Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function

The alpha 1-adrenergic receptors activate a phospholipase C enzyme by coupling to members of the large molecular size (approximately 74 to 80 kilodaltons) G alpha h family of guanosine triphosphate (GTP)-binding proteins. Rat liver G alpha h is now shown to be a tissue transglutaminase type II (TGase II). The transglutaminase activity of rat liver TGase II expressed in COS-1 cells was inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) or by alpha 1-adrenergic receptor activation. Rat liver TGase II also mediated alpha 1-adrenergic receptor stimulation of phospholipase C activity. Thus, G alpha h represents a new class of GTP-binding proteins that participate in receptor signaling and may be a component of a complex regulatory network in which receptor-stimulated GTP binding switches the function of G alpha h from transglutamination to receptor signaling.     

The phytofluors: a new class of fluorescent protein probes

BACKGROUND: Biologically compatible fluorescent protein probes, particularly the self-assembling green fluorescent protein (GFP) from the jellyfish Aequorea victoria, have revolutionized research in cell, molecular and developmental biology because they allow visualization of biochemical events in living cells. Additional fluorescent proteins that could be reconstituted in vivo while extending the useful wavelength range towards the orange and red regions of the light spectrum would increase the range of applications currently available with fluorescent protein probes. RESULTS: Intensely orange fluorescent adducts, which we designate phytofluors, are spontaneously formed upon incubation of recombinant plant phytochrome apoproteins with phycoerythrobilin, the linear tetrapyrrole precursor of the phycoerythrin chromophore. Phytofluors have large molar absorption coefficients, fluorescence quantum yields greater than 0.7, excellent photostability, stability over a wide range of pH, and can be reconstituted in living plant cells. CONCLUSIONS: The phytofluors constitute a new class of fluorophore that can potentially be produced upon bilin uptake by any living cell expressing an apophytochrome cDNA. Mutagenesis of the phytochrome apoprotein and/or alteration of the linear tetrapyrrole precursor by chemical synthesis are expected to afford new phytofluors with fluorescence excitation and emission spectra spanning the visible to near-infrared light spectrum.     

The common marmoset: a new world primate species with limited Mhc class II variability

The common marmoset (Callithrix jacchus) is a New World primate species that is highly susceptible to fatal infections caused by various strains of bacteria. We present here a first step in the molecular characterization of the common marmoset's Mhc class II genes by nucleotide sequence analysis of the polymorphic exon 2 segments. For this study, genetic material was obtained from animals bred in captivity as well as in the wild. The results demonstrate that the common marmoset has, like other primates, apparently functional Mhc-DR and -DQ regions, but the Mhc-DP region has been inactivated. At the -DR and -DQ loci, only a limited number of lineages were detected. On the basis of the number of alleles found, the -DQA and -B loci appear to be oligomorphic, whereas only a moderate degree of polymorphism was observed for two of three Mhc-DRB loci. The contact residues in the peptide-binding site of the Caja-DRB1*03 lineage members are highly conserved, whereas the -DRB*W16 lineage members show more divergence in that respect. The latter locus encodes five oligomorphic lineages whose members are not observed in any other primate species studied, suggesting rapid evolution, as illustrated by frequent exchange of polymorphic motifs. All common marmosets tested were found to share one monomorphic type of Caja-DRB*W12 allele probably encoded by a separate locus. Common marmosets apparently lack haplotype polymorphism because the number of Caja-DRB loci present per haplotype appears to be constant. Despite this, however, an unexpectedly high number of allelic combinations are observed at the haplotypic level, suggesting that Caja-DRB alleles are exchanged frequently between chromosomes by recombination, promoting an optimal distribution of limited Mhc polymorphisms among individuals of a given population. This peculiar genetic make up, in combination with the limited variability of the major histocompatability complex class II repertoire, may contribute to the common marmoset's susceptibility to particular bacterial infections.     

Synthesis and biological evaluation of aeroplysinin analogues: a new class of receptor tyrosine kinase inhibitors

Receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR), are critically involved in the transduction of mitogenic signals across the plasma membrane and therefore in the regulation of cell growth and proliferation. Enhanced RTK activity is associated with proliferative diseases such as cancer, psoriasis and atherosclerosis, while decreased function may be associated for instance with diabetes. EGFR and PDGFR are selectively inhibited by analogues of the marine natural product aeroplysinin. The synthetic inhibitors display IC50 values in the low micromolar range and in contrast to the natural product show pronounced inhibitory activity in cultured cells in vivo. The mechanism of inhibition is likely based on a covalent modification of the target enzymes by reaction of epoxy ketone 8 with various nucleophiles.     

A new class of antihypertensive therapy: angiotensin II receptor antagonists

Despite the availability of safe and efficacious antihypertensive agents, hypertension continues to be a major source of morbidity and mortality in the United States. Losartan, the first of a new class of agents, the angiotensin II receptor antagonists, can be administered as monotherapy in the treatment of hypertension or to complement existing therapy. The angiotensin II receptor antagonists block the effects of angiotensin II through preferential binding to angiotensin II receptor subtype AT1 on the cell membrane. Compared with angiotensin-converting enzyme inhibitors, they may provide more complete blockade of the renin-angiotensin system and be associated with a lower frequency of cough as a side effect.     

Ran/TC4: a small nuclear GTP-binding protein that regulates DNA synthesis

Ran/TC4, first identified as a well-conserved gene distantly related to H-RAS, encodes a protein which has recently been shown in yeast and mammalian systems to interact with RCC1, a protein whose function is required for the normal coupling of the completion of DNA synthesis and the initiation of mitosis. Here, we present data indicating that the nuclear localization of Ran/TC4 requires the presence of RCC1. Transient expression of a Ran/TC4 protein with mutations expected to perturb GTP hydrolysis disrupts host cell DNA synthesis. These results suggest that Ran/TC4 and RCC1 are components of a GTPase switch that monitors the progress of DNA synthesis and couples the completion of DNA synthesis to the onset of mitosis.     

GTP-binding proteins in plants: new members of an old family

Regulatory guanine nucleotide-binding proteins (G proteins) have been studied extensively in animal and microbial organisms, and they are divided into the heterotrimeric and the small (monomeric) classes. Heterotrimeric G proteins are known to mediate signal responses in a variety of pathways in animals and simple eukaryotes, while small G proteins perform diverse functions including signal transduction, secretion, and regulation of cytoskeleton. In recent years, biochemical analyses have produced a large amount of information on the presence and possible functions of G proteins in plants. Further, molecular cloning has clearly demonstrated that plants have both heterotrimeric and small G proteins. Although the functions of the plant heterotrimeric G proteins are yet to be determined, expression analysis of an Arabidopsis G alpha protein suggests that it may be involved in the regulation of cell division and differentiation. In contrast to the very few genes cloned thus far that encode heterotrimeric G proteins in plants, a large number of small G proteins have been identified by molecular cloning from various plants. In addition, several plant small G proteins have been shown to be functional homologues of their counterparts in animals and yeasts. Future studies using a number of approaches are likely to yield insights into the role plant G proteins play.     

Identification of a specific effector of the small GTP-binding protein Rap2

The coupled processes of the chloroplast trans-envelope transport of malate and oxaloacetate and their interconversion as catalyzed by the stromal NADP-linked malate dehydrogenase are quantitatively analyzed by means of a steady-state model. The equation for the NADP-malate dehydrogenase reaction is developed. The empirical dependence of enzyme activity on NADPH and NADP+ is used to determine its actual activity. The trans-envelope counter exchange of malate and oxaloacetate is described by a kinetic model of the translocator. Kinetic parameters are derived from known data, except for the Km value and the maximum rate for oxaloacetate transport, which are estimated from oxaloacetate-dependent malate formation in isolated intact chloroplasts. Using the kinetic properties of the system and the known metabolite concentrations, the model demonstrates that photosynthetically generated NADPH can be exported efficiently from the chloroplasts to the cytosol by the malate-valve system. The transfer capacity of the malate valve is estimated not to exceed 20 mumol (mg Chl)-1 h-1 (or 5% of the electron transport) under normal physiological conditions. The possible role of the malate valve in leaf cells under normal conditions and during stress is discussed.     

Helicobacter pylori infection in immunized mice lacking major histocompatibility complex class I and class II functions

The role of major histocompatibility complex (MHC) class I- and class II-restricted functions in Helicobacter pylori infection and immunity upon oral immunization was examined in vivo. Experimental challenge with H. pylori SS1 resulted in significantly greater (P 相似文献   

Characterization of a GTP-binding protein in the ADP-ribosylation factor subfamily from Leishmania tarentolae

We report the cloning and characterization of a gene, LtARL, which encodes a small GTP-binding protein, from the protozoan Leishmania tarentolae. Hybridization analysis of genomic DNA under high stringency conditions indicates the single-copy nature of LtARL. LtARL is transcribed and yields a approximately 0.9 kb mRNA that is processed at the 5' end by trans-splicing. When expressed in Escherichia coli, LtArl binds GTP with a low stoichiometry and in a phospholipid-independent manner. Based on the greatest sequence identity with Homo sapiens Arl3 and lipid-independent binding of guanine nucleotides we designate this gene LtARL and the encoded protein LtArl.     

A new class of pyrrolidin-2-ones: synthesis and pharmacological study. II

OBJECTIVE: The measurements of transient evoked otoacoustic emissions and distortion-product otoacoustic emissions are being used increasingly, both as an objective hearing test clinically, and as a research tool to investigate the micromechanical aspects of cochlear function. We hypothesized that localized damage in the apical or middle cochlear turns may have an influence on the micromechanics and the function of adjacent, apparently normal cochlea. For that purpose, we used an animal model of localized apical and middle-turn cochlear lesions. METHOD: Extent of damage was assessed by scanning electron microscopy and the function of the damaged cochlea by change in the otoacoustic emission (OAE) levels. RESULTS: We found that localized damage to the apical or middle turn may be accompanied by an increase in OAE measured from adjacent apparently normal cochlea. CONCLUSION: Explanations to this phenomenon are suggested, and possible clinical associations such as to Meniere's disease and to sudden hearing loss are reviewed.     

Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein

Rap1 small GTP-binding protein has the same amino acid sequence at its effector domain as that of Ras. Rap1 has been shown to antagonize the Ras functions, such as the Ras-induced transformation of NIH 3T3 cells and the Ras-induced activation of the c-Raf-1 protein kinase-dependent mitogen-activated protein (MAP) kinase cascade in Rat-1 cells, whereas we have shown that Rap1 as well as Ras stimulates DNA synthesis in Swiss 3T3 cells. We have established a cell-free assay system in which Ras activates bovine brain B-Raf protein kinase. Here we have used this assay system and examined the effect of Rap1 on the B-Raf activity to phosphorylate recombinant MAP kinase kinase (MEK). Recombinant Rap1B stimulated the activity of B-Raf, which was partially purified from bovine brain and immunoprecipitated by an anti-B-Raf antibody. The GTP-bound form was active, but the GDP-bound form was inactive. The fully post-translationally lipid-modified form was active, but the unmodified form was nearly inactive. The maximum B-Raf activity stimulated by Rap1B was nearly the same as that stimulated by Ki-Ras. Rap1B enhanced the Ki-Ras-stimulated B-Raf activity in an additive manner. These results indicate that not only Ras but also Rap1 is involved in the activation of the B-Raf-dependent MAP kinase cascade.     

Intermolecular and interdomain interactions of a dynamin-related GTP-binding protein, Dnm1p/Vps1p-like protein

Dnm1p/Vps1p-like protein (DVLP) is a mammalian member of the dynamin GTPase family, which is classified into subfamilies on the basis of the structural similarity. Mammalian dynamins constitute the dynamin subfamily. DVLP belongs to the Vps1 subfamily, which also includes yeast Vps1p and Dnm1p. Typical structural features that discriminate between members of the Vps1 and dynamin subfamilies are that the former lacks the pleckstrin homology and Pro-rich domains. Dynamin exists as tetramers under physiological salt conditions, whereas under low salt conditions, it can polymerize into spirals that resemble the collar structures seen at the necks of constricted coated pits. In this study, we found that DVLP is also oligomeric, probably tetrameric, under physiological salt conditions and forms sedimentable large aggregates under low salt conditions. The data indicate that neither the pleckstrin homology nor Pro-rich domain is required for the self-assembly. Analyses using the two-hybrid system and co-immunoprecipitation show that the N-terminal region containing the GTPase domain and a domain (DVH1) conserved across members of the dynamin and Vps1 subfamilies, can interact with the C-terminal region containing another conserved domain (DVH2). The data on the interdomain interaction of DVLP is compatible with the previous reports on the interdomain interaction of dynamin. Thus, the self-assembly mechanism of DVLP appears to resemble that of dynamin, suggesting that DVLP may also be involved in the formation of transport vesicles.     

Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase

The several hundred members of the eukaryotic protein kinase superfamily characterized to date share a similar catalytic domain structure, consisting of 12 conserved subdomains. Here we report the existence and wide occurrence in eukaryotes of a protein kinase with a completely different structure. We cloned and sequenced the human, mouse, rat, and Caenorhabditis elegans eukaryotic elongation factor-2 kinase (eEF-2 kinase) and found that with the exception of the ATP-binding site, they do not contain any sequence motifs characteristic of the eukaryotic protein kinase superfamily. Comparison of different eEF-2 kinase sequences reveals a highly conserved region of approximately 200 amino acids which was found to be homologous to the catalytic domain of the recently described myosin heavy chain kinase A (MHCK A) from Dictyostelium. This suggests that eEF-2 kinase and MHCK A are members of a new class of protein kinases with a novel catalytic domain structure.     

Characterization of a GTP-binding protein implicated in both memory storage and interorganelle vesicle transport

The phosphorylation state of cp20, a low molecular weight GTP-binding protein that is a high-affinity substrate for protein kinase C, was previously shown to change after associative conditioning of molluscs and mammals and to induce many of the biophysical and structural modifications that accompany memory retention. Here, cp20 was purified from squid optic lobes and biochemically characterized. A monoclonal antibody prepared against squid cp20 reacted with Hermissenda cp20 and a 20-kDa protein in rabbit hippocampus, while a polyclonal antibody also cross-reacted with Sar1p and ADP-ribosylation factor (ARF). A partial peptide sequence of squid cp20 was 50% identical (23/46 amino acids) with Sar1p, a yeast GTP-binding protein involved in vesicle transport, indicating that cp20 is probably a new member of the ARF family. This classification is consistent with our recent demonstration that cp20 affects retrograde movement of intraaxonal organelles or particles and suggests a possible role for particle traffic between intraneuronal organelles in memory acquisition.