Search

To accomplish key physiological processes ranging from drug metabolism to steroidogenesis, human microsomal cytochrome P450 enzymes require the sequential input of two electrons delivered by the FMN domain of NADPH-cytochrome P450 reductase. Although some human microsomal P450 enzymes can instead accept the second electron from cytochrome b5, for human steroidogenic CYP17A1, the cytochrome P450 reductase FMN domain delivers both electrons, and b5 is an allosteric modulator. The structural basis of these key but poorly understood protein interactions was probed by solution NMR using the catalytically competent soluble domains of each protein. Formation of the CYP17A1.FMN domain complex induced differential line broadening of the NMR signal for each protein. Alterations in the exchange dynamics generally occurred for residues near the surface of the flavin mononucleotide, including 87-90 (loop 1), and for key CYP17A1 active site residues. These interactions were modulated by the identity of the substrate in the buried CYP17A1 active site and by b5. The FMN domain outcompetes b5 for binding to CYP17A1 in the threecomponent system. These results and comparison with previous NMR studies of the CYP17A1.b5 complex suggest a model of CYP17A1 enzyme regulation. [ABSTRACT FROM AUTHOR]

R3434 --> 492.8 --> CYP121 is an essential cytochrome P450 enzyme (CYP) from Mycobacterium tuberculosis.CYP121 mediates an atypical phenol‐coupling reaction that converts cyclodityrosine (cYY) into mycocylosin. Our lab has previously employed a 19F nuclear magnetic resonance (NMR) label, 3‐bromo‐1,1,1‐trifluoroacetone (BTFA), to study dynamics of the FG‐loop, a region connecting the F and G‐helices. In other CYPs, the FG‐loop region has been implicated in active site access and substrate recognition. The 19F‐NMR spectrum for the FG‐loop in CYP121 is heterogeneous, suggesting that in solution there are multiple conformations. Upon titration of cYY, the signal converges to a single peak representing a bound state. Interestingly, this labeling site exists approximately 20 angstroms away from the active site heme, where ligand binding is typically measured in CYPs by UV‐visible spectroscopy. Crystal structures show an invariable protein structure with ligands clustered near the FG‐helices, away from the heme. Here we investigate the specificity of this interaction site by expanding our use of 19F labeling. Supplementing cells with 5‐fluoroindole (5‐FI) during recombinant protein expression, 19F was incorporated into each tryptophan residue of CYP121. This method of labeling allowed for four tryptophan residues to be monitored, which span the entire protein structure. Notably, two residues, Trp‐182 and Trp‐380, are located adjacent to the CYP121 homodimer interface. The 19F‐NMR spectrum contains three distinct signals, two of which are equal in intensity, and a third which has been determined to be two overlapping resonances. Addition of cYY causes a shift of equilibrium in the spectra, with the overlapped resonances resolving and resulting in four peaks of roughly equal area and intensity. These results suggest that only the active site residue Trp‐182 is affected by the addition of cYY. Using this labeling scheme, we also tested alanine substitutions of Trp‐182 and another active site aromatic residue, Phe‐168. 19F‐NMR spectra from these mutants indicate a general perturbation of the structure, with only one Trp residue left unaffected. Since Trp‐182 extends into a hydrophobic pocket of CYP121 (suggesting a structural role), additional substitutions were made using tyrosine and leucine side chains. The conservative W182Y mutant yielded similar disorder as seen when replaced with alanine, while W182L results a partially restored spectrum. Interestingly, both mutants bind substrate in a similar manner as wild‐type when measured by UV‐visible spectroscopy, although with weakened affinity. These findings indicate that while Phe‐168 and Trp‐182 are involved in substrate interaction, they also play a role in the fold of the protein, with mutagenesis causing long range changes in structure. [ABSTRACT FROM AUTHOR]

Hantaviruses are distributed worldwide and can cause a hemorrhagic fever or a cardiopulmonary syndrome in humans. Mature virions consist of RNA genome, nucleocapsid protein, RNA polymerase, and two transmembrane glycoproteins, Gi and G2. The ectodomain of Gi is surface-exposed; however, it has a 142-residue C-terminal cytoplasmic tail that plays important roles in viral assembly and host-pathogen interaction. Here we show by NMR, circular dichroism spectroscopy, and mutagenesis that a highly conserved cysteine/histidine-rich region in the Gi tail of hantaviruses forms two CCHC-type classical zinc fingers. Unlike classical zinc fingers, however, the two Gi zinc fingers are intimately joined together, forming a compact domain with a unique fold. We discuss the implication of the hantaviral G1 zinc fingers in viral assembly and host-pathogen interaction. [ABSTRACT FROM AUTHOR]

Local diminution of the neural growth factor (NGF) contributes to the apparition of diabetic neuropathy. All-trans retinoic acid (RA) increases the expression of neural growth factor and its receptor participating in translation pathways. This study evaluates RA as a treatment of diabetic neuropathy: 120 mice were assigned randomly to 4 groups. Group A (n = 30) was taken as control; group B (n = 30) received 50 mg/kg intraperitoneal streptozotocin (STZ); group C (n = 30) received STZ, and after diabetic neuropathy developed, they were treated with subcutaneous RA 20 mg/kg daily during 60 days; and group D (n = 30) only received RA. Plasma glucose, thermosensitive tests, serum, and the nerve contents of NGF were measured in all animals. Evaluation by electron microscopy was performed in search of morphologic changes secondary to neuropathy and nerve regeneration. Diabetic mice had an increased threshold to pain. Treatment with RA in diabetic mice reverted changes in sensitivity as compared with diabetic mice that received placebo (P < 0.001). No differences in pain threshold among controls, RA, and diabetes mellitus (DM) + RA groups were found. Glucose levels were not affected by the treatment with RA. NGF diminished significantly in the sciatic nerve in diabetic mice as compared with controls and with the RA group. Animals with DM + RA had a significant increase of NGF in nerves as compared with the other groups. RA also regressed the ultrastructural changes induced by diabetes that showed increased neural regeneration. RA can revert functional and ultrastructural changes and induce neural regeneration after the establishment of diabetic neuropathy, possibly because of the increased of NGF concentrations in nerve terminals. [ABSTRACT FROM AUTHOR]

The current study explored the potential impact of a training program on graduate counseling students' competency to serve lesbian, gay, and bisexual (LGB) clients. In this pilot study, the Sexual Orientation Counselor Competency Scale (SOCCS; Bidell, 2005) was administered to students enrolled in a counselor education program. Results indicated that the training program had a positive impact upon the competency areas of knowledge and skills as measured by the SOCCS. The benefits of incorporating an LGB counselor competency component in training modules to serve the LGB community are discussed. Ways for counselor educators and supervisors to enhance their own LGB competencies and help their student's ability to meet the needs of LGB clients are also presented. [ABSTRACT FROM AUTHOR]

The authors discuss the importance of social advocacy and empowerment models in working with lesbian clients. They also explore the multiple contexts that impact lesbian clients and their families. A case illustration involving a lesbian client facing issues of internalized homophobia, heterosexism, sexism, and legal issues is presented. Suggestions for applying a feminist and social advocacy approach are offered. [ABSTRACT FROM AUTHOR]

R2327 --> Understanding conformational dynamics is an integral part of understanding function in cytochrome P450 enzymes (CYPs), a ubiquitous class of heme‐coordinated enzymes that are important for xenobiotic and endogenous metabolism. While nuclear magnetic resonance (NMR) is well suited to detect changes in the structures of CYPs and their associated redox partners, use of traditional isotopic labels are difficult to apply to such large enzymes and complexes. In this work, we have employed a thiol‐reactive 19F label, 3‐bromo‐1,1,1‐trifluoracetone (BTFA), to probe various aspects of Class‐I CYP systems in a targeted approach. 19F is well suited for these studies due in part to its enhanced sensitivity over traditional NMR observable nuclei, as well as the fact that it does not naturally occur in proteins, which reduces the overall complexity of the resulting spectra. Here we deploy this strategy to study different aspects of CYP function, including details of redox partner interaction in a mammalian CYP and dynamics of substrate binding in a bacterial CYP. Our lab has previously used traditional 15N NMR to study the interaction between the vitamin D metabolizing enzyme, CYP24A1, and its redox partner, adrenodoxin (Adx). Using labeled Adx, we were able to titrate unlabeled CYP24A1 and monitor changes in the spectra, but were limited by the extent of signal loss attributed to the size of the complex. Substituting 15N labeling with two selectively placed 19F probes on α‐helix 1 and α‐helix 3 of Adx, we were able to probe biologically relevant complexes of these enzymes. Upon addition of CYP24A1 to 19F labeled Adx, we observe differential line broadening on α‐helix 3, consistent with other studies implicating this region in CYP recognition. We also observe changes in the heterogeneity of the signal at α‐helix 3, suggesting that this site undergoes conformational selection upon binding. The same probe was also applied to study the solution structure of CYP121 from Mycobacterium tuberculosis. Crystal structures of CYP121 show ligands clustered to a pocket near the F‐G helices, which may play an important role in substrate recognition and control access to the active site. A probe was attached to the loop connecting these two helices (FG‐loop) and monitored during titration with the substrate, cyclodityrosine (cYY). The 19F NMR spectrum for the FG‐loop is heterogeneous suggesting multiple conformations exist in solution, but converges to a single peak upon cYY addition, representing a bound form. Utilizing an engineered monomer, our data also suggest that the dimer interface confers structural rigidity at this site, and in the dimeric form a population of enzyme exists in the 'bound' conformation prior to ligand introduction. Interestingly, the ratios of CYP121:cYY that are required to saturate the observed NMR response do not match with ratios required to detect binding using other techniques, thereby suggesting the 19F probe on the FG‐loop is monitoring a 'pre‐catalytic' binding event. These findings highlight the utility of employing an underutilized NMR label to understand function in an important class of enzymes. [ABSTRACT FROM AUTHOR]

Cytochromes P450 are versatile enzymes that function in endobiotic and xenobiotic metabolism and undergo meaningful structural changes that relate to their function. However, the way in which conformational changes inform the specific recognition of the substrate is often unknown. Here, we demonstrate the utility of fluorine (19F)-NMR spectroscopy to monitor structural changes in CYP121A1, an essential enzyme from Mycobacterium tuberculosis. CYP121A1 forms functional dimers that catalyze the phenol-coupling reaction of the dipeptide dicyclotyrosine. The thiol-reactive compound 3-bromo-1,1,1-trifluoroacetone was used to label an S171C mutation of the enzyme FG loop, which is located adjacent to the homodimer interface. Substrate titrations and inhibitor-bound 19F-NMR spectra indicate that ligand binding reduces conformational heterogeneity at the FG loop in both the dimer and in an engineered monomer of CYP121A1. However, only the dimer was found to promote a substrate-bound conformation that was preexisting in the substrate-free spectra, thus confirming a role for the dimer interface in dicyclotyrosine recognition. Moreover, 19F-NMR spectra in the presence of substrate analogs indicate the hydrogen-bonding feature of the dipeptide aromatic side chain as a dicyclotyrosine specificity criterion. This study demonstrates the utility of 19F-NMR as applied to a multimeric cytochrome P450, while also revealing mechanistic insights for an essential M. tuberculosis enzyme. [ABSTRACT FROM AUTHOR]