In vitro evolution of horse heart myoglobin to increase peroxidase activity

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k1 (for H2O2 oxidation of metmyoglobin) of 1.34 × 104 M−1 s−1 (≈25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2′-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 × 106 M−1 s−1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1°C vs. 76.5°C). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 × 10−6 M−1 s−1 vs. 22.3 × 10−6 M−1 s−1; koff, 24.3 s−1 vs. 24.2 s−1; KO2, 0.91 × 10−6 M−1 vs. 0.92 × 10−6 M−1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 × 10−6 M−1s−1 vs. 0.51 × 10−6 M−1s−1; koff, 5.08 s−1 vs. 3.51 s−1; KCO, 1.77 × 10−7 M−1 vs. 1.45 × 10−7 M−1). All four substitutions are in the heme pocket and within 5 Å of the heme group.

Inhibition of MHC class I-restricted antigen presentation by γ2-herpesviruses

The γ-herpesviruses, in contrast to the α- and β-herpesviruses, are not known to inhibit antigen presentation to CD8+ cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine γ-herpesvirus 68 causes a chronic lytic infection in CD4+ T cell-deficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, γ-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two γ-herpesviruses allows continued lytic infection in the face of strong CTL immunity.

Angiostatin gene transfer: Inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest

The antitumoral effects that follow the local delivery of the N-terminal fragment of human plasminogen (angiostatin K3) have been studied in two xenograft murine models. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molecule from the cytomegalovirus promoter (AdK3). In in vitro studies, AdK3 selectively inhibited endothelial cell proliferation and disrupted the G2/M transition induced by M-phase-promoting factors. AdK3-infected endothelial cells showed a marked mitosis arrest that correlated with the down-regulation of the M-phase phosphoproteins. A single intratumoral injection of AdK3 into preestablished rat C6 glioma or human MDA-MB-231 breast carcinoma grown in athymic mice was followed by a significant arrest of tumor growth, which was associated with a suppression of neovascularization within and at the vicinity of the tumors. AdK3 therapy also induced a 10-fold increase in apoptotic tumor cells as compared with a control adenovirus. Furthermore, we showed that systemic injection of AdK3 delayed C6 tumor establishment and growth, confirming that angiostatin can function in a paracrin manner. Our data support the concept that targeted antiangiogenesis, using adenovirus-mediated gene transfer, represents a promising alternative strategy for delivering antiangiogenic factors as their bolus injections present unsolved pharmacological problems.