Biochemical and biophysical aspects of molecular recognition and signalling by neurotrophins

Sammanfattning: Neurotrophins are members of a family of structurally and functionally relatedneurotrophic factors that control the development and maintenance of the nervoussystem. There are currently 5 members which make up this family: nerve growth factor(NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4(NT-4) and neurotrophin-6 (NT-6), which so far is only described in fish. Neurotrophinmediated effects, (e.g. cell survival and differentiation) are caused by ligand specificbinding to the trk family of tyrosine kinase receptors. NGF binds to TrkA, BDNF andNT 4 bind TrkB and NT-3 preferentially binds to TrkC. Due to the similarity in activitiesbetween NT-6 and NGF, the former is believed to act through a fish homologue of TrkA.Furthermore, neurotrophins bind with equal affinities to the p75 neurotrophin receptor(p75NTR) which is related to members of the tumour necrocis factor (TNF) receptorsuperfamily. Members of this family were initially grouped according to similaritiesin their extracellular domains. However, recent sequence analysis revealed that somemembers also share similar intracellular domain of about 80 residues involved inapoptosis, called the "death domain" (DD). Using site-directed mutagenesis and various biological and biochemical assaysthe critical domains and residues determining neurotrophin specificity towards bindingand receptor activation were identified. Results defined discontinous stretches ofamino acids in the primary structure of these proteins which, upon inspection ofthe prototypic three dimensional structure of NGF, delineated a continuous surfaceextending approximately parallel to the two-fold symmetry axis of the molecule. Usinginformation from this structure-function analysis, chimaeric neurotrophins with novelproperties were constructed such as a heparin-binding NGF and a multifunctional pan-neurotrophin-1(PNT-1). To gain insight on the possible mechanisms of p75NTR signalling, the structureof the p75 intracellular domain (p75ICD) was determined using nuclear magnetic resonance(NMR) spectroscopy. The only structured region in our ICD construct was the conservedDD module which reveals a novel fold (shared only with Fas receptor) consisting oftwo perpendicular sets of three helices packed into a globular structure. A surfacearea devoid of charged residues (hydrophobic patch) in the DD indicated a potentialsite of interaction with downstream targets. Using the Selectively Infective Phage(SIP) display technique, several peptides binding to the ICD were selected. A peptide(CFFRGGFFNHNPRYC) that interacted with the DD, was further studied by NMR and wasfound to bind to the above-mentioned hydrophobic patch. These selected peptides shouldprovide leads to the natural targets of the p75ICD and also should prove to be usefulreagents in probing the signalling mechanism of this receptor. Taken together, this study combines genetic, biochemical and biophysical approachesto understand the molecular basis of recognition and signalling by neurotrophins,and would help in the design of agonsists and antagonists that could mediate in disorderslike neurodegeneration.

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