Biomedical subjects

R J Kaufman

Publications and source records attributed to R J Kaufman.

At least 91 records · Page 5Linked to original sources

PubMed1996

Inhibition of translational initiation by activators of the glucose-regulated stress protein and heat shock protein stress response systems. Role of the interferon-inducible double-stranded RNA-activated eukaryotic initiation factor 2alpha kinase.

Depletion of endoplasmic reticulum (ER) Ca2+ perturbs protein folding and processing within the organelle while inhibiting translational initiation through activation of the double-stranded RNA-activated eukaryotic initiation factor (eIF)-2alpha kinase (PKR) (Prostko, C. R., Dholakia, J. N., Brostrom, M. A., and Brostrom, C. O. (1995) J. Biol. Chem. 270, 6211-6215). The glucose-regulated stress protein (GRP) chaperones are subsequently induced. We now report that sodium arsenite, a prototype for stressors fostering cytoplasmic protein misfolding, also inhibits translational initiation through activation of PKR while subsequently inducing the heat shock protein (HSP) chaperones. Arsenite neither mobilized ER-associated Ca2+ nor slowed peptide chain elongation. Various HSP-inducing chemicals caused rapid phosphorylation of eIF-2alpha. When incubated with double-stranded RNA, extracts derived from arsenite-treated cells displayed greater degrees of phosphorylation of PKR and eIF-2alpha than did control extracts. Cells overexpressing a dominant negative PKR mutation resisted translational inhibition and eIF-2alpha phosphorylation in response to ER or cytoplasmic stressors. Induction of either the HSP or GRP chaperones was accompanied by development of translational tolerance to either Ca2+-mobilizing agents or arsenite. Following induction of the HSPs by arsenite, cells remained susceptible to induction of the GRPs by Ca2+-mobilizing agents. Conversely, cells possessing induced GRP contents in response to Ca2+-mobilizing agents readily induced the HSPs in response to arsenite. It is concluded that the two chaperone systems function independently except for their mutual suppression of PKR.

3T3 Cells↗

PubMed1996

Moderation of hemophilia A phenotype by the factor V R506Q mutation.

Although many examples of unrelated hemophilia A patients carrying identical point mutations in the factor VIII (FVIII) gene have been reported, the clinical phenotype is not always the same among patients sharing the same molecular defect. Possible explanations for this discrepancy include undetected additional mutations in the FVIII gene or coinheritance of mutations at other genetic loci that modulate FVIII function. We report molecular genetic analysis of potential modifying genes in two sets of unrelated patients carrying common FVIII missense mutations but exhibiting different levels of clinical severity. Both mutations (FVIII R1689C and R2209Q) are associated with severe hemophilia A in some patients and mild/moderate disease in others. The common von Willebrand disease type 2N mutation (R91Q) was excluded as a modifying factor in these groups of patients. However, analysis of the recently described factor V (FV) R506Q mutation (leading to activated protein C resistance) identified a correlation of inheritance of this defect with reduced hemophilia A severity. Two moderately affected hemophilia A patients, each with either of two FVIII gene mutations, were heterozygous for FV R506Q, whereas two severely affected patients and two moderately affected patients were homozygous normal at the FV locus. Our results suggest that coinheritance of the FV R506Q mutation may be an important determinant of clinical phenotype in hemophilia A and that modification of the protein C pathway may offer a new strategy for the treatment of FVIII deficiency.

Alleles↗

PubMed1996

The unfolded protein response pathway in Saccharomyces cerevisiae. Oligomerization and trans-phosphorylation of Ire1p (Ern1p) are required for kinase activation.

In eukaryotic cells, accumulation of unfolded proteins in the endoplasmic reticulum (ER) results in a transcriptional induction of a number of ER chaperone proteins. In Saccharomyces cerevisiae, the putative transmembrane receptor kinase, Ire1p (Ern1p), has been implicated as the sensor of unfolded proteins in the ER that initiates transmittance of the unfolded protein signal from the ER to the nucleus. We have shown that the cytoplasmic domain of Ire1p receptor indeed has intrinsic Ser/Thr kinase activity and contains Ser/Thr phosphorylation sites as well. The cytoplasmic domain is also shown to form oligomers in vivo and in vitro. The ability to form oligomers primarily resides within the last 130 amino acids of the cytoplasmic domain, a region that is dispensable for in vitro kinase activity of the receptor. Oligomerization of the cytoplasmic domains is required for receptor trans-phosphorylation and subsequent activation of the kinase function. The activated kinase may transmit the unfolded protein signal from the ER to the nucleus to activate the transcription of the chaperone genes in the nucleus.

Cytoplasm↗

PubMed1996

A eukaryotic translation initiation factor 2-associated 67 kDa glycoprotein partially reverses protein synthesis inhibition by activated double-stranded RNA-dependent protein kinase in intact cells.

A eukaryotic translation initiation factor 2 (eIF-2)-associated 67 kDa glycoprotein (p67) protects the eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, and this promotes protein synthesis in the presence of active eIF-2 alpha kinases in vitro [Ray, M. K., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543]. We have now examined the effect of overexpression of this cellular eIF-2 kinase inhibitor in an in vivo system using transiently transfected COS-l cells. In this system, coexpression of genes that inhibit PKR activity restores translation of plasmid-derived mRNA. We now report the following. (1) Transient transfection of COS-1 cells with a p67 expression vector increased p67 synthesis by 20-fold over endogenous levels in the isolated subpopulation of transfected cells. (2) Cotransfection of p67 cDNA increased translation of plasmid-derived mRNAs. (3) Overexpression of p67 reduced phosphorylation of coexpressed eIF-2 alpha. (4) p67 synthesis was inhibited by cotransfection with an eIF-2 alpha mutant S51D, a mutant that mimics phosphorylated eIF-2 alpha, indicating that p67 cannot bypass translational inhibition mediated by phosphorylation of the eIF-2 alpha-subunit. These results show that the cellular protein p67 can reverse PKR-mediated translational inhibition in intact cells.

Aminopeptidases↗

PubMed1996

Model for the factor VIIIa-dependent decay of the intrinsic factor Xase. Role of subunit dissociation and factor IXa-catalyzed proteolysis.

The intrinsic factor Xase complex (FXase) is comprised of a serine protease, FIXa, and a protein cofactor, FVIIIa, assembled on a phospholipid surface. Activity of FXase decays with time and reflects the lability of FVIIIa. Two mechanisms potentially contribute to this decay: (i) a weak affinity interaction between the FVIIIa A2 subunit and Al/A3-Cl-C2 dimer and (ii) FVIIIa inactivation resulting from FIXa-catalyzed proteolysis of the Al subunit. At low reactant concentrations (0.5 nm FVIIIa; 5 nm FIXa), FXase decay is governed by the inter-FVIIIa subunit affinity and residual activity approaches a value consistent with this equilibrium, as judged by reactions containing exogenous A2 subunit. Analysis using a mutant form of FVIII (FVIIIR336I) possessing an altered FIXa cleavage site, showed similar rates of FXase decay (0.12 min(-1)) and confirmed the lack of contribution of proteolysis under these conditions. When the concentration of FIXa was increased 10-fold, the initial rate of decay of FXase containing native FVIIIa increased (0.82 min(-1)) and paralleled the rate of proteolysis of Al subunit. However, the rate of decay of FXase containing the FVIIIaR336I was reduced (0.048 min(-1)) consistent with the elevated concentration of FIXa stabilizing the labile subunit structure of the cofactor. Reconstitution of FVIII with FIXa-cleaved light chain showed that cleavage at the alternate FIXa site (A3 domain) was not inhibitory to FXase. The presence of substrate FX resulted in a 10-fold reduction in the rate of FIXa-catalyzed proteolysis of FVIIIa. These results suggest a model whereby decay of FXase results from both FVIIIa subunit dissociation and FIXa-catalyzed cleavage, dependent upon the relative concentration of reactants, with greater contribution of the former at low values and, in the absence of substrate, greater contribution of the latter at high values.

Amino Acid Sequence↗

PubMed1996

Double-stranded (ds) RNA binding and not dimerization correlates with the activation of the dsRNA-dependent protein kinase (PKR).

Upon binding to double-stranded (ds) RNA, the dsRNA-dependent protein kinase (PKR) sequentially undergoes autophosphorylation and activation. Activated PKR may exist as a dimer and phosphorylates the eukaryotic translation initiation factor 2 alpha subunit (cIF-2 alpha) to inhibit polypeptide chain initiation. Transfection of COS-1 cells with a plasmid cDNA expression vector encoding a marker gene, activates endogenous PKR, and selectively inhibits translation of the marker mRNA, dihydrofolate reductase (DHFR). This system was used to study the dsRNA binding and dimerization requirements for over-expressed PKR mutants and subdomains to affect DHFR translation. DHFR translation was rescued by expression of either an ATP hydrolysis defective mutant PKR K296P, the amino-terminal 1-243 fragment containing two dsRNA binding motifs, or the isolated first RNA binding motif (amino acids 1-123). Mutation of K64E within the dsRNA binding motif 1 destroyed dsRNA binding and the ability to rescue DHFR translation. Immunoprecipitation of T7 epitope-tagged PKR derivatives from cell lysates detected interaction between intact PKR and the amino-terminal 1-243 fragment as well as a 1-243 fragment harboring the K64E mutation. Expression of adenovirus VAI RNA, a potent inhibitor of PKR activity, did not disrupt this interaction. In contrast, intact PKR did not interact with fragments containing the first dsRNA binding motif (1-123), the second dsRNA binding motif (98-243), or the isolated PKR kinase catalytic domain (228-551). These results demonstrate that the translational stimulation mediated by the dominant negative PKR mutant does not require dimerization, but requires the ability to bind dsRNA and indicate these mutants act by competition for binding to activators.

Adenoviridae↗

PubMed1996

A common frameshift mutation in von Willebrand factor does not alter mRNA stability but interferes with normal propeptide processing.

Quantitative defects in von Willebrand factor (VWF) result in type 1 and type 3 von Willebrand disease (VWD). This study characterizes the defect in VWF expression resulting from a single nucleotide deletion in VWF exon 18, a mutation previously reported to be common among type 3 VWD patients. A severely affected (type 3) VWD patient in the current pedigree is homozygous for the mutation, whereas heterozygous individuals exhibit variable expression of type 1 VWD. In contrast to the previously reported high frequency of the exon 18 deletion in Sweden and Germany, this mutation appears to be infrequent among type 3 VWD patients in the United States. Although this frameshift mutation results in proximal premature termination of VWF translation, the abnormal VWF mRNA is stable. The mutant truncated recombinant VWF protein is retained within the transfected cell, and no propeptide processing is observed, suggesting a defect in protein folding. Cotransfection of mutant and wild-type recombinant VWF fails to demonstrate a dominant effect of the mutant on the normal allele. Consistent with these results, plasma VWF propeptide of the homozygous individual was markedly reduced whereas heterozygotes exhibited moderately reduced levels. In contrast to type 2A VWD (group 1), the misfolded mutant protein does not appear to exert a dominant-negative effect on normal VWF subunits expressed from the wild-type allele.

Exons↗

PubMed1996

Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo.

The bone morphogenetic proteins (BMPs), a subgroup of the TGF-beta gene super-family, are dimeric molecules involved in the growth, differentiation and repair of a wide variety of tissues. Based on the observation that several of the BMPs co-purify when isolated from bovine bone and that a pattern of co-localization exists during mouse embryogenesis, we co-expressed various combinations of BMPs in Chinese hamster ovary cells to test for possible heterodimer formation and activity. Transient co-expression of BMP-2 with either BMP-5, BMP-6 or BMP-7, or BMP-4 transiently co-expressed with BMP-7, resulted in more BMP activity than expression of any single BMP. Stable cell lines were then made in order to purify and characterize co-expressed BMPs in more detail. Co-expression of BMP-2 with BMP-7 yielded heterodimeric BMP-2/7 with a specific activity about 20-fold higher than BMP homodimers in an in vitro alkaline phosphatase induction assay. These heterodimers were also 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo assay. Similar results were obtained with BMP-2/6 heterodimer. These experiments demonstrate the increased potency of several BMP heterodimers relative to BMP homodimers and support the hypothesis that such heterodimeric forms are likely to have natural biological functions.

Alkaline Phosphatase↗

PubMed1995

Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.

Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

Amino Acid Sequence↗

PubMed1995

The factor V B-domain provides two functions to facilitate thrombin cleavage and release of the light chain.

Blood coagulation factors V and VIII are homologous proteins that have the domain organization A1-A2-B-A3-C1-C2. Upon thrombin activation, the B-domains of both molecules are released. Previous studies on factor VIII showed that the B-domain was not required for thrombin cleavage or activity. In contrast, deletion of the factor V B-domain (residues 709 to 1545) yielded a molecule with sevenfold reduced procoagulant activity that was not cleaved by thrombin. However, this factor V B-domain deletion molecule was activated by factor Xa, although the fold-activation was 85% that of wild-type factor V. Thrombin cleavage of factor V occurs initially after residue 709 and subsequently after residues 1018 and 1545. The requirement for thrombin cleavage within the B-domain at residue 1018 was evaluated by mutagenesis of Arg1018 to Ile. In the resultant R1018I mutant, the rate of thrombin activation and appearance of maximal cofactor activity was delayed and was consistent with delayed cleavage of the light chain at residue 1545. In contrast, the rate of factor Xa activation in the R1018I mutant was not altered. This finding suggests that thrombin cleavage at 1018 facilitates subsequent thrombin cleavage at 1545. Further mutagenesis was used to study the requirement for sequences within the factor V B-domain for thrombin cleavage at residue 1545. Whereas the factor V deletion molecule removing residues 709 to 1545 was not cleaved by thrombin, a smaller B-domain deletion molecule (residues 709 to 1476) containing an acidic amino acid-rich region (residues 1490 to 1520) was effectively cleaved by thrombin. These results show that residues 1476 to 1545, which contain an acidic amino acid-rich region, were required for thrombin cleavage of the light chain. Introduction of an acidic amino acid-rich region from factor VIII (residues 337 to 372) into the factor V 709 to 1545 deletion also restored thrombin cleavage of the light chain. In contrast, similar replacement with the acidic region from the factor VIII light chain (residues 1649 to 1689) was significantly less effective in promoting thrombin cleavage of the light chain. This finding suggests that the different acidic regions in factors V and VIII are not functionally equivalent in their interaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Calcium depletion from the endoplasmic reticulum activates the double-stranded RNA-dependent protein kinase (PKR) to inhibit protein synthesis.

Calcium depletion from the endoplasmic reticulum inhibits protein synthesis and correlates with increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) by a mechanism that does not require ongoing protein synthesis. To elucidate whether protein synthesis inhibition requires eIF-2 alpha phosphorylation and whether eIF-2 alpha phosphorylation is mediated by the double-stranded RNA-dependent protein kinase (PKR), we studied protein synthesis in response to calcium depletion mediated by calcium ionophore A23187 in cell lines overexpressing wild-type eIF-2 alpha, a mutant eIF-2 alpha (S51A) that is resistant to phosphorylation, or a dominant negative mutant PKR (K296P in catalytic subdomain II). Expression of either mutant eIF-2 alpha or mutant PKR partially protected NIH3T3 cells from inhibition of protein synthesis upon A23187 treatment. In contrast, overexpression of wild-type PKR increased sensitivity to protein synthesis inhibition mediated by A23187 treatment. In a COS-1 monkey cell transient transfection system, increased eIF-2 alpha phosphorylation in response to A23187 treatment was inhibited by expression of the dominant negative PKR mutant. Overexpression of the PKR regulatory RNA binding domain, independent of the PKR catalytic domain, was sufficient to inhibit increased phosphorylation of eIF-2 alpha upon A23187 treatment. In addition, overexpression of the HIV TAR RNA binding protein also inhibited eIF-2 alpha phosphorylation upon A23187 treatment. Taken together, our data show that calcium depletion activates PKR to phosphorylate eIF-2 alpha, and this activation is likely mediated through the PKR RNA binding domain.

3T3 Cells↗

PubMed1995

A 110-amino acid region within the A1-domain of coagulation factor VIII inhibits secretion from mammalian cells.

Factor VIII is the coagulation factor deficient in the X-chromosome-linked bleeding disorder hemophilia A. Factor VIII is homologous to blood coagulation factor V, both having a domain structure of A1-A2-B-A3-C1-C2. Previous transfection studies demonstrated that factor VIII is 10-fold less efficiently expressed than the homologous coagulation factor, factor V. The inefficient expression correlated with interaction of the factor VIII primary translation product with the protein chaperonin BiP in the lumen of the endoplasmic reticulum. In contrast, factor V was not detected in association with BiP and was secreted efficiently. To determine whether specific amino acid sequences within factor VIII inhibit secretion, we have studied the secretion of factor VIII deletion and factor VIII/factor V chimeric proteins upon transient transfection of COS-1 monkey cells. A chimeric factor VIII protein that contained the A1- and A2-domains of factor V was secreted with a similar efficiency as wild-type factor V, whereas the complementary chimera having the A1- and A2-domains of factor VIII was secreted with low efficiency, similar to wild-type factor VIII. These results suggested that sequences within the A1- and A2-domains were responsible for the low secretion efficiency of factor VIII. Secretion of A1-domain-deleted factor VIII was increased approximately 10-fold compared to wild-type factor VIII or A2-domain-deleted factor VIII. Expression of the factor VIII A1-domain alone did not yield secreted protein, whereas expression of the factor VIII A2-domain alone or the factor V A1-domain or A2-domain alone directed synthesis of secreted protein. Secretion of a hybrid in which the carboxyl-terminal 110 amino acids of the A1-domain were replaced by homologous sequences from the factor V A1-domain was also increased 10-fold compared to wild-type factor VIII, however, the secreted protein was not functional and the heavy and light chains were not associated. These results localize a 110-amino acid region within the A1-domain that inhibits factor VIII secretion. This region is clustered with multiple short peptide sequences that have potential to bind BiP.

Amino Acid Sequence↗

PubMed1994

Role of the B domain for factor VIII and factor V expression and function.

Factor V and factor VIII are homologous cofactors in the blood coagulation cascade that have the domain structure A1-A2-B-A3-C1-C2, of which the B domain has extensively diverged. In transfected COS-1 monkey cells, expression of factor VIII is approximately 10-fold less efficient than that of factor V, primarily because of inefficient protein secretion and, to a lesser extent, reduced mRNA expression. To study the functional significance and effect of the B domain on expression and activity, chimeric cDNAs were constructed in which the B domains of factor V and factor VIII were exchanged. Expression of a factor VIII chimera harboring the B-domain of factor V yielded a fully functional factor VIII molecule that was expressed twofold more efficiently than wild-type factor VIII because of increased mRNA expression. Thus, sequences within the factor VIII B domain were not responsible for the inefficient secretion of factor VIII compared with factor V. Expression of a factor V chimera harboring the B domain of factor VIII was slightly reduced compared with wild-type factor V, although the secreted molecule had significantly reduced procoagulant activity correlating with dissociated heavy and light chains and resistance to thrombin activation. Interestingly, the factor V chimera containing the factor VIII B domain was efficiently activated by Russell's viper venum (RVV). A factor V B domain deletion (residues 710-1545) molecule also exhibited significantly reduced procoagulant activity caused by resistance to thrombin cleavage and activation, although this molecule was activatable by RVV. These results show that, in contrast to factor VIII, thrombin activation of factor V requires sequences within the B domain. In addition, thrombin activation of factor V occurs through a different mechanism than activation by RVV.

Animals↗

PubMed1994

Depletion of manganese within the secretory pathway inhibits O-linked glycosylation in mammalian cells.

Proteins transiting the secretory pathway are posttranslationally modified by addition of oligosaccharides to asparagine N-linked and serine and threonine O-linked residues. The effects of divalent cation depletion on oligosaccharide processing of erythropoietin (EPO) and macrophage colony stimulating factor (M-CSF) were studied in Chinese hamster ovary cells. Treatment with A23187 did not inhibit M-CSF or EPO secretion but did inhibit addition of complex N-linked and O-linked oligosaccharides to both molecules. Similar results were obtained by treatment with thapsigargin, a potent inhibitor of the Ca(2+)-activated microsomal ATPase, indicating that the effect was due to depletion of divalent cations within the secretory pathway. Whereas addition of extracellular calcium chloride did not reverse the inhibition in complex N-linked and O-linked glycosylation, addition of manganese chloride partially reversed both defects. These results are consistent with a specific manganese requirement within the secretory pathway for the processing of complex N-linked oligosaccharides and the addition of O-linked oligosaccharides. Since there are no known specific inhibitors of O-linked glycosylation, the use of ionophores should significantly facilitate studies on the requirement and role of O-linked oligosaccharides in protein structure and function.

Animals↗

PubMed1994

Identification of individual tyrosine sulfation sites within factor VIII required for optimal activity and efficient thrombin cleavage.

Factor VIII functions as an essential cofactor in the blood coagulation cascade for the factor IXa-mediated activation of factor X. Factor VIII contains 6 tyrosine residues at positions 346, 718, 719, 723, 1664, and 1680 that are modified by post-translational sulfation. This modification is required for full factor VIII procoagulant activity. We have employed site-directed mutagenesis to identify the individual sulfated tyrosines within factor VIII that influence activity. The molecules were expressed in COS-1 monkey cells by transient transfection, and the resultant proteins were characterized. Metabolic incorporation of [35S]sulfate demonstrated that all 6 tyrosine residues are sulfated in factor VIII. Sulfation at residues 346 and 1664 was required for full activity in a factor VIII clotting assay but did not affect factor VIII activity monitored by a factor Xa generation assay. The Tyr346-->Phe and Tyr1664-->Phe mutants displayed delayed thrombin activation that correlated with delayed cleavage at residues 372 and 1689, respectively. In contrast, these mutants were efficiently activated by factor Xa. A triple Tyr to Phe mutant at residues 718, 719, and 723 displayed both reduced factor VIII clotting activity and factor Xa generation activity. Finally, a Tyr1680-->Phe mutant factor VIII displayed a 5-fold reduced affinity for von Willebrand factor. The results demonstrate that 1) sulfation at tyrosine residues 346 and 1664 increases factor VIII activity by increasing the rate of thrombin activation and cleavage; 2) sulfation at tyrosine residues 718, 719, and 723 increases the intrinsic activity of factor VIIIa; and 3) sulfation at tyrosine residue 1680 increases the affinity for vWF. In addition, the results implicate that thrombin interacts with three distinct sites within factor VIII, two of which are required for proteolytic activation. The results demonstrate that the six sites of tyrosine sulfation modulate factor VIII activity through different mechanisms.

Amino Acid Sequence↗

PubMed1994

Post-translational requirements for functional factor V and factor VIII secretion in mammalian cells.

Coagulation factors V and VIII are homologous glycosylated plasma proteins that provide essential functions for hemostasis. Factor V is secreted as a single chain polypeptide, whereas factor VIII is processed intracellularly to yield a metal-ion-associated heterodimer that is stabilized through interaction with von Willebrand factor. In transfected mammalian cells, factor V is more efficiently secreted than factor VIII. To provide insight into the different secretion efficiencies, we compared the post-translational processing requirements for factor V and factor VIII expressed in mammalian cells. In contrast to factor VIII, factor V was not detected in association with the immunoglobulin-binding protein (BiP), a chaperonin protein of the endoplasmic reticulum (ER). Depletion of intracellular ATP levels by treatment of cells with low concentrations of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), protonophore that uncouples oxidative phosphorylation, inhibited secretion of factor VIII but had no effect on the secretion of factor V. Inhibition of N-linked oligosaccharide addition by treatment with tunicamycin prevented secretion of both factor V and factor VIII, whereas treatment with an inhibitor of complex oligosaccharide addition, deoxymannojirimycin, did not affect secretion, although the specific activities of both factor V and factor VIII were slightly increased. Thus, complex oligosaccharide addition was not required for secretion or functional activity of either factor V or factor VIII. Depletion of intralumenal calcium with the ionophore A23187 did not affect secretion of either factor V or factor VIII. In the presence of A23187, the secreted factor V was fully functional, whereas the factor VIII heavy and light chains were not associated and the secreted molecule was inactive. In addition, A23187 treatment inhibited addition of serine/threonine (O)-linked oligosaccharides to factor V and factor VIII. The differences between factor V and factor VIII were further evaluated by characterization of a single chain mutant factor VIII. The single chain factor VIII was secreted with an efficiency similar to wild-type factor VIII and also required von Willebrand factor for stabilization. In addition, the activity of single chain factor VIII was also inhibited by A23187 treatment, suggesting a unique metal-ion requirement within the secretory pathway for functional factor VIII folding. The differences identified in BiP association, ATP requirements, and metal-ion dependence for effective functional secretion of these two molecules may underlie mechanisms accounting for their different secretion efficiencies.

Adenosine Triphosphate↗

PubMed1994

Posttranslational sulfation of factor V is required for efficient thrombin cleavage and activation and for full procoagulant activity.

Factor VIII and factor V function as cofactors in the blood coagulation cascade to accelerate the rate of activation of factor X and prothrombin, respectively. Both cofactors require proteolytic activation by either activated factor X or thrombin for functional activity. Human factor VIII and factor V expressed in mammalian cells are both modified by posttranslational sulfation of tyrosine residues. In the present study, the posttranslational addition of sulfate in factor V expressed in transfected Chinese hamster ovary (CHO) cells was demonstrated by [35S]sulfate incorporation into the thrombin-cleaved 94-kDa heavy chain and the 150-kDa activation peptide. The presence of tyrosine sulfate in recombinant factor V was confirmed by barium hydroxide hydrolysis and two-dimensional thin-layer electrophoresis. The importance of sulfation for factor V secretion and activity was evaluated by characterizing factor V expressed in Chinese hamster ovary cells grown in the presence of sodium chlorate, a potent inhibitor of posttranslational sulfation in intact cells. Increasing concentrations of sodium chlorate inhibited the incorporation of [35S]sulfate into factor V but did not inhibit the synthesis or secretion of factor V. However, the specific activity of factor V secreted in the presence of sodium chlorate was reduced 5-fold. The reduced activity was attributed to (1) slower cleavage and activation by thrombin and (2) a reduced intrinsic activity of factor Va. In contrast, sulfation of factor V did not affect the rate of activation mediated by factor Xa. These results show that sulfation of factor V is required for efficient thrombin activation but not for activation by factor Xa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR.

A cDNA encoding a double-stranded-RNA (dsRNA)-binding protein was isolated by screening a HeLa cell cDNA expression library for proteins that bind the HIV-1 Rev-responsive-element RNA. The cDNA encoded a protein that was identical to TRBP, the previously reported cellular protein that binds the transactivation response element (TAR) RNA of human immunodeficiency virus type 1. TRBP inhibited phosphorylation of the interferon-induced ribosome-associated protein kinase PKR and of the eukaryotic translation initiation factor eIF-2 alpha in a transient-expression system in which the translation of a reporter gene was inhibited by the localized activation of PKR. TRBP expression in HeLa cells complemented the growth and protein-synthesis defect of a vaccinia virus mutant lacking the expression of the dsRNA-binding protein E3L. These results implicate TRBP as a cellular regulatory protein that binds RNAs containing specific secondary structure(s) to mediate the inhibition of PKR activation and stimulate translation in a localized manner.

Animals↗