The Factor VII Variant p.A354V-p.P464Hfs: Clinical versus Intracellular and Biochemical Phenotypes Induced by Chemical Chaperones

(1) Background: Congenital factor (F) VII deficiency is caused by mutations in the F7 gene. Patients with modest differences in FVII levels may display large differences in clinical severity. The variant p.A354V-p.P464Hfs is associated with reduced FVII antigen and activity. The aim of the study was to investigate the clinical manifestation of this variant and the underlying molecular mechanisms. (2) Methods: Analyses were conducted in 37 homozygous patients. The recombinant variant was produced in mammalian cells. (3) Results: We report a large variation in clinical phenotypes, which points out genetic and acquired components beyond F7 mutations as a source of variability. In contrast, patients displayed similarly reduced FVII plasma levels with antigen higher than its activity. Comparative analysis of the recombinant variant and of plasma samples from a subset of patients indicated the presence of an elongated variant with indistinguishable migration. Treatment of cells with the chemical chaperone 4-phenylbutyrate (4-PBA) improved the intracellular trafficking of the variant and increased its secretion to the conditioned medium up to 2-fold. However, the effect of 4-PBA on biological activity was marginal. (4) Conclusions: Chemical chaperones can be used as biochemical tools to study the intracellular fate of a trafficking-defective FVII variant.

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The Chemical Chaperone 4-Phenylbutyrate Increases Secretion and Activity of Missense and Elongated Factor VII Mutants

Blood ◽

10.1182/blood-2018-99-118246 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3777-3777

Author(s):

Elisabeth Andersen ◽

Maria Eugenia Chollet ◽

Marcello Baroni ◽

Mirko Pinotti ◽

Francesco Bernardi ◽

...

Keyword(s):

Er Stress ◽

Histone Acetylation ◽

Conditioned Medium ◽

Intracellular Trafficking ◽

Luciferase Assay ◽

Factor Vii ◽

Mrna Levels ◽

Assay Sensitivity ◽

Chemical Chaperone ◽

Chemical Chaperones

Abstract The F7 gene mutations p.Q160R and p.A354V-p.P464Hfs are associated with very low factor VII (FVII) activity and antigen levels and a bleeding phenotype. We have previously demonstrated reduced secretion of the recombinant (r) variants rFVII-160R and rFVII-354V-464Hfs in vitro, possibly caused by a misfolding mechanism resulting in intracellular retention and increased endoplasmic reticulum (ER) stress. Chemical chaperones are compounds that can enable proteins to recover from a misfolded state and rescue intracellular processing. The aim of the study was to determine whether chemical chaperones could increase the secretion and enhance the biological activity of the above mentioned FVII mutants. Chinese hamster ovary (CHO-K1) cells stably expressing rFVII-160R or rFVII-354V-464Hfs were treated with the chemical chaperones 4-phenylbutyrate (4-PBA), betaine, taurine, taurourosdeoxycholic acid (TUDCA), trimethylamine N-oxide (TMAO) or lumacaftor (VX-809). The intracellular and secreted levels of FVII antigen were measured by ELISA and F7 mRNA levels were assessed by quantitative RT-PCR. Treatment of cells with 4-PBA for 48 hours increased F7 mRNA levels and increased the ratio of secreted/intracellular of both FVII mutants by ~2-2.5-fold, indicating an effect of 4-PBA FVII expression and secretion. 4-PBA is known to inhibit the activity of histone deacetylases, thereby inducing hyperacetylation of histones and increasing gene transcription. To measure histone acetylation, we next performed Western blot analysis of acetylated histone H3 (AcH3). We found that the levels of AcH3 were significantly increased after treatment with 4-PBA, indicating that induced transcription of F7 by 4-PBA could be a result of increased global histone acetylation. Since 4-PBA treatment increased the secretion of the FVII mutants, we questioned whether this also would alleviate ER stress. Using an ER stress luciferase assay, we observed that the ER stress levels in 4-PBA treated cells were significantly lower than in non-treated cells. To determine whether 4-PBA treatment could restore the intracellular trafficking of the FVII mutants from the ER to Golgi, confocal immunofluorescence microscopy was utilized The studies revealed that upon treatment with 4-PBA, the p.Q160R mutant had a strong co-localization with the Golgi marker GM130 and with the Golgi stacking protein GRASP55, known to be associated with unconventional protein secretion. A vesicular pattern of staining could be observed in cells expressing the p.A354V-p.P464Hfs. However, these structures did not overlap with endosomal markers or markers of the ER-Golgi transport remaining the secretory route of this FVII mutant elusive. Finally, to assess the activity of FVII mutants, we measured the generation of activated (a) factor X (FXaG) in FVII deficient plasma supplemented with conditioned medium from cells stably expressing rFVII mutants before and after 4-PBA treatment. To increase the assay sensitivity to very low FVII activity levels we also boosted the coagulation initiation phase by inhibiting the tissue factor pathway inhibitor (TFPI) through an anti-TFPI RNA aptamer. We found that treatment of cells with 4-PBA gave rise to a modest, but measurable FXaG activity in conditioned medium for both FVII mutants. Furthermore, inhibition of TFPI proportionally potentiated FXaG activity of both 4-PBA-induced FVII variants. The present study demonstrates that chemical chaperones, such as 4-PBA, can increase intracellular trafficking through the secretory pathway of misfolded FVII mutants. Importantly, the secreted mutant proteins were found to be biologically regulated and active in the coagulation pathway. Thus, 4-PBA could have therapeutic effect in FVII deficiency caused by misfolded FVII mutants. Disclosures No relevant conflicts of interest to declare.

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Chemical Chaperones Reduce Endoplasmic Reticulum Stress and Prevent Mutant HFE Aggregate Formation

Journal of Biological Chemistry ◽

10.1074/jbc.m702672200 ◽

2007 ◽

Vol 282 (38) ◽

pp. 27905-27912 ◽

Cited By ~ 111

Author(s):

Sérgio F. de Almeida ◽

Gonçalo Picarote ◽

John V. Fleming ◽

Maria Carmo-Fonseca ◽

Jorge E. Azevedo ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Molecular Mechanisms ◽

Hereditary Hemochromatosis ◽

Signal Transduction Pathway ◽

Mutant Protein ◽

Chemical Chaperone ◽

Chemical Chaperones ◽

Unfolded Protein ◽

The Stability ◽

Intracellular Aggregates

HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.

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Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157760 ◽

1990 ◽

Vol 48 (3) ◽

pp. 44-45

Author(s):

G-A. Keller ◽

S. J. Gould ◽

S. Subramani ◽

S. Krisans

Keyword(s):

Mammalian Cells ◽

Molecular Mechanisms ◽

Protein Targeting ◽

Firefly Luciferase ◽

Peroxisomal Enzyme ◽

Transfected Cells ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Series Of Experiments ◽

Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

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Two Typical Hereditary Charts of Congenital Factor VII Deficiency

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654905 ◽

1961 ◽

Vol 05 (01) ◽

pp. 087-092 ◽

Cited By ~ 11

Author(s):

F. J Cleton ◽

E. A Loeliger

Keyword(s):

Factor Vii ◽

Autosomal Gene ◽

Homozygous State ◽

Recessive Character ◽

Factor Vii Deficiency ◽

Complete Penetrance ◽

Coagulation Defect ◽

Haemorrhagic Diathesis ◽

Intermediate Expression ◽

Congenital Factor

SummaryThe inheritance of congenital factor VII deficiency was investigated in 2 unrelated families. Out of 68 individuals, 4 (3 proven and 1 highly probable) were found to have severe factor VII deficiency (<C 0.1% factor VII), and 29 appeared to be heterozygotes (30—60% factor VII). The coagulation defect is due to an autosomal gene of intermediate expression and complete penetrance. The recessive character of the haemorrhagic diathesis due to the homozygous state for the abnormal gene is clearly demonstrated.

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A Possible Modulation Mechanism of Intramolecular and Intermolecular Interactions for NCAM Polysialylation and Cell Migration

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666191018094805 ◽

2019 ◽

Vol 19 (25) ◽

pp. 2271-2282 ◽

Cited By ~ 5

Author(s):

Bo Lu ◽

Xue-Hui Liu ◽

Si-Ming Liao ◽

Zhi-Long Lu ◽

Dong Chen ◽

...

Keyword(s):

Cell Migration ◽

Intermolecular Interactions ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Intramolecular Interaction ◽

Polysialic Acid ◽

Tumor Cell Migration ◽

Neural Cell Adhesion ◽

Polybasic Domains ◽

Novel Model

Polysialic acid (polySia) is a novel glycan that posttranslationally modifies neural cell adhesion molecules (NCAMs) in mammalian cells. Up-regulation of polySia-NCAM expression or NCAM polysialylation is associated with tumor cell migration and progression in many metastatic cancers and neurocognition. It has been known that two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST), can catalyze polysialylation of NCAM, and two polybasic domains, polybasic region (PBR) and polysialyltransferase domain (PSTD) in polySTs play key roles in affecting polyST activity or NCAM polysialylation. However, the molecular mechanisms of NCAM polysialylation and cell migration are still not entirely clear. In this minireview, the recent research results about the intermolecular interactions between the PBR and NCAM, the PSTD and cytidine monophosphate-sialic acid (CMP-Sia), the PSTD and polySia, and as well as the intramolecular interaction between the PBR and the PSTD within the polyST, are summarized. Based on these cooperative interactions, we have built a novel model of NCAM polysialylation and cell migration mechanisms, which may be helpful to design and develop new polysialyltransferase inhibitors.

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The Saccharomyces cerevisiae RAD6 Group Is Composed of an Error-Prone and Two Error-Free Postreplication Repair Pathways

Genetics ◽

10.1093/genetics/155.4.1633 ◽

2000 ◽

Vol 155 (4) ◽

pp. 1633-1641 ◽

Cited By ~ 11

Author(s):

Wei Xiao ◽

Barbara L Chow ◽

Stacey Broomfield ◽

Michelle Hanna

Keyword(s):

Mammalian Cells ◽

Molecular Mechanisms ◽

Signal Transducer ◽

Repair Pathway ◽

Polyubiquitin Chain ◽

Postreplication Repair ◽

Translesion Dna Synthesis ◽

Radiation Repair ◽

Repair Pathways ◽

High Degree

Abstract The RAD6 postreplication repair and mutagenesis pathway is the only major radiation repair pathway yet to be extensively characterized. It has been previously speculated that the RAD6 pathway consists of two parallel subpathways, one error free and another error prone (mutagenic). Here we show that the RAD6 group genes can be exclusively divided into three rather than two independent subpathways represented by the RAD5, POL30, and REV3 genes; the REV3 pathway is largely mutagenic, whereas the RAD5 and the POL30 pathways are deemed error free. Mutants carrying characteristic mutations in each of the three subpathways are phenotypically indistinguishable from a single mutant such as rad18, which is defective in the entire RAD6 postreplication repair/tolerance pathway. Furthermore, the rad18 mutation is epistatic to all single or combined mutations in any of the above three subpathways. Our data also suggest that MMS2 and UBC13 play a key role in coordinating the response of the error-free subpathways; Mms2 and Ubc13 form a complex required for a novel polyubiquitin chain assembly, which probably serves as a signal transducer to promote both RAD5 and POL30 error-free postreplication repair pathways. The model established by this study will facilitate further research into the molecular mechanisms of postreplication repair and translesion DNA synthesis. In view of the high degree of sequence conservation of the RAD6 pathway genes among all eukaryotes, the model presented in this study may also apply to mammalian cells and predicts links to human diseases.

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Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5208 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5208-5218 ◽

Cited By ~ 431

Author(s):

Michael Gale ◽

Collin M. Blakely ◽

Bart Kwieciszewski ◽

Seng-Lai Tan ◽

Michelle Dossett ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Kinase ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Functional Assay ◽

Binding Region ◽

Dimerization Domain ◽

Antiviral Effects

ABSTRACT The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

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Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4185 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4185-4189 ◽

Cited By ~ 7

Author(s):

J A Greenspan ◽

F M Xu ◽

R L Davidson

Keyword(s):

Cell Line ◽

Cell Lines ◽

Dna Sequences ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Shuttle Vector ◽

Ethyl Methanesulfonate ◽

Wild Type ◽

Single Base ◽

Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

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