Biomedical subjects

W J Henzel

Publications and source records attributed to W J Henzel.

At least 37 records · Page 2Linked to original sources

PubMed1996

Mouse RAD50 has limited epitopic homology to p53 and is expressed in the adult myocardium.

Previous studies have identified a 180-kDa mouse cardiomyocyte phosphoprotein with limited epitopic homology to p53. In this study, the protein was purified and partially sequenced. Oligonucleotide probes based on the available amino acid sequence data were used to isolate cDNA clones. Sequence analyses revealed that the clones encoded a protein with regional homology to the yeast RAD50 gene product. Expression of the mouse cDNA rescued the methyl methanesulfonate-sensitive phenotype in rad50 mutant yeast, indicating that the cardiomyocyte phosphoprotein is the mammalian homologue of the yeast RAD50 gene product. Fluorescence in situ hybridization analyses localized the mouse RAD50 gene to the A5-B1 region of chromosome 11. Northern blot analyses demonstrated a complex pattern of RAD50 expression during mouse development which was further complicated by the presence of several alternatively spliced transcripts. High levels of RAD50 expression was evident in the adult myocardium, a somewhat surprising observation given the absence of DNA synthesis in adult cardiomyocytes.

3T3 Cells↗

PubMed1996

IRAK: a kinase associated with the interleukin-1 receptor.

The pleiotropic biological activities of interleukin-1 (IL-1) are mediated by its type I receptor (IL-1RI). When the ligand binds, IL-1RI initiates a signaling cascade that results in the activation of the transcription regulator nuclear factor kappa B (NF-kappa B). A protein kinase designated IRAK (IL-1 receptor-associated kinase) was purified, and its complementary DNA was molecularly cloned. When human embryonic kidney cells (cell line 293) over-expressing IL-1RI or HeLa cells were exposed to IL-1, IRAK rapidly associated with the IL-1RI complex and was phosphorylated. The primary amino acid sequence of IRAK shares similarity with that of Pelle, a protein kinase that is essential for the activation of a NF-kappa B homolog in Drosophila.

Amino Acid Sequence↗

PubMed1995

The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins.

The 75 kDa tumor necrosis factor receptor (TNFR2) transduces extracellular signals via receptor-associated cytoplasmic proteins. Two of these signal transducers, TRAF1 and TRAF2, were isolated and characterized previously. We report here the biochemical purification and subsequent molecular cloning of two novel TNFR2-associated proteins, designated c-IAP1 and c-IAP2, that are closely related mammalian members of the inhibitor of apoptosis protein (IAP) family originally identified in baculoviruses. The viral and cellular IAPs contain N-terminal baculovirus IAP repeat (BIR) motifs and a C-terminal RING finger. The c-IAPs do not directly contact TNFR2, but rather associate with TRAF1 and TRAF2 through their N-terminal BIR motif-comprising domain. The recruitment of c-IAP1 or c-IAP2 to the TNFR2 signaling complex requires a TRAF2-TRAF1 heterocomplex.

Animals↗

PubMed1995

Cloning, sequencing, and expression of a 24-kDa Ca(2+)-binding protein activating photoreceptor guanylyl cyclase.

Two vertebrate photoreceptor-specific membrane guanylyl cyclases, RetGC-1 and RetGC-2, are activated by a soluble 24-kDa retinal protein, p24, in a Ca(2+)-sensitive manner (Dizhoor, A.M., Lowe, D.G., Olshevskaya, E.V., Laura, R.P., and Hurley, J.B. (1994) Neuron 12, 1345-1352; Lowe, D.G., Dizhoor, A.M., Liu, K., Gu, O., Laura, R., Lu, L., and Hurley, J.B. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5535-5539). The primary structure of bovine p24 has been derived from peptide sequencing and from its cDNA. p24 is a new EF-hand-type Ca(2+)-binding protein, related but not identical to another guanylyl cyclase-activating protein, GCAP (Palczewski, K., Subbaraya, I., Gorczyca, W.A., Helekar, B.S., Ruiz, C.C., Ohguro, H. Huang, J., Zhao, X., Crabb, J.W., Johnson, R.S., Walsh, K.A., Gray-Keller, M.P., Detwiler, P.B., and Baehr, W. (1994) Neuron 13, 395-404) and other members of the recovering family of Ca(2+)-binding proteins. Antibodies against a truncated fusion protein and against a p24-specific synthetic peptide specifically recognize retinal p24 on immunoblot. Both antibodies inhibit activation of photoreceptor membrane guanylyl cyclase by purified p24. p24 is found only in retina, and it copurifies with outer segment membranes. Immunocytochemical analysis shows that it is present in rod photoreceptor cells. An immobilized antibody column was used to purify p24 from a heat-treated retinal extract. Purified p24 appears on SDS-polyacrylamide gel electrophoresis as a homogeneous protein not contaminated with GCAP, and it activates photoreceptor guanylyl cyclase in vitro at submicromolar concentrations. Ca2+ inhibits this activation with an EC50 near 200 nM and a Hill coefficient of 1.7. Recombinant p24 expressed in 293 cells effectively stimulates photoreceptor guanylyl cyclase. These findings demonstrate that p24, like GCAP, imparts Ca2+ sensitivity to photoreceptor membrane guanylyl cyclase. We propose that p24 be referred to as GCAP-2 and that GCAP be referred to as GCAP-1.

Amino Acid Sequence↗

PubMed1995

Distinct regions of c-Mpl cytoplasmic domain are coupled to the JAK-STAT signal transduction pathway and Shc phosphorylation.

c-Mpl, a member of the hematopoietic cytokine receptor family, is the receptor for thrombopoietin. To investigate signal transduction by c-Mpl, a chimeric receptor, composed of the extracellular domain of human growth hormone receptor and the intracellular domain of c-Mpl, was introduced into the interleukin 3-dependent cell line Ba/F3. In response to growth hormone, this chimeric receptor induced growth in the absence of interleukin 3. Deletion analysis of the 123-amino acid intracellular domain indicated that the elements responsible for this effect are present within the 63 amino acids proximal to the transmembrane domain. Mutation of the recently described box 1 motif abrogated the proliferative response. Tyrosine phosphorylation of the tyrosine kinase JAK-2 and activation of STAT proteins were dependent on box 1 and sequences within 63 amino acids of the plasma membrane. STAT proteins activated by thrombopoietin in a megakaryocytic cell line were purified and shown to be STAT1 and STAT3. A separate region located at the C terminus of the c-Mpl intracellular domain was found to be required for induction of Shc phosphorylation and c-fos mRNA accumulation, suggesting involvement of the Ras signal transduction pathway. Thus, at least two distinct regions are involved in signal transduction by the c-Mpl.

Amino Acid Sequence↗

PubMed1995

Cloning of AL-1, a ligand for an Eph-related tyrosine kinase receptor involved in axon bundle formation.

REK7 is an Eph-related tyrosine kinase receptor expressed exclusively in the nervous system, predominantly in hippocampus and cortex. A soluble REK7-IgG fusion protein, produced to analyze the biological role of REK7, prevents axon bundling in cocultures of cortical neurons with astrocytes, a model of late stage nervous system development and differentiation. Using REK7-IgG as an affinity reagent, we purified and cloned a novel REK7 ligand called AL-1, a GPI-linked protein homologous to other members of an emerging ligand family. Membrane attachment of AL-1 appears necessary for receptor activation, since REK7 on cortical neurons is efficiently activated by transfected cells expressing GPI-linked AL-1, but not by soluble AL-1. Consistent with this, soluble AL-1 blocks axon bundling. Our findings, together with the observation that both molecules are expressed in the brain, suggest a role in the formation of neuronal pathways, a crucial feature of nervous system development and regeneration.

Amino Acid Sequence↗

PubMed1995

Identification and purification of human Stat proteins activated in response to interleukin-2.

A key cytokine induced during the immune response is IL-2. Following T cell activation, the genes encoding IL-2 and the various chains of its receptor are transcriptionally induced. In turn, secreted IL-2 serves to stimulate the proliferation and differentiation of T lymphocytes. Several recent studies have implicated Jak kinases in the signaling pathway induced by IL-2. Following this lead, we set out to identify transcription factors induced in response to IL-2. Human peripheral blood lymphocytes were observed to contain several IL-2-inducible DNA binding activities. Similar activities were also observed in a transformed human lymphocyte line, termed YT. We have purified these activities and found that the principal IL-2-inducible component bears significant relatedness to a prolactin-induced transcription factor first identified in sheep mammary gland tissue. We hypothesize that activation of this protein, designated hStat5, helps govern the biological effects of IL-2 during the immune response.

Amino Acid Sequence↗

PubMed1994

An interleukin-4-induced transcription factor: IL-4 Stat.

Interleukin-4 (IL-4) is an immunomodulatory cytokine secreted by activated T lymphocytes, basophils, and mast cells. It plays an important role in modulating the balance of T helper (Th) cell subsets, favoring expansion of the Th2 lineage relative to Th1. Imbalance of these T lymphocyte subsets has been implicated in immunological diseases including allergy, inflammation, and autoimmune disease. IL-4 may mediate its biological effects, at least in part, by activating a tyrosine-phosphorylated DNA binding protein. This protein has now been purified and its encoding gene cloned. Examination of the primary amino acid sequence of this protein indicates that it is a member of the signal transducers and activators of transcription (Stat) family of DNA binding proteins, hereby designated IL-4 Stat. Study of the inhibitory activities of phosphotyrosine-containing peptides derived from the intracellular domain of the IL-4 receptor provided evidence for direct coupling of receptor and transcription factor during the IL-4 Stat activation cycle. Such observations indicate that IL-4 Stat has the same functional domain for both receptor coupling and dimerization.

Amino Acid Sequence↗

PubMed1994

A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor.

Mutational analysis identified a C-terminal region of 78 amino acids within the cytoplasmic domain of the human 75 kDa tumor necrosis factor receptor (TNF-R2) that is required for signal transduction. This region was subsequently shown to mediate the interaction of cytoplasmic factors with TNF-R2. Two of these factors were isolated and molecularly cloned using biochemical purification and the yeast two-hybrid system. TNF receptor-associated factor 1 (TRAF1) and TRAF2 are the first two members of a novel protein family containing a novel C-terminal homology region, the TRAF domain. In addition, TRAF2 contains an N-terminal RING finger motif. TRAF1 and TRAF2 can form homo- and heterotypic dimers. Our analysis indicates that TRAF1 and TRAF2 are associated with the cytoplasmic domain of TNF-R2 in a heterodimeric complex in which TRAF2 contacts the receptor directly. TRAF1 interacts with TNF-R2 indirectly through heterodimer formation with TRAF2.

Amino Acid Sequence↗

PubMed1994

Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand.

Physiological platelet synthesis is thought to require the humoral activities of meg-CSF and thrombopoietin, which respectively promote proliferation and maturation of megakaryocytic cells. A meg-CSF/thrombopoietin-like protein that is present in plasma of irradiated pigs has been purified and cloned. This protein binds to and activates the c-mpl protein, a member of the cytokine receptor superfamily. The isolated Mpl ligand shares homology with erythropoietin and stimulates both megakaryocytopoiesis and thrombopoiesis.

Amino Acid Sequence↗

PubMed1994

3-Carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa: an alternate cycloisomerase motif.

3-Carboxy-cis,cis-muconate lactonizing enzyme (CMLE; EC 5.5.1.5) from Neurospora crassa catalyzes the reversible gamma-lactonization of 3-carboxy-cis,cis-muconate by a syn-1,2 addition-elimination reaction. The stereochemical and regiochemical course of the reaction is (i) opposite that of CMLE from Pseudomonas putida (EC 5.5.1.2) and (ii) identical to that of cis,cis-muconate lactonizing enzyme (MLE; EC 5.5.1.1) from P. putida. In order to determine the mechanistic and evolutionary relationships between N. crassa CMLE and the procaryotic cycloisomerases, we have purified CMLE from N. crassa to homogeneity and determined its nucleotide sequence from a cDNA clone isolated from a p-hydroxybenzoate-induced N. crassa cDNA library. The deduced amino acid sequence predicts a protein of 41.2 kDa (365 residues) which does not exhibit sequence similarity with any of the bacterial cycloisomerases. The cDNA encoding N. crassa CMLE was expressed in Escherichia coli, and the purified recombinant protein exhibits physical and kinetic properties equivalent to those found for the isolated N. crassa enzyme. We also report that N. crassa CMLE possesses substantially reduced yet significant levels of MLE activity with cis,cis-muconate and, furthermore, does not appear to be dependent on divalent metals for activity. These data suggest that the N. crassa CMLE may represent a novel eucaryotic motif in the cycloisomerase enzyme family.

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of an intracellular serine proteinase inhibitor from a monkey kidney epithelial cell line.

A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.

Amino Acid Sequence↗

PubMed1993

Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases.

A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of two-dimensional gel spots. The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity.

Amino Acid Sequence↗

PubMed1993

In vitro aging of calmodulin generates isoaspartate at multiple Asn-Gly and Asp-Gly sites in calcium-binding domains II, III, and IV.

We have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein L-isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyl-3H from S-adenosyl-L-[methyl-3H]methionine to the isoaspartyl (alpha-carboxyl) side chain. More than 1.2 mol of methyl-acceptor sites per mol of calmodulin accumulated during a 2-week incubation without calcium at pH 7.4, 37 degrees C. Analysis of proteolytic peptides of aged calmodulin revealed that > 95% of the methylation capacity is restricted to residues in the four calcium-binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains III, IV, and II accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn-97-Gly-98 sequence (domain III) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp-131-Gly-132 and Asp-133-Gly-134 in domain IV, and Asn-60-Gly-61 in domain II. Significant isoaspartate formation was also localized to Asp-20, Asp-22, and/or Asp-24 in domain I, to Asp-56 and/or Asp-58 in domain II, and to Asp-93 and/or Asp-95 in domain III. All of these residues are calcium ligands in the highly conserved EF-hand calcium-binding motif. Thus, other EF-hand proteins may also be subject to isoaspartate formation at these ligands. The results support the idea that isoaspartate formation in structured proteins is strongly influenced by both the C-flanking residue and by local flexibility.

Amino Acid Sequence↗

PubMed1992

Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA.

Methylation of mammalian DNA can lead to repression of transcription and alteration of chromatin structure. Recent evidence suggests that both effects are the result of an interaction between the methylated sites and methyl-CpG-binding proteins (MeCPs). MeCP1 has previously been detected in crude nuclear extracts. Here we report the identification, purification, and cDNA cloning of a novel MeCP called MeCP2. Unlike MeCP1, the new protein is able to bind to DNA that contains a single methyl-CpG pair. By staining with an antibody, we show that the distribution of MeCP2 along the chromosomes parallels that of methyl-CpG. In mouse, for example, MeCP2 is concentrated in pericentromeric heterochromatin, which contains a large fraction (about 40%) of all genomic 5-methylcytosine.

Amino Acid Sequence↗

PubMed1992

An endothelial ligand for L-selectin is a novel mucin-like molecule.

The adhesive interaction between circulating lymphocytes and the high endothelial venules (HEV) of lymph nodes (LN) is mediated by lymphocyte L-selectin, a member of the selectin family of cell adhesion proteins. Previous work has identified a sulfated 50 kd glycoprotein (Sgp50) as an HEV ligand for L-selectin. We now report the purification of this glycoprotein and the utilization of the derived N-terminal amino acid sequence to clone a cDNA. The predicted sequence reveals a novel, mucin-like molecule containing two serine/threonine-rich domains. The mRNA encoding this glycoprotein is preferentially expressed in LN. Antibodies against predicted peptides immunoprecipitate Sgp50 and stain the apical surface of LN HEV. These results thus define a tissue-specific mucin-like endothelial glycoprotein that appears to function as a scaffold that presents carbohydrates to the L-selectin lectin domain.

Amino Acid Sequence↗

PubMed1992

Identification of heregulin, a specific activator of p185erbB2.

The proto-oncogene designated erbB2 or HER2 encodes a 185-kilodalton transmembrane tyrosine kinase (p185erbB2), whose overexpression has been correlated with a poor prognosis in several human malignancies. A 45-kilodalton protein heregulin-alpha (HRG-alpha) that specifically induced phosphorylation of p185erbB2 was purified from the conditioned medium of a human breast tumor cell line. Several complementary DNA clones encoding related HRGs were identified, all of which are similar to proteins in the epidermal growth factor family. Scatchard analysis of the binding of recombinant HRG to a breast tumor cell line expressing p185erbB2 showed a single high affinity binding site [dissociation constant (Kd) = 105 +/- 15 picomolar]. Heregulin transcripts were identified in several normal tissues and cancer cell lines. The HRGs may represent the natural ligands for p185erbB2.

Amino Acid Sequence↗

PubMed1992

Identity of a differentiation inhibiting factor for mouse myeloid leukemia cells with NM23/nucleoside diphosphate kinase.

Mouse myeloid leukemic line M1 cells can be induced to differentiate into the monocyte/macrophage pathway by various inducers. The induction of differentiation of M1 cells can be inhibited by protein inhibitors termed differentiation inhibiting factors (I-factors) in a cell lysate and conditioned medium of differentiation resistant M1 cells. Production of the I-factor activity in resistant M1 cells is well associated with development of resistance of M1 cells to differentiation inducers. We have now purified one of the I-factors from conditioned medium of differentiation resistant M1 cells. The purified I-factor has a relative molecular mass of approximately 16000-17000 Da (16K I-factor). The amino acid sequence of all fragments of the 16K I-factor we have found are identical with Nm23/nucleoside diphosphate kinase (EC2.7.4.6) protein involved in tumor metastasis. The findings indicate that the I-factor, a candidate suppressor protein for differentiation of leukemic cells, is Nm23/nucleoside diphosphate kinase protein.

Amino Acid Sequence↗