The Calmodulin-interacting peptide Pcp4a regulates feeding state-dependent behavioral choice in zebrafish.

Animals constantly need to judge the valence of an object in their environment: is it potential food or a threat? The brain makes fundamental decisions on the appropriate behavioral strategy by integrating external information from sensory organs and internal signals related to physiological needs. For example, a hungry animal may take more risks than a satiated one when deciding to approach or avoid an object. Using a proteomic profiling approach, we identified the Calmodulin-interacting peptide Pcp4a as a key regulator of foraging-related decisions. Food intake reduced abundance of protein and mRNA of pcp4a via dopamine D2-like receptor-mediated repression of adenylate cyclase. Accordingly, deleting the pcp4a gene made zebrafish larvae more risk averse in a binary decision assay. Strikingly, neurons in the tectum became less responsive to prey-like visual stimuli in pcp4a mutants, thus biasing the behavior toward avoidance. This study pinpoints a molecular mechanism modulating behavioral choice according to internal state.

PEAK1 Y635 phosphorylation regulates cell migration through association with Tensin3 and integrins.

Integrins mediate cell adhesion by connecting the extracellular matrix to the intracellular cytoskeleton and orchestrate signal transduction in response to chemical and mechanical stimuli by interacting with many cytoplasmic proteins. We used BioID to interrogate the interactomes of ß1 and ß3 integrins in epithelial cells and identified PEAK1 as an interactor of the RGD-binding integrins α5ß1, αVß3, and αVß5 in focal adhesions. We demonstrate that the interaction between integrins and PEAK1 occurs indirectly through Tensin3, requiring both the membrane-proximal NPxY motif on the integrin ß tail and binding of the SH2 domain of Tensin3 to phosphorylated Tyr-635 on PEAK1. Phosphorylation of Tyr-635 is mediated by Src and regulates cell migration. Additionally, we found that Shc1 localizes in focal adhesions in a PEAK1 phosphorylated Tyr-1188-dependent fashion. Besides binding Shc1, PEAK1 also associates with a protein cluster that mediates late EGFR/Shc1 signaling. We propose a model in which PEAK1 binds Tensin3 and Shc1 to converge integrin and growth factor receptor signal transduction.

Accurate and Automated High-Coverage Identification of Chemically Cross-Linked Peptides with MaxLynx.

Cross-linking combined with mass spectrometry (XL-MS) provides a wealth of information about the three-dimensional (3D) structure of proteins and their interactions. We introduce MaxLynx, a novel computational proteomics workflow for XL-MS integrated into the MaxQuant environment. It is applicable to noncleavable and MS-cleavable cross-linkers. For both, we have generalized the Andromeda peptide database search engine to efficiently identify cross-linked peptides. For noncleavable peptides, we implemented a novel dipeptide Andromeda score, which is the basis for a computationally efficient N-squared search engine. Additionally, partial scores summarize the evidence for the two constituents of the dipeptide individually. A posterior error probability (PEP) based on total and partial scores is used to control false discovery rates (FDRs). For MS-cleavable cross-linkers, a score of signature peaks is combined with the conventional Andromeda score on the cleavage products. The MaxQuant 3D peak detection was improved to ensure more accurate determination of the monoisotopic peak of isotope patterns for heavy molecules, which cross-linked peptides typically are. A wide selection of filtering parameters can replace the manual filtering of identifications, which is often necessary when using other pipelines. On benchmark data sets of synthetic peptides, MaxLynx outperforms all other tested software on data for both types of cross-linkers and on a proteome-wide data set of cross-linked Drosophila melanogaster cell lysate. The workflow also supports ion mobility-enhanced MS data. MaxLynx runs on Windows and Linux, contains an interactive viewer for displaying annotated cross-linked spectra, and is freely available at https://www.maxquant.org/.

Signatures of muscle disuse in spaceflight and bed rest revealed by single muscle fiber proteomics.

Astronauts experience dramatic loss of muscle mass, decreased strength, and insulin resistance, despite performing daily intense physical exercise that would lead to muscle growth on Earth. Partially mimicking spaceflight, prolonged bed rest causes muscle atrophy, loss of force, and glucose intolerance. To unravel the underlying mechanisms, we employed highly sensitive single fiber proteomics to detail the molecular remodeling caused by unloading and inactivity during bed rest and changes of the muscle proteome of astronauts before and after a mission on the International Space Station. Muscle focal adhesions, involved in fiber-matrix interaction and insulin receptor stabilization, are prominently downregulated in both bed rest and spaceflight and restored upon reloading. Pathways of antioxidant response increased strongly in slow but not in fast muscle fibers. Unloading alone upregulated markers of neuromuscular damage and the pathway controlling EIF5A hypusination. These proteomic signatures of mechanical unloading in muscle fiber subtypes contribute to disentangle the effect of microgravity from the pleiotropic challenges of spaceflight.

MaxDIA enables library-based and library-free data-independent acquisition proteomics.

MaxDIA is a software platform for analyzing data-independent acquisition (DIA) proteomics data within the MaxQuant software environment. Using spectral libraries, MaxDIA achieves deep proteome coverage with substantially better coefficients of variation in protein quantification than other software. MaxDIA is equipped with accurate false discovery rate (FDR) estimates on both library-to-DIA match and protein levels, including when using whole-proteome predicted spectral libraries. This is the foundation of discovery DIA-hypothesis-free analysis of DIA samples without library and with reliable FDR control. MaxDIA performs three- or four-dimensional feature detection of fragment data, and scoring of matches is augmented by machine learning on the features of an identification. MaxDIA's bootstrap DIA workflow performs multiple rounds of matching with increasing quality of recalibration and stringency of matching to the library. Combining MaxDIA with two new technologies-BoxCar acquisition and trapped ion mobility spectrometry-both lead to deep and accurate proteome quantification.

Tissue-specific modulation of gene expression in response to lowered insulin signalling in Drosophila.

Reduced activity of the insulin/IGF signalling network increases health during ageing in multiple species. Diverse and tissue-specific mechanisms drive the health improvement. Here, we performed tissue-specific transcriptional and proteomic profiling of long-lived Drosophila dilp2-3,5 mutants, and identified tissue-specific regulation of >3600 transcripts and >3700 proteins. Most expression changes were regulated post-transcriptionally in the fat body, and only in mutants infected with the endosymbiotic bacteria, Wolbachia pipientis, which increases their lifespan. Bioinformatic analysis identified reduced co-translational ER targeting of secreted and membrane-associated proteins and increased DNA damage/repair response proteins. Accordingly, age-related DNA damage and genome instability were lower in fat body of the mutant, and overexpression of a minichromosome maintenance protein subunit extended lifespan. Proteins involved in carbohydrate metabolism showed altered expression in the mutant intestine, and gut-specific overexpression of a lysosomal mannosidase increased autophagy, gut homeostasis, and lifespan. These processes are candidates for combatting ageing-related decline in other organisms.

Loss of the psychiatric risk factor SLC6A15 is associated with increased metabolic functions in primary hippocampal neurons.

Major depressive disorder (MDD) is one of the most severe global health problems with millions of people affected, however, the mechanisms underlying this disorder is still poorly understood. Genome-wide association studies have highlighted a link between the neutral amino acid transporter SLC6A15 and MDD. Additionally, a number of preclinical studies support the function of this transporter in modulating levels of brain neurotransmitters, stress system regulation and behavioural phenotypes related to MDD. However, the molecular and functional mechanisms involved in this interaction are still unresolved. Therefore, to investigate the effects of the SLC6A15 transporter, we used hippocampal tissue from Slc6a15-KO and wild-type mice, together with several in-vitro assays in primary hippocampal neurons. Utilizing a proteomics approach we identified differentially regulated proteins that formed a regulatory network and pathway analysis indicated significantly affected cellular domains, including metabolic, mitochondrial and structural functions. Furthermore, we observed reduced release probability at glutamatergic synapses, increased mitochondrial function, higher GSH/GSSG redox ratio and an improved neurite outgrowth in primary neurons lacking SLC6A15. In summary, we hypothesize that by controlling the intracellular concentrations of neutral amino acids, SLC6A15 affects mitochondrial activity, which could lead to alterations in neuronal structure and activity. These data provide further indication that a pharmacological or genetic reduction of SLC6A15 activity may indeed be a promising approach for antidepressant therapy.

Identification of signal peptide features for substrate specificity in human Sec62/Sec63-dependent ER protein import.

In mammalian cells, one-third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61 channel. While the Sec61 complex facilitates ER import of most precursor polypeptides, the Sec61-associated Sec62/Sec63 complex supports ER import in a substrate-specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63 complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified 22 novel Sec62/Sec63 substrates under these in vivo-like conditions. As a common feature, those previously unknown substrates share signal peptides (SP) with comparatively longer but less hydrophobic hydrophobic region of SP and lower carboxy-terminal region of SP (C-region) polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly gating SP and a downstream translocation-disruptive positively charged cluster of amino acid residues as decisive for the Sec62/Sec63 requirement. In the case of ERj3, these features were found to be responsible for an additional immunoglobulin heavy-chain binding protein (BiP) requirement and to correlate with sensitivity toward the Sec61-channel inhibitor CAM741. Thus, the human Sec62/Sec63 complex may support Sec61-channel opening for precursor polypeptides with slowly gating SPs by direct interaction with the cytosolic amino-terminal peptide of Sec61α or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61α. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63-linked polycystic liver disease. DATABASES: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/projects/Identifiers) with the dataset identifiers: PXD008178, PXD011993, and PXD012078. Supplementary information was deposited at Mendeley Data (https://data.mendeley.com/datasets/6s5hn73jcv/2).

SILAC-based quantitative proteomics using mass spectrometry quantifies endoplasmic reticulum stress in whole HeLa cells.

The unfolded protein response (UPR) involves extensive proteome remodeling in many cellular compartments. To date, a comprehensive analysis of the UPR has not been possible because of technological limitations. Here, we employ stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics to quantify the response of over 6200 proteins to increasing concentrations of tunicamycin in HeLa cells. We further compare the effects of tunicamycin (5 µg/ml) to those of thapsigargin (1 µM) and DTT (2 mM), both activating the UPR through different mechanisms. This systematic quantification of the proteome-wide expression changes that follow proteostatic stress is a resource for the scientific community, enabling the discovery of novel players involved in the pathophysiology of the broad range of disorders linked to proteostasis. We identified increased expression in 38 proteins not previously linked to the UPR, of which 15 likely remediate ER stress, and the remainder may contribute to pathological outcomes. Unexpectedly, there are few strongly downregulated proteins, despite expression of the pro-apoptotic transcription factor CHOP, suggesting that IRE1-dependent mRNA decay (RIDD) has a limited contribution to ER stress-mediated cell death in our system.

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

A proteomic atlas of insulin signalling reveals tissue-specific mechanisms of longevity assurance.

Lowered activity of the insulin/IGF signalling (IIS) network can ameliorate the effects of ageing in laboratory animals and, possibly, humans. Although transcriptome remodelling in long-lived IIS mutants has been extensively documented, the causal mechanisms contributing to extended lifespan, particularly in specific tissues, remain unclear. We have characterized the proteomes of four key insulin-sensitive tissues in a long-lived Drosophila IIS mutant and control, and detected 44% of the predicted proteome (6,085 proteins). Expression of ribosome-associated proteins in the fat body was reduced in the mutant, with a corresponding, tissue-specific reduction in translation. Expression of mitochondrial electron transport chain proteins in fat body was increased, leading to increased respiration, which was necessary for IIS-mediated lifespan extension, and alone sufficient to mediate it. Proteasomal subunits showed altered expression in IIS mutant gut, and gut-specific over-expression of the RPN6 proteasomal subunit, was sufficient to increase proteasomal activity and extend lifespan, whilst inhibition of proteasome activity abolished IIS-mediated longevity. Our study thus uncovered strikingly tissue-specific responses of cellular processes to lowered IIS acting in concert to ameliorate ageing.

Single Muscle Fiber Proteomics Reveals Fiber-Type-Specific Features of Human Muscle Aging.

Skeletal muscle is a key tissue in human aging, which affects different muscle fiber types unequally. We developed a highly sensitive single muscle fiber proteomics workflow to study human aging and show that the senescence of slow and fast muscle fibers is characterized by diverging metabolic and protein quality control adaptations. Whereas mitochondrial content declines with aging in both fiber types, glycolysis and glycogen metabolism are upregulated in slow but downregulated in fast muscle fibers. Aging mitochondria decrease expression of the redox enzyme monoamine oxidase A. Slow fibers upregulate a subset of actin and myosin chaperones, whereas an opposite change happens in fast fibers. These changes in metabolism and sarcomere quality control may be related to the ability of slow, but not fast, muscle fibers to maintain their mass during aging. We conclude that single muscle fiber analysis by proteomics can elucidate pathophysiology in a sub-type-specific manner.

Calsequestrins in skeletal and cardiac muscle from adult Danio rerio.

Calsequestrin (Casq) is a high capacity, low affinity Ca(2+)-binding protein, critical for Ca(2+)-buffering in cardiac and skeletal muscle sarcoplasmic reticulum. All vertebrates have multiple genes encoding for different Casq isoforms. Increasing interest has been focused on mammalian and human Casq genes since mutations of both cardiac (Casq2) and skeletal muscle (Casq1) isoforms cause different, and sometime severe, human pathologies. Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work, expression, biochemical properties cellular and sub-cellular localization of D. rerio native Casq isoforms are investigated. By quantitative PCR, three mRNAs were detected in skeletal muscle and heart with different abundances. Three zebrafish Casqs: Casq1a, Casq1b and Casq2 were identified by mass spectrometry (Data are available via ProteomeXchange with identifier PXD002455). Skeletal and cardiac zebrafish calsequestrins share properties with mammalian Casq1 and Casq2. Skeletal Casqs were found primarily, but not exclusively, at the sarcomere Z-line level where terminal cisternae of sarcoplasmic reticulum are located.

A human interactome in three quantitative dimensions organized by stoichiometries and abundances.

The organization of a cell emerges from the interactions in protein networks. The interactome is critically dependent on the strengths of interactions and the cellular abundances of the connected proteins, both of which span orders of magnitude. However, these aspects have not yet been analyzed globally. Here, we have generated a library of HeLa cell lines expressing 1,125 GFP-tagged proteins under near-endogenous control, which we used as input for a next-generation interaction survey. Using quantitative proteomics, we detect specific interactions, estimate interaction stoichiometries, and measure cellular abundances of interacting proteins. These three quantitative dimensions reveal that the protein network is dominated by weak, substoichiometric interactions that play a pivotal role in defining network topology. The minority of stable complexes can be identified by their unique stoichiometry signature. This study provides a rich interaction dataset connecting thousands of proteins and introduces a framework for quantitative network analysis.

Parasite-induced ER stress response in hepatocytes facilitates Plasmodium liver stage infection.

Upon infection of a mammalian host, Plasmodium parasites first replicate inside hepatocytes, generating thousands of new parasites. Although Plasmodium intra-hepatic development represents a substantial metabolic challenge to the host hepatocyte, how infected cells respond to and integrate this stress remains poorly understood. Here, we present proteomic and transcriptomic analyses, revealing that the endoplasmic reticulum (ER)-resident unfolded protein response (UPR) is activated in host hepatocytes upon Plasmodium berghei infection. The expression of XBP1s--the active form of the UPR mediator XBP1--and the liver-specific UPR mediator CREBH is induced by P. berghei infection in vivo. Furthermore, this UPR induction increases parasite liver burden. Altogether, our data suggest that ER stress is a central feature of P. berghei intra-hepatic development, contributing to the success of infection.

DNA repair. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links.

DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we identified SLF1 and SLF2, which form a complex with RAD18 and together define a pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides a global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells.

Single muscle fiber proteomics reveals unexpected mitochondrial specialization.

Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high-sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity.

Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog.

Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.

Phosphorylation of TET proteins is regulated via O-GlcNAcylation by the O-linked N-acetylglucosamine transferase (OGT).

TET proteins oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine and thus provide a possible means for active DNA demethylation in mammals. Although their catalytic mechanism is well characterized and the catalytic dioxygenase domain is highly conserved, the function of the regulatory regions (the N terminus and the low-complexity insert between the two parts of the dioxygenase domains) is only poorly understood. Here, we demonstrate that TET proteins are subject to a variety of post-translational modifications that mostly occur at these regulatory regions. We mapped TET modification sites at amino acid resolution and show for the first time that TET1, TET2, and TET3 are highly phosphorylated. The O-linked GlcNAc transferase, which we identified as a strong interactor with all three TET proteins, catalyzes the addition of a GlcNAc group to serine and threonine residues of TET proteins and thereby decreases both the number of phosphorylation sites and site occupancy. Interestingly, the different TET proteins display unique post-translational modification patterns, and some modifications occur in distinct combinations. In summary, our results provide a novel potential mechanism for TET protein regulation based on a dynamic interplay of phosphorylation and O-GlcNAcylation at the N terminus and the low-complexity insert region. Our data suggest strong cross-talk between the modification sites that could allow rapid adaption of TET protein localization, activity, or targeting due to changing environmental conditions as well as in response to external stimuli.

Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.