MTORC2 is a physiological hydrophobic motif kinase of S6 Kinase 1.

Ribosomal protein S6 kinase 1 (S6K1), a major downstream effector molecule of mTORC1, regulates cell growth and proliferation by modulating protein translation and ribosome biogenesis. We have recently identified eIF4E as an intermediate in transducing signals from mTORC1 to S6K1 and further demonstrated that the role of mTORC1 is restricted to inducing eIF4E phosphorylation and interaction with S6K1. This interaction relieves S6K1 auto-inhibition and facilitates its hydrophobic motif (HM) phosphorylation and activation as a consequence. These observations underscore a possible involvement of mTORC1 independent kinase in mediating HM phosphorylation. Here, we report mTORC2 as an in-vivo/physiological HM kinase of S6K1. We show that rapamycin-resistant S6K1 truncation mutant ∆NH∆CT continues to display HM phosphorylation with selective sensitivity toward Torin-1. We also show that HM phosphorylation of wildtype S6K1and ∆NH∆CT depends on the presence of mTORC2 regulatory subunit-rictor. Furthermore, truncation mutagenesis and molecular docking analysis reveal the involvement of a conserved 19 amino acid stretch of S6K1 in mediating interaction with rictor. We finally show that deletion of the 19 amino acid region from wildtype S6K1 results in loss of interaction with rictor, with a resultant loss of HM phosphorylation regardless of the presence of functional TOS motif. Our data demonstrate that mTORC2 acts as a physiological HM kinase that can activate S6K1 after its auto-inhibition is overcome by mTORC1. We, therefore, propose a novel mechanism for S6K1 regulation where mTOR complexes 1 and 2 act in tandem to activate the enzyme.

Expanding the view of the molecular mechanisms of autophagy pathway.

Autophagy is an evolutionarily conserved multistep degradation mechanism in eukaryotes, that maintains cellular homoeostasis by replenishing cells with nutrients through catabolic lysis of the cytoplasmic components. This critically coordinated pathway involves sequential processing events that begin with initiation, nucleation, and elongation of phagophores, followed by the formation of  double-membrane vesicles known as autophagosomes. Finally, autophagosomes migrate towards and fuse with lysosomes in mammals and vacuoles in yeast and plants, for the eventual degradation of the intravesicular cargo. Here, we review the recent advances in our understanding of the molecular events that define the process of autophagy.

TGF-ß signaling: A recap of SMAD-independent and SMAD-dependent pathways.

Transforming growth factor-ß (TGF-ß) is a proinflammatory cytokine known to control a diverse array of pathological and physiological conditions during normal development and tumorigenesis. TGF-ß-mediated physiological effects are heterogeneous and vary among different types of cells and environmental conditions. TGF-ß serves as an antiproliferative agent and inhibits tumor development during primary stages of tumor progression; however, during the later stages, it encourages tumor development and mediates metastatic progression and chemoresistance. The fundamental elements of TGF-ß signaling have been divulged more than a decade ago; however, the process by which the signals are relayed from cell surface to nucleus is very complex with additional layers added in tumor cell niches. Although the intricate understanding of TGF-ß-mediated signaling pathways and their regulation are still evolving, we tried to make an attempt to summarize the TGF-ß-mediated SMAD-dependent andSMAD-independent pathways. This manuscript emphasizes the functions of TGF-ß as a metastatic promoter and tumor suppressor during the later and initial phases of tumor progression respectively.

mTORC1 induces eukaryotic translation initiation factor 4E interaction with TOS-S6 kinase 1 and its activation.

Eukaryotic translation initiation factor 4E was recently shown to be a substrate of mTORC1, suggesting it may be a mediator of mTORC1 signaling. Here, we present evidence that eIF4E phosphorylated at S209 interacts with TOS motif of S6 Kinase1 (S6K1). We also show that this interaction is sufficient to overcome rapamycin sensitivity and mTORC1 dependence of S6K1. Furthermore, we show that eIF4E-TOS interaction relieves S6K1 from auto-inhibition due to carboxy terminal domain (CTD) and primes it for hydrophobic motif (HM) phosphorylation and activation in mTORC1 independent manner. We conclude that the role of mTORC1 is restricted to engaging eIF4E with S6K1-TOS motif to influence its state of HM phosphorylation and inducing its activation.

MYP2 locus genes: Sequence variations, genetic association studies and haplotypic association in patients with High Myopia.

High Myopia (HM) is a common complex-trait eye disorder. There is essential evidence that genetic factors play a significant role in the development of nonsyndromic high myopia. Identification of susceptibility genes of high myopia will shed light on the pathophysiological mechanism underlying their genesis. This was a case control study examining the prospect of association of DLGAP1, EMILIN2 & MYOM1 genes on MYP2 locus in purely ethnic (Kashmiri) population representing a homogeneous cohort. Genomic DNA was extracted using phenol chloroform and salting out method. Extracted DNA was genotyped for polymorphic variations in MYOM1, EMILIN2 and DLGAP1 genes involving Sanger di-deoxy method. Allele frequencies were tested for Hardy-Weinberg disequilibrium in 224 cases and compared with 220 emmetropic controls. In DLGAP1, documented single nucleotide polymorphism (SNP); Pro517Pro was observed. A previously reported Asn451Asn SNP was observed in EMILIN2. MYOM1 showed five polymorphic variations; two in coding region (Gly333Gly & Gly341Ala) and three intronic (c.1022+23, G>A; c.3418+44 G>T & c.3418+65; C>G). All of the elucidated SNPs were having statistical significant role in increasing or decreasing the risk of disease. Although not statistically significant, a novel Glu507Lys SNP was observed in DLGAP1 (P>0.05). In silico predictions showed MYOM1 Gly341Ala to be benign & tolerated substitution while as DLGAP1 Glu507Lys to be possibly damaging substitution. The studied SNPs followed Over-Dominant, Recessive and Co-Dominant mode of inheritance with specific haplotypes associated with the disease. Our study reveals the involvement of MYP2 locus candidate gene polymorphism in the pathogenesis of HM.

Improved pulmonary function test (PFT) after 1 one year of Sublingual Immunotherapy (SLIT) in unison with pharmacotherapy in mild allergic asthmatics.

BACKGROUND: Allergen immunotherapy (AIT) is a promising treatment for allergic disease that induces immunological tolerance through the administration of specific allergens. The study of AIT is in its early stage and its clinical effects are not well elucidated. The present study was aimed at determining the effect of AIT on pulmonary function and serum variables of mild allergic asthma patients. METHODS: A total of 80 patients with mild allergic asthma were recruited for the study. Allergen Specific Immunotherapy was administered in the form of Sublingual Immunotherapy and consisted of a build up phase followed by a maintenance phase (six months each respectively). Total serum IgE and vitamin D levels were quantified by ELISA. The percent eosinophill count was determined by cell analyzers. Pulmonary function test was performed at the baseline and after the end of study period. Subjective symptom score was recorded in the form of asthma control questionnaire score. RESULTS: There was a significant increase in the pre FEV1% and pre FEV1/FVC post AIT administration. A significant decrease in the total serum IgE was found post AIT. A decrease in Asthma control Questionnaire (ACQ) scores indicated an improvement in clinical symptoms. Besides there was a significant effect on ICS discontinuation after AIT. CONCLUSION: The study supports SLIT as an effective treatment for Immunomodulation in mild allergic asthmatics besides it gives us significant information regarding the safety and efficacy of SLIT in such patients.

Expansion of the CRISPR/Cas Genome-Sculpting Toolbox: Innovations, Applications and Challenges.

The emergence of the versatile gene-editing technology using programmable sequence-specific endonuclease system (CRISPR-Cas9) has instigated a major upheaval in biomedical research. In a brief span of time, CRISPR/Cas has been adopted by research labs around the globe because of its potential for significant progress and applicability in terms of efficiency, versatility and simplicity. It is a breakthrough technique for systematic genetic engineering, genome labelling, epigenetic and transcriptional modulation, and multiplexed gene editing, amongst others. This review provides an illustrative overview of the current research trends using CRISPR/Cas technology. We highlight the latest developments in CRISPR/Cas technique including CRISPR imaging, discovery of novel CRISPR systems, and applications in altering the genome, epigenome or RNA in different organisms. Finally, we address the potential challenges of this technique for its future use. Development of new CRISPR/Cas systems.

Eukaryotic Initiation Factor 4E phosphorylation acts a switch for its binding to 4E-BP1 and mRNA cap assembly.

Translational regulation has invited considerable interest consequent of its circumstantial dysregulation during cancer genesis. eIF4E (Eukaryotic Initiation Factor 4E) has been identified as an important factor involved in tumor progression by way of instrumenting the convergence of oncogenic signals for up-regulation of Cap-dependent translation. In the backdrop of dramatic eIF4E over-expression in a large population of human cancers, we suggest that the tumorigenic property of eIF4E is strictly attributed to its phosphorylation state. We provide evidence that while phosphorylated eIF4E fails to be sequestered by 4E-BP1, its dephosphorylated form shows overwhelming binding with 4E-BP1 without any consideration to the state of 4E-BP1 phosphorylation to suggest that eIF4E-4EBP1 binding is governed by eIF4E phosphorylation instead of 4E-BP1. We also show that eIF4E engages in Cap-assembly formation preferably in a phosphorylation-dependent manner to suggest that eIF4E phosphorylation rather than 4E-BP1 regulates its availability for Cap-assembly.

RET/PTC Gene Rearrangements in Thyroid Carcinogenesis: Assessment and Clinico-Pathological Correlations.

Rearranged during transfection (RET) is a proto oncogene implicated in thyroid carcinogenesis of papillary type (PTC). The RET proto-oncogene in PTC is constitutively activated by fusion of its tyrosine kinase domain with the 5 ´region of another gene thereby generating chimeric products collectively named RET/PTCs. RET/PTC1 and RET/PTC3 are best characterized among all RET/PTC rearrangements. Kashmir valley has witnessed an alarming increase in thyroid cancer incidence in young women. Therefore, we investigated the occurrence of RET/PTC 1 & 3 rearrangements by semi quantitative and qPCR in thyroid cancer patients (n = 48) of Kashmiri population and interrelated results with various clinicopathological characteristics. We observed that all the RET/PTC rearrangements were confined to PTC cases (10/40). Presence of RET/PTC rearrangement significantly correlated with gender, elevated TSH levels and lymph node metastasis. Overall, our study advocates that RET/PTC3 rearrangement is a frequent event in the carcinogenesis of thyroid gland in Kashmiri population although a study with a larger sample size is needed to get a clear scenario.

Eukaryotic initiation factor 4E is a novel effector of mTORC1 signaling pathway in cross talk with Mnk1.

Cellular signals that influence Cap-dependent translation have assumed significant relevance in the backdrop of their enforced dysregulation during oncogenesis. Eukaryotic initiation factor 4E(eIF4E), the mRNA cap-binding protein, has emerged as a key player to facilitate tumor progression through upregulated cap-dependent translation synchronized with enhanced cell division. We provide evidence that eIF4E phosphorylation is regulated by mTORC1 by virtue of its interaction with Raptor through a novel TPTPNPP motif and consequent phosphorylation invitro and in vivo in a Rapamycin-sensitive manner. While we show that phosphorylation pattern of eIF4E responds faithfully to Rapamycin inhibition, the prolonged exposure to Rapamycin rescues the loss of eIF4E phosphorylation through Mnk1 activation. We also present evidence that eIF4E interacts with the amino terminal domain of S6K1 in a phospho-dependent manner, and this interaction is instrumental in overriding Rapamycin inhibition of S6K1. The data endorses eIF4E as a regulatory subunit that modulates the functional attributes of mTOR effectors to synchronize cap-dependent translation with growth assertion.

Eukaryotic initiation factor 4E (eIF4E): A recap of the cap-binding protein.

Eukaryotic initiation factor 4E (eIF4E), a fundamental effector and rate limiting element of protein synthesis, binds the 7-methylguanosine cap at the 5' end of eukaryotic messenger RNA (mRNA) specifically as a constituent of eIF4F translation initiation complex thus facilitating the recruitment of mRNA to the ribosomes. This review focusses on the engagement of signals contributing to growth factor originated maxim and their role in the activation of eIF4E to achieve a collective influence on cellular growth, with a key focus on conjuring vital processes like protein synthesis. The review invites considerable interest in elevating the appeal of eIF4E beyond its role in regulating translation viz a viz cancer genesis, attributed to its phosphorylation state that improves the prospect for the growth of the cancerous cell. This review highlights the latest studies that have envisioned to target these pathways and ultimately the translational machinery for therapeutic intervention. The review also brings forward the prospect of eIF4E to act as a converging juncture for signaling pathways like mTOR/PI3K and Mnk/MAPK to promote tumorigenesis.

Polymorphic Analysis of Leptin Promoter in Obese/diabetic Subjects in Kashmiri Population.

BACKGROUND: The role of common variants in leptin promoter has already been established to play a major role in obesity and diabetes in humans. The study was accordingly focused on leptin promoter variants and their potential association with diabetes and obesity in ethnic population from Kashmir, India. METHODS: Allele frequencies of 620 Kashmiri subjects with diabetes (200), obese subjects (200), and ethnically matched healthy controls (200) were tested for the Hardy-Weinberg disequilibrium. Among 200 obese subjects, a total of 50 persons were with diabetes. The genotype and allele frequencies were evaluated using the Chi-square or Fisher's exact tests. RESULTS: Sequence analysis revealed two reported variations i.e., rs72563764C>T and rs7799039G>A in promoter region. Both variants show homozygous as well as heterozygous genotypes. These variations indicated significant difference with respect to allelic and genotypic frequencies in all groups i.e., persons with diabetes, obese, and obese persons with diabetes (P < 0.05). We also analyzed the association of these variations with biochemical characteristics and found significant association of rs72563764C>T with triglycerides (TG) in obese patients and fasting plasma glucose (FPG) and random blood sugar (RBS) in obese/persons with diabetes. Also rs7799039G>A showed association with postprandial plasma sugar (PPPS) in obese patients and FPG and resting plasma glucose (RPG) in obese persons with diabetes. CONCLUSIONS: Our results are suggestive of the association of leptin promoter gene variations i.e., rs72563764C>T and rs7799039G>A with both diabetes and obesity.

TAK1 mediates convergence of cellular signals for death and survival.

TGF-ß activated kinase 1, a MAPK kinase kinase family serine threonine kinase has been implicated in regulating diverse range of cellular processes that include embryonic development, differentiation, autophagy, apoptosis and cell survival. TAK1 along with its binding partners TAB1, TAB2 and TAB3 displays a complex pattern of regulation that includes serious crosstalk with major signaling pathways including the C-Jun N-terminal kinase (JNK), p38 MAPK, and I-kappa B kinase complex (IKK) involved in establishing cellular commitments for death and survival. This review also highlights how TAK1 orchestrates regulation of energy homeostasis via AMPK and its emerging role in influencing mTORC1 pathway to regulate death or survival in tandem.

Eukaryotic Initiation Factor 4E (eIF4E) sequestration mediates 4E-BP1 response to rapamycin.

The cap dependent translation initiation is a tightly controlled process of cooperative ternary complex formation by 4E-BP1, eIF4E and the 5' cap of eukaryotic mRNA in response to environmental cues like glucose, nutrients and growth factor levels. Based on the well-described effects of mTORC1/rapamycin complex on 4E-BP1 phosphorylation/s, it is generally accepted that rapamycin is a global inhibitor of cap-dependent translation. We have previously shown that 4E-BP1 resistance to rapamycin was overcome by the stoichiometric abundance of S6K1. Now we present evidence that the TOS-bearing amino terminal domain of S6K1 is sufficient to relieve the rapamycin resistance of 4E-BP1 as TOS deleted variants of S6K1, active or inactive with regard to S6K1 activity failed to bring about relief of 4E-BP1 resistance to rapamycin. We also show that the reciprocal inactivation of S6K1 by abundance of 4E-BP1 gets accomplished only with intact TOS motif in the protein. The data presented in this study identifies eIF4E and not Raptor as a cellular factor responsible to regulate rapamycin sensitivity of 4E-BP1 suggesting that the phosphorylation dynamics and rapamycin sensitivity of 4E-BP1 and S6K1 are regulated independently.

Reappraisal to the study of 4E-BP1 as an mTOR substrate - A normative critique.

mTOR-4E-BP1 axis is regarded as the best oncogenic circuitry impinging on translational control whereby mTORC1 dictates post-translational regulation of 4E-BP1. This review provides new insights into the molecular network of signalling pathways highlighting the recent explosion of studies in respect to the deviant behaviour of 4E-BP1 towards mTORC1. Despite the striking conservation of mTOR nexus, the eccentric phosphorylation dynamics of 4E-BP1 negate the apparent linear architecture of mTORC1 attesting the importance of other kinases that may evoke cross-talks with the conventional frame, most of which are enlisted in the manuscript. We also throw light on the tenuous role of rapamycin in 4E-BP1 regulation, which further necessitates the evaluation of 4E-BP1 to envisage the underlying molecular mechanisms in the discovery of novel drugs of 4E-BP1 for new treatment strategies. Finally, the review brings forward comprehensive studies delineating the redundancy of 4E-BP isoforms in regulating translational control.

Mutations in OTOF, CLDN14 & SLC26A4 genes as major causes of hearing impairment in Dhadkai village, Jammu & Kashmir, India.

Background & objectives: A high incidence of hearing impairment is reported from the village of Dhadkai in the State of Jammu and Kashmir, India. Prevalence of endogamy in this community suggested a common genetic basis for the disorder. A genetic study was undertaken to ascertain the basis for the high incidence of hearing impairment in this region. Methods: In a two-step approach to identify the causative mutation/s, a whole-genome-based linkage analysis of an extended family of 45 members was carried out, which included 23 affected and 22 unaffected members. Mutational analysis for the candidate deafness genes helped reveal causative mutations in the family. In addition, seven deafness-causing genes, Cx26, SLC26A4, CLDN14, TMPRSS3, TMC1, TMIE and USH1C, were analyzed in smaller families with hearing impairment. Results: In the 45-member extended family, the critical chromosomal region mapped to 2p24-p22.The c.2122C>T (p.R708X) mutation in OTOF in 2p24-p22was identified as being the causal change. Linkage to 2p24-p22 locus was not observed in a particular branch of this extended family. Analysis of seven known deafness-causing genes in this branch revealed a mutation, c.254T>A (p.V85D), in CLDN14. Among seven small families unrelated to the 45-member extended family, hearing loss was attributable to p.R708X in OTOF in three families and to p.V85D in CLDN14 in one family; a new mutation c.1668T>A (p.Y556X) SLC26A4 was identified in two families and the causative change could not be identified in one family. Interpretation & conclusions: This study suggested considerable genetic heterogeneity in the causation of hearing loss in Dhadkai. Recessive mutations were observed in at least three genes causing hearing loss: OTOF (p.R708X), SLC26A4 (p.Y556X) and CLDN14 (p.V85D). Mutation p.R708X appeared to be the major cause of hearing impairment in Dhadkai.

Expression Profiling and Cellular Localization of Stress Responsive Proteins in Squamous Cell Carcinoma of Human Esophagus.

BACKGROUND: The ambiguity in relating expression dynamics of stress response proteins with human esophageal squamous cell carcinoma (ESCC) has sidelined the potential of stress proteins as therapeutic targets. This study was an attempt to unequivocally relate the stress protein dynamics with stage and propensity of ESCC. METHODS: Surgically resected tumor and adjacent histologically normal tissue from 46 patients with esophageal squamous cell carcinoma were investigated in the present study. Expression of HSPs was analyzed by Western blotting and immunohistochemistry. RESULTS: HSP expression was observed in all 46 cases both in adjacent normal and tumor tissues. The expression and the localization of individual HSP showed no significant correlation with depth of invasion, tumor grade, and pathological stage of the tumor. HSP 27 was the most abundant protein followed by HSP 90 and HSP 70. The HSP 27 localized exclusively in the cytoplasm of adjacent normal and tumor cells. HSP 70 showed dispersed expression with predominating nuclear localization in both normal and tumor tissue cells and HSP 90 was localized in cytoplasm of adjacent normal and in nucleus of tumor cells in majority of the cases. CONCLUSION: Our data advocate lack of relationship between stress protein expression and the progression of ESCC. The data renew the prospect of anti-HSP drugs as therapeutic resources in light of the possibility that their use would continue to sensitize cancer cells towards drug induced apoptosis for tumor regression.

Clinical relevance of genetic polymorphism in CYP2C9 gene to pharmacodynamics and pharmacokinetics of phenytoin in epileptic patients: validatory pharmacogenomic approach to pharmacovigilance.

AIMS AND OBJECTIVES: Variations in drug metabolizing genes are known to have a clinical impact on AED therapy. We genotyped normal and epileptic patient cohorts of monoethnic population of Kashmir valley for CYP2C9 gene and allelic polymorphism and investigated the effect of CYP2C9*2 and *3 polymorphism on the Pharmacokinetic and therapeutic and/or adverse pharmacodynamic responses to Phenytoin in the idiopathic epilepsy patients. METHODS: PCR-RFLP methods were used for genotyping of 121 normal controls and 92 idiopathic epilepsy patients for CYP2C9*2 and *3 polymorphism, the results were validated by direct sequencing. Phenytoin pharmacokinetic (PK) analysis in idiopathic epilepsy patients was done using a validated EMIT assay technique. Pharmacodynamic analysis was done by evaluating clinical response to phenytoin therapy and ADR monitoring. RESULTS: The respective frequencies of CYP2C9 *1, *2, and *3 alleles were 64%, 6.6%, 29.3%, and 58%, 9.8%, 32.6% in controls and idiopathic epilepsy patients from Kashmir valley. PK analysis revealed that AUC0–4 was a better surrogate biomarker of CYP2C9 metabolizer status compared to C4 and C0 concentrations alone. A comparison of “phenytoin response categories” among CYP2C9 Wild and Heterozygous groups did not reveal any significant difference between the groups (p=0.3800). CONCLUSION: CYP2C9* 3 was the most frequent mutant allele found in healthy controls and idiopathic epilepsy patients of ethnic Kashmiri population. CYP2C9 genotype based phenytoin therapy is highly relevant in Kashmiri population due to a high incidence of genetic variations associated with therapeutic and adverse responses to phenytoin. Phenytoin AUC0–4 tends to correlate better with genetic polymorphism of CYP2C9.

mTOR Signaling in Protein Translation Regulation: Implications in Cancer Genesis and Therapeutic Interventions.

mTOR is a central nutrient sensor that signals a cell to grow and proliferate. Through distinct protein complexes it regulates different levels of available cellular energy substrates required for cell growth. One of the important functions of the complex is to maintain available amino acid pool by regulating protein translation. Dysregulation of mTOR pathway leads to aberrant protein translation which manifests into various pathological states. Our review focuses on the role mTOR signaling plays in protein translation and its physiological role. It also throws some light on available data that show translation dysregulation as a cause of pathological complexities like cancer and the available drugs that target the pathway for cancer treatment.