Robert R. Rando, Ph.D. - US grants

Protein kinase C (PKC) is an important regulatory enzyme whose physiological activation requires it to become membrane-bound. This occurs via the simultaneous presence of calcium, an acidic phospholipid such as phosphatidyl serine (PS), and a diglyceride. The presence of the latter is of great regulatory significance because it is the product of polyphosphatidylinositol turnover. The diglyceride requirement can be satisfied by structurally diverse tumor promoters such as the phorbol esters, teleocidin, and the debromoaplysiatoxins. It is likely that PKC is an important target for the tumor promoters. In addition, PKC plays an important role in platelet aggregation, cardiac function, and neural function. The major focus of this grant is to understand the mechanism of the novel activation process of PKC. Of particular interest is the unravelling the structural basis for PKC activator function. On the one hand, PKC is exceedingly selective with respect to the chemical structure of diglyceride activators. At the same time, structurally diverse tumor promoters belonging to the diterpene, peptide, and macrolide series , can all potently activate the enzyme. These observations are reconciled here with the presentation of a new unifying structural hypothesis on PKC activators which are based on experiments described in the progress report. This new hypothesis makes predictions concerning novel diglyceride and peptide analogs which should potently interact with the kinase. These molecules will be synthesized and studied as putative PKC activators and inhibitors. Finally, novel cyclic PS analogs will be prepared to study the specificity of the phospholipid binding-site of PKC. Complimentary studies on the nature of activator binding-site are also anticipated. The PKC regulatory domain binding-site will be covalently labeled with activator-based photoaffinity probes and the labeled region will be sequenced. Taken together, these studies should bring us closer to an understanding of the regulation of PKC at a molecular level and the general issue of how proteins can interact with and bind to membranes. Finally, the structural information obtained here will serve to map the PKC diglyceride binding-site and can be used as a starting point to prepare potent inhibitors of the enzyme.

The studies described here are concerned with elucidating the molecular and physiological basis of rhodopsin regeneration. Specifically, the studies are focused on understanding the process by which 11-cis-retinol, the ultimate precursor of the visual chromophore, is synthesized in the vertebrate pigment epithelium. We have shown that the membranes of the pigment epithelium contain a novel biosynthetic enzyme system capable of transforming free all-trans-retinol into its 11-cis congener. This thermodynamically uphill process derives its energy from the phospholipids of the membranes in a novel process which utilizes the free energy of hydrolysis of the ester bonds to drive the isomerization. The mechanism employed involves the transesterification of all-trans-retinol with a phospholipid followed by enzymatic processing of the retinyl ester directly to 11-cis-retinol. We have solubilized and partially purified the enzymes involved. In this grant request, we propose to completely purify the enzyme components of this isomerization process, namely, the retinyl ester synthetase and isomerase. We propose to study the mechanisms of action of these enzymes and prepare mechanism-based enzyme inhibitors of them to be used to study their physiological roles. Furthermore, monoclonal and monospecific polyclonal antibodies will be prepared against the enzymes. These antibodies will be used to determine the steady state levels, the turnover rates, and the possible post-translational modification of the enzymes. The mechanisms by which the enzymes might be regulated as well as the mechanism by which the enzymes interact with the membrane are of exceptional interest here. The availability of sequence information on the enzymes and the availability of specific antibodies to the enzymes should enable us to clone them and fully sequence them. This information is important for determining what these enzymes are related to, what their major structural motifs might be, and how the enzymes interact with the membrane. Sequence information will also be of interest clinically for determining whether or not these enzymes, which are the sine qua non of vertebrate vision, are altered in major diseases of vision.

The regulation of the fluxes and metabolism of vitamin A in the retina and retinal pigment epithelium is not yet well understood and is the subject of this grant request. Central to this problem are quantitative studies on certain enzymes and binding proteins which process vitamin A in the eye and which could be points of regulation. In this grant request we propose to study by chemical and biochemical means retinal ester esterase, synthetase, rhodopsin and certain of the retinol(al) binding proteins. The two enzymes will be purified to homogeniety and their mechanisms of action determined. In order to study these mechanisms a new quantitative assay system for these enzymes will be developed involving the transfer of the retinoids from small unilamellar vesicles (SUV's) to the protein. This assay will allow for the precise calculation of retinoid concentrations as well as providing a known and stable environment for them which allows for a meaningful calculation of thermodynamic and kinetic constants. This assay system should be generally useful for binding studies involving hydrophobic ligands. Having a quantitative assay for the enzymes in hand, we will determine whether they are regulated by light in the albino rat and frog. Highly specific, irreversible, mechanism-based inhibitors will be synthesized for these enzymes and they will be tested in vivo to determine their physiological roles. The same assay system mentioned above will be used to quantitatively measure the binding of the retinol(al)s to ascertain the retinol binding proteins and determine whether the activity of these proteins is regulated by light. Specific irreversible inactivators of these proteins will also be prepared and studied in vivo. Finally, new specific mechanism-based inactivators for opsin have been prepared. These will be primarily used to study how rhodopsins activity is regulated by light. Quantitative studies on the visible absorption spectra of these inhibitor(s) - opsin complexes will also be of interest in determining the mechanism(s) by which opsin perturbs the absorption spectra of bound chromophores.

Our overall interest is to understand the molecular basis of signal transduction in the vertebrate visual system. G proteins are essential elements in diverse signal transducing systems, including the visual system. Indeed, the transducin-rhodopsin couple remains the most well understood of all the G protein mediated signal transducing systems. Recent studies have shown that these G proteins are post-translationally modified by the covalent linkage of a hydrophobic isoprenoid moiety to a carboxyl terminal cysteine residue followed by the reversible carboxymethylation of the same residue in a process termed prenylation. It appears that these modifications are critical for the physiological function of the G proteins, although the mechanisms by which these modifications might affect function is unknown. We have found that the visual system heterotrimeric G proteins transducin is prenylated and methylated, as are the rod outer segment (ROS) 'small' G proteins. We propose to study the biochemical basis and the role of the G protein methylation, which is reversible, and prenylation in ROS at the molecular level. In other signal transducing systems, such as in bacterial chemotaxis, reversible methylation reactions have key regulatory significance. The proposed investigation will initially entail studying the interactions between methylated and demethylated transducin and activated rhodopsin to understand the biochemical role of the reversible methylation step. We will also quantitatively study the membrane binding properties of methylated and demethylated G proteins and model compounds, including synthetic peptides, and the possible role that specific lipid-lipid interactions might play in membrane targeting. Finally, the ROS associated methylase and demethylase enzymes will be purified and characterized. Specific inhibitors will be designed for these enzymes, based on their novel mechanistic properties. These inhibitors will be used to further elucidate the physiologic role(s) of G protein methylation and demethylation in signal transduction in the visual system. It is expected that the studies reported herein on G protein processing and regulation in the visual system will serve as a general model.

RNA molecules are able to form precise three-dimensional structures which can be binding-sites for small organic molecules. An understanding of the rules that govern RNA/small molecule recognition processes rules would allow for an approach to our long-term goal of the de novo design of antagonists directed at particular RNA structures, in much the same way that inhibitors are designed for protein-based enzymes and receptors. Specific inhibitors designed to inhibit RNA molecules could be of enormous interest in ophthalmology and generally in medicine, in the design, for example, of small-molecules that can specifically interfere with the expression of mutant proteins that can lead to retinal degeneration, and in the design of small molecules that can antagonize RNA molecules from infectious disease producing organisms. This proposal describes approaches to gaining an understanding of the rules by which certain classes of naturally occurring RNA antagonists, the aminoglycosides, are recognized by specific RNA molecules. Random RNA molecules are selected by column methods to bind to defined aminoglycoside containing antibiotics with high affinity and specificity. New quantitative fluorescence methods are developed to determine the affinities and stoichiometries of antibiotic binding to the selected RNA aptamers. Novel chemical approaches are developed to reveal the regions of the aptamers which define the binding-sites for the specific antibiotic. High field NMR structural studies on the high affinity binding aptamers are planned. Those motifs in the specific RNA aptamers which recognize particular aminoglycosides will be determined and used as a guide for the future design of specific antagonists directed against naturally occurring RNA molecules. Two biologically occurring RNA molecules of particular interest in this context are the procaryotic 16S rRNA decoding region and the HIV-RRE transcriptional activator region. Quantitative structure-activity studies on aminoglycoside binding to the decoding and RRE regions leads to the design of novel aminoglycosidemimetic diversity libraries containing l,3(2)-hydroxylamine moieties. Specific antagonists directed against the decoding and RRE regions of RNA will be prepared and studies quantitatively. These antagonists are expected to be starting points for the design of drugs useful in the treatment of corneal infections. Overall, the studies described herein will serve as the basis for a general program on the design of specific RNA antagonists.

DESCRIPTION (Adapted from applicant's abstract): The vertebrate visual cycle is comprised of biochemical reactions involved in processing all-trans retinal produced by the photoisomerisation of 11-cis retinal in rhodopsin. The visual cycle is essential for vision and visual adaptation. The goal of this project is to characterize on a molecular level essential components of the visual cycle and to learn how the cycle is regulated. Two of the key reactions in the visual cycle are catalyzed by the retinal pigment epithelium membrane bound enzymes, lecithin retinol acyl transferase (LRAT)and the isomerohydrolase. LRAT transfers an acyl group from lecithin to vitamin A to generate all-trans retinyl esters and the omerohydrolase processes the esters to produce 11-cis retinol. Both enzymes are essential for vision. An understanding of LRAT at the molecular level is of major interest in this grant proposal. LRAT has a unique sequence which does not reveal the mechanistic class to which it belongs. Biochemical studies including chemical mapping studies using the novel technique of biotin affinity labeling and site-specific mutagenic studies are proposed to both map elements of the active-site structure of LRAT and define its molecular mechanism of action. Chemical mapping studies are also proposed to begin to elucidate the structure of LRAT in the membrane and to reveal nearest neighbor proteins in RPE membranes. One of the LRAT associated proteins may be the isomerohydrolase. Identification and characterization of isomerohydrolase is another important aspect of this proposal. Approaches to the identification of this enzyme system will involve both exploiting interactions with LRAT to either affinity purify or cross-link isomerohydrolase and photoaffinity labeling approaches to label the enzyme. When isomerohydrolase is identified it will be coned sequenced and expressed in LRAT transfected HEK cells. The structure, mechanism of action and regulation of the isomerohydrolase will be explored as will possible relationships to diseases of vision caused by mutations in the enzyme.

[unreadable] DESCRIPTION (provided by applicant): The visual cycle is completed in the retinal pigment epithelium (RPE) by those biochemical reactions involved in the processing of all-trans-retinol (vitamin A) into 11-cis-retinol(al). Some of the critical steps include the lecithin retinol acyl transferase (LRAT) mediated esterification of vitamin A using lecithin as the acyl donor to generate hydrophobic all-trans-retinyl esters followed by the processing of these esters to form 11-cis-retinol by isomerohydrolase. Significant questions concerning the operation of the visual cycle include the identification of the full inventory of retinoid binding proteins (RBPs) involved in the cycle, an understanding of how it is regulated, and an understanding of how the highly hydrophobic long-chain fatty acid retinyl esters are mobilized and processed. RPE65 has been shown to be essential for the binding and mobilization of the hydrophobic all-trans-retinyl esters (tREs) for processing by isomerohydrolase (IMH). Mutations in RPE65 are known to cause a form of retinyl degeneration. It is only the membrane associated form (mRPE65) which stereospecifically binds tREs and the soluble form of this protein (sRPE65) is shown to stereospecifically bind vitamin A with high affinity. The two forms of RPE65 are interconverted by LRAT, acting here as a palmitoyl transferase, and transferring a palmitoyl group from mRPE65 to vitamin A or 11-cis-retinol. We propose an RPE65 epicycle as an essential regulatory switch in the operation of the visual cycle. The control element reveals new roles for palmitoylated proteins and functions here by directing retinoid flow in the visual cycle depending on the relative levels of mRPE65/sRPE65. This model will be rigorously tested using biochemical and functional approaches. For example, the nature of the post-translational modifications of m and sRPE65 will be described and the molecular enzymology of the LRAT mediated interconversion will be elucidated. The chemical biological basis of mRPE65 and sRPE65 recognition of retinoids will be explored. The roles of sRPE65 and mRPE65 in visual cycle function will be determined. Aside from the known feed-back inhibition of IMH by 11-cis-retinoids, the proposed RPE65 cycle represents the only other known control element in the operation of the visual cycle. Finally, the demonstration of the stereospecific binding of tREs by mRPE65 suggests that there will be cognate11-cis-RE binding proteins. Specific affinity biotinylation methods, which proved to be successful in the identification of mRPE65 as a tRE binding protein, will be adapted to characterize the 11-cis-RE binding cognates and 11-cis-retinyl ester hydrolase(s). [unreadable] [unreadable]