Protein Kinase Phosphorylation Site Research Articles

Chapter Eleven - SLC41 Transporters—Molecular Identification and Functional Role

The solute carrier family 41 (SLC41) encompasses three members A1, A2, and A3. Based on their distant homology to the bacterial Mg²⁺ channel MgtE, all have been linked to Mg²⁺ transport. There is only very limited knowledge on the molecular biology and exact functions of SLC41A2 and SLC41A3. SLC41A1 is ubiquitously expressed and data on its functional and molecular properties, regulation, complex-forming ability, and spectrum of binding partners are available. SLC41A1 was recently identified as being the Na⁺/Mg²⁺ exchanger (NME)-a predominant Mg²⁺ efflux system. Mg²⁺-dependent and hormonal regulation of NME activity is now known to depend on the intracellular N terminus of SLC41A1 that is involved in Mg²⁺ sensing and contains phosphorylation sites for protein kinase (PK) A and PKC. Data showing a link between SLC41A1 and human disorders such as Parkinson's disease, nephronophthisis (induced by the null mutation c.698G>T in renal SLC41A1), and preeclampsia make the protein a candidate therapeutic target.

Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3

Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein implicated in Parkinson disease (PD); however, the molecular mechanism and mode of action of this protein remain elusive. cAMP-dependent protein kinase (PKA), along with other kinases, has been suggested to be an upstream kinase regulating LRRK2 function. Using MS, we detected several sites phosphorylated by PKA, including phosphorylation sites within the Ras of complex proteins (ROC) GTPase domain as well as some previously described sites (S910 and S935). We systematically mapped those sites within LRRK2 and investigated their functional consequences. S1444 in the ROC domain was confirmed as a target for PKA phosphorylation using ROC single-domain constructs and through site-directed mutagenesis. Phosphorylation at S1444 is strikingly reduced in the major PD-related LRRK2 mutations R1441C/G/H, which are part of a consensus PKA recognition site ((1441)RASpS(1444)). Furthermore, our work establishes S1444 as a PKA-regulated 14-3-3 docking site. Experiments of direct binding to the three 14-3-3 isotypes gamma, theta, and zeta with phosphopeptides encompassing pS910, pS935, or pS1444 demonstrated the highest affinities to phospho-S1444. Strikingly, 14-3-3 binding to phospho-S1444 decreased LRRK2 kinase activity in vitro. Moreover, substitution of S1444 by alanine or by introducing the mutations R1441C/G/H, abrogating PKA phosphorylation and 14-3-3 binding, resulted in increased LRRK2 kinase activity. In conclusion, these data clearly demonstrate that LRRK2 kinase activity is modulated by PKA-mediated binding of 14-3-3 to S1444 and suggest that 14-3-3 interaction with LRRK2 is hampered in R1441C/G/H-mediated PD pathogenesis.

Gap junctions

In vertebrates and invertebrates, signaling among neurons is most commonly mediated by chemical synapses. At these synapses neurotransmitter released by presynaptic neurons is detected by receptors on the postsynaptic neurons, leading to an influx of ions through the receptors themselves or through channels activated by intracellular signaling downstream of the receptors. But neurons can communicate with each other in a more direct way, by passing signals composed of small molecules and ions through pores called gap junctions. Gap junctions that transmit electrical signals are called electrical synapses. Unlike most chemical synapses, electrical synapses interact through axon-to-axon or dendrite-to-dendrite contacts. Found throughout the nervous system, they are probably best known for linking the relatively few inhibitory, GABAergic, neurons into large, effective networks within vertebrate brains. They are particularly important early in development before the formation of most chemical synapses, but recent work shows gap junctions play important roles in the adult nervous system, too. Gap junctions are sometimes thought to be mere passageways between cells. But, as recent work shows, their properties can be complex and surprising. Gap junctions help generate, propagate, and regulate neural oscillations, can filter electrical signals, and can be modulated in a variety of ways. Here we discuss recent work highlighting the diversity and importance of gap junctions throughout the nervous system.

Cyclic AMP-Dependent Protein Kinase Enhances SC35-Promoted Tau Exon 10 Inclusion

Alternative splicing of tau exon 10 generates tau with three or four microtubule-binding repeats (3R-tau or 4R-tau). The ratio of 3R-tau to 4R-tau is approximately 1:1 in the adult normal human brain. Disturbances in the ratio result in neurodegenerative tauopathies. Splicing factor SC35 acts on a SC35-like element located at the 5' end of tau exon 10 and promotes tau exon 10 inclusion. Here, we report that protein kinase (PKA) was able to interact and phosphorylate SC35. Activation or overexpression of PKA catalytic subunits promoted SC35-mediated tau exon 10 inclusion. Four PKA catalytic subunits, α1, α2, β1, and β2, all enhanced SC35-promoted tau exon 10 inclusion. SC35 has four putative PKA phosphorylation sites, Ser121, Ser128, Ser130, and Ser171. Pseudophosphorylation (SC354E) and blockage (SC354A) of phosphorylation of SC35 at these four sites increased and decreased, respectively, SC35's ability to promote tau exon 10 inclusion. Moreover, PKA catalytic subunits no longer further enhanced tau exon 10 inclusion when these four were mutated to either alanine or glutamate. These results suggest that PKA interacts with and phosphorylates SC35 and enhances SC35-promoted tau exon 10 inclusion. In Alzheimer's brain, down-regulation of the PKA pathway could lead to dysregulation of tau exon 10, contributing to tau pathogenesis.

Calcium and Calmodulin-dependent Serine/Threonine Protein Kinase Type II (CaMKII)-mediated Intramolecular Opening of Integrin Cytoplasmic Domain-associated Protein-1 (ICAP-1α) Negatively Regulates β1 Integrins

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state, which is regulated by the direct recruitment of proteins onto the β integrin chain cytoplasmic domain. We previously reported that ICAP-1α, a specific cytoplasmic partner of β1A integrins, limits both talin and kindlin interaction with β1 integrin, thereby restraining focal adhesion assembly. Here we provide evidence that the calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII) is an important regulator of ICAP-1α for controlling focal adhesion dynamics. CaMKII directly phosphorylates ICAP-1α and disrupts an intramolecular interaction between the N- and the C-terminal domains of ICAP-1α, unmasking the PTB domain, thereby permitting ICAP-1α binding onto the β1 integrin tail. ICAP-1α direct interaction with the β1 integrin tail and the modulation of β1 integrin affinity state are required for down-regulating focal adhesion assembly. Our results point to a molecular mechanism for the phosphorylation-dependent control of ICAP-1α function by CaMKII, allowing the dynamic control of β1 integrin activation and cell adhesion.

Role of serine 249 of ezrin in the regulation of sodium-dependent phosphate transporter NaPi-IIa activity in renal proximal tubular cells

Type IIa sodium-dependent phosphate transporter (NaPi-IIa) is responsible for renal phosphate reabsorption and maintenance of systemic phosphate homeostasis in mammals. Macromolecular complex formation of NaPi-IIa with sodium-proton exchanger related factor-1 (NHERF-1) and ezrin is important for apical membrane localization in the proximal tubular cells. Here, we investigated the interactions of the ezrin phosphomimetic mutation of serine to aspartic acid at 249 with NHERF-1 and the inhibition of apical membrane localization of NaPi-IIa. In vitro phosphorylation analysis revealed that serine 249 of human ezrin serves as a phosphorylation site for protein kinase A. The N-terminal half of ezrin had a dominant negative effect on the phosphate transport activity and inhibited the apical localization of NaPi-IIa in renal proximal tubular cells. We found that the phosphomimetic S249D mutant interfered with the inhibitory effects of the dominant negative mutant on the transport and localization of NaPi-IIa. The S249D mutant also inhibited the interaction with NHERF-1. Therefore, serine 249 of ezrin can play important roles in the regulation of the complex formation and membrane localization of NaPi-IIa.

Complete Nucleotide Sequence Analysis of Outer Membrane Efflux Protein Gene of 1347bp ORF from <i>Riemerella anatipestifer</i>

A 1347bp complete open reading frame of the Riemerella anatipestifer outer membrane efflux protein﹙GenBank No.CP003388﹚was analyzed by bioinformatics software. The gene encoded a protein of 448 amino acids, Molecular weight was 50811.1. The formula of the outer membrane efflux protein gene is C2265H3639N609O689S12. There was no transmembrane region and signal peptide. six N-glycosylation sites, one cAMP- and cGMP-dependent protein kinase phosphorylation site, eight Protein kinase C phosphorylation sites, ten Casein kinase II phosphorylation sites, four Tyrosine kinase phosphorylation sites, five N-myristoylation sites, one Cell attachment sequence. The sequence of Riemerella anatipestifer outer membrane efflux protein shared high homology with other members of flavobacterium. These results will lay the foundation for further study.

Bioinformatics Analysis of OmpA/MotB Protein Encoded by OmpA/MotB Gene(ORF 648bp) of <i>Riemerella anatipestifer</i>

The OmpA/MotB gene from RA by our lab was sequenced. And the molecular characteristic of this gene was analyzed with bioinformatics software. The results indicated that this gene encodes an estimated 215 protein, contains the conserved domain of the OmpA-like protein. The relative molecular weight of the protein was 23.34525kDa, and there is no signal peptide and transmembrane region of the protein. One cAMP-and cGMP-dependent protein kinase phosphorylation site, six Protein kinase C phosphorylation sites, five Casein kinase II phosphorylation sites, five N-myristoylation sites and antigenic determinants were searched in the protein. Most of the total proteins were located in the Outer membrane and cytoplasmic. Phylogenetic tree of the amino acids sequences showed this gene has a close evolutionary relationship with Elizabethkingia anopheles Ag1 and Weeksella virosa DSM16922, indicating that the RA OmpA/MotB protein may have some similar functions with them. These results provided rational data to elucidate biological function and physiological features of the protein.

Disease-causing R1185C mutation of WNK4 disrupts a regulatory mechanism involving calmodulin binding and SGK1 phosphorylation sites

The R1185C mutation in WNK4 is associated with pseudohypoaldosteronism type II (PHAII). Unlike other PHAII-causing mutations in the acidic motif, the R1185C mutation is located in the COOH-terminal region of WNK4. The goal of the study is to determine what properties of WNK4 are disrupted by the R1185C mutation. We found that the R1185C mutation is situated in the middle of a calmodulin (CaM) binding site and the mutation reduces the binding of WNK4 to Ca(2+)/CaM. The R1185C mutation is also close to serum- and glucocorticoid-induced protein kinase (SGK1) phosphorylation sites S1190 and S1217. In addition, we identified a novel SGK1 phosphorylation site (S1201) in WNK4, and phosphorylation at this site is reduced by Ca(2+)/CaM. In the wild-type WNK4, the level of phosphorylation at S1190 is the lowest and that at S1217 is the highest. In the R1185C mutant, phosphorylation at S1190 is eliminated and that at S1201 becomes the strongest. The R1185C mutation enhances the positive effect of WNK4 on the Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2) as tested in Xenopus laevis oocytes. Deletion of the CaM binding site or phospho-mimicking at two or three of the SGK1 sites enhances the WNK4 effects on NKCC2. These results indicate that the R1185C mutation disrupts an inhibitory domain as part of the suppression mechanism of WNK4, leading to an elevated WNK4 activity at baseline. The presence of CaM binding and SGK1 phosphorylation sites in or close to the inhibitory domain suggests that WNK4 activity is subject to the regulation by intracellular Ca(2+) and phosphorylation.

Dopamine- and cAMP-regulated Phosphoprotein of 32-kDa (DARPP-32)-dependent Activation of Extracellular Signal-regulated Kinase (ERK) and Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Experimental Parkinsonism

Dyskinesia, a motor complication caused by prolonged administration of the antiparkinsonian drug l-3,4-dihydroxyphenylalanine (l-DOPA), is accompanied by activation of cAMP signaling and hyperphosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). Here, we show that the abnormal phosphorylation of DARPP-32 occurs specifically in medium spiny neurons (MSNs) expressing dopamine D1 receptors (D1R). Using mice in which DARPP-32 is selectively deleted in D1R-expressing MSNs, we demonstrate that this protein is required for l-DOPA-induced activation of the extracellular signal-regulated protein kinases 1 and 2 and the mammalian target of rapamycin complex 1 (mTORC1) pathways, which are implicated in dyskinesia. We also show that mutation of the phosphorylation site for cAMP-dependent protein kinase on DARPP-32 attenuates l-DOPA-induced dyskinesia and reduces the concomitant activations of ERK and mTORC1 signaling. These studies demonstrate that, in D1R-expressing MSNs, l-DOPA-induced activation of ERK and mTORC1 requires DARPP-32 and indicates the importance of the cAMP/DARPP-32 signaling cascade in dyskinesia.

Gene cloning and function analysis of complement B factor-2 of Apostichopus japonicus

In this study, a homologue of complement B factor (AjBf-2, GenBank ID: JN634069.1) was cloned and characterized from Apostichopus japonicus by using bioinformatics methods and molecular biotechnologies including homology cloning and RACE. The full-length cDNA of AjBf-2 was composed of 3261bp. The sequence shows 268bp in the 5′UT region, 395bp in the 3′UT region, and 2595 bp in the open reading frame. AjBf-2 gene encodes 865 amino acids. The deduced amino acids sequence and domain structure of AjBf-2 gene show significant similarity to the vertebrate Bf/C2 family protein. AjBf-2 is a mosaic protein. It has a deduced molecular mass of 96.8 kDa, with a conserved site for a D factor. AjBf-2 is composed of five short consensus repeats, a von Willebrand Factor domain, a serine protease domain and an Mg2+ binding site. It has eight consensus recognition sites for N-linked glycosylation and four cAMP- and cGMP-dependent protein kinase phosphorylation sites. Phylogenetic analysis of AjBf-2 compared with other species Bf shows that A. japonicus has a close evolutionary relationship with Strongylocentrotus purpuratus and Carcinoscorpius rotundicaud. It can be speculated that Bf in invertebrate is the ancestor of Bf in vertebrate. The result of RT-PCR shows that the AjBf-2 gene is expressed in every tested tissue of A. japonicus, and is especially high in the coelomocyte and the body wall. The expression tendency in coelomocyte and the body wall are approximately the same. After LPS induction, the expression of AjBf-2 gene peaks at 12 h in coelomocyte and 3 h in the body wall.

Deletion of the C-terminal Phosphorylation Sites in the Cardiac β-Subunit Does Not Affect the Basic β-Adrenergic Response of the Heart and the Cav1.2 Channel

Phosphorylation of the cardiac β subunit (Ca(v)β(2)) of the Ca(v)1.2 L-type Ca(2+) channel complex has been proposed as a mechanism for regulation of L-type Ca(2+) channels by various protein kinases including PKA, CaMKII, Akt/PKB, and PKG. To test this hypothesis directly in vivo, we generated a knock-in mouse line with targeted mutation of the Ca(v)β(2) gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; βStop mouse). This mutation prevented translation of the Ca(v)β(2) C terminus that contains the relevant phosphorylation sites for the above protein kinases. Homozygous cardiac βStop mice were born at Mendelian ratio, had a normal life expectancy, and normal basal L-type I(Ca). The regulation of the L-type current by stimulation of the β-adrenergic receptor was unaffected in vivo and in cardiomyocytes (CMs). βStop mice were cross-bred with mice expressing the Ca(v)1.2 gene containing the mutation S1928A (SAβStop) or S1512A and S1570A (SFβStop) in the C terminus of the α(1C) subunit. The β-adrenergic regulation of the cardiac I(Ca) was unaltered in these mouse lines. In contrast, truncation of the Ca(v)1.2 at Asp(1904) abolished β-adrenergic up-regulation of I(Ca) in murine embryonic CMs. We conclude that phosphorylation of the C-terminal sites in Ca(v)β(2), Ser(1928), Ser(1512), and Ser(1570) of the Ca(v)1.2 protein is functionally not involved in the adrenergic regulation of the murine cardiac Ca(v)1.2 channel.

Site-Specific Synapsin I Phosphorylation Participates in the Expression of Post-Tetanic Potentiation and Its Enhancement by BDNF

A large amount of experimental evidence has highlighted the rapid changes in synaptic efficacy induced by high-frequency stimulation and BDNF at central excitatory synapses. We clarified the quantal mechanisms and the involvement of Synapsin I (SynI) phosphorylation in the expression of post-tetanic potentiation (PTP) and in its modulation by BDNF in mouse glutamatergic autapses. We found that PTP is associated with an elevation in the probability of release and a concomitant increase in the size of the readily releasable pool (RRP). The latter component was virtually absent in SynI knock-out (KO) neurons, which indeed displayed impaired PTP. PTP was fully rescued by the expression of wild-type SynI, but not of its dephosphomimetic mutants in the phosphorylation sites for cAMP-dependent protein kinase and Ca²⁺/calmodulin-dependent protein kinases I/II. BDNF potently enhanced PTP through a further increase in the RRP size, which was missing in SynI KO neurons. In these neurons, the BDNF-induced PTP enhancement was rescued by the expression of wild-type SynI, but not of its dephosphomimetic mutant at the mitogen-dependent protein kinase sites. The results indicate that the increase in RRP size necessary for the full expression of PTP, and its sensitivity to BDNF, involve phosphorylation of SynI at distinct sites, thus implicating SynI as an essential downstream effector for the expression of PTP and for its enhancement by BDNF.

MGlu5R promotes glutamate AMPA receptor phosphorylation via activation of PKA/DARPP-32 signaling in striatopallidal medium spiny neurons

Group I metabotropic glutamate receptors (mGluRs), which comprise mGlu1Rs and mGlu5Rs, are enriched in striatal medium spiny neurons (MSNs), where they modulate glutamatergic transmission. Here, we have examined the effect of group I mGluRs on the regulation of the state of phosphorylation of the GluA1 subunit of the AMPA glutamate receptor. We found that incubation of mouse striatal slices with the group I mGluR agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) promotes GluA1 phosphorylation at the cAMP-dependent protein kinase (PKA) site, Ser845. This effect is prevented by 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), a selective mGlu5R antagonist. The increase in GluA1 phosphorylation produced by DHPG is also prevented by blockade of adenosine A2A receptors (A2ARs), which are known to promote cAMP signaling specifically in striatopallidal MSNs, as well as by enzymatic degradation of endogenous adenosine, achieved with adenosine deaminase. The ability of DHPG to increase PKA-dependent phosphorylation of GluA1 depends on concomitant activation of the dopamine- and cAMP-regulated phosphoprotein of 32kDa (DARPP-32). Thus, inactivation of the PKA phosphorylation site of DARPP-32 abolishes the effect of DHPG. Moreover, cell-specific knock out of DARPP-32 in striatopallidal, but not in striatonigral, MSNs prevents the increase in Ser845 phosphorylation induced by DHPG. These results indicate that activation of mGlu5Rs promotes PKA/DARPP-32-dependent phosphorylation of downstream target proteins in striatopallidal MSNs and that this effect is exerted via potentiation of tonic A2AR transmission.This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’.

S gene mutations of HBV in children with HBV-associated glomerulonephritis

BackgroundHepatitis B virus-associated glomerulonephritis (HBV-GN) is a kind of immune complex-induced glomerulonephritis. The present study was designed to determine whether mutation of Hepatitis B virus (HBV) S gene is associated with glomerulonephritis in Chinese children.MethodsTotal 53 subjects, including 30 HBV-GN, 5 nephrosis with HBV carriers (control group 1), and 18 HBV carriers (control group 2) were included in this study. Polymerase chain reaction (PCR) was used to detect the HBV-GN S gene mutation.Results(1) The serotype of HBV was adw in the majority (52/53) of subjects, and was adr in only 1 subject in the control group 2; (2) the genotype of HBV was the type B in 51 subjects, the type E in 1 HBV-GN child, and the type C in 1 HBV carrier; (3) Seventeen point mutations in the S gene of HBV were identified in 21 of 30 (70%) HBV-GN patients. Among them, 16 of 21 (76.2%) mutations may cause amino acid substitutions of the HBV proteins, which occur predominantly (11/16 mutations) at threonine, serine or tyrosine phosphorylation sites of mitogen-activated protein kinase (MAPK) or protein tyrosine kinase (PTK). (4) In addition, single nucleotide mutations without amino acid substitutions (same sense mutation) were found in 2 subjects in each control group and 5 subjects in HBV-GN group.ConclusionsHBV S gene mutations and the subsequent amino acid substitutions in HBV proteins were found in most children with HBV-GN, suggesting that these mutations may play an important role in the pathogenesis of HBV-GN.

Nemo phosphorylates Eyes absent and enhances output from the Eya-Sine oculis transcriptional complex during Drosophila retinal determination

The retinal determination gene network comprises a collection of transcription factors that respond to multiple signaling inputs to direct Drosophila eye development. Previous genetic studies have shown that nemo (nmo), a gene encoding a proline-directed serine/threonine kinase, can promote retinal specification through interactions with the retinal determination gene network, although the molecular point of cross-talk was not defined. Here, we report that the Nemo kinase positively and directly regulates Eyes absent (Eya). Genetic assays show that Nmo catalytic activity enhances Eya-mediated ectopic eye formation and potentiates induction of the Eya-Sine oculis (So) transcriptional targets dachshund and lozenge. Biochemical analyses demonstrate that Nmo forms a complex with and phosphorylates Eya at two consensus mitogen-activated protein kinase (MAPK) phosphorylation sites. These same sites appear crucial for Nmo-mediated activation of Eya function in vivo. Thus, we propose that Nmo phosphorylation of Eya potentiates its transactivation function to enhance transcription of Eya-So target genes during eye specification and development.

Spatial distribution pattern analysis of Dof1 transcription factor in different tissues of three Eleusine coracana genotypes differing in their grain colour, yield and photosynthetic efficiency

In the present study Dof1 gene of finger millet was cloned and sequenced. In silico analysis reveals 61% identity with the Sorghum bicolor and 57% identity with the Oryza sativa Dof1 sequence. A comparative analysis of gene sequences from different crops and three finger millet genotypes {Brown (PRM-1), Golden (PRM-701) and White (PRM-801)} differing in grain colour, yield and photosynthetic efficiency showed a high degree of sequence identity of Dof1 sequence gene ranging from 22 to 70% as evident from distance matrix of the built phylogenetic tree showing two major clusters. A total of five conserved motifs were observed in Dof1 sequences of different cereals. Motif 1 with multilevel consensus sequence CKNCRRYWTKGGAMRNVPVG contains zinc finger Dof domain. Motif 3 and motif 5 contains protein kinase phosphorylation site. Motif 2 contains Dof domain and zinc finger N-glycosylation site while motif 4 is involved in Zinc finger type profiling. Further, we studied the spatial distribution of Dof1 gene in three vegetative tissues (root, stem and flag leaf) as well as four stages of developing spikes (S1, S2, S3 and S4) of the three finger millet genotypes using qualitative and quantitative PCR based approaches. Physiological parameters (plant height, leaf area, chlorophyll content, SPAD value and photosynthetic efficiency) at the time of flowering was found to be highest in white (PRM-801) genotype followed by golden (PRM-701) and brown (PRM-1) genotype. Semi-quantitative RT-PCR and quantitative real-time PCR analysis revealed that the expression of Dof1 is highest in leaves and lowest in roots, which suggests its role in regulation of photosynthesis-related genes and carbon skeleton synthesis. Also at grain maturity stage, expression of Dof1 was higher in white (PRM-801) genotype followed by golden (PRM-701) and brown (PRM-1) genotype. The result is suggestive of Dof1 role in the accumulation of grain protein and yield attribute through regulation of key enzymes involved in source to sink relationship during grain filling stage.

CDNA, Genomic sequence cloning and over-expression of ribosomal protein L15 gene (RPL15) from the giant panda

RPL15 is a component of the 60S large ribosomal subunit encoded by RPL15 gene and belongs to the L15e family of ribosomal proteins, which is located in the cytoplasm. The cDNA and genomic sequence of RPL15 was cloned successfully from the giant panda using RT-PCR and Touchdown-PCR technology, respectively. These two sequences were analyzed preliminarily and the cDNA of the RPL15 gene was also over expressed inEscherichia coli BL21. The length of fragment cloned is 669 bp, containing an open-reading frame (ORF) of 615 bp encoding 204 amino acids. The length of the genomic sequence is 1,835 bp, with three exons and two introns. Primary structure analysis revealed that, the molecular weight of the putative RPL15 protein is 24.142 kDa with a theoretical pI 12.11. Topology prediction shows that there are two cAMP- and cGMP-dependent protein kinase phosphorylation sites, four N-myristoylation site, two Protein kinase C phosphorylation site, two Casein kinase II phosphorylation sites, two Amidation site and one Ribosomal protein L15e signature in the RPL15 protein of the giant panda. Alignment analysis indicates that, the nucleotide sequence of the coding sequence shows a high homology to other knownRPL15 sequences of Homo sapiens, Bos taurus, Mus musculus, Rattus norvegicus, Canis familiaris and Danio rerio; as determined by Blast analysis, 94.31, 89.92, 91.54, 91.22, 96.59 and 79.02%, respectively. The homologies for deduced amino acid sequence are all 100% compared with the first five animals and share a high homology with that of D. rerio by 95.59%. The cDNA of RPL15 was cloned successfully from the giant panda in this study. It provides scientific material for enriching and improving the RPL15 gene database. TheRPL15 gene can be really expressed in E. coli and the RPL15 protein fusioned with the N-terminally His-tagged protein gave rise to the accumulation of an expected 30 KDa polypeptide, in good agreement with the predicted molecular weight. The expression product obtained could be used for purification and study of its function further. Key words: Giant panda, RT-PCR, RPL15, genomic sequences, cloning, over-express.

Molecular Modeling of Cathepsin B protein in different Leishmania strains

Cathepsin B like cysteine proteases representing a major component of the lysosomal proteolytic repertoire plays an important role in intracellular protein degradation. Comparative models of cathepsin B (CatB) protein of six different Leishmania strains were developed using MODELLER. The modeled three-dimensional (3-D) structure has the correct stereochemistry as gauged from the Ramachandran plot and good 3-D structure compatibility as assessed by PROCHECK and the DOPE score ( DS2.1, Accelrys ). T he modeled proteins were energy minimized and validated using standard dynamic cascade protocol (DS 2.1). Seven different disulfide bonding sites are predicted in CatB protein of Leishmania . Two domains were identified and different motifs are present in catB protein of Leishmania like aspargine glycosylation site, protein kinase phosphorylation site, Protein kinase C activation site, N-myristoylation site. Considering that cathepsin B is essential for survival of Leishmania , including for virulence to the mammalian host, it may be viewed as an attractive drug target.

Characterization of human cytomegalovirus UL145 and UL136 genes in low-passage clinical isolates from infected Chinese infants

SummaryBackgroundHuman cytomegalovirus (HCMV) is a leading cause of morbidity and mortality in immunocompromised individuals. The unique long b’ (ULB’) region of HCMV contains at least 19 open reading frames (ORFs); however, little is known about the function of UL145 and UL136. We characterized UL145 and UL136 in low-passage clinical isolates from Chinese infants.Material/MethodsThe clinical strains of HCMV were recovered from the urine from HCMV-infected infants. Human embryonic lung fibroblasts (HELFs) were infected with clinical isolates of HCMV, and the viral DNA and mRNA for UL145 and UL136 were analyzed by polymerase chain reaction (PCR) and sequencing techniques. We also predicted the structure and function of UL145 and UL136 proteins.ResultsSixty-two Chinese infants infected with HCMV were recruited into this study and the clinical isolates were recovered from the urine. Two strains among the low-passage isolates, D2 and D3, were obtained. The UL145 and UL136 sequences were deposited with GenBank under accession numbers of DQ180367, DQ180381, DQ180377, and DQ180389. The mRNA expression of both UL145 and UL136 was confirmed by reverse transcription (RT-PCR) assays. UL145 was predicted to contain 1 protein kinase C phosphorylation site, 2 casein kinase II phosphorylation sites and a zinc finger structure. UL136 was predicted to contain a protein kinase C phosphorylation site, N-myristoylation site, cAMP- and cGMP-dependent protein kinase phosphorylation site and tyrosine kinase II phosphorylation site. Both UL145 and UL136 are highly conserved.ConclusionsUL145 may act as an intranuclear regulating factor by direct binding to DNA, while UL136 may be a membrane receptor involving signal transduction.