Crystal structures of ADP and AMPPNP-bound propionate kinase (TdcD) from Salmonella typhimurium: comparison with members of acetate and sugar kinase/heat shock cognate 70/actin superfamily

Recently, it has been shown that l-threonine can be catabolized non-oxidatively to propionate via 2-ketobutyrate. Propionate kinase (TdcD; EC 2.7.2.-) catalyses the last step of this metabolic process by enabling the conversion of propionyl phosphate and ADP to propionate and ATP. To provide insights into the substrate-binding pocket and catalytic mechanism of TdcD, the crystal structures of the enzyme from Salmonella typhimurium in complex with ADP and AMPPNP have been determined to resolutions of 2.2A and 2.3A, respectively, by molecular replacement using Methanosarcina thermophila acetate kinase (MAK; EC 2.7.2.1). Propionate kinase, like acetate kinase, contains a fold with the topology betabetabetaalphabetaalphabetaalpha, identical with that of glycerol kinase, hexokinase, heat shock cognaten 70 (Hsc70) and actin, the superfamily of phosphotransferases. The structure consists of two domains with the active site contained in a cleft at the domain interface. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate. This was further confirmed by kinetic studies with the purified enzyme, which showed about ten times lower K(m) for propionate (2.3 mM) than for acetate (26.9 mM). Comparison of TdcD complex structures with those of acetate and sugar kinase/Hsc70/actin obtained with different ligands has permitted the identification of catalytically essential residues involved in substrate binding and catalysis, and points to both structural and mechanistic similarities. In the well-characterized members of this superfamily, ATP phosphoryl transfer or hydrolysis is coupled to a large conformational change in which the two domains close around the active site cleft. The significant amino acid sequence similarity between TdcD and MAK has facilitated study of domain movement, which indicates that the conformation assumed by the two domains in the nucleotide-bound structure of TdcD may represent an intermediate point in the pathway of domain closure.     

A novel gyrB mutation in a fluoroquinolone-resistant clinical isolate of Salmonella typhimurium

Abstract In order to study the role of gyrB in antibiotic resistance in post-ciprofloxacin therapy fluoroquinolone-resistant clinical isolates of Salmonella typhimurium , plasmid pBP548, which contains the Escherichia coli gyrB gene, was used in complementation studies. In a heterodiploid strain, the wild-type (quinolone sensitive) allele is dominant over the resistant allele therefore, eleven clinical isolates were complemented with gyrB encoded on pBP548. Only one transformant, L18pBP548, exhibited increased susceptibility to the quinolones nalidixic acid, ciprofloxacin and sparfloxacin. The amino acid sequence of the gyrase B protein from a wild-type and the pre-therapy S. typhimurium (deduced from the nucleotide sequence) was identical to that of E. coli from codons 436 to 470; however, a point mutation was identified in codon 463 of gyrB of the quinolone-resistant post-therapy isolate L18, giving rise to an amino acid substitution of serine to tyrosine.     

Crystal structures of GMP kinase in complex with ganciclovir monophosphate and Ap5G

Guanosine monophosphate kinases (GMPK), by catalyzing the phosphorylation of GMP or dGMP, are of dual potential in assisting the activation of anti-viral prodrugs or as candidates for antibiotic strategies. Human GMPK is an obligate step for the activation of acyclic guanosine analogs, such as ganciclovir, which necessitate efficient phosphorylation, while GMPK from bacterial pathogens, in which this enzyme is essential, are potential targets for therapeutic inhibition. Here we analyze these two aspects of GMPK activity with the crystal structures of Escherichia coli GMPK in complex with ganciclovir-monophosphate (GCV-MP) and with a bi-substrate inhibitor, Ap5G. GCV-MP binds as GMP to the GMP-binding domain, which is identical in E. coli and human GMPKs, but unlike the natural substrate fails to stabilize the closed, catalytically-competent conformation of this domain. Comparison with GMP- and GDP-bound GMPK structures identifies the 2'hydroxyl of the ribose moiety as responsible for hooking the GMP-binding domain onto the CORE domain. Absence of this hydroxyl in GCV-MP impairs the stabilization of the active conformation, and explains why GCV-MP is phosphorylated less efficiently than GMP, but as efficiently as dGMP. In contrast, Ap5G is an efficient inhibitor of GMPK. The crystal structure shows that Ap5G locks an incompletely closed conformation of the enzyme, in which the adenine moiety is located outside its expected binding site. Instead, it binds at a subunit interface that is unique to the bacterial enzyme, which is in equilibrium between a dimeric and an hexameric form in solution. This suggests that inhibitors could be designed to bind at this interface such as to prevent nucleotide-induced domain closure. Altogether, these complexes point to domain motions as critical components to be evaluated in therapeutic strategies targeting NMP kinases, with opposite effects depending on whether efficient phosphorylation or inhibition is being sought after.     

Surface display compared to periplasmic expression of a malarial antigen in Salmonella typhimurium and its implications for immunogenicity

Abstract Two different expression systems were investigated for the production of an 80 amino acid polypeptide, M3, from the C-terminus of the Plasmodium falciparum blood stage antigen Pf155/RESA in an attenuated Salmonella typhimurium vaccine strain. Upon expression, the malarial polypeptide was targeted either to the periplasm as a soluble fusion protein containing two IgG-binding domains (ZZ) from the staphylococcal protein A or, to the bacterial surface as an insert within a chimeric outer membrane protein A (OmpA) derived from Escherichia coli and Shigella dysenteriae . Both the ZZM3 and the OmpAM3 proteins were stably expressed in the periplasm or on the surface of Salmonella , respectively. The ZZ expression system yielded 10–100 times more malarial immunogen than did the OmpA system. Live recombinant Salmonella expressing ZZM3 or OmpAM3 were used to immunize mice intraperitoneally. Both the ZZM3 and OmpAM3 genes persisted for up to three weeks in bacteria isolated from different lymphoid organs. Bacteria expressing ZZM3 induced antibodies to M3, ZZ and to the Pf155/RESA antigen whereas, bacteria producing OmpAM3 induced similar levels of antibodies reactive with M3 but not with Pf155/RESA. Both recombinants induced a memory response of antibodies reactive with both M3 and Pf155/RESA. The high levels of M3 produced by the ZZ expression system make it suitable for the expression of heterologous antigens in Salmonella . Nevertheless, in spite of the quantitative difference in M3 expression, the ZZ and OmpA constructs elicited comparable immune responses to M3.     

Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model

Two strains of Salmonella typhimurium presenting increased mutation rates, either spontaneous or mediated by DNA damage, have been constructed. One of the strains carries a null mutS mutation, while the other harbors plasmid pRW30, which contains the Escherichia coli umuDC operon. The virulence of these strains has been determined by inoculating BALB/c or Swiss mice. The 50% lethal dose of both strains is identical to that obtained for the wild-type. Likewise, the two strains and the wild-type contribute equally to animal death in mixed infections. The frequency of Nal(R) mutants recovered from animals inoculated with either wild-type or MutS(-) cells was not affected by the presence of pRW30. These results indicate that the DNA damage which S. typhimurium cells can suffer during the infectious process by host cell metabolites does not cause induction of the SOS response at levels able to trigger the error-prone DNA repair pathway.     

Integration of minitransposons for expression of the Escherichia coli elt genes at a preferred site in Salmonella typhimurium identifies a novel putative fimbrial locus

An asd-complementing mini-Tn5 transposon was constructed for random insertion of the Escherichia coli LT enterotoxin genes (elt) into the genome of Δasd attenuated strains of Salmonella typhimurium. Transfer of the minitransposon to different S. typhimurium strains resulted in random integration only in strain χ4072, while in strain χ3987, which harbours the virulence plasmid, over 20% of the insertions occurred at the same site. Expression of elt was found to be highest in Salmonella isolates carrying the mini-Tn5 integrated at the preferred site, which was mapped to an uncharacterised region of the virulence plasmid. Sequence analysis of the integration site showed that it lies within an open reading frame with sequence similarity to E. coli leuO and contiguous to a novel fimbrial locus. Received: 26 August 1998 / Accepted: 9 November 1998     

Nuclear Location of a Diadenosine 5′,5′”-P1,P4Tetraphosphate (Ap4A) Hydrolase in Tomato Cells Grown in Suspension Cultures1

Diadenosine 5′,5′”-P¹,P⁴-tetraphosphate (Ap₄A) cleaving enzymes are assumed to regulate intracellular levels of Ap₄A, a compound known to affect cell proliferation and stress responses. From plants an Ap₄A hydrolase was recently purified using tomato cells grown in suspension. It was partially sequenced and a peptide antibody was prepared (Feussner et al., 1996). Using this polyclonal monospecific antibody, an abundant nuclear location of Ap₄A hydrolase in 4-day-old cells of atomato cell suspension culture is demonstrated here by means of immunocytochemical techniques using FITC (fluorescein-5-isothiocyanate) labeled secondary antibodies. The microscopic analysis of the occurrence of Ap₄A hydrolase performed for different stages of the cell cycle visualized by parallel DAPI (4,6-diamidino-2-phenylindole) staining revealed that the protein accumulates within nuclei of cells in the interphase, but is absent in the nucleus as well as cytoplasm during all stages of mitosis. This first intracellular localization of an Ap₄A degrading enzyme within the nucleus and its pattern of appearance during the cell cycle is discussed in relation to the suggested role of Ap₄A in triggering DNA synthesis and cell proliferation.     

Intra-arterial administration of tumor-targeting Salmonella typhimurium A1-R regresses a cisplatin-resistant relapsed osteosarcoma in a patient-derived orthotopic xenograft (PDOX) mouse model

Previously, a patient-derived orthotopic xenograft (PDOX) model was established with a lung metastasis from an osteosarcoma patient which developed after adjuvant cisplatinum (CDDP) treatment. In this model, we previously demonstrated the efficacy of trabectedin (TRAB) and temozolomide (TEM) compared with CDDP. In the present report, osteosarcoma tissue was implanted orthotopically in the distal femur of mice which were randomized into the following groups when tumor volume reached approximately 100 mm³; On day 14 after initiation of treatment, all but CDDP significantly inhibited tumor volume growth compared with untreated controls. Control (G1): 793.7 ± 215.0 mm³; CDDP (G2): 588.1 ± 176.9 mm³; Salmonella typhimurium A1-R (S. typhimurium A1-R) intravenous (i.v.) (G3): 269.7 ± 72.7 mm³; S. typhimurium A1-R intra-arterial (i.a.) (G4): 70.2 ± 18.9 mm³ (CDDP: p = 0.056; S. typhimurium A1-R i.v.: p = 0.0001; S. typhimurium A1-R i.a.: p = 0.00003, all vs. untreated controls). i.a. administration of S. typhimurium A1-R was significantly more effective than either CDDP (p = 0.00007), or i.v. administration of S. typhimurium A1-R (p = 0.00007) and significantly regressed the tumor volume compared with day 0 (p = 0.001). The new model of i.a. administration of S. typhimurium A1-R has great promise for the treatment of recalcitrant osteosarcoma.     

Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): Insights from kinetic and crystallographic studies

Short-chain fatty acids (SCFAs) play a major role in carbon cycle and can be utilized as a source of carbon and energy by bacteria. Salmonella typhimurium propionate kinase (StTdcD) catalyzes reversible transfer of the γ-phosphate of ATP to propionate during l-threonine degradation to propionate. Kinetic analysis revealed that StTdcD possesses broad ligand specificity and could be activated by various SCFAs (propionate > acetate ≈ butyrate), nucleotides (ATP ≈ GTP > CTP ≈ TTP; dATP > dGTP > dCTP) and metal ions (Mg^2 + ≈ Mn^2 + > Co^2 +). Inhibition of StTdcD by tricarboxylic acid (TCA) cycle intermediates such as citrate, succinate, α-ketoglutarate and malate suggests that the enzyme could be under plausible feedback regulation. Crystal structures of StTdcD bound to PO₄ (phosphate), AMP, ATP, Ap4 (adenosine tetraphosphate), GMP, GDP, GTP, CMP and CTP revealed that binding of nucleotide mainly involves hydrophobic interactions with the base moiety and could account for the broad biochemical specificity observed between the enzyme and nucleotides. Modeling and site-directed mutagenesis studies suggest Ala88 to be an important residue involved in determining the rate of catalysis with SCFA substrates. Molecular dynamics simulations on monomeric and dimeric forms of StTdcD revealed plausible open and closed states, and also suggested role for dimerization in stabilizing segment 235–290 involved in interfacial interactions and ligand binding. Observation of an ethylene glycol molecule bound sufficiently close to the γ-phosphate in StTdcD complexes with triphosphate nucleotides supports direct in-line phosphoryl transfer.     

Crystal structures of eosinophil-derived neurotoxin (EDN) in complex with the inhibitors 5'-ATP, Ap3A, Ap4A, and Ap5A

Eosinophil-derived neurotoxin (EDN) is a catalytically proficient member of the pancreatic ribonuclease superfamily secreted along with other eosinophil granule proteins during innate host defense responses and various eosinophil-related inflammatory and allergic diseases. The ribonucleolytic activity of EDN is central to its antiviral and neurotoxic activities and possibly to other facets of its biological activity. To probe the importance of this enzymatic activity further, specific inhibitors will be of great aid. Derivatives of 5'-ADP are among the most potent inhibitors currently known. Here, we use X-ray crystallography to investigate the binding of four natural nucleotides containing this moiety. 5'-ATP binds in two alternative orientations, one occupying the B2 subsite in a conventional manner and one being a retro orientation with no ordered adenosine moiety. Diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) bind with one adenine positioned at the B2 subsite, the polyphosphate chain extending across the P1 subsite in an ill-defined conformation, and a disordered second adenosine moiety. Diadenosine pentaphosphate (Ap5A), the most avid inhibitor of this series, binds in a completely ordered fashion with one adenine positioned conventionally at the B2 subsite, the polyphosphate chain occupying the P1 and putative P(-1) subsites, and the other adenine bound in a retro-like manner at the edge of the B1 subsite. The binding mode of each of these inhibitors has features seen in previously determined structures of adenosine diphosphates. We examine the structure-affinity relationships of these inhibitors and discuss the implications for the design of improved inhibitors.     

Crystal structure of Salmonella typhimurium 2-methylisocitrate lyase (PrpB) and its complex with pyruvate and Mg(2+)

Propionate metabolism in Salmonella typhimurium occurs via 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (PrpB). Here we report the X-ray crystal structure of the native and the pyruvate/Mg(2+) bound PrpB from S. typhimurium, determined at 2.1 and 2.3A, respectively. The structure closely resembles that of the Escherichia coli enzyme. Unlike the E. coli PrpB, Mg(2+) could not be located in the native Salmonella PrpB. Only in pyruvate bound PrpB structure, Mg(2+) was found coordinated with pyruvate. Binding of pyruvate to PrpB seems to induce movement of the Mg(2+) by 2.5A from its position found in E. coli native PrpB. In both the native enzyme and pyruvate/Mg(2+) bound forms, the active site loop is completely disordered. Examination of the pocket in which pyruvate and glyoxalate bind to 2-methylisocitrate lyase and isocitrate lyase, respectively, reveals plausible rationale for different substrate specificities of these two enzymes. Structural similarities in substrate and metal atom binding site as well as presence of similar residues in the active site suggest possible similarities in the reaction mechanism.     

Crystal structures of Salmonella typhimurium biodegradative threonine deaminase and its complex with CMP provide structural insights into ligand-induced oligomerization and enzyme activation

Two different pyridoxal 5'-phosphate-containing l-threonine deaminases (EC 4.3.1.19), biosynthetic and biodegradative, which catalyze the deamination of l-threonine to alpha-ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of l-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to l-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB.CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for l-threonine. Comparison of TdcB with related pyridoxal 5'-phosphate-dependent enzymes points to structural and mechanistic similarities.     

Crystal structures of IRAK-4 kinase in complex with inhibitors: a serine/threonine kinase with tyrosine as a gatekeeper

Interleukin-1 (IL-1) receptor-associated kinase-4 (IRAK-4) is a serine/threonine kinase that plays an essential role in signal transduction by Toll/IL-1 receptors (TIRs). Here, we report the crystal structures of the phosphorylated human IRAK-4 kinase domain in complex with a potent inhibitor and with staurosporine to 2.0 and 2.2 A, respectively. The structures reveal that IRAK-4 has a unique tyrosine gatekeeper residue that interacts with the conserved glutamate from helix alphaC. Consequently, helix alphaC is "pulled in" to maintain the active orientation, and the usual pre-existing hydrophobic back pocket of the ATP-binding site is abolished. The peptide substrate-binding site is more open when compared with other protein kinases due to a marked movement of helix alphaG. The pattern of phosphate ligand interactions in the activation loop bears a close resemblance to that of a tyrosine kinase. Our results provide insights into IRAK-4 function and the design of selective inhibitors.     

Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap5A,Mg2+ Ap5A,and Mn2+ Ap5A reveal an intermediate lid position and six coordinate octahedral geometry for bound Mg2+ and Mn2+

Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap₅A, Mn²⁺ Ap₅A, and Mg²⁺ Ap₅A have been determined by X-ray crystallography to resolutions of 1.6 Å, 1.85 Å, and 1.96 Å, respectively. The protein's lid domain is partially open, being both rotated and translated away from bound Ap₅A. The flexibility of the lid domain in the ternary state and its ability to transfer force directly to the the active site is discussed in light of our proposed entropic mechanism for catalytic turnover. The bound Zn²⁺ atom is demonstrably structural in nature, with no contacts other than its ligating cysteine residues within 5 Å. The B. stearothermophilus adenylate kinase lid appears to be a truncated zinc finger domain, lacking the DNA binding finger, which we have termed a zinc knuckle domain. In the Mg²⁺ Ap₅A and Mn²⁺ Ap₅A structures, Mg²⁺ and Mn²⁺ demonstrate six coordinate octahedral geometry. The interactions of the Mg²⁺-coordinated water molecules with the protein and Ap₅A phosphate chain demonstrate their involvement in catalyzing phosphate transfer. The protein selects for β-γ (preferred by Mg²⁺) rather than α-γ (preferred by Mn²⁺) metal ion coordination by forcing the ATP phosphate chain to have an extended conformation. Proteins 32:276–288, 1998. © 1998 Wiley-Liss, Inc.     

Rice Crinkly4 receptor-like kinase positively regulates culm elongation and amino acid K532 is not essential for its kinase activity

Receptor-like kinases (RLKs) play important roles in multiple aspects of plant growth and development. As a member of the TNFR-like RLK subfamily, rice Crinkly4 (OsCR4) functions mainly in epidermal cell differentiation in many organs. Here we show that in addition to its essential role in epidermal cell differentiation in the palea and lemma, OsCR4 positively regulates rice culm elongation, similar to maize CR4. Although OsCR4 is an active kinase, like CR4 in maize and ACR4 in Arabidopsis, the conserved amino acid K532 in OsCR4 is not essential for its kinase activity in vitro. Whether other conserved amino acids are required for its kinase activity and the relationship between its activity and function in plant development remain to be investigated.     

Tumor-targeting Salmonella typhimurium A1-R combined with recombinant methioninase and cisplatinum eradicates an osteosarcoma cisplatinum-resistant lung metastasis in a patient-derived orthotopic xenograft (PDOX) mouse model: decoy,trap and kill chemotherapy moves toward the clinic

In the present study, a patient-derived orthotopic xenograft (PDOX) model of recurrent cisplatinum (CDDP)-resistant metastatic osteosarcoma was treated with Salmonella typhimurium A1-R (S. typhimurium A1-R), which decoys chemoresistant quiescent cancer cells to cycle, and recombinant methioninase (rMETase), which selectively traps cancer cells in late S/G₂, and chemotherapy. The PDOX models were randomized into the following groups 14 days after implantation: G1, control without treatment; G2, CDDP (6 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, rMETase (100 unit/mouse, i.p., daily, for 2 weeks). G4, S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., weekly, for 2 weeks); G5, S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks); G6, S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks) and CDDP (6 mg/kg, i.p. injection, weekly, for 2 weeks). On day 14 after initiation, all treatments except CDDP alone, significantly inhibited tumor growth compared to untreated control: (CDDP: p = 0.586; rMETase: p = 0.002; S. typhimurium A1-R: p = 0.002; S. typhimurium A1-R combined with rMETase: p = 0.0004; rMETase combined with both S. typhimurium A1-R and CDDP: p = 0.0001). The decoy, trap and kill combination of S. typhimurium A1-R, rMETase and CDDP was the most effective of all therapies and was able to eradicate the metastatic osteosarcoma PDOX.     

Salmonella typhimurium A1-R targeting of a chemotherapy-resistant BRAF-V600E melanoma in a patient-derived orthotopic xenograft (PDOX) model is enhanced in combination with either vemurafenib or temozolomide

A metastatic melanoma obtained from the right chest wall of a patient was previously established orthotopically in the right chest wall of nude mice as a patient-derived orthotopic xenograft (PDOX) model. We previously showed that the combination of tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) and chemotherapy was highly effective against the melanoma PDOX. In the present study, we investigated the mechanism of the high efficacy of this combination. Two weeks after implantation, 40 PDOX mouse models were randomized into 4 groups of 10 mice each: untreated control (n = 10); treated with S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks, n = 10); treated with temozolomide (TEM) (25 mg/kg, p.o. for 14 consecutive days) combined with S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks, n = 10); treated with vemurafenib (VEM) (30 mg/kg, p.o., for 14 consecutive days) combined with S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks) (n = 10). On day 14 from initiation, all treatments significantly inhibited tumor growth compared with untreated control (S. typhimurium A1-R: p < 0.01; TEM combined with S. typhimurium A1-R: p < 0.01; VEM combined with S. typhimurium A1-R: p < 0.01). Combination therapy with S. typhimurium A1-R was significantly more effective on tumor growth than S. typhimurium A1-R alone (with TEM: p < 0.01; with VEM: p < 0.01). Combination therapy significantly increased S. typhimurium A1-R tumor targeting alone (S. typhimurium A1-R + TEM: p < 0.01, S. typhimurium A1-R + VEM: p < 0.01), relative to S. typhimurium A1-R alone, respectively. In conclusion, chemotherapy drugs promoted targeting of S. typhimurium A1-R of melanoma, thereby enhancing efficacy against the melanoma PDOX.     

AU4S: A novel synthetic peptide to measure the activity of ATG4 in living cells

ATG4 plays a key role in autophagy induction, but the methods for monitoring ATG4 activity in living cells are limited. Here we designed a novel fluorescent peptide named AU4S for noninvasive detection of ATG4 activity in living cells, which consists of the cell-penetrating peptide (CPP), ATG4-recognized sequence “GTFG,” and the fluorophore FITC. Additionally, an ATG4-resistant peptide AG4R was used as a control. CPP can help AU4S or AG4R to penetrate cell membrane efficiently. AU4S but not AG4R can be recognized and cleaved by ATG4, leading to the change of fluorescence intensity. Therefore, the difference between AU4S- and AG4R-measured fluorescence values in the same sample, defined as “F-D value,” can reflect ATG4 activity. By detecting the F-D values, we found that ATG4 activity paralleled LC3B-II levels in rapamycin-treated cells, but neither paralleled LC3B-II levels in starved cells nor presented a correlation with LC3B-II accumulation in WBCs from healthy donors or leukemia patients. However, when DTT was added to the system, ATG4 activity not only paralleled LC3B-II levels in starved cells in the presence or absence of autophagy inhibitors, but also presented a positive correlation with LC3B-II accumulation in WBCs from leukemia patients (R² = 0.5288). In conclusion, this study provides a convenient, rapid, and quantitative method to monitor ATG4 activity in living cells, which may be beneficial to basic and clinical research on autophagy.     

Catabolism of Ap3A and Ap4A in human plasma. Purification and characterization of a glycoprotein complex with 5'-nucleotide phosphodiesterase activity

A hydrolase splitting adenosine(5')triphospho(5')adenosine (Ap3A) to AMP and ADP has recently been detected in human plasma [Lüthje, J. and Ogilvie, A. (1984) Biochem. Biophys. Res. Commun. 118, 704-709]. The enzyme has been purified to apparent homogeneity, as stained in a native polyacrylamide gel. From gel filtration data a Stokes radius of 5.9 nm was calculated, suggesting a molecular mass of about 230 kDa. The presence of the non-ionic detergent Triton X-100 did not change the molecular mass. The hydrolase dissociated to three major protein components (66 kDa; 45 kDa; 16 kDa) during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol. Binding of the native enzyme to concanavalin-A--Sepharose and specific inhibition of binding by methyl mannoside indicated that the hydrolase is a glycoprotein. Two of the subunits (66 kDa; 45 kDa) could be affinity-labeled with radioiodinated concanavalin A. Active hydrolase could be prepared in buffers without added metal ions. Treatment with EDTA, however, completely abolished the hydrolytic activity. The enzyme could be reactivated by incubation with Ca2+, Co2+ and, at best, with Zn2+, whereas Mg2+ was ineffective. The affinity of the enzyme for Ap3A was high (Km = 1 microM), with normal Michaelis-Menten kinetics. The homolog dinucleotide Ap4A was also substrate (Km = 0.6 microM) yielding AMP and ATP as products after the asymmetric split. Other dinucleotides, such as NAD, and also mononucleotides (ATP,UTP) were degraded to nucleoside monophosphates indicating a broad specificity of the enzyme. The synthetic compound thymidine 5'-monophosphate p-nitrophenyl ester was substrate with low affinity whereas its 3'-homolog was not hydrolyzed. Optimal activity of the hydrolase was found at pH 8.5.     

Direct evidence of a heterotrimeric complex of human interleukin-4 with its receptors.

The mode of binding of interleukin-4 (IL-4) to its two known receptors, specific receptor IL-4R and a shared receptor gamma c, was investigated using gel filtration and gel electrophoresis. A ternary complex between IL-4 and the soluble domains of the two receptors was shown to exist in solution. The association constant between gamma c and the stable complex of IL-4/sIL-4R is in the millimolar range, making the ternary complex a feasible target for crystallization studies.