these proteins, and the oldest examples of advanced glycation end-products and carbamylation yet known. The persistence of protein sequences within dense enamel tissues in one of the persistently warmest places on Earth promises the discovery of far older proteomes that will aid in the study of the biology and evolutionary relationships of extinct taxa.
Project description:The tooth enamel proteome is enzymatically broken down during development, which leads to a heterogenous distribution of remaining peptides of a protein sequence in a grown individual's enamelome. After death of the organism, the heterogeneous distribution of remaining peptides may be enhanced, as the proteome further degrades. The pattern of this degradation is poorly understood, but it might happen at different rates depending on the site within the protein sequence, due to different chemical properties of the peptides and their interaction with the environment. To learn more about this process, we recovered six ancient enamel proteomes from Equidae and Proboscidea fossils from Spain. The fossils span an age range of <100 ka to 11.6 Ma.
Project description:Cellular protein concentrations are maintained through a balance of synthesis and clearance. Clearance occurs through both protein degradation and growth-dependent dilution. At slow growth, clearance is dominated by degradation, which leads to the accumulation of long lived proteins. At fast growth, however, it is dominated by dilution, preventing this accumulation. Thus, the concentration of long lived proteins will be reduced unless cells compensate by preferentially increasing synthesis rates. To determine the dominant regulatory mechanisms, we quantified the degree of compensation between activated and resting human B cells and across mouse tissues. The results indicate that growth-dependent dilution is insufficiently compensated for by changes in protein synthesis, and it accounts for over a third of the concentration changes between high and low growth conditions. Furthermore, we find that about 25 % of the differences in protein concentration across all tissues are controlled by protein clearance. When comparing only slowly growing tissues such as the brain and pancreas, clearance differences explain as much as 42 %. Within a tissue or cell type, clearance variation is sufficient to account for 50 % of the abundance variation for all measured proteins at slow growth, contrasted with 7 % at fast growth. Thus, our model unifies previous observations with our new results and highlights a context-dependent and larger than previously appreciated contribution of protein degradation in shaping protein variation both across the proteome and across cell states.
Project description:Examination of multiple proteomics datasets within or between species increases the reliability of protein identification. We report here proteomes of inner-ear hair bundles from three species (chick, mouse, and rat), which were collected on LTQ or LTQ Velos ion-trap mass spectrometers; the constituent proteins were quantified using MS2 intensities, which are the summed intensities of all peptide fragmentation spectra matched to a protein. The data are available via ProteomeXchange with identifiers PXD002410 (chick LTQ), PXD002414 (chick Velos), PXD002415 (mouse Velos), and PXD002416 (rat LTQ). The two chick bundle datasets compared favourably to a third, already-described chick bundle dataset, which was quantified using MS1 peak intensities, the summed intensities of peptides identified by high-resolution mass spectrometry (PXD000104; updated analysis in PXD002445). The mouse bundle dataset described here was comparable to a different mouse bundle dataset quantified using MS1 intensities (PXD002167). These six datasets will be useful for identifying the core proteome of vestibular hair bundles.
Project description:Calcium ions play important roles in nearly every biological process, yet whole-proteome analysis of calcium effectors has been hindered by lack of high-throughput, unbiased, and quantitative methods to identify proteins-calcium engagement. To address this, we adapted protein thermostability assays in the budding yeast, human cells, and mouse mitochondria. Based on calcium-dependent thermostability, we identified 2884 putative calcium-regulated proteins across human, mouse, and yeast proteomes. These data revealed calcium engagement of novel signaling hubs and cellular processes, including metabolic enzymes and the spliceosome. Cross-species comparison of calcium-protein engagement and mutagenesis experiments identified residue-specific cation engagement, even within well-known EF-hand domains. Additionally, we found that the dienoyl-CoA reductase DECR1 binds calcium at physiologically-relevant concentrations with substrate-specific affinity, suggesting direct calcium regulation of mitochondrial fatty acid oxidation. These unbiased, proteomic analyses of calcium effectors establish a key resource to dissect cation engagement and its mechanistic effects across multiple species and diverse biological processes.
Project description:The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. Here we present a suite of techniques, based on ribosome profiling (the deep-sequencing of ribosome-protected mRNA fragments), to provide genome-wide maps of protein synthesis as well as a pulse-chase strategy for determining rates of translation elongation. We exploit the propensity of harringtonine to cause ribosomes to accumulate at sites of translation initiation together with a machine learning algorithm to define protein products systematically. Analysis of translation in mouse embryonic stem cells reveals thousands of strong pause sites and novel translation products. These include amino-terminal extensions and truncations and upstream open reading frames with regulatory potential, initiated at both AUG and non-AUG codons, whose translation changes after differentiation. We also define a new class of short, polycistronic ribosome-associated coding RNAs (sprcRNAs) that encode small proteins. Our studies reveal an unanticipated complexity to mammalian proteomes.
Project description:Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where "x" is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.
Project description:Since 1992 PredictProtein (https://predictprotein.org) is a one-stop online resource for protein sequence analysis with its main site hosted at the Luxembourg Centre for Systems Biomedicine (LCSB) and queried monthly by over 3,000 users in 2020. PredictProtein was the first Internet server for protein predictions. It pioneered combining evolutionary information and machine learning. Given a protein sequence as input, the server outputs multiple sequence alignments, predictions of protein structure in 1D and 2D (secondary structure, solvent accessibility, transmembrane segments, disordered regions, protein flexibility, and disulfide bridges) and predictions of protein function (functional effects of sequence variation or point mutations, Gene Ontology (GO) terms, subcellular localization, and protein-, RNA-, and DNA binding). PredictProtein's infrastructure has moved to the LCSB increasing throughput; the use of MMseqs2 sequence search reduced runtime five-fold (apparently without lowering performance of prediction methods); user interface elements improved usability, and new prediction methods were added. PredictProtein recently included predictions from deep learning embeddings (GO and secondary structure) and a method for the prediction of proteins and residues binding DNA, RNA, or other proteins. PredictProtein.org aspires to provide reliable predictions to computational and experimental biologists alike. All scripts and methods are freely available for offline execution in high-throughput settings.
Project description:Class-switching to IgG2a/c in mice is a hallmark response to intracellular pathogens. T cells can promote class-switching and the predominant pathway for induction of IgG2a/c antibody responses has been suggested to be via stimulation from Th1 cells. We previously formulated CAF®01 (cationic liposomes containing dimethyldioctadecylammonium bromide (DDA) and Trehalose-6,6-dibehenate (TDB)) with the lipidated TLR7/8 agonist 3M-052 (DDA/TDB/3M-052), which promoted robust Th1 immunity in newborn mice. When testing this adjuvant in adult mice using the recombinant Chlamydia trachomatis (C.t.) vaccine antigen CTH522, it similarly enhanced IgG2a/c responses compared to DDA/TDB, but surprisingly reduced the magnitude of the IFN-g+ Th1 response in a TLR7 agonist dose-dependent manner. Single cell RNA-sequencing revealed that DDA/TDB/3M-052 liposomes initiated early transcription of class-switch regulating genes directly in pre-germinal center B cells. Mixed bone marrow chimeras further demonstrated that this adjuvant did not require Th1 cells for IgG2a/c switching, but rather facilitated TLR7-dependent T-bet programming directly in B cells. This study underlines that adjuvant-directed IgG2a/c class-switching in vivo can occur in the absence of T cell help, via direct activation of TLR7 on B cells and positions DDA/TDB/3M-052 as a powerful adjuvant capable of eliciting type I-like immunity in B cells without strong induction of Th1 responses.
Project description:The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. Here we present a suite of techniques, based on ribosome profiling (the deep-sequencing of ribosome-protected mRNA fragments), to provide genome-wide maps of protein synthesis as well as a pulse-chase strategy for determining rates of translation elongation. We exploit the propensity of harringtonine to cause ribosomes to accumulate at sites of translation initiation together with a machine learning algorithm to define protein products systematically. Analysis of translation in mouse embryonic stem cells reveals thousands of strong pause sites and novel translation products. These include amino-terminal extensions and truncations and upstream open reading frames with regulatory potential, initiated at both AUG and non-AUG codons, whose translation changes after differentiation. We also define a new class of short, polycistronic ribosome-associated coding RNAs (sprcRNAs) that encode small proteins. Our studies reveal an unanticipated complexity to mammalian proteomes. Examination of translation in mouse embryonic stem cells and during differentiation into embryoid bodies