Binding of protein S to C4b-binding protein. Mutagenesis of protein S.

Protein S and C4b-binding protein (C4BP) form a tight complex (Kd approximately 0.6 nM) the physiologic purpose of which is unknown. The participation of protein S in this complex was investigated using site-specific mutagenesis. Normal recombinant human protein S (rHPS) and five specifically mutated protein S analogs were expressed in transformed human kidney 293 cells and the following properties were characterized: solution-phase C4BP binding, ability to be cleaved by thrombin, ability to act as a cofactor in the activated protein C-catalyzed inactivation of factor Va, and gamma-carboxyglutamic acid content. In some cases, beta-hydroxyaspartic acid plus beta-hydroxyasparagine content was also determined. Binding studies indicated that while clearly important for a high affinity interaction, the amino acid sequence Gly605-Ile614 identified by Walker (Walker, F J. (1989) J. Biol. Chem. 264, 17645-17648) does not account for all the binding energy of the HPS-C4BP interaction. All mutants perturbed in this region or lacking it altogether displayed reduced C4BP binding, and some retained anticoagulant cofactor function. Neither human factor X nor human steroid-binding protein had any measurable ability to compete with plasma HPS for C4BP binding. Furthermore, bovine protein S and a rHPS analog with bovine sequence from Gly597-Trp629 bound to human C4BP with the same affinity as did HPS, and both proteins substituted effectively for HPS as a cofactor for activated protein C in an otherwise human anticoagulation system. Together these results suggest that optimal binding of protein S to C4BP requires the putative alpha-helix Gly605-Ile614, as well as other undetermined regions of protein S, and that the regions of HPS responsible for C4BP binding and activated protein C cofactor function are structurally isolated.     

Malaria circumsporozoite protein inhibits protein synthesis in mammalian cells.

Native Plasmodium circumsporozoite (CS) protein, translocated by sporozoites into the cytosol of host cells, as well as recombinant CS constructs introduced into the cytoplasm by liposome fusion or transient transfection, all lead to inhibition of protein synthesis in mammalian cells. The following findings suggest that this inhibition of translation is caused by a binding of the CS protein to ribosomes. (i) The distribution of native CS protein translocated by sporozoites into the cytoplasm as well as microinjected recombinant CS protein suggests association with ribosomes. (ii) Recombinant CS protein binds to RNase-sensitive sites on rough microsomes. (iii) Synthetic peptides representing the conserved regions I and II-plus of the P.falciparum CS protein displace recombinant CS protein from rough microsomes with dissociation constants in the nanomolar range. (iv) Synthetic peptides representing region I from the P.falciparum CS protein and region II-plus from the P.falciparum, P.berghei or P.vivax CS protein inhibit in vitro translation. We propose that Plasmodium manipulates hepatocyte protein synthesis to meet the requirements of a rapidly developing schizont. Since macrophages appear to be particularly sensitive to the presence of CS protein in the cytosol, inhibition of translation may represent a novel immune evasion mechanism of Plasmodium.     

Effect of RecF protein on reactions catalyzed by RecA protein.

RecF protein is one of at least three single strand DNA (ssDNA) binding proteins which act in recombination and repair in Escherichia coli. In this paper we show that our RecF protein preparation complexes with ssDNA so as to retard its electrophoretic movement in an agarose gel. The apparent stoichiometry of RecF-ssDNA-binding measured in this way is one RecF molecule for every 15 nucleotides and the binding appears to be cooperative. Interaction of the other two ssDNA-binding proteins, RecA and Ssb proteins, has been studied extensively; so in this paper we begin the study of the interaction of RecF and RecA proteins. We found that the RecF protein preparation inhibits the activity of RecA protein in the formation of joint molecules whether added before or after addition of RecA protein to ssDNA. It, therefore, differs from Ssb protein which stimulates joint molecule formation when added to ssDNA after RecA protein. We found that our RecF protein preparation inhibits two steps prior to joint molecule formation: RecA protein binding to ssDNA and coaggregate formation between ssDNA-RecA complexes and dsDNA. We found that it required a much higher ratio of RecF to RecA protein than normally occurs in vivo to inhibit joint molecule formation. The insight that these data give to the normal functioning of RecF protein is discussed.     

Opsonic characteristics of chylomicron-bound protein.

A blood protein in complex with native chylomicrons was labeled with radiogold colloid, thus indirectly identifying the protein material in the complex as opsonin and suggesting the possibility that native chylomicrons may utilize the opsonin system for its clearance from the circulation. The protein in question has been shown to be at least divalent, in that the protein in combination with one type of particle could still react with another. It is felt that these studies provide further insight into the complex surface events that result during the process of opsonization.     

Synthesis of protein in the pancreas. II. The role of ribonucleoprotein in protein synthesis

1. The ribonucleoprotein of the microsome fraction which sediments at 40,000 R.P.M. as a pellet (and which is referred to as the pellet material) has been studied with reference to its role in protein synthesis in the pancreas. 2. In pellet material nucleic acid and protein form a definite complex as shown by its electrophoretic behavior and unchanging composition under various conditions. 3. Protein of pellet material is not especially rich in the diamino acids. 4. Evidence is brought forward indicating that the protein component of pellet material takes part in the general process of protein synthesis in the cell. (a) The well known correlation between quantity of RNA and rate of protein synthesis in a tissue implicates the protein of the pellet material, for most of the RNA in the pancreas and other tissues is in this material. (b) Uptake of isotopically labelled glycine by the pellet material, confirming results of previous workers, is for short periods greater than in other protein fractions. (c) Comparing the pellet materials of pancreas, liver, and kidney-three tissues with vastly different rates of protein synthesis, in the sequence given-there is a correlation between the quantity of RNA in the pellet and the rate of protein synthesis in the tissue; a similar correlation between quantity of RNA in the pellet material and rate of N(15)-glycine uptake by the protein component of the pellet; and finally, the level of uptake by total protein varies with the tissue and is related to the uptake of N(15)-glycine by protein of the pellet. 5. In the pancreas a distinction can be made between proteins synthesized for secretion and the nucleoprotein of the pellet (not found in the secretion) which, however, takes part in the synthetic process, as shown by the fact that the N(15) uptake by protein of the pellet is increased when the synthesis of digestive enzymes is stimulated by secretion. 6. The time course of N(15) uptake by proteins of the pancreas indicates that pellet protein serves as precursor material in the synthesis of the secretory proteins. 7. Rate of uptake of N(15)-glycine by the purines of RNA of the pellet material is not correlated with uptake by the protein. 8. The uptake of C(14)-alanine by an in vitro system of microsomes + mitochondria is impaired by preincubation of the microsomes with ribonuclease. This is direct experimental evidence for the dependence of protein synthesis upon the presence or intactness of ribonucleic acid in the microsomes.     

Identification of a new protein involved in the regulation of the anticoagulant activity of activated protein C. Protein S-binding protein

The apparent molecular weight of functional protein S in citrated plasma was observed to be between 115,000 and 130,000 as measured by sedimentation equilibrium in the air-driven ultracentrifuge. The molecular weight of the functional protein decreased to approximately 62,000 when copper ions were added to the plasma. This suggested the presence of a protein S-binding protein in plasma, which was confirmed by gel filtration experiments. Frontal analysis of plasma indicated that functional protein S could exist in as many as three forms. Addition of copper ions to plasma reduced the number of forms to one. In order to isolate the binding protein, plasma was fractionated first on a column of immobilized iminodiacetic acid that had been equilibrated with copper ions. The proteins that eluted in a 0.6 M NaCl wash were passed over a column of protein S immobilized on agarose beads. A protein, eluted in the 0.6 M NaCl wash, was observed to bind to protein S in gel filtration experiments. When added to plasma depleted of both protein S and the binding protein, the binding protein was observed to enhance the anticoagulant activity of activated protein C only in the presence of protein S. Protein S-binding protein was also observed to enhance the rate of factor Va inactivation by activated protein C and protein S.     

Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain.     

Specificity of protein phosphatases in the dephosphorylation of protein kinase C.

Protein kinase C can autophosphorylate in vitro and has also been shown to be phosphorylated in vivo. In order to investigate the factors that may determine the phosphorylation state of protein kinase C in vivo, we determined the ability of the ATP + Mg2+-dependent phosphatase and the polycation-stimulated (PCS) phosphatases to dephosphorylate protein kinase C in vitro. These studies show that all the oligomeric forms of the PCS phosphatases (PCSH1, PCSH2, PCSM and PCSL phosphatases) are effective in the dephosphorylation of protein kinase C, showing 34-82% of the activity displayed with phosphorylase a as substrate. In contrast both the catalytic subunit of the PCS phosphatase and that of the ATP+Mg2+-dependent phosphatase showed only weak activity with protein kinase C as substrate. All these phosphatases, however, were activated by protamine (Ka 14-16 micrograms/ml) through what appears to be a substrate-directed effect. The relative role of these phosphatases in the control of protein kinase C is discussed.     

Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.     

Membrane orientation of laminin binding protein.

Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end.     

Polyamine-stimulated phosphorylation of prostatic spermine-binding protein is mediated only by cyclic AMP-independent protein kinases.

Spermine-binding protein (a rat ventral prostatic protein with high affinity for spermine) was phosphorylated in situ through the action of intrinsic cellular protein kinase(s), suggesting it to be a phosphoprotein in vivo. The purified protein served as a substrate in a number of cyclic AMP-independent protein kinase reactions in vitro, but not for cyclic AMP-dependent, Ca2+ + calmodulin-dependent or Ca2+ + phospholipid-dependent protein kinases. Available data indicate that at least one of the cyclic AMP-independent protein kinases (cytosolic protein kinase C2) may be physiologically relevant in mediating the phosphorylation of this protein. The phosphorylation reaction was stimulated several-fold in the presence of spermine. Spermidine was somewhat less effective, whereas putrescine, cadaverine and 1,6-hexanediamine were minimally active. Phospho amino acid analysis of 32P-labelled spermine-binding protein indicated that phosphoserine was the only labelled phospho amino acid. Spermine-binding protein did not undergo autophosphorylation, or modify the stimulative effect of spermine on the phosphorylation of other substrates such as non-histone proteins. In situ the phosphorylation of spermine-binding protein in tissue from castrated rats was markedly diminished as compared with the normal. Since the phosphorylation of spermine-binding protein appears to be mediated by cyclic AMP-independent protein kinase(s) whose activity in the prostate is under androgenic control, it is suggested that androgen-dependent modulation of the protein kinase(s) exerts a regulatory control (via phosphorylation-dephosphorylation) on the spermine-binding activity and stability of this protein in vivo. Further, since this protein is a substrate for only the cyclic AMP-independent protein kinases, it could serve as a tool for the investigation of such kinases.     

The modulator-dependent protein kinase. A multifunctional protein kinase activatable by the Ca2+-dependent modulator protein of the cyclic nucleotide system.

A protein kinase which depends on the simultaneous presence of Ca2+ and the modulator protein for its histone phosphorylation activity has been demonstrated in rabbit skeletal muscle and partially purified. The purified enzyme was not activated by cAMP, cGMP, or incubation with trypsin. Nor was the enzyme inhibited by the protein inhibitor of cAMP-dependent protein kinase. In addition to histone, myosin light chains and phosphorylase kinase served as substrates for the protein kinase, and their phosphorylation also depended on the presence of Ca2+ and the modulator protein. The phosphorylation of phosphorylase kinase was accompanied with a marked activation of the enzyme. The results suggest that the protein kinase has multiple functions and may be involved in the mediation of Ca2+ effects in many biological processes. It is proposed that this enzyme be designated as the modulator-dependent protein kinase. The modulator-dependent protein kinase may be identical to the myosin light chain kinase; chicken gizzard light chain kinase has been shown activatable by the modulator protein (Dabrowska, R., Sherry, J. M. F., Aramatorio, D. K., and Hartshorne, D. J. (1978) Biochemistry 17, 253-258).     

Association of the Drosophila melanogaster engrailed protein with specific soluble nuclear protein complexes.

The Drosophila engrailed protein which contains a homeobox domain and specific DNA binding activity is believed to function in the regulation of gene expression during embryogenesis. Here we show that the engrailed protein interacts stably with specific complexes of soluble nuclear proteins when expressed artificially in a cell line and in the developing embryo. The engrailed complexes have molecular masses between 10(7) and 10(8) which suggests they contain a polymeric protein component. The complex is able to bind reversibly to DNA and a definitive purification shows it to be constituted of 12 distinct protein species, two of which are predominant. Purified, bacterially produced engrailed protein can be reconstituted with both culture cell and embryo nuclear protein fractions to form complexes of the same and related composition respectively. On the basis of these results we propose that protein--protein interactions as well as DNA binding are important for correct engrailed protein function in vivo.     

A dnaB-like protein of Pseudomonas aeruginosa.

A dnaB-like protein from P. aeruginosa was purified to near homogeneity using as an assay the immunoprecipitation by E. coli dnaB antiserum in a solid-phase. In the chromatographic characteristics including the affinity to immobilized ATP the dnaB-like protein of P. aeruginosa is similar to the dnaB protein of E. coli with the exception that it does not bind to heparin-Sepharose. The dnaB-like protein has a native molecular weight of about 320,000 as determined by glycerol gradient sedimentation. It consists of several identical subunits of molecular weight of 56,000 as measured in a denaturing SDS gel. Associated with the enzyme is a DNA-dependent ATPase- and helicase activity. The dnaB-like protein is similar to the E. coli dnaB protein with regard to the binding of ATP gamma S and the formation of a ternary complex consisting of the enzyme, ATP gamma S, and phi X174 DNA. However, the enzyme of P. aeruginosa is inactive in a phi X174 DNA-dependent in vitro dnaB complementation assay using an E. coli dnaBts extract.     

Biosynthesis of adenovirus type 2 i-leader protein.

The i-leader is a 440-base-pair sequence located between 21.8 and 23.0 map units on the adenovirus type 2 genome and is spliced between the second and third segments of the major tripartite leader in certain viral mRNA molecules. The i-leader contains an open translational reading frame for a hypothetical protein of Mr about 16,600, and a 16,000-Mr polypeptide (16K protein) has been translated in vitro on mRNA selected with DNA containing the i-leader (A. Virtanen, P. Alestr?m, H. Persson, M. G. Katze, and U. Pettersson, Nucleic Acids Res. 10:2539-2548, 1982). To determine whether the i-leader protein is synthesized during productive infection and to provide an immunological reagent to study the properties and functions of the i-leader protein, we prepared antipeptide antibodies directed to a 16-amino acid synthetic peptide which is encoded near the N terminus of the hypothetical i-leader protein and contains a high acidic amino acid and proline content. Antipeptide antibodies immunoprecipitated from extracts of adenovirus type 2-infected cells a major 16K protein that comigrated with a 16K protein translated in vitro. Partial N-terminal amino acid sequence analysis by Edman degradation of radiolabeled 16K antigen showed that methionine is present at residue 1 and leucine is present at residues 8 and 10, as predicted from the DNA sequence, establishing that the 16K protein precipitated by this antibody is indeed the i-leader protein. Thus, the i-leader protein is a prominent species that is synthesized during productive infection. The i-leader protein is often seen as a doublet on polyacrylamide gels, suggesting that either two related forms of i-leader protein are synthesized in infected cells or that a posttranslational modification occurs. Time course studies using immunoprecipitation analysis with antipeptide antibodies revealed that the E1A 289R T antigen and the E1B-19K (175R) T antigen are synthesized beginning at 2 to 3 and 4 to 5 h postinfection, respectively, whereas the i-leader protein is synthesized starting at about 8 h postinfection and continues unabated until at least 25 h postinfection. The i-leader protein is very stable, as determined by pulse-chase labeling experiments, and accumulates continuously from 8 to 25 h postinfection, as shown by immunoblot analysis. The synthesis of i-leader protein does not depend upon viral DNA replication. Thus, the i-leader protein is a viral gene product of unknown function and high stability that is made in large quantities at intermediate times of productive infection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase.

Epidermal growth factor stimulates the activity of several cytosolic serine/threonine protein kinases in quiescent Swiss 3T3 cells. Two of these, which use myelin basic protein (MBP) as substrate, act as kinase kinases in that they are able to activate a separate peptide kinase activity in vitro by a mechanism involving protein phosphorylation. In this study, we have identified two activities from extracts of epidermal growth factor-treated cells that stimulate an ATP-dependent activation of both of the MBP kinases, derived in their inactive precursor forms from extracts of untreated cells. The resulting MBP kinase activities are stable to further purification and can be inactivated with either tyrosine or serine/threonine protein phosphatases and then reactivated to their original levels of activity. Thus, we propose that the in vitro activation involves protein phosphorylation, stimulated by the action of novel MBP kinase activating factors that represent intermediate components in a growth factor-stimulated kinase cascade.     

The transthyretin-related protein family.

A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretin-related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand-binding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used double-stranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity.     

Evolution of protein sequences and structures.

The relationship between sequence similarity and structural similarity has been examined in 36 protein families with five or more diverse members whose structures are known. The structural similarity within a family (as determined with the DALI structure comparison program) is linearly related to sequence similarity (as determined by a Smith-Waterman search of the protein sequences in the structure database). The correlation between structural similarity and sequence similarity is very high; 18 of the 36 families had linear correlation coefficients r>/=0.878, and only nine had correlation coefficients r相似文献   

Specificity of interferon action in protein synthesis.

Inhibitors of elongation steps in protein synthesis such as cycloheximide and anisomycin mimic interferon treatment in that they specifically inhibit the synthesis of certain viral proteins. These specific effects are seen only at very low concentrations of the antibiotics, under conditions where host cellular protein synthesis, as well as cell viability, are not severely reduced. A qualitatively as well as quantitatively close correlation between the effects of the two types of agents has been established for encephalomyocarditis virus, vesicular stomatitis virus and murine leukemia virus protein synthesis. It is concluded that one of the primary mechanisms of interferon action may be a nonspecific retardation of one or more elongation steps, and that this may be sufficient to account for its effects on the replication of certain viruses such as encephalomyocarditis and vesicular stomatitis viruses.     

Prostatic binding protein. A steriod-binding protein secreted by rat prostate.

Rat prostatic cytosol contains a high concentration of a prostatic binding protein with peculiar steroid-binding properties. Indeed, in spite of a relatively low affinity, charcoal adsorption can be used for its measurement. Furthermore, the binding is not specific for particular steroids and increases very strongly after delipidation. In delipidated cytosol the concentration of the binding site is 3.1 micronmol/g protein and the apparent affinity for pregenolone 1.7 X 10(6) M-1. The high concentration of prostatic binding protein in prostatic fluid shows that this substance is secreted by the prostate. Prostatic binding protein has the following physicochemical characteristics: it is precipitated by ammonium sulfate between 50 and 70% saturation; the elution position from a Sephadex G-100 column corresponds to a molecular weight of 51000; it sediments in sucrose density gradients at 3.7 S and is eluted from DEAE-cellulose columns at about 0.25 M KCl. On polyacrylamide gel electrophoresis the binding activity coincides with the major cytosolic protein band. This band has the same mobility as serum albumin in 7% gels, but a higher mobility in more concentrated gels.