Stable Isotope Labeling With Amino Acids In Cell Culture Research Articles

SILAC and LC-MS/MS identification of Streptococcus equi ssp. zooepidemicus proteins that contribute to mouse brain microvascular endothelial cell infection.

Streptococcus equi ssp. zooepidemicus (SEZ) causes meningitis in both humans and animals. Some dissociative proteins of SEZ are cytotoxic to mouse brain microvascular endothelial cells (mBMECs) and may contribute to the penetration of SEZ across the blood-brain barrier (BBB). In this study, the ability of SEZ to penetrate across an in vitro BBB model was confirmed. We used stable isotope labeling with amino acids in cell culture (SILAC) to label SEZ proteins with heavy or light isotope-tagged amino acids, along with LC-MS/MS to determine which SEZ proteins were involved in interactions with mBMECs. The efficiency of SEZ protein isotope labeling was 94.7%, which was sufficient for further analysis. Forty-nine labeled peptides were identified as binding to mBMECs, which matched to 25 SEZ proteins. Bioinformatic analysis indicated that most of these proteins were cytoplasmic. These proteins may have functions in breaching the host BBB, and some of them are known virulence factors in other bacteria. Indirect immunofluorescence results indicated that SEZ enolase had binding activity toward mBMECs. Protective test results showed that enolase was a protective antigen against SEZ infection. This research is the first application of SILAC combined with LC-MS/MS to identify SEZ proteins that may contribute to the infection of mBMECs and potentially show functions related to breaching the BBB. The outcomes provide many future avenues for research into the mechanism of SEZ-induced meningitis.

Quantitative interactome reveals that porcine reproductive and respiratory syndrome virus nonstructural protein 2 forms a complex with viral nucleocapsid protein and cellular vimentin

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has heavily impacted the global swine industry. The PRRSV nonstructural protein 2 (nsp2) plays crucial roles in viral replication and host immune regulation, most likely by interacting with viral or cellular proteins that have not yet been identified. In this study, a quantitative interactome approach based on immunoprecipitation and stable isotope labeling with amino acids in cell culture (SILAC) was performed to identify nsp2-interacting proteins in PRRSV-infected cells with an nsp2-specific monoclonal antibody. Nine viral proteins and 62 cellular proteins were identified as potential nsp2-interacting partners. Our data demonstrate that the PRRSV nsp1α, nsp1β, and nucleocapsid proteins all interact directly with nsp2. Nsp2-interacting cellular proteins were classified into different functional groups and an interactome network of nsp2 was generated. Interestingly, cellular vimentin, a known receptor for PRRSV, forms a complex with nsp2 by using viral nucleocapsid protein as an intermediate. Taken together, the nsp2 interactome under the condition of virus infection clarifies a role of nsp2 in PRRSV replication and immune evasion. Biological significanceViral proteins must interact with other virus-encoded proteins and/or host cellular proteins to function, and interactome analysis is an ideal approach for identifying such interacting proteins. In this study, we used the quantitative interactome methodology to identify the viral and cellular proteins that potentially interact with the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) under virus infection conditions, thus providing a rich source of potential viral and cellular interaction partners for PRRSV nsp2. Based on the interactome data, we further demonstrated that PRRSV nsp2 and nucleocapsid protein together with cellular vimentin, form a complex that may be essential for viral attachment and replication, which partly explains the role of nsp2 in PRRSV replication and immune evasion.

Quantitative phosphotyrosine profiling of patient-derived xenografts identifies therapeutic targets in pediatric leukemia.

Activating mutations in tyrosine kinases (TKs) drive pediatric high-risk acute lymphoblastic leukemia (ALL) and confer resistance to standard chemotherapy. Therefore, there is urgent need to characterize dysregulated TK signaling axes in patients with ALL and identify actionable kinase targets for the development of therapeutic strategies. Here, we present the first study to quantitatively profile TK activity in xenografted patient biopsies of high-risk pediatric ALL. We integrated a quantitative phosphotyrosine profiling method with 'spike-in' stable isotope labeling with amino acids in cell culture (SILAC) and quantified 1394 class I phosphorylation sites in 16 ALL xenografts. Moreover, hierarchical clustering of phosphotyrosine sites could accurately classify these leukemias into either B or T-cell lineages with the high-risk early T-cell precursor (ETP) and Ph-like ALL clustering as a distinct group. Furthermore, we validated this approach by using specific kinase pathway inhibitors to perturb ABL1, FLT3, and JAK TK signaling in four xenografted patient samples. By quantitatively assessing the tyrosine phosphorylation status of activated kinases in xenograft models of ALL, we were able to identify and validate clinically relevant targets. Therefore, this study highlights the application and potential of phosphotyrosine profiling for identifying clinically relevant kinase targets in leukemia.

Freezing effects on the acute myeloid leukemia cell proteome and phosphoproteome revealed using optimal quantitative workflows

MS-based proteomic studies aiming for the discovery of acute myeloid leukemia (AML) biomarkers require sample processing that can assure an optimal proteome coverage and identification of PTMs. We evaluated different in-solution and filter-aided sample preparation (FASP) proteomic workflows and different enrichment strategies of phosphorylated peptides. The FASP protocols in the label-free and SILAC (stable isotope labelling with amino acids in cell culture) approaches were selected for producing the highest number of quantified proteins with reduced number of missed cleavages. The IMAC method was selected for producing the highest number of quantified phosphopeptides from SILAC-labelled peptides prepared with FASP. Using these selected workflows, we studied the effect of liquid nitrogen storage on the proteome and phosphoproteome of four AML patients. Our results showed that although there was not a major global proteome and phosphoproteome change when compared to their freshly processed counterparts, the freezing appeared to influence the abundance of mitochondrial proteins involved in the respiratory chain transport and affect the phosphorylation of apoptosis related proteins, cell surface interactors, ERK/MAPK targets and proteins involved in thrombin signalling. Our results encourage the assessment of current procedures of AML sample collection and preservation that could be used in future AML biomarker discovery studies. Biological significanceProteomic studies aiming to identify potential cancer biomarkers need to utilize the best sample preparation workflows on the samples of interest to achieve maximal proteome coverage. We have tested the most popular and recent proteomic and phosphoproteomic methods on cell lysates from patients with AML and systematically evaluated their performance. Our study shows the relevance of selecting the patient sample procedure giving the highest protein and PTM coverage. Moreover, we assessed how the proteome and phosphoproteome were affected by the conventional liquid nitrogen storage compared to cell lysis of fresh material, using the methods that worked best in our hands. For potential biomarkers that could be used for AML diagnostic and prognostic, it is of great importance to study the behaviour during sample conservation in order to avoid artefactual findings. Our results recommend caution in data interpretation when using different protocols of sample collection and conservation for proteomic and phosphosproteomic research.

Proteome-wide dataset supporting functional study of tyrosine kinases in breast cancer.

Tyrosine kinases (TKs) play an essential role in regulating various cellular activities and dysregulation of TK signaling contributes to oncogenesis. However, less than half of the TKs have been thoroughly studied. Through a combined use of RNAi and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics, a global functional proteomic landscape of TKs in breast cancer was recently revealed highlighting a comprehensive and highly integrated signaling network regulated by TKs (Stebbing et al., 2015) [1]. We collate the enormous amount of the proteomic data in an open access platform, providing a valuable resource for studying the function of TKs in cancer and benefiting the science community. Here we present a detailed description related to this study (Stebbing et al., 2015) [1] and the raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier PXD002065.

Nachbarschaftsstudien am Ribosom: proximity dependent biotin identification

Scaffolds mediate proximities of proteins and by that organize microenvironments. With proximity dependent biotin identification (BioID) the proteinaceous neighborhood of a bait is labeled in vivo and identified by mass spectrometry. The scaffold protein Asc1 of Saccharomyces cerevisiae locates to the head of the 40S ribosome and is involved in signaling and translation. To monitor Asc1-proximate proteins and their local dynamics we combine BioID with stable isotope labeling with amino acids in cell culture (SILAC).

Abstract P2-06-10: Loss of COX5B inhibits proliferation and promotes senescence via mitochondrial dysfunction in breast cancer

Abstract Background: COX5B, a peripheral subunit of the cytochrome c oxidase complex, is reported to maintain the stability of this complex and impact on cell viability. Recently, several studies demonstrated that cox5b may involve in cancer progression. In our study, we also found that COX5B was upregulated both in breast cancer tissues and cells. However, the role and mechanism of COX5B in breast cancer remain unclear. Therefore, the aim of this study is to validate COX5B functions and its underlying mechanisms in breast cancer cells. Methods: SILAC (stable isotope labeling with amino acids in cell culture) assay was conducted in mammary epithelial cells and its paired breast tumor cells, and the results was validated by other two paired tissues. Forty tumors and twenty normal or benign samples were implied for COX5B expression by IHC. Online Kaplan Meier plotting tool was used to confirm the association between COX5B expression and clinical outcome. Three Cell lines (MDA-MB-231, MDA-MB-468 and MCF-7) were constructed by sh-RNA and the efficiency was confirmed by qPCR and western blot and then migration, proliferation and senescence characteristics were evaluated. Furthermore, microarray was implemented to detect potential mechanisms and the associated genes were assessed by qPCR. ELISA was carried out for IL8 secretion. Condition medium was collected from stable knock down cells and used as attractant in lower chamber of tranwell assay to examine microenvironment influence. Finally, the intracellular ATP glucose uptake and lactate secretion were evaluated, as well as mitochondrial membrane potential and ROS production. Results: We found that the level of COX5B was elevated in breast cancer and cell lines. Patients with high levels of COX5B were likely to have a longer disease-free survival than patients with low levels. Furthermore, loss of COX5B inhibited proliferation and induced the senescence of breast cancer cells, which was accompanied by increases in the secretion of IL-8 and other cytokines. Additionally, conditioned medium form COX5B knockdown cells increased the migration of breast cancer cells compared with conditioned medium from control cells. The mechanisms underlying these processes were associated with mitochondrial dysfunction due to increased ROS production and decreased MMP and metabolism disorder. Conclusion: Our results show that loss of COX5B inhibits proliferation and promotes senescence in breast cancer cells, which is associated with mitochondrial dysfunction. Indeed, loss of COX5B has different functions in target cells and the surrounding environments. These findings may provide a new perspective for the design of anti-cancer therapy combining with anti-IL8 treatment. Citation Format: Gao S-P, Sun H-F, Jiang H-L, Li L-D, Jin W. Loss of COX5B inhibits proliferation and promotes senescence via mitochondrial dysfunction in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-06-10.

Analysis of Signaling Endosome Composition and Dynamics Using SILAC in Embryonic Stem Cell-Derived Neurons

Neurons require efficient transport mechanisms such as fast axonal transport to ensure neuronal homeostasis and survival. Neurotrophins and their receptors are conveyed via fast axonal retrograde transport of signaling endosomes to the soma, where they elicit transcriptional responses. Despite the essential roles of signaling endosomes in neuronal differentiation and survival, little is known about their molecular identity, dynamics, and regulation. Gaining a better mechanistic understanding of these organelles and their kinetics is crucial, given the growing evidence linking vesicular trafficking deficits to neurodegeneration. Here, we exploited an affinity purification strategy using the binding fragment of tetanus neurotoxin (HCT) conjugated to monocrystalline iron oxide nanoparticles (MIONs), which in motor neurons, is transported in the same carriers as neurotrophins and their receptors. To quantitatively assess the molecular composition of HCT-containing signaling endosomes, we have developed a protocol for triple Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in embryonic stem cell-derived motor neurons. After HCT internalization, retrograde carriers were magnetically isolated at different time points and subjected to mass-spectrometry and Gene Ontology analyses. This purification strategy is highly specific, as confirmed by the presence of essential regulators of fast axonal transport in the make-up of these organelles. Our results indicate that signaling endosomes undergo a rapid maturation with the acquisition of late endosome markers following a specific time-dependent kinetics. Strikingly, signaling endosomes are specifically enriched in proteins known to be involved in neurodegenerative diseases and neuroinfection. Moreover, we highlighted the presence of novel components, whose precise temporal recruitment on signaling endosomes might be essential for proper sorting and/or transport of these organelles. This study provides the first quantitative proteomic analysis of signaling endosomes isolated from motor neurons and allows the assembly of a functional map of these axonal carriers involved in long-range neuronal signaling.

Characterization of secretomes from a human blood brain barrier endothelial cells in-vitro model after ischemia by stable isotope labeling with aminoacids in cell culture (SILAC)

The human immortalized brain endothelial cell line hCMEC/D3 is considered a simple in-vitro model of the blood–brain-barrier. Our aim was to characterize changes in the secretome of hCMEC/D3 subjected to oxygen and glucose deprivation (OGD) to identify new proteins altered after ischemia and that might trigger blood–brain-barrier disruption and test their potential as blood biomarkers for ischemic stroke. Using a quantitative proteomic approach based on SILAC, 19 proteins were found differentially secreted between OGD and normoxia/normoglycemia conditions. Among the OGD-secreted proteins, protein folding was the main molecular function identified and for the main canonical pathways there was an enrichment in epithelial adherens junctions and aldosterone signaling. Western blot was used to verify the MS results in a set of 9 differentially secreted proteins and 5 of these were analyzed in serum samples of 38 ischemic stroke patients, 18 stroke-mimicking conditions and 18 healthy controls. Significance“We characterized changes in the secretome of hCMEC/D3 cells after an ischemic insult by SILAC and identified proteins associated with ischemia that might be involved in the disruption of the blood–brain barrier. Besides we analyzed the putative potential of the candidate proteins to become biomarkers for the diagnosis of ischemic stroke.

Cell-Surface Proteomics to Unravel Cytokine-Induced Endothelial Activation

Endothelial dysfunction is associated with a variety of vascular disease processes, including hypertension, hypercholesterolemia, diabetes and atherosclerosis. The molecular mechanisms that underlie endothelial dysfunction are poorly understood, but are associated with endothelial activation by pro-inflammatory cytokines. Although transcriptional profiling has revealed cytokine-induced regulation of mRNA levels of various proteins, including the cell surface adhesion molecules Vascular Cell Adhesion Molecule 1 (VCAM1) and Intercellular Adhesion Molecule 1 (ICAM1), cell surface alterations at the protein level have remained largely unexplored. Unraveling these cell surface alterations is key to identify novel inflammation-markers and putative therapeutic targets to treat endothelial dysfunction. Therefore, the aim of this study is to probe cytokine-induced cell surface changes on endothelial cells.We have developed a novel quantitative cell surface proteomics method by combining metabolic labeling and cell surface foot printing. Briefly, Blood Outgrowth Endothelial Cells (BOECs) were metabolically labeled using Stable Isotope Labeling with Amino acids in Cell culture (SILAC) and treated with Interleukin 1β (IL1ß) or Tumor necrosis factor α (TNFα) or mock treated for 24 hours. Lysine residues available on the cell surface were labeled using a non-membrane permeable N -hydroxysuccinimidobiotin label. Pooled cell lysates were processed into peptides, biotinylated peptides were enriched using streptavidin pull-down, desalted using Empore C18 StageTips, subjected to high resolution chromatography, measured at the Orbitrap Fusion Tribrid Mass Spectrometer and analyzed using the MaxQuant and Perseus computational platform.Using this approach, 2526 biotinylated peptides were identified. The majority of the biotinylated peptides showed a SILAC ratio close to 1, indicating an unaltered cell surface presence. However, for 397 biotinylated peptides, the SILAC ratio was more than 2-fold increased or decreased in the cytokine-stimulated samples and we considered these to be regulated. Gene Ontology (GO)-term enrichment analysis of the corresponding proteins was assessed using BioMart. This analysis revealed a clear enrichment of the GO terms 'extracellular region' and 'extracellular space', supporting the applied approach. Hierarchical clustering of the regulated biotinylated peptides revealed a cluster of proteins with an increased cell surface expression after induction by both IL1ß and TNFα. In addition, two clusters of proteins were identified that show a unique footprint for one of the cytokine-stimulated samples, indicating that IL1ß and TNFα induce specific cell surface alterations. As expected, the most prominent changes were detected in ICAM1 and VCAM1. For ICAM1 6 peptides were identified with SILAC ratios 13.5 ± 1.75 (mean ± standard deviation) upon TNFα and 6.6 ± 2.9 upon IL1ß stimulation. For VCAM1 4 peptides were identified with ratios of 20.8 ± 14.3 (TNFα) and 3.2 ± 0.5 (IL1ß). Secondly, an increased presence of Human Leukocyte Antigen (HLA) and Beta-2-Microglobulin (B2M), part of the self-antigen presenting complex, was detected. Six peptides of HLA were increased on average 4.0 times ± 2.5 (TNFα) and 1.8 ± 0.6 (IL1ß). B2M peptides identified showed comparable values: 3.3 ± 1.1 (TNFα) and 1.7 ± 0.2 (IL1ß). These proteins are present on the cell surface in complex, thereby confirming the validity of our approach. Moreover, all biotinylated peptides identified for ICAM1, VCAM1 and HLA are in the extracellular region, further validating our method. Intriguingly, our approach not only identified the established adhesion molecules ICAM1 and VCAM1, but also a variety of other adhesion molecules involved in cell-cell interactions. More importantly, we also identified increased expression of proteins with other biological functions, including basement membrane proteins, ion channels, tyrosine phosphatase receptors, enzymes and protease inhibitors.In conclusion, we developed a novel mass spectrometry-based cell surface proteomics method to quantify cytokine-induced cell surface alterations. Our approach not only enabled a detailed quantification at the protein level for established inflammatory cell surface markers, but also revealed a variety of potential novel targets for intervention of endothelial activation and dysfunction. DisclosuresNo relevant conflicts of interest to declare.

Towards a Targeted Therapy:Phosphoproteomics Reveals Signaling Pathways That Are Normalized in AML Cells Following Treatment with Anti-CD44 Antibodies

Acute myeloid leukemia (AML) is a clonal malignant disease characterized by a blockage in the differentiation of myeloid cells resulting in the accumulation of highly proliferating immature blast cells. With the success of All Trans Retinoic acid (ATRA) in acute promyelocytic leukemia (AML3), differentiation therapy has become a very attractive treatment option. Ligation of CD44 (a cell surface antigen) with anti-CD44 monoclonal antibodies (mAbs) is reported to reverse the blockage of differentiation and suppress the proliferation of blasts derived from most AML subtypes. However, the molecular mechanisms underlying this apparent 'normalization' (reversal) of AML cells induced by CD44 have not been fully elucidated. To expand our understanding of the cellular regulation and circuitry involved, we aimed to apply a quantitative phosphoproteomic approach using Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to monitor dynamic changes of phosphorylation states in HL60 cells following treatment with CD44 mAbs. Phosphoproteomic analysis identified differentially phosphorylated proteins among CD44 mAb treated and control HL60 cells that are involved in a number of major signaling pathways as determined by the Ingenuity Pathway analysis (IPA) platform. Among others, Rho signaling emerged as a major pathway significantly changed by CD44 mAb treatment. Rho GTPases are well-recognized regulators of the actin cytoskeleton but have also been implicated in diverse cellular events such as cell polarity, microtubule dynamics, membrane trafficking, transcriptional regulation, cell growth control and development. An interesting Rho family member, PAK-2 was identified in our search. PAK-2 is a ubiquitously expressed serine/threonine protein kinase, which is a direct target for small GTPases and has been identified as a switch between cell survival and cell death signaling depending on its mode of activation. Western-blot analysis of cell lysates of CD44 mAb treated and control HL60 cells confirmed that the phosphorylation of Pak-2 was altered as early as 5 minutes following treatment. Further validation and characterization of the activation mode, phosphorylation dynamics and protein-protein interactions of PAK-2 are essential in understanding its role in AML. DisclosuresNo relevant conflicts of interest to declare.

Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique.

The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the non-hydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATP-binding proteins in the Saccharomyces Genome Database (SGD). The 14 non-annotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins. Graphical Abstract ᅟ.

C-Abl-p38α signaling plays an important role in MPTP-induced neuronal death.

Oxidative stress is a major cause of sporadic Parkinson's disease (PD). Here, we demonstrated that c-Abl plays an important role in oxidative stress-induced neuronal cell death. C-Abl, a nonreceptor tyrosine kinase, was activated in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP)-induced acute PD model. Conditional knockout of c-Abl in neurons or treatment of mice with STI571, a c-Abl family kinase inhibitor, reduced the loss of dopaminergic neurons and ameliorated the locomotive defects induced by short-term MPTP treatment. By combining the SILAC (stable isotope labeling with amino acids in cell culture) technique with other biochemical methods, we identified p38α as a major substrate of c-Abl both in vitro and in vivo and c-Abl-mediated phosphorylation is critical for the dimerization of p38α. Furthermore, p38α inhibition mitigated the MPTP-induced loss of dopaminergic neurons. Taken together, these data suggested that c-Abl-p38α signaling may represent a therapeutic target for PD.

Hypoxia Strongly Affects Mitochondrial Ribosomal Proteins and Translocases, as Shown by Quantitative Proteomics of HeLa Cells.

Hypoxia is an important and common characteristic of many human tumors. It is a challenge clinically due to the correlation with poor prognosis and resistance to radiation and chemotherapy. Understanding the biochemical response to hypoxia would facilitate the development of novel therapeutics for cancer treatment. Here, we investigate alterations in gene expression in response to hypoxia by quantitative proteome analysis using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with LCMS/MS. Human HeLa cells were kept either in a hypoxic environment or under normoxic conditions. 125 proteins were found to be regulated, with maximum alteration of 18-fold. In particular, three clusters of differentially regulated proteins were identified, showing significant upregulation of glycolysis and downregulation of mitochondrial ribosomal proteins and translocases. This interaction is likely orchestrated by HIF-1. We also investigated the effect of hypoxia on the cell cycle, which shows accumulation in G1 and a prolonged S phase under these conditions. Implications. This work not only improves our understanding of the response to hypoxia, but also reveals proteins important for malignant progression, which may be targeted in future therapies.

Characterization of the Tyrosine Kinase-Regulated Proteome in Breast Cancer by Combined use of RNA interference (RNAi) and Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantitative Proteomics

Tyrosine kinases (TKs) are central regulators in cellular activities and perturbations of TK signaling contribute to oncogenesis. However, less than half of the TKs have been thoroughly studied and a global functional analysis of their proteomic portrait is lacking. Here we conducted a combined approach of RNA interference (RNAi) and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics to decode the TK-regulated proteome and associated signaling dynamics. As a result, a broad proteomic repertoire modulated by TKs was revealed, upon silencing of the 65 TKs expressed in MCF7 breast cancer cells. This yielded 10 new distinctive TK clusters according to similarity in TK-regulated proteome, each characterized by a unique signaling signature in contrast to previous classifications. We provide functional analyses and identify critical pathways for each cluster based on their common downstream targets. Analysis of different breast cancer subtypes showed distinct correlations of each cluster with clinical outcome. From the significantly up- and down-regulated proteins, we identified a number of markers of drug sensitivity and resistance. These data supports the role of TKs in regulating major aspects of cellular activity, but also reveals redundancy in signaling, explaining why kinase inhibitors alone often fail to achieve their clinical aims. The TK-SILACepedia provides a comprehensive resource for studying the global function of TKs in cancer.

Abstract 1225: Proteome-wide analysis of gain-of-function mutant p53 targets in breast cancer implicates PARP, PCNA and MCM4 as oncogenic drivers

Abstract Gain-of-function mutant p53 (mtp53) changes the cancer cell transcriptome. However no analysis of mtp53-associated proteome diversity has been carried out. We coupled cell fractionation with stable isotope labeling with amino acids in cell culture (SILAC) and inducible knockdown of endogenous mtp53 to determine the mtp53 driven proteome. Using SILAC followed by tandem mass spectrometry (LC-MS/MS) we identified that R273H mtp53 expression in MDA-MB-468 breast cancer cells both up and down-regulated multiple proteins and metabolic pathways. We sequenced 73,154 peptide pairs that corresponded to 3010 proteins detected under reciprocal labeling conditions. Importantly the high impact regulated targets included the previously identified transcriptionally regulated mevalonate pathway proteins but also identified two new levels of mtp53 protein regulation for non-transcriptional targets. Interestingly, mtp53 depletion profoundly influenced poly (ADP-ribose) polymerase 1 (PARP1) localization, with increased cytoplasmic and decreased chromatin-associated PARP1. An enzymatic PARP shift occurred with high mtp53 expression resulting in increased poly-ADP-ribosylated proteins in the nucleus. Mtp53 increased the level of PCNA and MCM4 protein without changing the amount of pcna or mcm4 transcript. Pathway enrichment analysis ranked the DNA replication pathway above the cholesterol biosynthesis pathway as an R273H mtp53 activated proteomic target. Knowledge of the proteome diversity driven by mtp53 suggests DNA replication and repair pathways are major targets of mtp53 and highlights consideration of combination chemotherapeutic strategies targeting cholesterol biosynthesis and PARP inhibition. Acknowledgements. This work was supported by a grant to JB from the Breast Cancer Research Foundation. Citation Format: Alla Polotskaia, Gu Xiao, Katherine Reynoso, Che Martin, Wei-Gang Qiu, Ronald Hendrickson, Jill Bargonetti. Proteome-wide analysis of gain-of-function mutant p53 targets in breast cancer implicates PARP, PCNA and MCM4 as oncogenic drivers. [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 1225. doi:10.1158/1538-7445.AM2015-1225

Abstract 50: Elucidation of B cell receptor signaling in Burkitt's lymphoma reveals novel signaling nodes with potential therapeutic relevance

Abstract B cell antigen receptors (BCR) control important cell fate decisions of the B-lineage including differentiation, proliferation and B cell survival. Recently, it was demonstrated that beside chronic lymphocytic leukemia and diffuse large B cell lymphomas of the activated B cell-type also major subgroups of Burkitt lymphomas (BL) critically depend on BCR expression. As intensive chemotherapy and anti-CD20 antibodies are the current treatment strategies, pharmacological interference with critical BCR signaling pathways might open up the possibility for targeted therapies in BL. Such targeted therapeutic approaches are needed in particular for elderly patients and patients with endemic BL in developing countries. In order to (i) identify putative drug targets in BL and (ii) to elucidate BCR induced processes, we characterized BCR signaling in BL by quantitative phosphoproteomics and transcriptome sequencing. We identified about 10.000 phospho-sites in various BL cell-types, of which more than 1000 in almost 600 proteins were regulated in a BCR-dependent manner leading to dynamic transcription of more than 100 genes. The identified BCR signaling effectors belong to various functional groups, of which kinases, transcription factors and cytoskeleton regulators are most represented and bioinformatic analyses revealed that 83% of the identified BCR effectors were not described in the context of B cells or lymphoma before. For instance, we identified a cell surface receptor that is highly expressed in BL and whose expression is regulated by the transcription factor c-Myc which is known to be frequently dysregulated in BL. ShRNA mediated knockdown of the cell surface receptor in BL cells impaired phosphorylation of spleen tyrosine kinase (Syk) and B-cell linker protein (BLNK) upon BCR stimulation, most probably due to dysregulation of the cytoskeleton. By using stable isotope labeling with amino acids in cell culture (SILAC) we investigated in a comprehensive manner, how the newly identified BCR co-receptor influences BCR signaling. Our study is a considerable complement to recent genetic studies that elucidated the mutational landscape in BL as it reveals mechanisms of signaling addiction to non-mutated BCR effector proteins that have to be validated in the future. Citation Format: Carmen Doebele, Jasmin Corso, Anjali Cremer, Silvia Muench, Julia Beck, Christof Lenz, Hanibal Bohnenberger, Astrid Wachter, Tim Beissbarth, Ekkehard Schütz, Hubert Serve, Henning Urlaub, Thomas Oellerich. Elucidation of B cell receptor signaling in Burkitt's lymphoma reveals novel signaling nodes with potential therapeutic relevance. [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 50. doi:10.1158/1538-7445.AM2015-50

Quantitative Proteomic Analysis of BHK-21 Cells Infected with Foot-and-Mouth Disease Virus Serotype Asia 1.

Stable isotope labeling with amino acids in cell culture (SILAC) was used to quantitatively study the host cell gene expression profile, in order to achieve an unbiased overview of the protein expression changes in BHK-21 cells infected with FMDV serotype Asia 1. The SILAC-based approach identified overall 2,141 proteins, 153 of which showed significant alteration in the expression level 6 h post FMDV infection (57 up-regulated and 96 down-regulated). Among these proteins, six cellular proteins, including three down-regulated (VPS28, PKR, EVI5) and three up-regulated (LYPLA1, SEC62 and DARs), were selected according to the significance of the changes and/or the relationship with PKR. The expression level and pattern of the selected proteins were validated by immunoblotting and confocal microscopy. Furthermore, the functions of these cellular proteins were assessed by small interfering RNA-mediated depletion, and their functional importance in the replication of FMDV was demonstrated by western blot, reverse transcript PCR (RT-PCR) and 50% Tissue Culture Infective Dose (TCID50). The results suggest that FMDV infection may have effects on the expression of specific cellular proteins to create more favorable conditions for FMDV infection. This study provides novel data that can be utilized to understand the interactions between FMDV and the host cell.

Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage*

The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2–7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage.

Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1)

Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving patient outcome, and may result in the identification of novel therapeutic targets. Thus, we attempted to gain insight into the proteomic changes that occur during anchorage-independent cancer cell aggregation. As such, an ovarian cancer cell line, OV-90, was cultured in adherent and non-adherent conditions using stable isotope labeling with amino acids in cell culture (SILAC). Anchorage-dependent cells (OV-90AD) were grown in tissue culture flasks, whereas anchorage-independent cells (OV-90AI) were grown in suspension using the hanging-drop method. Cellular proteins from both conditions were then identified using LC-MS/MS, which resulted in the quantification of 1533 proteins. Of these, 13 and 6 proteins were up-regulated and down-regulated, respectively, in aggregate-forming cells compared with cells grown as monolayers. Relative gene expression and protein expression of candidates were examined in other cell line models of aggregate formation (TOV-112D and ES-2), which revealed an increased expression of calcium-activated chloride channel regulator 1 (CLCA1). Moreover, inhibitor and siRNA transfection studies demonstrated an apparent effect of CLCA1 on cancer cell aggregation. Further elucidation of the role of CLCA1 in the pathogenesis of ovarian cancer is warranted.