Laser-induced transient grating analysis of dynamics of interaction between sensory rhodopsin II D75N and the HtrII transducer.

The interaction between sensory rhodopsin II (SRII) and its transducer HtrII was studied by the time-resolved laser-induced transient grating method using the D75N mutant of SRII, which exhibits minimal visible light absorption changes during its photocycle, but mediates normal phototaxis responses. Flash-induced transient absorption spectra of transducer-free D75N and D75N joined to 120 amino-acid residues of the N-terminal part of the SRII transducer protein HtrII (DeltaHtrII) showed only one spectrally distinct K-like intermediate in their photocycles, but the transient grating method resolved four intermediates (K(1)-K(4)) distinct in their volumes. D75N bound to HtrII exhibited one additional slower kinetic species, which persists after complete recovery of the initial state as assessed by absorption changes in the UV-visible region. The kinetics indicate a conformationally changed form of the transducer portion (designated Tr*), which persists after the photoreceptor returns to the unphotolyzed state. The largest conformational change in the DeltaHtrII portion was found to cause a DeltaHtrII-dependent increase in volume rising in 8 micros in the K(4) state and a drastic decrease in the diffusion coefficient (D) of K(4) relatively to those of the unphotolyzed state and Tr*. The magnitude of the decrease in D indicates a large structural change, presumably in the solvent-exposed HAMP domain of DeltaHtrII, where rearrangement of interacting molecules in the solvent would substantially change friction between the protein and the solvent.

NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system.

Type IV secretion (T4S) systems translocate DNA and protein effectors through the double membrane of Gram-negative bacteria. The paradigmatic T4S system in Agrobacterium tumefaciens is assembled from 11 VirB subunits and VirD4. Two subunits, VirB9 and VirB7, form an important stabilizing complex in the outer membrane. We describe here the NMR structure of a complex between the C-terminal domain of the VirB9 homolog TraO (TraO(CT)), bound to VirB7-like TraN from plasmid pKM101. TraO(CT) forms a beta-sandwich around which TraN winds. Structure-based mutations in VirB7 and VirB9 of A. tumefaciens show that the heterodimer interface is conserved. Opposite this interface, the TraO structure shows a protruding three-stranded beta-appendage, and here, we supply evidence that the corresponding region of VirB9 of A. tumefaciens inserts in the membrane and protrudes extracellularly. This complex structure elucidates the molecular basis for the interaction between two essential components of a T4S system.

Interaction between cell division proteins FtsE and FtsZ.

FtsE and FtsX, which are widely conserved homologs of ABC transporters and interact with each other, have important but unknown functions in bacterial cell division. Coimmunoprecipitation of Escherichia coli cell extracts revealed that a functional FLAG-tagged version of FtsE, the putative ATP-binding component, interacts with FtsZ, the bacterial tubulin homolog required to assemble the cytokinetic Z ring and recruit the components of the divisome. This interaction is independent of FtsX, the predicted membrane component of the ABC transporter, which has been shown previously to interact with FtsE. The interaction also occurred independently of FtsA or ZipA, two other E. coli cell division proteins that interact with FtsZ. In addition, FtsZ copurified with FLAG-FtsE. Surprisingly, the conserved C-terminal tail of FtsZ, which interacts with other cell division proteins, such as FtsA and ZipA, was dispensable for interaction with FtsE. In support of a direct interaction with FtsZ, targeting of a green fluorescent protein (GFP)-FtsE fusion to Z rings required FtsZ, but not FtsA. Although GFP-FtsE failed to target Z rings in the absence of ZipA, its localization was restored in the presence of the ftsA* bypass suppressor, indicating that the requirement for ZipA is indirect. Coexpression of FLAG-FtsE and FtsX under certain conditions resulted in efficient formation of minicells, also consistent with an FtsE-FtsZ interaction and with the idea that FtsE and FtsX regulate the activity of the divisome.

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli.

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.

Gene by BMI Interactions Influencing C-Reactive Protein Levels in European-Americans

C-Reactive Protein (CRP) is a biomarker indicating tissue damage, inflammation, and infection. High-sensitivity CRP (hsCRP) is an emerging biomarker often used to estimate an individual’s risk for future coronary heart disease (CHD). hsCRP levels falling below 1.00 mg/l indicate a low risk for developing CHD, levels ranging between 1.00 mg/l and 3.00 mg/l indicate an elevated risk, and levels exceeding 3.00 mg/l indicate high risk. Multiple Genome-Wide Association Studies (GWAS) have identified a number of genetic polymorphisms which influence CRP levels. SNPs implicated in such studies have been found in or near genes of interest including: CRP, APOE, APOC, IL-6, HNF1A, LEPR, and GCKR. A strong positive correlation has also been found to exist between CRP levels and BMI, a known risk factor for CHD and a state of chronic inflammation. We conducted a series of analyses designed to identify loci which interact with BMI to influence CRP levels in a subsample of European-Americans in the ARIC cohort. In a stratified GWA analysis, 15 genetic regions were identified as having significantly (p-value < 2.00*10-3) distinct effects on hsCRP levels between the two obesity strata: lean (18.50 kg/m2 < BMI < 24.99 kg/m2) and obese (BMI ≥ 30.00 kg/m2). A GWA analysis performed on all individuals combined (i.e. not a priori stratified for obesity status) with the inclusion of an additional parameter for BMI by gene interaction, identified 11 regions which interact with BMI to influence hsCRP levels. Two regions containing the genes GJA5 and GJA8 (on chromosome 1) and FBXO11 (on chromosome 2) were identified in both methods of analysis suggesting that these genes possibly interact with BMI to influence hsCRP levels. We speculate that atrial fibrillation (AF), age-related cataracts and the TGF-β pathway may be the biological processes influenced by the interaction of GJA5, GJA8 and FBXO11, respectively, with BMI to cause changes in hsCRP levels. Future studies should focus on the influence of gene x bmi interaction on AF, age-related cataracts and TGF-β.

Dissecting the Interaction between p53 and TRIM24

Dissecting the Interaction of p53 and TRIM24 Aundrietta DeVan Duncan Supervisory Professor, Michelle Barton, Ph.D. p53, the “guardian of the genome”, plays an important role in multiple biological processes including cell cycle, angiogenesis, DNA repair and apoptosis. Because it is mutated in over 50% of cancers, p53 has been widely studied in established cancer cell lines. However, little is known about the function of p53 in a normal cell. We focused on characterizing p53 in normal cells and during differentiation. Our lab recently identified a novel binding partner of p53, Tripartite Motif 24 protein (TRIM24). TRIM24 is a member of the TRIM family of proteins, defined by their conserved RING, B-box, and coiled coil domains. Specifically, TRIM24 is a member of the TIF1 subfamily, which is characterized by PHD and Bromo domains in the C-terminus. Between the Coiled-coil and PHD domain is a linker region, 437 amino acids in length. This linker region houses important functions of TRIM24 including it’s site of interaction with nuclear receptors. TRIM24 is an E3-ubiquitin ligase, recently discovered to negatively regulate p53 by targeting it for degradation. Though it is known that Trim24 and p53 interact, it is not known if the interaction is direct and what effect this interaction has on the function of TRIM24 and p53. My study aims to elucidate the specific interaction domains of p53 and TRIM24. To determine the specific domains of p53 required for interaction with TRIM24, we performed co-immuoprecipitation (Co-IP) with recombinant full-length Flag-tagged TRIM24 protein and various deletion constructs of in vitro translated GST-p53, as well as the reverse. I found that TRIM24 binds both the carboxy terminus and DNA binding domain of p53. Furthermore, my results show that binding is altered when post-translational modifications of p53 are present, suggesting that the interaction between p53 and TRIM24 may be affected by these post-translational modifications. To determine the specific domains of TRIM24 required for p53 interaction, we performed GST pull-downs with in vitro translated, Flag-TRIM24 protein constructs and recombinant GST-p53 protein purified from E. coli. We found that the Linker region is sufficient for interaction of p53 and TRIM24. Taken together, these data indicate that the interaction between p53 and TRIM24 does occur in vitro and that interaction may be influenced by post-translational modifications of the proteins.

Molecular signals in B lymphocyte activation: The role of intracellular calcium ion concentration, protein kinase C activation, and autocrine interleukin-2

Calcium ionophore, ionomycin, and phorbol myristate acetate (PMA) were used to activate rabbit peripheral blood B cells to study the role of increased intracellular calcium ion concentration ( (Ca$\sp2+\rbrack\sb{\rm i}$), protein kinase C (PKC) activation, and autocrine interleukin (IL-2) in inducing cell cycle entry and maintaining activation to DNA synthesis. When stimulated with a combination of ionomycin and PMA the B cells produced a soluble factor that supported the IL-2 dependent cell line, CTLL-2. The identity of the factor was established as IL-2 and its source was proved to be B cells in further experiments. Absorption studies and limiting dilution analysis indicated that IL-2 produced by B cells can act as an autocrine growth factor. Next, the effect of complete and incomplete signalling on B lymphocyte activation leading to cell cycle entry, IL-2 production, functional IL-2 receptor (IL-2R) expression, and DNA synthesis was examined. It was observed that cell cycle entry could be induced by signals provided by each reagent alone, but IL-2 production, IL-2R expression, and progression to DNA synthesis required activation with both reagents. Incomplete activation with ionomycin or PMA alone altered the responsiveness of B cells to further stimulation only in the case of ionomycin, and the unresponsiveness of these cells was apparently due to a lack of functional IL-2R expression on these cells, even though IL-2 production was maintained. The requirement of IL-2 for maintenance of activation to DNA synthesis was then investigated. The hypothesis that IL-2, acts in late G$\sb1$ and is required for DNA synthesis in B cells was supported by comparing IL-2 production and DNA synthesis in peripheral blood cells and purified B cells, kinetic analysis of these events in B cells, effects of anti-IL-2 antibody and PKC inhibitors, and by the response of G$\sb1$ B cells. Additional signals transduced by the interaction of autocrine IL-2 and functional IL-2 receptor on rabbit B cells were found to be necessary to drive these cells to S phase, after initial activation caused by simultaneous increase in (Ca$\sp2+\rbrack\sb{\rm i}$ and PKC activation had induced cell cycle entry, IL-2 production, and functional IL-2 receptor expression. ^

The interaction of P160 BCR with BCR-ABL gene products unique to Philadelphia chromosome leukemias

The BCR gene is involved in the pathogenesis of Philadelphia chromosome-positive (Ph$\sp1$) leukemias. Typically, the 5$\sp\prime$ portion of BCR on chromosome 22 becomes fused to a 5$\sp\prime$ truncated ABL gene from chromosome 9 resulting in a chimeric BCR-ABL gene. To investigate the role of the BCR gene product, a number of BCR peptide sequences were used to generate anti-BCR antibodies for detection of BCR and BCR-ABL proteins. Since both BCR and ABL proteins have kinase activity, the anti-BCR antibodies were tested for their ability to immunoprecipitate BCR and BCR-ABL proteins from cellular lysates by use of an immunokinase assay. Antisera directed towards the C-terminal portions of P160 BCR, sequences not present in BCR-ABL proteins, were capable of co-immunoprecipitating P210 BCR-ABL from the Ph$\sp1$- positive cell line K562. Re-immunoprecipitation studies following complete denaturation showed that C-terminal BCR antisera specifically recognized P160 BCR but not P210 BCR-ABL. These and other results indicated the presence of a P160 BCR/P210 BCR-ABL protein complex in K562 cells. Experiments performed with Ph$\sp1$-positive ALL cells and uncultured Ph$\sp1$-positive patient white blood cells established the general presence of BCR/BCR-ABL protein complexes in BCR-ABL expressing cells. However, two cell lines derived from Ph$\sp1$-positive patients lacked P160 BCR/P210 BCR-ABL complexes. Lysates from one of these cell lines mixed with lysates from a cell line that expresses only P160 BCR failed to generate BCR/BCR-ABL protein complexes in vitro indicating that P160 BCR and P210 BCR-ABL do not simply oligomerize.^ Two-dimensional tryptic maps were performed on both BCR and BCR-ABL proteins labeled in vitro with $\sp{32}$P. These maps indicate that the autophosphorylation sites in BCR-ABL proteins are primarily located within BCR exon 1 sequences in both P210 and P185 BCR-ABL, and that P160 BCR is phosphorylated in trans in similar sites by the activated ABL kinase of both BCR-ABL proteins. These results provide strong evidence that P160 BCR serves as a target for the BCR-ABL oncoprotein.^ K562 cells, induced to terminally differentiate with the tumor promoter TPA, show a loss of P210 BCR-ABL kinase activity 12-18 hours after addition of TPA. This loss coincides with the loss of activity in P160 BCR/P210 BCR-ABL complexes but not with the loss of the P210 BCR-ABL, suggesting the existence of an inactive form of P210 BCR-ABL. However, a degraded BCR-ABL protein served as the kinase active form preferentially sequestered within the remaining BCR/BCR-ABL protein complex.^ The results described in this thesis form the basis for a model for BCR-ABL induced leukemias which is presented and discussed. ^

The inhibition of lipid transfer protein by negatively charged liposomes modifies the therapeutic index of amphotericin B

We have shown that liposomal amphotericin B (L-AmpB) decreased renal toxicity and maintains the antifungal activity of amphotericin B (AmpB). We have also observed that L-AmpB is predominantly associated with high density lipoproteins (HDL) as compared to Fungizone (AmpB + deoxycholate). The present experiments were designed to assess the biological relevance of transferring AmpB to HDL. We observed that AmpB was less toxic to kidney cells when associated with HDL, however AmpB toxicity was maintained when associated with LDL. To further understand how HDL-associated AmpB reduces renal cell toxicity the presence of HDL and LDL receptors in this cell line was determined. We observed that these cells expressed high and low affinity LDL receptors, but only low affinity HDL receptors. The reduced renal cell toxicity of HDL-associated AmpB may be due to its lack of interaction with renal cells because of the absence of HDL receptors. Since AmpB interacts with cholesteryl esters whose transfer among lipoproteins is regulated by Lipid transfer Protein (LTP), the role of LTP on the distribution of AmpB to HDL and LDL was next examined. We found that negatively charged liposomes significantly reduced LTP-mediated transfer of CE between HDL and LDL, independent of the presence of AmpB, while Fungizone only significantly inhibited CE transfer at one concentration tested (20$\mu$g/ml). Therefore, we believe that the decreased renal toxicity of L-AmpB is related to its predominant distribution to HDL which is regulated by the inhibition of LTP activity. ^

The roles of electrostatic charge pairing in the interaction between cytochrome P-450 and NADPH-cytochrome P-450 reductase

The cytochrome P450 enzyme catalysis requires two electrons transferred from NADPH-cytochrome P450 reductase (reductase) to P450. Electrostatic charge-pairing has been proposed to be one of the major forces in the interaction between P450 and reductase. In order to obtain further insight into the molecular basis for the protein interaction, I used two methods, chemical modification and specific anti-peptide antibodies, to study the involvement and importance of charged amino acid residues. Acetylation of lysine residues of P450c and P450b by acetic anhydride dramatically inhibited the reductase-supported P450c-dependent ethoxycoumarin hydroxylation activity, but P450 activity supported by cumene hydroperoxide is relatively unchanged. The modification of lysine residues of P450c and P450b did not grossly disturb the protein conformation as revealed by several spectral studies. This differential effect of lysine modification on the P450 activity in the system reconstituted with reductase versus the system supported by cumene hydroperoxide suggested an important role for P450 lysine residues in the interaction with reductase. Using $\rm\sp{14}C$-acetic anhydride, P450 lysine residues were labelled and further identified on P450c and P450b. Those lysine residues are at position 97, 271, 279, and 407 for P450c, and 251, 384, 422, 433, and 473 for P450b. Alignment of those identified lysine residues on P450c and P450b with amino acid residues identified in other studies indicated those residues reside in three major sequence areas. Modification of arginine residues of P450b by phenylglyoxal and 2, 3-butanedione have no significant effect on P450 activity either supported by NADPH and reductase or supported by cumene hydroperoxide. Further studies using $\rm\sp{14}C$-phenylglyoxal reveals that no incorporation of phenylglyoxal into P450b was found. These results demonstrated a predominant role of lysine residues of P450 in the electrostatic interaction with reductase. To understand the protein binding sites on each of P450 and reductase, I generated three anti-peptide antibodies against regions on reductase and five anti-peptide antibodies against five putative reductase binding sites on P450c. These anti-peptide antibodies were affinity purified and characterized on ELISA and by Western blot analysis. Inhibition experiments using these antibodies demonstrated that regions 109-120 and 204-220 of reductase are probably the two major binding sites for P450. The association of reductase with cytochromes P450 and cytochrome c may rely on different mechanisms. The data from experiments using anti-peptide (P450c) antibodies supports the important role of P450c lysine residues 271/279 and 458/460 in the interaction with reductase. ^

Cloning and characterization of the mouse ret finger protein (rfp), a B box zinc finger protein

An important question in biology is to understand the role of specific gene products in regulating embryogenesis and cellular differentiation. Many of the regulatory proteins possess specific motifs, such as the homeodomain, basic helix-loop-helix structure, zinc finger, and leucine zipper. These sequence motifs participate in specific protein-DNA, protein-RNA, and protein-protein interactions, and are important for the function of these regulatory proteins.^ The human rfp (ret finger protein) belongs to a novel zinc finger protein family, the B box zinc finger family. Most of the B box proteins, including rfp, have a conserved tripartite motif, consisting of two novel zinc fingers (the RING finger and the B box) and a coiled-coil domain. Interestingly, a fusion protein between the tripartite motif of rfp and the tyrosine kinase domain of c-ret has transforming activity. In this study, we examined the expression of rfp during mouse development, and characterized the role of the tripartite motif in rfp function.^ We cloned the mouse rfp cDNA, which shares a 98.4% homology with the human sequence at amino acid level. Such strikingly high degree of homology indicates the high evolutionary pressure on the conservation of the sequence, suggesting that rfp may have an important function. Using the somatic cell hybrid system, we assigned the rfp gene to mouse chromosome 13 and human chromosome 6. Rfp transcripts and protein were ubiquitous in day 10.5-13.5 mouse embryos; however, they were restricted in adult mice, with the highest level of expression in the testis. Rfp expression in the testis is detected only in late pachytene spermatocytes and round spermatids. In both embryos and spermatogenic cells, rfp protein was distributed within cell nuclei in a punctate pattern, similar to the PODs (PML oncogenic domains) observed with another B box protein, PML. In cultured mammalian cells, we found that rfp was indeed co-localized to the PODs with PML. Using the yeast two-hybrid system, we showed that the rfp could specifically interact with PML, and that the interaction was dependent on the distal portion of the rfp coiled-coil domain.^ We also showed that rfp could form homodimers, and both the B box and coiled-coil domain were required for proper dimerization. It seems that the proximal portion of the coiled-coil domain provides the interacting interface, while the B box zinc finger orients the coil and maintains the correct structure of the whole molecule. Our data are consistent with the zinc-binding property and structural analysis of the B box. The RING finger seems to be involved in rfp nuclear localization through interaction with other proteins. We believe that homodimerization and interaction with PML are important for the normal interaction of rfp during development and differentiation. In addition, rfp homodimerization may also be essential for the oncogenic activation of the rfp-ret fusion protein. ^

Structural studies of cytochrome P4501A1: Identification of substrate and cumene hydroperoxide binding regions in the active site of P4501A1 and characterization of the P4501A1 interaction with cytochrome P450 reductase

Cytochromes P450 are a superfamily of heme-thiolate proteins that function in a concert with another protein, cytochrome P450 reductase, as terminal oxidases of an enzymatic system catalyzing the metabolism of a variety of foreign compounds and endogenous substrates. In order to better understand P450s catalytic mechanism and substrate specificity, information about the structure of the active site is necessary. Given the lack of a crystal structure of mammalian P450, other methods have been used to elucidate the substrate recognition and binding site structure in the active center. In this project I utilized the photoaffinity labeling technique and site-directed mutagenesis approach to gain further structural insight into the active site of mammalian cytochrome P4501AI and examine the role of surface residues in the interaction of P4501A1 with the reductase. ^ Four crosslinked peptides were identified by photoaffinity labeling using diazido benzphetamine as a substrate analog. Alignment of the primary structure of cytochrome P4501A1 with that of bacterial cytochrome P450102 (the crystal structure of which is known) revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and β10-β11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were proposed to be involved in reductase-P450 interaction. ^ Alternatively, it has been shown that some of the organic hydroperoxides can support P450 catalyzed reactions in the absence of NADPH, O2 and reductase. By means of photoaffinity labeling the cumene hydroperoxide binding region was identified. Using azidocumene as the photoaffinity label, the tripeptide T501-L502-K503 was shown to be the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. The role of Thr501 in the cumene hydroperoxide binding was confirmed by mutations of this residue and kinetic analysis of the effects of the mutations. ^ In addition, the role of two lysine residues, Lys271 and Lys279, in the interaction with reductase was examined by means of site-directed mutagenesis. The lysine residues were substituted with isoleucine and enzymatic activity of the wild type and the mutants were compared in reductase- and cumene hydroperoxide-supported systems. The lysine 279 residue has been shown to play a critical role in the P4501A1-reductase interaction. ^

BIOCHEMICAL PROPERTIES OF GABA (B) RECEPTORS (BACLOFEN, ADENYLATE CYCLASE, CYCLIC-AMP, PHOSPHOLIPASE, PROTEIN KINASE C)

(gamma)-Aminobutyric acid (GABA), a neurotransmitter in the mammalian central nervous system, influences neuronal activity by interacting with at least two pharmacologically and functionally distinct receptors. GABA(,A) receptors are sensitive to blockade by bicuculline, are associated with benzodiazepine and barbiturate binding sites, and mediate chloride flux. The biochemical and pharmacolocal properties of GABA(,B) receptors, which are stereoselectively activated by (beta)-p-chlorophenyl GABA (baclofen), are less well understood. The aim of this study was to define these features of GABA(,B) receptors, with particular emphasis on their possible relationship to the adenylate cyclase system in brain.^ By themselves, GABA agonists have no effect on cAMP accumulation in rat brain slices. However, some GABA agonists markedly enhance the cAMP accumulation that results from exposure to norepinephrine, adenosine, VIP, and cholera toxin. Evidence that this response is mediated by the GABA(,B) system is provided by the finding that it is bicuculline-insensitive, and by the fact that only those agents that interact with GABA(,B) binding sites are active in this regard. GABA(,B) agonists are able to enhance neurotransmitter-stimulated cAMP accumulation in only certain brain regions, and the response is not influenced by phosphodiesterase inhibitors, although is totally dependent on the availability of extracellular calcium. Furthermore, data suggest that inhibition of phospholipase A(,2), a calcium-dependent enzyme, decreases the augmenting response to baclofen, although inhibitors of arachidonic acid metabolism are without effect. These findings indicate that either arachidonic acid or lysophospholipid, products of PLA(,2)-mediated degradation of phospholipids, mediates the augmentation. Moreover, phorbol esters, compounds which directly activate protein kinase C, were also found to enhance neurotransmitter-stimulated cAMP accumulation in rat brain slices. Since this enzyme is known to be stimulated by unsaturated fatty acids such as arachidonate, it is proposed that GABA(,B) agonists enhance cAMP accumulation by fostering the production of arachidonic acid which stimulates protein kinase C, leading to the phosphorylation of some component of the adenylate cyclase system. Thus, GABA, through an interaction with GABA(,B) receptors, modulates neurotransmitter receptor responsiveness in brain. The pharmocological manipulation of this response could lead to the development of therapeutic agents having a more subtle influence than current drugs on central nervous system function. ^