C9orf72 nucleotide repeat structures initiate molecular cascades of disease.

A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat-length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Specifically, nucleolin, an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases.

A draft map of the human proteome.

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling.

Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD002411 (http://proteomecentral.proteomexchange.org/dataset/PXD002411).

Plasma Proteome Database as a resource for proteomics research: 2014 update.

Plasma Proteome Database (PPD; http://www.plasmaproteomedatabase.org/) was initially described in the year 2005 as a part of Human Proteome Organization's (HUPO's) pilot initiative on Human Plasma Proteome Project. Since then, improvements in proteomic technologies and increased throughput have led to identification of a large number of novel plasma proteins. To keep up with this increase in data, we have significantly enriched the proteomic information in PPD. This database currently contains information on 10,546 proteins detected in serum/plasma of which 3784 have been reported in two or more studies. The latest version of the database also incorporates mass spectrometry-derived data including experimentally verified proteotypic peptides used for multiple reaction monitoring assays. Other novel features include published plasma/serum concentrations for 1278 proteins along with a separate category of plasma-derived extracellular vesicle proteins. As plasma proteins have become a major thrust in the field of biomarkers, we have enabled a batch-based query designated Plasma Proteome Explorer, which will permit the users in screening a list of proteins or peptides against known plasma proteins to assess novelty of their data set. We believe that PPD will facilitate both clinical and basic research by serving as a comprehensive reference of plasma proteins in humans and accelerate biomarker discovery and translation efforts.

Identifying novel targets of oncogenic EGF receptor signaling in lung cancer through global phosphoproteomics.

Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook SILAC strategy, phosphopeptide enrichment, and quantitative MS to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFR(L858R) and EGFR(L858R/T790M) , the TKI-sensitive, and TKI-resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, insulin receptor, hepatocyte growth factor, mitogen-activated protein kinase, mechanistic target of rapamycin, ribosomal protein S6 kinase beta 1, and Janus kinase/signal transducer and activator of transcription signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinases from, AGC and Calcium and calmodulin-dependent kinase groups, as well as STE group kinases were significantly enriched and those of proline-directed kinases from, CMGC and Casein kinase groups were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma. All MS data have been deposited in the ProteomeXchange with identifier PXD001101 (http://proteomecentral.proteomexchange.org/dataset/PXD001101).

Inhibitors of histone demethylation and histone deacetylation cooperate in regulating gene expression and inhibiting growth in human breast cancer cells.

Abnormal activities of histone lysine demethylases (KDMs) and lysine deacetylases (HDACs) are associated with aberrant gene expression in breast cancer development. However, the precise molecular mechanisms underlying the crosstalk between KDMs and HDACs in chromatin remodeling and regulation of gene transcription are still elusive. In this study, we showed that treatment of human breast cancer cells with inhibitors targeting the zinc cofactor dependent class I/II HDAC, but not NAD(+) dependent class III HDAC, led to significant increase of H3K4me2 which is a specific substrate of histone lysine-specific demethylase 1 (LSD1) and a key chromatin mark promoting transcriptional activation. We also demonstrated that inhibition of LSD1 activity by a pharmacological inhibitor, pargyline, or siRNA resulted in increased acetylation of H3K9 (AcH3K9). However, siRNA knockdown of LSD2, a homolog of LSD1, failed to alter the level of AcH3K9, suggesting that LSD2 activity may not be functionally connected with HDAC activity. Combined treatment with LSD1 and HDAC inhibitors resulted in enhanced levels of H3K4me2 and AcH3K9, and exhibited synergistic growth inhibition of breast cancer cells. Finally, microarray screening identified a unique subset of genes whose expression was significantly changed by combination treatment with inhibitors of LSD1 and HDAC. Our study suggests that LSD1 intimately interacts with histone deacetylases in human breast cancer cells. Inhibition of histone demethylation and deacetylation exhibits cooperation and synergy in regulating gene expression and growth inhibition, and may represent a promising and novel approach for epigenetic therapy of breast cancer.

Transcriptomic and proteomic profiling of KEAP1 disrupted and sulforaphane-treated human breast epithelial cells reveals common expression profiles.

Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a potent inhibitor of experimental mammary carcinogenesis and may be an effective, safe chemopreventive agent for use in humans. SFN acts in part on the Keap1/Nrf2 pathway to regulate a battery of cytoprotective genes. In this study, transcriptomic and proteomic changes in the estrogen receptor negative, non-tumorigenic human breast epithelial MCF10A cell line were analyzed following SFN treatment or KEAP1 knockdown with siRNA using microarray and stable isotopic labeling with amino acids in culture (SILAC), respectively. Changes in selected transcripts and proteins were confirmed by PCR and Western blot in MCF10A and MCF12A cells. There was strong correlation between the transcriptomic and proteomic responses in both the SFN treatment (R = 0.679, P < 0.05) and KEAP1 knockdown (R = 0.853, P < 0.05) experiments. Common pathways for SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidants, glutathione metabolism, carbohydrate metabolism, and NADH/NADPH regeneration. Moreover, these pathways were most prominent in both the transcriptomic and the proteomic analyses. The aldo-keto reductase family members, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, as well as NQO1 and ALDH3A1, were highly upregulated at both the transcriptomic and the proteomic levels. Collectively, these studies served to identify potential biomarkers that can be used in clinical trials to investigate the initial pharmacodynamic action of SFN in the breast.

Monoclonal antibody cocktail as an enrichment tool for acetylome analysis.

The availability and robustness of methods to analyze phosphorylated proteins has greatly expanded our knowledge of phosphorylation based cell signaling. A key ingredient to the success of these studies is the ability to enrich phosphopeptides using antibodies or other chemical approaches. Most other post-translational modifications, such as lysine acetylation, are still poorly characterized because of the lack of availability of such enrichment methods. Recently, some groups have reported identification of acetylation sites in a global fashion by enriching acetylated peptides with a polyclonal antibody from a single source that was raised against pan-acetylated lysine. Instead of the use of this polyclonal antibody, we used a cocktail of monoclonal antibodies where each was directed against acetylated lysine in different contexts. Using high resolution Fourier transform mass spectrometry, we observed that the majority of acetylated lysine residues identified using the monoclonal antibody cocktail were distinct from those enriched by the polyclonal antibody used by the other groups. Our study demonstrates that immunoaffinity enrichment of acetylated peptides is somewhat limited by substrate specificity and that an optimal yield of enrichment can be achieved by employing a broader array of affinity reagents.

Screening for therapeutic targets of vorinostat by SILAC-based proteomic analysis in human breast cancer cells.

Histone deacetylases (HDACs) play critical roles in silencing tumor suppressor genes. HDAC inhibitors reactivate tumor suppressor genes and inhibit tumor cell growth in vitro and in vivo, and several HDAC inhibitors are currently being evaluated in clinical trials for cancer therapy. A comprehensive analysis of proteins regulated by HDAC inhibitors would enhance our ability to define and characterize their essential therapeutic targets. Here, we employed stable isotope labeling with amino acids in cell culture-based quantitative proteomics to identify acetylated proteins in human breast cancer cells. Treatment with the clinically relevant HDAC inhibitor, suberoylanilide hydroxamic acid (vorinostat), induces lysine acetylation of 61 proteins in MDA-MB-231 human breast cancer cells. Suberoylanilide hydroxamic acid not only induces lysine acetylation in chromatin-associated proteins, but also acetylates previously unrecognized nonhistone proteins, including transcriptional factors and regulators, chaperones, cell structure proteins, and glycolytic enzymes in a time-dependent manner. Knowledge of the full repertoire of acetylated proteins will provide a foundation for further defining the functions of HDACs in cancer cells.

Inhibition of histone deacetylase suppresses EGF signaling pathways by destabilizing EGFR mRNA in ER-negative human breast cancer cells.

Estrogen receptor alpha (ER)-negative human breast cancer cells frequently overexpress epidermal growth factor receptor (EGFR) and respond poorly to endocrine therapies. Our previous studies demonstrate that histone deacetylation plays a key role in ER gene silencing, and ER expression can be restored with histone deacetylase (HDAC) inhibitors in ER-negative human breast cancer cells. Whether inhibition of HDAC also alters epidermal growth factor (EGF) signaling pathways is not defined. Here we present evidence that reexpression of ER protein by a clinically available HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA or vorinostat), is coupled with loss of EGFR in ER-negative human breast cancer cells. Consistent with this observation, MDA-MB-231 cells, which are ER-negative and overexpress EGFR, that are engineered to express ER show a decrease in EGFR protein expression. Down-regulation of EGFR by SAHA results from attenuation of its mRNA stability. We also confirm that new protein synthesis is required for maintaining EGFR mRNA stability. Further experiments indicate that a decrease in EGFR abolished EGF-initiated signaling pathways including phosphorylated PAK1, p38MAPK and AKT. Thus, SAHA may not only reactivate silenced ER, but also simultaneously deplete EGFR expression. These data suggest that inhibition of HDAC is a promising epigenetic therapy for ER-negative human breast cancer.

A multi-omic analysis of human naïve CD4+ T cells.

BACKGROUND: Cellular function and diversity are orchestrated by complex interactions of fundamental biomolecules including DNA, RNA and proteins. Technological advances in genomics, epigenomics, transcriptomics and proteomics have enabled massively parallel and unbiased measurements. Such high-throughput technologies have been extensively used to carry out broad, unbiased studies, particularly in the context of human diseases. Nevertheless, a unified analysis of the genome, epigenome, transcriptome and proteome of a single human cell type to obtain a coherent view of the complex interplay between various biomolecules has not yet been undertaken. Here, we report the first multi-omic analysis of human primary naïve CD4+ T cells isolated from a single individual. RESULTS: Integrating multi-omics datasets allowed us to investigate genome-wide methylation and its effect on mRNA/protein expression patterns, extent of RNA editing under normal physiological conditions and allele specific expression in naïve CD4+ T cells. In addition, we carried out a multi-omic comparative analysis of naïve with primary resting memory CD4+ T cells to identify molecular changes underlying T cell differentiation. This analysis provided mechanistic insights into how several molecules involved in T cell receptor signaling are regulated at the DNA, RNA and protein levels. Phosphoproteomics revealed downstream signaling events that regulate these two cellular states. Availability of multi-omics data from an identical genetic background also allowed us to employ novel proteogenomics approaches to identify individual-specific variants and putative novel protein coding regions in the human genome. CONCLUSIONS: We utilized multiple high-throughput technologies to derive a comprehensive profile of two primary human cell types, naïve CD4+ T cells and memory CD4+ T cells, from a single donor. Through vertical as well as horizontal integration of whole genome sequencing, methylation arrays, RNA-Seq, miRNA-Seq, proteomics, and phosphoproteomics, we derived an integrated and comparative map of these two closely related immune cells and identified potential molecular effectors of immune cell differentiation following antigen encounter.

Integrated proteomic and metabolic analysis of breast cancer progression.

One of the most persistent hallmarks of cancer biology is the preference of tumor cells to derive energy through glycolysis as opposed to the more efficient process of oxidative phosphorylation (OXPHOS). However, little is known about the molecular cascades by which oncogenic pathways bring about this metabolic switch. We carried out a quantitative proteomic and metabolic analysis of the MCF10A derived cell line model of breast cancer progression that includes parental cells and derivatives representing three different tumor grades of Ras-driven cancer with a common genetic background. A SILAC (Stable Isotope Labeling by Amino acids in Cell culture) labeling strategy was used to quantify protein expression in conjunction with subcellular fractionation to measure dynamic subcellular localization in the nucleus, cytosol and mitochondria. Protein expression and localization across cell lines were compared to cellular metabolic rates as a measure of oxidative phosphorylation (OXPHOS), glycolysis and cellular ATP. Investigation of the metabolic capacity of the four cell lines revealed that cellular OXPHOS decreased with breast cancer progression independently of mitochondrial copy number or electron transport chain protein expression. Furthermore, glycolytic lactate secretion did not increase in accordance with cancer progression and decreasing OXPHOS capacity. However, the relative expression and subcellular enrichment of enzymes critical to lactate and pyruvate metabolism supported the observed extracellular acidification profiles. This analysis of metabolic dysfunction in cancer progression integrated with global protein expression and subcellular localization is a novel and useful technique for determining organelle-specific roles of proteins in disease.

Inhibition of histone deacetylases.

Lysine acetylation of histones is one of the major epigenetic regulators of chromatin conformation and gene expression. The dynamic nature of histone acetylation is determined by the counterbalancing activity of histone acetyltransferase and histone deacetylase (HDAC) enzymes. Acetylation of histones is generally associated with open and transcriptionally active chromatin, whereas the activity of HDACs leads to histone deacetylation, condensation of chromatin, and inhibition of transcription. Aberrant silencing of tumor suppressors and other genes has been found in different types of cancer. Abnormal activity of HDACs has been implicated in tumorigenesis and therefore considerable effort has been put into the development of HDAC inhibitors as a means of modifying histone acetylation status and reexpressing aberrantly silenced tumor suppressor genes. This has led to the generation of a number of structurally diverse compounds that can effectively inhibit HDAC activity, thus altering chromatin structure in cancer cells. This unit discusses the methods and recent technological developments with respect to the studies of HDAC inhibition in cancer.

Mechanistic and structural analyses of the roles of Arg409 and Asp402 in the reaction of the flavoprotein nitroalkane oxidase.

The flavoprotein nitroalkane oxidase (NAO) catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones. The enzyme is a homologue of acyl-CoA dehydrogenase. Asp402 in NAO has been proposed to be the active site base responsible for removing the substrate proton in the first catalytic step; structurally it corresponds to the glutamate which acts as the base in medium chain acyl-CoA dehydrogenase. In the active site of NAO, the carboxylate of Asp402 forms an ionic interaction with the side chain of Arg409. The R409K enzyme has now been characterized kinetically and structurally. The mutation results in a decrease in the rate constant for proton abstraction of 100-fold. Analysis of the three-dimensional structure of the R409K enzyme, determined by X-ray crystallography to a resolution of 2.65 A, shows that the critical structural change is an increase in the distance between the carboxylate of Asp402 and the positively charged nitrogen in the side chain of the residue at position 409. The D402E mutation results in a smaller decrease in the rate constant for proton abstraction of 18-fold. The structure of the D402E enzyme, determined at 2.4 A resolution, shows that there is a smaller increase in the distance between Arg409 and the carboxylate at position 402, and the interaction of this residue with Ser276 is perturbed. These results establish the critical importance of the interaction between Asp402 and Arg409 for proton abstraction by nitroalkane oxidase.