Paul Taylor

Global proteomic assessment of the classical protein-tyrosine phosphatome and "Redoxome".

Protein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases, play key roles in cellular signaling. All Class I PTPs contain an essential active site cysteinyl residue, which executes a nucleophilic attack on substrate phosphotyrosyl residues. The high reactivity of the catalytic cysteine also predisposes PTPs to oxidation by reactive oxygen species, such as H(2)O(2). Reversible PTP oxidation is emerging as an important cellular regulatory mechanism and might contribute to diseases such as cancer. We exploited these unique features of PTP enzymology to develop proteomic methods, broadly applicable to cell and tissue samples, that enable the comprehensive identification and quantification of expressed classical PTPs (PTPome) and the oxidized subset of the PTPome (oxPTPome). We find that mouse and human cells and tissues, including cancer cells, display distinctive PTPomes and oxPTPomes, revealing additional levels of complexity in the regulation of protein-tyrosine phosphorylation in normal and malignant cells.

Multiple myeloma phosphotyrosine proteomic profile associated with FGFR3 expression, ligand activation, and drug inhibition

Signaling by growth factor receptor tyrosine kinases is manifest through networks of proteins that are substrates and/or bind to the activated receptors. FGF receptor-3 (FGFR3) is a drug target in a subset of human multiple myelomas (MM) and is mutationally activated in some cervical and colon and many bladder cancers and in certain skeletal dysplasias. To define the FGFR3 network in multiple myeloma, mass spectrometry was used to identify and quantify phosphotyrosine (pY) sites modulated by FGFR3 activation and inhibition in myeloma-derived KMS11 cells. Label-free quantification of peptide ion currents indicated the activation of FGFR3 by phosphorylation of tandem tyrosines in the kinase domain activation loop when cellular pY phosphatases were inhibited by pervanadate. Among the 175 proteins that accumulated pY in response to pervanadate was a subset of 52 including FGFR3 that contained a total of 61 pY sites that were sensitive to inhibition by the FGFR3 inhibitor PD173074. The FGFR3 isoform containing the tandem pY motif in its activation loop was targeted by PD173074. Forty of the drug-sensitive pY sites, including two located within the 35-residue cytoplasmic domain of the transmembrane growth factor binding proteoglycan (and multiple myeloma biomarker) Syndecan-1/CD138, were also stimulated in cells treated with the ligand FGF1, providing additional validation of their link to FGFR3. The identification of these overlapping sets of co-modulated tyrosine phosphorylations presents an outline of an FGFR3 network in the MM model and demonstrates the potential for pharmacodynamic monitoring by label-free quantitative phospho-proteomics.

Distinct properties of murine α5 γ-aminobutyric acid type a receptors revealed by biochemical fractionation and mass spectroscopy

γ‐Aminobutyric acid type A receptors (GABAARs) that contain the α5 subunit are expressed predominantly in the hippocampus, where they regulate learning and memory processes. Unlike conventional postsynaptic receptors, GABAARs containing the α5 subunit (α5 GABAARs) are localized primarily to extrasynaptic regions of neurons, where they generate a tonic inhibitory conductance. The unique characteristics of α5 GABAARs have been examined with pharmacological, immunostaining, and electrophysiological techniques; however, little is known about their biochemical properties. The aim of this study was to modify existing purification and enrichment techniques to isolate α5 GABAARs preferentially from the mouse hippocampus and to identify the α5 subunit by using tandem mass spectroscopy (MS/MS). The results showed that the detergent solubility of the α5 subunits was distinct from that of α1 and α2 subunits, and the relative distribution of the α5 subunits in Triton X‐100‐soluble fractions was correlated with that of the extracellular protein radixin but not with that of the postsynaptic protein gephyrin. Mass spectrometry identified the α5 subunit and showed that this subunit associates with multiple α, β, and γ subunits, but most frequently the β3 subunit. Thus, the α5 subunits coassemble with similar subunits as their synaptic counterparts yet have a distinct detergent solubility profile. Mass spectroscopy now offers a method for detecting and characterizing factors that confer the unique detergent solubility and possibly cellular location of α5 GABAARs in hippocampal neurons.

The ubiquitin ligase HERC4 mediates c-Maf ubiquitination and delays the growth of multiple myeloma xenografts in nude mice

The transcription factor c-Maf is extensively involved in the pathophysiology of multiple myeloma (MM), a fatal malignancy of plasma cells. In the present study, affinity chromatography and mass spectrometry were used to identify c-Maf ubiquitination-associated proteins, from which the E3 ligase HERC4 was found to interact with c-Maf and catalyzed its polyubiquitination and subsequent proteasome-mediated degradation. HERC4 mediated polyubiquitination at K85 and K297 in c-Maf, and this polyubiquitination could be prevented by the isopeptidase USP5. Further analysis on the NCI-60 cell line collection revealed that RPMI 8226, a MM-derived cell line, expressed the lowest level of HERC4. Primary bone marrow analysis revealed HERC4 expression was high in normal bone marrow, but was steadily decreased during myelomagenesis. These findings suggested HERC4 played an important role in MM progression. Moreover, ectopic HERC4 expression decreased MM proliferation in vitro, and delayed xenograft tumor growth in vivo. Therefore, modulation of c-Maf ubiquitination by targeting HERC4 may represent a new therapeutic modality for MM.

The ubiquitin-conjugating enzyme UBE2O modulates c-Maf stability and induces myeloma cell apoptosis

BackgroundUBE2O is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown.MethodsMass spectrometry was applied to identify c-Maf ubiquitination-associated proteins. Immunoprecipitation was applied for c-Maf and UBE2O interaction. Immunoblotting was used for Maf protein stability. Luciferase assay was used for c-Maf transcriptional activity. Lentiviral infections were applied for UBE2O function in multiple myeloma (MM) cells. Flow cytometry and nude mice xenografts were applied for MM cell apoptosis and tumor growth assay, respectively.ResultsUBE2O was found to interact with c-Maf, a critical transcription factor in MM, by the affinity purification/tandem mass spectrometry assay and co-immunoprecipitation assays. Subsequent studies showed that UBE2O mediated c-Maf polyubiquitination and degradation. Moreover, UBE2O downregulated the transcriptional activity of c-Maf and the expression of cyclin D2, a typical gene modulated by c-Maf. DNA microarray revealed that UBE2O was expressed in normal bone marrow cells but downregulated in MGUS, smoldering MM and MM cells, which was confirmed by RT-PCR in primary MM cells, suggesting its potential role in myeloma pathophysiology. When UBE2O was restored, c-Maf protein in MM cells was significantly decreased and MM cells underwent apoptosis. Furthermore, the human MM xenograft in nude mice showed that re-expression of UBE2O delayed the growth of myeloma xenografts in nude mice in association with c-Maf downregulation and activation of the apoptotic pathway.ConclusionsUBE2O mediates c-Maf polyubiquitination and degradation, induces MM cell apoptosis, and suppresses myeloma tumor growth, which provides a novel insight in understanding myelomagenesis and UBE2O biology.

Abstract SY33-04: Integrated omic analysis of lung cancer reveals metabolism-proteome signatures with prognostic impact

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Cancer results from processes prone to selective pressure and dysregulation acting along the sequence-to- phenotype continuum DNA→RNA→Protein→Disease. An Omics Array integrating DNA gene copy number, mRNA transcriptome, and quantified proteome was assembled into a genetic map representing non-small cell lung carcinoma (NSCLC). Data were collected from patient-matched normal lung, primary tumors, and patient tumor-derived xenograft (PDX) tumors. Dysregulated proteins not previously implicated as cancer drivers were found encoded throughout the genome including but not limited to regions of recurrent DNA amplification/deletion in NSCLC. Unsupervised clustering revealed signatures comprising metabolism proteins particularly highly recapitulated between matched primary and PDX tumors, and which distinguished between the major NSCLC histological subtypes adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Interrogation of The Cancer Genome Atlas (TCGA) revealed sizeable cohorts of NSCLC patients with DNA alterations in genes encoding the metabolism proteome signatures, and accompanied by differences in survival. Similar to the proteome signatures from which they were extrapolated, the gene mutation signatures with prognostic impact discriminated between the lung ADC and SCC subtypes. Serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in serine/glycine and folate- dependent one-carbon metabolism, is upregulated in the proteomes of NSCLC primary and PDX tumours, and is implicated as a driver of recurrent chromosome 12q14.1 amplification in NSCLC. SHMT2, along with other enzymes implicated as anti-folate targets, is also part of a metabolism proteome signature associated with poor outcome in lung ADC. The interrogation of cancer genomes and proteomes for alterations that are related products of selective pressures driving the cancer phenotype may be a general approach to uncover and group together cryptic, polygenic cancer drivers, which might represent new anti-cancer therapeutic targets. Citation Format: Michael F. Moran, Lei Li, Yuhong Wei, Paul Taylor, Christine To, Jiefei Tong, Vladimir Ignatchenko, Melania Pintilie, Nhu-An Pham, Wen Zhang, Lakshmi Muthuswamy, Frances A. Shepherd, Thomas Kislinger, Ming S. Tsao. Integrated omic analysis of lung cancer reveals metabolism-proteome signatures with prognostic impact. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY33-04. doi:10.1158/1538-7445.AM2015-SY33-04

Abstract 3753: Quantitation of EGFR and phosphoEGFR in FFPE tissue

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The epidermal growth factor receptor (EGFR) is a drug target for both small molecule and antibody therapeutic approaches for several cancers; however, current methods of selecting patients that will most likely respond to anti-EGR therapy are not effective. Better methods for patient stratification are needed. To this end we have developed an approach which can determine both absolute EGFR levels and the phosphorylation status of EGFR directly in formalin-fixed paraffin-embedded (FFPE) patient tissue. This approach is based on the Liquid Tissue®-SRM technology platform, a combination of tissue microdissection, Liquid Tissue® processing which turns dissected tissue to a complete solubilized tryptic digest, and mass spectrometry-based selected reaction monitoring (SRM). This approach was used to measure the EGFR protein and its phosphorylation status in formalin fixed tissue culture cells, xenograft tumors, and patient tumor tissue. For assay development, 3 distinct tryptic peptides were assessed for absolute protein quantitation and multiple peptides where specific residues are known to become phosphorylated (pT693 and pY1197) were assessed for assaying the phosphorylation status of the EGFR protein. We demonstrate the ability to detect and quantify the EGFR protein and to monitor its phosphorylation status directly in patient tumor tissue. This approach offers a dynamic range and quantification which is superior to traditional IHC methods, and that could be used to identify and stratify patients most likely to benefit from anti-EGFR therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3753.