A Sample Preparation Procedure for Isobaric Labeling-Based Single-Cell Proteomics.

Mass spectrometry-based proteomics has traditionally been limited by the amount of input material for analysis. Single-cell proteomics has emerged as a challenging discipline due to the ultra-high sensitivity required. Isobaric labeling-based multiplex strategies with a carrier proteome offer an approach to overcome the sensitivity limitations.

Deciphering the Role and Signaling Pathways of PKCα in Luminal A Breast Cancer Cells.

Protein kinase C (PKC) comprises a family of highly related serine/threonine protein kinases involved in multiple signaling pathways, which control cell proliferation, survival, and differentiation. The role of PKCα in cancer has been studied for many years. However, it has been impossible to establish whether PKCα acts as an oncogene or a tumor suppressor.

Identification and Validation of Stage-Associated Serum Biomarkers in Colorectal Cancer Using MS-Based Procedures.

Purpose: Successful prevention of colorectal cancer (CRC) would benefit from a rapid serum screening for early detection. Here, a novel strategy for CRC biomarker discovery and validation exclusively based on MS procedures is reported.

Experimental Design: Identification of CRC serum biomarkers is initially made using label-free quantification on pooled serum samples from different CRC stages followed by two consecutive steps of targeted parallel reaction monitoring assays in different serum cohorts.

Proteomic Characterization of Transcription and Splicing Factors Associated with a Metastatic Phenotype in Colorectal Cancer.

We investigated new transcription and splicing factors associated with the metastatic phenotype in colorectal cancer. A concatenated tandem array of consensus transcription factor (TF)-response elements was used to pull down nuclear extracts in two different pairs of colorectal cancer cells, KM12SM/KM12C and SW620/480, genetically related but differing in metastatic ability. Proteins were analyzed by label-free LC-MS and quantified with MaxLFQ.

Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach.

The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments.

Functional Identification of Target by Expression Proteomics (FITExP) reveals protein targets and highlights mechanisms of action of small molecule drugs.

Phenomenological screening of small molecule libraries for anticancer activity yields potentially interesting candidate molecules, with a bottleneck in the determination of drug targets and the mechanism of anticancer action. We have found that, for the protein target of a small-molecule drug, the abundance change in late apoptosis is exceptional compared to the expectations based on the abundances of co-regulated proteins. Based on this finding, a novel method to drug target deconvolution is proposed.

RRP6/EXOSC10 is required for the repair of DNA double-strand breaks by homologous recombination.

The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively.

The effects of 5-fluorouracil on the proteome of colon cancer cells.

The pyrimidine analogue 5-fluorouracil (5FU) is used as a treatment for solid tumors, but its mechanism of action is not fully understood. We have used mass spectrometry to study the mechanism of action of 5FU, and we have measured the effects of this drug on the composition and on the turnover of the proteome of RKO cancer cells. We have identified novel potential targets of 5FU that are affected after very short exposure times.

ATP enhances neuronal differentiation of PC12 cells by activating PKCα interactions with cytoskeletal proteins.

PKCα is a key mediator of the neuronal differentiation controlled by NGF and ATP. However, its downstream signaling pathways remain to be elucidated. To identify the signaling partners of PKCα, we analyzed proteins coimmunoprecipitated with this enzyme in PC12 cells differentiated with NGF and ATP and compared them with those obtained with NGF alone or growing media.

Are the majority of a(2)-ions cyclic?

Detailed analysis of >18 400 high-mass accuracy tandem mass spectra resulting from higher energy collisional dissociation yields further evidence of the cyclic nature of a(2)-ions.

Structural and mechanistic insights into the association of PKCalpha-C2 domain to PtdIns(4,5)P2.

C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca(2+)-binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCalpha-C2 domain in complex with Ca(2+), 1,2-dihexanoyl-sn-glycero-3-[phospho-L-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P(2)] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns(4,5)P(2) occupies the concave surface of strands beta3 and beta4.

The PtdIns(4,5)P2 ligand itself influences the localization of PKCalpha in the plasma membrane of intact living cells.

Rapamycin-triggered heterodimerization strategy is becoming an excellent tool for rapidly modifying phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P2] levels at the plasma membrane and for studying their influence in different processes. In this work, we studied the effect of modulation of the PtdIns(4,5)P2 concentration on protein kinase C (PKC) alpha membrane localization in intact living cells. We showed that an increase in the PtdIns(4,5)P2 concentration enlarges the permanence of PKCalpha in the plasma membrane when PC12 cells are stimulated with ATP, independently of the diacylglycerol generated.

The C2 domains of classical PKCs are specific PtdIns(4,5)P2-sensing domains with different affinities for membrane binding.

C2 domains are conserved protein modules in many eukaryotic signaling proteins, including the protein kinase (PKCs). The C2 domains of classical PKCs bind to membranes in a Ca(2+)-dependent manner and thereby act as cellular Ca(2+) effectors. Recent findings suggest that the C2 domain of PKCalpha interacts specifically with phosphatidylinositols 4,5-bisphosphate (PtdIns(4,5)P(2)) through its lysine rich cluster, for which it shows higher affinity than for POPS.

The C2 domain of PKCalpha is a Ca2+ -dependent PtdIns(4,5)P2 sensing domain: a new insight into an old pathway.

The C2 domain is a targeting domain that responds to intracellular Ca2+ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P2 specifically interacts with this motif.

Molecular mechanisms of PKCalpha localization and activation by arachidonic acid. The C2 domain also plays a role.

Arachidonic acid, one of the major unsaturated fatty acids released during cell stimulation, participates in the signaling necessary for activation of different enzymes, including protein kinase C (PKC). Here, we demonstrate that arachidonic acid is a direct activator of PKCalpha, but needs the cooperation of Ca(2+) to exert its function. By using several mutants of the C2 and C1 domains, we were able to determine the molecular mechanism of this activation.

The ATP-dependent membrane localization of protein kinase Calpha is regulated by Ca2+ influx and phosphatidylinositol 4,5-bisphosphate in differentiated PC12 cells.

Signal transduction through protein kinase Cs (PKCs) strongly depends on their subcellular localization. Here, we investigate the molecular determinants of PKCalpha localization by using a model system of neural growth factor (NGF)-differentiated pheochromocytoma (PC12) cells and extracellular stimulation with ATP. Strikingly, the Ca2+ influx, initiated by the ATP stimulation of P2X receptors, rather than the Ca2+ released from the intracellular stores, was the driving force behind the translocation of PKCalpha to the plasma membrane.