Clustered syk tyrosine kinase domains trigger phagocytosis.

Phagocytosis is a phylogenetically primitive mechanism adapted by specialized cells of the immune system to ingest particulate pathogens. Recent evidence suggests that the program of specific cytoskeletal rearrangements that underlies phagocytosis may share elements with the antigen receptor signaling pathway in lymphocytes. Tyrosine phosphorylation, necessary for both lymphocyte effector function and phagocytosis, is thought to allow cytoskeletal elements to couple to the intracellular domains of antigen and Fc receptor subunits. We show here that the intracellular domains of the receptors are not inherently required for cytoskeletal coupling. Chimeric transmembrane proteins bearing syk but not src family tyrosine kinase domains are capable of autonomously triggering phagocytosis and redistribution of filamentous actin in COS cells. These responses cannot be initiated by a receptor chimera bearing a point mutation in the syk catalytic domain, and the kinase domain alone is sufficient for initiating cytoskeletal coupling.

A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana.

We describe here the cloning and characterization of a cDNA encoding a protein kinase that has high sequence homology to members of the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK or MEKK) family; this cDNA is named cATMEKKI (Arabidopsis thaliana MAP kinase or ERK kinase kinase 1). The catalytic domain of the putative ATMEKK1 protein shows approximately 40% identity with the amino acid sequences of the catalytic domains of MAPKKKs (such as Byr2 from Schizosaccharomyces pombe, Ste11 from Saccharomyces cerevisiae, Bck1 from S. cerevisiae, MEKK from mouse, and NPK1 from tobacco). In yeast cells that overexpress ATMEKK1, the protein kinase replaces Ste11 in responding to mating pheromone. In this study, the expression of three protein kinases was examined by Northern blot analyses: ATMEKK1 (structurally related to MAPKKK), ATMPK3 (structurally related to MAPK), and ATPK19 (structurally related to ribosomal S6 kinase). The mRNA levels of these three protein kinases increased markedly and simultaneously in response to touch, cold, and salinity stress. These results suggest that MAP kinase cascades, which are thought to respond to a variety of extracellular signals, are regulated not only at the posttranslational level but also at the transcriptional level in plants and that MAP kinase cascades in plants may function in transducing signals in the presence of environmental stress.

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

Identification of a 95-kDa WEE1-like tyrosine kinase in HeLa cells.

Human WEE1 (WEE1Hu) was cloned on the basis of its ability to rescue wee1+ mutants in fission yeast [Igarashi, M., Nagata, A., Jinno, S., Suto, K. & Okayama, H. (1991) Nature (London) 353, 80-83]. Biochemical studies carried out in vitro with recombinant protein demonstrated that WEE1Hu encodes a tyrosine kinase of approximately 49 kDa that phosphorylates p34cdc2 on Tyr-15 [Parker, L. L. & Piwnica-Worms, H. (1992) Science 257, 1955-1957]. To study the regulation of WEE1Hu in human cells, two polyclonal antibodies to bacterially produced p49WEE1Hu were generated. In addition, a peptide antibody generated against amino acids 361-388 of p49WEE1Hu was also used. Unexpectantly, these antibodies recognized a protein with an apparent molecular mass of 95 kDa in HeLa cells, rather than one of 49 kDa. Immunoprecipitates of p95 phosphorylated p34cdc2 on Tyr-15, indicating that p95 is functionally related to p49WEEIHu, and mapping studies demonstrated that p95 is structurally related to p49WEE1Hu. In addition, the substrate specificity of p95 was more similar to that of fission yeast p107wee1 than to that of human p49WEE1. Finally, the kinase activity of p95 toward p34cdc2/cyclin B was severely impaired during mitosis. Taken together, these results indicate that the original WEE1Hu clone isolated in genetic screens encodes only the catalytic domain of human WEE1 and that the authentic human WEE1 protein has an apparent molecular mass of approximately 95 kDa.

The hepatitis C virus NS3 serine proteinase and NS4A cofactor: establishment of a cell-free trans-processing assay.

The hepatitis C virus RNA genome encodes a long polyprotein that is proteolytically processed into at least 10 products. The order of these cleavage products in the polyprotein is NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. A serine proteinase domain located in the N-terminal one-third of nonstructural protein NS3 mediates cleavage at four downstream sites (the 3/4A, 4A/4B, 4B/5A, and 5A/5B sites). In addition to the proteinase catalytic domain, the NS4A protein is required for processing at the 4B/5A site but not at the 5A/5B site. These cleavage events are likely to be essential for virus replication, making the serine proteinase an attractive antiviral target. Here we describe an in vitro assay where the NS3-4A polyprotein, NS3, the serine proteinase domain (the N-terminal 181 residues of NS3), and the NS4A cofactor were produced by cell-free translation and tested for trans-processing of radiolabeled substrates. Polyprotein substrates, NS4A-4B or truncated NS5A-5B, were cleaved in trans by all forms of the proteinase, whereas NS4A was also required for NS4B-5A processing. Proteolysis was abolished by substitution mutations previously shown to inactivate the proteinase or block cleavage at specific sites in vivo. Furthermore, N-terminal sequence analysis established that cleavage in vitro occurred at the authentic 4A/4B site. Translation in the presence of microsomal membranes enhanced processing for some, but not all, proteinase-substrate combinations. Trans-processing was both time and temperature dependent and was eliminated by treatment with a variety of detergents above their critical micelle concentrations. Among many common proteinase inhibitors tested, only high (millimolar) concentrations of serine proteinase inhibitors tosyllysyl chloromethyl ketone and 4-(2-aminoethyl)benzenesulfonyl fluoride inactivated the NS3 proteinase. This in vitro assay should facilitate purification and further characterization of the viral serine proteinase and identification of molecules which selectively inhibit its activity.

Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Using partial amino acid sequence data derived from porcine methionyl aminopeptidase (MetAP; methionine aminopeptidase, peptidase M; EC 3.4.11.18), a full-length clone of the homologous human enzyme has been obtained. The cDNA sequence contains 2569 nt with a single open reading frame corresponding to a protein of 478 amino acids. The C-terminal portion representing the catalytic domain shows limited identity with MetAP sequences from various prokaryotes and yeast, while the N terminus is rich in charged amino acids, including extended strings of basic and acidic residues. These highly polar stretches likely result in the spuriously high observed molecular mass (67 kDa). This cDNA sequence is highly similar to a rat protein, termed p67, which was identified as an inhibitor of phosphorylation of initiation factor eIF2 alpha and was previously predicted to be a metallopeptidase based on limited sequence homology. Model building established that human MetAP (p67) could be readily accommodated into the Escherichia coli MetAP structure and that the Co2+ ligands were fully preserved. However, human MetAP was found to be much more similar to a yeast open reading frame that differed markedly from the previously reported yeast MetAP. A similar partial sequence from Methanothermus fervidus suggests that this p67-like sequence is also found in prokaryotes. These findings suggest that there are two cobalt-dependent MetAP families, presently composed of the prokaryote and yeast sequences (and represented by the E. coli structure) (type I), on the one hand, and by human MetAP, the yeast open reading frame, and the partial prokaryotic sequence (type II), on the other.

MEKK1 phosphorylates MEK1 and MEK2 but does not cause activation of mitogen-activated protein kinase.

A constitutively active fragment of rat MEK kinase 1 (MEKK1) consisting of only its catalytic domain (MEKK-C) expressed in bacteria quantitatively activates recombinant mitogen-activated protein (MAP) kinase/extracellular signal-regulated protein kinase (ERK) kinases 1 and 2 (MEK1 and MEK2) in vitro. Activation of MEK1 by MEKK-C is accompanied by phosphorylation of S218 and S222, which are also phosphorylated by the protein kinases c-Mos and Raf-1. MEKK1 has been implicated in regulation of a parallel but distinct cascade that leads to phosphorylation of N-terminal sites on c-Jun; thus, its role in the MAP kinase pathway has been questioned. However, in addition to its capacity to phosphorylate MEK1 in vitro, MEKK-C interacts with MEK1 in the two-hybrid system, and expression of mouse MEKK1 or MEKK-C in mammalian cells causes constitutive activation of both MEK1 and MEK2. Neither cotransfected nor endogenous ERK2 is highly activated by MEKK1 compared to its stimulation by epidermal growth factor in spite of significant activation of endogenous MEK. Thus, other as yet undefined mechanisms may be involved in determining information flow through the MAP kinase and related pathways.

Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation.

The p70 S6 kinase is activated by insulin and mitogens through multisite phosphorylation of the enzyme. One set of activating phosphorylations occurs in a putative autoinhibitory domain in the noncatalytic carboxyl-terminal tail. Deletion of this tail yields a variant (p70 delta CT104) that nevertheless continues to be mitogen regulated. Coexpression with a recombinant constitutively active phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) gives substantial activation of both full-length p70 and p70 delta CT104 but not Rsk. Activation of p70 delta CT104 by PI 3-kinase and inhibition by wortmannin are each accompanied by parallel and selective changes in the phosphorylation of p70 Thr-252. A Thr or Ser at this site, in subdomain VIII of the catalytic domain just amino-terminal to the APE motif, is necessary for p70 40S kinase activity. The inactive ATP-binding site mutant K123M p70 delta CT104 undergoes phosphorylation of Thr-252 in situ but does not undergo direct phosphorylation by the active PI 3-kinase in vitro. PI 3-kinase provides a signal necessary for the mitogen activation of the p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain, through a distinctive signal transduction pathway.

Mutation spectrum of the gene encoding the beta subunit of rod phosphodiesterase among patients with autosomal recessive retinitis pigmentosa.

Mutations in the gene encoding the beta subunit of rod cGMP phosphodiesterase are known causes of photoreceptor degeneration in two animal models of retinitis pigmentosa, the rd (retinal degeneration) mouse and the Irish setter dog with rod/cone dysplasia. Here we report a screen of 92 unrelated patients with autosomal recessive retinitis pigmentosa for defects in the human homologue of this gene. We identified seven different mutations that cosegregate with the disease. They were found among four patients with each patient heterozygously carrying two mutations. All of these mutations are predicted to affect the putative catalytic domain, probably leading to a decrease in phosphodiesterase activity and an increase in cGMP levels within rod photoreceptors. Mutations in the gene encoding the beta subunit of rod phosphodiesterase are the most common identified cause of autosomal recessive retinitis pigmentosa, accounting for approximately 4% of cases in North America.

Molecular cloning and characterization of chitinase genes from Candida albicans.

Chitinase (EC 3.2.1.14) is an important enzyme for the remodeling of chitin in the cell wall of fungi. We have cloned three chitinase genes (CHT1, CHT2, and CHT3) from the dimorphic human pathogen Candida albicans. CHT2 and CHT3 have been sequenced in full and their primary structures have been analyzed: CHT2 encodes a protein of 583 aa with a predicted size of 60.8 kDa; CHT3 encodes a protein of 567 aa with a predicted size of 60 kDa. All three genes show striking similarity to other chitinase genes in the literature, especially in the proposed catalytic domain. Transcription of CHT2 and CHT3 was greater when C. albicans was grown in a yeast phase as compared to a mycelial phase. A transcript of CHT1 could not be detected in either growth condition.

Clonagem molecular, expressão, purificação e caracterização estrutural da endoglucanase de Trichoderma harzianum visando o desenvolvimento de coquetéis enzimáticos para a produção de etanol lignocelulósico

O aumento na demanda mundial por energia, a perspectiva de encolhimento dos recursos energéticos e a preocupação global com a questão ambiental, despertaram o interesse por fontes alternativas de energia. A biomassa lignocelulósica é abundante e de baixo custo, com potencial para complementar a produção em larga escala de combustíveis. A degradação das moléculas constituintes da parede celular à açúcares fermentescíveis e então à etanol, ocorre através da hidrólise enzimática da biomassa. Contudo, a utilização de enzimas para esse fim encontra-se em estágio exploratório e representa um gargalo na implementação de tecnologias de etanol 2G em escala industrial, desencadeando a busca de celulases bioquimicamente mais ativas, estáveis e economicamente viáveis. O presente trabalho visou a caracterização da endoglucanase I do fungo Trichoderma harzianum, e para isso foi realizada expressão, ensaios bioquímicos e biofísicos do domínio catalítico (ThCel7B-CCD) e da proteína inteira (ThCel7B-full). A enzima exibiu um perfil acidofílico, com atividade ótima em pH 3,0 a 55°C. A proteína também se mostrou capaz de hidrolisar uma variedade de substratos, sendo a maior atividade hidrolítica em β-glucano (75 U mg-1). Ao analisar a estabilidade térmica medida a 55°C em pH 5, a atividade residual manteve-se intacta por mais de 2 meses. Outra característica relevante foi o elevado grau de sinergismo entre ThCel7B e ThCel7A. Análises de microscopia eletrônica de flocos de aveia submetidas à hidrólise com ThCel7B evidenciaram os efeitos de degradação do substrato em relação às amostras controle. O conjunto desses resultados, além de importante para a compreensão do mecanismo molecular de ThCel7B e de outras endoglucanases da família GH7, também revelou uma enzima de interesse biotecnológicos uma vez que o comportamento ácido e sua estabilidade térmica são características relevantes para aplicações industriais sob condições extremamente ácidas.

Immunization with a circumsporozoite epitope fused to Bordetella pertussis adenylate cyclase in conjunction with cytotoxic T-lymphocyte-associated antigen 4 blockade confers protection against Plasmodium berghei liver-stage malaria

The adenylate cyclase toxoid (ACT) of Bordetella pertussis is capable of delivering its N-terminal catalytic domain into the cytosol of CD11b-expressing professional antigen-presenting cells such as myeloid dendritic cells. This allows delivery of CD8+ T-cell epitopes to the major histocompatibility complex (MHC) class I presentation pathway. Recombinant detoxified ACT containing an epitope of the Plasmodium berghei circumsporozoite protein (CSP), indeed, induced a specific CD8+ T-cell response in immunized mice after a single application, as detected by MHC multimer staining and gamma interferon (IFN-gamma) ELISPOT assay. This CSP-specific response could be significantly enhanced by prime-boost immunization with recombinant ACT in combination with anti-CTLA-4 during the boost immunization. This increased response was accompanied by complete protection in a number of mice after a challenge with P. berghei sporozoites. Transient blockade of CTLA-4 may overcome negative regulation and hence provide a strategy to enhance the efficacy of a vaccine by amplifying the number of responding T cells.

Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane

The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.

The Role of TET Proteins in the Epigenetic Regulation of Neural Gene Expression and Behavior

Understanding how genes affect behavior is critical to develop precise therapies for human behavioral disorders. The ability to investigate the relationship between genes and behavior has been greatly advanced over the last few decades due to progress in gene-targeting technology. Recently, the Tet gene family was discovered and implicated in epigenetic modification of DNA methylation by converting 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). 5hmC and its catalysts, the TET proteins, are highly abundant in the postnatal brain but with unclear functions. To investigate their neural functions, we generated new lines of Tet1 and Tet3 mutant mice using a gene targeting approach. We designed both mutations to cause a frameshift by deleting the largest coding exon of Tet1 (Tet1Δe4) and the catalytic domain of Tet3 (Tet3Δe7-9). As Tet1 is also highly expressed in embryonic stem cells (ESCs), we generated Tet1 homozygous deleted ESCs through sequential targeting to compare the function of Tet1 in the brain to its role in ESCs. To test our hypothesis that TET proteins epigenetically regulate transcription of key neural genes important for normal brain function, we examined transcriptional and epigenetic differences in the Tet1Δe4 mouse brain. The oxytocin receptor (OXTR), a neural gene implicated in social behaviors, is suggested to be epigenetically regulated by an unknown mechanism. Interestingly, several human studies have found associations between OXTR DNA hypermethylation and a wide spectrum of behavioral traits and neuropsychiatric disorders including autism spectrum disorders. Here we report the first evidence for an epigenetic mechanism of Oxtr transcription as expression of Oxtr is reduced in the brains of Tet1Δe4-/- mice. Likewise, the CpG island overlapping the promoter of Oxtr is hypermethylated during early embryonic development and persists into adulthood. We also discovered altered histone modifications at the hypermethylated regions, indicating the loss of TET1 has broad effects on the chromatin structure at Oxtr. Unexpectedly, we discovered an array of novel mRNA isoforms of Oxtr that are selectively reduced in Tet1Δe4-/- mice. Additionally, Tet1Δe4-/- mice display increased agonistic behaviors and impaired maternal care and short-term memory. Our findings support a novel role for TET1 in regulating Oxtr expression by preventing DNA hypermethylation and implicate TET1 in social behaviors, offering novel insight into Oxtr epigenetic regulation and its role in neuropsychiatric disorders.

Dynamic coupling between the LID and NMP domain motions in the catalytic conversion of ATP and AMP to ADP by adenylate kinase

The catalytic conversion of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP) by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of ATP binding domain (LID) and AMP binding domain (NMP) domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motion of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis, and multidimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two-dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed root mean square deviation variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder-type theoretical model to explain the observed dynamic coupling between the motion of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations. (C) 2011 American Institute of Physics. doi:10.1063/1.3516588]