The identification of specific biomarkers will improve the early diagnosis of disease, facilitate the development of targeted therapies, and provide an accurate method to monitor treatment response. ...A major challenge in the process of verifying biomarker candidates in blood plasma is the complexity and high dynamic range of proteins. This article reviews the current, targeted proteomic strategies that are capable of quantifying biomarker candidates at concentration ranges where biomarkers are expected in plasma (i.e. at the ng/ml level). In addition, a workflow is presented that allows the fast and definitive generation of targeted mass spectrometry-based assays for most biomarker candidate proteins. These assays are stored in publicly accessible databases and have the potential to greatly impact the throughput of biomarker verification studies.
Gap junction channels (GJCs) mediate intercellular communication by connecting two neighbouring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) ...GJCs is important in a wide range of cellular processes in health and disease (Churko and Laird, 2013; Liang et al., 2020; Poelzing and Rosenbaum, 2004), yet the structural basis of Cx43 function and regulation has not been determined until now. Here, we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Å resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels (HCs) in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and HC cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state.
The rise of systems biology implied a growing demand for highly sensitive techniques for the fast and consistent detection and quantification of target sets of proteins across multiple samples. This ...is only partly achieved by classical mass spectrometry or affinity-based methods. We applied a targeted proteomics approach based on selected reaction monitoring (SRM) to detect and quantify proteins expressed to a concentration below 50 copies/cell in total S. cerevisiae digests. The detection range can be extended to single-digit copies/cell and to proteins undetected by classical methods. We illustrate the power of the technique by the consistent and fast measurement of a network of proteins spanning the entire abundance range over a growth time course of S. cerevisiae transiting through a series of metabolic phases. We therefore demonstrate the potential of SRM-based proteomics to provide assays for the measurement of any set of proteins of interest in yeast at high-throughput and quantitative accuracy.
Metabolic activity is intimately linked to T cell fate and function. Using high-resolution mass spectrometry, we generated dynamic metabolome and proteome profiles of human primary naive T cells ...following activation. We discovered critical changes in the arginine metabolism that led to a drop in intracellular L-arginine concentration. Elevating L-arginine levels induced global metabolic changes including a shift from glycolysis to oxidative phosphorylation in activated T cells and promoted the generation of central memory-like cells endowed with higher survival capacity and, in a mouse model, anti-tumor activity. Proteome-wide probing of structural alterations, validated by the analysis of knockout T cell clones, identified three transcriptional regulators (BAZ1B, PSIP1, and TSN) that sensed L-arginine levels and promoted T cell survival. Thus, intracellular L-arginine concentrations directly impact the metabolic fitness and survival capacity of T cells that are crucial for anti-tumor responses.
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•Dataset on dynamic metabolome/proteome profiles of activated human naive T cells•Intracellular L-arginine levels regulate several metabolic pathways in T cells•T cells with increased L-arginine display enhanced survival and anti-tumor activity•LiP-MS identified proteins that are structurally modified by high L-arginine levels
Metabolomic and proteomic profiling unveil intracellular L-arginine as a crucial regulator of metabolic fitness, survival capacity, and anti-tumor activity of central memory T cells.
Microtremor measurements and the horizontal-to-vertical spectral ratio (HVSR)
technique, generally used for site effect studies as well as to determine the
thickness of soft sedimentary layers, can ...effectively be applied to map the
thickness of glaciers. In this work the radio-echo sounding, geoelectric and
active seismic methods, widely employed to image the earth interior, are applied
to verify the reliability of the HVSR technique in Alpine and Antarctic glacial
environments. The technique has been used to analyze passive seismic data from
glaciers of the Adamello and Ortles-Cevedale massifs (Italy), the Bernese
Oberland Alps (Switzerland) and from the Whillans Ice Stream (West Antarctica).
Comparing with the results obtained from the different geophysical imaging
methods, we show that the resonance frequency in the HVSR spectra correlates
well with the ice thickness at the site, in a wide range from a few tens of
meters to more than 800 m. The reliability of the method mainly depends
on the coupling of sensors at the glacier surface and on the basal impedance
contrast. This passive seismic technique offers a logistically efficient and
cost effective method to map glacier and ice-sheet thicknesses. Moreover, under
certain conditions, it allows reliable estimations of the basal seismic
properties.
Molecular profiling of small molecules offers invaluable insights into the function of compounds and allows for hypothesis generation about small-molecule direct targets and secondary effects. ...However, current profiling methods are limited in either the number of measurable parameters or throughput. Here we developed a multiplexed, unbiased framework that, by linking genetic to drug-induced changes in nearly a thousand metabolites, allows for high-throughput functional annotation of compound libraries in Escherichia coli. First, we generated a reference map of metabolic changes from CRISPR interference (CRISPRi) with 352 genes in all major essential biological processes. Next, on the basis of the comparison of genetic changes with 1,342 drug-induced metabolic changes, we made de novo predictions of compound functionality and revealed antibacterials with unconventional modes of action (MoAs). We show that our framework, combining dynamic gene silencing with metabolomics, can be adapted as a general strategy for comprehensive high-throughput analysis of compound functionality from bacteria to human cell lines.
Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of people with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). ...Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTLD cases into at least four subtypes, which are correlated with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for biochemical isolation of pathological TDP-43. By combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43 that become abnormally insoluble in a disease subtype-specific manner. We show that pTDP-43 extracted from brain forms stable assemblies of distinct densities and morphologies that are associated with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies showed differential neurotoxicity and seeding that were correlated with disease duration of FTLD subjects. Our data are consistent with the notion that disease heterogeneity could originate from alternate pathological TDP-43 conformations, which are reminiscent of prion strains.
As a frequent post‐translational modification, protein phosphorylation regulates many cellular processes. Although several hundred phosphorylation sites have been mapped to metabolic enzymes in ...Saccharomyces cerevisiae, functionality was demonstrated for few of them. Here, we describe a novel approach to identify in vivo functionality of enzyme phosphorylation by combining flux analysis with proteomics and phosphoproteomics. Focusing on the network of 204 enzymes that constitute the yeast central carbon and amino‐acid metabolism, we combined protein and phosphoprotein levels to identify 35 enzymes that change their degree of phosphorylation during growth under five conditions. Correlations between previously determined intracellular fluxes and phosphoprotein abundances provided first functional evidence for five novel phosphoregulated enzymes in this network, adding to nine known phosphoenzymes. For the pyruvate dehydrogenase complex E1 α subunit Pda1 and the newly identified phosphoregulated glycerol‐3‐phosphate dehydrogenase Gpd1 and phosphofructose‐1‐kinase complex β subunit Pfk2, we then validated functionality of specific phosphosites through absolute peptide quantification by targeted mass spectrometry, metabolomics and physiological flux analysis in mutants with genetically removed phosphosites. These results demonstrate the role of phosphorylation in controlling the metabolic flux realised by these three enzymes.
A strategy is presented that combines metabolic fluxes with targeted phosphoproteomics measurements to drive testable hypotheses for the functionality of post‐translational regulation in S. cerevisiae central metabolism.
Synopsis
A strategy is presented that combines metabolic fluxes with targeted phosphoproteomics measurements to drive testable hypotheses for the functionality of post‐translational regulation in S. cerevisiae central metabolism.
Discovery‐driven mass spectrometry phosphoproteomics identified 35 differentially phosphorylated enzymes of yeast central metabolism.
Phosphoenzymes are predominant in upper glycolysis, around the pyruvate node and in carbohydrate storage pathways.
A targeted phosphoproteomics method was developed to quantify total, phospho and non‐phosphoprotein directly from crude cell extracts.
Correlation of phosphoprotein levels with metabolic fluxes across conditions provided functional evidence for five novel phosphoregulated enzymes.
Functional follow‐ups demonstrated the inhibitory role of phosphorylation in controlling metabolic fluxes realised by Gpd1, Pda1 and Pfk2.
Mycobacterium tuberculosis
adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the ...vital role of ACs in cellular signalling is well established, the function of their transmembrane (TM) regions remains unknown. Here, we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 Å resolution. The TM helices 1–5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.
Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S ribosomal subunits. This involves final 18S rRNA processing and release of residual ...biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S subunits, we characterized pre-ribosomal subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors. We demonstrate that USP16 constitutes a component of late cytoplasmic pre-40S subunits that promotes the removal of ubiquitin from an internal lysine of ribosomal protein RPS27a/eS31.
deletion leads to late 40S subunit maturation defects, manifesting in incomplete processing of 18S rRNA and retarded recycling of late-acting ribosome biogenesis factors, revealing an unexpected contribution of USP16 to the ultimate step of 40S synthesis. Finally, ubiquitination of RPS27a appears to depend on active translation, pointing at a potential connection between 40S maturation and protein synthesis.
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