Working title: Molecular involvement of p53-MDM2 interactome in gastrointestinal cancers.

The interaction between murine double minute 2 (MDM2) and p53, marked by transcriptional induction and feedback inhibition, orchestrates a functional loop dictating cellular fate. The functional loop comprising p53-MDM2 axis is made up of an interactome consisting of approximately 81 proteins, which are spatio-temporally regulated and involved in DNA repair mechanisms. Biochemical and genetic alterations of the interactome result in dysregulation of the p53-mdm2 axis that leads to gastrointestinal (GI) cancers. A large subset of interactome is well known and it consists of proteins that either stabilize p53 or MDM2 and proteins that target the p53-MDM2 complex for ubiquitin-mediated destruction. Upstream signaling events brought about by growth factors and chemical messengers invoke a wide variety of posttranslational modifications in p53-MDM2 axis. Biochemical changes in the transactivation domain of p53 impact the energy landscape, induce conformational switching, alter interaction potential and could change solubility of p53 to redefine its co-localization, translocation and activity. A diverse set of chemical compounds mimic physiological effectors and simulate biochemical modifications of the p53-MDM2 interactome. p53-MDM2 interactome plays a crucial role in DNA damage and repair process. Genetic aberrations in the interactome, have resulted in cancers of GI tract (pancreas, liver, colorectal, gastric, biliary, and esophageal). We present in this article a review of the overall changes in the p53-MDM2 interactors and the effectors that form an epicenter for the development of next-generation molecules for understanding and targeting GI cancers.

Plasma Metabolite Profiling Identifies Nondiabetic Chronic Pancreatitis Patients With Metabolic Alterations Progressing to Prediabetes Before HbA1c.

INTRODUCTION: Diabetes (T3cDM) secondary to chronic pancreatitis (CP) arises due to endocrine dysfunction and metabolic dysregulations. Currently, diagnostic tests are not available to identify patients who may progress from normoglycemia to hyperglycemia in CP. We conducted plasma metabolomic profiling to diagnose glycemic alterations early in the course of disease. METHODS: Liquid chromatography-tandem mass spectrometry was used to generate untargeted, targeted plasma metabolomic profiles in patients with CP, controls (n = 445) following TRIPOD guidelines. Patients were stratified based on glucose tolerance tests following ADA guidelines. Multivariate analysis was performed using partial least squares discriminant analysis to assess discriminatory ability of metabolites among stratified groups. COMBIROC and logistic regression were used to derive biomarker signatures. AI-ML tool (Rapidminer) was used to verify these preliminary results. RESULTS: Ceramide, lysophosphatidylethanolamine, phosphatidylcholine, lysophosphatidic acid (LPA), phosphatidylethanolamine, carnitine, and lysophosphatidylcholine discriminated T3cDM CP patients from healthy controls with AUC 93% (95% CI 0.81-0.98, P < 0.0001), and integration with pancreatic morphology improved AUC to 100% (95% CI 0.93-1.00, P < 0.0001). LPA, phosphatidylinositol, and ceramide discriminated nondiabetic CP with glycemic alterations (pre-diabetic CP); AUC 66% (95% CI 0.55-0.76, P = 0.1), and integration enhanced AUC to 74% (95% CI 0.55-0.88, P = 0.86). T3cDM was distinguished from prediabetic by LPA, phosphatidylinositol, and sphinganine (AUC 70%; 95% CI 0.54-0.83, P = 0.08), and integration improved AUC to 83% (95% CI 0.68-0.93, P = 0.05). CombiROC cutoff identified 75% and 78% prediabetes in validation 1 and 2 cohorts. Random forest algorithm assessed performance of integrated panel demonstrating AUC of 72% in predicting glycemic alterations. DISCUSSION: We report for the first time that a panel of metabolites integrated with pancreatic morphology detects glycemia progression before HbA1c in patients with CP.

Clastogenic, aneugenic, and tubulin polymerization properties of di-(2-ethylhexyl) phthalate and dibutyl phthalate.

Phthalate compounds were found to disrupt the endocrine system and alter transcriptomes during human embryonic development. In our previous work, we have isolated and reported two such phthalates di-(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP) from Brevibacterium mcbrellneri bacteria and evaluated their bioactive properties. Naturally derived phthalates might be less toxic compared with synthesized molecules. We have investigated biologically isolated phthalates to understand the possible genotoxic effects in mice and further investigated in silico binding and polymerization of ß-tubulin. Three sub-lethal concentrations of DEHP (150 µM, 175 µM, and 200 µM) and DBP (10 µM, 15 µM, and 30 µM) were studied. The results showed that the phthalates were found to be highly genotoxic in nature. However, the pattern of genotoxic effects was not found to be dose-dependent in the induction of chromosome aberrations (CA), micronuclei (MN), and changes in the mitotic index (MI) in cells. In silico studies of phthalates on polymerization of ß-tubulin suggested that both DBP and DEHP were able to interact with the hydrogen bonds and make strong van der Waals interactions with ß-tubulin thereby possibly causing destabilization of microtubule network. Our study suggests that these phthalates might be playing an important role in normal cell division thereby showing highly genotoxic effects.

Iron mediated reductive cyclization/oxidation for the generation of chemically diverse scaffolds: An approach in drug discovery.

Chemically diverse scaffolds represent a main source of biologically important starting points in drug discovery. Herein, we report the development of such diverse scaffolds from nitroarene/ nitro(hetero)arenes using a key synthetic strategy. In a pilot-scale study, the synthesis of 10 diverse scaffolds was achieved. The 1,7-phenanthroline, thiazolo[5,4-f]quinoline, 2,3-dihydro-1H-pyrrolo[2,3-g]quinoline, pyrrolo[3,2-f]quinoline, 1H-[1,4]oxazino[3,2-g]quinolin-2(3H)-one, [1,2,5]oxadiazolo[3,4-h]quinoline, 7H-pyrido[2,3-c]carbazole, 3H-pyrazolo[4,3-f]quinoline, pyrido[3,2-f]quinoxaline were obtained from nitro hetero arenes in ethanol using iron-acetic acid treatment followed by reaction under oxygen atmosphere. This diverse library is compliant with the rule of five for drug-likeness. The mapping of chemical space represented by these scaffolds revealed a significant contribution to the underrepresented chemical diversity. Crucial to the development of this approach was the mapping of biological space covered by these scaffolds which revealed neurotropic and prophylactic anti-inflammatory activities. In vitro, neuro-biological assays revealed that compounds 14a and 15a showed excellent neurotropic potential and neurite growth compared to controls. Further, anti-inflammatory assays (in vitro and in vivo models) exhibited that Compound 16 showed significant anti-inflammatory activity by attenuating the LPS-induced TNF-α and CD68 levels by modulating the NFkB pathway. In addition, treatment with compound 16 significantly ameliorated the LPS-induced sepsis conditions, and pathological abnormalities (in lung and liver tissues) and improved the survival of the rats compared to LPS control. Owing to their chemical diversity along with bioactivities, it is envisaged that new quality pre-clinical candidates will be generated in the above therapeutic areas using identified leads.

Integration of metabolites from meta-analysis with transcriptome reveals enhanced SPHK1 in PDAC with a background of pancreatitis.

BACKGROUND: Pathophysiology of transformation of inflammatory lesions in chronic pancreatitis (CP) to pancreatic ductal adenocarcinoma (PDAC) is not clear. METHODS: We conducted a systematic review, meta-analysis of circulating metabolites, integrated this data with transcriptome analysis of human pancreatic tissues and validated using immunohistochemistry. Our aim was to establish biomarker signatures for early malignant transformation in patients with underlying CP and identify therapeutic targets. RESULTS: Analysis of 19 studies revealed AUC of 0.86 (95% CI 0.81-0.91, P < 0.0001) for all the altered metabolites (n = 88). Among them, lipids showed higher differentiating efficacy between PDAC and CP; P-value (< 0.0001). Pathway enrichment analysis identified sphingomyelin metabolism (impact value-0.29, FDR of 0.45) and TCA cycle (impact value-0.18, FDR of 0.06) to be prominent pathways in differentiating PDAC from CP. Mapping circulating metabolites to corresponding genes revealed 517 altered genes. Integration of these genes with transcriptome data of CP and PDAC with a background of CP (PDAC-CP) identified three upregulated genes; PIGC, PPIB, PKM and three downregulated genes; AZGP1, EGLN1, GNMT. Comparison of CP to PDAC-CP and PDAC-CP to PDAC identified upregulation of SPHK1, a known oncogene. CONCLUSIONS: Our analysis suggests plausible role for SPHK1 in development of pancreatic adenocarcinoma in long standing CP patients. SPHK1 could be further explored as diagnostic and potential therapeutic target.

Identification, characterization and evaluation of novel antifungal cyclic peptides from Neobacillus drentensis.

Marine microbes secrete exopolymeric substances (EPS), which surrounds the biofilm and inhibits the fungal growth. Elucidation of the structure and function of the extracellular exopolymeric substances is of vital relevance therapeutically. The active compound responsible for bioactivity was purified and characterized using TLC, LC/MS/MS, GC/MS and FT-IR. Bioactivity of the characterized cyclic peptides (CLPs) against azole resistant and susceptible Candida strains were examined for growth and biofilm formation using scanning electron microscopy, flow cytometry, confocal microscopy. In the present study we identified bioactive cyclic peptides from marine isolated Neobacillus drentensis that exhibited promising tensio-active properties and antifungal efficacy against azole resistant and susceptible Candida albicans. The cluster is composed of five CLP isoforms which were sequenced and identified as new peptides with compositional and structural variations in the amino acid sequence and fatty acid chain. In vitro cytotoxic activity of CLPs was tested in human fibroblast normal cells. We have observed that the CLPs repressed the Candida albicans growth and multiplication by inhibiting the biofilm formation and disruption of branching filamentous hyphae. CLPs have been found to arrest the C. albicans cell cycle by a block at G1-S transition followed by apoptotic cell death. The current studies suggest these natural marine derived CLPs function as potential anti-biofilm agents against azole C. albicans resistant strains.

A variant in TMPRSS2 is associated with decreased disease severity in COVID-19.

BACKGROUND: Mortality due to COVID-19 caused by SARS-CoV-2 infection varies among populations. Functional relevance of genetic variations in Angiotensin-converting enzyme 2 (ACE2) and Transmembrane serine protease 2 (TMPRSS2), two crucial host factors for viral entry, might explain some of this variation. METHODS: In this comparative study in Indian subjects, we recruited 510 COVID-19 patients and retrieved DNA from 520 controls from a repository. Associations between variants in ACE2 and TMPRSS2 with disease severity were identified by whole exome sequencing (WES, n = 20) and targeted genotyping (n = 1010). Molecular dynamic simulations (MDS) were performed to explore functional relevance of the variants. Cleavage of spike glycoprotein by wild and variant TMPRSS2 was determined in HEK293T cells. Potential effects of confounders on the association between genotype and disease severity were tested (Mantel-Haenszel test). RESULTS: WES identified deleterious variant in TMPRSS2 (rs12329760, G > A, p. V160M). The minor allele frequency (MAF) was 0·27 in controls, 0·31 in asymptomatic, 0·21 in mild-to-moderately affected and 0·19 in severely affected COVID-19 patients. Risk of severity increased with decreasing MAF: Asymptomatic: Odds ratio-0·69 (95% CI-0·52-0·93; p = 0·01); mild-to-moderate: Odds ratio-1·89 (95% CI-1·22-2.92;p = 0·004) and severe: Odds ratio-1·79 (95% CI-1·11-2.88;p = 0·01). No confounding effect of diabetes and hypertension were observed on the risk of developing severe COVID-19 disease with respect to genotype. MDS revealed decreased stability of TMPRSS2 with 160 M variant. Spike glycoprotein cleavage by TMPRSS2 reduced ~2·4-fold in cells expressing 160 M variant. CONCLUSION: We demonstrate association of TMPRSS2 variant rs12329760 with decreased disease severity in COVID-19 patients from India.

In-silico driven design and development of spirobenzimidazo-quinazolines as potential DNA gyrase inhibitors.

DNA gyrase and Topoisomerase IV are promising antibacterial drug targets as they regulate bacterial DNA replication and topology. In a quest for novel DNA topoisomerase inhibitors, a multidisciplinary approach was adopted that involves computational prediction of binding sites and molecular modelling followed by green synthesis and biological evaluation of antibacterial activity of spirobenzimidazo quinazolines derivatives. Using basic quantum chemistry principles, we evaluated spirobenzimidazo quinazolines derivatives with their pharmacokinetic profiles. Based on the results of the aforesaid in-silico studies, we synthesized a series of titled compounds using green synthetic methodology that were validated as potential antimicrobial agents. Quantum chemoinformatics based predicted activity for the synthesized compounds 9b, 9c, and 9j was concomitant with biological evaluation of broadspectrum antibacterial activity. Biological evaluation revealed that inhibition of biofilm formation was due to their potential antibacterial activity. We believe that the novel spirobenzimidazo quinazolines have the potential to be alternatives to aminocoumarins and classical quinazolines upon detailed target specific biological studies.

Identification, synthesis and evaluation of CSF1R inhibitors using fragment based drug design.

Colony-stimulating factor 1 receptor is a type III receptor protein tyrosine kinase belonging to PDGFR family. CSF1R signaling is essential for differentiation, proliferation and survival of macrophages. Aberrant expression of CSF1R appears to be an attractive target in several cancer types. Higher expression of CSF1R ligands correlates to tumor progression. CSF1R inhibitors have been shown to suppress cancers. We have attempted an in silico fragment derived drug discovery approach by screening ˜25,000 in-house compounds as potential CSF1R inhibitors. Using FBDD approach we have identified six diverse fragments that exhibit affinity towards hinge region of CSF1R. Some of the fragments 5-nitroindole and 7-azaindole and their derivatives were synthesized for further evaluation. The in silico and in vitro enzyme activity studies reveal moderate inhibition of CSF1R kinase activity by 5-nitroindole and good inhibition by 7-azaindole fragments. Bio and chemiinformatics studies have shown that 7-azaindole compounds have better membrane permeability and enzyme inhibition properties. Molecular docking studies show that the amino acid residues 664-666 in the hinge region of the cytosolic domain of CSF1R to be the preferred region of binding for nitroindole and azaindole derivatives. Further optimization and biological analysis would identify these fragments as potential and promising leads as CSF1R inhibitors.

Molecular diagnostics of disorders of sexual development: an Indian survey and systems biology perspective.

We aimed to survey the monogenic causes of disorders of sex development (DSD) and thereby its prevalence in India. This study revealed mutations resulting in androgen insensitivity syndrome, 5α-reductase type 2 deficiency, and gonadal dysgenesis were commonly reported. Intriguingly, AR deficits were the most prevalent (32 mutations) and of 11/26 missense mutations were in exons 4-8 (encoding ligand binding domain). The unique features of SRD5A2 defects were p.R246Q (most prevalent) and p.G196S could be mutational hotspots, dual gene defects (p.A596T in AR and p.G196S in SRD5A2) in a patient with hypospadias and novel 8 nucleotide deletion (exon 1) found in a patient with perineal hypospadias. Deficits in SRY, WT1, DHH, NR5A1, and DMRT1 caused 46,XY gonadal dysgenesis. Notably, mutations in AR, SRD5A2, MAMLD1, WT1, and MAP3K1 have led to hypospadias and only one CYP19A1 mutation caused aromatase deficiency was reported to date. Data mining from various databases has not only reinforced the role of well-established genes (e.g., SRY, WT1, DHH, NR5A1, DMRT1, AR, SRD5A2, MAMLD1) involved in DSD but also provided us 12 more potential candidate genes (ACVR1, AMHR2, CTNNB1, CYP11A1, CYP19A1, FGFR2, FGF9, PRKACA, PRKACG, SMAD9, TERT, ZFPM2), which benefit from a close association with the well-established genes involved in DSD and might be useful to screen owing to their direct gene-phenotype relationship or through direct functional interaction. As more genes have been revealed in relation to DSD, we believe ultimately it holds a better scenario for therapeutic regimen. Despite the advances in translational medicine, hospitals are yet to adopt genetic testing and counseling facilities in India that shall have potential impact on clinical diagnosis. Abbreviations: 5α-RD2: 5α-Reductase type 2; AIS: androgen insensitivity syndrome; AMH: antimullerian hormone; AMHR: antimullerian hormone receptor; AR: androgen receptor gene; CAH: congenital adrenal hyperplasia; CAIS: complete AIS; CAH: congenital adrenal hyperplasia; CHH: congenital hypogonadotropic hypogonadism; CXORF6: chromosome X open reading frame 6 gene; CYP19A1: cytochrome P450 family 19 subfamily A member 1 gene; DHT: dihydrotestosterone; DMRT1: double sex and mab-3 related transcription factor 1 gene; DSD: disorders of sexual development; GD: gonadal dysgenesis; HGMD: human gene mutation database; IH: isolated hypospadias; MAMLD1: mastermind like domain containing 1 gene; MIS: mullerian inhibiting substance; NTD: N-terminal domain; OT DSD: ovotesticular DSD; PAIS: partial AIS; SOX9: SRY-related HMG-box 9 gene; SRY: sex-determining region Y gene; STAR: steroidogenic acute regulatory protein gene; SRD5A2: steroid 5 alpha-reductase 2 gene; T DSD: testicular DSD; T: testosterone; WNT4: Wnt family member 4 gene; WT1: Wilms tumor 1 gene; Δ4: androstenedione.

Aspergillus Secondary Metabolite Database, a resource to understand the Secondary metabolome of Aspergillus genus.

Aspergillus is a genus of ubiquitous fungi that are pathologically & therapeutically important. Aspergillus Secondary Metabolites Database (A2MDB) is a curated compendium of information on Aspergillus & its secondary metabolome. A2MDB catalogs 807 unique non-redundantsecondary metabolites derived from 675 Aspergillus species. A2MDB has a compilation of 100 cellular targets of secondary metabolites, 44 secondary metabolic pathways, 150 electron and light microscopy images of various Aspergillus species. A phylogenetic representation of over 2500 strains has been provided. A2MDB presents a detailed chemical information of secondary metabolites and their mycotoxins. Molecular docking models of metabolite-target protein interactions have been put together. A2MDB also has epidemiological data representing Aspergillosis and global occurrence of Aspergillus species. Furthermore a novel classification of Aspergillosis along with 370 case reports with images, were made available. For each metabolite catalogued, external links to related databases have been provided. All this data is available on A2MDB, launched through Indian Institute of Chemical Technology, Hyderabad, India, as an open resource http://www.iictindia.org/A2MDB . We believe A2MDB is of practical relevance to the scientific community that is in pursuit of novel therapeutics.

Isolation and characterization of phthalates from Brevibacterium mcbrellneri that cause cytotoxicity and cell cycle arrest.

Bacteria belonging to the family Brevibacterieae are ubiquitous Gram positive organisms that are responsible for the feet odour and cheese aroma. Brevibacterium mcbrellneri is a relatively new member belonging to Brevibacterieae. In the current manuscript we discuss isolation of biologically active metabolites from Brevibacterium mcbrellneri. Two aromatic esters were isolated from Brevibacterium mcbrellneri by "Bioassay guided fractionation strategy" and identified as di-(2-ethylhexyl) phthalate and dibutyl phthalate by chemical characterization using biophysical techniques. The phthalate compounds show broad spectrum antibacterial activity and mosquito larvicidal activity. Mosquito larvicidal activity has been attributed to inhibition of acetylcholinesterase enzyme activity. These compounds were found to be cytotoxic in multiple cell lines causing cell cycle arrest in G1 phase.

Acetylcholinesterase inhibitory activity of stigmasterol & hexacosanol is responsible for larvicidal and repellent properties of Chromolaena odorata.

BACKGROUND: Chromolaena odorata, has been traditionally known for its insect repellent property. Aim of this study was to determine larvicidal tendency of C. odorata on Culex quinquefasciatus and isolate compounds responsible for this activity and to determine the mechanism of action of these compounds. METHODS: C. odorata plant extract was screened for mosquito larvicidal activity. The extract was fractionated using chromatography and the bioactive fraction showing larvicidal activity was identified. The chemical nature of the compounds in the bioactive fraction was determined using NMR and Mass spectrometry. RESULTS: We identified phytosterols and alkanols to be the compounds regulating larvicidal activity in the bioactive fraction of the plant extract. Stigmasterol and 1-hexacosanol were identified to be the chief orchestrators of larvicidal activity and their mode of action has been observed to be neurotoxicity. At a molecular level both stigmasterol and 1-hexacosanol were found to be inhibiting acetylcholinesterase activity in C. quinquefasciatus & A. aegypti. The acetylcholinesterase inhibitory effect was validated in vitro using recombinant acetylcholinesterase and ex vivo in larval homogenates of Culex and Aedes. Electrophysiological studies using electroantennography have shown enhanced neural response to these compounds. CONCLUSIONS: Neurotoxic effect of C. odorata derived stigmasterol and 1-hexacosanol, exerted through acetylcholinesterase inhibition was responsible for the mortality of C. quinquefasciatus, A. aegypti &Chironomus riparius. EAG studies pointed out hyper-excitability of the olfactory system by these compounds. GENERAL SIGNIFICANCE: These compounds are natural agents for mosquito control that can be used in vector control as larvicidal compounds, pending further investigations.

Endophytic Fungi as Novel Resources of natural Therapeutics

ABSTRACT Fungal endophytes constitute a major part of the unexplored fungal diversity. Endophytic fungi (EF) are an important source for novel, potential and active metabolites. Plant-endophyte interaction and endophyte -endophyte interactions study provide insights into mutualism and metabolite production by fungi. Bioactive compounds produced by endophytes main function are helping the host plants to resist external biotic and abiotic stress, which benefit the host survival in return. These organisms mainly consist of members of the Ascomycota, Basidiomycota, Zygomycota and Oomycota. Recently, the genome sequencing technology has emerged as one of the most efficient tools that can provide whole information of a genome in a small period of time. Endophytes are fertile ground for drug discovery. EFare considered as the hidden members of the microbial world and represent an underutilized resource for new therapeutics and compounds. Endophytes are rich source of natural products displaying broad spectrum of biological activities like anticancer, antibacterial, antiviral, immunomodulatory, antidiabetic, antioxidant, anti-arthritis and anti-inflammatory.

Phytofabrication of silver nanoparticles using Myriostachya wightiana as a novel bioresource, and evaluation of their biological activities

ABSTRACT Nanobiotechnology deals with the properties of nanomaterials and their potential uses. Here we report for the first time novel, cost-effective and eco-friendly method for the rapid green synthesis of silver nanoparticles (AgNPs) using leaf extracts of Myriostachya wightiana. The growth of silver nanoparticles was monitored by UV-vis spectroscopy complemented by Zeta potential, dynamic light scattering technique (DLS), Fourier-transform infrared spectroscopy (FTIR), Transmission electron microscope (TEM) and X-ray diffraction (XRD). The surface plasmon resonance (SPR) band found at 434 nm confirmed the reduction of AgNO3 to AgNPs. TEM micrographs revealed that AgNPs are irregular in shape with the size range from 15-65 nm. The functional groups responsible for bio-reduction of silver nitrate into silver were analyzed by FTIR and confirmed by X-ray photoelectron spectrum (XPS). Further these biogenic AgNP were evaluated for insecticidal activities against stored product pests, Tribolium castaneum (Flour beetle), Rhyzopertha dominica (F.)(Lesser grain borer) and Sitophilus oryzae L (Rice weevil). The fabricated AgNPs showed moderate activity on stored pests and strong antibacterial activity with varying degrees against Xanthomonas campestris and Ralstonia solanacearum as evidenced by their zone of inhibition at all concentrations. Hence, these AgNP can be used as control agents against agricultural pests and pathogens in future.

p62 is a key regulator of nutrient sensing in the mTORC1 pathway.

The signaling adaptor p62 is a critical mediator of important cellular functions, owing to its ability to establish interactions with various signaling intermediaries. Here, we identify raptor as an interacting partner of p62. Thus, p62 is an integral part of the mTORC1 complex and is necessary to mediate amino acid signaling for the activation of S6K1 and 4EBP1. p62 interacts in an amino acid-dependent manner with mTOR and raptor. In addition, p62 binds the Rags proteins and favors formation of the active Rag heterodimer that is further stabilized by raptor. Interestingly, p62 colocalizes with Rags at the lysosomal compartment and is required for the interaction of mTOR with Rag GTPases in vivo and for translocation of the mTORC1 complex to the lysosome, a crucial step for mTOR activation.

Identification of Novel Phosphorylation Motifs Through an Integrative Computational and Experimental Analysis of the Human Phosphoproteome.

Protein phosphorylation occurs in certain sequence/structural contexts that are still incompletely understood. The amino acids surrounding the phosphorylated residues are important in determining the binding of the kinase to the protein sequence. Upon phosphorylation these sequences also determine the binding of certain domains that specifically bind to phosphorylated sequences. Thus far, such 'motifs' have been identified through alignment of a limited number of well identified kinase substrates. RESULTS: Experimentally determined phosphorylation sites from Human Protein Reference Database were used to identify 1,167 novel serine/threonine or tyrosine phosphorylation motifs using a computational approach. We were able to statistically validate a number of these novel motifs based on their enrichment in known phosphopeptides datasets over phosphoserine/threonine/tyrosine peptides in the human proteome. There were 299 novel serine/threonine or tyrosine phosphorylation motifs that were found to be statistically significant. Several of the novel motifs that we identified computationally have subsequently appeared in large datasets of experimentally determined phosphorylation sites since we initiated our analysis. Using a peptide microarray platform, we have experimentally evaluated the ability of casein kinase I to phosphorylate a subset of the novel motifs discovered in this study. Our results demonstrate that it is feasible to identify novel phosphorylation motifs through large phosphorylation datasets. Our study also establishes peptide microarrays as a novel platform for high throughput kinase assays and for the validation of consensus motifs. Finally, this extended catalog of phosphorylation motifs should assist in a systematic study of phosphorylation networks in signal transduction pathways.

Phosphorylation of p62 by cdk1 controls the timely transit of cells through mitosis and tumor cell proliferation.

The protein scaffold and signaling regulator p62 is important in critical cellular functions, including bone homeostasis, obesity, and cancer, because of its interactions with various signaling intermediaries. p62 is overexpressed in human cancers and is induced during cell transformation. Its genetic ablation inhibits lung tumorigenesis in vivo and cell proliferation in culture by regulating the TRAF6/NF-κB signaling cascade to control reactive oxygen species (ROS) production and apoptosis. Here we show that cdk1 phosphorylates p62 in vitro and in vivo at T269 and S272, which is necessary for the maintenance of appropriate cyclin B1 levels and the levels of cdk1 activity necessary to allow cells to properly enter and exit mitosis. The lack of cdk1-mediated phosphorylation of p62 leads to a faster exit from mitosis, which translates into enhanced cell proliferation and tumorigenesis in response to Ras-induced transformation. Therefore, p62 emerges as a node for the control of not only cell survival but also cell transit through mitosis.