Heiko Horn

Proteome-Wide Mapping of the Drosophila Acetylome Demonstrates a High Degree of Conservation of Lysine Acetylation

Comparing the acetylomes with the phosphoproteomes of flies and humans suggests that phosphorylation sites may have evolved faster than did acetylation sites. Age of the Acetylome Acetylation and phosphorylation are regulatory posttranslational modifications that occur on proteins. With proteome-wide data in divergent species, insights regarding the evolution of these two regulatory processes can be revealed. Weinert et al. report the proteome-wide analysis of acetylated proteins in the fruit fly. Comparing the data on acetylated proteins in humans and flies with proteome sequences of nematodes and zebrafish indicated that acetylated sites were more conserved than were nonacetylated sites, and comparison of the human and fly acetylomes with their phosphoproteomes indicated that acetylation sites were more conserved than were phosphorylation sites. Acetylation intersected with another posttranslational modification, ubiquitylation: Acetylation occurred on one-third of human ubiquitin-conjugating E2 enzymes and influenced the activity of these enzymes, suggesting that acetylation provides another regulatory layer for this other type of posttranslational modification. Posttranslational modification of proteins by acetylation and phosphorylation regulates most cellular processes in living organisms. Surprisingly, the evolutionary conservation of phosphorylated serine and threonine residues is only marginally higher than that of unmodified serines and threonines. With high-resolution mass spectrometry, we identified 1981 lysine acetylation sites in the proteome of Drosophila melanogaster. We used data sets of experimentally identified acetylation and phosphorylation sites in Drosophila and humans to analyze the evolutionary conservation of these modification sites between flies and humans. Site-level conservation analysis revealed that acetylation sites are highly conserved, significantly more so than phosphorylation sites. Furthermore, comparison of lysine conservation in Drosophila and humans with that in nematodes and zebrafish revealed that acetylated lysines were significantly more conserved than were nonacetylated lysines. Bioinformatics analysis using Gene Ontology terms suggested that the proteins with conserved acetylation control cellular processes such as protein translation, protein folding, DNA packaging, and mitochondrial metabolism. We found that acetylation of ubiquitin-conjugating E2 enzymes was evolutionarily conserved, and mutation of a conserved acetylation site impaired the function of the human E2 enzyme UBE2D3. This systems-level analysis of comparative posttranslational modification showed that acetylation is an anciently conserved modification and suggests that phosphorylation sites may have evolved faster than acetylation sites.

Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense.

Significance Although advances in human genetics have shaped our understanding of many complex diseases, little is known about the mechanism of action of alleles that influence disease. By using mice expressing a Crohn disease (CD)-associated risk polymorphism (Atg16L1 T300A), we show that Atg16L1 T300A-expressing mice demonstrate abnormalities in Paneth cells (similar to patients with the risk polymorphism) and goblet cells. We show that Atg16L1 T300A protein is more susceptible to caspase-mediated cleavage than WT autophagy related 16-like 1 (Atg16L1), resulting in decreased protein stability and effects on antibacterial autophagy and inflammatory cytokine production. We also identify interacting proteins that contribute to autophagy-dependent immune responses. Understanding how ATG16L1 T300A modulates autophagy-dependent immune responses sheds light on the mechanisms that underlie initiation and progression of CD. A coding polymorphism (Thr300Ala) in the essential autophagy gene, autophagy related 16-like 1 (ATG16L1), confers increased risk for the development of Crohn disease, although the mechanisms by which single disease-associated polymorphisms contribute to pathogenesis have been difficult to dissect given that environmental factors likely influence disease initiation in these patients. Here we introduce a knock-in mouse model expressing the Atg16L1 T300A variant. Consistent with the human polymorphism, T300A knock-in mice do not develop spontaneous intestinal inflammation, but exhibit morphological defects in Paneth and goblet cells. Selective autophagy is reduced in multiple cell types from T300A knock-in mice compared with WT mice. The T300A polymorphism significantly increases caspase 3- and caspase 7-mediated cleavage of Atg16L1, resulting in lower levels of full-length Atg16Ll T300A protein. Moreover, Atg16L1 T300A is associated with decreased antibacterial autophagy and increased IL-1β production in primary cells and in vivo. Quantitative proteomics for protein interactors of ATG16L1 identified previously unknown nonoverlapping sets of proteins involved in ATG16L1-dependent antibacterial autophagy or IL-1β production. These findings demonstrate how the T300A polymorphism leads to cell type- and pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease.

KinomeXplorer: an integrated platform for kinome biology studies

or even impossible to be captured by cellular or in vivo experiments alone. Furthermore, it is difficult to design kinase perturbation experiments, because the kinome-wide selectivity and specificity of many kinase inhibitors is unknown3,4. As a result, knowledge is lacking on which of the ~540 human kinases phosphorylate a given site: of the 42,914 phosphorylation sites currently annotated in the Phospho.ELM database5, only ~20% have been linked to a kinase. Technological advances in mass spectrometry–based phosphoproteomics have accelerated the ability to identify phosphorylation sites but not to determine which kinases phosphorylate them. To systematically identify these dynamic interactions, computational methods to guide experiments must be deployed. We have shown that combining computational algorithms with quantitative mass spectrometry is a powerful approach to validate kinase-substrate relationships6. Notably, we have shown that kinase specificity can be described in terms of two main contributing elements: the recognition motif of the individual kinase (for example, X-S/T-Q-X for the ATM kinase) and proteins that can be functionally associated with it (i.e., not just proteins that directly interact with the kinase). The network context of kinases is crucial, as exemplified by the discovery that the phenotypic role of the JNK kinase depends entirely on the state of the cellular signaling networks before its activation7. In other words, it is crucial to assess the protein networks embedding kinases and how these are dynamically modulated (for example, through time or perturbations) to predict cell behavior8. KinomeXplorer (Fig. 1) provides workflows that enable researchers to efficiently analyze phosphorylationd e p e n d e nt i n t e r a c t i o n n e t w o r k s (Supplementary Fig. 1) and aids them in designing follow-up perturbation experiments. The platform includes improved versions of NetworKIN (an algorithm that integrates cellular context information and motif-based predictions)6 and NetPhorest (a phylogenetic tree–based algorithm to classify phosphorylation sites in terms of kinases and phosphobinding domains)9, conferring increased prediction accuracy through a novel Bayesian scoring scheme, broader kinome coverage, new phosphatome coverage and a redesigned unifying web interface. The framework also integrates the new KinomeSelector tool, which enables the user to select an optimal kinase panel to functionally perturb the predicted phosphorylation signaling networks. We re-engineered the NetworKIN algorithm to improve its performance and usability (Supplementary Note). To calculate the NetworKIN score, we combined the NetPhorest probability and the STRING-derived proximity score using KinomeXplorer: an integrated platform for kinome biology studies

Reflect: augmented browsing for the life scientist.

Anyone who regularly reads life science literature often comes across names of genes, proteins, or small molecules that they would like to know more about. To make this process easier, we have developed a new, free service called Reflect (http://reflect.ws) that can be installed as a plug-in to Firefox or Internet Explorer. Reflect tags gene, protein, and small molecule names in any web page, typically within a few seconds, and without affecting document layout. Clicking on a tagged gene or protein name opens a popup showing a concise summary that includes synonyms, database identifiers, sequence, domains, 3D structure, interaction partners, subcellular location, and related literature. Clicking on a tagged small molecule name opens a popup showing 2D structure and interaction partners. The popups also allow navigation to commonly used databases. In the future we plan to add further entity types to Reflect, including outside the life sciences.

In Vivo Phosphoproteomics Analysis Reveals the Cardiac Targets of β-Adrenergic Receptor Signaling

Analysis of phosphorylated proteins from the hearts of mice given drugs targeting β-adrenergic receptors may aid in treating heart disease. Getting to the Heart of Signaling Patients with high blood pressure and other heart-related conditions routinely take inhibitors of β-adrenergic receptors (βARs) to prevent cardiac dysfunction. βAR signaling leads to the increased contractility of cardiomyocytes, among other effects; however, the number of downstream targets of βARs is unclear. Lundby et al. treated mice with combinations of specific β1AR and β2AR agonists and antagonists to activate each receptor isoform individually before harvesting the hearts and subjecting them to phosphoproteomics analysis. The authors identified previously uncharacterized peptides and sites phosphorylated in response to β1AR signaling, as well as characterized the activation of a potassium channel important for increasing heart rate. This in vivo approach provides insights into βAR signaling pathways that may help in understanding how heart diseases develop and how they may be treated. β-Blockers are widely used to prevent cardiac arrhythmias and to treat hypertension by inhibiting β-adrenergic receptors (βARs) and thus decreasing contractility and heart rate. βARs initiate phosphorylation-dependent signaling cascades, but only a small number of the target proteins are known. We used quantitative in vivo phosphoproteomics to identify 670 site-specific phosphorylation changes in murine hearts in response to acute treatment with specific βAR agonists. The residues adjacent to the regulated phosphorylation sites exhibited a sequence-specific preference (R-X-X-pS/T), and integrative analysis of sequence motifs and interaction networks suggested that the kinases AMPK (adenosine 5′-monophosphate–activated protein kinase), Akt, and mTOR (mammalian target of rapamycin) mediate βAR signaling, in addition to the well-established pathways mediated by PKA (cyclic adenosine monophosphate–dependent protein kinase) and CaMKII (calcium/calmodulin-dependent protein kinase type II). We found specific regulation of phosphorylation sites on six ion channels and transporters that mediate increased ion fluxes at higher heart rates, and we showed that phosphorylation of one of these, Ser92 of the potassium channel KV7.1, increased current amplitude. Our data set represents a quantitative analysis of phosphorylated proteins regulated in vivo upon stimulation of seven-transmembrane receptors, and our findings reveal previously unknown phosphorylation sites that regulate myocardial contractility, suggesting new potential targets for the treatment of heart disease and hypertension.

Proteomic Analysis of Arginine Methylation Sites in Human Cells Reveals Dynamic Regulation During Transcriptional Arrest

The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function, and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein arginine methyltransferases; however, very little is known about which arginine residues become methylated on target substrates. Here we describe a proteomics methodology that combines single-step immunoenrichment of methylated peptides with high-resolution mass spectrometry to identify endogenous arginine mono-methylation (MMA) sites. We thereby identify 1027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared with the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers strong site-specific regulation of MMA sites during transcriptional arrest. Interestingly, several MMA sites are down-regulated after a few hours of transcriptional arrest. In contrast, the corresponding di-methylation or protein expression levels are not altered, confirming that MMA sites contain regulated functions on their own. Collectively, we present a site-specific MMA data set in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase.

Systematic functional interrogation of rare cancer variants identifies oncogenic alleles

UNLABELLED Cancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency, and the function of these alleles remains undefined. We have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. We subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles, including two in genes (PIK3CB, POT1) that have not been shown to be tumorigenic. One rare KRAS allele, D33E, displayed tumorigenicity and constitutive activation of known RAS effector pathways. By comparing gene expression changes induced upon expression of wild-type and mutant alleles, we inferred the activity of specific alleles. Because alleles found to be mutated only once in 5,338 tumors rendered cells tumorigenic, these observations underscore the value of integrating genomic information with functional studies. SIGNIFICANCE Experimentally inferring the functional status of cancer-associated mutations facilitates the interpretation of genomic information in cancer. Pooled in vivo screen and gene expression profiling identified functional variants and demonstrated that expression of rare variants induced tumorigenesis. Variant phenotyping through functional studies will facilitate defining key somatic events in cancer. Cancer Discov; 6(7); 714-26. ©2016 AACR.See related commentary by Cho and Collisson, p. 694This article is highlighted in the In This Issue feature, p. 681.

Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation

Proteomic analysis of neurotrophin signaling identifies an inhibitory E3 ubiquitin ligase. Proteomic analysis reveals an inhibitor Neurotrophins, such as nerve growth factor (NGF), control the differentiation and proliferation of neuronal precursors, and the outcome depends on the duration of the signal and neurotrophin-receptor pair. NGF binding to the receptor tyrosine kinase TrkA induces neuronal differentiation and neurite outgrowth. By mediating the attachment of ubiquitin chains, E3 ubiquitin ligases stimulate the internalization and degradation, and hence reduce the activity, of various receptors. Emdal et al. performed a temporal analysis by mass spectrometry of changes in the proteome in response to NGF in neuroblastoma cells and found that NGF not only promoted the activation of TrkA but also signaled its degradation by promoting the interaction of TrkA with the E3 ubiquitin ligase Cbl-b, which resulted in the ubiquitylation and degradation of both proteins. Neuroblastoma cells with reduced Cbl-b had increased TrkA signaling and produced longer neurites. In addition to identifying this inhibitory role for Cbl-b, the proteomic data are a resource for further investigation of TrkA signaling dynamics. SH-SY5Y neuroblastoma cells respond to nerve growth factor (NGF)–mediated activation of the tropomyosin-related kinase A (TrkA) with neurite outgrowth, thereby providing a model to study neuronal differentiation. We performed a time-resolved analysis of NGF-TrkA signaling in neuroblastoma cells using mass spectrometry–based quantitative proteomics. The combination of interactome, phosphoproteome, and proteome data provided temporal insights into the molecular events downstream of NGF binding to TrkA. We showed that upon NGF stimulation, TrkA recruits the E3 ubiquitin ligase Cbl-b, which then becomes phosphorylated and ubiquitylated and decreases in abundance. We also found that recruitment of Cbl-b promotes TrkA ubiquitylation and degradation. Furthermore, the amount of phosphorylation of the kinase ERK and neurite outgrowth increased upon Cbl-b depletion in several neuroblastoma cell lines. Our findings suggest that Cbl-b limits NGF-TrkA signaling to control the length of neurites.

DistiLD Database: diseases and traits in linkage disequilibrium blocks

Genome-wide association studies (GWAS) have identified thousands of single nucleotide polymorphisms (SNPs) associated with the risk of hundreds of diseases. However, there is currently no database that enables non-specialists to answer the following simple questions: which SNPs associated with diseases are in linkage disequilibrium (LD) with a gene of interest? Which chromosomal regions have been associated with a given disease, and which are the potentially causal genes in each region? To answer these questions, we use data from the HapMap Project to partition each chromosome into so-called LD blocks, so that SNPs in LD with each other are preferentially in the same block, whereas SNPs not in LD are in different blocks. By projecting SNPs and genes onto LD blocks, the DistiLD database aims to increase usage of existing GWAS results by making it easy to query and visualize disease-associated SNPs and genes in their chromosomal context. The database is available at http://distild.jensenlab.org/.

TIMP-1 increases expression and phosphorylation of proteins associated with drug resistance in breast cancer cells.

Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. To elucidate the unknown molecular mechanisms underlying the association between high TIMP-1 levels and increased chemotherapy resistance, we employed SILAC-based quantitative mass spectrometry to analyze global proteome and phosphoproteome differences of MCF-7 breast cancer cells expressing high or low levels of TIMP-1. In TIMP-1 high expressing cells, 312 proteins and 452 phosphorylation sites were up-regulated. Among these were the cancer drug targets topoisomerase 1, 2A, and 2B, which may explain the resistance phenotype to topoisomerase inhibitors that was observed in cells with high TIMP-1 levels. Pathway analysis showed an enrichment of proteins from functional categories such as apoptosis, cell cycle, DNA repair, transcription factors, drug targets and proteins associated with drug resistance or sensitivity, and drug transportation. The NetworKIN algorithm predicted the protein kinases CK2a, CDK1, PLK1, and ATM as likely candidates involved in the hyperphosphorylation of the topoisomerases. Up-regulation of protein and/or phosphorylation levels of topoisomerases in TIMP-1 high expressing cells may be part of the mechanisms by which TIMP-1 confers resistance to treatment with the widely used topoisomerase inhibitors in breast and colorectal cancer.