Molecular cloning and expression analyses of mouse betaklotho, which encodes a novel Klotho family protein

We report here the identification of mouse betaklotho (betakl), which encodes a type I membrane protein with high resemblance to Klotho (KL). Both betaKL and KL consist of two internal repeats with homology to family 1 glycosidases, while these essential glutamates for the enzymatic activities were not conserved. The identical pattern of substitution and variation in the substituted amino acids between these two proteins indicate that they likely to form a unique family within the glycosidase family 1 superfamily. During mouse embryonic development, strong betakl expression was detected in the yolk sac, gut, brown and white adipose tissues, liver and pancreas, and in the adult, predominantly in the liver and pancreas. Despite the high structural similarity between betaKL and KL, their expression profiles were considerably different and betakl expression was not induced in kl-deficient mouse mutants.     

Identification of an atypical interaction site in the BTB domain of the MYC-interacting zinc-finger protein 1

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Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.     

Identification of a novel testis-specific gene mtLR1, which is expressed at specific stages of mouse spermatogenesis

A novel testis-specific gene termed mtLR1 was identified by digital differential display. Sequence analyses revealed that mtLR1 protein contains an amino terminus leucine-rich repeat domain and shows 33% similarities to PIDD which functions in p53-mediated apoptosis. Northern blot analysis showed that mtLR1 mRNA was specifically expressed in adult mouse testis, and RT-PCR results also showed that mtLR1 was exclusively expressed in adult testis and not in spermatogonial cells. The expression of mtLR1 mRNA was developmentally upregulated in the testes during sexual maturation and was, conversely, downregulated by experimental cryptorchidism in vivo. We also showed that the expression of mtLR1 mRNA was relatively highly sensitive to heat stress in vitro. The green fluorescent protein produced by pEGFP-C3/mtLR1 was only detected in the cytoplasm of spermatogonia cell line GC-1 after 24 h posttransfection. Immunohistochemical analysis revealed that the protein is most abundant in the cytoplasm of spermatocytes and round spermatids within seminiferous tubules of the adult testis. The time-dependent expression pattern of mtLR1 in postnatal mouse testes suggested that mtLR1 gene might be involved in the regulation of spermatogenesis and sperm maturation.     

Identification of an elicitor active site within the three-dimensional structure of the tobacco mosaic tobamovirus coat protein.

The coat protein (CP) of tobacco mosaic tobamovirus (TMV) elicits the hypersensitive response (HR) conferred by the N' gene from Nicotiana sylvestris. This study presents evidence demonstrating a critical role for a specific CP structural site in eliciting this HR. Based on the known structure of the TMV CP, specific substitutions were created within the CP of the elicitor strain P20L to identify structural areas essential for host recognition. Of 32 substitutions made, 14 conferred either a temperature-sensitive (loss of the HR at 29 degrees C) or a knockout (loss of the HR at 25 degrees C) HR phenotype in N.sylvestris. These essential residues were noncontiguous in position; however, within the three-dimensional CP structure, all resided primarily along the right face of the molecule's helical bundle. Substitutions that did not affect the HR phenotype either were located outside of this area or were conservative in change. In addition, placing two temperature-sensitive substitutions within the same CP resulted in lowering temperature sensitivity from 29 to 27 degrees C. This additive effect suggests that residues essential for HR elicitation contribute independently to host recognition. This feature is characteristic of recognition surfaces. The presence of a specific elicitor active site within the three-dimensional structure of the TMV CP is consistent with binding of a host-encoded receptor and demonstrates the importance of CP structure in HR specificity.     

Identification of Nd1, a novel murine kelch family protein,involved in stabilization of actin filaments

We isolated Nd1, a novel kelch family gene that encodes two forms of proteins, Nd1-L and Nd1-S. Nd1-L contains a BTB/POZ domain in its N terminus and six kelch repeats in the C terminus. Nd1-S has the BTB/POZ domain but lacks the six kelch repeats. Nd1-L but not Nd1-S mRNA is detected ubiquitously in normal mouse tissues. Nd1-L and Nd1-S proteins can form a dimer through the BTB/POZ domain. Nd1-L colocalizes with actin filaments detected using a confocal microscope, and its kelch repeats bind to them in vitro. Overexpression of Nd1-L in NIH3T3 cells delayed cell growth by affecting the transition of cytokinesis. Furthermore, the overexpression prevented NIH3T3 cells from cell death induced by actin destabilization but not by microtubule dysfunction. These data suggest that Nd1-L functions as a stabilizer of actin filaments as an actin-binding protein and may play a role in the dynamic organization of the actin cytoskeleton.     

Pak6 protein kinase is a novel effector of an atypical Rho family GTPase Chp/RhoV

Chp/RhoV is an atypical Rho GTPase whose functions are far from being fully understood. To date several effector proteins of Chp have been identified, including p21-activated kinases Pak1, Pak2, and Pak4. Using a yeast two-hybrid system and co-immunoprecipitation, here we show that another p21-activated kinase, Pak6, is a novel Chp-binding protein. Interaction between Chp and Pak6 depends on the activation state of the GTPase, suggesting that Pak6 is an effector protein for Chp. Point mutations in the effector domain of Chp or in the CRIB motif of Pak6 significantly impair the interaction between Chp and Pak6 upon co-immunoprecipitation, suggesting that the binding interface involves the effector domain of Chp and the CRIB motif in Pak6. We found that Chp does not affect the phosphorylation status of the S560 residue in the catalytic domain of Pak6 when Chp and Pak6 are co-expressed in HEK293 cells. Therefore, similarly to Cdc42, Chp is not likely to activate Pak6. In NCI-H1299 cells, Chp co-localizes with Pak6 on vesicular structures in activation state-dependent manner. Taking the data together, we report here the identification of p21-activated kinase Pak6 as a novel effector of the atypical Rho GTPase Chp. Our data suggest further directions in elucidating biological functions of these proteins.     

Identification of a novel phosphorylation site in ataxin-1

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease resulting from an expanded CAG repeat in the SCA1 gene that leads to an expanded polyglutamine tract in the gene product. Previous studies have demonstrated that serine at site 776 is phosphorylated [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Studies of ataxin-1 S776 and serine mutated to an alanine, A776, have also shown differential protein-protein interactions and reduced neurodegeneration [H.K. Chen, P. Fernandez-Funez, S.F. Acevedo, Y.C. Lam, M.D. Kaytor, M.H. Fernandez, A. Aitken, E.M. Skoulakis, H.T. Orr, J. Botas, H.Y. Zoghbi, Interaction of Akt_phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1.]. However, mutation of the site serine 776 to an alanine did not abolish all phosphorylation of the protein ataxin-1, suggesting the presence of additional phosphorylation sites [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and mutational analysis demonstrated a novel phosphorylation site at serine 239 of ataxin-1.     

Identification of a novel domain--DIM, which defines a new family composed mainly of bacterial membrane proteins

We report here the identification of a novel domain - DIM (N-terminal domain in bacterial membrane proteins and other proteins) present exclusively in bacterial species including mycobacteria, revealed by PSI-BLAST iterative searches. DIM comprises about 53 amino acids in length with conserved Leu, Ile and Gly residues. Secondary structure prediction indicated that this domain contains two alpha-helices. DIM occurs at the N-terminus of proteins, and was found particularly but not exclusively in proteins with a transmembrane domain, and also in proteins with a FHA domain or RPT repeats. DIM-containing proteins have been reported to be involved in pathogenicity, signal transduction or small solute transport.     

The nucleotide binding site of F1-ATPase which carries out uni-site catalysis is one of the alternating active sites of the enzyme

Nucleotide-depleted mitochondrial F1-ATPase binds 3'-(2')-O-(2-nitro-4-azidobenzoyl)-derivatives of ATP (NAB-ATP) and GTP (NAB-GTP) when these nucleotide analogues are added to the enzyme in equimolar quantities in the presence of Mg2+ (uni-site catalysis conditions). The binding of NAB-ATP is accompanied by its hydrolysis and inorganic phosphate dissociation from the enzyme; NAB-ADP remains bound to F1-ATPase. The F1-ATPase X NAB-ADP complex has no ATPase activity and its reactivation in the presence of an excess of ATP is accompanied by NAB-ADP release. The illumination of the F1-ATPase complexes with NAB-ADP or NAB-GDP leads to the covalent binding of one nucleotide analogue molecule to the enzyme and to the irreversible inactivation of F1-ATPase. It follows from the results obtained that the modification of just one of the F1-ATPase catalytic sites is sufficient to complete the inhibition of ATPase activity.     

pAMβ1 resolvase has an atypical recombination site and requires a histone-like protein HU

The broad-host-range plasmid pAMβ1 from Gram-positive bacteria encodes a resolvase, designated Resβ, which shares homology with the proteins of the resolvase—invertase family. Here we report the purification and in vitro characterization of Resβ. This resolvase is particular in two aspects: it has an atypical binding site and requires a cofactor to promote resolution in vitro . Resβ binds to two regions within its resolution site res . One contains two inverted repeats (R1 and R2), the other contains only one repeat (R3). The cofactor required for resolution in vitro is present in crude extracts of both Bacillus subtilis and Escherichia coli and can be substituted by the E. coli histone-like protein HU. The possible mode of action of HU in the resolution process is discussed.     

Identification of a ubiquitin family protein as a novel neutrophil chemotactic factor

Neutrophil chemotaxis is a process that is essential for the recruitment of neutrophils to an inflamed site. In the present study, we found a remarkable increase in neutrophil chemotactic activity in the lysate of red blood cells (RBC) of mice infected with murine malaria, Plasmodium yoelii. A neutrophil chemotactic factor with an apparent molecular weight of 17 kDa (IP17) was isolated from RBC by a combination of anion-exchange chromatography on DE52 and cation-exchange chromatography on Mono S. A comprehensive GenBank database search of N-terminal amino acid sequences and MALDI-TOF mass analysis of IP17 revealed that IP17 is identical to a murine homologue of ISG15/UCRP, a member of the ubiquitin family of proteins that are inducible by interferon-beta. Recombinant mouse ISG15 showed neutrophil chemotactic activity comparable to that of natural IP17. IP17 showed specific chemotactic activity forward neutrophils and activated neutrophils to induce the release of eosinophil chemotactic factors. These results suggest that the ubiquitin family protein ISG15/UCRP has novel functions in neutrophil-mediated immune mechanisms.     

Identification and characterization of a novel member of olfactomedin-related protein family, hGC-1, expressed during myeloid lineage development

We have cloned a novel hematopoietic granulocyte colony-stimulating factor (G-CSF)-induced olfactomedin-related glycoprotein, termed hGC-1 (human G-CSF-stimulated clone-1). mRNA differential display was used in conjunction with a modified two-phase liquid culture system. Cultures were enriched for early precursors of erythroid, myeloid, and megakaryocytic lineages, which were isolated after induction with erythropoietin, G-CSF, and thrombopoietin, respectively. RNA from the enriched cells was subjected to differential display analysis to identify lineage-specific expressed genes. One clone specifically induced by G-CSF, hGC-1, was characterized. The 2861 bp cDNA clone of hGC-1 contained an open reading frame of 1530 nucleotides, translating into a protein of 510 amino acids with a signal peptide and six N-linked glycosylation motifs. The protein sequence of hGC-1 showed it to be a glycoprotein of the olfactomedin family, which includes olfactomedin, TIGR, Noelin-2 and latrophilin-1. Olfactomedin-like genes show characteristic tissue-restricted patterns of expression; the specific tissues expressing these genes differ among the family members. hGC-1 was strongly expressed in the prostate, small intestine, and colon, moderately expressed in the bone marrow and stomach, and not detectable in other tissues. In vitro translation and ex vivo expression showed hGC-1 to be an N-linked glycoprotein. The hGC-1 gene locus mapped to chromosome 13q14.3. Together, our findings indicate that hGC-1 is primarily expressed as an extracellular olfactomedin-related glycoprotein during normal myeloid-specific lineage differentiation, suggesting the possibility of a matrix-related function for hGC-1 in differentiation.