Synthesis of Amyloidogenic Peptides and Endosomolytic Peptides for Functional and Intracellular Protein Delivery Studies.

Abstract

Over the past few decades, peptide research gained lot of prominence due to their extensive usage in and across domains ranging from biomedical to bio-material science. This thesis deals with two different types of peptides having unique structural or functional properties – amyloidogenic peptides and endosomolytic peptides. The following aspects will be discussed:

(i) The first part describes microwave assisted solid phase peptide synthesis (SPPS) of amyloid-β (Aβ) 1-42 peptide. Amyloid-β peptide is widely regarded as the causative agent of the AD. However, a major bottleneck in doing research with amyloid-β peptide is synthesizing the same efficiently with reasonable yield and good purity. Aβ 1-42 is rightly considered as a “difficult sequence” for solid phase peptide synthesis. This section describes an efficient method for synthesizing and obtaining Aβ 1-42 peptide. Here microwave assisted SPPS was applied to synthesize Aβ 1-42 having a covalently attached hydrophilic tail, which aids the process of synthesis and purification and can be removed easily to produce the desired peptide. The tail was attached at the C-terminal of the peptide using a double linker system. (ii) The following part describes development of catalytic amyloid scaffolds based on amyloid-β peptide sequence. The “amyloid-world” hypothesis identifies short self-assembling amyloids as the common ancestral protein (CA) fold, and this has prompted many scientists to pursue research towards discovering or designing amyloids having catalytic behavior. This part describes the design and formation of catalytic amyloids based on small peptide fragments, derived from the Aβ peptide sequence, which can perform hydrolysis of model substrate p-nitrophenyl acetate (p-NPA). Two of the peptides studied were found to possess metal independent catalytic amyloid behavior towards the substrate. (iii) The last part investigates the effects of various endosomolytic peptides on cellular uptake and endosomal release of halogenated green fluorescent protein (GFP). Most therapeutic macromolecules are internalized by the cells via endocytosis, and eventually end up inside the endosomes. Timely release of these cargoes from the endosomes is extremely essential since, otherwise, they end up being hydrolyzed once the endosomes mature and fuse with lysosomes. Endosomolytic peptides are known to act as efficient endosome disrupting agents which can help to mediate release of the cargo from the endosomes before their lysosomal degradation. Recently, it has been reported from our group that cellular uptake of a halogenated green fluorescent protein (GFP) occurs via receptor-mediated endocytosis. This part discusses the syntheses of various types of endosomolytic peptides and presents a comparative analysis of how effective they are in mediating endosomal release of halogenated GFP. Studies were performed using two different types of mammalian cell lines.