Stimulation of intralysosomal proteolysis by cysteinyl-glycine,a product of the action of γ-glutamyl transpeptidase on glutathione

The mechanism of the stimulatory effect of glutathione on proteolysis in mouse kidney lysosomes and a lack of an effect in lysomes from the liver was investigated. The stimulation in kidney lysosomes was inhibited by serine plus borate, a reversible inhibitor of γ-glutamyl transpeptidase. Treatment of mouse kidney lysosome suspensions with $\text{l}\text{-(αS,5S)-α-}\text{amino}\text{-3-}\text{chloro}\text{-4,5-}\text{dihydro}\text{-5-}\text{isoxazoleacetic}$ acid (acivicin), an irreversible inhibitor of the transpeptidase, also inhibited the effect of glutathione, but this inhibition was completely relieved by washing and addition of freshly prepated kidney membranes or purified γ-glutamyl transpeptidase to the incubation mixtures. Cysteinyl-glycine, a product of the action of γ-glutamyl transpeptidase, stimulated proteolysis in acivicin-inhibited kidney lysosome preparations similarly to glutathione, and cysteine had no effect at equivalent concentrations. Glutathione also stimulated proteolysis in liver lysosomes in the presence of washed kidney membranes or γ-glutamyl transpeptidase, but the effect was similar to that produced by equivalent concentrations of cysteine. These results suggest that the stimulatory effect of glutathione was mediated by the action of γ-glutamyl transpeptidase present in contaminating cell membrane fragments in the lysosome preparations, and that glutathione does not take part in intralysosomal proteolysis. However, the possibility that cysteinyl-glycine is a physiological intralysosomal disulfide reductant in kidney lysosomes has not been excluded.     

Purification and properties of a novel recombinant human hybrid interferon, σ-4 α2/α1

The human interferon (huIFN) σ-4 α2_5–62/α1_64–166 is a genetically engineered hybrid that consists of residues 5–62 of huIFN α2 and residues 64–166 of huIFN α1. This variant contains four cysteine residues at positions 29, 86, 99 and 139, but does not contain the cysteine at position 1 that is characteristic of naturally occurring huIFN α subtypes. This novel recombinant hybrid was purified fromEscherichia coli to greater than 95% homogeneity. The purification was based on ethanol extraction of a trichloroacetic acid precipitate and Matrex Gel Blue A chromatography followed by either a selective precipitation or DEAE-Sepharose chromatography. The purified protein that was treated with 2-mercaptoethanol exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 17 800 and 17 100, both of which exhibited antiviral activity. Electrophoresis performed without prior reduction with 2-mercaptoethanol indicated only a minor extent of intermolecular disulfide bonding. The purified protein exhibited a high specific antiviral activity of 7·10⁷ units/mg when assayed on human fibroblast cells and, in distinction to the parental huIFN α2, it also demonstrated antiviral activity on murine L929 cells. The level of antiproliferative activity of huIFN δ-4 α2_5–62/α1_64–166 on various cell lines of different histological origin appeared to be more comparable to that of huIFN α1 than huIFN α2. The data suggest that huIFN δ-4 α2_5–62/α1_64–166 hybrid may be a useful tool for understanding huIFN structure-function relations.     

Mechanistic flexibility in the reduction of copper(II) complexes of aliphatic polyamines by mercapto amino acids

The reactions of copper(II)-ahphatic polyamine complexes with cysteine, cysteine methyl ester, penicillamine. and glutathione have been investigated, with the goal of understanding the relationship between RS^?-Cu(II) adduct structure and preferred redox decay pathway. Considerable mechanistic flexibility exists within this class of mercapto ammo acid oxidations, as changes in the rate law could be induced by modest variations in reductant concentration (at fixed [Cu(II)]_o), pH, and the structure of the redox partners. With excess cysteine present at 25°C, pH 5 0, I = 0 2 M (NaOAc), decay of 1:1 cys-S^?-Cu(II) transient adducts was found to be first order in both cys-SH and transient. Second-order rate constants characteristic of Cu(dien)²⁺ (6 1 × 10³M^?1sec^?1), Cu(Me₅dien)²⁺ (2.7 × 10³M^?1 sec^?1), Cu(en)₂²⁺ (2.1 × 10³M^?1 sec^?1), and Cu(dien)₂²⁺ (4.7 × 10³ M^?1 sec ^?1) are remarkably similar, considering substantial differences in the composition and geometry of the oxidant first coordination sphere. A mechanism involving attack of cysteine on the coordinated sulfur atom of the transient, giving a disulfide anion radical intermediate, is proposed to account for these results Moderate reactivity decreases in the cysteine-Cu(dien)²⁺, Cu(Me₅dien)²⁺ reactions with increasing [H⁺] (pH 4–6) reflect partial protonation of the polyamine ligands. A very different rate law, second order in the RS^?-Cu(II) transient and approximately zeroth order in mercaptan, applies in the pH 5.0 oxidations of cysteine methyl ester, penicillamine, and glutathione by Cu(dien)²⁺ and Cu(Me₅dien)²⁺. This behavior suggests the mtermediacy of di-μ-mercapto-bridged binuclear Cu(II) species, in which a concerted two-electron change yields the disulfide and Cu(I) products. Similar hydroxo-bridged intermediates are proposed to account for the transition from first- to second-order transient dependence in cysteine oxidations by Cu(dien)²⁺ and Cu(Me₅dien)²⁺ as the pH is increased from 5 to 7. Yet another rate law, second order in transient and first order in cysteine, applies in the pH 5.0 oxidation of cysteine by Cu(Me₆tren)²⁺ (k(25°C) 7.5 × 10⁷ M^?2 sec^?1, I = 0.2 M). Steric rigidity of this trigonal bipyramidal oxidant evidently protects the coordinated sulfur atom from attack in a RSSR^?-forming pathway. Formation of a coordinated disulfide in the rate-determining step is purposed, coupled with attack of a noncoordinated cysteine molecule on a vacated coordination position to stabilize the (Me₆(tren)Cu(I) product.     

Calmodulin and IQGAP1 activation of PI3Kα and Akt in KRAS,HRAS and NRAS-driven cancers

Calmodulin (CaM) binds only oncogenic KRas, but not HRas or NRas, and thus contributes only to KRAS-driven cancers. How CaM interacts with KRas and how it boosts KRAS cancers are among the most coveted aims in cancer biology. Here we address this question, and further ask: Are there proteins that can substitute for CaM in HRAS- and NRAS-driven cancers? Can scaffolding protein IQGAP1 be one? Data suggest that formation of a CaM–KRas–PI3Kα ternary complex promotes full PI3Kα activation, and thereby potent PI3Kα/Akt/mTOR proliferative signaling. CaM binds PI3Kα at the cSH2 and nSH2 domains of its regulatory p85 subunit; the WW domain of IQGAP1 binds cSH2. This raises the question whether IQGAP1, together with an oncogenic Ras isoform, can partially activate PI3Kα. Activated, membrane-bound PI3Kα generates PIP₃. CaM shuttles Akt to the plasma membrane; CaM's release and concomitant phosphoinositide binding stimulates Akt activation. Notably, IQGAP1 directly interacts with, and helps juxtapose, PI3Kα and Akt as well as mTOR. Our mechanistic review aims to illuminate CaM's actions, and help decipher how oncogenic Ras isoforms – not only KRas4B – can activate the PI3Kα/Akt/mTOR pathway at the membrane and innovate drug discovery, including blocking the PI3Kα–IQGAP1 interaction in HRAS- and NRAS-driven cancers.     

Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation

Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (I _CALHM1). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as I _CALHM1. Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect I _CALHM1 for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both I _CALHM1 and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane.     

Control of Periplasmic Interdomain Thiol:Disulfide Exchange in the Transmembrane Oxidoreductase DsbD

The bacterial protein DsbD transfers reductant from the cytoplasm to the otherwise oxidizing environment of the periplasm. This reducing power is required for several essential pathways, including disulfide bond formation and cytochrome c maturation. DsbD includes a transmembrane domain (tmDsbD) flanked by two globular periplasmic domains (nDsbD/cDsbD); each contains a cysteine pair involved in electron transfer via a disulfide exchange cascade. The final step in the cascade involves reduction of the Cys¹⁰³-Cys¹⁰⁹ disulfide of nDsbD by Cys⁴⁶¹ of cDsbD. Here we show that a complex between the globular periplasmic domains is trapped in vivo only when both are linked by tmDsbD. We have found previously (Mavridou, D. A., Stevens, J. M., Ferguson, S. J., & Redfield, C. (2007) J. Mol. Biol. 370 ,643 -658) that the attacking cysteine (Cys⁴⁶¹) in isolated cDsbD has a high pK_a value (10.5) that makes this thiol relatively unreactive toward the target disulfide in nDsbD. Here we show using NMR that active-site pK_a values change significantly when cDsbD forms a complex with nDsbD. This modulation of pK_a values is critical for the specificity and function of cDsbD. Uncomplexed cDsbD is a poor nucleophile, allowing it to avoid nonspecific reoxidation; however, in complex with nDsbD, the nucleophilicity of cDsbD increases permitting reductant transfer. The observation of significant changes in active-site pK_a values upon complex formation has wider implications for understanding reactivity in thiol:disulfide oxidoreductases.DsbD is a unique protein that transfers reductant across the cytoplasmic membrane to the periplasm in many Gram-negative bacteria (1, 2). Provision of reductant to the periplasm is required because this compartment is otherwise considered to be an oxidizing environment (2). DsbD includes three domains, each containing a pair of cysteine residues that perform a series of disulfide exchange reactions (Fig. 1A). In the first step, the transmembrane domain (tmDsbD) accepts electrons from thioredoxin in the cytoplasm; these are then transferred to the periplasmic C-terminal domain (cDsbD) and finally to the N-terminal domain (nDsbD), which is also located in the periplasm (3-5). nDsbD acts as a junction point for several pathways that require reductant, including the general disulfide isomerase system and the pathway that is thought to reduce the cysteine thiols of apocytochromes in the cytochrome c biogenesis pathway (6). In Gram-positive bacteria, CcdA, an integral membrane protein, and ResA, which has a thioredoxin fold, provide the reductant required for cytochrome c maturation (7).Open in a separate windowFIGURE 1.Schematic representation of DsbD. A, proposed pathway of electron flow from thioredoxin (TrxA) in the cytoplasm, via the three domains of DsbD, to the cytochrome c maturation (Ccm) and disulfide bond isomerization pathways in the periplasm is shown. The crystal structure of nDsbD is from Protein Data Bank code 1L6P (8), cDsbD from Protein Data Bank code 1UC7 (11), and the nDsbD-cDsbD complex from Protein Data Bank code 1VRS (12). The cyan boxes indicate the thrombin cleavage sites introduced into full-length DsbD to allow detection of the nDsbD-cDsbD complex following its formation in vivo. The cysteine residues are shown in yellow. B, schematic representation of the active site of cDsbD in the covalent complex with nDsbD (12). Some active-site residues of cDsbD are indicated in stick representation and the inter-domain disulfide (Cys⁴⁶¹-SS-Cys¹⁰⁹) is shown in yellow.Structural studies have sought to explain how DsbD functions and interacts with its various partners. The structures of the two soluble periplasmic domains have been determined (Fig. 1A, left). nDsbD has an immunoglobulin-like structure (8, 9) and is the only known thiol:disulfide oxidoreductase with this fold. cDsbD has the more typical thioredoxin fold found in many oxidoreductases; this has the characteristic active-site CXXC motif (10, 11). A covalent complex between single-cysteine variants of each of these two domains was produced in vitro and its x-ray structure solved (12), revealing the interface between the two domains (Fig. 1A, right). Although this mixed disulfide is accepted as a physiological intermediate in the function of DsbD, an in vivo complex between the two soluble domains has not been reported previously (3). Further complexes between nDsbD and its other physiological partners have also been trapped and their structures examined (9, 13). Interestingly, all of the interaction partners of nDsbD are thioredoxin-like proteins; similarities in their folds are congruous with common interaction interfaces (14). However, only cDsbD will reduce nDsbD, whereas nDsbD will reduce several partners. This raises questions about how the direction of reductant flow is maintained and controlled within the series of disulfide-exchange reactions.As part of our structural and mechanistic characterization of DsbD and its domains in solution, we have previously measured by NMR the pK_a values of the active-site cysteine pair, Cys⁴⁶¹ and Cys⁴⁶⁴, of cDsbD (numbered according to the full-length Escherichia coli DsbD sequence) (15). An unusually high pK_a value of 10.5 was measured for the N-terminal cysteine of the CXXC motif, Cys⁴⁶¹, and the pK_a value of the second cysteine, Cys⁴⁶⁴, was significantly higher than the maximum pH value that was studied (pH 12.2). The pK_a value of 10.5 is the highest reported for the N-terminal cysteine of the CXXC motif in a thioredoxin fold. The striking consequence of the elevated pK_a value is that the active-site cysteine of cDsbD, Cys⁴⁶¹, is not strongly nucleophilic, raising critical questions about how this cysteine reacts with the disulfide in nDsbD. It was demonstrated using site-directed mutagenesis that the negatively charged side chains of Asp⁴⁵⁵ and Glu⁴⁶⁸, which are located close to the CXXC motif (Fig. 1B), are responsible for the unusually high pK_a value of Cys⁴⁶¹; mutation of one or both of these residues to Asn and Gln, respectively, resulted in decreases in the pK_a value of Cys⁴⁶¹ from 10.5 to 9.9 (E468Q), to 9.3 (D455N), and to 8.6 (D455N/E468Q). The pK_a values for Asp⁴⁵⁵ were found to be 5.9 and 6.6 in oxidized and reduced cDsbD; these values are significantly higher than the value of ∼4 for an unperturbed aspartic acid. We postulated that the properties of the amino acid side chains in the immediate environment of the cysteines in cDsbD would change upon complex formation with nDsbD, changing the reactivity of the cysteines and explaining how the reaction between the two domains is initiated (15). Specifically, we proposed that an increase in the pK_a value of Asp⁴⁵⁵ upon complex formation would lead to a decrease in the pK_a value of Cys⁴⁶¹, thereby making it a better nucleophile. Stirnimann et al. (10) previously presented pK_a calculations suggesting an increase in the Asp⁴⁵⁵ pK_a value upon complex formation.The aim of this work has been to determine the molecular basis of the control of the reactivity of the active-site cysteine residues in cDsbD, using NMR to compare the active-site properties of cDsbD alone and in its physiological complex with nDsbD. We demonstrate that the pK_a value of Asp⁴⁵⁵ is elevated by at least 1.1 pH units when cDsbD forms a complex with nDsbD. This modulation of the pK_a value is critical for the specificity and function of cDsbD. These in vitro studies are complemented by in vivo studies on complex formation, in which we have trapped the nDsbD-cDsbD complex for the first time. The results of our experiments explain how the intramolecular disulfide cascade within the soluble domains of DsbD functions, and demonstrate the importance of the transmembrane domain in controlling and facilitating complex formation between the soluble domains.     

Disulfide Bond Formation and Cysteine Exclusion in Gram-positive Bacteria

Most secretion pathways in bacteria and eukaryotic cells are challenged by the requirement for their substrate proteins to mature after they traverse a membrane barrier and enter a reactive oxidizing environment. For Gram-positive bacteria, the mechanisms that protect their exported proteins from misoxidation during their post-translocation maturation are poorly understood. To address this, we separated numerous bacterial species according to their tolerance for oxygen and divided their proteomes based on the predicted subcellular localization of their proteins. We then applied a previously established computational approach that utilizes cysteine incorporation patterns in proteins as an indicator of enzymatic systems that may exist in each species. The Sec-dependent exported proteins from aerobic Gram-positive Actinobacteria were found to encode cysteines in an even-biased pattern indicative of a functional disulfide bond formation system. In contrast, aerobic Gram-positive Firmicutes favor the exclusion of cysteines from both their cytoplasmic proteins and their substantially longer exported proteins. Supporting these findings, we show that Firmicutes, but not Actinobacteria, tolerate growth in reductant. We further demonstrate that the actinobacterium Corynebacterium glutamicum possesses disulfide-bonded proteins and two dimeric Dsb-like enzymes that can efficiently catalyze the formation of disulfide bonds. Our results suggest that cysteine exclusion is an important adaptive strategy against the challenges presented by oxidative environments.     

Characterization of Microtubule-Binding and Dimerization Activity of Giardia lamblia End-Binding 1 Protein

End-binding 1 (EB1) proteins are evolutionarily conserved components of microtubule (MT) plus-end tracking protein that regulate MT dynamics. Giardia lamblia, with two nuclei and cytoskeletal structures, requires accurate MT distribution for division. In this study, we show that a single EB1 homolog gene of G. lamblia regulates MT dynamics in mitosis. The haemagglutinin-tagged G. lamblia EB1 (GlEB1) localizes to the nuclear envelopes and median bodies, and is transiently present in mitotic spindles of dividing cells. Knockdown of GlEB1 expression using the morpholinos-based anti-EB1 oligonucleotides, resulted in a significant defect in mitosis of Giardia trophozoites. The MT-binding assays using recombinant GlEB1 (rGlEB1) proteins demonstrated that rGlEB1_102–238, but not rGlEB1_1–184, maintains an MT-binding ability comparable with that of the full length protein, rGlEB1_1–238. Size exclusion chromatography showed that rGlEB1 is present as a dimer formed by its C-terminal domain and a disulfide bond. In vitro-mutagenesis of GlEB1 indicated that an intermolecular disulfide bond is made between cysteine #13 of the two monomers. Complementation assay using the BIM1 knockout mutant yeast, the yeast homolog of mammalian EB1, indicated that expression of the C13S mutant GlEB1 protein cannot rescue the mitotic defect of the BIM1 mutant yeast. These results suggest that dimerization of GlEB1 via the 13th cysteine residues plays a role during mitosis in Giardia.     

Radioactive labeling of alpha1-antitrypsin, trypsin, and chymotrypsin by reductive methylation: properties of the labeled derivatives.

Human plasma α₁-antitrypsin (α₁-AT), bovine trypsin, and α-chymotrypsin were labeled with either ¹⁴C or ³H by reductive methylation. The labeled inhibitor retained the capacity to inactivate and to form 1:1 molar complexes with either the unlabeled or labeled trypsin and α-chymotrypsin. After intravenous injection of reductively methylated α₁-AT into rats, the labeled glycoprotein showed a circulating half-life of 12 h. When the N-acetylneuraminic acid residues were removed from the labeled α₁-AT by neuraminidase in vitro, injection into rats of this product resulted in a rapid (half-life of about 5 min) and almost complete disappearance of the label from the circulation in 30 min. There was a concomitant accumulation of radioactivity in the liver of over 75% of the injected dose. The reductively methylated radioactively labeled trypsin and chymotrypsin experienced no loss of enzymatic activities. They showed the ability to form complexes in vivo with the two major plasma inhibitors, namely, α₁-AT and α₂-macroglobulin. High-voltage paper electrophoretic separation of acid hydrolysates of the labeled proteins revealed that ?-N-monomethyllysine and ?N,N-dimethyllysine are the only residues found to be radioactive.     

The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C‐terminal cysteine of the CXXC motif

Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N‐terminal cysteine interacts with substrates, whereas the C‐terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C⁸⁶P⁸⁷D⁸⁸C⁸⁹ catalytic motif. In vitro, SdbA single cysteine variants at the N or C‐terminal position (SdbA_C86P and SdbA_C89A) were active but displayed different susceptibility to oxidation, and N‐terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N‐terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C‐terminal cysteine alone (in the SdbA_C86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C‐terminal cysteine.     

Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys peroxide sensor

OhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented by Bacillus subtilis OhrR) and 2-Cys (represented by Xanthomonas campestris OhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. The B. subtilis 1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, the X. campestris 2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-Cys B. subtilis OhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like the X. campestris OhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols.     

Proteome of Acidic Phospholipid-binding Proteins: SPATIAL AND TEMPORAL REGULATION OF CORONIN 1A BY PHOSPHOINOSITIDES*

Reversible interactions between acidic phospholipids in the cellular membrane and proteins in the cytosol play fundamental roles in a wide variety of physiological events. Here, we present a novel approach to the identification of acidic phospholipid-binding proteins using nano-liquid chromatography-tandem mass spectrometry. We found more than 400 proteins, including proteins with previously known acidic phospholipid-binding properties, and confirmed that several candidates, such as Coronin 1A, mDia1 (Diaphanous-related formin-1), PIR121/CYFIP2, EB2 (end plus binding protein-2), KIF21A (kinesin family member 21A), eEF1A1 (translation elongation factor 1α1), and TRIM2, directly bind to acidic phospholipids. Among such novel proteins, we provide evidence that Coronin 1A activity, which disassembles Arp2/3-containing actin filament branches, is spatially and temporally regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Whereas Coronin 1A co-localizes with PI(4,5)P₂ at the plasma membrane in resting cells, it is dissociated from the plasma membrane during lamellipodia formation where the PI(4,5)P₂ signal is significantly reduced. Our in vitro experiments show that Coronin 1A preferentially binds to PI(4,5)P₂-containing liposomes and that PI(4,5)P₂ antagonizes the ability of Coronin 1A to disassemble actin filament branches, indicating a spatiotemporal regulation of Coronin 1A via a direct interaction with the plasma membrane lipid. Collectively, our proteomics data provide a list of potential acidic phospholipid-binding protein candidates ranging from the actin regulatory proteins to translational regulators.     

Cystine lyase: β-cystathionase from turnip roots

Cystine lyase (EC 4.4.1.-) was purified 277-fold by a combination of ammonium sulfate precipitation, chromatography on calcium phosphate and DEAE-cellulose with a 6% recovery. The MW as measured by gel filtration on Biogel p-300 was ca 150 000. The enzyme catalysed the pyridoxal phosphate-dependent degradation of cystine to pyruvate, ammonia and cysteine persulfide. Cysteine persulfide normally degraded spontaneously to elemental sulfur and cysteine, that further reacted to yield cystine and H₂S. Pyridoxal phosphate stabilized the enzyme. The K_m value for cystine was 0.94 mM. The enzyme was insensitive to thiol reagents but was inhibited by some thiols (which may have reduced the cystine). Cystine lyase degraded many compounds having the L-α-amino propionic acid group with a thioether or disulfide linkage attached to the β-carbon but was inactive towards D-configuration at the α-carbon or L-homocystine. The cystine lyase was also a β-cystathionase as indicated by (1) a constant ratio of β-cystathionase activity to cystine lyase activity throughout a 277-fold purification, (2) the inhibition of cystine lyase activity by cystathionine and inhibition of β-cystathionase activity by cystine and (3) similarity in sensitivity to heat, cyanide and hydroxylamine. Using DL-cystathionine as substrate, the K_m value was 4 mM.     

Studies on 125I-labeled proteins in rat plasma using an air-driven ultracentrifuge: protein-protein interactions and nonideality

The molecular weights of a number of ¹²⁵I-labeled plasma proteins have been determined from an analysis of their sedimentation equilibrium behavior in an air-driven ultracentrifuge. The values obtained agree well with results obtained by other methods. Molecular weights obtained for ¹²⁵I-labeled bovine serum albumin and the rat serum proteins albumin, α₁-acid glycoprotein, and major acute-phase α₁-protein were unaffected by the addition of 7% rat plasma. Direct evidence for protein-protein interactions was obtained for mixtures of ¹²⁵I-labeled rat α₁-acid glycoprotein and the plant lectin concanavalin A and for mixtures of ¹²⁵I-labeled protein A from Staphylococcus aureus and 7% rat plasma. Interactions of a different type were observed when the sedimentation equilibrium profiles of ¹²⁵I-labeled proteins were determined in concentrated solutions of other proteins. Under these conditions the effects of molecular exclusion or nonideality became significant and low estimates were obtained for the molecular weights of the labeled proteins. Analysis of the data obtained for ¹²⁵I-labeled bovine serum albumin in concentrated solutions of bovine serum albumin (20–80 mg/ ml) yielded nonideality coefficients in good agreement with literature values. Analysis of the behavior of ¹²⁵I-labeled rat serum albumin, transferrin, and α₁-acid glycoprotein yielded nonideality coefficients and hence activities of these proteins in undiluted rat plasma.     

Rapid purification of a phospholipase-free α-bungarotoxin: Maintenance of cation barriers of acetylcholine receptor membranes upon preincubation with purified toxin

The purification of highly homogeneous, phospholipase-free α-bungarotoxin (α-Bgt) from the venom of the elapid Bungarus multicinctus or from commercial samples of α-Bgt is described. The method combines a conventional procedure for the purification of α-Bgt [D. Mebs, K. Narita, S. Iwanaga, Y. Samejima, and C. Y. Lee (1972) Hoppe-Seyler's Z. Physiol. Chem.353, 243–262] with high-resolution gel-filtration and cation-exchange chromatography steps to remove membrane-damaging, contaminating phospholipase activity. The procedure also removes contaminating radioactive peptides from commercial preparations of ¹²⁵I-α-Bgt. Apparent homogeneity of the purified α-Bgt (referred to as fraction D in the text), as well as the absence of contaminating phospholipase A₂ activity, is assessed by (i) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, (ii) gel-filtration and cation-exchange high-performance liquid chromatography, (iii) direct measurements of phospholipase A₂ activity under conditions where very low enzymatic levels should be detected, (iv) lack of interference with the passive cation permeability properties of acetylcholine receptor membranes, (v) competitive inhibition of ¹²⁵I-α-Bgt binding to the acetylcholine receptor membranes, and (vi) amino acid analysis and end-group (C- and N-terminus) determination. α-Bgt preparations subjected to these criteria do not exert the increase in membrane passive permeability to cations detected with other laboratory or commercial samples of α-Bgt. Availability of the new α-Bgt preparation allows for an assessment of the inertness of α-Bgt on lipid membrane properties while preventing cholinergic ligand binding to nicotinic acetylcholine receptor-rich membranes. These conditions are necessary for experiments requiring maintenance of the physical and phospholipid integrity of membranes.     

Domain Features of the Peripheral Stalk Subunit H of the Methanogenic A1AO ATP Synthase and the NMR Solution Structure of H1-47

A series of truncated forms of subunit H were generated to establish the domain features of that protein. Circular dichroism analysis demonstrated that H is divided at least into a C-terminal coiled-coil domain within residues 54-104, and an N-terminal domain formed by adjacent α-helices. With a cysteine at the C-terminus of each of the truncated proteins (H_1-47, H_1-54, H_1-59, H_1-61, H_1-67, H_1-69, H_1-71, H_1-78, H_1-80, H_1-91, and H_47-105), the residues involved in formation of the coiled-coil interface were determined. Proteins H_1-54, H_1-61, H_1-69, and H_1-80 showed strong cross-link formation, which was weaker in H_1-47, H_1-59, H_1-71, and H_1-91. A shift in disulfide formation between cysteins at positions 71 and 80 reflected an interruption in the periodicity of hydrophobic residues in the region ₇₁AEKILEETEKE₈₁. To understand how the N-terminal domain of H is formed, we determined for the first time, to our knowledge, the solution NMR structure of H_1-47, which revealed an α-helix between residues 15-42 and a flexible N-terminal stretch. The α-helix includes a kink that would bring the two helices of the C-terminus into the coiled-coil arrangement. H_1-47 revealed a strip of alanines involved in dimerization, which were tested by exchange to single cysteines in subunit H mutants.     

CYTOCHEMISTRY OF GOLGI FRACTIONS PREPARED FROM RAT LIVER

Cytochemical tests for several marker enzymes were applied to liver tissue and to the three Golgi fractions (GF₁, GF₂, GF₃) separated by the procedure of Ehrenreich et al. from liver homogenates of alcohol-treated rats. 5'-Nucleotidase (AMPase) reaction product was found in all three fractions but in different locations: It occurred along the inside of the membrane of VLDL-filled vacuoles in GF₁ and GF₂, and along the outside of the cisternal membranes in GF₃. In the latter it was restricted to the dilated cisternal rims and was absent from the cisternal centers. The AMPase activity found in the fractions by biochemical assay is therefore indigenous to Golgi components and is not due to contamination by plasma membrane. Acid phosphatase (AcPase) reaction product was detected within lysosomal contaminants in GF₁ and within many VLDL-filled vacuoles in GF₁ and GF₂, indicating that AcPase activity is due not only to contaminating lysosomes, but also to enzyme indigenous to Golgi secretory vacuoles. G-6-Pase reaction product was present in GF₃ and within contaminating endoplasmic reticulum fragments, but not in other fractions. Thiamine pyrophosphatase (TPPase) was localized to some of the VLDL-filled vacuoles and cisternae in GF₁ and GF₂, and was not found in the cisternae in GF₃. The results demonstrate the usefulness of cytochemical methods in monitoring the fractionation procedure: They have (a) allowed a reliable identification of contaminants, (b) made possible a distinction between indigenous and contaminating activities, and (c) shown, primarily by the results of the TPPase test, that the procedure achieves a meaningful subfractionation of Golgi elements, with GF₁ and GF₃, representing primarily trans-Golgi elements from the secretory Golgi face, and GF₃ consisting largely of cis-Golgi components from the opposite face.     

Mycothiol/Mycoredoxin 1-dependent Reduction of the Peroxiredoxin AhpE from Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H₂O₂-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria.     

Real-time Monitoring of Intermediates Reveals the Reaction Pathway in the Thiol-Disulfide Exchange between Disulfide Bond Formation Protein A (DsbA) and B (DsbB) on a Membrane-immobilized Quartz Crystal Microbalance (QCM) System

Disulfide bond formation protein B (DsbB_S-S,S-S) is an inner membrane protein in Escherichia coli that has two disulfide bonds (S-S, S-S) that play a role in oxidization of a pair of cysteine residues (SH, SH) in disulfide bond formation protein A (DsbA_SH,SH). The oxidized DsbA_S-S, with one disulfide bond (S-S), can oxidize proteins with SH groups for maturation of a folding preprotein. Here, we have described the transient kinetics of the oxidation reaction between DsbA_SH,SH and DsbB_S-S,S-S. We immobilized DsbB_S-S,S-S embedded in lipid bilayers on the surface of a 27-MHz quartz crystal microbalance (QCM) device to detect both formation and degradation of the reaction intermediate (DsbA-DsbB), formed via intermolecular disulfide bonds, as a mass change in real time. The obtained kinetic parameters (intermediate formation, reverse, and oxidation rate constants (k_f, k_r, and k_cat, respectively) indicated that the two pairs of cysteine residues in DsbB_S-S,S-S were more important for the stability of the DsbA-DsbB intermediate than ubiquinone, an electron acceptor for DsbB_S-S,S-S. Our data suggested that the reaction pathway of almost all DsbA_SH,SH oxidation processes would proceed through this stable intermediate, avoiding the requirement for ubiquinone.     

Intracellular precursor forms of transferrin, α1-acid glycoprotein,and α1-antitrypsin in human liver

Ion-exchange chromatography of dialyzed human plasma and of buffer extracts of acetone-dried powder from human liver was used to analyze 13 different plasma proteins which are synthesized in the liver. Specific intracellular forms which differ from the plasma forms were found for transferrin, α₁-acid glycoprotein, α₁-antitrypsin, and albumin. The intracellular forms were labeled earlier than the plasma forms, when liver slices were incubated with radioactive leucine, suggesting that they are precursor forms of the proteins in the bloodstream. The liver form of transferrin was found to have the same molecular weight and N-terminus as the plasma form, but it differed from the plasma form by the absence of sialic acid. For α₁-acid glycoprotein two different liver forms were observed, both of which had lower molecular weights than the plasma form. One of these liver forms was analyzed further. Its polypeptide chain was found to have a blocked N-terminus, as does the plasma form. However, in contrast to the plasma form, it did not contain sialic acid. Its content of N-acetyl glucosamine was about one-third and the content of neutral hexoses about two-thirds of that found in the plasma form. Circular dichroism spectra were similar for liver and plasma forms and indicated a predominant β structure with very little α-helix content for both.