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R R Rando

Publications and source records attributed to R R Rando.

At least 37 records · Page 2Linked to original sources
PubMed1998

Regulation of isomerohydrolase activity in the visual cycle.

While the overall biosynthetic pathway leading from all-trans-retinoids to 11-cis-retinoids in the visual cycle is understood, little is known about which step(s) may be rate-limiting and how control is exerted. One possible target for control is the isomerohydrolase, which processes all-trans-retinyl esters into 11-cis-retinol. The basal rate of 11-cis-retinol synthesis from all-trans-retinyl esters is extremely slow using bovine retinal pigment epithelial membranes [3.5 pmol of 11-cis-retinol min-1 (mg of protein)-1], and only small amounts of 11-cis-retinyl ester are formed. However, the addition of retinol binding proteins stimulates 11-cis-retinol formation by a factor of approximately 13. Specific protein-protein interactions are probably unimportant because bovine serum albumin and the physiologically relevant cellular retinaldehyde binding protein (CRALBP) both stimulate 11-cis-retinol formation to the same extent, although CRALBP does so at much lower concentrations. The relatively rapid rate of isomerization in the presence of binding proteins [44.3 pmol of 11-cis-retinol min-1 (mg of protein)-1] suggests that the rate-limiting enzyme in the visual cycle need not be the isomerohydrolase. Also, 11-cis-retinol is shown to inhibit isomerohydrolase, providing a simple mechanism for regulation of the visual cycle and the stimulating effect of binding proteins.

Animals↗
PubMed1998

Minimal RNA constructs that specifically bind aminoglycoside antibiotics with high affinities.

RNA molecules are the functional targets for aminoglycosides. In order to approach an understanding of the rules which underlie aminoglycoside-RNA recognition, high-affinity RNA aptamers have been prepared which discriminate among various aminoglycosides [Wang et al. (1996) Biochemistry 35, 12338-12346]. One of these aptamers, J6, which is 109 nts in length, binds the aminoglycoside tobramycin stoichiometrically with a dissociation constant of 0.77 +/- 0.03 nM. Aminoglycosides, similar in structure to tobramycin, bind with affinities diminished by 10(3)-10(4) compared to tobramycin. Experiments are reported here which are designed to reveal the nature of the tobramycin binding domain of J6. A small (40 nts) stem-loop derivative of J6, containing a 3 nt and a 1 nt bulge, stoichiometrically binds tobramycin with a dissociation constant of approximately 5 nM. This construct can strongly discriminate between similar aminoglycosides with respect to binding. Elimination of either the three or the single nucleotide bulge eliminates specific aminoglycoside binding. The structure of the loop region is also critical. These studies demonstrate that simplified RNA molecules can be generated which bind aminoglycosides specifically and with high affinities.

Aminoglycosides↗
PubMed1997

Specific binding of aminoglycosides to a human rRNA construct based on a DNA polymorphism which causes aminoglycoside-induced deafness.

RNA constructs prepared from wild-type and mutant (1555(G)) human mitochondrial 12S RNA were studied with respect to their abilities to specifically bind aminoglycoside antibiotics. The 1555(G) point mutation had previously been found to be associated with hereditary deafness induced by aminoglycosides. It is shown here that the 1555(G) RNA analog stoichiometrically binds aminoglycosides with high affinities, while the wild-type construct does not bind aminoglycosides at all. Analogous mutations in a 16S bacterial rRNA construct show the opposite behavior. Bacterial 16S rRNA constitutes the functional target for aminoglycoside antibiotics. While the wild-type 16S rRNA decoding region construct binds aminoglycosides stoichiometrically with binding affinities in the micromolar range, the mutant is unable to specifically bind aminoglycosides. These results demonstrate the importance of a specific GC base pair in aminoglycoside binding in both the human and the bacterial rRNA constructs. These studies also provide quantitative evidence in support of the hypothesis that the 1555(G) point mutation in human mitochondrial 12S RNA causes aminoglycoside induced deafness.

Aminoglycosides↗
PubMed1997

On the mechanism of the inhibition of transducin function by farnesylcysteine analogs.

The gamma subunits of heterotrimeric G proteins are isoprenylated/methylated on their carboxy termini. The photoreceptor G protein, transducin, is farnesylated/methylated at this position. Since the isoprenyl group is required for G protein function, it is of great interest to determine the mechanism by which the farnesyl group of Tgamma interacts with the other transducin subunits and/or the activated photoreceptor, rhodopsin. Farnesylcysteine derivatives (N-acetyl-S-farnesyl-L-cysteine and farnesylated peptides) have been previously shown to have effects on transducin activity at high concentrations. Here, an extensive survey is done of farnesylcysteine analogs and other lipid molecules, which are tested for their ability to inhibit GTP/GDP exchange in transducin catalyzed by photolyzed rhodopsin. These studies are carried out to determine the nature of the inhibition process. While it does not appear that these molecules exhibit the specificity which would characterize a ligand-receptor type mechanism, the results suggest that these compounds are not acting in a nonspecific detergent-like manner either. The most likely mode of action of famesylcysteine analogs is that they interfere with the lipid-lipid based association of Talpha and Tbetagamma through the lipid modifications present on each subunit.

Acetylcysteine↗
PubMed1997

Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region.

Aminoglycoside antibiotics can bind to many different types of RNA molecules. It was of interest to determine the nature of the selectivity of binding of aminoglycosides to important, biologically relevant RNA targets. Fluorescence anisotropy methods were developed to quantitatively measure aminoglycoside affinities to constructs of the HIV-1 RRE transcriptional activation region and the prokaryotic rRNA decoding region which is the natural antibacterial target of the aminoglycosides. A fluorescent analog of Rev34-50 (Fl-Rev34-50) was prepared and shown by fluorescence anisotropy measurements to bind to the HIV-1 RRE region with a stoichiometry of 1 and a dissociation constant of 7.6 nM. RRE RNA is a target for the arginine rich Rev protein, and the binding is known to be mimicked by Rev34-50. The binding is driven by a strongly negative enthalpic term. Aminoglycosides compete with Fl-Rev34-50 binding and competition experiments with semisynthetic aminoglycosides and neomycin B and tobramycin show binding affinities in the 1-2 microM range. The binding of aminoglycosides to this construct is thus not highly selective. A prokaryotic rRNA construct was also prepared and shown to bind a fluorescent dye labeled derivative of the antibiotic paromomycin (CRP) stoichiometrically with a dissociation constant of 0.16 microM. Competition experiments with other aminoglycosides showed binding in the micromolar range, with limited specificity for aminoglycoside type, suggesting that much of the aminoglycoside molecule is not involved in binding. The relatively modest specificity in the binding of aminoglycoside described above is to be contrasted to the subnanomolar affinities and specificity of aminoglycoside binding found using in vitro selected RNA molecules (Wang et al., 1996).

Amino Acid Sequence↗
PubMed1997

RNA aptamers that specifically bind to a K Ras-derived farnesylated peptide.

RNA aptamers were selected against an affinity column containing a farnesylated peptide modeled after the carboxyl terminus of K ras, the major oncogenic form of this small G protein family. After 10-rounds of selection, 25% of the RNA applied to the column could be specifically eluted. Sequence analysis of the binding RNA aptamers revealed two consensus sequences--GGGUGGG and GGGAGG. Quantitative fluorescence binding studies on two of the high-affinity aptamers, showed a binding affinities of 139 nM and 0.93 microM, respectively for the farnesylated peptide. Binding to the nonfarnesylated peptide was at least 10-fold weaker, showing that the aptamers can recognize the hydrophobic farnesyl moiety. High affinity aptamers could be useful in specifically interfering with oncogenic ras function in particular, and G proteins in general.

Base Sequence↗
PubMed1997

Retinal tubulin binds macular carotenoids.

PURPOSE: To investigate the biochemical mechanisms responsible for the specific uptake, concentration, and stabilization of the carotenoids lutein and zeaxanthin in the macula. METHODS: Soluble extracts of bovine retina mixed with radioactive carotenoids were purified by hydrophobic interaction, ion exchange, and gel filtration chromatography. Carotenoid-associated proteins in these purified preparations were identified through photoaffinity labeling and protein microsequencing. Similar purifications on human macular tissue without the addition of exogenous carotenoids also were performed. RESULTS: Experiments on bovine retinal tissue demonstrated that tubulin is the major soluble carotenoid-binding protein. When soluble extracts of human macular protein were examined, the endogenous carotenoids lutein and zeaxanthin were found to copurify with tubulin. CONCLUSIONS: Tubulin is found in abundance in the receptor axon layer of the fovea, where it can serve as a locus for the deposition of the high concentrations of macular carotenoids found there. The binding interaction of carotenoids and tubulin in the Henle's fiber layer could play an important role in the photoprotective effects of the macular carotenoids against the progression of age-related macular degeneration. The association of carotenoids with tubulin, a protein that can form highly ordered linear arrays, may provide an explanation for the dichroic phenomenon of Haidinger's brushes.

Affinity Labels↗
PubMed1996

RNA molecules that specifically and stoichiometrically bind aminoglycoside antibiotics with high affinities.

RNA aptamers had previously been selected which were able to bind to the aminoglycoside antibiotic tobramycin with high affinity (Wang & Rando, 1995). Consensus sequences are found in a variety of constructs, and these sequences were mapped to stem-loop regions by Mfold secondary structure prediction. A tobramycin-based affinity cleavage reagent specifically cleaves the aptamers in their consensus regions. A fluorescence depolarization method is developed to accurately measure the affinity and stoichiometry of aminoglycoside binding to RNA constructs. An RNA aptamer (J6RNA) selected to bind to the aminoglycoside antibiotic tobramycin is shown to do so with an affinity of 0.77 nM and a stoichiometry of 1:1. (Fluorescently labeled) 5-carboxytetramethylrhodamine tobramycin (CRT) is used as a ligand in the fluorescence depolarization studies. J6RNA binding is quite specific for tobramycin, and weakly binds structurally related aminoglycosides with affinities 10(3)-10(4)-fold lower than that for tobramycin. Specific aminoglycoside binding aptamers of this type should be useful for revealing the rules of RNA-aminoglycoside recognition.

Anti-Bacterial Agents↗
PubMed1996

The role of G protein methylation in the function of a geranylgeranylated beta gamma isoform.

The gamma subunit of heterotrimeric G proteins is isoprenylated and methylated on its carboxyl terminal cysteine residue. While retinal transducin is farnesylated, all other gamma subunits are modified by geranylgeranylation. An immobilized form of pig liver esterase (iPLE) is able to hydrolyze the methyl ester of a geranylgeranylated beta gamma isoform (beta 1 gamma 2). Since methylation is the only reversible reaction in the isoprenylation pathway, it could be a site of regulation of G protein activity. With both the methylated and demethylated beta 1 gamma 2 now available, the role of methylation for a geranylgeranylated heterotrimeric G protein may be addressed. Here, it is reported that methylation has no effect on the ability of beta gamma to interact with an alpha subunit, as probed by ADP-ribosylation studies with pertussis toxin, and has a small effect (less than 2-fold) on the ability of geranylgeranylated beta gamma to activate phosphatidylinositol-specific phospholipase C (PIPLC) and phosphoinositide 3 kinase (PI3K). In binding studies, demethylation only slightly decreased the ability of beta 1 gamma 2 to adhere to azolectin vesicles. Therefore, methylation of heterotrimeric G proteins appears to have only a minor effect in signal transduction processes which can be correlated to a decrease in hydrophobicity of the beta gamma subunit.

Adenosine Diphosphate Ribose↗
PubMed1996

Chemical biology of protein isoprenylation/methylation.

Isoprenylation/methylation is an important dual hydrophobic post-translational modification which occurs at or near a carboxyl terminal cysteine residue. All known G proteins are modified in this way, making the pathway of central interest for an understanding of signal transduction. In this review, aspects of the molecular enzymology of isoprenylation/methylation are reviewed. The functional significance of these modifications is discussed, with special reference to the signal transducing G proteins. Of further interest is the possible regulatory role of methylation, since this step is the only reversible one in the pathway. The biochemical and functional consequences of isoprenylation/methylation are of especial interest. Isoprenylation/methylation is generally assumed to enhance the abilities of modified proteins to associate with membranes. This can be due either to hydrophobic lipid-lipid or lipid-protein interactions. Available evidence, taken largely from studies on visual signal transduction and ras signalling pathways, strongly points to enhanced membrane binding being a consequence of hydrophobic lipid-lipid interactions. An exciting possibility that also emerges is concerned with whether isoprenylation may also have additional roles, in addition to enhancing the membrane partitioning ability of the modified protein. In a simple mechanism of this type, the isoprenylated/methylated cysteine residue would be specifically recognized by another protein. While no compelling case can yet be made for an effector role for the isoprenylated/methylated cysteine moiety mediating protein-protein interactions, recent studies on the pharmacology of isoprenylated cysteine analogs suggests the possibility of such a role.

Dimethylallyltranstransferase↗
PubMed1996

Solubilization, partial purification, and affinity labeling of the membrane-bound isoprenylated protein endoprotease.

A previously described [Ma, Y.-T., & Rando, R. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6275-6279] membrane-associated isoprenylated protein endoprotease is important in the processing of isoprenylated proteins terminating with CAAX. The enzyme is of substantial interest because specific inhibitors of it block the processing and functioning of ras in vivo. The enzyme appears to be an integral membrane protein, as it can only be removed from microsomal membranes with detergent. The enzyme is effectively solubilized by the detergent CHAPSO and can be partially purified (approximately 10-fold) by anion ion exchange and size exclusion chromatography. Attempts to further purify the enzyme by other column means, including affinity chromatography, were unsuccessful. The partially purified enzyme is very sensitive to thiol reagents but insensitive to other kinds of protease inhibitors, suggesting that the enzyme is a thiol protease. Potent and specific chloroketone containing affinity labeling agents have been developed. These novel inactivators owe their potency to an S-farnesylcysteine moiety which is recognized by the enzyme. Specific inhibitors of this type should allow for the identification and cloning of this protease, which is important for signal transduction.

Amino Acid Chloromethyl Ketones↗
PubMed1996

Polyenes and vision.

Polyenes are important for vision in all sighted species. The visual pigments (the rhodopsins) all use 11-cis-retinal as the chromophore; some possible reasons for the importance of this isomer are now emerging. New results on the involvement of xanthophils in the maintenance of the retina are also discussed.

Humans↗
PubMed1996

Inhibition of capacitative Ca2+ entry into cells by farnesylcysteine analogs.

Capacitative Ca2+ influx, which occurs in response to mobilization of intracellular Ca2+ stores, is a general feature of many cell types. Although the mechanism of capacitative Ca2+ entry is not known, evidence suggests the involvement of small G proteins that are prenylated on a cysteine residue near their carboxyl termini. We have investigated the actions of farnesyl-cysteine analogs on capacitative Ca2+ influx. Using human embryonic kidney 293 cells, we found that S-farnesylthioacetic acid, N-acetyl-S-farnesyl-L-cysteine, N-pivaloyl-S-farnesyl-L-cysteine, and N-acetyl-S-gernylgernyl-L-cysteine blocked the activation of capacitative Ca2+ influx, whereas N-benzoyl-S-farnesyl-S-cysteine had no effect on capacitative Ca2+ entry. Inhibition by S-farnesylthioacetic acid was concentration dependent (5-20 microM) and specific for Ca2+ influx through non-voltage-gated Ca2+ channels. A single protein band of 26-28 kDa was labeled specifically with a photoaffinity analog of farnesylcysteine. GTP binding to the photoaffinity-labeled band was demonstrated. These findings suggest, but do not prove, that a prenylated substrate, possibly a small G protein, is linked functionally to capacitative Ca2+ entry in human embryonic kidney 293 cells.

Affinity Labels↗
PubMed1995

A novel endoprotease responsible for the specific cleavage of transducin gamma subunit.

Isoprenylated/methylated heterotrimeric G proteins play important roles in a large number of signal transduction processes. While the enzymology of isoprenylated/methylated protein biosynthesis is well understood, nothing is known about how these proteins are degraded. In this article, a novel endoproteolytic activity has been identified from bovine retina and is shown specifically to remove the glycylfarnesylcysteine moiety from the carboxyl terminus of T gamma. When tested in a GTP binding assay, freshly prepared proteolyzed T beta gamma was unable to catalyze the binding of guanosine 5'-(gamma-thio)triphosphate (GTP-gamma-S) to T alpha in the presence of detergent solubilized rhodopsin. The optimum pH for this proteolytic activity is approximately 6, and the pH profile corresponds to an enzyme having pKa's of 4.4 +/- 0.1 and 7.7 +/- 0.1 for its active site residues. After analyzing a series of protease inhibitors, we found E-64, a specific thiol protease inhibitor, to be the most effective irreversible inhibitor of this enzyme, suggesting that the endoprotease might be a thiol protease. Affinity labeling studies using biotinylated affinity labeling probes have identified a 35 kDa protein as a candidate for the endoprotease.

Amino Acid Sequence↗
PubMed1995

Functional significance of beta gamma-subunit carboxymethylation for the activation of phospholipase C and phosphoinositide 3-kinase.

The gamma subunits of heterotrimeric G proteins are isoprenylated and methylated at their carboxyl-terminal cysteine residues. Since methylation is the only reversible reaction in the isoprenylation pathway, it could be a site of regulation of G protein activity. beta gamma subunits have been shown to activate a number of effectors involved in signal transduction pathways. The methyl group of retinal transducin (T) can be hydrolyzed by an immobilized form of pig liver esterase, allowing for a direct determination of the activities of methylated and demethylated T beta gamma. The abilities of methylated and demethylated T beta gamma to stimulate G protein regulated phosphatidylinositol-specific phospholipase C (PIPLC) and phosphoinositide 3-kinase (PI3K) were determined. It is reported here that there is a strong dependence on methylation for activating both PIPLC and PI3K. Demethylated T beta gamma is at least 10-fold less active than its methylated counterpart. Therefore, methylation may play an important role in the regulation of these effectors and of signal transduction processes in general.

Animals↗
PubMed1995

Farnesyl thiotriazole, a potent neutrophil agonist and structurally novel activator of protein kinase C.

Farnesylcysteine derivatives can initiate or inhibit superoxide (O2-) release in neutrophils. The mechanism by which one of these derivatives, farnesyl thiotriazole (FTT), initiates O2- release in neutrophils is the subject of this paper. Treatment of guinea pig neutrophils with FTT results in the rapid release of O2- by a route shown to be independent of the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) receptor. The signal transduction pathway utilized by the chemoattractant fMLP is generally accepted as the paradigm for receptor-mediated stimulation of O2- production. Antagonists of fMLP had no effect on FTT-induced O2- release, and pretreatment of neutrophils with fMLP had no effect on the ability of FTT to trigger further O2- generation. In fact, FTT behaves like a typical protein kinase C (PKC) activator. It promotes phosphorylation of the 47-kDa subunit of the NADH oxidase complex (p47-phox) in neutrophils, and this phosphorylation is specifically blocked by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an antagonist of PKC. FTT is also shown to activate PKC in vitro in a specific and saturable fashion. FTT is approximately equipotent with (S)-diolein, a physiologically relevant activator of this kinase. FTT represents a new, and quite novel, structure for a PKC activator. PKC activators include diglycerides and the structurally diverse tumor promoters.

Animals↗