1,721,067 research outputs found
Improved intracellular processing of protein variants as a personalized therapeutic approach for Haemophilia
No full textSevere bleeding disorders such as Haemophilia B are mainly caused by missense mutations that may affect protein folding, thus leading to the production of structurally-altered proteins associated with low or very low circulating levels. The research activity was aimed at characterizing the mechanism underlying the defective intracellular biosynthesis (misfolding), premature degradation and/or stress of the endoplasmic reticulum in cellular models of sever Haemophilia B and to evaluate the effects of small molecules or drugs acting as chemical/pharmacological chaperones in restoring the molecular defect. Reference: Pignani S, Todaro A, Ferrarese M, Marchi S, Lombardi S, Balestra D, Pinton P, Bernardi F, Pinotti M, Branchini A. The chaperone-like sodium phenylbutyrate improves factor IX intracellular trafficking and activity impaired by the frequent p.R294Q mutation. J Thromb Haemost. 2018 Oct;16(10):2035-2043
The carboxyl-terminal region of coagulation factors: role in biosynthesis and function of FVII and FX
No full text-BACKGROUND-
Factor VII (FVII), factor IX (FIX), factor X (FX) and protein C (PC), belonging to the family of coagulation vitamin K-dependent serine proteases, share high sequence and structural homology at the gene and protein level. However, their carboxyl-terminal region displays striking differences in length and aminoacid composition. While this region of FIX and PC has been demonstrated to be essential for efficient biosynthesis and secretion, little is shown for FVII and FX.
-AIMS-
The main aim was to investigate the carboxyl-terminal region of FVII and FX as determinant of biosynthesis/secretion and/or function.
In the study we took advantage of the characterization of i) a natural variant of FVII characterized by a nonsense mutation (R402X) leading to a slightly truncated protein (-4 residues), and ii) natural anti-FVII inhibitory antibodies developed in a patient with an altered carboxyl-terminal region.
The study was approached both by assays in patient’s plasma and by expression of the recombinant FVII variants in eukaryotic cells.
To address the issue of the role of the carboxyl-terminal region of FX, a panel of progressively truncated FX variants has been expressed and characterized.
-MAIN RESULTS AND CONCLUSIONS -
The main results of the studies can be summarized as follows.
i) Demonstration that the truncated FVII-402X molecule, albeit poorly secreted, possesses an increased specific activity, which explains the association of the R402X nonsense mutation with an asymptomatic phenotype;
ii) identification of an anti-FVII inhibitory antibody in a patient homozygous for a frequent FVII frameshift mutation (11125delC) and data supporting the FVII carboxyl-terminal region as the main epitope of this antibody;
iii) demonstration that the carboxyl-terminal region of FVII is essential for efficient biosynthesis and secretion, and the presence of an inverse relationship between the extent of the deletion of the carboxy-terminus and secretion levels. The deleted variants however possess a normal specific activity, thus not supporting a functional role for the very last residues of FVII;
iv) demonstration that efficient secretion of FX, at variance from its highly homologous FVII, FIX and PC proteins, is not affected by short deletions (up to 21 residues) of the carboxyl-terminal region, which seems to have a functional role.
Taken together these data indicate a differential role of the carboxyl-terminal region of FVII and FX, which might have contributed to divergence and evolution of these serine proteases from the common ancestor enzyme.
-METHODOLOGICAL APPROACHES-
Production and expression of recombinant proteins, ELISA-based and functional assays have been exploited in this study to investigate the expression and the activity of different natural or recombinant variants of coagulation FVII and FX
"An integrated multitool analysis contributes elements to interpreting unclassified factor IX missense variants associated with hemophilia B": reply
No full textNot availabl
MOLECULAR MECHANISMS AND THERAPEUTIC APROACHES FOR RESTORATION OF mRNA TRANSCRIPTION, MATURATION AND TRANSLATION IN INHERITED COAGULATION FACTOR DEFICIENCIES
No full textAlthough substitutive therapy in coagulation factor deficiencies has recently evolved towards proteins with extended half-life and reduced risk for complications, the quality of life of patients would be significantly ameliorated by innovative approaches, based on alternative strategies and able to provide prolonged/permanent expression of therapeutic levels of the defective factor.
Among these, rescuing the altered pre-RNA processing/maturation and mRNA translation has received particular attention because it would in principle permit to restore expression of the mutated gene still maintaining its physiological regulation in the competent tissues, especially hepatocytes which efficiently deliver several coagulation proteins.
More recently, gene editing approaches permit specific DNA sequence recognition, cleavage and thus repair/correction of mutated genes in the appropriate cells.
Here we focus on molecular mechanisms supporting innovative approaches for restoring transcription, maturation and translation of mRNAs in inherited coagulation factor deficiencies:
By engineering transcription activator-like effectors fused with an activation domain (TALE-TFs), able to specifically rescue the coagulation factor VII promoter activity impaired by severe disease-causing mutations. In turn, this triggers synthesis of factor VII mRNA and secretion of functional factor VII protein.
By engineering the key component of the spliceosome, the small nuclear RNA U1 (U1 snRNA), able to prevent exon skipping in mutated factor VII and factor IX pre-mRNA exon-intron junctions. In turn, this triggers synthesis of correct mRNA and secretion of functional factors.
By aminoglycoside drugs inducing ribosome readthrough on premature translation termination codons affecting factor VII. This permits synthesis of full length protein with procoagulant function instead of truncated non-functional molecules.
Depending on the approach and mutations affecting patients' mRNA, we report in cellular and animal models expression levels ranging from negligible to the rescue of potentially therapeutic amounts of coagulation factor activity. Our data support further studies aimed at evaluating clinical translatability of specific molecules in selected groups of patients
An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes
No full textTailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the -94C > G or -61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20-40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations
Translational readthrough of GLA nonsense mutations suggests dominant-negative effects exerted by the interaction of wild-type and missense variants
No full textNonsense mutations are relatively frequent in the rare X-linked lysosomal α-galactosidase A (α-Gal) deficiency (Fabry disease; FD), but have been poorly investigated. Here, we evaluated the responsiveness of a wide panel (n = 14) of GLA premature termination codons (PTCs) to the RNA-based approach of drug-induced readthrough through expression of recombinant α-Gal (rGal) nonsense and missense variants. We identified four high-responders to the readthrough-inducing aminoglycoside G418 in terms of full-length protein (C56X/W209X, ≥10% of wild-type rGal) and/or activity (Q119X/W209X/Q321X, ~5-7%), resulting in normal (Q119X/Q321X) or reduced (C56X, 0.27 ± 0.11; W209X, 0.35 ± 0.1) specific activity. To provide mechanistic insights we investigated the predicted amino acid substitutions mediated by readthrough (W209C/R, C56W/R), which resulted in correct lysosomal localization and appreciable protein/activity levels for the W209C/R variants. Differently, the C56W/R variants, albeit appreciably produced and localized into lysosomes, were inactive, thus indicating detrimental effects of substitutions at this position. Noticeably, when co-expressed with the functional W209C or W209R variants, the wild-type rGal displayed a reduced specific activity (0.5 ± 0.2 and 0.6 ± 0.2, respectively) that, considering the dimeric features of the α-Gal enzyme, suggested dominant-negative effects of missense variants through their interaction with the wild-type. Overall, we provide a novel mechanism through which amino acids inserted during readthrough might impact on the functional protein output. Our findings may also have implications for the interpretation of pathological phenotypes in heterozygous FD females, and for other human disorders involving dimeric or oligomeric proteins
Naturally occurring truncated proteins: decreased protein secretion and increased activity result in asymptomatic coagulation factor deficiency
No full textFactor VII (FVII), IX (FIX), X and protein C (PC), having distinct protein properties and specificities, belong to the coagulation serine protease family that evolved from a common ancestor. In spite of high homology at the gene and protein levels, they show remarkable differences in the carboxyl-terminal region. The C-terminus of FIX and PC has been shown to be essential for secretion but not function. To address this issue in FVII we took advantage from the identification of the homozygous R402Stop nonsense mutation in two asymptomatic FVII deficient patients. FVII protein and coagulant activity levels in patients’ plasma were 0,8% and 5% of normal, respectively, thus suggesting the presence of truncated molecules (FVII-R402Stop, natural stop codon at 407 position), poorly secreted but with improved procoagulant activity. Functional FXa and Thrombin generation assays in plasma confirmed these observations. To investigate these features, we expressed the naturally truncated FVII-402Stop and the FVII variants 403-406Stop. Similarly to the natural mutant, the rFVII-403Stop and rFVII-402Stop variants were poorly secreted (~1%). We found an inverse relationship between secreted protein levels in media and the extent of the deletion for the rFVII-406Stop (50-60% of WT), rFVII-405Stop (15-20%) and rFVII-404Stop (9-12%). FXa generation and coagulation assays revealed a normal specific activity for the rFVII-406Stop, rFVII-405Stop, rFVII-404Stop variants. Intriguingly, upon concentration of media, the specific activity of the rFVII-402Stop appeared to be 387±11% of rFVII-wt, thus recapitulating the mild coagulation phenotype of patients. Altogether these findings point toward multiple functional roles of the FVII carboxyl-terminal region, whose variation might have contributed to the divergence of this protein from the other coagulation serine proteases
Differential functional readthrough over homozygous nonsense mutations contributes to the bleeding phenotype in coagulation factor VII deficiency
No full textBackground Whereas the rare homozygous nonsense mutations causing factor (F)VII deficiency may predict null conditions that are almost completely incompatible with life, they are associated with appreciable differences in hemorrhagic symptoms. The misrecognition of premature stop codons (readthrough) may account for variable levels of functional full-length proteins.
Objectives To experimentally evaluate the basal and drug-induced levels of FVII resulting from the homozygous p.Cys132X and p.Ser112X nonsense mutations that are associated with moderate (132X) or life-threatening (112X) symptoms, and that are predicted to undergo readthrough with (132X) or without (112X) production of wild-type FVII.
Methods We transiently expressed recombinant FVII (rFVII) nonsense and missense variants in human embryonic kidney 293 cells, and evaluated secreted FVII protein and functional levels by ELISA, activated FX generation, and coagulation assays.
Results The levels of functional FVII produced by p.Cys132X and p.Ser112X mutants (rFVII-132X, 1.1% 0.2% of wild-type rFVII; rFVII-112X, 0.5% +/- 0.1% of wild-type rFVII) were compatible with the occurrence of spontaneous readthrough, which was magnified by the addition of G418 - up to 12% of the wild-type value for the rFVII-132X nonsense variant. The predicted missense variants arising from readthrough abolished (rFVII-132Trp/Arg) or reduced (rFVII-112Trp/Cys/Arg, 22-45% of wild-type levels) secretion and function. These data suggest that the appreciable rescue of p.Cys132X function was driven by reinsertion of the wild-type residue, whereas the minimal p.Ser112X function was explained by missense changes permitting FVII secretion and function.
Conclusions The extent of functional readthrough might explain differences in the bleeding phenotype of patients homozygous for F7 nonsense mutations, and prevent null conditions even for the most readthrough-unfavorable mutations
Asymmetric processing of mutant factor X Arg386Cys reveals differences between intrinsic and extrinsic pathway activation
No full textAlterations in coagulation factor X (FX) activation, mediated by the extrinsic VIIa/tissue factor (FVIIa/TF) or the intrinsic factor IXa/factor VIIIa (FIXa/FVIIIa) complexes, can result in hemorrhagic/prothrombotic tendencies. However, the molecular determinants involved in substrate recognition by these enzymes are poorly defined. Here, we investigated the role of arginine 386 (chymotrypsin numbering c202), a surface-exposed residue on the FX catalytic domain. The naturally occurring FX386Cys mutant and FX386Ala variant were characterized. Despite the unpaired cysteine, recombinant (r)FX386Cys was efficiently secreted (88.6±21.3% of rFXwt) and possessed normal clearance in mice. rFX386Cys was also normally activated by FVIIa/TF and displayed intact amidolytic activity. In contrast, rFX386Cys activation by the FIXa/FVIIIa complex was 4.5-fold reduced, which was driven by a decrease in the kcat (1.6∗10(-4)s(-1) vs 5.8∗10(-4)s(-1), rFXwt). The virtually unaltered Km (70.6nM vs 55.6nM, rFXwt) suggested no major alterations in the FX substrate exosite. Functional assays in plasma supplemented with rFX386Cys indicated a remarkable reduction in the thrombin generation rate and thus in coagulation efficiency. Consistently, the rFX386Ala variant displayed similar biochemical features suggesting that global changes at position 386 impact the intrinsic pathway activation. These data indicate that the FXArg386 is involved in FIXa/FVIIIa-mediated FX activation and help in elucidating the bleeding tendency associated with the FX386Cys in a rare FX deficiency case. Taking advantage of the unpaired cysteine, the rFX386Cys mutant may be efficiently targeted by thiol-specific ligands and represent a valuable tool to study FX structure-function relationships both in vitro and in vivo
Responsiveness of hemophilia B- causing non sense mutations to ribosome readthrough-inducing drugs strictly depends on the nucleotide and prrotein context
No full textBACKGRUOUND
Nonsense mutations, caused by premature termination codons, are relatively frequent in Haemophilia (>10%) and are considered as “null mutations”. However, they have been found also in moderate/mild Haemophiliacs and, as compared with large gene deletions, are associated with lower risk for developing inhibitors. This observation point to the presence of residual expression levels arising from “ribosome readthrough” over nonsense triplets, whose sequence context has an impact on readthrough efficiency, as we have shown in a very small cohort of Haemophilia B (HB) patients. Drugs inducing readthrough such as aminoglycosides are proposed as potential therapy.
AIM
To investigate secreted and functional levels of factor IX (FIX) produced by an extended panel of HB nonsense mutations.
METHODS
Recombinant FIX (rFIX) nonsense variants were expressed in HEK293 cells, and secreted and intracellular protein levels, as well as protein isoforms, were evaluated by ELISA and Western Blotting analysis. Residual activity was assessed by coagulant assays.
RESULTS
We investigated a panel of F9 mutations (R75X, L103X, R162X, W240X, R294X, R298X, Y330X, Q370X, R379X, R384X), associated to severe/moderate HB, representing the vast majority of patients with nonsense mutations in HB (324 patients out of 467, corresponding to ~70%). Appreciable levels of secreted FIX were detected for the R162X, W240X, R294X, R298X, Y330X mutants, with truncated isoforms being the large predominance. Truncated forms for Q370X, R379X and R384X were observed in cell lysates only, which suggests misfolding and intracellular retention. Noticeably, the full-length FIX form was appreciable for the R162X variant, indicating the occurrence of spontaneous readthrough, which was significantly increased by the use of aminoglycosides. Moreover, aminoglycosides promoted the synthesis of full-length FIX in the presence of the R75X, Y330X, Q370X, R379X, R384X mutations, not undergoing appreciable spontaneous readthrough. Intriguingly, we investigated the peculiar W240X nonsense mutation, caused by two different termination codons, which showed a differential impact of readthrough in restoring the full-length protein depending on the sequence context. Preliminary coagulant and functional assays revealed that only a few variants were prone to undergo efficient readthrough in terms of secretion and coagulant function.
CONCLUSIONS
These data demonstrate that nonsense mutations can be associated to residual FIX levels through a mechanism of “productive” readthrough. The identification of “leaky” nonsense mutations, and thus of patients with trace FIX levels and potentially “high responders” to readthrough-inducing drugs, might help diagnosis and treatment
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