Substrate trapping proteomics reveals targets of the ßTrCP2/FBXW11 ubiquitin ligase.

Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study ßTrCP2/FBXW11, a substrate adaptor for the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to "trap" ubiquitylated substrates on the SCF(FBXW11) E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCF(FBXW11) bound, polyubiquitylated, and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and, consequently, multinucleation. Surprisingly, this occurred independently of the guanine nucleotide exchange factor (GEF) catalytic activity and of the presence of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this report supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062.

An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain.

Proper centromere function is critical to maintain genomic stability and to prevent aneuploidy, a hallmark of tumors and birth defects. A conserved feature of all eukaryotic centromeres is an essential histone H3 variant called CENP-A that requires a centromere targeting domain (CATD) for its localization. Although proteolysis prevents CENP-A from mislocalizing to euchromatin, regulatory factors have not been identified. Here, we identify an E3 ubiquitin ligase called Psh1 that leads to the degradation of Cse4, the budding yeast CENP-A homolog. Cse4 overexpression is toxic to psh1Δ cells and results in euchromatic localization. Strikingly, the Cse4 CATD is a key regulator of its stability and helps Psh1 discriminate Cse4 from histone H3. Taken together, we propose that the CATD has a previously unknown role in maintaining the exclusive localization of Cse4 by preventing its mislocalization to euchromatin via Psh1-mediated degradation.

Proteomics and comparative genomic investigations reveal heterogeneity in evolutionary rate of male reproductive proteins in mice (Mus domesticus).

Male reproductive fitness is strongly affected by seminal fluid. In addition to interacting with the female environment, seminal fluid mediates important physiological characteristics of sperm, including capacitation and motility. In mammals, the male reproductive tract shows a striking degree of compartmentalization, with at least six distinct tissue types contributing material that is combined with sperm in an ejaculate. Although studies of whole ejaculates have been undertaken in some species, we lack a comprehensive picture of the specific proteins produced by different accessory tissues. Here, we perform proteomic investigations of six regions of the male reproductive tract in mice -- seminal vesicles, anterior prostate, dorsolateral prostate, ventral prostate, bulbourethral gland, and bulbourethral diverticulum. We identify 766 proteins that could be mapped to 506 unique genes and compare them with a high-quality human seminal fluid data set. We find that Gene Ontology functions of seminal proteins are largely conserved between mice and humans. By placing these data in an evolutionary framework, we show that seminal vesicle proteins have experienced a significantly higher rate of nonsynonymous substitution compared with the genome, which could be the result of adaptive evolution. In contrast, proteins from the other five tissues showed significantly lower nonsynonymous substitution, revealing a previously unappreciated level of evolutionary constraint acting on the majority of male reproductive proteins.

Bruton's tyrosine kinase revealed as a negative regulator of Wnt-beta-catenin signaling.

Wnts are secreted ligands that activate several receptor-mediated signal transduction cascades. Homeostatic Wnt signaling through beta-catenin is required in adults, because either elevation or attenuation of beta-catenin function has been linked to diverse diseases. To contribute to the identification of both protein and pharmacological regulators of this pathway, we describe a combinatorial screen that merged data from a high-throughput screen of known bioactive compounds with an independent focused small interfering RNA screen. Each screen independently revealed Bruton's tyrosine kinase (BTK) as an inhibitor of Wnt-beta-catenin signaling. Loss of BTK function in human colorectal cancer cells, human B cells, zebrafish embryos, and cells derived from X-linked agammaglobulinemia patients with a mutant BTK gene resulted in elevated Wnt-beta-catenin signaling, confirming that BTK acts as a negative regulator of this pathway. From affinity purification-mass spectrometry and biochemical binding studies, we found that BTK directly interacts with a nuclear component of Wnt-beta-catenin signaling, CDC73. Further, we show that BTK increased the abundance of CDC73 in the absence of stimulation and that CDC73 acted as a repressor of beta-catenin-mediated transcription in human colorectal cancer cells and B cells.

Long-term outcomes of drug-eluting versus bare-metal stent implantation in patients with chronic total coronary artery occlusions.

BACKGROUND: There are limited data on the efficacy of drug-eluting stents (DES) for treatment of chronic total occlusions (CTO). The aim of the study was to evaluate the long-term clinical outcomes of DES implantation for CTO compared with bare-metal stent (BMS) implantation. METHODS: Between June 1995 and December 2006, a total of 1184 patients with successful recanalization of at least one de novo CTO lesion were consecutively registered, including 660 (55.7%) who underwent DES and 524 (44.3%) who underwent BMS implantation. All patients were followed up for up to 5 years for occurrence of major adverse cardiac events (MACE). Long-term survival rates were estimated with the Kaplan-Meier method. RESULTS: Baseline clinical and angiographic characteristics were comparable between the two groups except that patients in the DES group received longer dual antiplatelet therapy ((7.4 +/- 2.5) months vs (1.7 +/- 0.8) months, P < 0.001). Average follow-up periods were (4.7 +/- 0.89) and (3.2 +/- 1.3) years for the BMS and DES groups, respectively. There was no significant difference in 5-year survival rates between the two groups (90.3% for DES group vs 89.6% for BMS group, Log-rank P = 0.38), but the 5-year target vessel revascularization (TVR)-free survival rate in the DES group was significantly higher than that in the BMS group (81.6% vs 73.5%, Log-rank P < 0.001). The cumulative MACE-free survival in the DES group was also significantly higher than that in the BMS group (80.6% vs 71.5%, Log-rank P < 0.001). The rates of re-admission caused by cardiovascular disease (27.0% vs 37.8%, P < 0.001) and the need for bypass surgery were significantly lower in the DES group (1.5% vs 3.4%, P < 0.05). By multivariable analysis, DES implantation could significantly lower the long-term MACE risk of PCI for CTO patients (HR: 0.492; 95% CI 0.396 - 0.656, P < 0.001). Left ventricular ejection fraction < 50% and elderly (> or = 65 years) were identified as independent predictors of long-term MACE during follow-up. CONCLUSION: This study demonstrates the long-term (up to 5 years) efficacy of DES for treatment of CTO, which is superior to BMS implantation in reducing the rates of TVR and MACE, as well as the need of re-admission and bypass surgery.

[Long-term clinical outcomes of patients undergoing successful or failed percutaneous coronary intervention for chronic total occlusions of coronary arteries].

OBJECTIVE: To evaluate the long-term outcomes of successful or failed revascularization in patients with chronic total occlusions (CTO). METHODS: The clinical data of 1332 consecutive patients underwent percutaneous coronary intervention (PCI) for CTO between June 1993 and December 2006 in our hospital were analyzed. These patients were divided into two groups according to the procedural success (n = 1202) or failure (n = 130). RESULTS: Overall success rate of procedure was 90.2% (1202/1332). The patients in CTO success group experienced a superior 10-year survival rate (76.9% vs. 64.6%, log rank P = 0.012) and a significantly higher no major adverse cardiovascular event (MACE) survival rate (41.8% vs. 27.6%, log rank P < 0.001) compared to the patients in CTO failure group. During the long-term follow-up, the proportion of patients who accepted coronary artery bypass grafting (CABG) was significantly lower in CTO success group than that in the CTO failure group (4.3% vs. 14.6%, P < 0.001). CONCLUSION: Successful PCI procedure leads to increased long-term survival and MACE-free survival and the reduced need for CABG for patients with CTO lesions.

New regulators of Wnt/beta-catenin signaling revealed by integrative molecular screening.

The identification and characterization of previously unidentified signal transduction molecules has expanded our understanding of biological systems and facilitated the development of mechanism-based therapeutics. We present a highly validated small interfering RNA (siRNA) screen that functionally annotates the human genome for modulation of the Wnt/beta-catenin signal transduction pathway. Merging these functional data with an extensive Wnt/beta-catenin protein interaction network produces an integrated physical and functional map of the pathway. The power of this approach is illustrated by the positioning of siRNA screen hits into discrete physical complexes of proteins. Similarly, this approach allows one to filter discoveries made through protein-protein interaction screens for functional contribution to the phenotype of interest. Using this methodology, we characterized AGGF1 as a nuclear chromatin-associated protein that participates in beta-catenin-mediated transcription in human colon cancer cells.

Proteomics reveals novel Drosophila seminal fluid proteins transferred at mating.

Across diverse taxa, seminal fluid proteins (Sfps) transferred at mating affect the reproductive success of both sexes. Such reproductive proteins often evolve under positive selection between species; because of this rapid divergence, Sfps are hypothesized to play a role in speciation by contributing to reproductive isolation between populations. In Drosophila, individual Sfps have been characterized and are known to alter male sperm competitive ability and female post-mating behavior, but a proteomic-scale view of the transferred Sfps has been missing. Here we describe a novel proteomic method that uses whole-organism isotopic labeling to detect transferred Sfps in mated female D. melanogaster. We identified 63 proteins, which were previously unknown to function in reproduction, and confirmed the transfer of dozens of predicted Sfps. Relative quantification of protein abundance revealed that several of these novel Sfps are abundant in seminal fluid. Positive selection and tandem gene duplication are the prevailing forces of Sfp evolution, and comparative proteomics with additional species revealed lineage-specific changes in seminal fluid content. We also report a proteomic-based gene discovery method that uncovered 19 previously unannotated genes in D. melanogaster. Our results demonstrate an experimental method to identify transferred proteins in any system that is amenable to isotopic labeling, and they underscore the power of combining proteomic and evolutionary analyses to shed light on the complex process of Drosophila reproduction.

Assessing the dynamic range and peak capacity of nanoflow LC-FAIMS-MS on an ion trap mass spectrometer for proteomics.

Proteomics experiments on complex mixtures have benefited greatly from the advent of fast-scanning ion trap mass spectrometers. However, the complexity and dynamic range of mixtures analyzed using shotgun proteomics is still beyond what can be sampled by data-dependent acquisition. Furthermore, the total liquid chromatography-mass spectrometry (LC-MS) peak capacity is not sufficient to resolve the precursors within these mixtures, let alone acquire tandem mass spectra on all of them. Here we describe the application of a high-field asymmetric waveform ion mobility spectrometry (FAIMS) device as an interface to an ion trap mass spectrometer. The dynamic range and peak capacity of the nanoflow LC-FAIMS-MS analysis was assessed using a complex tryptic digest of S. cerevisiae proteins. By adding this relatively simple device to the front of the mass spectrometer, we obtain an increase in peak capacity >8-fold and an increase in dynamic range of >5-fold, without increasing the length of the LC-MS analysis. Thus, the addition of FAIMS to the front of a table-top mass spectrometer can obtain the peak capacity of multidimensional protein identification technology (MudPIT) while increasing the throughput by a factor of 12.

Improving tandem mass spectrum identification using peptide retention time prediction across diverse chromatography conditions.

Most algorithms for identifying peptides from tandem mass spectra use information only from the final spectrum, ignoring non-mass-based information acquired routinely in liquid chromatography tandem mass spectrometry analyses. One physiochemical property that is always obtained but rarely exploited is peptide chromatographic retention time. Efforts to use chromatographic retention time to improve peptide identification are complicated because of the variability of retention time in different experimental conditions-making retention time calculations nongeneralizable. We show that peptide retention time can be reliably predicted by training and testing a support vector regressor on a small collection of data from a single liquid chromatography run. This model can be used to filter peptide identifications with observed retention time that deviates from predicted retention time. After filtering, positive peptide identifications increase by as much as 50% at a false discovery rate of 3%. We demonstrate that our dynamically trained model generalizes well across diverse chromatography conditions and methods for generating peptides, in particular improving peptide identification using nonspecific proteases.

Heat shock protein 70 interacts with aquaporin-2 and regulates its trafficking.

The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking.

Duplication and selection on abalone sperm lysin in an allopatric population.

While gene duplication is a major source of evolutionary novelty, the importance of this process in reproductive protein evolution has not been widely investigated. Here, we report the first known case of gene duplication of abalone sperm lysin in an allopatric subspecies found in the Eastern Atlantic, Haliotis tuberculata coccinea. Mass spectrometry identified both copies of the lysin protein in testis tissue, and 3-dimensional structural modeling suggests that both proteins remain functional. We also detected positive selection acting on both paralogs after duplication and found evidence of a recent selective sweep. Because H. t. coccinea occurs in geographic isolation from other abalone species, these findings suggest that the evolution of lysin is not driven to create reproductive barriers to unfit hybrid formation with an overlapping species. Instead, sexual selection or sexual conflict acting during abalone fertilization could be responsible for the recent positive selection on this protein. The presence of multiple, rapidly evolving lysin genes in H. tuberculata presents an opportunity to study the early stages of diversification of a protein whose function is well understood.

Wilms tumor suppressor WTX negatively regulates WNT/beta-catenin signaling.

Aberrant WNT signal transduction is involved in many diseases. In colorectal cancer and melanoma, mutational disruption of proteins involved in the degradation of beta-catenin, the key effector of the WNT signaling pathway, results in stabilization of beta-catenin and, in turn, activation of transcription. We have used tandem-affinity protein purification and mass spectrometry to define the protein interaction network of the beta-catenin destruction complex. This assay revealed that WTX, a protein encoded by a gene mutated in Wilms tumors, forms a complex with beta-catenin, AXIN1, beta-TrCP2 (beta-transducin repeat-containing protein 2), and APC (adenomatous polyposis coli). Functional analyses in cultured cells, Xenopus, and zebrafish demonstrate that WTX promotes beta-catenin ubiquitination and degradation, which antagonize WNT/beta-catenin signaling. These data provide a possible mechanistic explanation for the tumor suppressor activity of WTX.

Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R.

The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK(1) epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.

Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs.

Proteins harboring a zona pellucida (ZP) domain are prominent components of vertebrate egg coats. Although less well characterized, the egg coat of the non-vertebrate marine gastropod abalone (Haliotis spp.) is also known to contain a ZP domain protein, raising the possibility of a common molecular basis of metazoan egg coat structures. Egg coat proteins from vertebrate as well as non-vertebrate taxa have been shown to evolve under positive selection. Studied most extensively in the abalone system, coevolution between adaptively diverging egg coat and sperm proteins may contribute to the rapid development of reproductive isolation. Thus, identifying the pattern of evolution among egg coat proteins is important in understanding the role these genes may play in the speciation process. The purpose of the present study is to characterize the constituent proteins of the egg coat [vitelline envelope (VE)] of abalone eggs and to provide preliminary evidence regarding how selection has acted on VE proteins during abalone evolution. A proteomic approach is used to match tandem mass spectra of peptides from purified VE proteins with abalone ovary EST sequences, identifying 9 of 10 ZP domain proteins as components of the VE. Maximum likelihood models of codon evolution suggest positive selection has acted among a subset of amino acids for 6 of these genes. This work provides further evidence of the prominence of ZP proteins as constituents of the egg coat, as well as the prominent role of positive selection in diversification of these reproductive proteins.

Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery.

Protein ubiquitination is a common form of post-translational modification that regulates a broad spectrum of protein substrates in diverse cellular pathways. Through a three-enzyme (E1-E2-E3) cascade, the attachment of ubiquitin to proteins is catalysed by the E3 ubiquitin ligase, which is best represented by the superfamily of the cullin-RING complexes. Conserved from yeast to human, the DDB1-CUL4-ROC1 complex is a recently identified cullin-RING ubiquitin ligase, which regulates DNA repair, DNA replication and transcription, and can also be subverted by pathogenic viruses to benefit viral infection. Lacking a canonical SKP1-like cullin adaptor and a defined substrate recruitment module, how the DDB1-CUL4-ROC1 E3 apparatus is assembled for ubiquitinating various substrates remains unclear. Here we present crystallographic analyses of the virally hijacked form of the human DDB1-CUL4A-ROC1 machinery, which show that DDB1 uses one beta-propeller domain for cullin scaffold binding and a variably attached separate double-beta-propeller fold for substrate presentation. Through tandem-affinity purification of human DDB1 and CUL4A complexes followed by mass spectrometry analysis, we then identify a novel family of WD40-repeat proteins, which directly bind to the double-propeller fold of DDB1 and serve as the substrate-recruiting module of the E3. Together, our structural and proteomic results reveal the structural mechanisms and molecular logic underlying the assembly and versatility of a new family of cullin-RING E3 complexes.