Thesis advisor: Jianmin Gao / Thesis advisor: Eranthie Weerapana / Cysteine residues are known to perform an array of functional roles in proteins, including nucleophilic and redox catalysis, regulation, metal binding, and structural stabilization, on proteins across diverse functional classes. These functional cysteine residues often display hyperreactivity, and electrophilic chemical probes can be utilized to modify reactive cysteines and modulate their protein functions. A particular focus was placed on three peptide-based cysteine-reactive chemical probes (NJP2, NJP14. and NJP15) and their particular biological applications. NJP2 was discovered to be an apoptotic cell-selective inhibitor of glutathione S-transferase omega 1 and shows additional utility as an imaging agent of apoptosis. NJP14 aided in the development of a chemical-proteomic platform to detect Zn2+-cysteine complexes. This platform identified both known and unknown Zn2+-cysteine complexes across diverse protein classes and should serve as a valuable complement to existing methods to characterize functional Zn2+-cysteine complexes. Finally, NJP15 was part of a panel of site-selective cysteine-reactive inhibitors of protein disulfide isomerase A1 (PDIA1). These inhibitors show promise in clarifying the unique and redundant properties of PDIA1's dual active-sites, as well as interrogating the protein's role in cancer. Together, these case studies illustrate the potential of cysteine-reactive chemical probes to modulate protein activities, interrogate biological systems, and aid in the development of powerful therapeutic drugs. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Alzheimer’s disease (AD), the most common form of dementia is associated with fibril formation of amyloid-ß peptides (Aß). Aß, proteolytically derived from Aß precursor protein (AßPP), is the major component of amyloid plaques in AD brains. Familial British and Danish dementias (FBD and FDD) share pathological and clinical characteristics with AD, and the underlying mechanisms are associated with amyloid formation of mutant peptides released from the Bri2 protein. Bri2 interacts with AßPP and its BRICHOS domain has been shown to delay Aß40 and Aß42 fibril formation and toxicity in vitro and in vivo. This makes Bri2 BRICHOS a promising anti-amyloid chaperone and a potential treatment strategy for AD. Furthermore, Bri2 BRICHOS possesses a general chaperone activity as it suppresses non-fibrillar aggregation of destabilized citrate synthase (CS). Recent findings show that Bri2 BRICHOS produced in E.coli can form different molecular weight assemblies, ranging from monomers to dimers and poly-disperse oligomers. The oligomers inhibit CS aggregation, whereas the monomers and dimers are more efficient against Aß42 fibrillation and neurotoxicity, respectively. The work in this thesis shows that similar Bri2 BRICHOS quaternary structures are formed in eukaryotic cells as in E.coli. Larger BRICHOS oligomers were found in cell media, derived from proteolytically processed endogenous Bri2 in SH-SY5Y cells, as well as in human embryonic kidney (HEK293) cells transfected with a Bri2 BRICHOS construct. Recombinant human Bri2 BRICHOS mutants with one or none of the two strictly conserved cysteine residues were studied. All mutant monomers become proteolytically degraded during purification, but form stable oligomers. Single Cys to Ser mutants form stable disulfide-dependent dimers that differ in ability to prevent Aß42 fibrillation, the most stable mutant (C164S) being even more efficient than the wildtype Bri2 BRICHOS dimer. This result suggests that intra or intermolecular disulfide(s) and oligomerization affect Bri2 BRICHOS stability and activity towards Aß42 fibril formation.