Publications by Year: 2010

Reactive molecules have diverse effects on cells and contribute to several pathological conditions. Cells have evolved complex protective systems to neutralize these molecules and restore redox homeostasis. Previously, we showed that association of nuclear factor (NF)-erythroid-derived 2 (E2)-related factor 2 (NRF2) with the nuclear matrix protein NRP/B was essential for the transcriptional activity of NRF2 target genes in tumor cells. The present study demonstrates the molecular mechanism by which NRP/B, via NRF2, modulates the transcriptional activity of antioxidant response element (ARE)-driven genes. NRP/B is localized in the nucleus of primary brain tissue and human neuroblastoma (SH-SY5Y) cells. Treatment with hydrogen peroxide (H(2)O(2)) enhances the nuclear colocalization of NRF2 and NRP/B and induces heme oxygenase 1 (HO1). Treatment of NRP/B or NRF2 knockdowns with H(2)O(2) induced apoptosis. Co-expression of NRF2 with members of the Kelch protein family, NRP/B, MAYVEN, or MAYVEN-related protein 2 (MRP2), revealed that the NRF2-NRP/B complex is important for the transcriptional activity of ARE-driven genes HO1 and NAD(P)H:quinine oxidoreductase 1 (NQO1). NRP/B interaction with Nrf2 was mapped to NRF2 ECH homology 4 (Neh4)/Neh5 regions of NRF2. NRP/B mutations that resulted in low binding affinity to NRF2 were unable to activate NRF2-modulated transcriptional activity of the ARE-driven genes, HO1 and NQO1. Thus, the interaction of NRP/B with the Neh4/Neh5 domains of NRF2 is indispensable for activation of NRF2-mediated ARE-driven antioxidant and detoxifying genes that confer cellular defense against oxidative stress-induced damage.

The network organization of type IV collagen consisting of α3, α4, and α5 chains in the glomerular basement membrane (GBM) is speculated to involve interactions of the triple helical and NC1 domain of individual α-chains, but in vivo evidence is lacking. To specifically address the contribution of the NC1 domain in the GBM collagen network organization, we generated a mouse with specific loss of α3NC1 domain while keeping the triple helical α3 chain intact by connecting it to the human α5NC1 domain. The absence of α3NC1 domain leads to the complete loss of the α4 chain. The α3 collagenous domain is incapable of incorporating the α5 chain, resulting in the impaired organization of the α3α4α5 chain-containing network. Although the α5 chain can assemble with the α1, α2, and α6 chains, such assembly is incapable of functionally replacing the α3α4α5 protomer. This novel approach to explore the assembly type IV collagen in vivo offers novel insights in the specific role of the NC1 domain in the assembly and function of GBM during health and disease.

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys(14)-Xaa(4)-Asn(19)-Tyr-Gly-Phe-Phe-Leu(24)), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI ((503)VLQEAKL(509)). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K(d)) of the K14A and F22A mutants were 3.2 and 4.0 μM, respectively, similar to 2.6 μM measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI ((505)QEAKL(509)) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10-20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.