Mushtaq A. Beigh

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.

Phosphorylation dynamics of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) is discordant with its potential to interact with eukaryotic initiation factor 4E (eIF4E).

Mammalian target of rapamycin (mTOR) controls cellular growth and proliferation by virtue of its ability to regulate protein translation. Eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) - a key mTOR substrate, binds and sequesters eIF4E to impede translation initiation that is supposedly overcome upon 4E-BP1 phosphorylation by mTOR. Ambiguity surrounding the precise identity of mTOR regulated sites in 4E-BP1 and their invariable resistance to mTOR inactivation raises concerns about phospho-regulated model proposed for 4E:4E-BP1 interaction. Our attempt to mimic dephosphorylation associated with rapamycin response by introducing phospho deficient mutants for sites implicated in regulating 4E:4E-BP1 interaction individually or globally highlighted no obvious difference in the quantum of their association with CAP bound 4E when compared with their phosphomimicked counterparts or the wild type 4E-BP1. TOS or RAIP motif deletion variants compromised for raptor binding and resultant phosphodeficiency did little to influence their association with CAP bound 4E. Interestingly ectopic expression of ribosomal protein S6 kinase 1 (S6K1) that restored 4E-BP1 sensitivity to rapamycin/Torin reflected by instant loss of 4E-BP1 phosphorylation, failed to bring about any obvious change in 4E:4E-BP1 stoichiometry. Our data clearly demonstrate a potential disconnect between rapamycin response of 4E-BP1 and its association with CAP bound 4E.

Rapamycin inhibition of baculovirus recombinant (BVr) ribosomal protein S6 kinase (S6K1) is mediated by an event other than phosphorylation

BackgroundRibosomal protein S6 kinase 1(S6K1) is an evolutionary conserved kinase that is activated in response to growth factors and viral stimuli to influence cellular growth and proliferation. This downstream effector of target of rapamycin (TOR) signaling cascade is known to be directly activated by TOR- kinase mediated hydrophobic motif (HM) phosphorylation at Threonine 412 (T412). Selective loss of this phosphorylation by inactivation of TOR kinase or activation/recruitment of a phosphatase has accordingly been implicated in mediating inhibition by rapamycin.FindingsWe present evidence that baculovirus driven expression of S6K1 in insect cells (Sf9) fails to activate the enzyme and instead renders it modestly active representing 4-6 folds less activity than its fully active mammalian counterpart. Contrary to the contention that viral infection activates TOR signaling pathway, we report that BVr enzyme fails to exhibit putative TOR dependent phosphorylation at the HM and the resultant phosphorylation at the activation loop (AL) of the enzyme, correlating with the level of activity observed. Surprisingly, the BVr enzyme continued to exhibit sensitivity to rapamycin that remained unaffected by mutations compromised for TOR phosphorylation (T412A) or deletions compromised for TOR binding (ΔNH 2-46/ΔCT104).ConclusionsThese data together with the ability of the BVr enzyme to resist inactivation by phosphatases indicate that inhibition by rapamycin is not mediated by any phosphorylation event in general and TOR dependent phosphorylation in particular.

Growth Inhibition by Bupivacaine Is Associated with Inactivation of Ribosomal Protein S6 Kinase 1

Bupivacaine is an amide type long acting local anesthetic used for epidural anesthesia and nerve blockade in patients. Use of bupivacaine is associated with severe cytotoxicity and apoptosis along with inhibition of cell growth and proliferation. Although inhibition of Erk, Akt, and AMPK seemingly appears to mediate some of the bupivacaine effects, potential downstream targets that mediate its effect remain unknown. S6 kinase 1 is a common downstream effector of several growth regulatory pathways involved in cell growth and proliferation known to be affected by bupivacaine. We have accordingly attempted to relate the growth inhibitory effects of bupivacaine with the status of S6K1 activity and we present evidence that decrease in cell growth and proliferation by bupivacaine is mediated through inactivation of S6 kinase 1 in a concentration and time dependent manner. We also show that ectopic expression of constitutively active S6 kinase 1 imparts substantial protection from bupivacaine induced cytotoxicity. Inactivation of S6K1 though associated with loss of putative mTOR mediated phosphorylation did not correspond with loss of similar phosphorylations in 4EBP1 indicating that S6K1 inhibition was not mediated through inactivation of mTORC1 signaling pathway or its down regulation.

Recent Trends in the Fabrication of Starch Nanofibers: Electrospinning and Non-electrospinning Routes and Their Applications in Biotechnology

Electrospinning a versatile and the most preferred technique for the fabrication of nanofibers has revolutionized by opening unlimited avenues in biomedical fields. Presently, the simultaneous functionalization and/or post-modification of as-spun nanofibers with biomolecules has been explored, to serve the distinct goals in the aforementioned field. Starch is one of the most abundant biopolymers on the earth. Besides, being biocompatible and biodegradable in nature, it has unprecedented properties of gelatinization and retrogradation. Therefore, starch has been used in numerous ways for wide range of applications. Keeping these properties in consideration, the present article summarizes the recent expansion in the fabrication of the pristine/modified starch-based composite scaffolds by electrospinning along with their possible applications. Apart from electrospinning technique, this review will also provide the comprehensive information on various other techniques employed in the fabrication of the starch-based nanofibers. Furthermore, we conclude with the challenges to be overcome in the fabrication of nanofibers by the electrospinning technique and future prospects of starch-based fabricated scaffolds for exploration of its applications.

Prospects of Natural Polymeric Scaffolds in Peripheral Nerve Tissue-Regeneration

Tissue-engineering is emerging field and can be considered as a novel therapeutic intervention in nerve tissue-regeneration. The various pitfalls associated with the use of autografts in nerve-regeneration after injuries have inspired researchers to explore the possibilities using various natural polymers. In this context, the present chapter summarizes the advances of the various types of natural polymeric scaffolds such as fibrous scaffolds, porous scaffolds, and hydrogels in nerve-regeneration and repair process. The functionalization of the scaffolds with wide-range of biomolecules and their biocompatibility analysis by employing various cells (e.g., mesenchymal, neural progenitor stem cells) along with the in vivo regeneration outcomes achieved upon implantation are discussed here. Besides, the various avenues that have been explored so far in nerve tissue-engineering, the use of the extracellular matrix in enhancing the functional polymeric scaffolds and their corresponding outcomes of regeneration are mentioned. We conclude with the present challenges and prospects of efficient exploration of natural polymeric scaffolds in the future to overcome the problems of nerve-regeneration associated with various nerve injuries and neurodegenerative disorders.

Scaffolds Fabricated from Natural Polymers/Composites by Electrospinning for Bone Tissue Regeneration

Naturally bone is a hierarchical and highly integrative dynamic tissue that is continuously remodeled by osteoblasts and osteoclasts. Deformities in bone due to trauma and/or disease are highly prevalent and mostly need surgical intervention. However, the methods of surgical treatments are associated with donor site morbidity, infection and/or complete rejection. Bone tissue-engineering provides a platform for growth of new bone tissue by fabricating scaffolds along with cells, growth factors and other dynamic forces. The polymeric materials especially natural polymers in their nanofibrous forms have been developed and introduced for bone tissue regeneration. At the nanoscale, natural polymers possess tunable properties and can be surface functionalized or blended with other polymers to provide juncture for cell-seeding, proliferation, differentiation and further resulting in regenerated tissue formation. These scaffolds fabricated from natural polymers and additives by electrospinning are bio-inspired to mimic the natural extracellular matrix resembling the native collagen of bone. This chapter focuses on the fabrication techniques as state of art nanofibrous scaffolds from natural polymers/additives during the recent years by the process of electrospinning for use in bone tissue regeneration. Further on, this chapter highlights the development in the scaffold fabrication from natural polymers like silk fibroin, chitosan, collagen, gelatin, cellulose, starch and, zein. The importance of add-on materials like stem cells, hydroxyapatite, apatite-wollanstonite, growth factors, osteogenic cells, bone morphogenic proteins and osteogenic drugs have been discussed and illustrated by various examples for enhancing the formation of new bone tissue. Furthermore, this chapter explains how these natural polymers influence the several signaling pathways to regulate bone regeneration.

Reconstructing nanofibers from natural polymers using surface functionalization approaches for applications in tissue engineering, drug delivery and biosensing devices

Previously, the nanofibers were predominantly fabricated from synthetic polymers due to their excellent mechanical properties. Understanding the different complex processes in fabrication and various process parameters involved have not only allowed the use of natural polymers for fabricating nanofibers but also broadened the scope of applications. To date, many of the natural polymeric composites have been fabricated by different functionalization techniques to increase their applicability. Nanofibers fabricated from natural polymers have been chemically functionalized by a variety of molecules like drugs, enzymes, metal ions etc. by techniques such as plasma treatment, wet chemical method, graft polymerization and co-electrospinning of surface-active molecules. Furthermore, the nanofibers derived from natural polymers have been surface-coated on the synthetic polymers to induce extracellular matrix mirroring properties like cell adhesion, migration, proliferation and differentiation. In this review, we have not only investigated the various novel and facile functionalization approaches but potential properties and applications are discussed as well. The various surface chemistry modifications of the natural polymeric nanofibers and their potential applications in drug delivery, enzymology, catalysis, filtration, biosensing and tissue engineering are discussed. In addition, a brief presentation of an overview of challenges and future scope with the aim of making them a clinical success has been presented.