Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The photoreceptor G-protein, transducin, belongs to the class of heterotrimeric GTP-binding proteins that transfer information from activated seven-span membrane receptors to effector enzymes or ion channels. Like other G-proteins, transducin acts as a molecular clock. It is activated by photoexcited rhodopsin which catalyzes the exchange of transducin-bound GDP for GTP and then stays active until bound GTP is hydrolyzed by an intrinsic GTPase activity. Our previous study on the components of the amphibian phototransduction cascade (Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416-417) has shown that transducin GTPase can be significantly accelerated by the target enzyme, cGMP phosphodiesterase (PDE), and more specifically its gamma-subunit (PDE gamma). Here we report that an analogous mechanism is present in bovine photoreceptors. Addition of recombinant PDE gamma to the test photoreceptor membranes which retain transducin but are depleted of endogenous PDE causes a significant acceleration of transducin GTPase activity. A similar effect was observed with the PDE holoenzyme, but not with the complex of PDE alpha- and beta-subunits prepared by a limited proteolysis of PDE with trypsin. The activating effect of PDE gamma is increased as test membrane concentration increases, exceeding 20-fold at rhodopsin concentrations over 80 microM and approaching the rate of the photoresponse turnoff. This suggests either that photoreceptor membranes contain a further factor which is essential for PDE-dependent regulation of transducin-bound GTP hydrolysis or that components of the phototransduction cascade interact in a cooperative manner. We also report that the GTPase-activating epitope is located within the C-terminal third of PDE gamma: the peptide corresponding to the 25 C-terminal amino acid residues of PDE gamma can accelerate transducin GTPase almost as well as the full-length PDE gamma. A part of the GTPase activating epitope is located within the 3 C-terminal amino acid residues: the truncation PDE gamma mutant lacking these residues accelerates transducin GTPase considerably less than the whole length PDE gamma.
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PMID:Regulation of transducin GTPase activity in bovine rod outer segments. 805 Oct 70

The cGMP phosphodiesterase (PDE) of retinal rod outer segments (ROS) is activated by the GTP-bound form of the G protein, transducin (Gt alpha). This activation can be reversed by the inhibitory gamma subunit of PDE through two distinct mechanisms: acceleration of GTP hydrolysis and direct inactivation independent of GTP hydrolysis. We have found that acceleration of Gt alpha GTPase by PDE gamma does not occur upon formation of a Gt alpha PDE gamma complex but rather reflects enhanced activity toward this complex of a membrane-bound GTPase accelerating protein. GTPase rate constants for Gt alpha in the presence of 3.3 microM PDE gamma were as high as 0.7 s-1 with hypotonically washed ROS membranes at 40 microM rhodopsin but were more than 10-fold lower when protein-free vesicles containing ROS lipids were substituted for ROS membranes. Acceleration of Gt alpha GTPase by PDE gamma was also barely detectable at low ROS concentrations (e.g. 4 microM rhodopsin) or if ROS treated with trypsin or urea were used. GTPase-independent inactivation by PDE gamma occurred efficiently at much lower membrane concentrations. Inhibition of Gt alpha-activated PDE was much slower than inhibition of PDE alpha beta by PDE gamma. Effects of PDE gamma upon successive additions of GTP suggested formation of a complex of PDE gamma and Gt alpha-GDP that is refractory to reactivation.
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PMID:Enhancement of rod outer segment GTPase accelerating protein activity by the inhibitory subunit of cGMP phosphodiesterase. 820 35

Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is composed of P alpha beta and two P gamma subunits. Interaction of P gamma with P alpha beta or with the alpha subunit (T alpha) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photoreceptors. Here we have investigated phosphorylation of P gamma by cAMP-dependent protein kinase and its functional effect on the P gamma interaction with P alpha beta or T alpha in vitro. P gamma, but not P gamma complexed with T alpha (both GTP and GDP forms), is phosphorylated. Measurement of 32P radioactivity in phosphorylated P gamma, analysis of phosphorylated P gamma by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of P gamma indicate that only threonine 35 in P gamma is phosphorylated. Phosphorylation of P gamma mutants also reveals that the C and N terminals of P gamma which are required for the regulation of P alpha beta functions are not involved in the P gamma phosphorylation but that arginine 33, which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated P gamma has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated P gamma, indicating that the P gamma-P alpha beta interaction is affected by P gamma phosphorylation. Nonphosphorylated P gamma inhibits both the GTPase activity of T alpha and the binding of a hydrolysis-resistant GTP analogue to T alpha, while P gamma phosphorylation reduces these inhibitory activities. These observations suggest that a P gamma domain containing threonine 35 is involved in the P gamma-T alpha interaction, and P gamma phosphorylation regulates the P gamma-T alpha interaction. Our observation suggests that P gamma phosphorylation by cAMP-dependent protein kinase may function for the regulation of phototransduction in vertebrate rod photoreceptors.
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PMID:Phosphorylation of the gamma subunit of the retinal photoreceptor cGMP phosphodiesterase by the cAMP-dependent protein kinase and its effect on the gamma subunit interaction with other proteins. 955 60

Because G protein-regulated cation channels in type II pneumocytes constitute the most likely pathway for alveolar Na+ entry, we explored the hypothesis that a G protein-coupled prostaglandin (PG) E2 receptor controls perinatal lung alveolar Na+ transport. [3H]PGE2 binding to the alveolar apical membrane was trypsin sensitive and showed a rank order of competitive inhibition: PGE2 = PGE1 > PGD2 > PGF2 alpha. Kinetic analysis demonstrated both high-affinity [dissociation constant (KD) = 2.1 +/- 0.7 nM; maximal binding (Bmax) = 27 +/- 7 fmol/mg protein] and low-affinity (KD = 28 +/- 2 nM; Bmax = 265 +/- 29 fmol/mg protein) binding sites. Modulation of high-affinity GTPase activity identified a similar potency order (IC50 = 11 mM for PGF2 alpha vs. 10-50 microM for other PGs), suggesting that the receptors are G protein coupled. Finally, 1 microM PGE2 (approximately IC25) increased conductive 22Na+ uptake into membrane vesicles only in the presence of 100 microM intravesicular GTP. The KD value for the high-affinity binding site together with the rank order of PG effect on ligand binding and G protein function places this PG receptor in the EP3 subtype, whereas Na+ uptake studies suggest that it helps maintain perinatal lung Na+ homeostasis.
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PMID:G protein-coupled prostaglandin receptor modulates conductive Na+ uptake in lung apical membrane vesicles. 957 75

Albright hereditary osteodystrophy (AHO), a disorder characterized by skeletal abnormalities and obesity, is associated with heterozygous inactivating mutations in the gene for Gsalpha. A novel Gsalpha mutation encoding the substitution of tryptophan for a nonconserved arginine within the switch 3 region (Gsalpha R258W) was identified in an AHO patient. Although reverse transcription-polymerase chain reaction studies demonstrated that mRNA expression from wild type and mutant alleles was similar, Gsalpha expression in erythrocyte membranes from the affected patient was reduced by 50%. A Gsalpha R258W cDNA, as well as one with arginine replaced by alanine (Gsalpha R258A), was generated, and the biochemical properties of in vitro transcription/translation products were examined. When reconstituted with cyc- membranes, both mutant proteins were able to stimulate adenylyl cyclase normally in the presence of guanosine- 5'-O-(3-thiotriphosphate) (GTPgammaS) but had decreased ability in the presence of isoproterenol or AlF4- (a mixture of 10 microM AlCl3 and 10 mM NaF). The ability of each mutant to bind and be activated by GTPgammaS or AlF4- was assessed by trypsin protection assays. Both mutants were protected normally by GTPgammaS but showed reduced protection in the presence of AlF4-. The addition of excess GDP (2 mM) was able to rescue the ability of AlF4- to protect the mutants, suggesting that they might have reduced affinity for GDP. A Gsalpha R258A mutant purified from Escherichia coli had decreased affinity for GDP and an apparent rate of GDP release that was 10-fold greater than that of wild type Gsalpha. Sucrose density gradient analysis demonstrated that both Gsalpha R258W and Gsalpha R258A were thermolabile at higher temperatures and that denaturation of both mutants was prevented by the presence of 0.1 mM GTPgammaS or 2 mM GDP. The crystal structure of Gsalpha demonstrates that Arg258 interacts with a conserved residue in the helical domain (Gln170). Arg258 substitutions would be predicted to open the cleft between the GTPase and helical domains, allowing for increased GDP release in the inactive state, resulting in enhanced thermolability and reduced AlF4--induced adenylyl cyclase stimulation and trypsin protection, since activation by AlF4- requires bound GDP.
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PMID:A novel mutation in the switch 3 region of Gsalpha in a patient with Albright hereditary osteodystrophy impairs GDP binding and receptor activation. 972 13

The alpha subunit (Galpha) of heterotrimeric G proteins is a major determinant of signaling selectivity. The Galpha structure essentially comprises a GTPase "Ras-like" domain (RasD) and a unique alpha-helical domain (HD). We used the vertebrate phototransduction model to test for potential functions of HD and found that the HD of the retinal transducin Galpha (Galphat) and the closely related gustducin (Galphag), but not Galphai1, Galphas, or Galphaq synergistically enhance guanosine 5'-gamma[-thio]triphosphate bound Galphat (GalphatGTPgammaS) activation of bovine rod cGMP phosphodiesterase (PDE). In addition, both HDt and HDg, but not HDi1, HDs, or HDq attenuate the trypsin-activated PDE. GalphatGDP and HDt attenuation of trypsin-activated PDE saturate with similar affinities and to an identical 38% of initial activity. These data suggest that interaction of intact Galphat with the PDE catalytic core may be caused by the HD moiety, and they indicate an independent site(s) for the HD moiety of Galphat within the PDE catalytic core in addition to the sites for the inhibitory Pgamma subunits. The HD moiety of GalphatGDP is an attenuator of the activated catalytic core, whereas in the presence of activated GalphatGTPgammaS the independently expressed HDt is a potent synergist. Rhodopsin catalysis of Galphat activation enhances the PDE activation produced by subsaturating levels of Galphat, suggesting a HD-moiety synergism from a transient conformation of Galphat. These results establish HD-selective regulations of vertebrate retinal PDE, and they provide evidence demonstrating that the HD is a modulatory domain. We suggest that the HD works in concert with the RasD, enhancing the efficiency of G protein signaling.
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PMID:The helical domain of a G protein alpha subunit is a regulator of its effector. 978 8

We previously reported that substitution of Arg258 within the switch 3 region of Gsalpha impaired activation and increased basal GDP release due to loss of an interaction between the helical and GTPase domains (Warner, D. R., Weng, G., Yu, S., Matalon, R., and Weinstein, L. S. (1998) J Biol. Chem. 273, 23976-23983). The adjacent residue (Glu259) is strictly conserved in G protein alpha-subunits and is predicted to be important in activation. To determine the importance of Glu259, this residue was mutated to Ala (Gsalpha-E259A), Gln (Gsalpha-E259Q), Asp (Gsalpha-E259D), or Val (Gsalpha-E259V), and the properties of in vitro translation products were examined. The Gsalpha-E259V was studied because this mutation was identified in a patient with Albright hereditary osteodystrophy. S49 cyc reconstitution assays demonstrated that Gsalpha-E259D stimulated adenylyl cyclase normally in the presence of GTPgammaS but was less efficient with isoproterenol or AlF4-. The other mutants had more severely impaired effector activation, particularly in response to AlF4-. In trypsin protection assays, GTPgammaS was a more effective activator than AlF4- for all mutants, with Gsalpha-E259D being the least severely impaired. For Gsalpha-E259D, the AlF4--induced activation defect was more pronounced at low Mg2+ concentrations. Gsalpha-E259D and Gsalpha-E259A purified from Escherichia coli had normal rates of GDP release (as assessed by the rate GTPgammaS binding). However, for both mutants, the ability of AlF4- to decrease the rate of GTPgammaS binding was impaired, suggesting that they bound AlF4- more poorly. GTPgammaS bound to purified Gsalpha-E259D irreversibly in the presence of 1 mM free Mg2+, but dissociated readily at micromolar concentrations. Sucrose density gradient analysis of in vitro translates demonstrated that all mutants except Gsalpha-E259V bind to beta gamma at 0 degreesC and were stable at higher temperatures. In the active conformation Glu259 interacts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4- in the guanine nucleotide binding pocket. Although both Gsalpha Arg258 and Glu259 are critical for activation, the mechanisms by which these residues affect Gsalpha protein activation are distinct.
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PMID:Mutagenesis of the conserved residue Glu259 of Gsalpha demonstrates the importance of interactions between switches 2 and 3 for activation. 998 42

G-protein alpha subunits consist of two domains: a Ras-like domain also called GTPase domain (GTPaseD), structurally homologous to monomeric G-proteins, and a more divergent domain, unique to heterotrimeric G-proteins, called helical domain (HD). G-protein activation, requires the exchange of bound GDP for GTP, and since the guanine nucleotide is buried in a deep cleft between both domains, it has been postulated that activation may involve a conformational change that will allow the opening of this cleft. Therefore, it has been proposed, that interdomain interactions are playing an important role in regulating the nucleotide exchange rate of the alpha subunit. While constructing different Gs(alpha) quimeras, we identified a Gs(alpha) random mutant, which was very inefficient in stimulating adenylyl cyclase activity. The introduced mutation corresponded to the substitution of Ser(111) for Asn (S111N), located in the carboxi terminal end of helix A of the HD, a region neither involved in AC interaction nor in the interdomain interface. In order to characterize this mutant, we expressed it in bacteria, purified it by niquel-agarose chromatography, and studied its nucleotide exchange properties. We demonstrated that the recombinant S111N Gs(alpha) was functional since it was able to undergo the characteristic conformational change upon GTP binding, detected by the acquisition of a trypsin-resistant conformation. When the biochemical properties were determined, the mutant protein exhibited a reduced GDP dissociation kinetics and as a consequence a slower GTPgammaS binding rate that was responsible for a diminished adenylyl cyclase activation when GTPgammaS was used as activator. These data provide new evidence that involves the HD as a regulator of Gs(alpha) function, in this case the alphaA helix, which is not directly involved with the nucleotide binding site nor the interdomain interface.
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PMID:S111N mutation in the helical domain of human Gs(alpha) reduces its GDP/GTP exchange rate. 1196 1

Recently, it was shown that Yersinia outer protein T (YopT) belongs to a new family of cysteine proteases containing invariant C, H, and D residues that are crucial for its activity. YopT cleaves RhoA, Rac, and Cdc42 at their C termini, thereby releasing them from the membrane. Moreover, YopT inhibits the Rho-rhotekin and Rho-guanine nucleotide dissociation inhibitor interactions. To characterize the active domain of YopT, we constructed N- and C-terminal truncations and expressed them as glutathione S-transferase fusion proteins in Escherichia coli. The toxin fragments were tested for stability by trypsin digestion. The activity of the proteins was studied by membrane release assay, rhotekin pulldown experiments, and microinjection. Whereas deletion of the first 74 N-terminal amino acids did not influence the activity of YopT, deletion of 8 amino acids from the C terminus led to complete loss of activity. N-terminal deletion of 100 amino acids led to an inactive protein, although it still contained the amino acids C139, H258, and D274, which are essential for catalysis. Loss of activity of the N-terminal deletions corresponded to the block of interaction with RhoA, indicating that residues 75 to 100 of YopT are essential for binding to the GTPase. By contrast, when up to 15 amino acids of the C terminus were deleted, the protein had no activity but was still able to interact with RhoA, suggesting a role for the C terminus in the enzyme activity of YopT.
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PMID:The C terminus of YopT is crucial for activity and the N terminus is crucial for substrate binding. 1287 42

Efficient hydrolysis of native poly(3-hydroxybutyrate) (nPHB) granules in vitro by soluble PHB depolymerase of Rhodospirillum rubrum requires pretreatment of nPHB with an activator compound present in R. rubrum cells (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). Edman sequencing of the purified activator (17.4 kDa; matrix-assisted laser desorption ionization-time of flight mass spectrometry) revealed identity to a hypothetical protein deduced from a partially sequenced R. rubrum genome. The complete activator gene, apdA (activator of polymer degradation), was cloned from genomic DNA, expressed as a six-His-tagged protein in recombinant Escherichia coli (M(r), 18.3 x 10(3)), and purified. The effect of ApdA on PHB metabolism was studied in vitro and in vivo. In vitro, the activity of the activator could be replaced by trypsin, but recombinant ApdA itself had no protease activity. Comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein patterns of trypsin- and ApdA-treated nPHB granules isolated from different PHB-accumulating bacteria showed that trypsin activated nPHB by removing proteins of the surface layer of nPHB regardless of the origin of nPHB, but ApdA bound to and interacted with the surface layer of nPHB in a nonproteolytic manner, thereby transforming nPHB into an activated form that was accessible to the depolymerase. In vivo, expression of ApdA in E. coli harboring the PHB biosynthetic genes, phaCBA, resulted in significant increases in the number and surface/volume ratio of accumulated PHB granules, which was comparable to the effect of phasin proteins, such as PhaP in Ralstonia eutropha. The amino acid sequence of ApdA was 55% identical to the amino acid sequence of Mms16, a magnetosome-associated protein in magnetotactic Magnetospirillum species. Mms16 was previously reported to be a GTPase with an essential function in magnetosome formation (Y. Okamura, H. Takeyama, and T. Matsunaga, J. Biol. Chem. 276:48183-48188, 2001). However, no GTPase activity of ApdA could be demonstrated. We obtained evidence that Mms16 of Magnetospirillum gryphiswaldense can functionally replace ApdA in R. rubrum. Fusions of apdA and mms16 to gfp or yfp were functionally expressed, and both fusions colocalized with PHB granules after conjugative transfer to R. rubrum. In conclusion, ApdA in vivo is a PHB-bound, phasin-like protein in R. rubrum. The function of Mms16 in magnetotactic bacteria requires further clarification.
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PMID:Unraveling the function of the Rhodospirillum rubrum activator of polyhydroxybutyrate (PHB) degradation: the activator is a PHB-granule-bound protein (phasin). 1506 50


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