Protein Engineering of Heat shock 27 and Neprilysin for therapeutic application to Alzheimer's disease

Abstract

Alzheimers disease (AD) is a progressive neurodegenerative disorder with neurofibrillary tangles (NFTs) and amyloid-beta (Aβ) plaques. The tau is a major component of NFTs, a microtubule-associated protein. In AD, tau is abnormally aggregated and hyperphosphorylated into NFTs in neurons. The pathological tau proteins lose the capability to rescue microtubules in transporting neuronal substance, leading to neuronal dysfunction and apoptosis. Phosphorylated tau would have a ubiquitin-independent pathway that can be facilitated by Hsp27 mechanism leading to its dephosphorylation or degradation. The cytoprotective effects of Hsp27 include its role as a molecular chaperone, the inhibition of caspase activation, the prevention of stress-induced disruption of the cytoskeleton, and modulation of the intracellular redox potential. I generated a fusion protein of Hsp27 and the protein transduction domain of the HIV Tat protein (Tat-Hsp27) to enhance the delivery of Hsp27 into cells. the recombinant Tat-Hsp27 reduced the level of hyperphosphorylated tau induced by okadaic acid in SH-SY5Y neuroblastoma cells and prevented the apoptosis induced by abnormal tau aggregates in our cellular model of AD. Aβ is derived from amyloid precursor protein (APP) by β-secretase and γ-secretase. Aβ is immediately catabolized under normal conditions, whereas dysmetabolism of Aβ seems to lead to pathological deposition upon aging. Neprilysin (NEP) is a rate-limiting peptidase involved in the physiological degradation of Aβ in the brain. Thus, the reduction of neprilysin activity will contribute to Aβ accumulation and consequently to AD development. NEP is a type II membrane metalloendopeptidase with an active site containing a zinc-binding motif at the extracellular carboxyl terminal domain and exists on plasma membrane as a noncovalently associated homodimer. I designed a gene encoding the extracellular domain (52?750) of human NEP. Recombinant soluble human NEP was secreted from insect cells transduced with baculovirus expressing the extracellular domain and purified by affinity chromatography on a histrap column and size exclusion chromatography on a superdex 200 column. The purified NEP could reduce a decrease of Aβ-mediated apoptosis of hippocampal cells and increase cell viability in vitro. The number of Aβ plaques and memory impairment was diminished by the presence of recombinant soluble neprilysin in the brain of AD model mice. These findings suggest that recombinant Tat-Hsp27 and soluble human neprilysin are a promising candidates as therapeutic protein for Alzheimers disease.