Application of Recombinant Proteins as Alzheimers disease Therapeutics and Protein Delivery Vehicle

Abstract

Therapeutic molecules such as small molecules (> 1 kDa) and gene are successfully achieved for various therapeutic researches. However, small molecules are resulted in off-target side effects due to its limited specificity and selectivity. Also, using genes as therapeutic molecules in many cases raises safety concerns while they used. Recombinant proteins should be alternative molecules for overcome those limitations. Recombinant proteins have several attractive features such as high specificity, selectivity, safety and the protein production process is very simple and systematic. Therefore, I used recombinant protein to apply on Alzheimers Disease (AD) therapeutic and protein delivery vehicle. <br/> AD is a neurodegeneration disorder that damages neurons and causes loss of brain functions that involve memory, learning and reasoning. There are two main pathological features of AD: intra-neuronal neurofibrillary tangles (NFTs) containing paired helical filaments (PHFs) comprised of hyper-phosphorylated tau and the accumulation of amyloid-β (Aβ) plaques in both intra- and extra-cellular neurons. <br/> Accumulation of senile plaques is comprised of 40 or 42 length of hydrophobic amyloid-β peptides (Aβ1– 40 or Aβ1– 42). The type II membrane-bound zinc metallo-endopeptidase, NEP, is the key enzyme for degrading Aβ peptides. Because NEP is capable of degrade only monomer form of Aβ peptides but also oligomer form of Aβ peptide. In order to degrade neuronal toxic Aβ peptides, recombinant human soluble NEP protein which removed cell membrane bound region was produced by mammalian cells (HEK293T) to demonstrate protective effect against Aβ peptides in neuronal cells in vitro. <br/> Neurofibrillary tangles (NFTs) are composed of aggregates of hyperphosphorylated tau, which is a microtubule-associated protein. As a microtubule-associated protein, aggregates of normal tau protect cells against toxic hyperphosphorylation. Therefore, reducing the level of hyperphosphorylated tau is crucial for treating AD. In previous studies, heat shock proteins (HSPs) can bind with tau and Hsp27 which, especially, directly associates with hyperphosphorylated tau or PHF and eliminates tau aggregates. I investigated recombinant Hsp27 protein to obtain the protective effect on AD. To enhance the efficiency for delivering the protein, cell penetrating peptide called Tat was applied. HIV Tat is 11 amino acids sequence (YGRKKRRQRRR) that can be effectively internalized with the fused protein into the cells at reasonable concentration. After recombinant fusion Hsp27 protein was applied on neuronal cells, tau-induced neurodegeneration therapeutic effect had been demonstrated.<br/> sNEP and Tat-Hsp27 recombinant proteins exhibited great potential as therapeutic molecules for AD.<br/> The use of cell penetrating peptides (CPPs) applied on recombinant proteins facilitates great opportunities for various biomedical applications. However, high extracellular concentration threshold (micromolar level) at which showing significant penetration of CPPs fusion protein causes one of the crucial weaknesses impeding the broad biomedical applications. <br/> In previous research, chemically synthesize 16 meric amphipathic α-helical LK peptide had been demonstrated enhancing effect of cell penetrating activity (nanomolar range) compared to conventional CPPs such as Tat or oligo-arginine. Furthermore, increased cell penetrating activity of LK peptide was obtained by multimerization. However, currently, with chemical synthesis could be elongated the peptide as only up to 50 amino acids. Therefore, recombinant protein technique should be better method for production of multimer form of LK peptide fusion proteins.<br/> I produced momoneric LK to hexameric LK (LK-1 to LK-6) fusion proteins in order to demonstrate the intracellular penetrating efficiency. The multimeric LK fused protein has demonstrated cell penetrating activity within a hundred nanomolar concentration which is about 100–1,000 fold lower concentrations than Tat-proteins. Interestingly, LK fusion proteins with monomer and multimer form underwent different penetration mechanism. The multimeric LK showed rapid cell penetration through macropinocytosis at low nanomolar concentrations, unlike the monomeric LK, which had slower penetrating kinetics at much higher concentrations. Mammalian cell membrane proteoglycan, heparin sulfate proteoglycan (HSPG), was seemed to be highly involved in the rapid penetration of multimeric fusion proteins. I delivered an adipogenic transcription factor, PPAR-γ2, into pre-adipocyte to demonstrate cell differentiation for biomedical applications.<br/> It is essential to discover or develop new CPPs that are able to deliver proteins into cells at clinically feasible concentrations for future practical CPP fusion protein-based therapeutic applications.<br/>