Phosphorylation of a gelatin-binding protein from L6 myoblasts by protein kinase C.

A gelatin-binding glycoprotein from L6 rat myoblasts, designated gp46, was shown to be phosphorylated in vivo. This phosphorylation was increased slightly (18%) by phorbol ester treatment of L6 suggesting protein kinase C involvement. Purified gp46 could be phosphorylated in vitro with protein kinase C, but not by the catalytic subunit of cAMP-dependent protein kinase. Comparison of the phosphotryptic peptide maps of in vitro and in vivo labeled gp46 suggested that in vivo phosphorylation of gp46 may be mediated by protein kinase C.

Binding of vascular heparan sulfate proteoglycan to Alzheimer's amyloid precursor protein is mediated in part by the N-terminal region of A4 peptide.

The exact mechanisms of deposition and accumulation of amyloid in senile plaques and in blood vessels in Alzheimer's disease remain unknown. Heparan sulfate proteoglycans may play an important role in amyloid deposition in Alzheimer's disease. Previous investigations have demonstrated high affinity binding between heparan sulfate proteoglycans and the amyloid precursor, as well as with the A4 peptide. In the current studies, a specific vascular heparan sulfate proteoglycan found in senile plaques bound with high affinity to two amyloid protein precursors (APP695 and APP770). Vascular heparan sulfate proteoglycan also bound the Alzheimer's amyloid A4 peptide, and not other amyloid protein precursor regions studied, with high affinity. Both heparan sulfate glycosaminoglycan chains and chemically deglycosylated vascular heparan sulfate proteoglycan protein core bound to A4. High affinity interactions between vascular heparan sulfate proteoglycan and the A4 peptide may play a role in the process of amyloidogenesis in Alzheimer's disease, by localizing the site of deposition of A4, protecting A4 from further proteolysis, or by promoting aggregation and fibril formation.

Localization of the basement membrane heparan sulfate proteoglycan in islet amyloid deposits in type II diabetes mellitus.

Amyloid in the islets of Langerhans is the uniform pathologic feature in the pancreata of patients with type II diabetes mellitus. Although the mechanisms of islet amyloid fibrillogenesis are unknown, the presence of heparan sulfate proteoglycan in many other forms of amyloid suggests a role for this proteoglycan in amyloidogenesis in general. In this study, islet amyloid was evaluated for the presence of the basement membrane heparan sulfate proteoglycan using histochemical and immunohistochemical techniques. Staining with sodium sulfate-alcian blue identified highly sulfated glycosaminoglycans within all islet amyloid deposits, and anti-basement membrane heparan sulfate proteoglycan antisera localized this specific proteoglycan within the islet amyloid. The presence of the basement membrane heparan sulfate proteoglycan links islet amyloid to other disparate forms of amyloid and further supports the hypothesis that it has a role in a common pathway of amyloid fibrillogenesis.

In vivo and in vitro iron-replaced zinc finger generates free radicals and causes DNA damage.

The estrogen receptor (ER) is a ligand-activated transcription factor whose DNA-binding domain (ERDBD) has eight cysteines, which coordinate two zinc atoms, forming two zinc finger-like structures. We demonstrate the capability of iron to replace zinc in zinc finger (hereby referred to as iron finger) both in vivo (using Escherichia coli BL21 (DE3)) and in vitro. Iron has the ability to substitute for zinc in the ERDBD as demonstrated by mobility shift and methylation interference assays of iron finger, which show specific recognition of the estrogen response element. The DNA binding constants for both in vivo and in vitro iron-replaced zinc fingers were similar to that of the native finger. Atomic absorption analysis revealed a ratio of 2:1 iron atoms/mol of ERDBD protein, as found for zinc in the crystal structure of native ERDBD. More importantly, we demonstrate that iron finger in the presence of H2O2 and ascorbate generates highly reactive free radicals, causing a reproducible cleavage pattern to the proximate DNA, the estrogen response element. The deoxyribose method, used to detect free radical species generated, and the resultant cleaved DNA ends, caused by iron finger, suggest that the free radicals generated are hydroxyl radicals. Due to the close proximity of the zinc finger to DNA, we postulate that iron-substituted zinc finger may generate free radicals while bound to genetic regulatory response elements, leading to adverse consequences such as iron-induced toxicity and/or carcinogenesis.

The designed protein M(II)-Gly-Lys-His-Fos(138-211) specifically cleaves the AP-1 binding site containing DNA.

A new specific DNA cleavage protein, Gly-Lys-His-Fos(138-211), was designed, expressed, and characterized. The DNA-binding component of the design uses the basic and leucine zipper regions of the leucine zipper Fos, which are represented by Fos(138-211). The DNA cleavage moiety was provided by the design of the amino-terminal Cu(II)-, Ni(II)-binding site GKH at the amino terminus of Fos(138-211). Binding of Cu(II) or Ni(II) by the protein activates its cleavage ability. The GKH motif was predicted to form a specific amino-terminal Cu(II)-, Ni(II)-binding motif as previously defined [Predki, P. F., Harford, C., Brar, P., & Sarkar, B. (1992) Biochem. J. 287, 211 -215]. This prediction was verified as the tripeptide, GKH, and the expressed protein, GKH-Fos(138-211), were both shown to be capable of binding Cu(II) and Ni(II). The designed protein upon heterodimerization with Jun(248-334) was shown to bind to and cleave several forms of DNA which contained an AP-1 binding site. The cleavage was shown to be specific. This design demonstrates the versatility of the amino-terminal Cu(II)-, Ni(II)-binding motif and the variety of motifs which can be generated. The site of cleavage by GKH-Fos(138-211) on DNA provides further information regarding the bending of DNA upon binding to Fos-Jun heterodimers.

Probes of the structure of phosphoenolpyruvate synthetase: effects of a transition state analogue on enzyme conformation.

Phosphoenolpyruvate synthetase of Escherichia coli is strongly inhibited by oxalate. The magnitude of the inhibition constant for oxalate suggests that this compound acts to produce a transition state analogue, in keeping with the suggestion of others that oxalate mimics the structure of enolpyruvate, a presumed catalytic intermediate in the enzymatic reaction. The addition of oxalate together with ATP results in a dramatic shielding of sensitive amino acid residues from reaction with both N-ethyl maleimide and phenylglyoxal. Thus, under conditions otherwise giving rise to almost complete inactivation by either reagent, no loss of activity is detectable in the presence of oxalate and ATP. These results indicate the formation of an enclosed structure during catalysis in which reactive groups are rendered quite inaccessible to solvent.

Phosphoenolypyruvate synthetase of Escherichia coli: molecular weight, subunit composition, and identification of phosphohistidine in phosphoenzyme intermediate.

Phosphoenolypyruvate synthetase of Escherichia coli has been shown to be a dimer of molecular weight 150,000. The constituent subunits appear to be identical. The enzyme tends to dissociate to monomers at low protein concentration, but the tendency is much diminished in the phosphoenzyme form, suggesting that enzyme phosphorylation is accompanied by a structural rearrangement in the subunit contact domain. The enzyme appears to show half of the sites reactivity with respect to its phosphorylation by ATP. Several lines of evidence, including identification of 3-phosphohistidine in alkaline digests of the phosphoenzyme, indicate that a histidyl residue is the site of phosphorylation.

Purification and polyamine activation of a high-affinity 3',5'-cAMP phosphodiesterase from rabbit muscle.

A high-affinity phosphodiesterase, termed PDE II, has been purified about 1400-fold from rabbit skeletal muscle. This enzyme is activated by treatment with proteases. It is also activated specifically by polyarginine and arginine-rich histones, but not by other polyanions. The activation is counteracted nonspecifically by polycations, such as heparin and chondroitin sulphate. When the enzyme is fully activated by polyarginine it is no longer susceptible to activation by proteases. A conformational or structural change must thus occur in the enzyme by the binding of the polyanions.

In vivo and in vitro models of demyelinating disease: activation of the adenylate cyclase system influences JHM virus expression in explanted rat oligodendrocytes.

The specificity of JHM virus (JHMV) tropism for rat oligodendrocytes, as one of the primary host cells in the central nervous system, is maintained after explanation (S. Beushausen and S. Dales, Virology 141:89-101, 1985). The temporal correlation between onset of resistance to JHMV infection in vivo, completion of myelination, and maturation of the central nervous system can be simulated in vitro by inducers of oligodendrocyte differentiation (Beushausen and Dales, Virology, 1985). Stimulation of differentiation through the elevation of intracellular cyclic AMP (cAMP) levels suggests a possible connection between activation of the adenylate cyclase system and coronavirus expression. Chromatographic analysis of cAMP-dependent protein kinase activity in cytosol extracts prepared from astrocytes or oligodendrocytes revealed that both glial cell types were deficient in protein kinase I, indicating that expression of coronavirus in differentiated cells was not contingent upon the presence of protein kinase I. However, treatment with N6,2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (dbcAMP) resulted in a severalfold enhancement of the free regulatory subunit (RI) in oligodendrocytes but not in astrocytes. The RII subunit in both neural cell types was relatively unaffected. Rapid increase in RI due to dbcAMP treatment was correlated with inhibition of JHMV expression. Other differentiation inducers, including 8-Br cAMP and forskolin which, by contrast, caused a decrease in detectable RI, also blocked JHMV expression. This apparent anomaly can be attributed to an increased turnover of RI due to destabilization of the molecule which occurs upon site-specific binding of the cyclic nucleotides. On the basis of these observations, we conclude that the state of oligodendrocyte differentiation manifested with the modulation of RI regulates JHMV expression. The differentiation process did not affect either virus adsorption or sequestration but appeared to inhibit the expression of viral RNA and proteins, implying that replication was inhibited at some step between penetration and initiation of genomic functions, perhaps at the stage of uncoating. We therefore examined the possibility that protein kinases and phosphatases, which influence cellular regulation during cAMP-induced differentiation, may be responsible for the phenomenon of coronavirus suppression in oligodendrocytes. Evidence was obtained indicating that normal processing of the phosphorylated nucleocapsid protein is inhibited in differentiated oligodendrocytes, consistent with the notion that JHMV replication might be arrested during uncoating.

Regulation of cyclic 3',5'-adenosine monophosphate phosphodiesterase in Friend erythroleukemia cells.

Friend erythroleukemia cells contain only a single form of cAMP phosphodiesterase with high affinity for substrate. It is activated by treatment with various proteases including those present in snake venom. The activity of this enzyme is increased about 2-fold when the cells are either cultivated in the presence of cAMP or in the presence of compounds which are capable of generating cAMP in vivo, such as isoproterenol, epinephrine and prostaglandins. The enzyme produced in the presence of cAMP is modified in such a way that it is no longer susceptible to activation by treatment with proteases. The activation and modification obtained in vivo can be duplicated in cell-free extracts of Friend cells, if they are incubated in the presence of cAMP and ATP. A possibility thus exists that the phosphodiesterase is activated by its substrate by phosphorylation.

The metabolism of retinyl methyl ether in the rat in vivo.

1. Retinyl methyl ether was converted into vitamin A in vitamin A-deficient rats regardless of whether administered by oral, intraperitoneal, intramuscular or subcutaneous route; intramuscular administration seemed to be the best for conversion as well as storage. 2. Significantly, unchanged retinyl methyl ether was also found in the liver after oral administration but not after administration by other routes. 3. Oral administration of 1mg of retinyl methyl ether led to a progressive increase in liver vitamin A with time reaching a value of 16% of administered dose after 24h. No retinyl methyl ether was detectable in liver at any time-interval in this experiment. 4. Conversely, oral administration of 4mg of retinyl methyl ether/day for 4 days led to the accumulation of 25% of the dose as unchanged retinyl methyl ether in the liver 1 day after the last dose; however, it was gradually but completely converted into vitamin A over a period of 18 days. 5. The significance of these findings with special reference to the fundamental metabolism of vitamin A, the site of conversion of retinyl methyl ether into vitamin A, the relative efficiency of various routes of administration and its biological activity are discussed.

The cyclic adenosine monophosphate phosphodiesterases of myoblasts, fibroblasts, and their somatic cell hybrids.

Three forms of cAMP phosphodiesterases are found in mouse L cells (fibroblasts) and rat skeletal myoblasts. The myoblast enzymes can be resolved by chromatography on DEAE-cellulose and the fibroblast enzymes by chromatography on DEAE-Biogel. The myoblast enzymes are "high affinity" cAMP specific forms and have different molecular weights, while all L-cell enzymes have an apparent molecular weight of 450,000. Only one of the L-cell enzymes is able to hydrolyze both cyclic guanosine monophosphate (cGMP) and cAMP. Hydrolysis of the latter is stimulated by micromolar amounts of cGMP. The myoblast x L cell hybrids possess at least five phosphodiesterases, three of which can be identified as being of myoblast or fibroblast origin. One of the fibroblast enzymes appears to be modified in hybrids. The entire phosphodiesterase regulatory system of the myoblasts is active in the hybrids.

Regulation of protein kinase C by cyclic adenosine 3':5'-monophosphate and a tumor promoter in skeletal myoblasts.

The specific activity of protein kinase C in rat skeletal myoblasts decreased when they were exposed for very short periods to isoproterenol, forskolin, dibutyryl cyclic AMP (Bt2cAMP), or the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of Bt2cAMP or forskolin only the cytosolic but not the membrane-bound kinase activity was found to decrease. Treatment with TPA, however, led to a decrease in the activity of the enzyme both in the cytosolic as well as the membrane fractions. The effects observed in vivo could be duplicated in crude extracts of myoblasts incubated with cAMP analogues or TPA. In the presence of ATP, protein kinase C activity decreased considerably in crude cytosolic fractions treated with the cAMP analogues, but a requirement for ATP was not evident for the decrease in activity brought about by TPA. For the cAMP analogues the decrease in protein kinase C was also prevented by incubation of the extracts with an inhibitor of cAMP-dependent protein kinase. The regulation of protein kinase C by Bt2cAMP (but not by TPA) was altered in Rous sarcoma virus-transformed myoblasts. It is considered likely that a component affected by cAMP (probably a substrate for cAMP-dependent protein kinase) participates in the regulation of protein kinase C activity, and it is altered in unknown ways in transformed myoblasts.

Copper-induced conformational changes in the N-terminal domain of the Wilson disease copper-transporting ATPase.

The Wilson disease copper-transporting ATPase plays a critical role in the intracellular trafficking of copper. Mutations in this protein lead to the accumulation of a toxic level of copper in the liver, kidney, and brain followed by extensive tissue damage and death. The ATPase has a novel amino-terminal domain ( approximately 70 kDa) which contains six repeats of the copper binding motif GMTCXXC. We have expressed and characterized this domain with respect to the copper binding sites and the conformational consequences of copper binding. A detailed analysis of this domain by X-ray absorption spectroscopy (XAS) has revealed that each binding site ligates copper in the +1 oxidation state using two cysteine side chains with distorted linear geometry. Analysis of copper-induced conformational changes in the amino-terminal domain indicates that both secondary and tertiary structure changes take place upon copper binding. These copper-induced conformational changes could play an important role in the function and regulation of the ATPase in vivo. In addition to providing important insights on copper binding to the protein, these results suggest a possible mechanism of copper trafficking by the Wilson disease ATPase.

An interaction between basement membrane and Alzheimer amyloid precursor proteins suggests a role in the pathogenesis of Alzheimer's disease.

BACKGROUND: Extracellular matrix proteins (ECMPs) of the basement membrane type, such as the heparan sulfate proteoglycan perlecan, laminin, entactin, collagen IV, and fibronectin are present in and have been implicated in the genesis of amyloids. As in many forms of amyloid, perlecan, laminin, collagen IV, and fibronectin are present in Alzheimer deposits. We have previously demonstrated high-affinity interactions between Alzheimer amyloid precursor proteins (beta PP-695, -751, and -770), and perlecan or laminin. With a view to examining our hypothesis that beta PP:ECMP interactions are involved in Alzheimer's amyloidogenesis, additional studies have now been performed examining the interactions of the beta PPs with entactin, fibronectin, and collagen IV, the influence each of the ECMPs has on the binding of the others to beta PPs, and the effect of beta PPs on interactions among the various ECMPs. EXPERIMENTAL DESIGN: A modified solid-phase enzyme-linked immunosorbent assay was used to assess the binding of the various ECMPs to the beta PPs. One element was immobilized on plastic, and another element, operationally defined as a ligand, was incubated in solution at various concentrations over the immobilized protein. To evaluate the effect of one ECMP on the binding of other ECMPs to beta PP, the beta PP was immobilized and the binding of the "ligand" ECMP was assessed in the presence of a single concentration of a second "competitor" ECMP. Similarly, in evaluating the effect of beta PPs on the binding of ECMPs to each other, one ECMP was immobilized and the binding of a second ECMP "ligand" was assessed in the presence of a fixed concentration of beta PP "competitor." RESULTS: As in the case of perlecan and laminin, each of the ECMPs bound to the beta PPs with high affinity (Kd values in the nanomolar range). The binding of entactin to beta PPs was stimulated by collagen IV but was markedly inhibited by laminin, perlecan, and fibronectin. Conversely, the presence of entactin inhibited the binding of perlecan, laminin, and fibronectin to beta PPs. Moreover, the presence of beta PPs usually interfered with the binding of ECMPs to each other. Generally, in all binding assays, beta PP-751 and -770, behaved in similar ways, but beta PP-695, the brain-specific form, exhibited unique characteristics. CONCLUSIONS: These binding data may reflect the normal interactions of beta PPs with ECMPs. However, the fact that beta PPs interfere with the normal interactions between ECMPs themselves, a process that spontaneously generates a basement membrane, suggests that aspects of ECMP:beta PP binding may be a pathologic part of the amyloidogenic process in Alzheimer's disease.

Functional analysis of chimeric proteins of the Wilson Cu(I)-ATPase (ATP7B) and ZntA, a Pb(II)/Zn(II)/Cd(II)-ATPase from Escherichia coli.

ATP7B, the Wilson disease-associated Cu(I)-transporter, and ZntA from Escherichia coli are soft metal P1-type ATPases with mutually exclusive metal ion substrates. P1-type ATPases have a distinctive amino-terminal domain containing the conserved metal-binding motif GXXCXXC. ZntA has one copy of this motif while ATP7B has six copies. The effect of interchanging the amino-terminal domains of ATP7B and ZntA was investigated. Chimeric proteins were constructed in which either the entire amino-terminal domain of ATP7B or only its sixth metal-binding motif replaced the amino-terminal domain of ZntA. Both chimeras conferred resistance to lead, zinc, and cadmium salts but not to copper salts. The purified chimeras displayed activity with lead, cadmium, zinc, and mercury, which are substrates of ZntA. There was no activity with copper or silver, which are substrates of ATP7B. The chimeras were 2-3-fold less active than ZntA. Thus, the amino-terminal domain of P1-type ATPases cannot alter the metal specificity determined by the transmembrane segment. Also, these results suggest that this domain interacts with the rest of the transporter in a metal ion-specific manner; the amino-terminal domain of ATP7B cannot replace that of ZntA in restoring full catalytic activity.

High affinity interactions between the Alzheimer's beta-amyloid precursor proteins and the basement membrane form of heparan sulfate proteoglycan.

High affinity interactions were studied between the basement membrane form of heparan sulfate proteoglycan (HSPG) and the 695-, 751-, and 770-amino acid Alzheimer amyloid precursor (AAP) proteins. Based on quantitative analyses of binding data, we identified single binding sites for the HSPG on AAP-695 (Kd = 9 x 10(-10) M), AAP-751 (Kd = 10 x 10(-9) M), and AAP-770 (Kd = 9 x 10(-9) M). It is postulated that the "Kunitz" protease inhibitor domain which is present in AAP-751 and -770 reduces the affinity of AAPs for the HSPG through steric hindrance and/or conformational alteration. HSPG binding was inhibited by heparin and dextran sulfate, but not by dermatan or chondroitin sulfate. HSPG protein core, obtained by heparitinase digestion, also bound to the beta-amyloid precursor proteins with high affinity, indicating that the high affinity binding site is constituted by the polypeptide chain rather than the carbohydrate moiety. The effects of various cations on these interactions were also studied. Our results suggest that specific interactions between the AAP proteins and the extracellular matrix may be involved in the nucleation stages of Alzheimer's disease type amyloidogenesis.

Regulation of cyclic adenosine 3':5'-monophosphate phosphodiesterases: altered pattern in transformed myoblasts.

Rat skeletal myoblasts, L6 and L8, have two major forms of phosphodiesterases, PDE II and PDE III. Only the former is activated by treatment with proteases. When the myoblasts are exposed to cAMP for 10-16 h, the activity of PDE III increases considerably. This increase is accompanied by a loss of activatability of PDE II by proteases. Leupeptin prevents the increase in the levels of PDE III suggesting that a protease in vivo may be responsible for the formation of PDE III from PDE II. Spontaneously or Rous sarcoma virus-transformed myoblasts, however, show altered regulation of the two forms of PDE. In the presence of cAMP in the medium, unlike the nontransformed cells, the levels of PDE III do not increase but the activity of PDE II rises. Simultaneously, PDE II becomes refractory to activation by proteases. The altered mode of PDE regulation in transformed cells is dominant in hybrids between normal and transformed myoblasts, which suggests that altered regulation is due to an "acquisition" of some new property by transformed cells.

Regulation of protein kinase and its regulatory subunits during skeletal myogenesis.

Rat skeletal myoblasts contain two cytosolic cAMP-dependent protein kinases, types I and II. Photoaffinity labeling with 8-azido-cAMP reveals the presence of regulatory subunits of Mr = 52,000, 47,000, and 36,000. The Mr = 52,000 and 47,000 subunits are very likely RII and RI, respectively, while the Mr = 36,000 subunit appears to be a proteolytic product of RI, as judged by its cross-reactivity to anti-RI antiserum. The total protein kinase activity increases about 3-fold during the fusion of myoblasts. In parallel with this increase, the concentration of RI subunit also increases, while the levels of RII remain unchanged. Myoblast mutants which lack the capability to differentiate both biochemically and morphologically also lack the ability to increase the concentration of RI subunit. This ability is restored in complementing somatic hybrids which regain the capability to differentiate.