Deletion mapping of N-terminal domains of surfactant protein A. The N-terminal segment is required for phospholipid aggregation and specific inhibition of surfactant secretion

The objective of the current study was to examine the functional importance of the N-terminal domains of surfactant protein A (SP-A) including the N-terminal segment from Asn1 to Ala7 (denoted domain 1), the N-terminal portion of the collagen domain from Gly8 to Gly44 (domain 2), and the C-terminal portion of the collagen-like domain from Gly45 to Pro80 (domain 3). Wild type recombinant SP-A (SP-Ahyp; where hyp indicates hydroxyproline-deficient) and truncated mutant (TM) SP-As containing deletions of domain(s) 1 (TM1), 2 (TM2), 1 and 2 (TM1-2), and 1, 2, and 3 (TM1-2-3) were synthesized in insect cells and purified by mannose-Sepharose affinity chromatography. N-terminal disulfide-dependent dimerization was preserved at near wild type levels in the TM1-2 (at Cys-1) and TM2 proteins (at Cys-1 and Cys6), and to a lesser extent in TM1 (at Cys-1), but not in TM1-2-3. Cross-linking analyses demonstrated that the neck + CRD was sufficient for assembly of monomers into noncovalent trimers and that the N-terminal segment was required for the association of trimers to form higher oligomers. All TM proteins except TM1-2-3 bound to phospholipid, but only the N-terminal segment containing TM proteins aggregated phospholipid vesicles. The TM1, TM1-2, and TM2 but not the TM1-2-3 inhibited the secretion of surfactant from type II cells as effectively as SP-Ahyp, but the inhibitory activity of each mutant was blocked by excess alpha-methylmannoside and therefore nonspecific. TM1 and TM1-2-3 did not enhance the uptake of phospholipids by isolated type II cells, but the TM1-2 and TM2 had activities that were 72 and 83% of SP-Ahyp, respectively. We conclude the following for SP-A: 1) trimerization does not require the collagen-like region or interchain disulfide linkage; 2) the N-terminal portion of the collagen-like domain is required for specific inhibition of surfactant secretion but not for binding to liposomes or for enhanced uptake of phospholipids into type II cells; 3) N-terminal interchain disulfide linkage can functionally replace the N-terminal segment for lipid binding, receptor binding, and enhancement of lipid uptake; 4) the N-terminal segment is required for the association of trimeric subunits into higher oligomers, for phospholipid aggregation, and for specific inhibition of surfactant secretion and cannot be functionally replaced by disulfide linkage alone for these activities.     

Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity

Small granules of horseshoe crab hemocytes contain two known major antimicrobial substances, tachyplesin and big defensin (S5), and at least five protein components (S1 to S6), with unknown functions. In the present study, we examined the biological properties and primary structure of a small granular component S2, named tachycitin. This component was purified from the acid extract of hemocyte debris by two steps of chromatography. The purified tachycitin was a single chain protein with an apparent M(r) = 8,500 on Tricine-SDS-polyacrylamide gel electrophoresis. Ultracentrifugation analysis revealed tachycitin to be present in monomer form in solution. Tachycitin inhibited the growth of both Gram-negative and -positive bacteria, and fungi, with a bacterial agglutinating property. Moreover, tachycitin and big defensin acted synergistically in antimicrobial activities. The amino acid sequence and intrachain disulfide bonds of tachycitin were determined by amino acid and sequence analyses of peptides produced by enzymatic cleavages. The mature tachycitin consisted of 73 amino acid residues containing five disulfide bonds with no N-linked sugar. A cDNA coding for tachycitin was isolated from a hemocyte cDNA library. The open reading frame coded for an NH2-terminal signal sequence followed by the mature peptide and an extension sequence of -Gly-Arg-Lys at the COOH-terminus, which is a putative amidating signal. The COOH-terminal threonine amide released after digestion of tachycitin with lysylendopeptidase was identified. The NH2-terminal 28 residues of tachycitin shows sequence homology to a part of chitin-binding regions found in antifungal chitin-binding peptides, chitin-binding lectins, and chitinases, all of which have been isolated from plants. Tachycitin showed a specific binding to chitin but did not bind with the polysaccharides cellulose, mannan, xylan, and laminarin. Tachycitin may represent a new class of chitin-binding protein family in animals.     

The gamma-carboxylation recognition site is sufficient to direct vitamin K-dependent carboxylation on an adjacent glutamate-rich region of thrombin in a propeptide-thrombin chimera

The propeptides of the vitamin K-dependent proteins contain a gamma-carboxylation recognition site that is required for gamma-glutamyl carboxylation. To determine whether the propeptide is sufficient to direct carboxylation, two mutant prothrombin species were expressed and characterized with regard to posttranslational gamma-carboxylation. A double point mutant, in which serine substituted for cysteines 17 and 22 disrupted a conserved loop formed by a disulfide bond, was fully carboxylated when expressed in Chinese hamster ovary cells. A propeptide/thrombin chimeric protein, constructed by deleting the Gla, aromatic amino acid stack, and kringle domains of prothrombin, has the signal peptide and propeptide juxtaposed to a glutamate-rich COOH-terminal region of prothrombin, residues 249-530. Of the 8 glutamic acid residues contained within the first 40 residues of the NH2 terminus adjacent to the propeptide, at least seven were fully carboxylated as demonstrated by direct gamma-carboxyglutamic acid analysis of the alkaline hydrolysate and by NH2-terminal sequence analysis. These results indicate that the gamma-carboxylation recognition site within the prothrombin propeptide in a prothrombin propeptide-thrombin chimeric protein is sufficient to direct gamma-carboxylase-catalyzed carboxylation of adjacent glutamic acid residues in a glutamate-rich region of thrombin that is not normally gamma-carboxylated. Furthermore, the disulfide loop in the Gla domain of prothrombin is not required for complete carboxylation.     

Aberrant intermolecular disulfide bonding in a mutant HLA-DM molecule: implications for assembly, maturation, and function

HLA-DM (abbreviated DM) is an MHC-encoded glycoprotein that catalyzes the selective release of peptides, including class II-associated invariant chain peptides, from MHC class II molecules. To perform its function, DM must assemble in the endoplasmic reticulum (ER), travel to endosomes, and interact productively with class II molecules. We have described previously an EBV-transformed B cell line, 7.12.6, which displays a partial Ag presentation defect and expresses a mutated DM beta-chain with Cys79 replaced by Tyr. In this study, we show that HLA-DR molecules in 7.12.6 have a defect in peptide loading and accumulate class II-associated invariant chain peptides (CLIP). Peptide loading is restored by transfection of wild-type DMB. The mutant DM molecules exit the ER slowly and are degraded rapidly, resulting in greatly reduced levels of mutant DM in post-Golgi compartments. Whereas wild-type DM forms noncovalent alphabeta dimers, such dimers form inefficiently in 7.12.6; many mutant DM beta-chains instead form a disulfide-bonded dimer with DM alpha. Homodimers of DM beta are also detected in 7.12.6 and in the alpha-chain defective mutant, 2.2.93. We conclude that during folding of wild-type DM, the native conformation is stabilized by a conserved disulfide bond involving Cys79beta and by noncovalent contacts with DM alpha. Without these interactions, DM beta can form malfolded structures containing interchain disulfide bonds; malfolding is correlated with ER retention and accelerated degradation.     

Disulfide bonds in recombinant human platelet-derived growth factor BB dimer: characterization of intermolecular and intramolecular disulfide linkages

Interchain cystines of PDGF-BB dimer were characterized by Edman reaction and by SDS-PAGE analysis on the protein which was chemically cleaved at Trp-40. It was found that Cys-43 has a key role in dimer formation, asymmetrically cross-linked to a cysteine residue of another identical subunit. The remaining cystines participate in the intramolecular disulfide linkages. Pepsin digestion of PDGF-BB dimer generated several small peptides and one ubiquitous Cys-containing peptide. Sequence analyses of several Cys-containing peptides indicated the existence of three intramolecular disulfide linkages including Cys-16--Cys-60, Cys-49--Cys-97, and Cys-53--Cys-99. Two interchain disulfide bonds of Cys-43--Cys-52 between two subunits were deduced from the partial reduction and alkylation of PDGF-BB. This study provides chemically determined disulfide linkages of PDGF-BB.     

Localization of an insulin-like growth factor (IGF) binding site of bovine IGF binding protein-2 using disulfide mapping and deletion mutation analysis of the C-terminal domain

We have investigated which region(s) of bovine insulin-like growth factor binding protein-2 (bIGFBP-2) interact with insulin-like growth factors (IGFs) using C-terminally truncated forms of bIGFBP-2. Initially to aid in mutant design, we defined the disulfide bonding pattern of bIGFBP-2 C-terminal region using enzymatic digestion. The pattern is Cys186-Cys220, Cys231-Cys242, and Cys244-Cys265. In addition, cyanogen bromide cleavage of bIGFBP-2 revealed that the N- and C-terminal cysteine-rich domains were not linked by disulfide bonds. Taking the disulfide bonding pattern into consideration, C-terminal truncation mutants were designed and expressed in COS-1 mammalian cells. Following IGF binding assays, a region between residues 222 and 236 was identified as important in IGF binding. Specifically, mutants truncated by 14, 36, and 48 residues from the C terminus bound IGFs to the same extent as wild type (WT) bIGFBP-2. Removal of 63 residues resulted in a greatly reduced (up to 80-fold) ability to bind IGF compared with WT bIGFBP-2. Interestingly this mutant lacked the IGF-II binding preference of WT bIGFBP-2. Residues 236-270 also appeared to play a role in determining IGF binding specificity as their removal resulted in mutants with higher IGF-II binding affinity.     

Combined aortorenal reconstruction: is there an optimum method of exposure?

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluorescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluorescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA-beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead-linked library and a peptide containing the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized.     

Synthesis and conformational properties of a recombinant C-propeptide of human type III procollagen

A cDNA was prepared that coded for the signal peptide of type III procollagen linked to the complete C-propeptide of the protein. The cDNA was then used to express the protein in a baculovirus recombinant system. Recombinant protein was recovered as a trimer from the medium of transfected cells in a yield of 1 to 2.5 mg per liter. Mapping of peptide fragments with and without reduction indicated that the protein contained the expected interchain disulfide bonds. Analysis by circular dichroism suggested that the conformation of the protein corresponded to the native conformation. Therefore, the protein should be appropriate for further tests of its biological function and analysis of structure by X-ray diffraction.     

Functional analysis of conserved cysteines in heparan sulfate N-deacetylase-N-sulfotransferases

N-Deacetylase-N-sulfotransferases (NDANST) catalyze the two initial modifications of the polysaccharide precursor in the biosynthesis of heparin and heparan sulfate. These modifications are the gating steps in establishing growth factor protein-binding domains of these glycosaminoglycans. We have undertaken a structure-activity analysis of the 841-amino acid Golgi-luminal portion of the rat liver NDANST to localize the two enzymatic functions. Each activity can be assayed in vitro independently of the other when provided with the appropriate substrate, and N-ethylmaleimide treatment selectively inactivates the deacetylase activity. In this study, dithiothreitol treatment of the rat liver NDANST was shown to inactivate the sulfotransferase function, while stimulating deacetylase activity 2-3-fold over the native protein. Site-directed mutagenesis of the eight cysteine (Cys) residues in the rat liver NDANST that are conserved in the mouse mastocytoma protein produced three important findings regarding the localization of each enzymatic function: 1) derivatization of Cys486 with N-ethylmaleimide resulted in total inactivation of the deacetylase activity based on steric hindrance of the active site (this residue was shown not to be involved in enzymatic catalysis), 2) substitution of either Cys159 or Cys486 with alanine resulted in enhanced activity of the deacetylase to the level obtained by dithiothreitol treatment, and 3) alanine substitution of Cys818 or Cys828 completely inactivated the sulfotransferase activity, while substitution of Cys586 or Cys601 resulted in a 90% loss in activity. These findings suggest that the two enzymatic domains within the NDANST localize to different portions of the protein, with two disulfide pairs toward the COOH-terminal half of the protein necessary for the sulfotransferase activity, and Cys residues within the NH2-terminal half influencing or located near the active site of the deacetylase functionality.     

Mutagenesis of cardiac troponin I. Role of the unique NH2-terminal peptide in myofilament activation

Phosphorylation of Ser residues in the NH2-terminal extension unique to cardiac troponin I (cTnI) is known to occur through protein kinase A and to alter myofilament Ca2+ activation (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Yet, how the NH2-terminal extension may itself affect thin filament Ca2+ signaling is unknown. To approach this question we have used molecular cloning, mutagenesis, and bacterial synthesis of a full-length cTnI and a truncated mutant (cTnI/NH2) missing the 32 amino acids. Using reconstituted preparations we could show no differences between cTnI and cTnI/NH2 either in inhibition of actomyosin ATPase activity, in Ca(2+)-reversible inhibitory activity, or in the relation between pCa and Ca2+ binding to the regulatory site of cTnC at either pH 7.0 or 6.5. There were also no significant differences at either pH in the pCa-MgATPase activity relation of myofibrils into which the various species of TnI has been exchanged. Our results indicate: 1) that phosphorylation most likely induces a new state of TnI activity rather than altering an intrinsic effect of the NH2-terminal peptide on Ca2+ activation; and 2) that domains outside the NH2-terminal extension are important with regard to differences in effects of acidic pH on Ca2+ activation on cardiac and skeletal myofilaments.     

Oxidative regeneration and selective reduction of native disulfide bonds in the N-terminal half-molecule of ovotransferrin

The denatured, disulfide-reduced form of the N-terminal half-molecule of ovotransferrin was reoxidized with either oxidized dithiothreitol or GSSG and analyzed for the localization of disulfide bonds. Chemical analyses of the reoxidized proteins revealed that the disulfide peptides corresponding to the six native protein disulfides (SS-I, SS-II, SS-III, SS-IV, SS-V, and SS-VI) are all regained in the reoxidized protein. The peptide recoveries from the reoxidized proteins were, however, about half of those from the native protein with respect to the two inner disulfides (SS-IV and SS-V) in the kringle bridges, but all the disulfide peptides corresponding to the remaining disulfides (SS-I, SS-II, SS-III, and SS-VI) were recovered at almost equivalent yields in the native and reoxidized proteins. In addition, on searching for a nonnative disulfide peptide, the two disulfides, Cys171-Cys174 and Cys174-Cys182, which can be accounted for by mispaired bridges of sulfhydryls in SS-IV and SS-V, were detected in the protein reoxidized with oxidized dithiothreitol. Upon disulfide reduction of the native protein with reduced dithiothreitol, both SS-IV and SS-V were selectively cleaved under the same buffer and temperature conditions as in the oxidative refolding. The lower stabilities of the two inner disulfide bonds in the kringle may be related to the lower recoveries of the disulfide peptides from SS-IV and SS-V and the generation of the nonnative disulfide bonds.     

Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium

The catalytic properties of cysteine residues Cys46 and Cys165, which form intersubunit disulfide bonds in the peroxidatic AhpC protein of the alkyl hydroperoxide reductase (AhpR) system from Salmonella typhimurium, have been investigated. The AhpR system, composed of AhpC and a flavoprotein reductase, AhpF, catalyzes the pyridine nucleotide-dependent reduction of organic hydroperoxides and hydrogen peroxide. Amino acid sequence analysis of the disulfide-containing tryptic peptide demonstrated the presence of two identical disulfide bonds per dimer of oxidized AhpC located between Cys46 on one subunit and Cys165 on the other. Mutant AhpC proteins containing only one (C46S and C165S) or no (C46,165S) cysteine residues were purified and shown by circular dichroism studies to exhibit no major disruptions in secondary structure. In NADH-dependent peroxidase assays in the presence of AhpF, the C165S mutant was fully active in comparison with wild-type AhpC, while C46S and C46,165S displayed no peroxidatic activity. In addition, only C165S was oxidized by 1 equiv of hydrogen peroxide, giving a species that was stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF. Oxidized C165S also reacted rapidly with a stoichiometric amount of the thiol-containing reagent 2-nitro-5-thiobenzoic acid to generate a mixed disulfide, and was susceptible to inactivation by hydrogen peroxide, strongly supporting its identification as a cysteine sulfenic acid (Cys46-SOH). The lack of reactivity of the C46S mutant toward peroxides was not a result of inaccessibility of the remaining thiol as demonstrated by its modification with 5, 5'-dithiobis(2-nitrobenzoic acid), but could be due to the lack of a proximal active-site base which would support catalysis through proton donation to the poor RO- leaving group. Our results clearly identify Cys46 as the peroxidatic center of AhpC and Cys165 as an important residue for preserving the activity of wild-type AhpC by reacting with the nascent sulfenic acid of the oxidized protein (Cys46-SOH) to generate a stable disulfide bond, thus preventing further oxidation of Cys46-SOH by substrate.     

Fatty acylation of the rat and human asialoglycoprotein receptors. A conserved cytoplasmic cysteine residue is acylated in all receptor subunits

Functional rat or human asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomeric integral membrane glycoproteins. Rat ASGP-R contains three subunits, designated rat hepatic lectins (RHL) 1, 2, and 3; human ASGP-R contains two subunits, HHL1 and HHL2. Both receptors are covalently modified by fatty acylation (Zeng, F.-Y., Kaphalia, B. S., Ansari, G. A. S., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21382-21387; Zeng, F.-Y., Oka, J. A., and Weigel, P. H. (1996) Biochem. Biophys. Res. Commun. 218, 325-330). We report here that the single Cys residue in the cytoplasmic domain of each RHL or HHL subunit is fatty acylated. The degree of acylation is >/=90% per subunit. Deacylation of affinity-purified ASGP-Rs with hydroxylamine results in the spontaneous formation of dimers through reversible disulfide bonds, indicating that deacylation concomitantly generates free thiol groups. Reaction of hydroxylamine-treated ASGP-R with [14C]iodoacetamide resulted in the specific incorporation of radioactivity into all RHL and HHL subunits, verifying that fatty acids are attached via thioester linkages. To identify the Cys residue involved in the thioester linkages, 14C-carboxyamidomethylated RHL subunits were separated by SDS-polyacrylamide gel electrophoresis and digested in-gel with trypsin, and the resulting peptides were separated by reverse-phase high performance liquid chromatography. Amino acid sequence of radioactive peptides revealed that Cys35 in RHL1 and Cys54 in RHL2 and RHL3 were radiolabeled and, therefore, are fatty acylation sites. Fatty acylation of HHL subunits was analyzed by site-directed mutagenesis. Metabolic labeling of Cos7 cells transfected with wild type HHL1 cDNA resulted in substantial incorporation of [3H]palmitate into purified HHL1. Incorporation of [3H]palmitate into a C36S mutant of HHL1 was negligible ( approximately 1%) compared with wild type. This result also shows that Cys57 within the transmembrane domain of HHL1 is not normally palmitoylated. We conclude that Cys35 in RHL1, Cys54 in RHL2 and RHL3, and Cys36 in HHL1 are fatty acylated. Cys57 in HHL1 and probably Cys56 in RHL1 are not palmitoylated.     

Opening of ATP-sensitive K+ channels responsible for adenosine A2 receptor-mediated vasodepression does not involve a pertussis toxin-sensitive G protein

The structure of recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor expressed in CHO cells was investigated. The bioactive protein ([-32-153]M-CSF), expressed from a nucleotide sequence that encoded a signal peptide of 32 amino acids and N-terminal amino acids numbers 1-153, was heterogeneous in terms of molecular mass, as analyzed by SDS-PAGE, because of the presence of N-linked sugar moieties. The primary structure of the polypeptide was determined by sequence analysis and amino acid analysis of the fragments obtained from lysylendopeptidase digests of reduced and alkylated M-CSF, and from pepsin digests of the intact molecule. A sugar chain was located only at Asn-122 of the two putative sites of N-glycosylation that were present per subunit. The homodimeric structure appeared to have seven disulfide bonds, formed by inter- or intra-molecular linkages, since there were no free thiol groups in the molecule. The assignment of disulfide bonds by sequence analysis using peptide fragments indicated the combinations of Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146. Gel-filtration analysis of Ser31[-32-153]M-CSF, in which the remaining Cys31 was replaced by Ser and which was expressed in COS cells, suggested that the mutein existed as a monomer. Our study shows that the disulfide-bond pairings of [-32-153]M-CSF that is expressed and post-translationally modified in mammalian cells are identical to those of Escherichia coli-derived [3-153]M-CSF with only one intermolecular disulfide bond, namely, Cys31-Cys31.     

Nonparametric estimation of covariance structure in longitudinal data

The three possible disulfide bonded isomers of alpha-conotoxin GI have been selectively synthesised and their structures determined by 1H NMR spectroscopy. alpha-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2-7;3-13), GI(2-13;3-7) and GI(2-3;7-13). The NMR structure for the native isomer GI(2-7;3-13) is of excellent quality, with a backbone pairwise RMSD of 0.16 A for a family of 35 structures, and comprises primarily a distorted 310 helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2-7;3-13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in alpha-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2-7;3-13)相似文献   

Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2

Cytochrome P4501A1 is a hepatic, microsomal membrane-bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from beta-naphthoflavone-induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30-amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33-44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.     

Assignment of the inter- and intramolecular disulfide linkages in recombinant human macrophage colony stimulating factor using fast atom bombardment mass spectrometry

The disulfide bridges in recombinant human macrophage colony stimulating factor (rhM-CSF), a 49-kDa homodimeric protein, were assigned. The 18 cysteines in the dimer form three intermolecular and two sets of three intramolecular disulfide bonds. The intermolecular disulfide bridges hold the dimer together and form symmetric bonds in which Cys31 and Cys157/Cys159 from one monomer unit are linked to the corresponding cysteines of the second monomer. The intramolecular disulfide bonds are located between Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146, respectively. The resistance of native M-CSF to proteolytic cleavage was overcome by an initial chemical cleavage reaction using BrCN. The close proximity of four cysteines (Cys139, Cys146, Cys157, and Cys159) results in a tight core complex that makes the protein undigestable for most proteases. Digestion using endoprotease Asp-N resulted in cleavage at Asp156 near the C-terminal end of this region, thereby opening the complex structure.     

A peptide corresponding to residues Asp177 to Asn208 of human cyclin A forms an alpha-helix

Protein disulfide isomerase (PDI), the product of the essential PDI1 gene of Saccharomyces cerevisiae catalyzes oxidization of thiols, reduction of disulfide bonds, and isomerization of disulfides. It can also act as a chaperone to facilitate folding of denatured proteins. The protein has 6 cysteine (Cys) residues. Four of these Cys are part of the 2 thioredoxin-like catalytic sites (-CGHC-), one of which is located near the N- and the other near the C-terminus. In addition, it has 2 non-active site Cys near the N-terminus. The function of these non-active site Cys of yeast PDI is poorly understood. Whereas in yeast PDI, these Cys residues are in the vicinity of the N-terminal-most active site, in mammalian PDI their position is closer to the C-terminal-most active site. We have examined their role and that of the active site cysteines by constructing an extensive set of mutants in which the Cys were systematically replaced by Ser. As reported earlier, the N-terminal Cys of the two active sites sequences of yeast PDI were found to be required for cell viability, but mutation of the C-terminal Cys to Ser in the two active sites was not lethal. We found that replacement of the two non-active site Cys with Ser did not affect cell viability, but in the case of the double mutant in which both Cys were replaced by Ser the processing and secretion of CPY was impaired.     

Model of maltose-binding protein/chemoreceptor complex supports intrasubunit signaling mechanism

The Tar protein of Escherichia coli is unique among known bacterial chemoreceptors in that it generates additive responses to two very disparate ligands, aspartate and maltose. Aspartate binds directly to the periplasmic (extracytoplasmic) domain of Tar. Maltose first binds to maltose-binding protein (MBP). MBP then assumes a closed conformation in which it can interact with the periplasmic domain of Tar. MBP residues critical for binding Tar were identified in a screen of mutations that cause specific defects in maltose chemotaxis. Mutations were introduced into a plasmid-borne malE gene that encodes a mutant form of MBP in which two engineered Cys residues spontaneously generate a disulfide bond in the oxidizing environment of the periplasmic space. This disulfide covalently crosslinks the NH3-terminal and COOH-terminal domains of MBP and locks the protein into a closed conformation. Double-Cys MBP confers a dominant-negative phenotype for maltose taxis, and we reasoned that third mutations that relieve this negative dominance probably alter residues that are important for the initial interaction of MBP with Tar. The published three-dimensional structures of MBP and the periplasmic domain of E. coli Tar were docked in a computer simulation that juxtaposed the residues in MBP identified in this way with residues in Tar that have been implicated in maltose taxis. The resulting model of the MBP-Tar complex exhibits good complementarity between the surfaces of the two proteins and supports the idea that aspartate and MBP may each initiate an attractant signal through Tar by inducing similar conformational changes in the chemoreceptor.     

The MKK7 gene encodes a group of c-Jun NH2-terminal kinase kinases

The c-Jun NH2-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH2 termini (alpha, beta, and gamma isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7alpha isoforms lack an NH2-terminal extension that is present in the other MKK7 isoforms. This NH2-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7alpha isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7beta and MKK7gamma isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus was not, however, required for JNK activation in vivo. These data establish that the MKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli.