Date of Award

2011-01-01

Degree Name

Doctor of Philosophy

Department

Chemistry

Advisor(s)

Mahesh Narayan

Abstract

Protein disulfide isomerase (PDI), the chief endoplasmic reticulum (ER)-resident oxidoreductase chaperone, is known to catalyze the maturation of disulfide-bond-containing proteins primarily through oxidation-reduction and isomerization functions. The rate-determining step in the oxidative regeneration path of disulfide-bond-containing proteins generally couples chemical thiol-disulfide-exchange reactions to a physical conformational folding reaction. I have determined the impact of PDI and its subdomains on the rate-determining step in ribonuclease A folding and on the physical structure-forming step of select ER-processed proteins including RNase A. This was facilitated through application of a novel chemical tool to exclusively populate native-disulfide-containing intermediates in unstructured forms. The described biochemical inroad permits a deconvoluted study of the physical half-process in the rate-determining step from its chemical counterpart. Analysis of folding kinetics of RNase A and other proteins reveal that the highly evolved oxidoreductase activity of PDI masks its chaperone-like activity, impedes conformational folding of ER-processed proteins, and limits its potential to accelerate the rate-determining step in oxidative regeneration. Implications of the heretofore unknown and anomalous self-limiting behavior of PDI are discussed in the context of oxidative maturation and misfolding in vivo.

Nitrosative stress has recently been demonstrated as a causal in a select sporadic variant of Parkinson's (PD) and Alzheimer's (AD) diseases. Specifically, elevated levels of Nitric Oxide (NO) disrupt the redox activity of protein disulfide isomerase by S-nitroso modification of its redox-active cysteines. This leads to accumulation of misfolded AD- and PD-specific proteins. I have recently demonstrated in vitro that polyphenolic phytochemicals, curcumin and masoprocol, can prevent S-nitroso-PDI formation by scavenging NO*. In this study, using dopaminergic SHSY-5Y cells, I have monitored the aggregation of green-fluorescent protein (GFP)-tagged synphilin-1 (a known constituent of PD Lewy neurites) as a function of rotenone-induced nitrosative stress. Importantly, I demonstrate a marked decrease in synphilin-1 aggregation when the cell line is previously incubated with 3, 5-bis (2-flurobenzylidene) piperidin-4-one (EF24), a curcumin analogue, prior to rotenone insult. Furthermore, my data also reveal that rotenone attenuates PDI expression in the same cell line, a phenomenon that can be mitigated through EF24 intervention. I was also interested to investigate the bioavailability of EF 24 through binding assay with a specific carrier protein, human serum albumin (HSA). With high affinity binding sites, HSA is a major transporter for delivering several endogenous compounds and drugs in vivo. In this dissertation, the binding parameters of EF 24 to HSA have been determined.

Together, these results suggest that polyphenolic phytochemical EF24 can exert neuroprotective effects by ameliorating nitrosative stress-linked damage to PDI and the associated onset of PD in tested models. Essentially, EF24 can serve as a scaffold for the design and development of PD and AD specific prophylactics.

Another aspect of this dissertation was to investigate the role of PDI in cancer. PDI can bind to estrogens as well as interact with its receptor protein (i.e., estrogen receptors (Er) α and β, respectively). It has previously been shown that PDI also acts as an intracellular 17β-estradiol (E2)-binding protein that transports and accumulates E2 in live cells. Intracellular PDI-bound E2 can be released from PDI upon a drop in E2 levels; the released E2 can augment estrogen receptor-mediated transcriptional activity and mitogenic action in cultured cells by modulating the Erβ/Erα ratio. In this dissertation, I observed a significant increase in Erβ/Erα ratio, upon rotenone-induced insult to PDI. Specifically, rotenone-induced insult to PDI leads to the down-regulation of ERα and up-regulation of ERβ proteins, respectively. My data also shoId that the PDI-dependent disruption of the estrogenic status of cells can be restored through intervention by the polyphenolic curcumin analog, diphenyl difluoroketone (EF24), which acts by rescuing PDI from reactive oxygen species-induced damage. My study indicates that EF24 can play a vital role in maintaining estrogenic status in target cells suggesting future applications in select cancers.

Language

en

Provenance

Received from ProQuest

File Size

148 pages

File Format

application/pdf

Rights Holder

Rituraj Pal

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