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Molecular cellular proteomics [journal]
- The Membrane Proteome of Sensory Cilia to the Depth of Olfactory Receptors. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Apr 18.
In the nasal cavity, the nonmotile cilium of olfactory sensory neurons (OSNs) constitutes the chemosensory interface between the ambient environment and the brain. The unique sensory organelle facilitates odor detection for which it comprises all necessary components of initial and downstream olfactory signal transduction. In addition to its function in olfaction, a more universal role in modulating different signaling pathways is implicated, for example, in neurogenesis, apoptosis and neural regeneration. To further extend our knowledge about this multifunctional signaling organelle, it is of high importance to establish a most detailed proteome map of the ciliary membrane compartment down to the level of transmembrane receptors. We detached cilia from mouse olfactory epithelia via Ca2+/K+-shock followed by the enrichment of ciliary membrane proteins at alkaline pH and identified a total of 4,403 proteins by gel-based and gel-free methods in conjunction with high-resolution LC/MS. This study is the first to report the detection of 62 native olfactory receptor proteins and to provide evidence for their heterogeneous expression at the protein level. Quantitative data evaluation revealed four ciliary membrane-associated candidate proteins (the annexins ANXA1, ANXA2, ANXA5 and S100A5) with a suggested function in the regulation of olfactory signal transduction and their presence in ciliary structures was confirmed by immunohistochemistry. Moreover, we corroborated the ciliary localization of the potassium-dependent Na+/Ca2+ exchanger (NCKX) 4 and the plasma membrane Ca2+-ATPase 1 (PMCA1) involved in olfactory signal termination and detected for the first time NCKX2 in olfactory cilia. Through comparison with transcriptome data specific for mature, ciliated OSNs, we finally delineated the membrane ciliome of OSNs. The membrane proteome of olfactory cilia established here is the most complete today allowing us to pave new avenues for the study of diverse molecular functions and signaling pathways in and out of olfactory cilia and, thus, to advance our understanding of the biology of sensory organelles in general.
- Ferritin Heavy Chain in Triple Negative Breast Cancer: A Favorable Prognostic Marker that Relates to a CD8+ Effector T-cell Response. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Apr 17.
Ferritin heavy chain (FTH1) is a 21-kDa subunit of the ferritin complex, known for its role in iron metabolism, and which has recently been identified as a favorable prognostic protein for triple negative breast cancer (TNBC) patients. Currently, it is not well understood how FTH1 contributes to an anti-tumor response. Here, we explored whether expression and cellular compartmentalization of FTH1 correlates to an effective immune response in TNBC patients. Analysis of the tumor tissue transcriptome, complemented with in silico pathway analysis, revealed that FTH1 was an integral part of an immunomodulatory network of cytokine signaling, adaptive immunity, and cell death. These findings were confirmed using mass spectrometry (MS)-derived proteomic data, and immunohistochemical staining of tissue microarrays. We observed that FTH1 is localized in both the cytoplasm and/or nucleus of cancer cells. However, high cytoplasmic (c) FTH1 was associated with favorable prognosis (Log-rank P=0.001), while nuclear (n) FTH1 staining was associated with adverse prognosis (Log-rank P=0.019). cFTH1 staining significantly correlated with total FTH1 expression in TNBC tissue samples, as measured by MS analysis (Rs=0.473, P=0.0007), but nFTH1 staining did not (Rs=0.197, P=0.1801). Notably, IFN gamma-producing CD8+ effector T-cells, but not CD4+ T cells, were preferentially enriched in tumors with high expression of cFTH1 (P=0.02). Collectively, our data provide evidence towards new immune regulatory properties of FTH1 in TNBC, which may facilitate development of novel therapeutic targets.
- Proteomic Analysis of Altered Extracellular Matrix Turnover in Bleomycin-Induced Pulmonary Fibrosis. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Apr 16.
Fibrotic disease is characterized by the pathological accumulation of extracellular matrix (ECM) proteins. Surprisingly, very little is known about the synthesis and degradation rates of the many proteins and proteoglycans that comprise healthy and pathological extracellular matrix. As such, a comprehensive understanding of altered ECM protein synthesis and degradation during onset and progression of fibrotic disease would be immensely valuable. We have developed a dynamic proteomics platform that quantifies the fractional synthesis rates (FSR) of large numbers of proteins via stable isotope labeling and LC/MS-based mass isotopomer analysis. Here, we present the first broad analysis of ECM protein kinetics during onset of experimental pulmonary fibrosis. Mice were labeled with heavy water for up to 21 days following induction of lung fibrosis with bleomycin. Lung tissue was subjected to sequential protein extraction to fractionate cellular, guanidine-soluble ECM proteins, and residual insoluble ECM proteins. FSRs were calculated for 34 ECM proteins or protein subunits including collagens, proteoglycans, and microfibrillar proteins. Overall, FSRs of guanidine-soluble ECM proteins were faster than insoluble ECM proteins, suggesting that the insoluble fraction reflects older, more mature matrix components. This was confirmed by quantitation of pyridinoline crosslinks in each protein fraction. In fibrotic lung tissue, there was a significant increase in the fractional synthesis of unique sets of matrix proteins during early (pre-1wk) and late (post-1wk) fibrotic response. Furthermore, we isolated fast turnover subpopulations of several ECM proteins (e.g. type I collagen) based on guanidine solubility, allowing for accelerated detection of increased synthesis of typically slow-turnover protein populations. This establishes the presence of multiple kinetic pools of pulmonary collagen in vivo with altered turnover rates during evolving fibrosis. These data demonstrate the utility of dynamic proteomics in analyzing changes in ECM protein turnover associated with the onset and progression of fibrotic disease.
- Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localisation and dynamics. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Apr 16.
Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. 9 of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and 8 novel proteins termed FLAM (FLAgellar Member). Remarkably, they localised to 5 different sub-domains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and FRAP (Fluorescence Recovery After Photobleaching) approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.
- Glycoproteomic analysis of prostate cancer tissues by SWATH mass spectrometry discovers N-acylethanolamine acid amidase and protein tyrosine kinase 7 as signatures for tumor aggressiveness. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Apr 22.
The identification of biomarkers indicating the level of aggressiveness of prostate cancer (PCa) will address an urgent clinical need to minimize the general over-treatment of patients with non-aggressive PCa, which account for the majority of PCa cases. Here, we isolated formerly N-linked glycopeptides from normal prostate (n=10), non-aggressive (n=24), aggressive (n=16) and metastatic PCa (n=25) tumor tissues and analyzed the samples by SWATH mass spectrometry (SWATH-MS), an emerging data independent acquisition method that generates a single file containing fragment ion spectra of all ionized species of a sample. The resulting datasets were searched using a targeted data analysis strategy where a priori spectral reference library representing known N-glycosites of the human proteome was used to identify groups of signals in the SWATH-MS data. On-average we identified 1430 N-glycosites from each sample. Out of those, 220 glycoproteins showed significant quantitative changes associated with diverse biological processes involved in PCa aggressiveness and metastasis and indicated functional relationships. Two glycoproteins, N-acylethanolamine acid amidase (NAAA) and protein tyrosine kinase 7 (PTK7) that were significantly associated with aggressive PCa in the initial sample cohort were further validated in an independent set of patient tissues using tissue microarray analysis (TMA). The results suggest that NAAA and PTK7 may be used as potential tissue biomarkers to avoid overtreatment of non-aggressive PCa.
- Large-scale analysis of co-existing post-translational modifications on histone tails reveals global fine-structure of crosstalk. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Apr 16.
Mass spectrometry (MS) is a powerful analytical method for the identification and quantification of co-existing post-translational modifications (PTMs) in histone proteins. One of the most important challenges in current chromatin biology is to characterize the relationships between co-existing histone marks, the order and hierarchy of their deposition and their distinct biological functions. We developed a database CrossTalkDB to organize observed and reported co-existing histone marks as revealed by MS experiments of histone proteins and their derived peptides. Statistical assessment revealed sample-specific patterns for the co-frequency of histone PTMs. We implemented a new method to identify positive and negative interplay between pairs of methylation and acetylation marks in proteins. Many of the detected features were conserved between different cell types, or exist across species, thereby revealing general rules for crosstalk between histone marks. The observed features are in accordance with previously reported examples of crosstalk. We observed novel types of interplay (1) among acetylated residues, revealing positive crosstalk between nearby acetylated sites but negative crosstalk for distant ones, and (2) for discrete methylation states at K9, K27 and K36 of histone H3, suggesting a more differentiated functional role of methylation beyond the general expectation of enhanced activity at higher methylation states.
- SILAC-Based Strategy for Proteome-Wide Thermodynamic Analysis of Protein-Ligand Binding Interactions. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Apr 16.
Described here is a quantitative mass spectrometry-based proteomics method for the large-scale thermodynamic analysis of protein-ligand binding interactions. The methodology utilizes a chemical modification strategy termed, Stability of Proteins from Rates of Oxidation (SPROX), in combination with a Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) approach to compare the equilibrium folding/unfolding properties of proteins in the absence and presence of target ligands. The method, which is general with respect to ligand, measures the ligand-induced changes in protein stability associated with protein-ligand binding. The methodology is demonstrated in a proof-of-principle study in which the well-characterized protein-drug interaction between cyclosporine A (CsA) and cyclophilin A was successfully analyzed in the context of a yeast cell lysate. A control experiment was also performed to assess the method's false positive rate of ligand discovery, which was found to be on the order of 0.4 - 3.5%. The new method was utilized to characterize the adenosine triphosphate (ATP)-interactome in Saccharomyces cerevisiae using the non-hydrolyzable ATP analogue, adenylyl imidodiphosphate (AMP-PNP), and the proteins in a yeast cell lysate. The new methodology enabled the interrogation of 526 yeast proteins for interactions with ATP using 2035 peptide probes. Ultimately, 325 peptide hits from 139 different proteins were identified. Approximately 70% of the hit proteins identified in this work were not previously annotated as ATP binding proteins. However, nearly two-thirds of the newly discovered ATP interacting proteins have known interactions with other nucleotides and co-factors (e.g., NAD and GTP), DNA, and RNA based on GO-term analyses. The current work is the first proteome-wide profile of the yeast ATP-interactome, and it is the largest proteome-wide profile of any ATP-interactome generated, to date, using an energetics-based method.
- Regulation dynamics of Leishmania differentiation: deconvoluting signals and identifying phosphorylation trends. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Apr 16.
Leishmania are obligatory intracellular parasitic protozoa that cause a wide range of disease in humans; cycling between extracellular promastigotes in the mid-gut of sand flies and intracellular amastigotes in the phagolysosome of mammalian macrophages. While much of the molecular mechanism of development inside macrophages remains a mystery, development of a host-free system that simulates phagolysosome conditions (37°C and pH 5.5) has provided new insights into these processes. The time-course of promastigote-to-amastigote differentiation can be divided into four morphologically distinct phases: I, signal perception (0-5 hours after exposure); II, movement cessation and aggregation (5-10 hours); III, amastigote morphogenesis (10-24 hours); and IV, maturation (24-120 hours). Transcriptomic and proteomic analyses indicated that differentiation is a coordinated process that results in adaptation to life inside phagolysosomes. Recent phosphoproteomic analysis revealed extensive differences in phosphorylation between promastigotes and amastigotes, and identified stage-specific phosphorylation motifs. We hypothesize that the differentiation signal activates a phosphorylation pathway, which initiates Leishmania transformation. Here we use Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) to interrogate the dynamics of changes in phosphorylation profile during L. donovani promastigote-to-amastigote differentiation. Analysis of 163 phosphopeptides (from 106 proteins) revealed six distinct kinetic profiles; with increases in phosphorylation predominating during phase I and III, whereas phase II and IV are characterized by greater dephosphorylation. Several proteins (including a protein kinase) were phosphorylated in phase I after exposure to the complete differentiation signal (i.e. signal-specific; 37°C and pH 5.5); but not after either of the physical parameters separately. Several other protein kinases (including regulatory subunits) and phosphatases also showed changes in phosphorylation during differentiation. This work constitutes the first genome-scale interrogation of phosphorylation dynamics in a parasitic protozoa; revealing the outline of a signaling pathway during Leishmania differentiation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (identiﬁer PXD000671).
- Elevated Plasma Albumin and Apolipoprotein A-I Oxidation under Suboptimal Specimen Storage Conditions. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Apr 15.
S-cysteinylated albumin and methionine oxidized apolipoprotein A-I (apoA-I) have been posed as candidate markers of diseases associated with oxidative stress. Here, a dilute-and-shoot form of LC-ESI-MS requiring half a microliter of blood plasma was employed to simultaneously quantify the relative abundance of these oxidized proteoforms in samples stored at -80 °C, -20 °C, room temperature, and exposed to multiple freeze-thaw cycles and other adverse conditions to assess the possibility that protein oxidation may occur as a result of poor sample storage and/or handling. Samples from a healthy donor and a participant with poorly controlled type 2 diabetes started at the same low level of protein oxidation and behaved similarly as significant increases in albumin oxidation via S-cysteinylation were found to occur within hours at room temperature and days at -20 °C. Methionine oxidation of apoA-I took place on a longer time scale, setting in after albumin oxidation reached a plateau. Freeze-thaw cycles had minimal effect on protein oxidation. In matched collections protein oxidation in serum was the same as that in plasma. Albumin and apoA-I oxidation were not affected by sample headspace or the degree to which vials were sealed. ApoA-I, however, was unexpectedly found to oxidize faster in samples with lower surface area to volume ratios. An initial survey of samples from patients with inflammatory conditions normally associated with elevated oxidative stress - including acute myocardial infarction and prostate cancer - demonstrated a lack of detectable apoA-I oxidation. Albumin S-cysteinylation in these samples was consistent with known but relatively brief exposures to temperatures above -30 °C (the freezing point of blood plasma). Given their properties and ease of analysis, these oxidized proteoforms, once fully validated, may represent the first markers of blood plasma specimen integrity based on direct measurement of oxidative molecular damage that can occur under suboptimal storage conditions.
- Modulation of the chromatin phosphoproteome by the Haspin protein kinase. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Apr 14.
Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr3 of the Histone H3 which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date Histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the haspin consensus motif and the co-crystal structure of the kinase with the Histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the Histone H3 residues Arg2 and Lys4 which are adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of haspin activity by methylation of Lys4 of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser137 of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser137 resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser137 might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing.