Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma.

Cancer cells must overcome anoikis (detachment-induced death) to successfully metastasize. Using proteomic screens, we found that distinct oncoproteins upregulate IL1 receptor accessory protein (IL1RAP) to suppress anoikis. IL1RAP is directly induced by oncogenic fusions of Ewing sarcoma, a highly metastatic childhood sarcoma. IL1RAP inactivation triggers anoikis and impedes metastatic dissemination of Ewing sarcoma cells. Mechanistically, IL1RAP binds the cell-surface system Xc - transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and the glutathione antioxidant. Under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Therefore, IL1RAP maintains cyst(e)ine and glutathione pools, which are vital for redox homeostasis and anoikis resistance. IL1RAP is minimally expressed in pediatric and adult normal tissues, and human anti-IL1RAP antibodies induce potent antibody-dependent cellular cytotoxicity of Ewing sarcoma cells. Therefore, we define IL1RAP as a new cell-surface target in Ewing sarcoma, which is potentially exploitable for immunotherapy. SIGNIFICANCE: Here, we identify cell-surface protein IL1RAP as a key driver of metastasis in Ewing sarcoma, a highly aggressive childhood sarcoma. Minimal expression in pediatric and adult normal tissues nominates IL1RAP as a promising target for immunotherapy.See related commentary by Yoon and DeNicola, p. 2679.This article is highlighted in the In This Issue feature, p. 2659.

Deep Profiling of the Cleavage Specificity and Human Substrates of Snake Venom Metalloprotease HF3 by Proteomic Identification of Cleavage Site Specificity (PICS) Using Proteome Derived Peptide Libraries and Terminal Amine Isotopic Labeling of Substrates (TAILS) N-Terminomics.

Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.

Melanocyte development in the mouse tail epidermis requires the Adamts9 metalloproteinase.

The mouse tail has an important role in the study of melanogenesis, because mouse tail skin can be used to model human skin pigmentation. To better understand the development of melanocytes in the mouse tail, we cloned two dominant ENU-generated mutations of the Adamts9 gene, Und3 and Und4, which cause an unpigmented ring of epidermis in the middle of the tail, but do not alter pigmentation in the rest of the mouse. Adamts9 encodes a widely expressed zinc metalloprotease with thrombospondin type 1 repeats with few known substrates. Melanocytes are lost in the Adamts9 mutant tail epidermis at a relatively late stage of development, around E18.5. Studies of our Adamts9 conditional allele suggest that there is a melanocyte cell-autonomous requirement for Adamts9. In addition, we used a proteomics approach, TAILS N-terminomics, to identify new Adamts9 candidate substrates in the extracellular matrix of the skin. The tail phenotype of Adamts9 mutants is strikingly similar to the unpigmented trunk belt in Adamts20 mutants, which suggests a particular requirement for Adamts family activity at certain positions along the anterior-posterior axis.

Interactome disassembly during apoptosis occurs independent of caspase cleavage.

Protein-protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas-mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome-wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases.

TAILS N-Terminomics and Proteomics Show Protein Degradation Dominates over Proteolytic Processing by Cathepsins in Pancreatic Tumors.

Deregulated cathepsin proteolysis occurs across numerous cancers, but in vivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS), a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Z in vivo substrates and cleavage sites with the use of six different cathepsin knockout genotypes in the Rip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114 N termini identified by TAILS, stable proteolytic products were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%-44% of 139 cleavages). This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%-83%) for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP78, and the oncoprotein prothymosin-alpha. Thus, the identification of cathepsin substrates in tumorigenesis improves the understanding of cathepsin functions in normal physiology and cancer.

Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products.

Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.

Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.

Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.

Metadegradomics: toward in vivo quantitative degradomics of proteolytic post-translational modifications of the cancer proteome.

Post-translational modifications enable extra layers of control of the proteome, and perhaps the most important is proteolysis, a major irreversible modification affecting every protein. The intersection of the protease web with a proteome sculpts that proteome, dynamically modifying its state and function. Protease expression is distorted in cancer, so perturbing signaling pathways and the secretome of the tumor and reactive stromal cells. Indeed many cancer biomarkers are stable proteolytic fragments. It is crucial to determine which proteases contribute to the pathology versus their roles in homeostasis and in mitigating cancer. Thus the full substrate repertoire of a protease, termed the substrate degradome, must be deciphered to define protease function and to identify drug targets. Degradomics has been used to identify many substrates of matrix metalloproteinases that are important proteases in cancer. Here we review recent degradomics technologies that allow for the broadly applicable identification and quantification of proteases (the protease degradome) and their activity state, substrates, and interactors. Quantitative proteomics using stable isotope labeling, such as ICAT, isobaric tags for relative and absolute quantification (iTRAQ), and stable isotope labeling by amino acids in cell culture (SILAC), can reveal protease substrates by taking advantage of the natural compartmentalization of membrane proteins that are shed into the extracellular space. Identifying the actual cleavage sites in a complex proteome relies on positional proteomics and utilizes selection strategies to enrich for protease-generated neo-N termini of proteins. In so doing, important functional information is generated. Finally protease substrates and interactors can be identified by interactomics based on affinity purification of protease complexes using exosite scanning and inactive catalytic domain capture strategies followed by mass spectrometry analysis. At the global level, the N terminome analysis of whole communities of proteases in tissues and organs in vivo provides a full scale understanding of the protease web and the web-sculpted proteome, so defining metadegradomics.

Testosterone regulation of homocysteine metabolism modulates redox status in human prostate cancer cells.

Clearance of homocysteine via the transsulfuration pathway provides an endogenous route for cysteine synthesis and represents a quantitatively significant source of this amino acid needed for glutathione synthesis. Men have higher plasma levels of total homocysteine than do women, but the mechanism of this sex-dependent difference is not known. In this study, we investigated regulation by testosterone of cystathionine beta-synthase (CBS), which catalyzes the committing step in the transsulfuration pathway. We report that testosterone downregulates CBS expression via a posttranscriptional mechanism in the androgen-responsive prostate cancer cell line, LNCaP. This diminution in CBS levels is accompanied by a decrease in flux through the transsulfuration pathway and by a lower intracellular glutathione concentration. The lower antioxidant capacity in testosterone-treated prostate cancer cells increases their susceptibility to oxidative stress conditions. These results demonstrate regulation of the homocysteine-clearing enzyme, CBS, by testosterone and suggest the potential utility of targeting this enzyme as a chemotherapeutic strategy.

Testosterone regulation of renal cystathionine beta-synthase: implications for sex-dependent differences in plasma homocysteine levels.

Elevated plasma total homocysteine (tHcy) is an independent risk factor for ischemic heart disease and stroke. Epidemiological studies reveal that men have higher tHcy levels than women, but the mechanism underlying this sex-dependent difference is unknown. One route for intracellular disposal of homocysteine is catalyzed by cystathionine beta-synthase (CBS). Renal function is known to be an important determinant of tHcy, and, in this study, we demonstrate that renal CBS expression and activity in mice diminished approximately twofold after castration, whereas ovariectomization was without effect. The higher renal CBS activity in males (22.7 +/- 3.1 mmol cystathionine.h(-1).kg kidney(-1)) vs. females (8.4 +/- 3.4 mmol cystathionine.h(-1).kg kidney(-1), P < or = 10(-6)) in C57Bl/6J mice was associated with lower plasma tHcy levels in males vs. females, and this difference was exacerbated in Cbs+/- mice (7.7 +/- 1.9 micromol/l in males vs. 13.8 +/- 6.4 micromol/l in females, P = 0.005). Surprisingly, mammals exhibit a diversity of regulatory patterns for kidney CBS, with females exhibiting lower CBS activity in mice, higher in rats and humans, and being indistinguishable from males in rabbit, hamster, and guinea pig. Our data suggest that testosterone-dependent regulation of human CBS in kidney may contribute to sex-dependent differences in homocysteine transsulfuration.

S-adenosylmethionine stabilizes cystathionine beta-synthase and modulates redox capacity.

The transsulfuration pathway converts homocysteine to cysteine and represents the metabolic link between antioxidant and methylation metabolism. The first and committing step in this pathway is catalyzed by cystathionine beta-synthase (CBS), which is subject to complex regulation, including allosteric activation by the methyl donor, S-adenosylmethionine (AdoMet). In this study, we demonstrate that methionine restriction leads to a >10-fold decrease in CBS protein levels, and pulse proteolysis studies reveal that binding of AdoMet stabilizes the protein against degradation by approximately 12 kcal/mol. These observations predict that under pathological conditions where AdoMet levels are diminished, CBS, and therefore glutathione levels, will be reduced. Indeed, we demonstrate this to be the case in a mouse model for spontaneous steatohepatitis in which the gene for the MAT1A isoenzyme encoding AdoMet synthetase has been disrupted, and in human hepatocellular carcinoma, where MAT1A is silenced. Furthermore, diminished CBS levels are associated with reduced cell viability in hepatoma cells challenged with tert-butyl hydroperoxide. This study uncovers a mechanism by which CBS is allosterically activated by AdoMet under normal conditions but is destabilized under pathological conditions, for redirecting the metabolic flux toward methionine conservation. A mechanistic basis for the coordinate changes in redox and methylation metabolism that are a hallmark of several complex diseases is explained by these observations.

Analysis of pathological defects in methionine metabolism using a simple mathematical model.

Derangements in methionine metabolism are a hallmark of cancers and homocystinuria, an inborn error of metabolism. In this study, the metabolic consequences of the pathological changes associated with the key pathway enzymes, methionine adenosyl transferase (MAT), glycine N-methyl transferase (GNMT) and cystathionine beta-synthase (CBS) as well as an activation of polyamine metabolism, were analyzed using a simple mathematical model describing methionine metabolism in liver. The model predicts that the mere loss of allosteric regulation of CBS by adenosylmethionine (AdoMet) leads to an increase in homocysteine concentration. This is consistent with the experimental data on the corresponding genetic defects, which specifically impair allosteric activation but not basal enzyme activity. Application of the characteristics of transformed hepatocytes to our model, i.e., substitution of the MAT I/III isozyme by MAT II, loss of GNMT activity and activation of polyamine biosynthesis, leads to the prediction of a significantly different dependence of methionine metabolism on methionine concentrations. The theoretical predictions were found to be in good agreement with experimental data obtained with the human hepatoma cell line, HepG2.