The CorA Mg2+ transport protein of Salmonella typhimurium. Mutagenesis of conserved residues in the third membrane domain identifies a Mg2+ pore

The CorA transport system is the major Mg2+ influx pathway for bacteria and the Archaea. CorA contains three C-terminal transmembrane segments. No conserved charged residues are apparent within the membrane, suggesting that Mg2+ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport. Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity. Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment. In contrast, mutation of Tyr292, Met299, and Tyr307 greatly altered the transport properties of CorA. Y292F, Y292S, Y292C, or Y292I mutations essentially abolished transport, without effect on expression or membrane insertion. M299C and M299A mutants exhibited a decrease in cation affinity for Mg2+, Co2+, or Ni2+ of 10-50-fold without a significant change in uptake capacity. Mutations at Tyr307 had no significant effect on cation uptake capacity; however, the affinity of Y307F and Y307A mutations for Mg2+ and Co2+ was decreased 3-10-fold, while affinity for Ni2+ was unchanged compared with the wild type CorA. In contrast, the affinity of the Y307S mutant for all three cations was decreased 2-5-fold. Projection of the third transmembrane segment as an alpha-helix suggests that Tyr292, Met299, and Tyr307 all reside on the same face of the alpha-helix. We interpret the transport data to suggest that a hydroxyl group is important at Tyr307, and that these three residues interact with Mg2+ during transport, forming part of the cation pore or channel within CorA.     

A randomized controlled study comparing elemental diet and steroid treatment in Crohn''s disease

To elucidate the mechanism underlying the interaction between the L-type Ca2+ channel and the dihydropyridines (DHPs), contribution of the repeat III was studied by constructing chimeras between the DHP-sensitive alpha1C and DHP-insensitive alpha1E subunits. The chimeras were transiently expressed in human embryonic kidney 293 cells and the whole-cell Ba2+ current (IBa) was recorded. Mutating Thr1061 to Tyr in IIIS5 of the alpha1C sequence completely abolished the inhibition and stimulation of IBa by the antagonist (+)-isradipine and agonist (-)-Bay K 8644, whereas mutating Gln1065 to Met in IIIS5 decreased the affinity for isradipine 100-fold without affecting the stimulating effect of Bay K 8644. The conserved amino acid residue Tyr1174 in IIIS6 of the alpha1C subunit was necessary for the high affinity DHP block. The DHP-dependent block and stimulation of IBa were transferred to the alpha1E channel by the mutation of two amino acid residues in IIIS5 (Y1295T, M1299Q), three residues in IIIS6 (F1406I, F1409I, V1414M) and three residues in IVS6 (I1706Y, F1707M, L1714I). The mutated alpha1E channel was stimulated 2.8-fold by 1 microM Bay K 8644 and blocked by isradipine with an IC50 value of 60 nM. These results show that mutation of Thr1061 in the alpha1C sequence results in a DHP-insensitive L-type channel and that transfer of the high affinity DHP sensitivity requires mutation of eight amino acid residues in the alpha1E sequence.     

Screening and characterization of microorganisms with glutaryl-7ADCA acylase activity

A screening of microorganisms producing glutaryl-7ADCA acylase, an enzyme able to hydrolyse glutaric acid selectively from glutaryl-3-deacetoxy-7-aminocephalosporanic acid (glutaryl-7ADCA), has been carried out in soil samples. Five microorganisms expressing acylase activity were isolated and classified as Bacillus cereus, Achromobacter xylosooxidans, Bacillus sp., Pseudomonas sp. and Pseudomonas paucimobilis. The screening was carried out by preparing enrichment cultures containing glutaryl-7-ADCA or cephalosporin C as the selective carbon source. Four model compounds (adipoyl-, glutamyl- and glutaryl-p-nitroanilide and glutarylcoumarin), mimicking the glutaryl-7ADCA beta-lactam moiety, were synthesized as substrates suitable for the rapid screening of the microorganisms (2500) isolated from the enrichment cultures. A total of 300 strains were active on the model substrates and only 5 displayed acylase activity on glutaryl-7ADCA. The fermentation parameters, such as pH and inducer concentration, for the optimal acylase expression and acylase specificity towards the model substrates were different for each strain.     

Diversity and properties of calcium channel types in NG108-15 hybrid cells

Several lysines (Lys) were determined to be involved in the regulation of the ADP-glucose (Glc) pyrophosphorylase from spinach leaf and the cyanobacterium Anabaena sp. PCC 7120 (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706-24711; Y. Charng, A.A. Iglesias, J. Preiss [1994] J Biol Chem 269: 24107-24113). Site-directed mutagenesis was used to investigate the relative roles of the conserved Lys in the heterotetrameric enzyme from potato (Solanum tuberosum L.) tubers. Mutations to alanine of Lys-404 and Lys-441 on the small subunit decreased the apparent affinity for the activator, 3-phosphoglycerate, by 3090- and 54-fold, respectively. The apparent affinity for the inhibitor, phosphate, decreased greater than 400-fold. Mutation of Lys-441 to glutamic acid showed even larger effects. When Lys-417 and Lys-455 on the large subunit were mutated to alanine, the phosphate inhibition was not altered and the apparent affinity for the activator decreased only 9- and 3-fold, respectively. Mutations of these residues to glutamic acid only decreased the affinity for the activator 12- and 5-fold, respectively. No significant changes were observed on other kinetic constants for the substrates ADP-Glc, pyrophosphate, and Mg2+. These data indicate that Lys-404 and Lys-441 on the small subunit are more important for the regulation of the ADP-Glc pyrophosphorylase than their homologous residues in the large subunit.     

Motif mutation of bradykinin B2 receptor second intracellular loop and proximal C terminus is critical for signal transduction, internalization, and resensitization

In the search for the structural elements participating in signal transduction, internalization, and resensitization of the bradykinin B2 receptor, we identified two critical motifs, one in the second intracellular loop (IC2), the other in the proximal C terminus. We previously described the contribution of tyrosines within each of the two motifs (Tyr131 and Tyr322) to signal transduction and receptor internalization (Prado, G. N., Taylor, L., and Polgar, P. (1997) J. Biol. Chem. 272, 14638-14642). Here, we investigate the effect of exchanging both tyrosine residues simultaneously for alanine, phenylalanine, or serine, termed YAYA (Y131A/Y322A), YFYF (Y131F/Y322F), and YSYS (Y131S/Y322S) receptors, respectively. All of these mutants bound bradykinin (BK) normally, with a Kd of approximately 1.1 nM. However, although phosphoinositide (PI) turnover in response to BK by Y131A and Y131S proved negligible, the YAYA mutant returned BK-activated PI turnover to wild type (WT). In contrast, PI turnover with YSYS remained unresponsive to BK. Importantly, the pattern of BK-activated arachidonate release differed markedly in the mutant receptors. For example, whereas Y131S ablated BK-activated arachidonic acid release, conversion of this mutant to YSYS returned the BK-activated receptor function to a level above that of WT. However, YAYA showed only a partial recovery from the poor BK response of Y131A. These and additional results suggest that Tyr131 and Tyr322 interact cooperatively in conjunction with at least two separate signaling functions. Given these results, a molecular model of the receptor was generated with the IC2 and the proximal C terminus in close spatial proximity. Conformations were identified to provide structural explanation for these observations. The conserved Thr137 in the IC2 was next substituted with proline (T137P) to prevent phosphorylation at this position or with aspartate (T137D) to emulate phosphorylation. The T137P mutant demonstrated no change from WT with respect to either BK-activated PI turnover or arachidonic acid release. However, the mutant exhibited a markedly reduced capacity to internalize. It also resensitized poorly. The T137D mutant lacked both BK responsive activities. However, it internalized and resensitized normally, as did WT. These final results suggest that Thr137 is functioning as a switch in termination of signal transduction and the initiation of internalization.     

Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells

Multidrug-resistant cancer cells display elevated levels of glucosylceramide (Lavie, Y., Cao, H. T., Volner, A., Lucci, A., Han, T. Y., Geffen, V., Giuliano, A. E., and Cabot, M. C. (1997) J. Biol. Chem. 272, 1682-1687). In this study, we have introduced glucosylceramide synthase (GCS) into wild type MCF-7 breast cancer cells using a retroviral tetracycline-on expression system, and we developed a cell line, MCF-7/GCS. MCF-7/GCS cells expressed an 11-fold higher level of GCS activity compared with the parental cell line. Interestingly, the transfected cells demonstrated strong resistance to adriamycin and to ceramide, whereas both agents were highly cytotoxic to MCF-7 cells. The EC50 values of adriamycin and ceramide were 11-fold (p < 0.0005) and 5-fold (p < 0.005) higher, respectively, in MCF-7/GCS cells compared with MCF-7 cells. Ceramide resistance displayed by MCF-7/GCS cells closely paralleled the activity of expressed GCS with a correlation coefficient of 0.99. In turn, cellular resistance and GCS activity were dependent upon the concentration of the expression mediator doxycycline. Adriamycin resistance in MCF-7/GCS cells was related to the hyperglycosylation of ceramide and was not related to shifts in the levels of either P-glycoprotein or Bcl-2. This work demonstrates that overexpression of GCS, which catalyzes ceramide glycosylation, induces resistance to adriamycin and ceramide in MCF-7 breast cancer cells.     

Kinetic and thermodynamic studies of purine repressor binding to corepressor and operator DNA

The kinetic and thermodynamic parameters for purine repressor (PurR)-operator and PurR-guanine binding were determined using fluorescence spectroscopy and nitrocellulose filter binding. Operator binding affinity was increased by the presence of guanine as demonstrated previously (Choi, K. Y., Lu, F., and Zalkin, H. (1994) J. Biol. Chem. 269, 24066-24072; Rolfes, R. J., and Zalkin, H. (1990) J. Bacteriol. 172, 5637-5642), and conversely guanine binding affinity was increased by the presence of operator. Guanine enhanced operator affinity by increasing the association rate constant and decreasing the dissociation rate constant for binding. Operator had minimal effect on the association rate constant for guanine binding; however, this DNA decreased the dissociation rate constant for corepressor by approximately 10-fold. Despite significant sequence and structural similarity between PurR and LacI proteins, PurR binds to its corepressor ligand with a lower association rate constant than LacI binds to its inducer ligand. However, the rate constant for PurR-guanine binding to operator is approximately 3-fold higher than for LacI binding to its cognate operator under the same solution conditions. The distinct metabolic roles of the enzymes under regulation by these two repressor proteins provide a rationale for the observed functional differences.     

Three-dimensional structure of a mutant HIV-1 protease displaying cross-resistance to all protease inhibitors in clinical trials

Analysis of mutational effects in the human immunodeficiency virus type-1 (HIV-1) provirus has revealed that as few as four amino acid side-chain substitutions in the HIV-1 protease (M46I/L63P/V82T/I84V) suffice to yield viral variants cross-resistant to a panel of protease inhibitors either in or being considered for clinical trials (Condra, J. H., Schleif, W. A., Blahy, O. M., Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). As an initial effort toward elucidation of the molecular mechanism of drug resistance in AIDS therapies, the three-dimensional structure of the HIV-1 protease mutant containing the four substitutions has been determined to 2.4-A resolution with an R factor of 17.1%. The structure of its complex with MK639, a protease inhibitor of the hydroxyaminopentane amide class of peptidomimetics currently in Phase III clinical trials, has been resolved at 2.0 A with an R factor of 17.0%. These structures are compared with those of the wild-type enzyme and its complex with MK639 (Chen, Z., Li, Y., Chen, E., Hall, D. L., Darke, P. L., Culberson, C., Shafer, J., and Kuo, L. C. (1994) J. Biol. Chem. 269, 26344-26348). There is no gross structural alteration of the protease due to the site-specific mutations. The C alpha tracings of the two native structures are identical with a root-mean-square deviation of 0.5 A, and the four substituted side chains are clearly revealed in the electron density map. In the MK639-bound form, the V82T substitution introduces an unfavorable hydrophilic moiety for binding in the active site and the I84V substitution creates a cavity (unoccupied by water) that should lead to a decrease in van der Waals contacts with the inhibitor. These changes are consistent with the observed 70-fold increase in the Ki value (approximately 2.5 kcal/mol) for MK639 as a result of the mutations in the HIV-1 protease. The role of the M46I and L63P substitutions in drug resistance is not obvious from the crystallographic data, but they induce conformational perturbations (0.9-1.1 A) in the flap domain of the native enzyme and may affect the stability and/or activity of the enzyme unrelated directly to binding.     

Effect of ligand conformation on melanoma cell alpha3beta1 integrin-mediated signal transduction events: implications for a collagen structural modulation mechanism of tumor cell invasion

The importance of three-dimensional interactions between receptors with their respective ligands has been extensively explored during the binding process, but considerably less so for postbinding events such as induction of signaling pathways. Tumor cell receptor association with basement membrane proteins is believed to facilitate the metastatic process. Melanoma and ovarian carcinoma cells have been shown to utilize the alpha3beta1 integrin to bind to models of the alpha1(IV)531-543 sequence from basement membrane (type IV) collagen [Miles, A. J., et al. (1994) J. Biol. Chem. 269, 30939-30945; Miles, A. J., et al. (1995) J. Biol. Chem. 270, 29047-29050]. In the present study, the effects of ligand three-dimensional structure on possible signal transduction pathways induced by alpha3beta1 integrin binding have been evaluated. Human melanoma cell binding to type IV collagen resulted in Tyr phosphorylation of p125(FAK), consistent with prior studies correlating beta1 integrin subunit binding to collagen and p125(FAK) Tyr phosphorylation. Cross-linking of an anti-alpha3 integrin subunit monoclonal antibody also induced p125(FAK) Tyr phosphorylation. Incubation of melanoma cells with single-stranded or triple-helical peptide models of alpha1(IV)531-543 induced Tyr phosphorylation of intracellular proteins. Immunoprecipitation analysis identified one of these proteins as pp125(FAK). Induction of p125(FAK) Tyr phosphorylation was enhanced and the time of induction was shortened when the ligand was used in triple-helical conformation. Subsequent clustering of either the single-stranded or the triple-helical ligand also increased the level of p125(FAK) phosphorylation compared to unclustered ligand. The clustered triple-helical peptide ligand induced more rapid paxillin Tyr phosphorylation than the single-stranded ligand. In addition, the induction of activated proteases was found to be more rapid due to ligand triple helicity. Overall, these studies have shown that (i) a model of an isolated sequence from type IV collagen, alpha1(IV)531-543, can induce alpha3beta1 integrin-mediated signal transduction in melanoma cells and (ii) ligand conformation (secondary, tertiary, and/or quaternary structure) can directly influence several alpha3beta1 integrin-mediated signal transduction events. The effects of ligand conformation suggest that a "collagen structural modulation" mechanism may exist for tumor cell invasion, whereby triple-helical collagen promotes cell binding and induction of signal transduction, subsequently leading to collagen dissolution by proteases, decreased signal transduction, and enhanced tumor cell motility.     

The axial tyrosinate Fe3+ ligand in protocatechuate 3,4-dioxygenase influences substrate binding and product release: evidence for new reaction cycle intermediates

The essential active site Fe3+ of protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown that Tyr447 is dissociated from the Fe3+ in the anaerobic 3,4-PCD complex with protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here by site-directed mutation of this residue to His (Y447H), the first such mutation reported for an aromatic ring cleavage dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181) is essentially unchanged from that of the native enzyme outside of the active site region. The side chain position of His447 is stabilized by a His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827 appears to be replaced by a CO32-, thereby retaining the overall charge neutrality and coordination number of the Fe3+ center. Quantitative metal and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than that of the native enzyme. Single-turnover kinetic analysis of the Y447H-catalyzed reaction reveals that slow substrate binding accounts for the decreased kcat. Three new kinetically competent intermediates in this process are revealed. Similarly, the product dissociation from Y447H is slow and occurs in two resolved steps, including a previously unreported intermediate. The final E.PCA complex (ES4) and the putative E.product complex (ESO2*) are found to have optical spectra that are indistinguishable from those of the analogous intermediates of the wild-type enzyme cycle, while all of the other observed intermediates have novel spectra. Once the E.S complex is formed, reaction with O2 is fast. These results suggest that dissociation of Tyr447 occurs during turnover of 3,4-PCD and is important in the substrate binding and product release processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex by either dissociation or mutagenesis, the O2 attack and insertion steps proceed efficiently, suggesting that Tyr447 does not have a large role in this phase of the reaction. This study demonstrates a novel role for Tyr in a biological system and allows evaluation and refinement of the proposed Fe3+ dioxygenase mechanism.     

Role of active site tyrosine residues in catalysis by human glutathione reductase

Tyr114 and Tyr197 are highly conserved residues in the active site of human glutathione reductase, Tyr114 in the glutathione disulfide (GSSG) binding site and Tyr197 in the NADPH site. Mutation of either residue has profound effects on catalysis. Y197S and Y114L have 17% and 14% the activity of the wild-type enzyme, respectively. Mutation of Tyr197, in the NADPH site, leads to a decrease in Km for GSSG, and mutation of Tyr114, in the GSSG site, leads to a decrease in Km for NADPH. This behavior is predicted for enzymes operating by a ping-pong mechanism where both half-reactions partially limit turnover. Titration of the wild-type enzyme or Y114L with NADPH proceeds in two phases, Eox to EH2 and EH2 to EH2-NADPH. In contrast, Y197S reacts monophasically, showing that excess NADPH fails to enhance the absorbance of the thiolate-FAD charge-transfer complex, the predominant EH2 form of glutathione reductase. The reductive half-reactions of the wild-type enzyme and of Y114L are similar; FAD reduction is fast (approximately 500 s-1 at 4 degreesC) and thiolate-FAD charge-transfer complex formation has a rate of 100 s-1. In Y197S, these rates are only 78 and 5 s-1, respectively. The oxidative half-reaction, the rate of reoxidation of EH2 by GSSG, of the wild-type enzyme is approximately 4-fold faster than that of Y114L. These results are consistent with Tyr197 serving as a gate in the binding of NADPH, and they indicate that Tyr114 assists the acid catalyst His467'.     

A new protein conjugation system in human. The counterpart of the yeast Apg12p conjugation system essential for autophagy

Autophagy is an intracellular process for bulk degradation of cytoplasmic components. We recently found a protein conjugation system essential for autophagy in the yeast, Saccharomyces cerevisiae. The C-terminal glycine of a novel modifier protein, Apg12p, is conjugated to a lysine residue of Apg5p via an isopeptide bond. This conjugation reaction is mediated by Apg7p, a ubiquitin activating enzyme (E1)-like enzyme, and Apg10p, suggesting that it is a ubiquitination-like system (Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George, M. D., Klionsky, D. J., Ohsumi, M. , and Ohsumi, Y. (1998) Nature 395, 395-398). Although autophagy is a ubiquitous process in eukaryotic cells, no molecule involved in autophagy has yet been identified in higher eukaryotes. We reasoned that this conjugation system could be conserved. Here we report cloning and characterization of the human homologue of Apg12 (hApg12). It is a 140-amino acid protein and possesses 27% identity and 48% similarity with the yeast Apg12p, but no apparent homology to ubiquitin. Northern blot analysis showed that its expression was ubiquitous in human tissues. We found that it was covalently attached to another protein. This target protein was identified to be the human Apg5 homologue (hApg5). Mutagenic analyses suggested that this conjugation was formed via an isopeptide bond between the C-terminal glycine of hApg12 and Lys-130 of hApg5. These findings indicate that the Apg12 system is well conserved and may function in autophagy also in human cells.     

Microalbuminuria, arterial hypertension and the cardiovascular risk

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc-derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.     

Localization of the glucosyltransferase activity of Clostridium difficile toxin B to the N-terminal part of the holotoxin

Clostridium difficile toxin B that is one of the largest cytotoxins (270 kDa) known acts on Rho subfamily proteins by monoglucosylation (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503). By deletion analysis we identified the enzyme and cytotoxic activity of the toxin to be located at the N terminus of the holotoxin. A 63-kDa fragment of toxin B covering the first 546 amino acid residues glucosylated Rho, Rac, and Cdc42, but not Ras, by using UDP-glucose as a cosubstrate. As known for the holotoxin, glucosylation by the toxin fragment was favored with the GDP-bound form of the low molecular mass GTPases. Microinjection of the toxin fragment into NIH-3T3 cells induced rounding up of cells and redistribution of the actin cytoskeleton. In contrast, a toxin fragment encompassing the first 516 amino acid residues was at least 1000-fold less active than toxin fragment 1-546 and cytotoxically inactive. The data give direct evidence for location of the enzyme activity of C. difficile toxin B at the N-terminal 546 amino acids residues and indicate a functionally and/or structurally important role of the region from amino acid residues 516 through 546 for enzyme and cytotoxic activities.     

Identification of tissue-type plasminogen activator-specific plasminogen activator inhibitor-1 mutants. Evidence that second sites of interaction contribute to target specificity

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of the plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). A library of PAI-1 mutants containing substitutions at the P1 and P1' positions was screened for functional activity against tPA and thrombin. Several PAI-1 variants that were inactive against uPA in a previous study (Sherman, P. M., Lawrence, D. A., Yang, A. Y., Vandenberg, E. T., Paielli, D., Olson, S. T., Shore, J. D., and Ginsburg, D. (1992) J. Biol. Chem. 267, 7588-7595) had significant inhibitory activity toward tPA. This set of tPA-specific PAI-1 mutants contained a wide range of amino acid substitutions at P1 including Asn, Gln, His, Ser, Thr, Leu, Met, and all the aromatic amino acids. This group of mutants also demonstrated a spectrum of substitutions at P1'. Kinetic analyses of selected variants identified P1Tyr and P1His as the most efficient tPA-specific inhibitors, with second-order rate constants (ki) of 4.0 x 10(5) M-1s-1 and 3.6 x 10(5) M-1s-1, respectively. Additional PA-specific PAI-1 variants containing substitutions at P3 through P1' were constructed. P3Tyr-P2Ser-P1Lys-P1'Trp and P3Tyr-P2Ser-P1Tyr-P1'Met had ki values of 1.7 x 10(6) M-1s-1 and 2.5 x 10(6) M-1s-1 against tPA, respectively, but both were inactive against uPA. In contrast, P2Arg-P1Lys-P1'Ala inhibited uPA 74-fold more rapidly than tPA. The mutant PAI-1 library was also screened for inhibitory activity toward thrombin in the presence and absence of the cofactor heparin. While wild-type PAI-1 and several P1Arg variants inhibited thrombin in the absence of heparin, a number of variants were thrombin inhibitors only in the presence of heparin. These results demonstrate the importance of the reactive center residues in determining PAI-1 target specificity and suggest that second sites of interaction between inhibitors and proteases can also contribute to target specificity. Finally, the PA-specific mutants described here should provide novel reagents for dissecting the physiological role of PAI-1 both in vitro and in vivo.     

Classification of amino acid spin systems using PFG HCC(CO)NH-TOCSY with constant-time aliphatic 13C frequency labeling

We have developed a useful strategy for identifying amino acid spin systems and side-chain carbon resonance assignments in small 15N-, 13C-enriched proteins. Multidimensional constant-time pulsed field gradient (PFG) HCC(CO)NH-TOCSY experiments provide side-chain resonance frequency information and establish connectivities between sequential amino acid spin systems. In PFG HCC(CO)NH-TOCSY experiments recorded with a properly tuned constant-time period for frequency labeling of aliphatic 13C resonances, phases of cross peaks provide information that is useful for identifying spin system types. When combined with 13C chemical shift information, these patterns allow identification of the following spin system types: Gly, Ala, Thr, Val, Leu, Ile, Lys, Arg, Pro, long-type (i.e., Gln, Glu and Met), Ser, and AMX-type (i.e., Asp, Asn, Cys, His, Phe, Trp and Tyr).     

Isolated systolic hypertension: an important cardiovascular risk factor

BACKGROUND: The rising number of vancomycin-resistant enterococci (VREs) is a major concern to modern medicine because vancomycin is currently the 'last resort' drug for life-threatening infections. The D-alanyl-D-X ligases (where X is an hydroxy or amino acid) of bacteria catalyze a critical step in bacterial cell-wall peptidoglycan assembly. In bacteria that produce glycopeptide antibiotics and in opportunistic pathogens, including VREs, D-, D-ligases serve as switches that confer antibiotic resistance on the bacteria themselves. Peptidoglycans in vancomycin-sensitive bacteria end in D-alanyl-D-alanine, whereas in vancomycin-resistant bacteria they end in D-alanyl-D-lactate or D-alanyl-D-serine. RESULTS: We demonstrate that the selective utilization of D-serine by the Enterococcus casseliflavus VanC2 ligase can be altered by mutagenesis of one of two residues identified by homology to the X-ray structure of the Escherichia coli D-alanyl-Dalanine ligase (DdlB). The Arg322-->Met (R322M) and Phe250-->Tyr (F250Y) ligase mutants show a 36-44-fold decrease in the use of D-serine, as well as broadened specificity for utilization of other D-amino acids in place of D-serine. The F250Y R322M double mutant is effectively disabled as a D-alanyl-D-serine ligase and retains 10% of the catalytic activity of wild-type D-alanyl-D-alanine ligases, reflecting a 6,000-fold switch to the D-alanyl-D-alanine peptide. Correspondingly, the Leu282-->Arg mutant of the wild-type E. coli DdlB produced a 560-fold switch towards D-alanyl-D-serine formation. CONCLUSIONS: Single-residue changes in the active-site regions of D-, D-ligases can cause substantial changes in recognition and activation of hydroxy or amino acids that have consequences for glycopeptide antibiotic efficacy. The observations reported here should provide an approach for combatting antibiotic-resistant bacteria.     

Sorting of two polytopic proteins, the gamma-aminobutyric acid and betaine transporters, in polarized epithelial cells

The gamma-aminobutyric acid transporter (GAT-1) isoform of the gamma-aminobutyric acid and the betaine (BGT) transporters exhibit distinct apical and basolateral distributions when introduced into Madin-Darby canine kidney cells (Pietrini, G., Suh, Y. J., Edelman, L., Rudnick, G., and Caplan, M. J. (1994) J. Biol. Chem. 269, 4668-4674). We have investigated the presence of sorting signals in their COOH-terminal cytosolic domains by expression in Madin-Darby canine kidney cells of mutated and chimeric transporters. Whereas truncated GAT-1 (DeltaC-GAT) maintained the original functional activity and apical localization, either the removal (DeltaC-myc BGT) or the substitution (BGS chimera) of the cytosolic tail of BGT generated proteins that accumulated in the endoplasmic reticulum. Moreover, we have found that the cytosolic tail of BGT redirected apical proteins, the polytopic GAT-1 (GBS chimera) and the monotopic human nerve growth factor receptor, to the basolateral surface. These results suggest the presence of basolateral sorting information in the cytosolic tail of BGT. We have further shown that information necessary for the exit of BGT from the endoplasmic reticulum and for the basolateral localization of the GBS chimera is contained in a short segment, rich in basic residues, within the cytosolic tail of BGT.