Proteomics of Asrij Perturbation in Drosophila Lymph Glands for Identification of New Regulators of Hematopoiesis.

Hematopoiesis is the process of differentiation of precursor blood cells into mature blood cells that is controlled by a complex set of molecular interactions. Understanding hematopoiesis is important for the study of hematological disorders. However, a comprehensive understanding of how physiological and genetic mechanisms regulate blood cell precursor maintenance and differentiation is lacking. Owing to simplicity and ease of genetic analysis, the Drosophila melanogaster lymph gland (LG) is an excellent model to study hematopoiesis. Here, we quantitatively analyzed the LG proteome under genetic conditions that either maintain precursors or promote their differentiation in vivo, by perturbing expression of Asrij, a conserved endosomal regulator of hematopoiesis. Using iTRAQ-based quantitative proteomics, we determined the relative expression levels of proteins in Asrij-knockout and overexpressing LGs from 1500 larval dissections compared with wild type. Our data showed that at least 6.5% of the Drosophila proteome is expressed in wild type LGs. Of the 2133 proteins identified, 780 and 208 proteins were common to previously reported cardiac tube and hemolymph proteomes, respectively, resulting in the identification of 1238 proteins exclusive to the LG. Perturbation of Asrij levels led to differential expression of 619 proteins, of which 27% have human homologs implicated in various diseases. Proteins regulating metabolism, immune system, signal transduction and vesicle-mediated transport were significantly enriched. Immunostaining of representative candidates from the enriched categories and previous reports confirmed 73% of our results, indicating the validity of our LG proteome. Our study provides, for the first time, an in vivo proteomics resource for identifying novel regulators of hematopoiesis that will also be applicable to understanding vertebrate blood cell development.

Dehydration-responsive nuclear proteome landscape of chickpea (Cicer arietinum L.) reveals phosphorylation-mediated regulation of stress response.

Nonavailability of water or dehydration remains recurring climatic disorder affecting yield of major food crops, legumes in particular. Nuclear proteins (NPs) and phosphoproteins (NPPs) execute crucial cellular functions that form the regulatory hub for coordinated stress response. Phosphoproteins hold enormous influence over cellular signalling. Four-week-old seedlings of a grain legume, chickpea, were subjected to gradual dehydration, and NPs were extracted from unstressed control and from 72- and 144-hr stressed tissues. We identified 4,832 NPs and 478 phosphosites, corresponding to 299 unique NPPs involved in multivariate cellular processes including protein modification and gene expression regulation, among others. The identified proteins included several novel kinases, phosphatases, and transcription factors, besides 660 uncharacterized proteins. Spliceosome complex and splicing related proteins were dominant among differentially regulated NPPs, indicating their dehydration modulated regulation. Phospho-motif analysis revealed stress-induced enrichment of proline-directed serine phosphorylation. Association mapping of NPPs revealed predominance of differential phosphorylation of spliceosome and splicing associated proteins. Also, regulatory proteins of key processes viz., protein degradation, regulation of flowering time, and circadian clock were observed to undergo dehydration-induced dephosphorylation. The characterization of novel regulatory proteins would provide new insights into stress adaptation and enable directed genetic manipulations for developing climate-resilient crops.

Proteomic approach and expression analysis revealed the differential expression of predicted leptospiral proteases capable of ECM degradation.

Leptospira, the causative agent of leptospirosis is known to have many proteases with potential to degrade extracellular matrix. However, a multipronged approach to identify, classify, characterize and elucidate their role has not been attempted. Our proteomic approach using high-resolution LC-MS/MS analysis of Triton X-114 fractions of Leptospira interrogans resulted in the identification of 104 proteases out of 130 proteases predicted by MEROPS. In Leptospira approximately 3.5% of the genome complements for proteases, which include catalytic types of metallo-, serine-, cysteine-, aspartic-, threonine- and asparagine- peptidases. Comparison of proteases from different serovars revealed that M04, M09B, M14A, M75, M28A, A01 and U73 protease families are exclusively present in pathogenic form. The M23 and S33 protease families are represented with >14 members in Leptospira. The differential expression under physiological temperature (37 °C) and osmolarity (300 mOsM) showed that proteases belonging to the catalytic type of Metallo-peptidases are upregulated significantly in pathogenic conditions. In silico prediction and characterization of the proteases revealed that several proteases are membrane anchored and secretory, classical as well as non-classical system. The study demonstrates the diversity and complexity of proteases, while maintaining conservation across the serovars in Leptospira and their differential expression under pathogenic conditions.

Synovial fluid proteome in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoinflammatory disorder that affects small joints. Despite intense efforts, there are currently no definitive markers for early diagnosis of RA and for monitoring the progression of this disease, though some of the markers like anti CCP antibodies and anti vimentin antibodies are promising. We sought to catalogue the proteins present in the synovial fluid of patients with RA. It was done with the aim of identifying newer biomarkers, if any, that might prove promising in future. METHODS: To enrich the low abundance proteins, we undertook two approaches-multiple affinity removal system (MARS14) to deplete some of the most abundant proteins and lectin affinity chromatography for enrichment of glycoproteins. The peptides were analyzed by LC-MS/MS on a high resolution Fourier transform mass spectrometer. RESULTS: This effort was the first total profiling of the synovial fluid proteome in RA that led to identification of 956 proteins. From the list, we identified a number of functionally significant proteins including vascular cell adhesion molecule-1, S100 proteins, AXL receptor protein tyrosine kinase, macrophage colony stimulating factor (M-CSF), programmed cell death ligand 2 (PDCD1LG2), TNF receptor 2, (TNFRSF1B) and many novel proteins including hyaluronan-binding protein 2, semaphorin 4A (SEMA4D) and osteoclast stimulating factor 1. Overall, our findings illustrate the complex and dynamic nature of RA in which multiple pathways seems to be participating actively. CONCLUSIONS: The use of high resolution mass spectrometry thus, enabled identification of proteins which might be critical to the progression of RA.

Proteomic profiling of retinoblastoma by high resolution mass spectrometry.

BACKGROUND: Retinoblastoma is an ocular neoplastic cancer caused primarily due to the mutation/deletion of RB1 gene. Due to the rarity of the disease very limited information is available on molecular changes in primary retinoblastoma. High throughput analysis of retinoblastoma transcriptome is available however the proteomic landscape of retinoblastoma remains unexplored. In the present study we used high resolution mass spectrometry-based quantitative proteomics to identify proteins associated with pathogenesis of retinoblastoma. METHODS: We used five pooled normal retina and five pooled retinoblastoma tissues to prepare tissue lysates. Equivalent amount of proteins from each group was trypsin digested and labeled with iTRAQ tags. The samples were analyzed on Orbitrap Velos mass spectrometer. We further validated few of the differentially expressed proteins by immunohistochemistry on primary tumors. RESULTS: We identified and quantified a total of 3587 proteins in retinoblastoma when compared with normal adult retina. In total, we identified 899 proteins that were differentially expressed in retinoblastoma with a fold change of ≥2 of which 402 proteins were upregulated and 497 were down regulated. Insulin growth factor 2 mRNA binding protein 1 (IGF2BP1), chromogranin A, fetuin A (ASHG), Rac GTPase-activating protein 1 and midkine that were found to be overexpressed in retinoblastoma were further confirmed by immunohistochemistry by staining 15 independent retinoblastoma tissue sections. We further verified the effect of IGF2BP1 on cell proliferation and migration capability of a retinoblastoma cell line using knockdown studies. CONCLUSIONS: In the present study mass spectrometry-based quantitative proteomic approach was applied to identify proteins differentially expressed in retinoblastoma tumor. This study identified the mitochondrial dysfunction and lipid metabolism pathways as the major pathways to be deregulated in retinoblastoma. Further knockdown studies of IGF2BP1 in retinoblastoma cell lines revealed it as a prospective therapeutic target for retinoblastoma.

A proteogenomic analysis of Anopheles gambiae using high-resolution Fourier transform mass spectrometry.

Anopheles gambiae is a major mosquito vector responsible for malaria transmission, whose genome sequence was reported in 2002. Genome annotation is a continuing effort, and many of the approximately 13,000 genes listed in VectorBase for Anopheles gambiae are predictions that have still not been validated by any other method. To identify protein-coding genes of An. gambiae based on its genomic sequence, we carried out a deep proteomic analysis using high-resolution Fourier transform mass spectrometry for both precursor and fragment ions. Based on peptide evidence, we were able to support or correct more than 6000 gene annotations including 80 novel gene structures and about 500 translational start sites. An additional validation by RT-PCR and cDNA sequencing was successfully performed for 105 selected genes. Our proteogenomic analysis led to the identification of 2682 genome search-specific peptides. Numerous cases of encoded proteins were documented in regions annotated as intergenic, introns, or untranslated regions. Using a database created to contain potential splice sites, we also identified 35 novel splice junctions. This is a first report to annotate the An. gambiae genome using high-accuracy mass spectrometry data as a complementary technology for genome annotation.

A network map of cytoskeleton-associated protein 4 (CKAP4) mediated signaling pathway in cancer.

Cytoskeleton-associated protein 4 (CKAP4) is a non-glycosylated type II transmembrane protein that serves as a cell surface-activated receptor. It is expressed primarily in the plasma membranes of bladder epithelial cells, type II alveolar pneumocytes, and vascular smooth muscle cells. CKAP4 is involved in various biological activities including cell proliferation, cell migration, keratinocyte differentiation, glycogenesis, fibrosis, thymic development, cardiogenesis, neuronal apoptosis, and cancer. CKAP4 has been described as a pro-tumor molecule that regulates the progression of various cancers, including lung cancer, breast cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, cervical cancer, oral cancer, bladder cancer, cholangiocarcinoma, pancreatic cancer, myeloma, renal cell carcinoma, melanoma, squamous cell carcinoma, colorectal cancer, and osteosarcoma. CKAP4 and its isoform bind to DKK1 or DKK3 (Dickkopf proteins) or antiproliferative factor (APF) and regulates several downstream signaling cascades. The CKAP4 complex plays a crucial role in regulating the signaling pathways including PI3K/AKT and MAPK1/3. Recently, CKAP4 has been recognized as a potential target for cancer therapy. Due to its biomedical importance, we integrated a network map of CKAP4. The available literature on CKAP4 signaling was manually curated according to the NetPath annotation criteria. The consolidated pathway map comprises 41 activation/inhibition events, 21 catalysis events, 35 molecular associations, 134 gene regulation events, 83 types of protein expression, and six protein translocation events. CKAP4 signaling pathway map data is freely accessible through the WikiPathways Database ( https://www.wikipathways.org/index.php/Pathway:WP5322 ). Generation of CKAP4 signaling pathway map.

A molecular network map of orexin-orexin receptor signaling system.

Orexins are excitatory neuropeptides, which are predominantly associated with feeding behavior, sleep-wake cycle and energy homeostasis. The orexinergic system comprises of HCRTR1 and HCRTR2, G-protein-coupled receptors of rhodopsin family and the endogenous ligands processed from HCRT pro-hormone, Orexin A and Orexin B. These neuropeptides are biosynthesized by the orexin neurons present in the lateral hypothalamus area, with dense projections to other brain regions. The orexin-receptor signaling is implicated in various metabolic as well as neurological disorders, making it a promising target for pharmacological interventions. However, there is limited information available on the collective representation of the signal transduction pathways pertaining to the orexin-orexin receptor signaling system. Here, we depict a compendium of the Orexin A/B stimulated reactions in the form of a basic signaling pathway map. This map catalogs the reactions into five categories: molecular association, activation/inhibition, catalysis, transport, and gene regulation. A total of 318 downstream molecules were annotated adhering to the guidelines of NetPath curation. This pathway map can be utilized for further assessment of signaling events associated with orexin-mediated physiological functions and is freely available on WikiPathways, an open-source pathway database ( https://www.wikipathways.org/index.php/Pathway:WP5094 ).

An assembly of TROP2-mediated signaling events.

Trophoblast cell surface antigen 2 (TROP2) is a calcium-transducing transmembrane protein mainly involved in embryo development. The aberrant expression of TROP2 is observed in numerous cancers, including triple-negative breast cancer, gastric, colorectal, pancreatic, squamous cell carcinoma of the oral cavity, and prostate cancers. The main signaling pathways mediated by TROP2 are calcium signaling, PI3K/AKT, JAK/STAT, MAPKs, and ß-catenin signaling. However, collective information about the TROP2-mediated signaling pathway is not available for visualization or analysis. In this study, we constructed a TROP2 signaling map with respect to its role in different cancers. The data curation was done manually by following the NetPath annotation criteria. The described map consists of different molecular events, including 8 activation/inhibition, 16 enzyme catalysis, 19 gene regulations, 12 molecular associations, 39 induced-protein expressions, and 2 protein translocation. The data of the TROP2 pathway map is made freely accessible through the WikiPathways Database ( https://www.wikipathways.org/index.php/Pathway:WP5300 ). Development of TROP2 signaling pathway map.

A network map of apelin-mediated signaling.

The apelin receptor (APLNR) is a class A (rhodopsin-like) G-protein coupled receptor with a wide distribution throughout the human body. Activation of the apelin/APLNR system regulates AMPK/PI3K/AKT/mTOR and RAF/ERK1/2 mediated signaling pathways. APLNR activation orchestrates several downstream signaling cascades, which play diverse roles in physiological effects, including effects upon vasoconstriction, heart muscle contractility, energy metabolism regulation, and fluid homeostasis angiogenesis. We consolidated a network map of the APLNR signaling map owing to its biomedical importance. The curation of literature data pertaining to the APLNR system was performed manually by the NetPath criteria. The described apelin receptor signaling map comprises 35 activation/inhibition events, 38 catalysis events, 4 molecular associations, 62 gene regulation events, 113 protein expression types, and 4 protein translocation events. The APLNR signaling pathway map data is made freely accessible through the WikiPathways Database ( https://www.wikipathways.org/index.php/Pathway:WP5067 ).

A modular map of Bradykinin-mediated inflammatory signaling network.

Bradykinin, a member of the kallikrein-kinin system (KKS), is associated with an inflammatory response pathway with diverse vascular permeability functions, including thrombosis and blood coagulation. In majority, bradykinin signals through Bradykinin Receptor B2 (B2R). B2R is a G protein-coupled receptor (GPCR) coupled to G protein family such as Gαqs, Gαq/Gα11, Gαi1, and Gß1γ2. B2R stimulation leads to the activation of a signaling cascade of downstream molecules such as phospholipases, protein kinase C, Ras/Raf-1/MAPK, and PI3K/AKT and secondary messengers such as inositol-1,4,5-trisphosphate, diacylglycerol and Ca2+ ions. These secondary messengers modulate the production of nitric oxide or prostaglandins. Bradykinin-mediated signaling is implicated in inflammation, chronic pain, vasculopathy, neuropathy, obesity, diabetes, and cancer. Despite the biomedical importance of bradykinin, a resource of bradykinin-mediated signaling pathway is currently not available. Here, we developed a pathway resource of signaling events mediated by bradykinin. By employing data mining strategies in the published literature, we describe an integrated pathway reaction map of bradykinin consisting of 233 reactions. Bradykinin signaling pathway events included 25 enzyme catalysis reactions, 12 translocations, 83 activation/inhibition reactions, 11 molecular associations, 45 protein expression and 57 gene regulation events. The pathway map is made publicly available on the WikiPathways Database with the ID URL: https://www.wikipathways.org/index.php/Pathway:WP5132 . The bradykinin-mediated signaling pathway map will facilitate the identification of novel candidates as therapeutic targets for diseases associated with dysregulated bradykinin signaling.

Human Proteinpedia: a unified discovery resource for proteomics research.

Sharing proteomic data with the biomedical community through a unified proteomic resource, especially in the context of individual proteins, is a challenging prospect. We have developed a community portal, designated as Human Proteinpedia (http://www.humanproteinpedia.org/), for sharing both unpublished and published human proteomic data through the use of a distributed annotation system designed specifically for this purpose. This system allows laboratories to contribute and maintain protein annotations, which are also mapped to the corresponding proteins through the Human Protein Reference Database (HPRD; http://www.hprd.org/). Thus, it is possible to visualize data pertaining to experimentally validated posttranslational modifications (PTMs), protein isoforms, protein-protein interactions (PPIs), tissue expression, expression in cell lines, subcellular localization and enzyme substrates in the context of individual proteins. With enthusiastic participation of the proteomics community, the past 15 months have witnessed data contributions from more than 75 labs around the world including 2710 distinct experiments, >1.9 million peptides, >4.8 million MS/MS spectra, 150,368 protein expression annotations, 17,410 PTMs, 34,624 PPIs and 2906 subcellular localization annotations. Human Proteinpedia should serve as an integrated platform to store, integrate and disseminate such proteomic data and is inching towards evolving into a unified human proteomics resource.

Human Protein Reference Database--2009 update.

Human Protein Reference Database (HPRD--http://www.hprd.org/), initially described in 2003, is a database of curated proteomic information pertaining to human proteins. We have recently added a number of new features in HPRD. These include PhosphoMotif Finder, which allows users to find the presence of over 320 experimentally verified phosphorylation motifs in proteins of interest. Another new feature is a protein distributed annotation system--Human Proteinpedia (http://www.humanproteinpedia.org/)--through which laboratories can submit their data, which is mapped onto protein entries in HPRD. Over 75 laboratories involved in proteomics research have already participated in this effort by submitting data for over 15,000 human proteins. The submitted data includes mass spectrometry and protein microarray-derived data, among other data types. Finally, HPRD is also linked to a compendium of human signaling pathways developed by our group, NetPath (http://www.netpath.org/), which currently contains annotations for several cancer and immune signaling pathways. Since the last update, more than 5500 new protein sequences have been added, making HPRD a comprehensive resource for studying the human proteome.

An assembly of galanin-galanin receptor signaling network.

The galanin receptor family of proteins is present throughout the central nervous system and endocrine system. It comprises of three subtypes-GalR1, GalR2, and GalR3; all of which are G-protein-coupled receptors. Galanin predominantly acts as an inhibitory, hyper-polarizing neuromodulator, which has several physiological as well as pathological functions. Galanin has a role in mediating food intake, memory, sexual behavior, nociception and is also associated with diseases such as Alzheimer's disease, epilepsy, diabetes mellitus, and chronic pain. However, the understanding of signaling mechanisms of the galanin family of neuropeptides is limited and an organized pathway map is not yet available. Therefore, a detailed literature mining of the publicly available articles pertaining to the galanin receptor was followed by manual curation of the reactions and their integration into a map. This resulted in the cataloging of molecular reactions involving 64 molecules into five categories such as molecular association, activation/inhibition, catalysis, transport, and gene regulation. For enabling easy access of biomedical researchers, the galanin-galanin receptor signaling pathway data was uploaded to WikiPathways ( https://www.wikipathways.org/index.php/Pathway:WP4970 ), a freely available database of biological pathways.

Differences in milk metabolites in Malnad Gidda (Bos indicus) cows reared under pasture-based feeding system.

The milk and milk products from cows reared under grazing system are believed to be healthier and hence have high demand compared to milk from cows reared in the non-grazing system. However, the effect of grazing on milk metabolites, specifically lipids has not been fully understood. In this study, we used acetonitrile precipitation and methanol:chloroform methods for extracting the milk metabolites followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) run to identify the different metabolites between the milk of grazing and non-grazing early lactating Malnad Gidda cows. Various carbohydrates, amino acids, nucleosides and vitamin derivatives were found to be differentially abundant in grazing cows. A total of 35 metabolites were differentially regulated (fold change above 1.5) between the two groups. Tyrosyl-threonine, histidinyl-cysteine, 1-methyladenine, L-cysteine and selenocysteine showed fold change above 3 in grazing cows. The lipid profile of milk showed a lesser difference between grazing and non-grazing cows as compared to polar metabolites. To the best of our knowledge, this is the largest inventory of milk metabolomics data of an Indian cattle (Bos indicus) breed. We believe that our study would help to emerge a field of Nutri-metabolomics and veterinary omics research.

Proteomics-based approach for differentiation of age-related macular degeneration sub-types.

Purpose: Age-related macular degeneration (AMD) is one of the leading causes of irreversible central vision loss in the elderly population. The current study aims to find non-invasive prognostic biomarkers in the urine specimens of the AMD patients. Methods: Peripheral blood and urine samples were collected from 23 controls and 61 AMD patients. Genomic DNA was extracted from the buffy coat of peripheral blood. Allele specific PCR was used to assay SNPs in complement factor H (CFH), complement component 3 (C3). Comparative proteomic analysis of urine samples from early AMD, choroidal neovascular membrane (CNVM), geographic atrophy (GA), and healthy controls was performed using isobaric labelling followed by mass spectrometry. Validation was performed using enzyme-linked immunosorbent assay (ELISA). Results: Comparative proteomic analysis of urine samples identified 751 proteins, of which 383 proteins were found to be differentially expressed in various groups of AMD patients. Gene ontology classification of differentially expressed proteins revealed the majority of them were involved in catalytic functions and binding activities. Pathway analysis showed cell adhesion molecule pathways (CAMs), Complement and coagulation cascades, to be significantly deregulated in AMD. Upon validation by ELISA, SERPINA-1 (Alpha1 antitrypsin), TIMP-1 (Tissue inhibitor of matrix metaloprotease-1), APOA-1 (Apolipoprotein A-1) were significantly over-expressed in AMD (n = 61) patients compared to controls (n = 23). A logistic model of APOA-1 in combination with CFH and C3 polymorphisms predicted the risk of developing AMD with 82% accuracy. Conclusion: This study gives us a preliminary data on non-invasive predictive biomarkers for AMD, which can be further validated in a large cohort and translated for diagnostic use.

PathBuilder--open source software for annotating and developing pathway resources.

SUMMARY: We have developed PathBuilder, an open-source web application to annotate biological information pertaining to signaling pathways and to create web-based pathway resources. PathBuilder enables annotation of molecular events including protein-protein interactions, enzyme-substrate relationships and protein translocation events either manually or through automated importing of data from other databases. Salient features of PathBuilder include automatic validation of data formats, built-in modules for visualization of pathways, automated import of data from other pathway resources, export of data in several standard data exchange formats and an application programming interface for retrieving existing pathway datasets. AVAILABILITY: PathBuilder is freely available for download at http://pathbuilder.sourceforge.net/ under the terms of GNU lesser general public license (LGPL: http://www.gnu.org/copyleft/lesser.html). The software is platform independent and has been tested on Windows and Linux platforms. CONTACT: pandey@jhmi.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Human muscle pathology is associated with altered phosphoprotein profile of mitochondrial proteins in the skeletal muscle.

Analysis of human muscle diseases highlights the role of mitochondrial dysfunction in the skeletal muscle. Our previous work revealed that diverse upstream events correlated with altered mitochondrial proteome in human muscle biopsies. However, several proteins showed relatively unchanged expression suggesting that post-translational modifications, mainly protein phosphorylation could influence their activity and regulate mitochondrial processes. We conducted mitochondrial phosphoprotein profiling, by proteomics approach, of healthy human skeletal muscle (n = 10) and three muscle diseases (n = 10 each): Dysferlinopathy, Polymyositis and Distal Myopathy with Rimmed Vacuoles. Healthy human muscle mitochondrial proteins displayed 253 phosphorylation sites (phosphosites), which contributed to metabolic and redox processes and mitochondrial organization etc. Electron transport chain complexes accounted for 84 phosphosites. Muscle pathologies displayed 33 hyperphosphorylated and 14 hypophorphorylated sites with only 5 common proteins, indicating varied phosphorylation profile across muscle pathologies. Molecular modelling revealed altered local structure in the phosphorylated sites of Voltage-Dependent Anion Channel 1 and complex V subunit ATP5B1. Molecular dynamics simulations in complex I subunits NDUFV1, NDUFS1 and NDUFV2 revealed that phosphorylation induced structural alterations thereby influencing electron transfer and potentially altering enzyme activity. We propose that altered phosphorylation at specific sites could regulate mitochondrial protein function in the skeletal muscle during physiological and pathological processes.

Mapping the protein phosphorylation sites in human mitochondrial complex I (NADH: Ubiquinone oxidoreductase): A bioinformatics study with implications for brain aging and neurodegeneration.

In eukaryotes, mitochondrial complex I (NADH: ubiquinone oxidoreductase; CI) is central to oxidative phosphorylation (OXPHOS). Mammalian CI is a 45 subunit complex that forms supercomplexes with other OXPHOS complexes. Since CI defects are associated with aging and neurodegeneration, it is pertinent to understand its structure-function relationship. Although genetic mutations could lower CI activity causing mitochondrial dysfunction in several pathologies, post-translational modifications (PTMs) have emerged as a key mechanism contributing to altered CI activity. Among non-oxidative PTMs, protein phosphorylation is the most intricate regulatory mechanism controlling CI structure and function during normal physiology, aging and neurodegeneration. To comprehend this, we carried out a comprehensive bioinformatics analysis of protein phosphorylation of human CI subunits using software-based prediction of phosphorylation (phospho) sites and associated kinases. Phosphorylation was higher among core subunits and active domains of the complex. Among the subunits, NDUFS1 displayed significantly higher number as well as percent phospho sites compared to others. Analysis of the subunits containing iron-sulfur (Fe-S) cluster, NADH and FMN binding sites and quinone binding sites indicated the presence of phospho sites in close proximity to the binding sites of these cofactors with potential functional implications. Phosphoproteomics experiment in rat and human muscle mitochondria identified specific phospho sites in CI subunits, thereby validating the bioinformatic analysis. Molecular modeling of CI subunits indicated structural implications following phosphorylation. We surmise that protein phosphorylation, a transient and regulatory event could influence the structure-function relationship of CI thereby impinging on bioenergetics and ultimately contributing to aging and neurodegeneration.

MAP2K1 is a potential therapeutic target in erlotinib resistant head and neck squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) targeted therapies have shown limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients despite its overexpression. Identifying molecular mechanisms associated with acquired resistance to EGFR-TKIs such as erlotinib remains an unmet need and a therapeutic challenge. In this study, we employed an integrated multi-omics approach to delineate mechanisms associated with acquired resistance to erlotinib by carrying out whole exome sequencing, quantitative proteomic and phosphoproteomic profiling. We observed amplification of several genes including AXL kinase and transcription factor YAP1 resulting in protein overexpression. We also observed expression of constitutively active mutant MAP2K1 (p.K57E) in erlotinib resistant SCC-R cells. An integrated analysis of genomic, proteomic and phosphoproteomic data revealed alterations in MAPK pathway and its downstream targets in SCC-R cells. We demonstrate that erlotinib-resistant cells are sensitive to MAPK pathway inhibition. This study revealed multiple genetic, proteomic and phosphoproteomic alterations associated with erlotinib resistant SCC-R cells. Our data indicates that therapeutic targeting of MAPK pathway is an effective strategy for treating erlotinib-resistant HNSCC tumors.