Requirement of GM2 ganglioside activator for phospholipase D activation

Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known GM2 ganglioside activator whose deficiency results in the AB-variant of GM2 gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of GM2 to GM3 ganglioside that is catalyzed by beta-hexosaminidase A. Inversely, GM2 ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987) Methods Enzymol. 138, 792-815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown.     

Dopamine quinone formation and protein modification associated with the striatal neurotoxicity of methamphetamine: evidence against a role for extracellular dopamine

Methamphetamine-induced toxicity has been shown to require striatal dopamine and to involve mechanisms associated with oxidative stress. Dopamine is a reactive molecule that can oxidize to form free radicals and reactive quinones. Although this has been suggested to contribute to the mechanism of toxicity, the oxidation of dopamine has never been directly measured after methamphetamine exposure. In this study we sought to determine whether methamphetamine-induced toxicity is associated with the oxidation of dopamine by measuring the binding of dopamine quinones to cysteinyl residues on protein. We observed that administration of neurotoxic doses of methamphetamine to rats resulted in a two- to threefold increase in protein cysteinyl-dopamine in the striatum 2, 4, and 8 hr after treatment. When methamphetamine was administered at an ambient temperature of 5 degreesC, no increase in dopamine oxidation products was observed, and toxicity was prevented. Furthermore, as shown by striatal microdialysis, animals treated with methamphetamine at 5 degreesC showed DA release identical to that of animals treated at room temperature. These data suggest that the toxicity of methamphetamine and the associated increase in dopamine oxidation are not exclusively the result of increases in extracellular dopamine. Because dopamine-induced modifications of protein structure and function may result in cellular toxicity, it is likely that dopamine oxidation contributes to methamphetamine-induced toxicity to dopamine terminals, adding support to the role of dopamine and the evidence of oxidative stress in this lesion model.     

Versican V2 is a major extracellular matrix component of the mature bovine brain

We have isolated and characterized the proteoglycan isoforms of versican from bovine brain extracts. Our approach included (i) cDNA cloning and sequencing of the entire open reading frame encoding the bovine versican splice variants; (ii) preparation of antibodies against bovine versican using recombinant core protein fragments and synthetic peptides; (iii) isolation of versican isoforms by ammonium sulfate precipitation followed by anion exchange and hyaluronan affinity chromatography; and (iv) characterization by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining or immunoblotting. Our results demonstrate that versican V2 is, together with brevican, a major component of the mature brain extracellular matrix. Versicans V0 and V1 are only present in relatively small amounts. Versican V2 migrates after chondroitinase ABC digestion with an apparent molecular mass of about 400 kDa, whereas it barely enters a 4-15% polyacrylamide gel without the enzyme treatment. The 400-kDa product is recognized by antibodies against the glycosaminoglycan-alpha domain and against synthetic NH2- and COOH-terminal peptides. Our preparations contain no major proteolytic products of versican, e.g. hyaluronectin or glial hyaluronate-binding protein. Having biochemical quantities of versican V2 available will allow us to test its putative modulatory role in neuronal cell adhesion and axonal growth.     

Two mechanisms in the action of repressor deltaEF1: binding site competition with an activator and active repression

BACKGROUND: Counteraction between activators and repressors is crucial for the regulation of a number of cell-specific enhancers, where an activator and a repressor are mutually competitive in binding to the same site. DeltaEF1 is a repressor protein of delta1-crystallin minimal enhancer DC5 binding at the CACCT site, and inhibits activator deltaEF3 from binding to the overlapped site. It has two zinc finger clusters N-fin and C-fin, close to N- and C-termini, respectively, and a homeodomain in the middle. deltaEF1 also binds to the E2-box sequence CACCTG, and represses E2-box-dependent enhancers. RESULTS: The mechanism of the repressor action of deltaEF1 was investigated by examining various deletion mutants of deltaEF1 for their activity to repress delta1-crystallin enhancer fragment HN which contained DC5 sequence and an additional activator site. Both zinc finger clusters were found to be essential for DNA binding and repression, but the homeodomain was not. In addition, the NR domain close to the N-terminus was required for full repression. The NR domain showed active repression when fused to the Gal4 DNA binding domain. Active repression by deltaEF1, dependent on the NR domain, was also demonstrated in a situation where the binding sites of deltaEF1 and deltaEF3 were separated. N-fin and C-fin in their isolated forms bind the 5'-(T/C)ACCTG-3' and 5'-(t/C)ACCT-3' sequences, respectively, while the homeodomain showed no DNA binding activity. An analysis of DNA binding of the delta(Int)F form, having both N-fin and C-fin, indicated that a single DNA binding domain is assembled from two zinc finger clusters. CONCLUSION: Two mechanisms are involved in the repressor action of deltaEF1. First, a binding site competition with an activator which depends on the integrity of both zinc finger clusters, and second, an active repression to silence an enhancer which is attributed to the NR domain.     

Cloning of a human multispanning membrane protein cDNA: evidence for a new protein family

We report the cloning of a human cDNA encoding a protein of calculated 68.8 kDa molecular mass, named hMP70. The deduced protein sequence shows a large N-terminal hydrophilic part and a C-terminal part with nine putative hydrophobic regions characteristic of integral transmembrane domains. Computer searches with sequence databases revealed homologies with three complete yeast proteins and with at least 19 human, 10 plant and one nematode short unidentified protein sequences translated from Expressed Sequence Tags (ESTs). Remarkably, this hMP70 protein retains between 27 and 31% overall sequence identity with the yeast proteins. We propose that hMP70 and related genes have evolved from a common ancestral gene and form a new multispanning membrane protein family which we call the MP70 protein family. Gene expression of hMP70 appears to be ubiquitous, as the mRNA is detectable in all human tissues analysed so far, as shown by Northern blot analysis. Furthermore, a protein of about 70 kDa is detectable in different mammalian cell lines, as shown by immunoblot analysis. From its widespread expression and conservation from yeast, plants to mammals, it is likely that hMP70 has a fundamental biological function in the cell.     

Solution structure of the DNA-binding domain of a human papillomavirus E2 protein: evidence for flexible DNA-binding regions

The three-dimensional structure of the DNA-binding domain of the E2 protein from human papillomavirus-31 was determined by using multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. A total of 1429 NMR-derived distance and dihedral angle restraints were obtained for each of the 83-residue subunits of this symmetric dimer. The average root mean square deviations of 20 structures calculated using a distance geometry-simulated annealing protocol are 0.59 and 0.90 angstroms for the backbone and all heavy atoms, respectively, for residues 2-83. The structure of the human virus protein free in solution consists of an eight-stranded beta-barrel and two pairs of alpha-helices. Although the overall fold of the protein is similar to the crystal structure of the bovine papillomavirus-1 E2 protein when complexed to DNA, several small but interesting differences were observed between these two structures at the subunit interface. In addition, a beta-hairpin that contacts DNA in the crystal structure of the protein-DNA complex is disordered in the NMR structures, and steady-state 1H-15N heteronuclear NOE measurements indicate that this region is highly mobile in the absence of DNA. The recognition helix also appears to be flexible, as evidenced by fast amide exchange rates. This phenomenon has also been observed for a number of other DNA-binding proteins and may constitute a common theme in protein/DNA recognition.     

Purification and characterization of a novel protein activator of Ca2+/calmodulin-dependent protein kinase I

A protein activator of Ca2+/calmodulin (CaM)-dependent protein kinase I was purified from rat brain. The activator was retained on a CaM-Sepharose column in the presence of Ca2+ and kinase assay of renatured gel revealed the 64 kDa molecule in the purified activator fraction to be autophosphorylated and to phosphorylate recombinant CaM kinase I in the presence of Ca2+/calmodulin. These results suggest that this activator of CaM kinase I is also a CaM-dependent protein kinase. Phosphorylation of CaM kinase I by the activator resulted in drastic potentiation of its CaM-dependent activity. Furthermore, kinetic analyses demonstrated that the activation decreases the Km values of CaM kinase I for both ATP and syntide-2 without a change in Vmax values. Considering the quite low enzymatic activity of recombinant CaM kinase I without activation, the 64 kDa species might be essential for CaM kinase I function in vivo.     

Age-related changes in adaptive mechanisms of macronutrient self-selection: evidence for a sexual dimorphism

A novel binding site for angiotensin II has been identified in certain murine tissues. This site, denoted ATm, is distinct from both the AT1 and AT2 sites, as well as the various atypical sites that have been described. The site has a low affinity for angiotensin II (100 nM) and is not blocked by losartan, PD123177 or saralasin. The site shows structural specificity for angiotensin II since both angiotensin III and angiotensin II (1-7) failed to inhibit the binding. Numerous standard drugs, including various receptor blockers, enzyme inhibitors and therapeutic agents, showed no affinity for the site. In murine tissues the site is associated with active tissue generation, as found in spleen, placenta and growing tumors, but its occurrence appears to be rare outside of the mouse.     

Two distinct mechanisms for differential positioning of gene expression borders involving the Drosophila gap protein giant

We have combined genetic experiments and a targeted misexpression approach to examine the role of the gap gene giant (gt) in patterning anterior regions of the Drosophila embryo. Our results suggest that gt functions in the repression of three target genes, the gap genes Krüppel (Kr) and hunchback (hb), and the pair-rule gene even-skipped (eve). The anterior border of Kr, which lies 4-5 nucleus diameters posterior to nuclei that express gt mRNA, is set by a threshold repression mechanism involving very low levels of gt protein. In contrast, gt activity is required, but not sufficient for formation of the anterior border of eve stripe 2, which lies adjacent to nuclei that express gt mRNA. We propose that gt's role in forming this border is to potentiate repressive interaction(s) mediated by other factor(s) that are also localized to anterior regions of the early embryo. Finally, gt is required for repression of zygotic hb expression in more anterior regions of the embryo. The differential responses of these target genes to gt repression are critical for the correct positioning and maintenance of segmentation stripes, and normal anterior development.     

C-reactive protein: a physiological activator of interleukin 6 receptor shedding

The soluble interleukin 6 receptor (sIL-6R) circulates at elevated levels in various diseases. This suggests that inflammatory mediators control sIL-6R release. Through examination of human neutrophils, it was found that the acute phase reactant C-reactive protein (CRP) activates a threefold increase in sIL-6R production. Maximal release occurred after 30-60 min exposure to CRP (50 micrograms/ml), and was mimicked by peptides corresponding to amino acid residues 174- 185 and 201-206 of native CRP. A third peptide fragment (77-82) had no effect. Differential mRNA splicing did not account for the CRP-mediated release of sIL-6R, since this isoform was not detected in conditioned media. Furthermore, stimulation of neutrophils with CRP or with peptides 174-185 or 201-206 promoted a loss of membrane-bound IL-6R, suggesting release by proteolytic shedding. The metalloprotease inhibitor TAPI had only a marginal effect on CRP-mediated sIL-6R release, suggesting that shedding occurs via a mechanism distinct from that previously reported. It well established that IL-6 stimulates the acute phase expression of CRP. Our current findings demonstrate a novel relationship between these two mediators, since CRP may affect IL-6-mediated inflammatory events by enabling formation of the sIL-6R/IL-6 complex.     

HRX leukemic fusion proteins form a heterocomplex with the leukemia-associated protein SET and protein phosphatase 2A

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene at chromosome locus 11q23, resulting in HRX fusion proteins. Using the yeast two-hybrid system, in vitro binding studies, and human cell culture coimmunoprecipitation experiments, we show here that a region of the HRX protein that is consistently retained in HRX leukemic fusion proteins interacts directly with SET, another protein implicated in leukemia. We have identified the binding sites on HRX for SET and show that these sequences are clustered near the A.T hooks that have been shown to bind DNA. We also show that carboxyl-terminal SET sequences, possibly the acidic tail of SET, bind to HRX. We have also found serine/threonine-specific protein phosphatase activity in anti-HRX coimmunoprecipitates. Using the phosphatase inhibitor okadaic acid and Western blotting, the phosphatase was identified as protein phosphatase 2A (PP2A). Mutation of a single amino acid in one of the SET binding sites of HRX resulted in lower amounts of both coimmunoprecipitated SET protein and coimmunoprecipitated PP2A. These results suggest that the leukemogenic effects of HRX fusion proteins may be related to interactions with SET and PP2A.     

Molecular cloning of the cDNA for a human amyloid precursor protein homolog: evidence for a multigene family

Alzheimer's disease is a degenerative neurological disorder characterized by neural loss and brain lesions associated with plaques containing large amounts of the beta/A4 amyloid peptide. Molecular cloning of the cDNA for this peptide from human brain has shown it to be derived by proteolysis from a much larger precursor called the amyloid precursor protein (APP). The biological role of the precursor is unknown, but it has been shown to be transcribed in many human tissues in addition to brain. In the present report, we describe the molecular cloning from a human placental library of a full-length cDNA for a molecule closely related to APP. This novel molecule, which we have called amyloid precursor protein homolog (APPH), shares overall domain organization with APP. It is 763 amino acids in length and appears to encode a signal peptide, a large apparent extracellular domain including a Kunitz inhibitor domain, a transmembrane region, and a short cytoplasmic domain. Northern analysis indicates that it occurs in at least two molecular forms and is transcribed in human brain, heart, lung, liver, and kidney, in addition to placenta. On the basis of its extensive sequence similarity and conservation of domain structure, APPH is the nearest relative of APP yet identified in an emerging multigene family.     

Enhanced glycosylation and sulfation of secretory proteoglycans is coupled to the expression of a basic secretory protein

BACKGROUND: Because the prognosis of patients with hepatocellular carcinoma is not fully understood, particularly regarding therapy, we have evaluated it in a series of patients with a homogeneous diagnostic and therapeutic work-up. METHODS: From 1985 to 1996, 42 variables were recorded prospectively in 178 constructive patients who had a diagnosis of hepatocellular carcinoma. Treatment consisted of liver transplantation ( n = 22), partial hepatectomy (n = 11), arterial, chemoembolization ( n = 52), systemic or regional chemotherapy (n = 51), and other therapies (n = 5); 37 patients received no specific therapy. Statistical analysis was performed according to a Cox model. RESULTS: There were no differences between the survival of patients receiving chemotherapy, other therapies, or no treatment (control group n = 93). survival rates a 1,3, and 5 years were 81%, 74%, and 74% for liver transplantation, 72%, 58%, and 58% for hepatectomy, 55%, 26%, and 13% for chemoembolization, and 13%, 3%, and 0% for the control group. Cirrhosis, systemic syndrome, bilobar involvement, Child's stage C disease, and treatment were independent predictors of survival. CONCLUSIONS: This series shows that certain easily accessible parameters may help establish individual prognosis and stratify patients in clinical trials and indicates that chemoembolization, partial resection, and liver transplantation can prolong life expectancy of patients with hepatocellular carcinoma.     

Two signaling mechanisms for activation of alphaM beta2 avidity in polymorphonuclear neutrophils

Circulating polymorphonuclear neutrophils (PMN) are quiescent, nonadherent cells that rapidly activate at sites of inflammation, where they develop the capacity to perform a repertoire of functions that are essential for host defense. Induction of integrin-mediated adhesion, which requires an increase in integrin avidity, is critical for the development of these effector functions. Although a variety of stimuli can activate integrins in PMN, the signaling cascades involved are unclear. Phosphatidylinositol (PI) 3-kinase has been implicated in integrin activation in a variety of cells, including PMN. In this work, we have examined activation of the PMN integrin alphaM beta2, assessing both adhesion and generation of the epitope recognized by the activation-specific antibody CBRM1/5. We have found that PI 3-kinase has a role in activation of alphaM beta2 by immune complexes, but we have found no role for it in alphaM beta2 activation by ligands for trimeric G protein-coupled receptors, including formylmethionylleucylphenylalanine (fMLP), interleukin-8, and C5a. Cytochalasin D inhibition suggests a role for the actin cytoskeleton in immune complex activation of alphaM beta2, but cytochalasin has no effect on fMLP-induced activation. Similarly, immune complex activation of the Rac/Cdc42-dependent serine/threonine kinase Pak1 is blocked by PI 3-kinase inhibitors, but fMLP-induced activation is not. These results demonstrate that two signaling pathways exist in PMN for activation of alphaM beta2. One, induced by FcgammaR ligation, is PI 3-kinase-dependent and requires the actin cytoskeleton. The second, initiated by G protein-linked receptors, is PI 3-kinase-independent and cytochalasin-insensitive. Pak1 may be in a final common pathway leading to activation of alphaM beta2.     

Crystal structure of the extracellular domain from P0, the major structural protein of peripheral nerve myelin

P0, the major protein of peripheral nerve myelin, mediates membrane adhesion in the spiral wraps of the myelin sheath. We have determined the crystal structure of the extracellular domain from P0 (P0ex) at 1.9 A resolution. P0ex is folded like a typical immunoglobulin variable-like domain; five residues at the C-terminus are disordered, suggesting a flexible linkage to the membrane. The requirements for crystallization of P0ex are similar to those for maintaining the native extracellular spacing of adjacent myelin lamellae; thus, given the self-adhesive character of P0ex, the crystal itself may reveal some of the natural interactions that occur between P0 molecules in myelin. The structure leads to the suggestion that P0 extracellular domains may emanate from the membrane surface as tetramers that link to tetramers on the opposing membrane surface, to result in the formation of networks of molecules. We report analytical ultracentrifugation data for P0ex that support this idea.     

Molecular and functional properties of a calpain activator protein specific for mu-isoforms

A natural calpain activator protein has been isolated from bovine brain and characterized in its properties and molecular structure. The protein is a homodimer with a molecular mass of about 30 kDa and results in being almost identical to UK114 goat liver protein. Significant similarities with mouse HR12 protein were also observed, whereas a lower degree of similarity was found with a family of heat-responsive proteins named YJGF and YABJ from Haemophilus influenzae and Bacillus subtilis, respectively. The brain activator expresses a strict specificity for the mu-calpain isoform, being completely ineffective on the m-calpain form. As expected, also UK114 was found to possess calpain-activating properties, indistinguishable from those of bovine brain activator. A protein showing the same calpain-activating activity has been also isolated from human red cells, indicating that this factor is widely expressed. All these activators are efficient on mu-calpain independently from the source of the proteinase. The high degree of specificity of the calpain activator for a single calpain isoform may be relevant for the understanding of sophisticated intracellular mechanisms underlying intracellular proteolysis. These data are indicating the existence of a new component of the Ca2+-dependent proteolytic system, constituted of members of a chaperonin-like protein family and capable of promoting intracellular calpain activation.     

Improved survival in stage III melanoma patients with GM2 antibodies: a randomized trial of adjuvant vaccination with GM2 ganglioside

PURPOSE: To perform a double-blind randomized trial with American Joint Commission on Cancer (AJCC) stage III melanoma patients for the following reasons: (1) to confirm our previous finding that patients with antibodies against the melanoma differentiation antigen GM2 have an improved prognosis, and (2) to demonstrate clinical benefit from GM2 antibody induction. PATIENTS AND METHODS: One hundred twenty-two patients with AJCC stage III melanoma who were free of disease after surgery were randomized: 58 to receive treatment with the GM2/BCG vaccine, and 64 to receive treatment with bacille Calmette-Guèrin (BCG) alone. All patients were pretreated with low-dose cyclophosphamide (Cy). RESULTS: GM2 antibody was detected in 50 of 58 patients treated with GM2/BCG and seven of 64 patients treated with BCG alone. With a minimum follow-up period of 51 months, there was a highly significant increase in the disease-free interval (P = .004) and a 17% increase in overall survival (P = .02) in these 57 antibody-positive patients, confirming our earlier experience. Exclusion of all patients with preexisting GM2 antibodies (one in the GM2/BCG group and five in the BCG group) from statistical analysis resulted in a 23% increase in disease-free interval (P = .02) and a 14% increase in overall survival (P = .15) at 51 months for patients treated with the GM2/BCG vaccine. However, when all patients in the two treatment groups were compared as randomized, these increases were 18% for disease-free interval and 11% for survival in the GM2/BCG treatment group, with neither result showing statistical significance. CONCLUSION: (1) Vaccination with GM2/BCG induced immunoglobulin M (IgM) antibodies in most patients. (2) GM2 antibody production was associated with a prolonged disease-free interval and survival. (3) Comparison of the two arms of this trial as randomized fails to show a statistically significant improvement in disease-free interval or survival for patients treated with GM2/BCG vaccines.