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

Effects of glucoprivation on the adrenal medulla: a time course study using shotgun proteomics

Theoretical thesis.Bibliography: pages 55-65.1. Introduction -- 2. Experimental procedures -- 3. Results -- 4. Discussion.The adrenal medulla is the sole endocrine gland responsible for catecholamine release inresponse to stress. While a time course of stress response has been described for rate-limitingenzyme in catecholamine synthesis; tyrosine hydroxylase (TH), none have yet described any other metabolic changes in this gland. Therefore the aims of this study are to map the proteome of the adrenal medulla and determine any changes over time following an acute episode of glucopenia, a potent stimulus thereof. In addition, the final aim is to determine whether or not Western blot analysis reflects the findings shown using mass spectrometry (MS). A single IP injection of 2-deoxyglucose was used to induce glucopenia in Sprague Dawley rats. The adrenal glands were harvested over at 0min, 20min, 50min, 4h ,8h and 24h after the stimulus (n=6 for each timepoint). Label-free LC-MS/MS was then performed on the samples (n=3) for each timepoint to obtain the proteomic profile. Profiles were quantitatively compared to the control (0min/unstimulated). Western blotting was then used in order to validate the MS data using specifically selected proteins. The protein profile was determined and comprises 973 proteins, with marked changes at each time point; 20min=78, 50min=138, 4h=163, 8h=136 and 24h=107. Western blot analysis regarding TH corresponded with MS data, however other proteins showed differing expression levels. From this we understand that cellular metabolic changes occur rapidly in response to stress and last longer than 24h. It is also apparent that Western blot analysis does not always correspond with the MS output.Mode of access: World wide web1 online resource (iv, 106 pages) diagrams, graphs, table

Impaired protease regulatory activity of neuroserpin is observed in human glaucoma eyes and a rodent model of experimental glaucoma

Purpose: The serine protease inhibitor, neuroserpin, is predominantly expressed in neural tissues and is well expressed in the vitreous and retina. There is evidence of involvement of proteases in matrix remodelling and mediating damage to the optic nerve head and RGCs in glaucoma. As neuroserpin is linked to selectively inhibiting proteases, mainly plasminogen activators and plasmin, we evaluated changes in its expression and inhibitory activity. Methods: Human retinal and vitreous tissues from donated eyes were examined from control (age: control 64.8 ± 10.16, n = 12) and glaucoma subjects (age: 71.6 ± 8.67, n = 12). Retinal samples from rats exposed to experimentally elevated intraocular pressure (IOP) by microbead injections were also examined and compared with the control eyes. Immunoprecipitation followed by immunoblotting were carried out to assess changes in the neuroserpin expression. Neuroserpin activity was also assessed by immuno-precipitation followed by in-gel-zymography. Results: The plasmin inhibitory activity of neuroserpin was significantly reduced in the vitreous samples in the human glaucoma group compared to controls as revealed by immunoprecipitation followed by in-gel-zymography (n = 12, P < 0.0025). No significant changes were however observed in the neuroserpin expression in the human vitreous samples. Significantly decreased neuroserpin activity was also observed in the rat retinas exposed to experimentally increased IOP (P < 0.03, n = 5) as well as in retinal tissues from human optic nerve head region (P < 0.04; n = 6). Conclusions: Impaired neuroserpin activity is observed in human glaucoma tissues as well as in rodent model of experimentally elevated IOP, indicating a potential reduction of proteolytic inhibition in glaucoma. This could facilitate increased structural damage to tissues.1 page(s

Molecular changes in the adrenal medulla and brain following glucoprivation

Glucoprivation or hypoglycaemia evokes a complex array of counterregulatory responses which include autonomic, neuroendocrine and behavioural changes in order to maintain blood glucose levels. Catecholaminergic neurons of the rostral ventrolateral medulla activate adrenal adrenergic chromaffin cells to secrete adrenaline which mobilises glucose. Our aim was to determine the molecular changes and their timecourse in the adrenal medulla and brainstem regions in order to understand the cellular mechanisms altered in response to glucoprivation.2 page(s

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

Age-related neurodegenerative disease associated pathways identified in retinal and vitreous proteome from human glaucoma eyes

Abstarct Glaucoma is a chronic disease that shares many similarities with other neurodegenerative disorders of the central nervous system. This study was designed to evaluate the association between glaucoma and other neurodegenerative disorders by investigating glaucoma-associated protein changes in the retina and vitreous humour. The multiplexed Tandem Mass Tag based proteomics (TMT-MS3) was carried out on retinal tissue and vitreous humour fluid collected from glaucoma patients and age-matched controls followed by functional pathway and protein network interaction analysis. About 5000 proteins were quantified from retinal tissue and vitreous fluid of glaucoma and control eyes. Of the differentially regulated proteins, 122 were found linked with pathophysiology of Alzheimer’s disease (AD). Pathway analyses of differentially regulated proteins indicate defects in mitochondrial oxidative phosphorylation machinery. The classical complement pathway associated proteins were activated in the glaucoma samples suggesting an innate inflammatory response. The majority of common differentially regulated proteins in both tissues were members of functional protein networks associated brain changes in AD and other chronic degenerative conditions. Identification of previously reported and novel pathways in glaucoma that overlap with other CNS neurodegenerative disorders promises to provide renewed understanding of the aetiology and pathogenesis of age related neurodegenerative diseases