Ronit Mor-Cohen

Specific Cysteines in β3 Are Involved in Disulfide Bond Exchange-dependent and -independent Activation of αIIbβ3

Disulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of β3 was suggested to be involved in activation of αIIbβ3. To investigate the role of specific β3 cysteines in αIIbβ3 expression and activation, we expressed in baby hamster kidney cells normal αIIb with normal β3 or β3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and β-tail domains and assessed αIIbβ3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of αIIbβ3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active αIIbβ3, implying that these bonds stabilize the inactive αIIbβ3 conformer. Mutants of the Cys-567–Cys-581 bond in EGF-4 were inactive even after exposure to αIIbβ3-activating antibodies, indicating that this bond is necessary for activating αIIbβ3. Disrupting Cys-560–Cys-583 in the EGF-3/EGF-4 or Cys-608–Cys-655 in β-tail domain resulted in αIIbβ3 activation only when Cys-560 or Cys-655 of each pair was mutated but not when their partners (Cys-583, Cys-608) or both cysteines were mutated, suggesting that free sulfhydryls of Cys-583 and Cys-608 participate in αIIbβ3 activation by a disulfide bond exchange-dependent mechanism. The free sulfhydryl blocker dithiobisnitrobenzoic acid inhibited 70% of anti-LIBS6 antibody-induced activation of wild-type αIIbβ3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in αIIbβ3 activation. These data suggest that different disulfide bonds in β3 EGF and β-tail domains play variable structural and regulatory roles in αIIbβ3.

Unique Disulfide Bonds in Epidermal Growth Factor (EGF) Domains of β3 Affect Structure and Function of αIIbβ3 and αvβ3 Integrins in Different Manner

Background: Disulfide bonds in β3 are involved in αIIbβ3 activation. Results: Disruptions of unique disulfide bonds in EGF domains of β3 yielded constitutively active αIIbβ3 and αvβ3 variably dependent on the presence of free sulfhydryls. Conclusion: Unique disulfide bonds regulate differently αIIbβ3 and αvβ3 function. Significance: The findings highlight the importance of unique disulfide bonds in the function of β3 integrins. The β3 subunit of αIIbβ3 and αvβ3 integrins contains four epidermal growth factor (EGF)-like domains. Each domain harbors four disulfide bonds of which one is unique for integrins. We previously discerned a regulatory role of the EGF-4 Cys-560–Cys-583 unique bond for αIIbβ3 activation. In this study we further investigated the role of all four integrin unique bonds in both αIIbβ3 and αvβ3. We created β3 mutants harboring serine substitutions of each or both cysteines that disrupt the four unique bonds (Cys-437–Cys-457 in EGF-1, Cys-473–Cys-503 in EGF-2, Cys-523–Cys-544 in EGF-3, and Cys-560–Cys-583 in EGF-4) and transfected them into baby hamster kidney cells together with normal αv or αIIb. Flow cytometry was used to measure surface expression of αIIbβ3 and αvβ3 and their activity state by soluble fibrinogen binding. Most cysteine substitutions caused similarly reduced surface expression of both receptors. Disrupting all four unique disulfide bonds by single cysteine substitutions resulted in variable constitutive activation of αIIbβ3 and αvβ3. In contrast, whereas double C437S/C457S and C473S/C503S mutations yielded constitutively active αIIbβ3 and αvβ3, the C560S/C583S mutation did not, and the C523S/C544S mutation only yielded constitutively active αIIbβ3. Activation of C523S/C544S αvβ3 mutant by activating antibody and dithiothreitol was also impaired. Molecular dynamics of C523S/C544S β3 in αIIbβ3 but not in αvβ3 displayed an altered stable conformation. Our findings indicate that unique disulfide bonds in β3 differently affect the function of αIIbβ3 and αvβ3 and suggest a free sulfhydryl-dependent regulatory role for Cys-560–Cys-583 in both αIIbβ3 and αvβ3 and for Cys-523–Cys-544 only in αvβ3.

A 13‐bp deletion in αIIb gene is a founder mutation that predominates in Palestinian‐Arab patients with Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder caused by lack or dysfunction of αIIbβ3 in platelets. GT is relatively frequent in highly inbred populations. We previously identified a 13‐bp deletion in the αIIb gene that causes in‐frame deletion of six amino acids in three Palestinian GT patients. In this study, we determined the molecular basis of GT in all known Palestinian patients, examined whether Jordanian patients harbor the same mutations, analyzed whether there is a founder effect for the 13‐bp deletion, and determined the mechanism by which the 13‐bp deletion abolishes αIIbβ3 surface expression. Of 11 unrelated Palestinian patients, eight were homozygous for the 13‐bp deletion that displayed common ancestry by haplotype analysis, and was estimated to have occurred 300–600 years ago. Expression studies in baby hamster kidney cells showed that substitution of Cys107 or Trp110 located within the deletion caused defective αIIbβ3 maturation. Substitution of Trp110, but not of Cys107, prevented fibrinogen binding. The other Palestinian patients harbored three novel mutations: G2374 deletion in αIIb gene, TT1616‐7 deletion in β3 gene, and IVS14: −3C → G in β3 gene. The latter mutation caused cryptic splicing predicting an extended cytoplasmic tail of β3 and was expressed as dysfunctional αIIbβ3. None of 15 unrelated Jordanian patients carried any of the described mutations.

E109K is a SEC23B founder mutation among Israeli Moroccan Jewish patients with congenital dyserythropoietic anemia type II.

Objective: Congenital dyserythropoietic anemia (CDA) is characterized by ineffective erythropoiesis, binuclearity of erythroid precursors and secondary hemochromatosis. Recently, the gene mutated in CDA type II (CDA II), SEC23B, was identified. All Israeli patients with CDA II are of North African (mainly Moroccan) Jewish descent. We investigated the molecular basis of CDA II in those patients. Methods: Participants included 11 patients with CDA II from 8 apparently unrelated families. Clinical data were retrieved from medical files, and blood was collected for DNA analysis. Results: The majority of patients (10/11) were homozygous for a common SEC23B mutation (E109K). Haplotype analysis revealed a common genetic background in all patients. One patient was a compound heterozygote for the E109K mutation and a novel mutation, T710M. All patients were transfusion independent, with increasing iron overload with age. We estimate the E109K mutation to be 2,400 years old, in line with Jewish migration history. Conclusions: Most CDA II patients in Israel are of Moroccan Jewish origin and carry a common SEC23B mutation, E109K, the first to be described as a founder mutation causing CDA II. As previously suggested, carrying 2 missense mutations is associated with a relatively nonsevere phenotype.

Age estimates of ancestral mutations causing factor VII deficiency and Dubin-Johnson syndrome in Iranian and Moroccan Jews are consistent with ancient Jewish migrations.

Factor VII (FVII) deficiency and Dubin-Johnson syndrome (DJS) are rare autosomal recessive disorders caused by mutations in F7 and MRP2 genes, respectively. Both disorders are relatively frequent among Iranian and Moroccan Jews. FVII deficiency in both populations is caused by a founder A244V mutation in the F7 gene and DJS is caused by two founder mutations, I1173F and R1150H in the MRP2 gene that are specific for Iranian and Moroccan Jewish patients, respectively. We estimated the age of FVII A244V and MRP2 I1173F by analysis of microsatellite markers flanking F7 and MRP2 genes, respectively, in 13 Iranian Jewish homozygotes for the I1173F mutation and 21 Iranian and Moroccan Jewish homozygotes for the A244V mutation. Dating of the mutations was estimated by the DMLE+2.0 program employing observed linkage disequilibria of multiple genetic markers. The estimated age of the I1173F mutation was approximately 1500 years, and the age of the A244V mutation was approximately 2600 years. These estimates suggest that I1173F causing DJS in Iranian Jews occurred after the separation of Iranian Jews from Moroccan Jews 2000–2600 years ago, while A244V causing FVII deficiency in Iranian and Moroccan Jews occurred prior to the divergence of these two populations.

An allosteric disulfide bond is involved in enhanced activation of factor XI by protein disulfide isomerase.

Essentials Reduction of three disulfide bonds in factor (F) XI enhances chromogenic substrate cleavage. We measured FXI activity upon reduction and identified a bond involved in the enhanced activity. Reduction of FXI augments FIX cleavage, probably by faster conversion of FXI to FXIa. The Cys362‐Cys482 disulfide bond is responsible for FXI enhanced activation upon its reduction.

A mutation in the β3 cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of αIIbβ3 to an active state

Summary.  Background: The cytoplasmic tails of αIIb and β3 regulate essential αIIbβ3 functions. We previously described a variant Glanzmann thrombasthenia mutation in the β3 cytoplasmic tail, IVS14: −3C>G, which causes a frameshift with an extension of β3 by 40 residues. Objectives: The aim of this study was to characterize the mechanism by which the mutation abrogates transition of αIIbβ3 from a resting state to an active state. Methods: We expressed the natural mutation, termed 742ins, and three artificial mutations in baby hamster kidney (BHK) cells along with wild‐type (WT) αIIb as follows: β3‐742stop, a truncated mutant to evaluate the effect of deleted residues; β3‐749stop, a truncated mutant that preserves the NPLY conserved sequence; and β3‐749ins, in which the aberrant tail begins after the conserved sequence. Flow cytometry was used to determine ligand binding to BHK cells. Results and conclusions: Surface expression of αIIbβ3 of all four mutants was at least 60% of WT expression, but there was almost no binding of soluble fibrinogen following activation with activating antibodies (anti‐ligand‐induced‐binding‐site 6 [antiLIBS6] or PT25‐2). Activation of the αIIbβ3 mutants was only achieved when both PT25‐2 and antiLIBS6 were used together or following treatment with dithiothreitol. These data suggest that the ectodomain of the four mutants is tightly locked in a resting conformation but can be forced to become active by strong stimuli. These data and those of others indicate that the middle part of the β3 tail is important for maintaining αIIbβ3 in a resting conformation.

Mechano-redox control of integrin de-adhesion

How proteins harness mechanical force to control function is a significant biological question. Here we describe a human cell surface receptor that couples ligand binding and force to trigger a chemical event which controls the adhesive properties of the receptor. Our studies of the secreted platelet oxidoreductase, ERp5, have revealed that it mediates release of fibrinogen from activated platelet αIIbβ3 integrin. Protein chemical studies show that ligand binding to extended αIIbβ3 integrin renders the βI-domain Cys177-Cys184 disulfide bond cleavable by ERp5. Fluid shear and force spectroscopy assays indicate that disulfide cleavage is enhanced by mechanical force. Cell adhesion assays and molecular dynamics simulations demonstrate that cleavage of the disulfide induces long-range allosteric effects within the βI-domain, mainly affecting the metal-binding sites, that results in release of fibrinogen. This coupling of ligand binding, force and redox events to control cell adhesion may be employed to regulate other protein-protein interactions.