Mitogenesis, cellular aggregation, and motility follow upon the interaction of fibrinogen with certain defined cell surface receptors. In addition to circulating platelets and vascular endothelium, monocytes express what appears to be a receptor for fibrinogen. Evidence is presented here that the leukocyte adhesion receptor Mac-1 can be specifically induced to bind fibrinogen with characteristics immunochemically and functionally distinct from the established Arg-Gly-Asp-directed fibrinogen receptors. The competence of Mac-1 as a fibrinogen receptor is a general property of cells of monocyte and myeloid lineage acquired after maturational changes of some regions of the alpha subunit of Mac-1 during the process of cell differentiation. This ligand recognition specificity of Mac-1 is lacking for the resting cell. Rather, induction of fibrinogen binding capacity of Mac-1 is due to a cellular response to selected agonists characterized by inducing rapid transients of cytosolic Ca2+. Although different in activation pathways and recognition specificity, Mac-1 exhibits an oligospecific ligand versatility characteristic of other homologous Arg-Gly-Asp-directed adhesion receptors.

Exogenous leucine affects the expression of a number of different operons in Escherichia coli. For at least some of these operons, the leucine-related effect is mediated by a protein called Lrp (Leucine-responsive regulatory protein). The purification of Lrp to near homogeneity is described. Lrp is a moderately abundant, basic protein composed of two subunits of molecular mass 18.8 kDa each. In addition, the corresponding protein was purified from a strain having a mutation within the gene that encodes Lrp (lrp). This mutation (lrp-1) causes high constitutive expression of ilvIH, one of the operons controlled by Lrp (Platko, J. V., Willins, D.A., and Calvo, J.M. (1990) J. Bacteriol. 172, 4563-4570). The Lrp-1 and Lrp proteins have similar physical properties, but they show some differences in the characteristics with which they bind DNA upstream of the ilvIH promoter. The nucleotide sequences of the lrp and lrp-1 genes differ by only a single nucleotide, a C to G change that would substitute a Glu for an Asp at amino acid 114. Lrp has some amino acid sequence similarity to AsnC, a protein that regulates asnA expression (Kolling, R., and Lother, H. (1985) J. Bacteriol. 164, 310-315).

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus.

Nine-day-old harlequin (Hq) mice carrying the hypomorphic apoptosis-inducing factor (AIF)(Hq) mutation expressed 60% less AIF, 18% less respiratory chain complex I and 30% less catalase than their wild-type (Wt) littermates. Compared with Wt, the infarct volume after hypoxia-ischemia (HI) was reduced by 53 and 43% in male (YX(Hq)) and female (X(Hq)X(Hq)) mice, respectively (P<0.001). The Hq mutation did not inhibit HI-induced mitochondrial release of cytochrome c or activation of calpain and caspase-3. The broad-spectrum caspase inhibitor quinoline-Val-Asp(OMe)-CH(2)-PH (Q-VD-OPh) decreased the activation of all detectable caspases after HI, both in Wt and Hq mice. Q-VD-OPh reduced the infarct volume equally in Hq and in Wt mice, and the combination of Hq mutation and Q-VD-OPh treatment showed an additive neuroprotective effect. Oxidative stress leading to nitrosylation and lipid peroxidation was more pronounced in ischemic brain areas from Hq than Wt mice. The antioxidant edaravone decreased oxidative stress in damaged brains, more pronounced in the Hq mice, and further reduced brain injury in Hq but not in Wt mice. Thus, two distinct strategies can enhance the neuroprotection conferred by the Hq mutation, antioxidants, presumably compensating for a defect in AIF-dependent redox detoxification, and caspase inhibitors, presumably interrupting a parallel pathway leading to cellular demise.

BID, a pro-apoptotic Bcl-2 family member, promotes cytochrome c release during apoptosis initiated by CD95L or TNF. Activation of caspase-8 in the latter pathways results in cleavage of BID, translocation of activated BID to mitochondria, followed by redistribution of cytochrome c to the cytosol. However, it is unclear whether BID participates in cytochrome c release in other (non-death receptor) cell death pathways. Here, we show that BID is cleaved in response to multiple death-inducing stimuli (staurosporine, UV radiation, cycloheximide, etoposide). However BID cleavage in these contexts was blocked by Bcl-2, suggesting that proteolysis of BID occurred distal to cytochrome c release. Furthermore, addition of cytochrome c to Jurkat post-nuclear extracts triggered breakdown of BID at Asp-59 which was catalysed by caspase-3 rather than caspase-8. We provide evidence that caspase-3 catalysed cleavage of BID represents a feedback loop for the amplification of mitochondrial cytochrome c release during cytotoxic drug and UV radiation-induced apoptosis.

The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D(2) dopamine receptors (D(2)R) has been reported. The high affinity of DA and other DAT substrates for the D(2)R, however, has complicated investigation of the intracellular mechanisms mediating this effect. The present studies used the fluorescent DAT substrate, 4-[4-(diethylamino)-styryl]-N-methylpyridinium iodide (ASP(+)) with live cell imaging techniques to identify the role of two D(2)R-linked signaling pathways, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and phosphoinositide 3 kinase (PI3K) in mediating D(2)R regulation of DAT. Addition of the D(2)/D(3) receptor agonist quinpirole (0.1-10 muM) to human embryonic kidney cells coexpressing human DAT and D(2) receptor (short splice variant, D(2S)R) induced a rapid, concentration-dependent and pertussis toxin-sensitive increase in ASP(+) accumulation. The D(2)/D(3) agonist (S)-(+)-(4aR, 10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride (PD128907) also increased ASP(+) accumulation. D(2S)R activation increased phosphorylation of ERK1/2 and Akt, a major target of PI3K. The mitogen-activated protein kinase kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) prevented the quinpirole-evoked increase in ASP(+) accumulation, whereas inhibition of PI3K was without effect. Fluorescence flow cytometry and biotinylation studies revealed a rapid increase in DAT cell-surface expression in response to D(2)R stimulation. These experiments demonstrate that D(2S)R stimulation increases DAT cell surface expression and therefore enhances substrate clearance. Furthermore, they show that the increase in DAT function is ERK1/2-dependent but PI3K-independent. Our data also suggest the possibility of a direct physical interaction between DAT and D(2)R. Together, these results suggest a novel mechanism by which D(2S)R autoreceptors may regulate DAT in the central nervous system.

Glycine markedly potentiates N-methyl-D-aspartate (N-Me-D-Asp) responses in mammalian neurons by an action at a modulatory site on the N-Me-D-Asp receptor-ionophore complex. Here we present evidence that 7-chlorokynurenic acid (7-Cl KYNA) inhibits N-Me-D-Asp responses by a selective antagonism of glycine at this modulatory site. In rat cortical slices 7-Cl KYNA (10-100 microM) noncompetitively inhibited N-Me-D-Asp responses, and this effect could be reversed by the addition of glycine (100 microM) or D-serine (100 microM). Radioligand binding experiments showed that 7-Cl KYNA had a much higher affinity for the strychnine-insensitive [3H]glycine binding site (IC50 = 0.56 microM) than for the N-Me-D-Asp (IC50 169 microM), quisqualate (IC50 = 153 microM), or kainate (IC50 greater than 1000 microM) recognition sites. In whole-cell patch-clamp recordings from rat cortical neurones in culture, the inhibitory effects of 7-Cl KYNA on N-Me-D-Asp-induced currents could not be overcome by increasing the N-Me-D-Asp concentration but could be reversed by increasing the glycine concentration. 7-Cl KYNA could completely abolish N-Me-D-Asp responses, including basal responses in the absence of added glycine, suggesting that it may possess negative modulatory effects at the glycine site. These findings indicate that the glycine modulatory site is functional in intact adult tissue and that 7-Cl KYNA should prove to be a selective tool for elucidating the involvement of this site in physiological and pathological events mediated by N-Me-D-Asp receptors.

Our aim was to correlate the activity of matrix metalloproteinases (MMPs) with denaturation and the turnover of collagen in normal and pathological human tendons. MMPs were extracted from ruptured supraspinatus tendons (n=10), macroscopically normal ("control") supraspinatus tendons (n=29) and normal short head of biceps brachii tendons (n=24). Enzyme activity was measured using fluorogenic substrates selective for MMP-1, MMP-3 and enzymes with gelatinolytic activity (MMP-2, MMP-9 and MMP-13). Collagen denaturation was determined by alpha-chymotrypsin digestion. Protein turnover was determined by measuring the percentage of D-aspartic acid (% D-Asp). Zymography was conducted to identity specific gelatinases. MMP-1 activity was higher in ruptured supraspinatus compared to control supraspinatus and normal biceps brachii tendons (70.9, 26.4 and 11.5 fmol/mg tendon, respectively; P<0.001). Gelatinolytic and MMP-3 activities were lower in normal biceps brachii and ruptured supraspinatus compared to control supraspinatus (gelatinase: 0.18, 0.23 and 0.82 RFU/s/mg tendon respectively; P<0.001; MMP-3: 9.0, 8.6 and 55 fmol/mg tendon, respectively; P<0.001). Most gelatinase activity was shown to be MMP-2 by zymography. Denatured collagen was increased in ruptured supraspinatus compared to control supraspinatus (20.4% and 9.9%, respectively; P<0.001). The % D-Asp content increased linearly with age in normal biceps brachii but not in control supraspinatus and was significantly lower in ruptured supraspinatus compared to age-matched control tendons (0.33 and 1.09% D-Asp, respectively; P<0.01). We conclude that the short head of biceps brachii tendons show little protein turnover, whereas control supraspinatus tendons show relatively high turnover mediated by the activity of MMP-2, MMP-3 and MMP-1. This activity is thought to represent a repair or maintenance function that may be associated with an underlying degenerative process caused by a history of repeated injury and/or mechanical strain. After tendon rupture, there was increased activity of MMP-1, reduced activity of MMP-2 and MMP-3, increased turnover and further deterioration in the quality of the collagen network. Tendon degeneration is shown to be an active, cell-mediated process that may result from a failure to regulate specific MMP activities in response to repeated injury or mechanical strain.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.

Multifunctional and water-soluble superparamagnetic iron oxide (SPIO) nanocarriers were developed for targeted drug delivery and positron emission tomography/magnetic resonance imaging (PET/MRI) dual-modality imaging of tumors with integrin α(v)β₃ expression. An anticancer drug was conjugated onto the PEGylated SPIO nanocarriers via pH-sensitive bonds. Tumor-targeting ligands, cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDfC)) peptides, and PET ⁶⁴Cu chelators, macrocyclic 1,4,7-triazacyclononane-N, N', N″-triacetic acid (NOTA), were conjugated onto the distal ends of the PEG arms. The effectiveness of the SPIO nanocarriers as an MRI contrast agent was evaluated via an in vitro r₂ MRI relaxivity measurement. cRGD-conjugated SPIO nanocarriers exhibited a higher level of cellular uptake than cRGD-free ones in vitro. Moreover, cRGD-conjugated SPIO nanocarriers showed a much higher level of tumor accumulation than cRGD-free ones according to non-invasive and quantitative PET imaging, and ex vivo biodistribution studies. Thus, these SPIO nanocarriers demonstrated promising properties for combined targeted anticancer drug delivery and PET/MRI dual-modality imaging of tumors.

The amphipathic helix hypothesis for the lipid-associating domains of exchangeable plasma apolipoproteins has been further studied by analysis of the structure of the complexes formed between four synthetic peptide analogs of the amphipathic helix and dimyristoyl phosphatidylcholine (DMPC). Density gradient ultracentrifugation, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, 1H NMR, high sensitivity differential scanning calorimetry, and circular dichroism were the techniques used in these studies. The two analogs Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) and 18A-Pro-18A whose sequences most strongly mimic native amphipathic sequences were found also most strongly to mimic apolipoprotein A-I in DMPC complex structure. The covalently linked dimer of the prototype amphipathic analog 18A, 18A-Pro-18A, appears to have greater lipid affinity than 18A. This presumably is the result of the cooperativity provided by two covalently linked lipid-associating domains in 18A-Pro-18A. The studies further suggest that the charge-reversed analog of the prototype 18A, reverse-18A, has the lowest lipid affinity of the four analogs studied and forms only marginally stable discoidal DMPC complexes. We postulate that this low lipid affinity is due predominantly, but not necessarily exclusively, to the lack of a hydrophobic contribution of lysine residues at the polar-nonpolar interface of reverse-18A versus 18A. The intermediate lipid affinity of des-Val10-18A, the fourth analog peptide, to produce a rank order of 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, supports this interpretation. Des-Val10-18A which has Val deleted from 18A has an amphipathic helical structure partially disrupted by the shift of 2 lysine residues away from the polar-nonpolar interface.

Neuron death and neuron degeneration occur in the CNS during the course of aging. Although multiple cellular alterations transpire during the aging process, those that mediate age-associated neuron death have not been identified. Recent evidence implicates oxidative stress as a possible means of neuron death and neuron degeneration during aging. In the present study, we demonstrate a marked decrease in multicatalytic proteasome activity in the spinal cord of Fisher 344 rats at 12, 24 and 28 months, compared with spinal cord tissue from 3-week- and 3-month-old animals. Application of oxidative injury (FeSO(4)) or the lipid peroxidation product 4-hydroxynonenal decreases multicatalytic proteasome activity in a time- and dose-dependent manner in a motor neuron cell line. Loss of multicatalytic proteasome activity occurs before the loss of multicatalytic proteasome immunoreactivity, with FeSO(4)- and 4-hydroxynonenal-mediated decreases ameliorated by the application of a cell permeable form of the antioxidant glutathione. Application of multicatalytic proteasome inhibitors, but not inhibitors of lysosomal proteases, induced neuron death that was attenuated by the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone or N-acetyl-Asp-Glu-Val-Asp-Cho (aldehyde). Together, these data suggest that multicatalytic proteasome inhibition occurs during aging of the spinal cord, possibly as the result of oxidative stress, and that multicatalytic proteasome inhibition may be causally related to neuron death.

Previous work has demonstrated that fleR, the gene for a transcriptional activator belonging to the NtrC subfamily of response regulators, is involved in the regulation of mucin adhesion and flagellar expression by Pseudomonas aeruginosa. This report describes the identification and characterization of fleQ, the gene for another transcriptional regulator which also regulates mucin adhesion and motility in this organism. The complete nucleotide sequence of the fleQ gene was determined on both DNA strands, and an open reading frame (ORF) consisting of 1,493 nucleotides was identified. This ORF coded for a gene product of predicted molecular weight, as confirmed by the overexpression of the fleQ gene as a fusion protein under an inducible promoter. The fleQ gene is flanked by a flagellar operon, fliDSorf126, at the 5' end and the fleSR operon on the 3' end. FleQ also had striking homology to a number of proteins belonging to the NtrC subfamily of response regulators, which work in concert with the alternate sigma factor RpoN (sigma54) to activate transcription. However, FleQ lacks the residues corresponding to Asp-54 and Lys-104 of the NtrC protein which are conserved in most of the members belonging to this subfamily of regulators. In addition, unlike some of the other transcriptional activators of this group, FleQ does not appear to have a cognate sensor kinase. A chromosomal insertional mutation in the fleQ gene abolished mucin adhesion and motility of P. aeruginosa PAK and PAK-NP. Both of these functions were regained by providing the complete fleQ gene on a multicopy plasmid. The location of fleQ immediately upstream of the fleSR operon, which is also necessary for the same process, suggested that these regulators may interact in some way. We therefore examined the regulation of the fleSR operon by fleQ and vice versa. Promoter fusion experiments showed that the fleSR operon was regulated by RpoN and FleQ. On the other hand, the fleQ promoter was independent of RpoN and FleR. FleQ, thus, adds another level of regulation to motility and adhesion in P. aeruginosa, above that of fleSR. We therefore propose the existence of a regulatory cascade which consists of at least two transcriptional regulators, FleQ and FleR, in the control of motility and adhesion in P. aeruginosa.

The interaction of the chemokine stromal cell-derived factor 1 (SDF-1) with its receptor CXCR4 is vital for cell trafficking during development, is capable of inhibiting human immunodeficiency virus type 1 (HIV-1) utilization of CXCR4 as a coreceptor, and has been implicated in delaying disease progression to AIDS in vivo. Because of the importance of this chemokine-chemokine receptor pair to both development and disease, we investigated the molecular basis of the interaction between CXCR4 and its ligands SDF-1 and HIV-1 envelope. Using CXCR4 chimeras and mutants, we determined that SDF-1 requires the CXCR4 amino terminus for binding and activates downstream signaling pathways by interacting with the second extracellular loop of CXCR4. SDF-1-mediated activation of CXCR4 required the Asp-Arg-Tyr motif in the second intracellular loop of CXCR4, was pertussis toxin sensitive, and did not require the distal C-terminal tail of CXCR4. Several CXCR4 mutants that were not capable of binding SDF-1 or signaling still supported HIV-1 infection, indicating that the ability of CXCR4 to function as a coreceptor is independent of its ability to signal. Direct binding studies using the X4 gp120s HXB, BH8, and MN demonstrated the ability of HIV-1 gp120 to bind directly and specifically to the chemokine receptor CXCR4 in a CD4-dependent manner, using a conformationally complex structure on CXCR4. Several CXCR4 variants that did not support binding of soluble gp120 could still function as viral coreceptors, indicating that detectable binding of monomeric gp120 is not always predictive of coreceptor function.

The chemotactic responses of bacteria such as Escherichia coli and Salmonella typhimurium are mediated by phosphorylation of the CheY protein. Phospho-CheY interacts with the flagellar motor switch to cause tumbly behavior. CheY belongs to a large family of phosphorylated response regulators that function in bacteria to control motility and regulate gene expression. Residues corresponding to <em>Asp</em>57, <em>Asp</em>13, and Lys109 in CheY are highly conserved among all of these proteins. The site of phosphorylation in CheY is <em>Asp</em>57, and in the three-dimensional structure of CheY the <em>Asp</em>57 carboxylate side chain is in close proximity to the beta-carboxylate of <em>Asp</em>13 and the epsilon-amin of Lys109. To further examine the roles of these residues in response regulator function, each has been mutated to a conservative substitution. Asn for <em>Asp</em> and Arg for Lys. All mutations abolished CheY function in vivo. Whereas the <em>Asp</em> to Asn mutations dramatically reduced levels of CheY phosphorylation, the Lys to Arg mutation had the opposite effect. The high level of phosphorylation in the Lys109 mutant results from a decreased autophosphatase activity as well as a lack of phosphatase stimulation by the phosphatase activating protein, CheZ. Despite its high level of phosphorylation, the Lys109 mutant protein cannot produce tumbly behavior. Thus, Lys109 is required for an event subsequent to phosphorylation. We propose that an interaction between the epsilon-amino of Lys109 and the phosphoryl group at <em>Asp</em>57 is essential for the conformational switch that leads to activation of CheY.

A cDNA library was prepared in lambda gt11 bacteriophage from poly(A)+ RNA isolated from primary cultures of endothelial cells from human umbilical vein. Approximately 2.5 million independent recombinants were screened and 2 of those were found to synthesize a fusion protein with beta-galactosidase that reacted with rabbit antibody against human von Willebrand factor. Comparison of the amino acid sequence translated from the cDNA insert of the two clones with the amino acid sequence determined by Edman degradation of the protein established that both phage isolates code for von Willebrand factor. The first clone (lambda HvWF1) contained an insert of 404 nucleotides that corresponded to amino acid residues 1-110 in the mature protein circulating in blood, in addition to a portion (24 amino acids) of a prepro leader sequence. The second cDNA clone (lambda HvWF3) contained an insert of 4.9 kilobases that coded for the carboxyl-terminal 1525 amino acids of von Willebrand factor, a stop codon of TGA, 134 nucleotides of 3' noncoding sequence, and a poly(A) tail of 150 nucleotides. The two clones together code for greater than 80% of the molecule circulating in blood. The same carboxyl-terminal lysine residue was identified in the mature protein as well as in the cDNA, indicating that all of the proteolytic processing that occurs during the biosynthesis and assembly of von Willebrand factor is associated with the amino-terminal portion of the precursor protein. The amino acid sequence of von Willebrand factor indicates the presence of two different internal gene duplications and one triplication. These repetitive amino acid sequences account for about one-half of the amino acids present in the mature protein. The tetrapeptide sequence of Arg-Gly-Asp-Ser, which mediates the cell attachment and platelet binding activity of fibronectin, was also identified in the carboxyl-terminal portion of von Willebrand factor.

Although it has been reported that activated platelets can adhere to intact endothelium, the receptors involved have not been fully characterized. Also, it is not clear whether activated platelets bind primarily to matrix proteins at sites of endothelial cell denudation or directly to endothelial cells. Thus, this study was designed to further clarify the mechanisms of activated platelet adhesion to endothelium. Unstimulated human umbilical vein endothelial cell (HUVEC) monolayers were incubated with washed, stained, and thrombin-activated human platelets. To exclude matrix involvement, HUVEC were harvested mechanically and platelet binding was measured by flow cytometry. Before the adhesion assay, platelets or HUVEC were treated with different receptor antagonists. Whereas blockade of platelet beta1 integrins, GPIbalpha, GPIV, P-selectin, and platelet-endothelial cell adhesion molecule (PECAM)-1 did not reduce platelet adhesion to HUVEC, blockade of platelet GPIIbIIIa by antibodies or Arg-Gly-Asp (RGD) peptides markedly decreased adhesion. Moreover, when platelets were treated with blocking antibodies to GPIIbIIIa-binding adhesive proteins, including fibrinogen and fibronectin, and von Willebrand factor (vWF), platelet binding was also reduced markedly. Addition of fibrinogen, fibronectin, or vWF further increased platelet adhesion, indicating that both endogenous platelet-exposed and exogenous adhesive proteins can participate in the binding process. Evaluation of the HUVEC receptors revealed predominant involvement of intercellular adhesion molecule (ICAM)-1 and alphavbeta3 integrin. Blockade of these two receptors by antibodies decreased platelet binding significantly. Also, there was evidence that a component of platelet adhesion was mediated by endothelial GPIbalpha. Blockade of beta1 integrins, E-selectin, P-selectin, PECAM-1, vascular cell adhesion molecule (VCAM)-1 and different matrix proteins on HUVEC did not affect platelet adhesion. In conclusion, we show that activated platelet binding to HUVEC monolayers is mediated by a GPIIbIIIa-dependent bridging mechanism involving platelet-bound adhesive proteins and the endothelial cell receptors ICAM-1, alphavbeta3 integrin, and, to a lesser extent, GPIbalpha.

Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyzes the addition of myristic acid to the amino-terminal glycine residues of a number of eukaryotic proteins. Recently, we developed a cell-free system for analyzing NMT activity and have begun to characterize the substrate specificity of this enzyme by using a series of synthetic peptides. We have now purified NMT from Saccharomyces cerevisiae to apparent homogeneity. The native enzyme is a 55-kDa protein, exhibits no requirement for divalent cation, and appears to contain a histidine residue critical for enzyme activity. A total of 42 synthetic peptides have been used to define structure/activity relationships in NMT substrates. An amino-terminal glycine is required for acylation; substitution with glycine analogues produces peptides that are inactive as substrates or inhibitors of NMT. A broad spectrum of amino acids is permitted at positions 3 and 4, while strict amino acid requirements are exhibited at position 5. Replacement of Ala5 in the peptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg with Asp ablates the peptide's myristoyl-accepting activity. A serine at this position results in a decrease by a factor of approximately equal to 500 in the apparent Km in the context of three different sequences. Penta- and hexa-peptides are substrates, but with decreased affinity. These studies establish that structural information important for NMT-ligand interaction exists beyond the first two amino acids in peptide substrates and that the side chains of residue 5 play a critical role in the binding of substrates to this enzyme.

Prokaryotic chitinases, class III plant chitinases, yeast chitinases, and endo-beta-N-acetylglucosaminidases share weak amino acid sequence similarities at the certain region of each enzyme. These regions have been assumed to be important for catalytic activities of the enzymes. To verify this assumption, three amino acid residues (Ser-160, Asp-200, Glu-204) in chitinase A1 of Bacillus circulans WL-12 were chosen, based on the amino acid sequence alignment of the regions sharing sequence similarity, and were replaced by site-directed mutagenesis. Kinetic parameters for 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and seven mutant chitinases. Chitinases with Glu-204->>Gln mutation and Glu-204->>Asp mutation were essentially inactive and kcat values of these chitinases were approximately 1/5,000 and 1/17,000 of that of wild-type chitinase, respectively. Asp-200->>Asn mutation decreased the kcat value to approximately 1/350 of that of the wild-type enzyme, while the Km value decreased only slightly. On the other hand, neither the kcat value nor the Km value was affected by Asp-200->>Glu mutation. Thus, it appeared that Glu-204 and Asp-200 are directly involved in the catalytic events of chitinase A1. The role of the carboxyl group of Asp-200 can be fully substituted by that of Glu residue. The Ser-160->>Ala mutant retained 10% activity of the wild-type chitinase indicating that the hydroxyl group of Ser-160 is not absolutely required for the catalytic activity. These results indicate a lysozyme-type catalytic mechanism of the chitinase.

While all basic region/helix-loop-helix (bHLH) proteins bind the consensus CANNTG motif, other factors must be involved in determining regulatory specificity. In this report we show that bases outside this core 6 bp are involved in determining the specificity of binding. Thus, binding of the yeast bHLH protein PHO4, but not CPF-1, is inhibited by the presence of a T residue immediately 5' to their common CACGTG recognition sequence. PHO4 binding specificity is altered by mutation at any of three different positions in the basic region, including a single Glu to Asp substitution. The significance of these data for DNA-binding and transcription regulation by the bHLH family of transcription factors is discussed.

LEAFY COTYLEDON1 (LEC1) is a central regulator that is required for many aspects of Arabidopsis embryogenesis and sufficient to induce embryo development in vegetative cells when expressed ectopically. We previously showed that LEC1 encodes an HAP3 subunit of the CCAAT binding factor and that the 10 Arabidopsis HAP3 (AHAP3) subunits can be divided into two classes, the LEC1-type and the non-LEC1-type, based on sequence similarity within their B domains. By analyzing the ability of chimeric HAP3 subunits containing different combinations of domains from LEC1 and a non-LEC1-type AHAP3 subunit to suppress genetically the lec1 mutation, we show that the B domain of LEC1 is necessary and sufficient within the context of the protein for its activity in embryogenesis. Moreover, we identify one amino acid residue, Asp-55, specific to the LEC1-type B domain that is required for LEC1 activity in embryogenesis and sufficient to confer partial LEC1 activity to a non-LEC1-type B domain. Based on structural similarities between the HAP3 B domain and histone fold motif, we discuss how the Asp-55 residue may functionally differentiate LEC1 from the non-LEC1-type AHAP3 subunits.

The major substrate for Ca2+/calmodulin-dependent protein kinase III in mammalian cells is a species of Mr 100,000 that has a primarily cytoplasmic localization. This substrate has now been identified as elongation factor-2 (EF-2), a protein that catalyzes the translocation of peptidyl-tRNA on the ribosome. The amino acid sequence of 18 residues from the N-terminal of the Mr 100,000 CaM-dependent protein kinase III substrate purified from rat pancreas was found to be identical to the N-terminal sequence of authentic rat EF-2 as previously deduced from nucleic acid sequencing of a cDNA (Kohno, K., Uchida, T., Ohkubo, H., Nakanishi, S., Nakanishi, T., Fukui, T., Ohtsuka, E., Ikehara, M., and Okada, Y. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4978-4982). CaM-dependent protein kinase III phosphorylated EF-2 in vitro with a stoichiometry of approximately 1 mol/mol on a threonine residue. Amino acid sequencing of the purified tryptic phosphopeptide revealed that this threonine residue lies within the sequence: Ala-Gly-Glu-Thr-Arg-Phe-Thr-Asp-Thr-Arg (residues 51-60 of EF-2). The Mr 100,000 protein was stoichiometrically ADP-ribosylated in vitro by the addition of diphtheria toxin and NAD. The Mr 100,000 protein was photoaffinity labeled with a GTP analog and the protein had an endogenous GTPase activity that could be stimulated by the addition of salt-washed ribosomes. These properties are all characteristic of EF-2. Dephospho-EF-2 could support poly(U)-directed polyphenylalanine synthesis in a reconstituted elongation system when combined with EF-1. In the same system, phospho-EF-2 was virtually inactive in supporting polypeptide synthesis; this effect could be reversed by dephosphorylation of phospho-EF-2. These results suggest that intracellular Ca2+ inhibits protein synthesis in mammalian cells via CaM-dependent protein kinase III-catalyzed phosphorylation of EF-2.

N-terminal acetylation can occur cotranslationally on the initiator methionine residue or on the penultimate residue if the methionine is cleaved. We investigated the three N-terminal acetyltransferases (NATs), Ard1p/Nat1p, Nat3p and Mak3p. Ard1p and Mak3p are significantly related to each other by amino acid sequence, as is Nat3p, which was uncovered in this study using programming alignment procedures. Mutants deleted in any one of these NAT genes were viable, but some exhibited diminished mating efficiency and reduced growth at 37 degrees C, and on glycerol and NaCl-containing media. The three NATs had the following substrate specificities as determined in vivo by examining acetylation of 14 altered forms of iso-1-cytochrome c and 55 abundant normal proteins in each of the deleted strains: Ard1p/Nat1p, subclasses with Ser-, Ala-, Gly- and Thr-termini; Nat3p, Met-Glu- and Met-Asp- and a subclass of Met-Asn-termini; and Mak3p subclasses with Met-Ile- and Met-Leu-termini. In addition, a special subclass of substrates with Ser-Glu- Phe-, Ala-Glu-Phe- and Gly-Glu-Phe-termini required all three NATs for acetylation.