A coil-helix instead of a helix-coil motif can be induced in a chloroplast transit peptide from Chlamydomonas reinhardtii.
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A synthetic peptide MQVTMKSSAVSGQRVGGARVATRSVRRAQLQV corresponding to the 32 amino acid chloroplast transit sequence of the ribulose bisphosphatase carboxylase/oxygenase activase preprotein from Chlamydomonas reinhardtii, required for translocation through the envelope of the chloroplast, has been characterized structurally using CD and NMR under the same experimental conditions as used previously for the 32 amino acid presequence of preferredoxin from the same organism [Lancelin, J.-M., Bally, I., Arlaud, G. J., Blackledge, M., Gans, P., Stein, M. & Jacquot, J.-P. (1994) FEBS Lett. 343, 261-266]. The peptide is found to undergo a conformational transition in aqueous 2,2,2-trifluoroethanol, characterized by three turns of amphiphilic alpha-helix in the C-terminal region preceded by a disordered coil in the N-terminal region. Compared with the preferredoxin transit peptide, the helical and coiled domains are arranged in the reverse order along the peptide sequence, but the positively charged groups are distributed analogously as well as the hydrophobic residues within the amphiphilic alpha-helix. It is proposed that such coil-helix or helix-coil motifs, occasionally repeated, could be an intrinsic structural feature of chloroplastic transit peptides, adapted to the proper translocase and possibly to each nuclear-encoded chloroplast preproteins. This feature may distinguish chloroplastic transit sequences from the other organelle-targeting peptides in the eukaryotic green alga C. reinhardtii, particularly the mitochondrial transit sequences. (+info)
Predicting conformational switches in proteins.
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We describe a new computational technique to predict conformationally switching elements in proteins from their amino acid sequences. The method, called ASP (Ambivalent Structure Predictor), analyzes results from a secondary structure prediction algorithm to identify regions of conformational ambivalence. ASP identifies ambivalent regions in 16 test protein sequences for which function involves substantial backbone rearrangements. In the test set, all sites previously described as conformational switches are correctly predicted to be structurally ambivalent regions. No such regions are predicted in three negative control protein sequences. ASP may be useful as a guide for experimental studies on protein function and motion in the absence of detailed three-dimensional structural data. (+info)
Role of the prosequence of guanylin.
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Guanylin is a guanylyl cyclase (GC)-activating peptide that is mainly secreted as the corresponding prohormone of 94 amino acid residues. In this study, we show that the originally isolated 15-residue guanylin, representing the COOH-terminal part of the prohormone, is released from the prohormone by cleavage of an Asp-Pro amide bond under conditions applied during the isolation procedures. Thus, the 15-residue guanylin is probably a non-native, chemically induced GC-activating peptide. This guanylin molecule contains two disulfide bonds that are absolutely necessary for receptor activation. We demonstrate that the folding of the reduced 15-residue guanylin results almost completely in the formation of the two inactive disulfide isomers. In contrast, the reduced form of proguanylin containing the entire prosequence folds to a product with the native cysteine connectivity. Because proguanylin lacking the 31 NH2-terminal residues of the prosequence folds only to a minor extent to guanylin with the native disulfide bonds, it is evident that this NH2-terminal region contributes significantly to the correct disulfide-coupled folding. Structural studies using CD and NMR spectroscopy show that native proguanylin contains a considerable amount of alpha-helical and, to a lesser extent, beta-sheet structural elements. In addition, a close proximity of the NH2- and the COOH-terminal regions was found by NOESY. It appears that this interaction is important for the constitution of the correct conformation and provides an explanation of the minor guanylyl cyclase activity of proguanylin by shielding the bioactive COOH-terminal domain from the receptor. (+info)
alpha3beta3gamma complex of F1-ATPase from thermophilic Bacillus PS3 can maintain steady-state ATP hydrolysis activity depending on the number of non-catalytic sites.
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Homogeneous preparations of alpha(3)beta(3)gamma complexes with one, two or three non-competent non-catalytic site(s) were performed as described [Amano, Hisabori, Muneyuki, and Yoshida (1996) J. Biol. Chem. 271, 18128-18133] and their properties were compared with those of the wild-type complex. The ATPase activity of the complex with three non-competent non-catalytic sites decayed rapidly to an inactivated state, as reported previously [Matsui, Muneyuki, Honda, Allison, Dou, and Yoshida (1997) J. Biol. Chem. 272, 8215-8221]. In contrast, the complex with one or two non-competent non-catalytic sites displayed a substantial steady-state phase activity depending on the number of non-competent non-catalytic sites in the complex. This result indicates that one competent non-catalytic site can maintain the continuous catalytic turnover of the enzyme and can potentially relieve all three catalytic sites from inhibition by MgADP(-). Furthermore, the results suggest that the interaction between three non-catalytic sites might not be as strong as that between catalytic sites, which are all strictly required for a continuous catalytic turnover. (+info)
Relationship between phosphatidic acid level and regulation of protein transit in colonic epithelial cell line HT29-cl19A.
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Colonic epithelial HT29-cl19A cells are polarized and secrete proteins among which alpha(1)-antitrypsin represents about 95%. Secretion occurs via a constitutive pathway, so that the rates of secretion directly reflect the rates of protein transit. In this paper we have demonstrated that: 1) in resting cells phospholipase D (PLD) is implicated in the control of apical protein transit; 2) phorbol esters stimulate apical protein transit (stimulation factor 2.2), which is correlated with a PLD-catalyzed production of phosphatidic acid (PA) (2.45-fold increase); 3) the stimulation of cholinergic receptors by carbachol results in an increase (stimulation factor 1.45) of apical protein transit which is independent of protein kinase C and PLD activities, but related to PA formation (1.7-fold increase) via phospholipase(s) C and diacylglycerol kinase activation; 4) an elevation of the cAMP level enhances apical protein transit by a PA-independent mechanism; 5) a trans-Golgi network or post-trans-Golgi network step of the transit is the target for the regulatory events. In conclusion, we have shown that PA can be produced by two independent signaling pathways; whatever the pathway followed, a close relationship between the amount of PA and the level of secretion was observed. (+info)
Insulin stimulates transepithelial sodium transport by activation of a protein phosphatase that increases Na-K ATPase activity in endometrial epithelial cells.
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The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na(+) transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na(+) transport two- to threefold by increasing Na(+)-K(+) ATPase transport activity and basolateral membrane K(+) conductance without increasing the apical membrane amiloride-sensitive Na(+) conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na(+) absorption with a further increase in Na(+)-K(+) ATPase transport activity and an increase in apical membrane amiloride-sensitive Na(+) conductance. The effect of insulin on the Na(+)-K(+) ATPase was the result of an increase in V(max) for extracellular K(+) and intracellular Na(+), and an increase in affinity of the pump for Na(+). Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of alpha-1 and alpha-2 isoforms, but not the alpha-3 isoform of Na(+)-K(+) ATPase, which did not change in the presence of insulin. Insulin-stimulated Na(+) transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)(3)], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na(+) transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3-kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na(+)-K(+) ATPase activation. (+info)
Reconstitution of protein kinase C-induced contractile Ca2+ sensitization in triton X-100-demembranated rabbit arterial smooth muscle.
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1. Triton X-100-demembranated smooth muscle loses Ca2+-sensitizing responsiveness to protein kinase C (PKC) activators while intact and alpha-toxin-permeabilized smooth muscles remain responsive. We attempted to reconstitute the contractile Ca2+ sensitization by PKC in the demembranated preparations. 2. Western blot analyses showed that the content of the PKC alpha-isoform (PKCalpha) was markedly reduced and that the smooth muscle-specific protein phosphatase-1 inhibitor protein CPI-17 was not detectable, while the amount of calponin and actin still remained similar to those of intact strips. 3. Unphosphorylated recombinant CPI-17 alone induced a small but significant contraction at constant Ca2+. Isoform-selective PKC inhibitors inhibited unphosphorylated but not pre-thiophosphorylated CPI-17-induced contraction, suggesting that in situ conventional PKC isoform(s) can phosphorylate CPI-17. 4. Exogenously replenishing PKCalpha alone did not induce potentiation of contraction and only slowly increased myosin light chain (MLC) phosphorylation at submaximal Ca2+. 5. PKC in the presence of CPI-17, but not the [T38A]-CPI mutant, markedly induced potentiation of both contraction and MLC phosphorylation. CPI-17 itself was phosphorylated. 6. In in vitro experiments, CPI-17 was a much better substrate for PKCalpha than calponin, caldesmon, MLC and myosin. 7. Our results indicate that PKC requires CPI-17 phosphorylation at Thr-38 but not calponin for reconstitution of the contractile Ca2+ sensitization in the demembranated arterial smooth muscle. (+info)
A low-Mr lipase activation factor cooperating with lipase modulator protein LimL in Pseudomonas sp. strain 109.
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Pseudomonas sp. strain 109 produces a unique lipase (LipL) which efficiently catalyses intramolecular transesterification of omega-hydroxyesters to form macrocyclic lactones. In vivo production of enzymically active LipL requires lipase modulator protein (LimL), which functions as a molecular chaperone for the correct folding of LipL. However, previous work has shown that LipL forms a tight complex with LimL in vitro and the resulting LipL-LimL complex is only partially active, suggesting an additional mechanism that facilitates the dissociation of the complex to form enzymically active LipL. In the present work, a low-Mr compound (lipase activation factor, LAF) was found in Pseudomonas sp. strain 109 that when added to the LipL-LimL complex resulted in the activation of LipL. Ca2+ ions also enhanced lipase activity, but the instantaneous activation by Ca2+ was different from the gradual and time-dependent activation by LAF, indicating the novel nature of this compound. LAF passed through an ultrafiltration membrane with an Mr cut-off of 3000 and showed an apparent Mr of 330+/-30 on Superdex Peptide gel-filtration chromatography. Treatment of the LipL-LimL complex with LAF liberated free active LipL, indicating that LAF was necessary to dissociate the LipL-LimL complex. (+info)