Paul T. Wingfield

TIMP-2 Mediated Inhibition of Angiogenesis: An MMP-Independent Mechanism

Tissue inhibitors of metalloproteinases (TIMPs) suppress matrix metalloproteinase (MMP) activity critical for extracellular matrix turnover associated with both physiologic and pathologic tissue remodeling. We demonstrate here that TIMP-2 abrogates angiogenic factor-induced endothelial cell proliferation in vitro and angiogenesis in vivo independent of MMP inhibition. These effects require alpha 3 beta 1 integrin-mediated binding of TIMP-2 to endothelial cells. Further, TIMP-2 induces a decrease in total protein tyrosine phosphatase (PTP) activity associated with beta1 integrin subunits as well as dissociation of the phosphatase SHP-1 from beta1. TIMP-2 treatment also results in a concomitant increase in PTP activity associated with tyrosine kinase receptors FGFR-1 and KDR. Our findings establish an unexpected, MMP-independent mechanism for TIMP-2 inhibition of endothelial cell proliferation in vitro and reveal an important component of the antiangiogenic effect of TIMP2 in vivo.

Three‐dimensional solution structure of the 44 kDa ectodomain of SIV gp41

The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e‐gp41), consisting of residues 27–149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e‐gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N‐terminal (residues 30–80) and C‐terminal (residues 107–147) helices connected by a 26 residue loop (residues 81–106). The N‐terminal helices of each subunit form a parallel coiled‐coil structure in the interior of the complex which is surrounded by the C‐terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non‐sequential intramolecular contacts. The helical core of SIV e‐gp41 is similar to recent X‐ray structures of truncated constructs of the helical core of HIV‐1 e‐gp41. The present structure establishes unambiguously the connectivity of the N‐ and C‐terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e‐gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e‐gp41 represents one of the largest protein structures determined by NMR to date.

Solution NMR of proteins within polyacrylamide gels: Diffusional properties and residual alignment by mechanical stress or embedding of oriented purple membranes

The diffusive properties of biomacromolecules within the aqueous phase of polyacrylamide gels are described. High quality NMR spectra can be obtained under such conditions. As compared to water, a fivefold reduction in the translational diffusion constant, but only a 1.6-fold decrease (1.4-fold increase) in amide-15N T2 (T1) are observed for human ubiquitin within a 10% acrylamide gel. Weak alignment of the solute macromolecules can be achieved within such gels by vertical or radial compression or by the embedding of magnetically oriented purple membrane fragments. The methods are applied to derive residual dipolar couplings for human HIV-1 Nef and ubiquitin.

Interleukin-35 induces regulatory B cells that suppress autoimmune disease

Interleukin-10 (IL-10)-producing regulatory B (Breg) cells suppress autoimmune disease, and increased numbers of Breg cells prevent host defense to infection and promote tumor growth and metastasis by converting resting CD4+ T cells to regulatory T (Treg) cells. The mechanisms mediating the induction and development of Breg cells remain unclear. Here we show that IL-35 induces Breg cells and promotes their conversion to a Breg subset that produces IL-35 as well as IL-10. Treatment of mice with IL-35 conferred protection from experimental autoimmune uveitis (EAU), and mice lacking IL-35 (p35 knockout (KO) mice) or defective in IL-35 signaling (IL-12Rβ2 KO mice) produced less Breg cells endogenously or after treatment with IL-35 and developed severe uveitis. Adoptive transfer of Breg cells induced by recombinant IL-35 suppressed EAU when transferred to mice with established disease, inhibiting pathogenic T helper type 17 (TH17) and TH1 cells while promoting Treg cell expansion. In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rβ2 and IL-27Rα subunits. As IL-35 also induced the conversion of human B cells into Breg cells, these findings suggest that IL-35 may be used to induce autologous Breg and IL-35+ Breg cells and treat autoimmune and inflammatory disease.

Flexibility and function in HIV-1 protease

HIV protease is a homodimeric protein whose activity is essential to viral function. We have investigated the molecular dynamics of the HIV protease, thought to be important for proteinase function, bound to high affinity inhibitors using NMR techniques. Analysis of 15N spin relaxation parameters, of all but 13 backbone amide sites, reveals the presence of significant internal motions of the protein backbone. In particular, the flaps that cover the proteins active site of the protein have terminal loops that undergo large amplitude motions on the ps to ns time scale, while the tips of the flaps undergo a conformational exchange on the μs time scale. This enforces the idea that the flaps of the proteinase are flexible structures that facilitate function by permitting substrate access to and product release from the active site of the enzyme.

A powerful method of sequential proton resonance assignment in proteins using relayed 15N-1H multiple quantum coherence spectroscopy

A powerful method of sequential resonance assignment of protein 1H‐NMR spectra is presented and illustrated with respect to the DNA‐binding protein ner from phage Mu. It is based on correlating proton‐proton through‐space and through‐bond connectivities with the chemical shift of the directly bonded 15N atom. By this means, ambiguities arising from chemical shift degeneracy of amide proton resonances can be resolved. The experiments described involve combining the 1H‐detected heteronuclear multiple quantum coherence correlation experiment with homonuclear nuclear Overhauser enhancement, J‐correlated or Hartmann‐Hahn experiments.

Anisotropic rotational diffusion of perdeuterated HIV protease from 15N NMR relaxation measurements at two magnetic fields

Summary15N NMR relaxation times in perdeuterated HIV-1 protease, complexed with the sub-nanomolar inhibitor DMP323, have been measured at 600 and 360 MHz 1H frequency. The relative magnitudes of the principal components of the inertia tensor, calculated from the X-ray coordinates of the protein-drug complex, are 1.0:0.85:0.44. The relation between the T1/T2 ratios observed for the individual backbone amides and their N-H orientation within the 3D structure of the protease dimer yields a rotational diffusion tensor oriented nearly collinear to the inertia tensor. The relative magnitudes of its principal components (1.00:1.11:1.42) are also in good agreement with hydrodynamic modeling results. The orientation and magnitude of the diffusion tensors derived from relaxation data obtained at 360 and 600 MHz are nearly identical. The anisotropic nature of the rotational diffusion has little influence on the order parameters derived from the 15N T1 and T2 relaxation times; however, if anisotropy is ignored, this can result in erroneous identification of either exchange broadening or internal motions on a nanosecond time scale. The average ratio of the T1 values measured at 360 and 600 MHz is 0.50±0.015, which is slightly larger than the value of 0.466 expected for an isotropic rigid rotor with τc = 10.7 ns. The average ratio of the T2 values measured at 360 and 600 MHz is 1.14±0.04, which is also slightly larger than the expected ratio of 1.11. This magnetic field dependence of the T1 and T2 relaxation times suggests that the spectral density contribution from fast internal motions is not negligible, and that the chemical shift anisotropy of peptide backbone amides, on average, is larger than the 160 ppm value commonly used in 15N relaxation studies of proteins.

High-resolution mass spectrometry of viral assemblies: molecular composition and stability of dimorphic hepatitis B virus capsids.

Hepatitis B virus (HBV) is a major human pathogen. In addition to its importance in human health, there is growing interest in adapting HBV and other viruses for drug delivery and other nanotechnological applications. In both contexts, precise biophysical characterization of these large macromolecular particles is fundamental. HBV capsids are unusual in that they exhibit two distinct icosahedral geometries, nominally composed of 90 and 120 dimers with masses of ≈3 and ≈4 MDa, respectively. Here, a mass spectrometric approach was used to determine the masses of both capsids to within 0.1%. It follows that both lattices are complete, consisting of exactly 180 and 240 subunits. Nanoindentation experiments by atomic-force microscopy indicate that both capsids have similar stabilities. The data yielded a Youngs modulus of ≈0.4 GPa. This experimental approach, anchored on very precise and accurate mass measurements, appears to hold considerable potential for elucidating the assembly of viruses and other macromolecular particles.

Three-dimensional structure of ubiquinol: Cytochrome C reductase from Neurospora mitochondria determined by electron microscopy of membrane crystals

Abstract Membrane crystals have been prepared from mitochondrial ubiquinol: cytochrome c reductase by mixing the enzyme-Triton complex with phospholipid-Triton micelles and subsequently removing the Triton. The electron micrographs of the negatively stained crystals diffract to 2·5 nm, with unit cell dimensions of 13·7 nm by 17·4 nm. The enzyme is arranged in a two-sided plane group P 22 1 2 1 , i.e. alternate molecules span the bilayer in an up and down manner. By combining tilted views of the membrane crystals, a low-resolution three-dimensional structure of the enzyme has been calculated. The structure shows that the enzyme is a dimer, the monomers being related by a 2-fold axis running perpendicular to the membrane. The monomeric units of the enzyme are elongated, extending approximately 15 nm across the membrane. The protein is unequally distributed with about 30% of the total mass located in the bilayer, 50% in a section which extends 7 nm from one side of the bilayer and 20% in a section which extends 3 nm from the opposite side of the bilayer. The two monomeric units are in contact only in the membraneous section. This structure is compared with a model of the enzyme which is derived from biochemical properties of the isolated subunits.

Hepatitis B virus antigens made in microbial cells immunise against viral infection.

Chimpanzees have been vaccinated successfully against hepatitis B virus with preparations of the viral antigens made in microbial cells by genetic engineering methods.