Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

WbpM is essential for the biosynthesis of B-band lipopolysaccharide (LPS) in many serotypes of Pseudomonas aeruginosa. Homologues that can functionally complement a wbpM null mutant and that are also necessary for virulence have been identified in numerous pathogenic bacteria. WbpM and most of its homologues are large membrane proteins, which has long hampered the elucidation of their biochemical function. This paper describes the detailed characterization of WbpM using both in vivo and in vitro approaches. LacZ and PhoA fusion experiments showed that WbpM was anchored to the inner membrane via four N-terminal transmembrane domains, whereas the C-terminal catalytic domain resided in the cytoplasm. Although the membrane domains did not have any catalytic activity, complementation experiments suggested that they were important for the polymerization of high-molecular-weight B-band LPS. The biochemical characterization of a soluble truncated form of WbpM, His-S262, showed that WbpM was a C6 dehydratase specific for UDP-GlcNAc. It exhibited unusual low temperature (25-30 degrees C) and high pH (pH 10) optima. Although WbpM possessed an altered catalytic triad composed of SMK as opposed to SYK commonly found in other dehydratases, its catalysis was very efficient, with a kcat of 168 min(-1) and a kcat/Km of 58 mM(-1) min(-1). These unusual physico-kinetic properties suggested a potentially different mechanism of C6 dehydration for WbpM and its large homologues. His-S262 is now a precious tool for further structure-function studies.
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PMID:Topological and functional characterization of WbpM, an inner membrane UDP-GlcNAc C6 dehydratase essential for lipopolysaccharide biosynthesis in Pseudomonas aeruginosa. 1158 Aug 35

In this paper, an approach is described that combines multiple structure alignments and multiple sequence alignments to generate sequence profiles for protein families. First, multiple sequence alignments are generated from sequences that are closely related to each sequence of known three-dimensional structure. These alignments then are merged through a multiple structure alignment of family members of known structure. The merged alignment is used to generate a Hidden Markov Model for the family in question. The Hidden Markov Model can be used to search for new family members or to improve alignments for distantly related family members that already have been identified. Application of a profile generated for SH2 domains indicates that the Janus family of nonreceptor protein tyrosine kinases contains SH2 domains. This conclusion is strongly supported by the results of secondary structure-prediction programs, threading calculations, and the analysis of comparative models generated for these domains. One of the Janus kinases, human TYK2, has an SH2 domain that contains a histidine instead of the conserved arginine at the key phosphotyrosine-binding position, betaB5. Calculations of the pK(a) values of the betaB5 arginines in a number of SH2 domains and of the betaB5 histidine in a homology model of TYK2 suggest that this histidine is likely to be neutral around pH 7, thus indicating that it may have lost the ability to bind phosphotyrosine. If this indeed is the case, TYK2 may contain a domain with an SH2 fold that has a modified binding specificity.
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PMID:Combining multiple structure and sequence alignments to improve sequence detection and alignment: application to the SH2 domains of Janus kinases. 1175 26

X-linked agammaglobulinemia (XLA) is characterized by a severe B-cell deficiency, resulting from a differentiation arrest in the bone marrow (BM). Because XLA is clinically and immunologically heterogeneous, we investigated whether the B-cell differentiation arrest in BM of XLA patients is heterogeneous as well. First, we analyzed BM samples from 19 healthy children by flow cytometry. This resulted in a normal B-cell differentiation model with eight consecutive stages. Subsequently, we analyzed BM samples from nine XLA patients. Eight patients had amino acid substitutions in the Bruton's tyrosine kinase (BTK) domain or premature stop codons, resulting in the absence of functional BTK proteins. In seven of these eight patients a major differentiation arrest was observed at the transition between cytoplasmic Ig(mu-) pre-B-I cells and cytoplasmic Ig(mu+) pre-B-II cells, consistent with a role for BTK in pre-B-cell receptor signaling. However, one patient exhibited a very early arrest at the transition between pro-B cells and pre-B-I cells, which could not be explained by a different nature of the BTK mutation. We conclude that the absence of functional BTK proteins generally leads to an almost complete arrest of B-cell development at the pre-B-I to pre-B-II transition. The ninth XLA patient had a splice site mutation associated with the presence of low levels of wild-type BTK mRNA. His BM showed an almost normal composition of the precursor B-cell compartment, suggesting that low levels of BTK can rescue the pre-B-cell receptor signaling defect, but do not lead to sufficient numbers of mature B lymphocytes in the peripheral blood.
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PMID:Composition of precursor B-cell compartment in bone marrow from patients with X-linked agammaglobulinemia compared with healthy children. 1180 9

Two subfamilies of UDP-GlcNAc C6 dehydratases were recently identified. FlaA1, a short soluble protein that exhibits a typical SYK catalytic triad, characterizes one of these subfamilies, and WbpM, a large membrane protein that harbors an altered SMK triad that was not predicted to sustain activity, represents the other subfamily. This study focuses on investigating the structure and function of these C6 dehydratases and the role of the altered triad as well as additional amino acid residues involved in catalysis. The significant activity retained by the FlaA1 Y141M triad mutant and the low activity of the WbpM M438Y mutant indicated that the methionine residue was involved in catalysis. A Glu(589) residue, which is conserved only within the large homologues, was shown to be essential for activity in WbpM. Introduction of this residue in FlaA1 enhanced the activity of the corresponding V266E mutant. Hence, this glutamate residue might be responsible for the retention of catalytic efficiency in the large homologues despite alteration of their catalytic triad. Mutations of residues specific for the short homologues (Asp(70), Asp(149)-Lys(150), Cys(103)) abolished the activity of FlaA1. Among them, C103M prevented dimerization but did not significantly affect the secondary structure. The fact that we could identify subfamily-specific residues that are essential for catalysis suggested an independent evolution for each subfamily of C6 dehydratases. Finally, the loss of activity of the FlaA1 G20A mutant provided evidence that a cofactor is involved in catalysis, and kinetic study of the FlaA1 H86A mutant revealed that this conserved histidine is involved in substrate binding. None of the mutations investigated altered the substrate, product, and function specificity of these enzymes.
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PMID:Structure-function studies of two novel UDP-GlcNAc C6 dehydratases/C4 reductases. Variation from the SYK dogma. 1200 63

ETV6, a member of the Ets family of transcription factors, is frequently rearranged to various translocation partners in human leukaemias. We previously described a CD3+/TCRalpha/beta+ mature T-cell acute lymphoblastic leukaemia (T-ALL) cell line, MT-ALL, carrying a t(1;10;12)(q25; p13;p13) with cytokine-inducible lineage switch into the myeloid lineage. Using reverse transcription polymerase chain reaction with primers complementary to ETV6 and ABL2, two ETV6-ABL2 fusion transcripts were identified in MT-ALL which resulted from alternative splicing of an ABL2 exon. The fusion transcripts code for putative ETV6-ABL2 fusion proteins containing the pointed domain of ETV6 and almost the complete ABL2 protein, including the SH2, SH3 domains and the protein tyrosine kinase domain (PTK). Identical ETV6-ABL2 fusion transcripts have been reported in an acute myeloid leukaemia (AML) M3 cell line, carrying both a t(15;17)(q22;q21) and a t(1;12)(q25;p13) with unusual inducible differentiation to eosinophils, and in a patient with AML-M4eo. Interestingly, the non-rearranged allele of ETV6 in the MT-ALL cell line carries an arginine to histidine (R399H) mutation which affects a conserved amino acid in the ets DNA binding domain.
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PMID:Identification of an ETV6-ABL2 fusion transcript in combination with an ETV6 point mutation in a T-cell acute lymphoblastic leukaemia cell line. 1240 85

The novel fusion protein DT(388)IL3, composed of the catalytic and translocation domains of diphtheria toxin (DT(388)) fused with a Met-His linker to human interleukin 3 (IL-3), was tested for anti-leukemia efficacy in an in vivo model of differentiated human acute myeloid leukemia (AML). Six-week-old female SCID mice were irradiated with 350 cGy, inoculated 24 h later with 20 million (i.v., i.p., or s.c.) TF1 cells transfected with the v-SRC oncogene, and treated i.p., starting 24 h later, with up to five daily injections of saline, DT(388)IL3 (2 microg), DT(388)GMCSF (2 microg), DAB(389)IL2 (2 microg), or cytarabine (80 microg) or two weekly injections of anti-CD33-calicheamicin conjugate (5 microg). Animals were monitored twice daily, and moribund animals killed and necropsied. Control animals had a median disease-free survival (DFS) of 37 days (i.v., n = 45), 35 days (i.p., n = 20), and 21 days (s.c., n = 20), respectively. Only 5/49 (10%) of the DT(388)IL3 treated i.v. inoculated animals died with leukemia. Median DFS with i.v., i.p. and s.c. tumor inoculated animals was prolonged by fusion protein treatment to >120 days, 66 days and 31 days (P < 0.001, = 0.0003, and = 0.0006), respectively. Median DFS with s.c. tumor inoculated animals was also prolonged by other active anti-leukemia agents (DT(388)GMCSF, cytarabine and anti-CD33-calicheamicin) relative to controls by 67%, 172% and 47% (P < 0.0001, <0.0001, and =0.0004), respectively. In contrast, median DFS with s.c. tumor inoculated animals treated with DAB(389)IL2 non-significantly reduced by 13% relative to controls (P = 0.21). Thus, DT(388)IL3 fusion protein demonstrates in vivo anti-leukemia efficacy and warrants further preclinical development for treatment of chemo-resistant, IL-3 receptor positive AML patients.
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PMID:Diphtheria toxin-interleukin-3 fusion protein (DT(388)IL3) prolongs disease-free survival of leukemic immunocompromised mice. 1252 73

The cDNA of guinea pig (Cavia porcellus) growth hormone receptor (gpGHR) was cloned using RT-PCR in our laboratory. By sequence alignment, substitutions of amino acids conserved in other mammalian GHRs were found. For example, histidine-168 and tyrosine-332 equivalent to positions 170 and 333 in other mammalian GHRs, which were considered to be necessary for the dimerization of GHR and the specific GH-stimulated functions respectively, were replaced by tyrosine and serine in gpGHR. Here, we report the functional expression of gpGHR and its mutants, gpGHRY168H and gpGHRS332Y, in COS-7 cells and/or Chinese hamster ovary (CHO) cells. It was shown that the COS-7 cells transfected with pcDNA3-gpGHR possessed high affinity to bovine GH [K(a) = 1.3 x10(9) (mol/L)(-1)] and a protein band with molecular weight around 92 kD was detected by anti-mouse GHR monoclonal antibody (mAb263). When CHO cells were transfected with the expression vectors, pcDNA3-gpGHR, pcDNA3-gpGHRY168H and pcDNA3-gpGHRS332Y, the gpGHR and its mutants were expressed and the ligand binding, phosphorylation of JAK2, protein synthesis, and lipogenesis were studied. It was found that the mutation of serine to tyrosine at position 332 greatly increased the GH-stimulated protein synthesis and the phosphorylation of JAK2, while the mutation of tyrosine to histidine at position 168 increased the protein synthesis and decreased the phosphorylation of JAK2 only weakly. However, both mutations decreased the GH-stimulated lipogenesis. Thus, our study provides the experimental evidence that gpGHR may mediate the metabolic actions of GH and the substitutions of some conserved amino acids in gpGHR result in the changes of post-binding signaling.
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PMID:Functional expression of guinea pig growth hormone receptor and its mutants in mammalian cells. 1276 99

Irreversible inhibitors of proteases have proven themselves useful tools for determining which proteases are active under given conditions in tissues or cells and for studying the functional role that a protease plays in physiological processes. The application of such techniques to the study of the activity and function of protein-protein interactions has been hindered by the lack of guiding principles for the mechanistic design of irreversible inhibitors targeting the "active site" of a protein interaction. We report herein the first example of a mechanism-based irreversible inhibitor of a protein interaction that has been specifically targeted to one member of the PDZ family of protein interaction domains: the second PDZ domain of the membrane-associated guanylate kinase MAGI3. This inhibitor was designed using rationally directed computational evaluation to take advantage of a conserved histidine in the PDZ domain by introducing an ionizable group that will be held in close proximity to that nucleophile during binding. The novel compound exhibits all of the characteristics of an irreversible inhibitor of the interaction of the tumor suppressor PTEN with MAGI3 in in vitro models. In cells, the inhibitor can be shown to release PTEN from sequestration by MAGI3 and consequently upregulate the PKB signaling pathway.
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PMID:A selective irreversible inhibitor targeting a PDZ protein interaction domain. 1451 76

Recently, we isolated the Dub-2A gene, which encodes a novel murine deubiquitinating enzyme subfamily member, from a bacterial artificial chromosome library clone by PCR amplification with degenerate PCR primers for the Dub-2 cDNA (Baek, K.-H., Mondoux, M. A., Jaster, R., Fire-Levin E., and D'Andrea, A. D. (2001) Blood 98, 636-642). In this study, we analyzed two more clones from the library to isolate genes encoding other deubiquitinating enzymes. Dub-1A, which encodes the shortest member of the DUB subfamily of deubiquitinating enzymes so far, has been identified in both clones and characterized. Sequence analysis showed that Dub-1A encodes a 468-amino acid protein that has a molecular mass of approximately 51 kDa and that contains a putative catalytic domain (Cys, His, and Asp) conserved among DUB proteins. The amino acid sequence of DUB-1A is 84.5, 84.7, and 85.3% identical to those of DUB-1, DUB-2, and DUB-2A, respectively. Reverse transcription-PCR revealed that Dub-1A is expressed not only in B-lymphocytes in response to interleukin-3 stimulation, but also in T-lymphocytes, brain, heart, liver, lung, kidney, ovary, and spleen. This suggests that Dub-1A may play essential roles in each of these organs. In vivo and in vitro deubiquitinating enzyme assays showed that DUB-1A has functional deubiquitinating activity and that the 5'-flanking sequence of Dub-1A has a functional enhancer domain as shown in Dub-1 and Dub-2A. Interestingly, immunoblot analysis revealed that DUB-1A is polyubiquitinated, indicating that it is degraded through proteasome-mediated degradation. In the absence of JAK2, Dub-1A was expressed at a lower level. This suggests that DUB-1A functions downstream of JAK2 kinase in the interleukin-3 signaling pathway.
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PMID:DUB-1A, a novel deubiquitinating enzyme subfamily member, is polyubiquitinated and cytokine-inducible in B-lymphocytes. 1458 20

We report a 19-year-old man with Ollier's disease with multiple orthopedic procedures performed for leg length discrepancy; who developed chronic myeloid leukemia presenting with intramuscular hematoma. His symptoms resolved with cytoreductive treatment by hydroxyurea. Cytogenetic and molecular investigations showed a complex Philadelphia translocation t(9;22;13) (q34;q11.2;q12), with predominance of ela2 BCR/ABL splicing and deletion of reciprocal der(9) ABL/BCR locus, all suggesting poor prognosis. The cumulative X-ray exposure from repeated operations from the age of 7 to 12 years was estimated to be around 16 mSv, approximately the dose of 720 chest X rays. Literature review showed two other cases of leukemia occurring in patients with multiple enchondromatosis. Although the development of CML in this young patient might be related partly to genetic defects, the repeated radiation exposure, especially at young age and directly on the marrow tissue in the long bones, might also be an important pathogenetic factor.
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PMID:Chronic myeloid leukemia in an adolescent with Ollier's disease after intensive X-ray exposure. 1516 Sep 27


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