Development of bioassays engaging principal protein targets in the diagnostic and mechanistic studies of Parkinson's Disease and tuberculosis

Abstract

The aggregation of the α-synuclein protein (⍺S) is associated with the development of Parkinson's Disease (PD), the second most common neurodegenerative diseases. Misfolded ⍺S adopts numerous quaternary structures with diverse functions, but also inherently unstable ex vivo. Naturally occurring autoantibodies targeting these aggregates are suggested as potential biomarkers in the early stages of PD, offering potential assistance in its diagnosis. Chapter 1 describes a multiplexed, easy-to-operate immunoassay that is generally applicable to quantify the levels of amyloid proteins and their binding partners, named Oxaziridine-Assisted Solid-phase Immunosorbent (OASIS) assay. The hydrophilic PEG scaffold which this assay was built upon can inhibit aggregate nucleation, potentially reducing assay variance which was a “pain-point” in the field. We validated the novel OASIS assay platform with patient-derived samples where levels of naturally occurring antibodies against αS monomer and oligomers were measured in PD and healthy donors. The OASIS assay is anticipated to be highly valuable for research endeavors focused on comprehending the mechanisms of protein misfolding, its associated pathology, and the symptoms observed in Parkinson's Disease (PD) and other neurodegenerative disorders. There is increasing evidence showing that αS oligomers confer toxicity through a variety of mechanisms that may explain heterogeneity in patient symptomology. Studies to-date have focused on high molecular weight αS oligomers because methods to stabilize and isolate low-molecular weight αS oligomers have not been widely accessible. We hypothesize that mechanistic heterogeneity is due to the action of a single predominant αS oligomer structural isoform. In Chapter 2, a bio-orthogonal photo-oxidative coupling method (PICUP) was used to covalently trap low molecular weight (“SMALL”) αS oligomers in their native conformations. Several “SMALL” αS oligomeric species were purified using tandem size-exclusion chromatography. We next investigated the structural and functional features of αS oligomers with biophysical characterization including circular dichroism and transmission electron microscopy, seeding propensity with ThT assay, and their cytotoxicity with SH-SY5Y cell line. Tuberculosis is the second leading infectious killer and ranks the 13th on leading cause of death worldwide. The emergence of new strains that are resistant to existing antibiotics underscores the urgent need for the discovery and development of novel antibacterial treatments. Chapter 3 introduced the development of high throughput screening assay for the assistance of inhibitor discovery of FadD32, an essential and vulnerable target in Mycobacterium tuberculosis survival catalyzing biosynthesis of mycolic acids. This fluorescence polarization-based assay platform is simple, robust, and compatible with automation, making it suitable for high-throughput screening. By facilitating the discovery of antimycobacterial agents, this platform has the potential to accelerate efforts in overcoming drug resistance and developing effective treatments for tuberculosis. Biotin is an essential cofactor in Mtb survival, making enzymes involved in its biosynthesis pathway valuable targets in new antibiotics discovery. Biotin synthase (BioB) is a radical S-adenosylmethionine (SAM) enzyme, catalyzing the last step synthesis of biotin from desthiobiotin (DTB). Chapter 4 demonstrated that unlike BioB in E.coli, activation of the function of BioB in Mtb (BioB.tb) requires a small (8.6 kD) protein, BsaP, to be active. That BsaP is required for growth without extrabacterial biotin was predicted by transposon sequencing (TnSeq) and confirmed by deleting bsaP in Mtb and Mycobacterium smegmatis. Chemical genetic complementation mapped the function of BsaP to the conversion of DTB into biotin. The reliance of BioB.tb on BsaP was also confirmed biochemically with in vitro assays. Homologs of BsaP occur in many actinobacteria and our findings open new opportunities to seek BioB inhibitors to treat infections with Mtb and other pathogens.