Characterizing the normal proteome of human ciliary body

BACKGROUND: The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body. RESULTS: In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis. CONCLUSIONS: More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia

Phosphoproteomics of retinoblastoma:A pilot study identifies aberrant kinases

Retinoblastoma is a malignant tumour of the retina which most often occurs in children. Earlier studies on retinoblastoma have concentrated on the identification of key players in the disease and have not provided information on activated/inhibited signalling pathways. The dysregulation of protein phosphorylation in cancer provides clues about the affected signalling cascades in cancer. Phosphoproteomics is an ideal tool for the study of phosphorylation changes in proteins. Hence, global phosphoproteomics of retinoblastoma (RB) was carried out to identify signalling events associated with this cancer. Over 350 proteins showed differential phosphorylation in RB compared to control retina. Our study identified stress response proteins to be hyperphosphorylated in RB which included H2A histone family member X (H2AFX) and sirtuin 1. In particular, Ser140 of H2AFX also known as gamma-H2AX was found to be hyperphosphorylated in retinoblastoma, which indicated the activation of DNA damage response pathways. We also observed the activation of anti-apoptosis in retinoblastoma compared to control. These observations showed the activation of survival pathways in retinoblastoma. The identification of hyperphosphorylated protein kinases including Bromodomain containing 4 (BRD4), Lysine deficient protein kinase 1 (WNK1), and Cyclin-dependent kinase 1 (CDK1) in RB opens new avenues for the treatment of RB. These kinases can be considered as probable therapeutic targets for RB, as small-molecule inhibitors for some of these kinases are already in clinical trials for the treatment other cancers

Surgery, Octreotide, Temozolomide, Bevacizumab, Radiotherapy, and Pegvisomant Treatment of an AIP Mutation-Positive Child

UK India Education Research Initiative and the British Council (75-2014; to P.D.)Council of Scientific and Industrial Research, University Grants Commission, for financial support (2061330632; to A.R.).Medical Research Council (MR/M018539/1; to M.K

Hotspot SF3B1 mutations induce metabolic reprogramming and vulnerability to serine deprivation.

Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1MUT xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1MUT cancers

Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes

© 2017 Wong et al.; Published by Cold Spring Harbor Laboratory Press. Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions, 192 novel protein start sites, 19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted noncoding RNAs to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes

Proteomic Signatures of Diffuse and Intestinal Subtypes of Gastric Cancer

Gastric cancer is a leading cause of death from cancer globally. Gastric cancer is classified into intestinal, diffuse and indeterminate subtypes based on histology according to the Laurén classification. The intestinal and diffuse subtypes, although different in histology, demographics and outcomes, are still treated in the same fashion. This study was designed to discover proteomic signatures of diffuse and intestinal subtypes. Mass spectrometry-based proteomics using tandem mass tags (TMT)-based multiplexed analysis was used to identify proteins in tumor tissues from patients with diffuse or intestinal gastric cancer with adjacent normal tissue control. A total of 7448 or 4846 proteins were identified from intestinal or diffuse subtype, respectively. This quantitative mass spectrometric analysis defined a proteomic signature of differential expression across the two subtypes, which included gremlin1 (GREM1), bcl-2-associated athanogene 2 (BAG2), olfactomedin 4 (OLFM4), thyroid hormone receptor interacting protein 6 (TRIP6) and melanoma-associated antigen 9 (MAGE-A9) proteins. Although GREM1, BAG2, OLFM4, TRIP6 and MAGE-A9 have all been previously implicated in tumor progression and metastasis, they have not been linked to intestinal or diffuse subtypes of gastric cancer. Using immunohistochemical labelling of a tissue microarray comprising of 124 cases of gastric cancer, we validated the proteomic signature obtained by mass spectrometry in the discovery cohort. Our findings should help investigate the pathogenesis of these gastric cancer subtypes and potentially lead to strategies for early diagnosis and treatment

PGTools: a software suite for proteogenomic data analysis and visualization

We describe PGTools, an open source software suite for analysis and visualization of proteogenomic data. PGTools comprises applications, libraries, customized databases, and visualization tools for analysis of mass-spectrometry data using combined proteomic and genomic backgrounds. A single command is sufficient to search databases, calculate false discovery rates, group and annotate proteins, generate peptide databases from RNA-Seq transcripts, identify altered proteins associated with cancer, and visualize genome scale peptide data sets using sophisticated visualization tools. We experimentally confirm a subset of proteogenomic peptides in human PANC-1 cells and demonstrate the utility of PGTools using a colorectal cancer data set that led to the identification of 203 novel protein coding regions missed by conventional proteomic approaches. PGTools should be equally useful for individual proteogenomic investigations as well as international initiatives such as chromosome-centric Human Proteome Project (C-HPP). PGTools is available at http://qcmg.org/bioinformatics/PGTools

PGTools: A software suite for proteogenomic data analysis and visualization

We describe PGTools, an open source software suite for analysis and visualization of proteogenomic data. PGTools comprises applications, libraries, customized databases, and visualization tools for analysis of mass-spectrometry data using combined proteomic and genomic backgrounds. A single command is sufficient to search databases, calculate false discovery rates, group and annotate proteins, generate peptide databases from RNA-Seq transcripts, identify altered proteins associated with cancer, and visualize genome scale peptide data sets using sophisticated visualization tools. We experimentally confirm a subset of proteogenomic peptides in human PANC-1 cells and demonstrate the utility of PGTools using a colorectal cancer data set that led to the identification of 203 novel protein coding regions missed by conventional proteomic approaches. PGTools should be equally useful for individual proteogenomic investigations as well as international initiatives such as chromosome-centric Human Proteome Project (C-HPP). PGTools is available at http://qcmg.org/bioinformatics/PGTools