Fatbardha Varfaj

Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface

Factor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. The charged residues Glu272, Asp519, Glu665, and Glu1984 appear buried at the interface of the A2 domain with either the A1 or A3 domain, and thus may impact protein stability. To determine the effects of these residues on procofactor/cofactor stability, these residues were individually replaced with either Ala or Val, and stable BHK cell lines expressing the B-domainless proteins were prepared. Specific activity and thrombin generation parameters for 7 of the 8 variants were more than 80% the wild-type value. Factor VIII activity at 52 degrees C to 60 degrees C and the decay of factor VIIIa activity after thrombin activation were monitored. Six of the 7 variants showing wild-type-like activity demonstrated enhanced stability, with the Glu1984Val variant showing a 2-fold increase in thermostability and an approximately 4- to 8-fold increase in stability of factor VIIIa. These results indicate that replacement of buried charged residues is an effective alternative to covalent modification in increasing factor VIII (VIIIa) stability.

Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa

APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate approximately 25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated approximately 9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated approximately 2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a K(m) value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than K(i) values (approximately 9-18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341-345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.

Role of Cysteine Residues in Heme Binding to Human Heme Oxygenase-2 Elucidated by Two-dimensional NMR Spectroscopy

Background: Heme oxygenase-2 (HO-2) containing three cysteines is involved in signaling pathways. Results: The cysteines in HO-2 interact with each other, but their redox state only modestly alters heme affinity. Conclusion: Changes in the redox state of the cysteines do not significantly control heme oxidation rates. Significance: The HO-2 cysteine residues play roles in interactions related to the role of HO-2 in regulatory processes. Human heme oxygenases 1 and 2 (HO-1 and HO-2) degrade heme in the presence of oxygen and NADPH-cytochrome P450 reductase, producing ferrous iron, CO, and biliverdin. HO-1 is an inducible enzyme, but HO-2 is constitutively expressed in selected tissues and is involved in signaling and regulatory processes. HO-2 has three cysteine residues that have been proposed to modulate the affinity for heme, whereas HO-1 has none. Here we use site-specific mutagenesis and two-dimensional NMR of l-[3-13C]cysteine-labeled proteins to determine the redox state of the individual cysteines in HO-2 and assess their roles in binding of heme. The results indicate that in the apoprotein, Cys282 and Cys265 are in the oxidized state, probably in an intramolecular disulfide bond. The addition of a reducing agent converts them to the reduced, free thiol state. Two-dimensional NMR of site-specific mutants reveals that the redox state of Cys265 and Cys282 varies with the presence or absence of other Cys residues, indicating that the microenvironments of the Cys residues are mutually interdependent. Cys265 appears to be in a relatively hydrophilic, oxidizable environment compared with Cys127 and Cys282. Chemical shift data indicate that none of the cysteines stably coordinates to the heme iron atom. In the oxidized state of the apoprotein, heme is bound 2.5-fold more tightly than in the reduced state. This small difference in heme affinity between the oxidized and reduced states of the protein is much less than previously reported, suggesting that it is not a significant factor in the physiological regulation of cellular heme levels.

Residues surrounding Arg336 and Arg562 contribute to the disparate rates of proteolysis of factor VIIIa catalyzed by activated protein C.

Activated Protein C (APC) inactivates factor VIIIa by cleavage at Arg336 and Arg562 within the A1 and A2 subunits, respectively, with reaction at the former site occurring at a rate ∼25-fold faster than the latter. Recombinant factor VIII variants possessing mutations within the P4-P3′ sequences were used to determine the contributions of these residues to the disparate cleavage rates at the two P1 sites. Specific activity values for 336(P4-P3′)562, 336(P4-P2)562, and 336(P1′-P3′)562 mutants, where indicated residues surrounding the Arg336 site were replaced with those surrounding Arg562, were similar to wild type (WT) factor VIII; whereas 562(P4-P3′)336 and 562(P4-P2)336 mutants showed specific activity values <1% the WT value. Inactivation rates for the 336 site mutants were reduced ∼6–11-fold compared with WT factor VIIIa, and approached values attributed to cleavage at Arg562. Cleavage rates at Arg336 were reduced ∼100-fold for 336(P4-P3′)562, and ∼9–16-fold for 336(P4-P2)562 and 336(P1′-P3′)562 mutants. Inhibition kinetics revealed similar affinities of APC for WT factor VIIIa and 336(P4-P3′)562 variant. Alternatively, the 562(P4-P3′)336 variant showed a modest increase in cleavage rate (∼4-fold) at Arg562 compared with WT, whereas these rates were increased by ∼27- and 6-fold for 562(P4-P3′)336 and 562(P4-P2)336, respectively, using the factor VIII procofactor form as substrate. Thus the P4-P3′ residues surrounding Arg336 and Arg562 make significant contributions to proteolysis rates at each site, apparently independent of binding affinity. Efficient cleavage at Arg336 by APC is attributed to favorable P4-P3′ residues at this site, whereas cleavage at Arg562 can be accelerated following replacement with more optimal P4-P3′ residues.

Carbon-Carbon Bond Cleavage in Activation of the Prodrug Nabumetone

Carbon-carbon bond cleavage reactions are catalyzed by, among others, lanosterol 14-demethylase (CYP51), cholesterol side-chain cleavage enzyme (CYP11), sterol 17β-lyase (CYP17), and aromatase (CYP19). Because of the high substrate specificities of these enzymes and the complex nature of their substrates, these reactions have been difficult to characterize. A CYP1A2-catalyzed carbon-carbon bond cleavage reaction is required for conversion of the prodrug nabumetone to its active form, 6-methoxy-2-naphthylacetic acid (6-MNA). Despite worldwide use of nabumetone as an anti-inflammatory agent, the mechanism of its carbon-carbon bond cleavage reaction remains obscure. With the help of authentic synthetic standards, we report here that the reaction involves 3-hydroxylation, carbon-carbon cleavage to the aldehyde, and oxidation of the aldehyde to the acid, all catalyzed by CYP1A2 or, less effectively, by other P450 enzymes. The data indicate that the carbon-carbon bond cleavage is mediated by the ferric peroxo anion rather than the ferryl species in the P450 catalytic cycle. CYP1A2 also catalyzes O-demethylation and alcohol to ketone transformations of nabumetone and its analogs.

A3 domain residue Glu1829 contributes to A2 subunit retention in factor VIIIa.

Summary.  Background: Factor VIII (FVIII) is activated by thrombin to the labile FVIIIa, a heterotrimer of A1, A2 and A3C1C2 subunits, which serves as a cofactor for FIXa. A primary reason for the instability of FVIIIa is the tendency for the A2 subunit to dissociate from FVIIIa leading to an inactive cofactor and consequent loss of FXase activity. Objective: Based on our finding of low‐specific activity and a fast decay rate for a FVIII point mutation of Glu1829 to Ala (E1829A), we examined whether residue Glu1829 in the A3 subunit is important for A2 subunit retention. Results: The rate of activity decay of E1829A was ∼fourteenfold faster than wild‐type (wt) FVIIIa and this rate was reduced in the presence of added A2 subunit. Specific activity values for E1829A measured by one‐stage and two‐stage assays were ∼14% and ∼11%, respectively, compared with wt FVIII. Binding affinity for the A1 subunit to E1829A‐A3C1C2 was comparable to wt A3C1C2 (Kd = 20.1 ± 3.4 nm for E1829A, 15.3 ± 3.7 nm for wt), whereas A2 subunit affinity for the A1/A3C1C2 dimer forms was reduced by ∼3.6‐fold as a result of the mutation (Kd = 526 ± 107 nm for E1829A, 144 ± 21 nm for wt). Conclusion: As modeling data suggest that Glu1829 is located at the A2‐A3 domain interface these results are consistent with Glu1829 contributing to the interactions involved with A2 subunit retention in FVIIIa.