Mapping protein–protein interaction by 13C′-detected heteronuclear NMR spectroscopy

The copper-mediated protein–protein interaction between yeast Atx1 and Ccc2 has been examined by protonless heteronuclear NMR and compared with the already available ¹H–¹⁵N HSQC information. The observed chemical shift variations are analyzed with respect to the actual solution structure, available through intermolecular NOEs. The advantage of using the CON-IPAP spectrum with respect to the ¹H–¹⁵N HSQC resides in the increased number of signals observed, including those of prolines. CBCACO-IPAP experiments allow us to focus on the interaction region and on side-chain carbonyls, while a newly designed CEN-IPAP experiment on side-chains of lysines. An attempt is made to rationalize the chemical shift variations on the basis of the structural data involving the interface between the proteins and the nearby regions. It is here proposed that protonless ¹³C direct-detection NMR is a useful complement to ¹H based NMR spectroscopy for monitoring protein–protein and protein–ligand interactions. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at     

Characterization of the guanosine 3′∶5′-monophosphate-dependent protein kinase from silkworm eggs and analysis of the endogenous protein substrates

Summary In a previous paper, two types of cyclic nucleotide-dependent protein kinases, namely cGMP dependent G-kinase and cAMP dependent A-kinase, in silkworm eggs has been reported (Takahashi et al. 1975; Takahashi 1976). One of these, G-kinase, has now been purified 2400-fold by means of ammonium sulfate fractionation, chromatography on hydroxylapatite, DEAE cellulose, and gel filtration.Some of the properties of the enzyme are described. The enzyme is highly dependent on cGMP; it is strongly inhibited by GTP in a noncompetitive manner not only for ATP but also for cGMP. GTP was found to be highly inhibitory on G-kinases from various tissues of the silkworm, but did not inhibit the A-kinase.Incubation of the egg extract with [-³²P]ATP and Mg²⁺ led to the formation of three major³²P-labelled proteins, with molecular weights of 42.000, 70.000 and 180.000 as analyzed by SDS polyacrylamide gel electrophoresis. Two of them corresponded to the subunits of vitellin.The silkworm vitellin was effectively phosphorylated both by the highly purified G-kinase and by the A-kinase. It is concluded that the G-kinase is involved in the phosphorylation of vitellin in developing silkworm eggs.Abbreviations cAMP adenosine 35-monophosphate - cGMP guanosine 35-monophosphate - A-kinase adenosine 35-monophosphate-dependent protein kinase - G-kinase guanosine 35-monophosphate-dependent protein kinase - MIX 1-methyl-3-isobutylxanthine     

Anti-anabolic effects of adenosine 3′:5′-cyclic monophosphate. Inhibition of protein synthesis

1. Evidence is presented that cyclic AMP inhibits the incorporation of l-[4,5-(3)H]leucine into protein in a cell-free system from rat liver. This inhibition occurs after aminoacyl-tRNA formation. 2. Microsomal fractions, isolated after the incubation of postmitochondrial supernatant with cyclic AMP and ATP, show a diminished ability to synthesize protein. Both cyclic AMP and ATP are required for this effect. 3. A possible physiological role for the anti-anabolic action of cyclic AMP is discussed in terms of the control of gluconeogenesis.     

Plasmodium falciparum DDX55 is a nucleocytoplasmic protein and a 3′-5′ direction-specific DNA helicase

Malaria is one of the major causes of mortality as well as morbidity in many tropical and subtropical countries around the world. Although artemisinin combination therapies (ACTs) are contributing to substantial decline in the worldwide malaria burden, it is becoming vulnerable by the emergence of artemisinin resistance in Plasmodium falciparum leading to clinical failure of ACTs in Southeast Asia. Helicases play important role in nucleic acid metabolic processes and have been also identified as therapeutic drug target for different diseases. Previously, it has been reported that P. falciparum contains a group of DEAD-box family of helicases which are homologous to Has1 family of yeast. Here, we present the characterization of a member of Has1 family (PlasmoDB number PF3D7_1419100) named as PfDDX55. The biochemical characterization of PfDDX55C revealed that it contains both DNA- and RNA-dependent ATPase activity. PfDDX55C unwinds partially duplex DNA in 3′ to 5′ direction and utilizes mainly ATP or dATP for its activity. The immunofluorescence assay and q-RT PCR analysis show that PfDDX55 is a nucleocytoplasmic protein expressed in all the intraerythrocytic development of P. falciparum 3D7 strain with maximum expression level in trophozoite stage. The LC-MS/MS experiment results and STRING analysis show that PfDDX55 interacts with AAA-ATPase which has been shown to be involved in ribosomal biogenesis.

     

Quantitative microspectrophotometrical determination of protein thiols and disulfides with 2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD)

Summary 2,2-dihydroxy-6,6-dinaphthyldisulfide (DDD) reacts with both protein thiol groups and with protein disulfides (Nöhammer 1977). By varying the pH of the DDD-reaction, as well as the reaction times, the complex reaction became specific with respect to the histochemical demonstration of protein-SH groups. Furthermore, the application of the histochemical DDD-reaction following quantitative blockade of the protein-SH groups enabled the demonstration of distinctive DDD-reactive disulfides. The specifity and the extent of the different histochemical DDD-staining methods were investigated by comparing macroscopically determined values of the protein-SH-contents, and the contents of the different kinds of disulfides in Ehrlich-ascites-tumor cells (EATC) (Modig 1968; Hofer 1975), with microspectrometrical values determined with the MCN-method of Nöhammer et al. (1981), and with microspectrometrical values measured on EATC after staining with the modified DDD-methods. Also, the method for the histochemical demonstration of protein-SH with DDD after the reduction of the disulfides with thioglycolate was investigated and conditions were found by which the protein-SH content could be determined quantitatively with DDD and Fast blue B after the reduction of the disulfides. With the aid of the MCN-method (Nöhammer et al. 1981), the intracellular disulfide interchange reaction was investigated, leading to pH-dependent changes of the SH-SS-ratio of fixed cells during their incubation in aqueous media. In addition the possibility of protein loss during the long incubation times of the fixed cells in the DDD-solutions was investigated. For the quantitative microscpecrometrical determination of the protein content of EATC the so-called tetrazonium-coupling method, optimized by Nöhmmer (1978) and calibrated by Nöhammer et al. (1981), was used.Dedicated to Prof. Dr. E. Ziegler on the occasion of his 70^th birthday     

Simultaneous inhibition of guinea pig brain 2′, 3′-cyclic nucleotide 3′-phosphohydrolase and myelin protein synthesis by 2′-adenosine monophosphate

Although the function of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) in myelin is unknown, the enzyme has been implicated in the metabolism of myelin proteins. Using 2′-AMP to inhibit CNPase, we examined the effect of reduced enzyme activity on the $\text{in vitro}$ incorporation of ¹⁴C-leucine into brain proteins. The results of this study revealed that (1) guinea pig brain homogenates incorporate leucine into protein from a sucrose medium in a linear fashion, (2) all brain fractions (cytosol, myelin, and microsomes) are labelled within 1 hr, (3) 2′-AMP inhibition of CNPase by 50% results in a similar inhibition of brain protein synthesis, and (4) the reduced protein synthesis is accompanied by a shift in label from myelin proteins to those found in the microsomes. These results are consistent with a role for CNPase in myelin protein synthesis.     

Studies of kidney plasma membrane adenosine-3′,5′-monophosphate-dependent protein kinase

Porcine kidney cortex was utilized for the preparation of plasma-membrane-enriched and soluble cytoplasmic (cytosol) fractions for the purpose of examining the relative properties of cyclic [³H]AMP receptor and cyclic AMP-dependent protein kinase activities of these preparations. The affinity, specificity and reversibility of cyclic [³H]AMP interaction with renal membrane and cytosol binding sites were indicative of physiological receptors.Binding sites of cytosol and deoxycholate-solubilized membranes were half-saturated at approx. 50nM and 100 nM cyclic [³H]AMP. Native plasma membranes exhibited multiple binding sites which were not saturated up to 1 mM cyclic [³H]AMP. Modification of the cyclic phosphate configuration or 2′-hydroxyl of the ribose moiety of cyclic AMP produced a marked reduction in the effectiveness of the cyclic AMP analogue as a competitor with cyclic [³H]AMP for renal receptors. The cyclic [³H]AMP interaction with membrane and cytosol fractions was reversible and the rate and extent of dissociation of bound cyclic [³H]AMP was temperature dependent. With the plasma-membrane preparation, dissociation of cyclic [³H]AMP was enhanced by ATP or AMP.Assay of both kidney subcellular fractions for protein kinase activity revealed that cyclic AMP enhanced the phosphorylation of protamine, lysine-rich and arginine-rich histones but not casein. The potency and efficacy of activation of renal membrane and cytosol protein kinase by cyclic AMP analogues such as N⁶-butyryl-adenosine cyclic 3′,5′-monophosphate or N⁶,O²-dibutyryl-adenosine cyclic 3′,5′-monophosphate supported the observations on the effectiveness of cyclic AMP analogues as competitors with cyclic [³H]AMP in competitive binding assays.This study suggested that the membrane cyclic [³H]AMP receptors may be closely associated with the membrane-bound catalytic moiety of the cyclic AMP-dependent protein kinase system of porcine kidney.     

Adenosine 3′:5′-cyclic monophosphate-dependent protein kinase(s) of rat ovarian cells. Gonadotropin regulation of adenosine 3′:5′-cyclic monophosphate-receptor activity

Regulation of cyclic AMP-dependent protein kinase, cyclic AMP-receptor activity and intracellular cyclic AMP concentrations by choriogonadotropin was studied in ovarian cells prepared from 26-day-old rats. A close correlation was observed between phospho-transferase activity and cyclic AMP-receptor activity in 12000g supernatant fractions from rat ovarian homogenate. The apparent activation constant (K(a)) and I(50) (concentration required to produce 50% inhibition) of different cyclic nucleotides for phosphotransferase and cyclic AMP receptor activities respectively were also determined. Cyclic AMP and 8-bromo cyclic AMP were most effective, giving K(a) values of 0.08 and 0.09mum and I(50) of 0.12 and 0.16mum respectively. Other nucleotides were also effective, but required higher concentrations to give a comparable effect. An increased concentration of cyclic AMP produced by choriogonadotropin (1mug/ml) treatment was accompanied by decreased cyclic AMP binding as early as 5min after hormone addition. Choriogonadotropin also stimulated the protein kinase activity ratio (-cyclic AMP/+cyclic AMP) under identical experimental conditions. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiated the action of choriogonadotropin on the three parameters measured in a dose- and time-dependent manner. The maximal cyclic AMP-binding capacity, as determined by cyclic AMP-exchange assay, remained unchanged before and after hormone addition. The endogenously bound cyclic AMP was determined from the difference between the maximal binding capacity and the exogenously bound cyclic AMP. With different choriogonadotropin concentrations, a quantitative correlation was established between maximal binding capacity, exogenous binding and endogenous binding activities. Approx. 60% of total binding sites were endogenously occupied in untreated cells, and choriogonadotropin (1mug/ml) treatment fully saturated available binding sites with a parallel 10-fold increase in cellular cyclic AMP. The present results provide evidence for a probable intracellular compartmentalization of cyclic AMP in the ovarian cell, and suggest that in the unstimulated state all cyclic AMP present in the ovarian cell may not be available for protein kinase activation.     

Isolation of a novel protein,P12—from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3′-5′- exonuclease activity

ABSTRACT

We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development from embryonic cells to adult flies and found declining Dm-dNK activity during development and no activity in adult flies. Surprisingly, the extract from adult flies exhibited a strong inhibitory effect on deoxyribonucloside kinase activity. The dNK-inhibitor was precipitable with ammonium sulfate, and was purified to a high degree by gel-filtration as indicated by LC-MS/MS analysis. Since the inhibitor eluted from G-200 gel-filtration with a size of 10–13 kDa, we named it P12. We tested the purified fraction for specificity towards various enzymes and found that both mammalian and bacterial dNKs were inhibited, whereas there was no effect on hexokinase and pyruvate kinases and acidic phosphatase. However, when tested against cyclin B-dependent kinase, we found a strong inhibitory effect. Both with human Cdk1/CycB and S. pombe Cdc2/B-type cyclin the purified fraction from Superdex 200 that inhibited Dm-dNK, also inhibited the two protein kinases to the same degree. Furthermore, testing P12 in a DNA polymerase based assay we found that the 3′-5′-exonuclease part of the DNA polymerase (Klenow polymerase) was activated.     

Adenosine 3′:5′-cyclic monophosphate-binding protein from the cellular slime mould Dictyostelium discoideum (Short Communication)

During the differentiation of myxamoebae of Dictyostelium discoideum strain Ax-2 grown in axenic medium there is a seven- to ten-fold increase in the specific activity of cyclic AMP-binding protein(s). Evidence is presented for the belief that cyclic AMP phosphodiesterase and cyclic AMP-binding protein are distinct molecular species.     

Initiation of the 3′:5′-AMP-induced protein kinase A Iα regulatory subunit conformational transition. Part II. Inhibition by Rp-3′:5′-AMPS

Protein-ligand docking and ab initio calculations have shown that the 3′:5′-AMP phosphorothioate analog (Rp-3′:5′-AMPS) blocks the A326 amide group displacement typical of transition from the H- to B-conformation within the B-domain of protein kinase A Iα R-subunit. This behavior of Rp-3′:5′-AMPS leads to the inhibition of initial stages of hydrophobic relay operation. In accordance with the proposed hypothesis, Rp-3′:5′-AMPS similarly to 3′:5′-AMP forms a hydrogen bond with the amide group of A326; however, the properties of this bond together with the position of the sulfur atom prevent the movement of A326. Finally, the Rp-3′:5′-AMPS-bound domain appears to be locked in the H-conformation, which is in agreement with the X-ray data.     

Fast protein liquid chromatographic purification and some properties of a partially O-methylated flavonol glucoside 2′-/5′-O-methyltransferase

A flavonol O-methyltransferase was partially purified from Chrysosplenium americanum by fractional precipitation with ammonium sulphate followed by gel filtration and chromatofocusing using an FPLC system. The enzyme which was purified 420-fold catalysed the transfer of the methyl group of SAM to the 2′- or 5′-positions of partially methylated flavonol glucosides, the two terminal methylation steps in the biosynthesis of Chrysosplenium flavonoids. The enzyme had a pH optimum of 7 in Pi buffer, a pI of < 5, an M, of 57 000, no Mg²⁺ requirement and was inhibited by both N-ethylmaleimide and phenylmercuriacetate. The K_m value for the flavonol substrate was 2 μM and that for SAM was 100 μM. The role of this enzyme is discussed in relation to the biosynthesis of polymethylated flavonols in this tissue.     

3′,5′-Cyclic-nucleotide-dependent protein kinase of squamous cell carcinoma of the prostate

An adenosine 3'5'-cyclic-monophosphate (Cyclic AMP)-dependent protein kinase has been identified and partially purified from the rat prostate tumor induced by 20-methylcholanthrene. This enzyme is stimulated 2- to 3-fold by the nucleotide. Equilibrium studies at pH 5.0 suggest the presence of a major class of binding site for cyclic AMP with an association constant of approximately 10(8) M-1. The concentration of binding site is about 1 pmol/mg of protein of the enzyme preparation. The enzyme is stimulated by other cyclic nucleotides as well, but only by higher concentrations. In comparing the ability of different histone subfractions, casein and protamine, to serve as substrate for this particular protein kinase, maximal cyclic-AMP-dependent enzyme activity was observed with histones. The results suggest that factors contributing to the malignant growth of the prostatic tissue do not directly involve changes in the characteristics of a cyclic-AMP-dependent protein kinase.     

A cyclic adenosine 3′,5′-monophosphate-dependent protein kinase mutant of Neurospora crassa

A cpk mutant of Neurospora crassa with morphological alteration was obtained spontaneously during the cross between the wild-type and a glycerol utilizing cr-l strain. The growth rate of cpk was intermediate between the wild-type and cr-1 mutant strains. The cpk conidia contained a reduced level of carotenoid pigments as compared to the wild-type conidia. The cpk mutant had no detectable amount of cyclic adenosine 3,5-monophosphate (cAMP)-binding protein at all stages of growth tested. On a DEAE-Sephacel column chromatogram, protein kinase activity of the wild type was eluted at two peaks; the first peak was cAMP-dependent, and the second one was not. In contrast, the cpk strain had two peaks of cAMP-independent enzymes. It is suggested that cAMP-dependent protein kinase may be altered in the cpk mutant into a cAMP-independent type by an alteration of the regulatory subunit of this enzyme.Abbreviations cAMP Cyclic adenosine 3,5-monophosphate - 8-N₃-[³H] cAMP 8-azido-[³H]cyclic adenosine 3,5-monophosphate     

RecJ-like protein from Pyrococcus furiosus has 3′–5′ exonuclease activity on RNA: implications for proofreading of 3′-mismatched RNA primers in DNA replication

Replicative DNA polymerases require an RNA primer for leading and lagging strand DNA synthesis, and primase is responsible for the de novo synthesis of this RNA primer. However, the archaeal primase from Pyrococcus furiosus (Pfu) frequently incorporates mismatched nucleoside monophosphate, which stops RNA synthesis. Pfu DNA polymerase (PolB) cannot elongate the resulting 3′-mismatched RNA primer because it cannot remove the 3′-mismatched ribonucleotide. This study demonstrates the potential role of a RecJ-like protein from P. furiosus (PfRecJ) in proofreading 3′-mismatched ribonucleotides. PfRecJ hydrolyzes single-stranded RNA and the RNA strand of RNA/DNA hybrids in the 3′–5′ direction, and the kinetic parameters (K_m and K_cat) of PfRecJ during RNA strand digestion are consistent with a role in proofreading 3′-mismatched RNA primers. Replication protein A, the single-stranded DNA–binding protein, stimulates the removal of 3′-mismatched ribonucleotides of the RNA strand in RNA/DNA hybrids, and Pfu DNA polymerase can extend the 3′-mismatched RNA primer after the 3′-mismatched ribonucleotide is removed by PfRecJ. Finally, we reconstituted the primer-proofreading reaction of a 3′-mismatched ribonucleotide RNA/DNA hybrid using PfRecJ, replication protein A, Proliferating cell nuclear antigen (PCNA) and PolB. Given that PfRecJ is associated with the GINS complex, a central nexus in archaeal DNA replication fork, we speculate that PfRecJ proofreads the RNA primer in vivo.