Strong impact of TGF-β1 gene polymorphisms on breast cancer risk in Indian women: a case-control and population-based study

Introduction: TGF-β1 is a multi-functional cytokine that plays an important role in breast carcinogenesis. Critical role of TGF-β1 signaling in breast cancer progression is well documented. Some TGF-β1 polymorphisms influence its expression; however, their impact on breast cancer risk is not clear. Methods: We analyzed 1222 samples in a candidate gene-based genetic association study on two distantly located and ethnically divergent case-control groups of Indian women, followed by a population-based genetic epidemiology study analyzing these polymorphisms in other Indian populations. The c.29C>T (Pro10Leu, rs1982073 or rs1800470) and c.74G>C (Arg25Pro, rs1800471) polymorphisms in the TGF-β1 gene were analyzed using direct DNA sequencing, and peripheral level of TGF-β1 were measured by ELISA. Results: c.29C>T substitution increased breast cancer risk, irrespective of ethnicity and menopausal status. On the other hand, c.74G>C substitution reduced breast cancer risk significantly in the north Indian group (p  =  0.0005) and only in the pre-menopausal women. The protective effect of c.74G>C polymorphism may be ethnicity-specific, as no association was seen in south Indian group. The polymorphic status of c.29C>T was comparable among Indo-Europeans, Dravidians and Tibeto-Burmans. Interestingly, we found that Tibeto-Burmans lack polymorphism at c.74G>C locus as true for the Chinese populations. However, the Brahmins of Nepal (Indo-Europeans) showed polymorphism in 2.08% of alleles. Mean TGF-β1 was significantly elevated in patients in comparison to controls (p<0.001). Conclusion: c.29C>T and c.74G>C polymorphisms in the TGF-β1 gene significantly affect breast cancer risk, which correlates with elevated TGF-β1 level in the patients. The c.29C>T locus is polymorphic across ethnically different populations, but c.74G>C locus is monomorphic in Tibeto-Burmans and polymorphic in other Indian populations

Free radical stress-induced Parkinsonian Lewy-like aggregation prevented through polyphenolic phytochemical analog intervention: Implications for subcellular trafficking and neurodegenerative disorders

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 ratedetermining 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 ratedetermining 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 halfprocess 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 rotenoneinduced 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

PDI, reactive oxygen species stress and polyphenolic phytochemicals: Implications for neurodegenerative diseases

PDI, protein disulfide isomerase, is one of the most versatile proteins and highly expressed in mammalian cells because there is a vast number of proteins that must undergo processing before secretion to their final destinations. PDI has several functions: oxidation of nascent proteins and isomerization of existing disulfide bonds. It also possesses chaperone activity and participates in protein degradation. Because of its structure, PDI can exist in a reduced or oxidized form. In mammalian cells it is mostly reduced due to the high demand of disulfide bond shuffling of secreted proteins within the ER, PDI a, a’, and b’ posses the ability to facilitate substrate folding. In our experiments we are using rat PDI and according to the sequence there are cysteine residues located in a and a’ domains. These cysteine residues may contribute to the catalytic activity of this domain and will be analyzed in future studies. Misfolded proteins, and the associated endoplasmic reticulum (ER) stress, are emerging as hallmarks of age- and neurodegeneration-related disorders such as Huntington’s disease (HD), Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis. Recent and compelling evidence has linked nitrosative stress and the ER-resident oxidoreductase, protein disulfide isomerase (PDI), to the pathogenesis of PD and AD. Overexpression of PDI has been found to reduce the formation of polyubiquitinated proteins, making the oxidoreductase an important target for therapeutic intervention in PD, AD and other age- and neurodegeneration-related disorders. We demonstrate the NO-scavenging ability of the biphenolic natural products curcumin and masoprocol and the concomitant prevention of S-nitrosylation of PDI by a model NO-donor. Furthermore, both ethnopharmaceuticals accelerate protein fold acquisition in their neat and nitrated forms, making them attractive candidates for prevention of age- and neurodegeneration-related diseases

Reshuffling Activity of Protein Disulfide Isomerase Reduces Refolding Yield in the Structure-Forming Step of the Oxidative Protein Folding Reaction

We have determined the impact of the oxidoreductase chaperone protein disulfide isomerase (PDI) on the critical structure-forming step during the oxidative maturation of model disulfide-bond-containing proteins. This is achieved by using a novel tool to trap and populate native-disulfide-containing intermediates in unstructured forms that are poised to fold. Our data reveals that PDI inhibits the conformational folding step of oxidative fold maturation and, therefore, has limited overall catalytic efficiency as an oxidoreductase chaperone. Such an anomalous behavior of PDI during a key step in oxidative regeneration may contribute to misfolding in the endoplasmic reticulum, aggregation, and neurodegenerative disease

Real-time imaging of NADPH oxidase activity in living cells using a novel fluorescent protein reporter.

Production of reactive oxygen species (ROS) has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP (roGFP) to the Nox organizer protein, p47(phox), we have developed a redox sensitive protein to specifically assess Nox activity (p47-roGFP). Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47(phox) deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors