No abstract

(1991) Vision Res. 31, 17-31] as recognizing a short peptide sequence at the N terminus, failed to bind to rhodopsin when the latter was solubilized in dodecylmaltoside (DM). Of the detergents tested thus far, DM affords maximum stability to rhodopsin. Solubilization of rhodopsin in cholate allowed binding of the antibody, but the binding caused destabilization as evidenced by the accelerated loss of absorbance at 500 nm. The result provides support for the earlier conclusion that the N-terminal segment is an integral part of a tertiary structure in the intradiscal domain of native rhodopsin coupled to a tertiary structure in the transmembrane domain. Additional comparative studies on the stability of rhodopsin in different detergents were carried out after direct solubilization from rod outer segments and after extensive treatments to remove the endogenous phospholipids. Purification of rhodopsin in DM resulted in essentially quantitative removal of endogenous phospholipids. When rhodopsin thus purified was treated with the above antibody in DM and in cholate, enhanced destabilization (5-fold) was observed in the latter detergent.11-cis retinal ͉ transmembrane domain ͉ retinitis pigmentosa ͉ cholate ͉ dodecylmaltoside S tructure function studies on rhodopsin require purification of mutants as expressed in mammalian cell lines. The antirhodopsin mAb rho1D4 with the C-terminal octapeptide sequence as the epitope ( Fig. 1; ref. 1) has been useful in single-step purification of all the mutants in which the epitope has been left intact. Many of our current studies of rhodopsin require amino acid replacements at the extreme C terminus or even truncation of rhodopsin with removal of parts of the C-terminal segment (Fig. 1). For purification of such mutants, a complementary approach with an antibody recognizing a peptide sequence at the N terminus is desirable. A mAb (B6-30N) recognizing the N-terminal sequence G 3 -S 14 (Fig. 1) has indeed been characterized by Hargrave and coworkers (2), and the antibody has proved useful in identification of rhodopsin fragments containing the N terminus (3, 4). When, in the present study, rhodopsin solubilized in dodecylmaltoside (DM) was tested, the antibody failed to bind. Solubilization of rhodopsin in deoxycholate allowed binding of the antibody, but the binding resulted in destabilization of rhodopsin. This result provided support for the previous important conclusion that a tertiary structure is present in the intradiscal domain of which the N-terminal segment forms an integral part. Additional comparative studies have been carried out on the stability of rhodopsin in different detergents; (i) after direct solubilization of rhodopsin from rod outer segments (ROS) and (ii) after extensive treatments to remove the endogenous phospholipids (PLs). Immunoaffinity purification of rhodopsin in DM resulted in practically quantitative removal of the endogenous PLs. The stability of purified rhodopsin in DM and in cholate was compared in the presence and absence of the B6-30N antibody. ...

The neuronal nuclear matrix protein, NRP/B, contains a BTB domain and kelch repeats and is expressed in primary neurons but not in primary glial cells. To examine the function of NRP/B in neurons, we analyzed the structure/function of the NRP/B-BTB domain and its role in neurite outgrowth. Based on three-dimensional modeling of NRP/B, we generated an NRP/B-BTB mutant containing three mutations in the conserved amino acids D47A, H60A and R61D that was termed BTB mutant A. BTB mutant A significantly reduced the dimerization of NRP/B compared to wild-type NRP/B. The NRP/B-BTB domain was required for nuclear localization and mediated the association of NRP/B with p110RB through the TR subdomain within the B pocket of p110RB. Overexpression of wild-type NRP/B and NRP/B-BTB domain significantly induced neurite outgrowth in PC12 cells and enhanced the G0-G1 cell population by ∼23% compared to the control cells, whereas NRP/B-BTB mutant A reduced neurite outgrowth by 70-80%, and inhibited NRP/B-p110RB association. Single cell microinjection of NRP/B-specific antibodies also blocked the neurite outgrowth of PC12 cells upon NGF stimulation. Interference of NRP/B expression by small interfering RNA (NRP/B-siRNA) inhibited neurite outgrowth and suppressed the NGF-induced outgrowth of neurites in PC12 cells. Additionally, p110RB phosphorylation at serine residue 795 was significantly reduced in PC12 cells treated with NRP/B siRNA compared to those treated with control GFP-siRNA, indicating that p110RB is a downstream target of NRP/B. Thus, the BTB domain of NRP/B regulates neurite outgrowth through its interaction with the TR subdomain within the B pocket of p110RB, and the conserved amino acids D47A, H60A and R61D within this domain of NRP/B are crucial residues for neurite extension in neuronal cells. These findings support a role for the BTB-domain of NRP/B as an important regulator of neuronal differentiation.

Structure-function studies of rhodopsin kinase (RK; EC 2.7.1.125) require a variety of mutants. Therefore, there is need for a suitable system for the expression of RK mutant genes. Here we report on a study of expression of the RK gene in baculovirus-infected Sf21 cells and characterization of the enzyme produced as purified to near homogeneity. Particular attention has been paid to the posttranslational modifications, autophosphorylation and isoprenylation, found in the native bovine RK. The protein produced has been purified using, successively, heparin-Sepharose, Mono Q, and Mono S FPLC (fast protein liquid chromatography) and was obtained in amounts of about 2 mg from 1 liter of cell culture. The enzyme from the last step of purification was obtained in two main fractions that differ in the level of phosphorylation. The protein peak eluted first carries two phosphate groups per protein, whereas the second protein peak is monophosphorylated. Further, while both peaks are isoprenylated, the isoprenyl groups consist of mixtures of C 5 , C 10 , C 15 , and C 20 isoprenyl moieties. From these results, we conclude that the above expression system is suitable for some but not all aspects of structure-function studies.

Background:NRP/B, a family member of the BTB/Kelch repeat proteins, is implicated in neuronal and cancer development, as well as the regulation of oxidative stress responses in breast and brain cancer. Our previous studies indicate that the NRP/B-BTB/POZ domain is involved in the dimerization of NRP/B and in a complex formation with the tumor suppressor, retinoblastoma protein. Although much evidence supports the potential role of NRP/B as a tumor suppressor, the molecular mechanisms of NRP/B action on E2F transcription factors have not been elucidated.Methods:Three-dimensional modeling of NRP/B was used to generate point mutations in the BTB/Kelch domains. Tet-on inducible NRP/B expression was established. The NRP/B deficient breast cancer cell line, MDA-MB-231, was generated using lentiviral shNRP/B to evaluate the effect of NRP/B on cell proliferation, invasion and migration. Immunoprecipitation was performed to verify the interaction of NRP/B with E2F and histone deacetylase (HDAC-1), and the expression level of NRP/B protein was analyzed by Western blot analysis. Changes in cell cycle were determined by flow cytometry. Transcriptional activities of E2F transcription factors were measured by chloramphenicol acetyltransferase (CAT) activity.Results:Ectopic overexpression of NRP/B demonstrated that the NRP/B-BTB/POZ domain plays a critical role in E2F-mediated transcriptional activity. Point mutations within the BTB/POZ domain restored E2-promoter activity inhibited by NRP/B. Loss of NRP/B enhanced the proliferation and migration of breast cancer cells. Endogenous NRP/B interacted with E2F and HDAC1. Treatement with an HDAC inhibitor, trichostatin A (TSA), abolished the NRP/B-mediated suppression of E2-promoter activity. Gain or loss of NRP/B in HeLa cells confirmed the transcriptional repressive capability of NRP/B on the E2F target genes, Cyclin E and HsORC (Homo sapiens Origin Recognition Complex).Conclusions:The present study shows that NRP/B acts as a transcriptional repressor by interacting with the co-repressors, HDAC1, providing new insight into the molecular mechanisms of NRP/B on tumor suppression.