Molecular determinants and interaction data of cyclic peptide inhibitor with the extracellular domain of TrkB receptor

TrkB is a high affinity receptor for the brain derived neurotrophic factor (BDNF) and its phosphorylation stimulates activation of several intracellular signalling pathways linked to cellular growth, differentiation and maintenance. Identification of various activators and inhibitors of the TrkB receptor and greater understanding their binding mechanisms is critical to elucidate the biochemical and pharmacological pathways and analyse various protein crystallization studies. The data presented here is related to the research article entitled Brain Derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling [1]. Cyclotraxin B (CTXB) is a disulphide bridge linked cyclic peptide molecule that interacts with TrkB receptor and inhibits the BDNF/TrkB downstream signalling. This article reports for the first time binding mechanism and interaction parameters of CTXB with the TrkB receptor. The molecular model of CTXB has been generated and it\u27s docking with TrkB domain carried out to determine the critical residues involved in the protein peptide interaction. © 2016 The Authors

Amyloid β Induces Early Changes in the Ribosomal Machinery, Cytoskeletal Organization and Oxidative Phosphorylation in Retinal Photoreceptor Cells

Amyloid β (Aβ) accumulation and its aggregation is characteristic molecular feature of the development of Alzheimer’s disease (AD). More recently, Aβ has been suggested to be associated with retinal pathology associated with AD, glaucoma and drusen deposits in age related macular degeneration (AMD). In this study, we investigated the proteins and biochemical networks that are affected by Aβ in the 661 W photoreceptor cells in culture. Time and dose dependent effects of Aβ on the photoreceptor cells were determined utilizing tandem mass tag (TMT) labeling-based quantitative mass-spectrometric approach. Bioinformatic analysis of the data revealed concentration and time dependent effects of the Aβ peptide stimulation on various key biochemical pathways that might be involved in mediating the toxicity effects of the peptide. We identified increased Tau phosphorylation, GSK3β dysregulation and reduced cell viability in cells treated with Aβ in a dose and time dependent manner. This study has delineated for the first-time molecular networks in photoreceptor cells that are impacted early upon Aβ treatment and contrasted the findings with a longer-term treatment effect. Proteins associated with ribosomal machinery homeostasis, mitochondrial function and cytoskeletal organization were affected in the initial stages of Aβ exposure, which may provide key insights into AD effects on the photoreceptors and specific molecular changes induced by Aβ peptide

Understanding the retinoid-x-receptor biology and manipulating it as a novel strategy to protect the retina

Theoretical thesis.Bibliography: pages 174-197.1. Introduction -- 2. Material and methods -- 3. Neuroprotective effects of RXR modulation in SH-SY5Y cells against ER stress -- 4. Retinoid-X-receptor modulation protects against ER stress response and rescues glaucoma phenotypes in adult mice -- 5. Pharmacological targeting of RXR receptors: in silico studies -- 6. Identification of age-related neurodegenerative disease associated pathways identified in retinal and vitreous proteome from human glaucoma eyes -- 7. Conclusion and future directions.Glaucoma is a degenerative optic neuropathy affecting nearly 80 million individuals by 2020 worldwide. Glaucoma is mainly manifested as alterations of the optic disc with progressive degeneration of retinal ganglion cells (RGCs) contributing to visual field loss. High intraocular pressure (IOP) is considered as the main risk factor of glaucoma. Unfortunately, a significant number of patients show disease progression despite treating with IOP lowering drugs. So, RGC degeneration cannot be prevented only by reducing eye pressure. There is need for development of more novel strategies targeting retinal neuroprotection. Within this context, this PhD project aimed to assess the potential neuroprotective effect of RXR activation by its agonist bexarotene both in vitro studies as well as in vivo acute and chronic glaucoma models. Retinoid X receptors (RXRs) are ligand-dependent transcription factors that belong to the nuclear receptor (NR) superfamily. RXRs have three different isoforms (α, β, and γ) and can form both homo- and heterodimers with other nuclear receptors. Numerous studies have linked RXR modulation with neuroprotection. This thesis was mainly focused to elucidate the role of bexarotene as RXR modulator in glaucoma pathology. In first part of this study I demonstrated the expression and regulation of RXR receptors in SH-SY5Y cells using different concentrations of bexarotene. I also studied the role of TrkB signalling with RXR pathway in regulating endoplasmic reticulum stress response and apoptotic pathway activation. The results obtained from in vitro studies demonstrated that optimum concentrations of bexarotene upregulated the expression of all the three isoforms of RXRs. Also my studies revealed that higher concentrations of bexarotene upregulates ER stress proteins and BAD which can be prevented by pharmacological targeting of the TrkB receptor. I further extended my studies to assess the neuroprotective effects of RXR activation in mice in preventing loss of retinal ganglion cells (RGCs) under experimental glaucoma conditions. Two models i.e increased excitotoxicity mediated glutamate model (acute model) and microbead induced increased intraocular pressure model (chronic 2 months model) of RGC degeneration were used for in vivo studies. Bexarotene treatment showed enhanced expression of RXRs as compared to control and glaucoma mice retinal sections. Furthermore it was seen that bexarotene maintained inner retinal functional and structural integrity confirmed by electroretinography, H and E staining and Bieschowlsky silver staining of optic nerve sections. Moreover, docking studies also validated binding of bexarotene to RXR receptors. This thesis represents an integration of three different methodologies i.e in vitro, in vivo, in silico. The results from this thesis provide evidence to the hypothesis that RXR activation can be neuroprotective to RGCs in preventing apoptosis and cell death. Bexarotene or other RXR agonists may have potential for future therapeutic management of glaucoma.1 online resource (xx, 198 pages : illustrations

Computational analysis unravels novel destructive single nucleotide polymorphisms in the non-synonymous region of human caveolin gene

The caveolin (Cav) family of proteins comprises key constituents of caveolar structures in cell membranes, which are involved in receptor-independent endocytosis and cellular signalling pathways. Three isoforms of caveolin viz. cav-1, cav-2 and cav-3 encoded by CAV1, CAV2 and CAV3 genes respectively, have been reported to be expressed in various tissues. Genetic polymorphism in CAV has been identified as associated with the development of pathological changes in the cardiovascular system, chronic kidney disease as well as neurodegenerative diseases of the brain and retina such as Alzheimer\u27s disease, Parkinson\u27s disease and glaucoma. In this study, we investigate and characterise various polymorphisms associated with CAV1, CAV2 and CAV3 by using a combination of in silico algorithms such as SIFT, Polyphen 2.0, I-Mutant, PROVEAN, PANTHER, SNP&Go, PhD-SNP, MutPred and SNPEffects. Three-dimensional comparative modelling was performed using Phyre2 server, ab initio modelling, using the I-TASSER and RaptorX program. The predicted models were evaluated using Ramachandran plot to establish the accuracy of the models generated. The resulting mutant and wild type proteins obtained were energy minimized in Swiss Deep Viewer and evaluated. The study has identified two of the non-synonymous single nucleotide polymorphism (nsSNP) in CAV3 gene that may have a damaging effect on the protein stability. The surface residues in the wild type and mutant forms highlight different accessible surface area (ASA) of amino acid residues in the corresponding proteins. Our analysis predicted that none of the known nsSNPs have a negative effect on the CAV1 and CAV2 protein structures. Phylogenetic analysis using ConSurf further identified that most of the disease-associated nsSNPs were within the conserved regions in human cav3

Bexarotene modulates retinoid-x-receptor expression and is protective against neurotoxic endoplasmic reticulum stress response and apoptotic pathway activation

Retinoid X-receptors (RXRs) are members of the ligand-dependent transcription factor family of nuclear receptors that have gained recent research focus as potential targets for neurodegenerative disorders. Bexarotene is an RXR pharmacological agonist that is shown to be neuroprotective through its effects in promoting amyloid beta (Aβ) uptake by the glial cells in the brain. This study aimed to evaluate the dose-dependent effects of bexarotene on RXR expression in SH-SY5Y neuroblastoma cells and validate the drug effects in the brain in vivo. The protein expression studies were carried out using a combination of various drug treatment paradigms followed by expression analysis using Western blotting and immunofluorescence. Our study demonstrated that bexarotene promoted the expression of RXR α, β and γ isoforms at optimal concentrations in the cells and in the mice brain. Interestingly, a decreased RXR expression was identified in Alzheimer\u27s disease mouse model and in the cells that were treated with Aβ. Bexarotene treatment not only rescued the RXR expression loss caused by Aβ treatment (p \u3c 0.05) but also protected the cells against Aβ-induced ER stress (p \u3c 0.05) and pro-apoptotic BAD protein activation (p \u3c 0.05). In contrast, higher concentrations of bexarotene upregulated the ER stress proteins and led to BAD activation. Our study revealed that these downstream neurotoxic effects of high drug concentrations could be prevented by pharmacological targeting of the TrkB receptor. The ER stress and BAD activation induced by high concentrations of bexarotene were rescued by the TrkB agonist, 7,8 dihydroxyflavone (p \u3c 0.05) while TrkB inhibitor CTX-B treatment further exacerbated these effects. Together, these findings suggest a cross-talk of TrkB signalling with downstream effects of bexarotene toxicity and indicate that therapeutic targeting of RXRs could prevent the Aβ-induced molecular neurotoxic effects

PTPN11 induces endoplasmic stress and apoptosis in SH-SY5Y cells

PTPN11 is associated with regulation of growth factor signaling pathways in neuronal cells. Using SH-SY5Y neuroblastoma cells, we showed that adeno-associated virus (AAV)-mediated PTPN11 upregulation was associated with TrkB antagonism, reduced neuritogenesis and enhanced endoplasmic reticulum (ER) stress response leading to apoptotic changes. Genetic knock-down of PTPN11 on the other hand leads to increased TrkB phosphorylation in SH-SY5Y cells. ER stress response induced by PTPN11 upregulation was alleviated pharmacologically by a TrkB agonist. Conversely the enhanced ER stress response induced by TrkB receptor antagonism was ameliorated by PTPN11 suppression, providing evidence of cross-talk of PTPN11 effects with TrkB actions. BDNF treatment of neuronal cells with PTPN11 upregulation also resulted in reduced expression of ER stress protein markers. This study provides evidence of molecular interactions between PTPN11 and the TrkB receptor in SH-SY5Y cells. The results reinforce the role played by PTPN11 in regulating neurotrophin protective signaling in neuronal cells and highlight that PTPN11 dysregulation promotes apoptotic activation. Based on these findings we suggest that blocking PTPN11 could have potential beneficial effects to limit the progression of neuronal loss in neurodegenerative disorders

Regulation of Brain-Derived Neurotrophic Factor and Growth Factor Signaling Pathways by Tyrosine Phosphatase Shp2 in the Retina: A Brief Review

SH2 domain-containing tyrosine phosphatase-2 (PTPN11 or Shp2) is a ubiquitously expressed protein that plays a key regulatory role in cell proliferation, differentiation and growth factor (GF) signaling. This enzyme is well expressed in various retinal neurons and has emerged as an important player in regulating survival signaling networks in the neuronal tissues. The non-receptor phosphatase can translocate to lipid rafts in the membrane and has been implicated to regulate several signaling modules including PI3K/Akt, JAK-STAT and Mitogen Activated Protein Kinase (MAPK) pathways in a wide range of biochemical processes in healthy and diseased states. This review focuses on the roles of Shp2 phosphatase in regulating brain-derived neurotrophic factor (BDNF) neurotrophin signaling pathways and discusses its cross-talk with various GF and downstream signaling pathways in the retina

Glaucoma is associated with plasmin proteolytic activation mediated through oxidative inactivation of neuroserpin

Neuroserpin is a serine protease inhibitor that regulates the activity of plasmin and its activators in the neuronal tissues. This study provides novel evidence of regulatory effect of the neuroserpin on plasmin proteolytic activity in the retina in glaucoma. Human retinal and vitreous tissues from control and glaucoma subjects as well as retinas from experimental glaucoma rats were analysed to establish changes in plasmin and neuroserpin activity. Neuroserpin undergoes oxidative inactivation in glaucoma which leads to augmentation of plasmin activity. Neuroserpin contains several methionine residues in addition to a conserved reactive site methionine and our study revealed enhanced oxidation of Met residues in the serpin under glaucoma conditions. Met oxidation was associated with loss of neuroserpin inhibitory activity and similar findings were observed in the retinas of superoxide dismutase (SOD) mutant mice that have increased oxidative stress. Treatment of purified neuroserpin with H2O2 further established that Met oxidation inversely correlated with its plasmin inhibitory activity. Dysregulation of the plasmin proteolytic system associated with increased degradation of the extracellular matrix (ECM) proteins in the retina. Collectively, these findings delineate a novel molecular basis of plasmin activation in glaucoma and potentially for other neuronal disorders with implications in disease associated ECM remodelling