Functional proteomic analysis reveals roles for PKCδ in regulation of cell survival and cell death: Implications for cancer pathogenesis and therapy.

Protein Kinase C-δ (PKCδ), regulates a broad group of biological functions and disease processes, including well-defined roles in immune function, cell survival and apoptosis. PKCδ primarily regulates apoptosis in normal tissues and non-transformed cells, and genetic disruption of the PRKCD gene in mice is protective in many diseases and tissue damage models. However pro-survival/pro-proliferative functions have also been described in some transformed cells and in mouse models of cancer. Recent evidence suggests that the contribution of PKCδ to specific cancers may depend in part on the oncogenic context of the tumor, consistent with its paradoxical role in cell survival and cell death. Here we will discuss what is currently known about biological functions of PKCδ and potential paradigms for PKCδ function in cancer. To further understand mechanisms of regulation by PKCδ, and to gain insight into the plasticity of PKCδ signaling, we have used functional proteomics to identify pathways that are dependent on PKCδ. Understanding how these distinct functions of PKCδ are regulated will be critical for the logical design of therapeutics to target this pathway.

Promoter Haplotypes of the ABCB1 Gene Encoding the P-Glycoprotein Differentially Affect Its Promoter Activity by Altering Transcription Factor Binding.

Promoter single nucleotide polymorphisms (SNPs) of the ABCB1 gene, encoding the placental efflux transporter P-glycoprotein, can alter its expression and affect fetal exposure to therapeutics and environmental xenobiotics. SNPs are not arrayed as independent variants but as combinations forming defined haplotypes. Recently, we defined the haplotypes encompassing ABCB1 promoter SNPs and found that ABCB1 haplotypes differentially affect its promoter activity. The mechanism(s) by which ABCB1 haplotypes alter its promoter activity are not known. We hypothesize that the haplotype-dependent differences in ABCB1 promoter activity are due to haplotype-specific alterations in transcription factor (TF) binding. To test our hypothesis, we used a TF binding profile array and determined whether differences in TF binding exist across different ABCB1 haplotypes. TFs showing significant haplotype binding differences were mechanistically evaluated using small interfering RNA (siRNA) in cultured human placental cells. Our data indicate significant haplotype-dependent differences in TF binding. Our siRNA studies showed that the regulatory effects of TFs on promoter activity are also haplotype dependent. Our data provide a mechanistic explanation for the differential effects of ABCB1 haplotypes on its promoter activity and underscore the importance of evaluating genetic variants in the context of haplotypes rather than individual SNPs when investigating their effects on gene/protein expression and disease risk.

Bisphenol A (BPA) and bisphenol S (BPS) alter the promoter activity of the ABCB1 gene encoding P-glycoprotein in the human placenta in a haplotype-dependent manner.

Exposure to bisphenols (BPA and BPS) during pregnancy can significantly affect fetal development and increase risk of adverse health consequences, however the underlying mechanisms are not fully elucidated. In human placenta, the efflux transporter P-glycoprotein (P-gp), encoded by the ABCB1 gene, extrudes its substrates from the trophoblasts back into the maternal circulation. Alterations in levels of placental P-gp could therefore significantly affect fetal exposure to xenobiotics that are P-gp substrates. The ABCB1 promoter contains many single nucleotide polymorphisms (SNPs). In the genome, SNPs are not arrayed as independent variants but as combinations forming defined haplotypes. Recently, we determined the haplotype sequences encompassing the ABCB1 promoter SNPs and found that promoter haplotypes differentially affect ABCB1 promoter activity. Here we investigate the effect of BPA and BPS on ABCB1 promoter activity by testing the hypothesis that BPA and BPS exposure affect ABCB1 promoter activity in a haplotype-dependent manner. Our data indicate that acute exposure to 50 nM BPA induced a significant haplotype-dependent increase in ABCB1 promoter activity (P < .05). However, acute exposure to 0.5 nM BPS induced a significant decrease (P < .05) in promoter activity that was haplotype-dependent. Chronic exposure to BPA and BPS individually (5 nM and 0.3 nM, respectively) or as a mixture (5 nM BPA:1.5 nM BPS) induced significant haplotype-dependent increases (P < .01) in ABCB1 promoter activity. Our data indicate that BPA and BPS significantly alter ABCB1 promoter activity in a haplotype- and exposure type- dependent manners. Such alteration could significantly impact placental P-gp levels and alter fetal exposure to many therapeutic and environmental xenobiotics.

Context-dependent autoprocessing of human immunodeficiency virus type 1 protease precursors.

HIV-1 protease autoprocessing is responsible for liberation of free mature protease (PR) from the Gag-Pol polyprotein precursor. A cell-based model system was previously developed to examine the autoprocessing mechanism of fusion precursors carrying the p6*-PR miniprecursor sandwiched between various proteins or epitopes. We here report that precursor autoprocessing is context-dependent as its activity and outcomes can be modulated by sequences upstream of p6*-PR. This was exemplified by the 26aa maltose binding protein (MBP) signal peptide (SigP) when placed at the N-terminus of a fusion precursor. The mature PRs released from SigP-carrying precursors are resistant to self-degradation whereas those released from SigP-lacking fusion precursors are prone to self-degradation. A H69D mutation in PR abolished autoprocessing of SigP-containing fusion precursors whereas it only partially suppressed autoprocessing of fusion precursors lacking SigP. An autoprocessing deficient GFP fusion precursor with SigP exhibited a subcellular distribution pattern distinct from the one without it in transfected HeLa cells. Furthermore, a SigP fusion precursor carrying a substitution at the P1 position released the mature PR and PR-containing fragments that were different from those released from the precursor carrying the same mutation but lacking SigP. We also examined autoprocessing outcomes in viral particles produced by a NL4-3 derived proviral construct and demonstrated the existence of several PR-containing fragments along with the mature PR. Some of these resembled the SigP precursor autoprocessing outcomes. This finding of context-dependent modulation reveals the complexity of precursor autoprocessing regulation that most likely accompanies sequence variation imposed by the evolution of the upstream Gag moiety.

Differential effect of ABCB1 haplotypes on promoter activity.

OBJECTIVE: Promoter single-nucleotide polymorphisms (SNPs) of the ABCB1 gene, encoding the placental efflux transporter P-glycoprotein, can affect its expression and alter xenobiotic transfer from the maternal to the fetal circulation. Because SNPs are arranged in specific combinations as defined haplotypes, the aims of this study were to: (i) determine the placental haplotype structure of the ABCB1 promoter and (ii) determine the differential effect of these haplotypes on placental ABCB1 promoter activity. MATERIALS AND METHODS: DNA samples from 100 healthy placentas were PCR-amplified and sequenced to identify existing SNPs in the proximal ABCB1 promoter. The haplotype structure encompassing these SNPs was inferred by PHASE analysis. Luciferase reporter constructs representing these haplotypes were generated and transfected into human placental 3A cells and their effect on ABCB1 promoter activity was determined using a dual-luciferase assay. RESULTS: We identified 12 ABCB1 promoter SNPs. These SNPs were predicted by PHASE to segregate into 28 haplotypes with frequencies ranging between 0.019 and 0.88. We found 12 of these haplotypes in our population in addition to two haplotypes not predicted by PHASE. We also generated two haplotypes to determine individual SNP effects for a total of 16 studied. Compared with the ancestral haplotype, three haplotypes significantly up-regulated (107-266% increase; P<0.05), one significantly down-regulated (95.4% decrease; P<0.01), and 12 had no statistically significant effect on ABCB1 promoter activity. DISCUSSION AND CONCLUSION: Our data show that the effect of SNPs on promoter activity depends on their presence in a specific haplotype. This indicates that haplotypes, rather than individual SNPs, could play a significant role in regulating placental P-glycoprotein expression and affect placental transfer and fetal exposure to xenobiotics.

MGMT DNA repair gene promoter/enhancer haplotypes alter transcription factor binding and gene expression.

BACKGROUND: The O6-methylguanine-DNA methyltransferase (MGMT) protein removes O6-alkyl-guanine adducts from DNA. MGMT expression can thus alter the sensitivity of cells and tissues to environmental and chemotherapeutic alkylating agents. Previously, we defined the haplotype structure encompassing single nucleotide polymorphisms (SNPs) in the MGMT promoter/enhancer (P/E) region and found that haplotypes, rather than individual SNPs, alter MGMT promoter activity. The exact mechanism(s) by which these haplotypes exert their effect on MGMT promoter activity is currently unknown, but we noted that many of the SNPs comprising the MGMT P/E haplotypes are located within or in close proximity to putative transcription factor binding sites. Thus, these haplotypes could potentially affect transcription factor binding and, subsequently, alter MGMT promoter activity. METHODS: In this study, we test the hypothesis that MGMT P/E haplotypes affect MGMT promoter activity by altering transcription factor (TF) binding to the P/E region. We used a promoter binding TF profiling array and a reporter assay to evaluate the effect of different P/E haplotypes on TF binding and MGMT expression, respectively. RESULTS: Our data revealed a significant difference in TF binding profiles between the different haplotypes evaluated. We identified TFs that consistently showed significant haplotype-dependent binding alterations (p ≤ 0.01) and revealed their role in regulating MGMT expression using siRNAs and a dual-luciferase reporter assay system. CONCLUSIONS: The data generated support our hypothesis that promoter haplotypes alter the binding of TFs to the MGMT P/E and, subsequently, affect their regulatory function on MGMT promoter activity and expression level.

Aligned electrospun ZnO nanofibers for simple and sensitive ultraviolet nanosensors.

The current of uni-axially aligned electrospun ZnO nanofibers is modulated reversibly under UV irradiation, with the sensitivity of the UV nanosensors depending on the surface coating of the nanofibers, due to the effect on the photo-generated current.