Joan Hare

The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy

The structure of the adeno-associated virus (AAV-2) has been determined to 3-Å resolution by x-ray crystallography. AAV is being developed as a vector for gene therapy to treat diseases including hemophilia, cancer, and cystic fibrosis. As in the distantly related autonomous parvoviruses, the capsid protein has a β-barrel fold, but long loops between the β-strands share little structural homology with other parvoviruses, leading to unique surface features. Most prominent are groups of threefold-related peaks, each an intimate association of loops from two neighboring subunits. Mutations affecting cell entry and receptor binding are clustered near the positively charged side of each peak, implicating the region in attachment to the cellular receptor, heparan sulfate proteoglycan. Amino acids involved in antibody binding are in the same general vicinity. The structure will guide rational engineering of vector capsids to tailor cellular targeting and to avoid immediate neutralization by an immune system sensitized by prior exposure to AAV.

Role of dietary iron restriction in a mouse model of Parkinson's disease

There is a growing body of evidence suggesting that iron chelation may be a useful therapy in the treatment of Parkinsons Disease (PD). Experiments were designed to test the impact of dietary iron availability on the pathogenic process and functional outcome in a mouse model of PD. Mice were fed diets containing low (4 ppm) or adequate (48 ppm) amounts of iron for 6 weeks before the administration of MPTP, a mitochondrial toxin that damages nigrostriatal dopaminergic neurons and induces Parkinson-like symptoms. Low dietary iron increased serum total iron binding capacity (P < 0.001). Consistent with neuronal protection, iron restriction increased sphingomyelin C16:0 and decreased ceramide C16:0. However, there was a 35% decrease in striatal dopamine (DA) in iron-restricted mice. Motor behavior was also impaired in these animals. In vitro studies suggested that severe iron restriction could lead to p53-mediated neuronal apoptosis. Administration of MPTP reduced striatal DA (P < 0.01) and impaired motor behavior in iron-adequate mice. However, in iron-restricted mice, striatal dopamine levels and motor behavior were unchanged compared to saline-treated mice. Thus, while reduced iron may provide protection against PD-inducing insults such as MPTP, the role of iron in the synthesis of DA and neuronal survival should be considered, particularly in the development of iron-chelating agents to be used chronically in the clinical setting.

Licania michauxii Prance root extract induces hsp 70 mRNA and necrotic cell death in cultured human hepatoma and colon carcinoma cell lines.

An extract of Licania michauxii Prance root was found to be cytotoxic to cultured human hepatoma (HepG2) and colon carcinoma (Caco-2) cells. Morphological and nuclear characteristics of treated cells were consistent with necrotic death. Increases in the chaperone protein hsp 70 and hsp 70 mRNA were dose dependent reaching peak mRNA levels (40-fold above control) at 6 h. Increases in nuclear localization of hsp 70 was also observed with treatment. Heat treatment of cells for 45 min to induce hsp 70 prior to treatment with the extract provided transient protection from the necrotic response.

Twinned crystals of adeno-associated virus serotype 3b prove suitable for structural studies.

Adeno-associated viruses (AAVs) are leading candidate vectors for gene-therapy applications. The AAV-3b capsid is closely related to the well characterized AAV-2 capsid (87% identity), but sequence and presumably structural differences lead to distinct cell-entry and immune-recognition properties. In an effort to understand these differences and to perhaps harness them, diffraction-quality crystals of purified infectious AAV-3b particles have been grown and several partial diffraction data sets have been recorded. The crystals displayed varying levels of merohedral twinning that in earlier times would have rendered them unsuitable for structure determination, but here is shown to be a tractable complication.

Crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 6

Adeno-associated viruses are being developed as vectors for gene therapy and have been used in a number of clinical trials. Vectors to date have been based on the type species AAV-2, the structure of which was published in 2002. There is growing interest in modulating the cellular tropism and immune neutralization of AAV-2 with variants inspired by the properties of other serotypes. Towards the determination of a structure for AAV type 6, this paper reports the high-yield production, purification, crystallization and preliminary diffraction studies of infectious AAV-6 virions. The crystals diffracted to 3.2 A resolution using synchrotron radiation. The most promising crystal form belonged to space group R3 and appeared to be suitable for initial structure determination.

Characterization of the Capsid Protein Glycosylation of Adeno-Associated Virus Type 2 by High-Resolution Mass Spectrometry

ABSTRACT Adeno-associated virus type 2 (AAV-2) capsid proteins have eight sequence motifs that are potential sites for O- or N-linked glycosylation. Three are in prominent surface locations, close to the sites of cellular receptor attachment and to neutralizing epitopes on or near protrusions surrounding the three-fold axes, raising the possibility that AAV-2 might use glycosylation as a means of immune escape or for preventing reattachment on release of progeny virus. Peptide mapping and structural analysis by Fourier transform ion cyclotron resonance mass spectrometry demonstrates, however, no glycosylation of the capsid protein for virus prepared in cultured HeLa cells.

Methylation in eucaryotes influences the repair of G/T and A/C DNA basepair mismatches

Although methylation of DNA at some sites regulates gene expression, 5mC at many sites does not appear to have any effect. We present evidence that hemimethylation at many different sites can act as a discrimination signal in mismatch repair. Deamination of 5mC in a symmetrically methylated doublet CpG yields the mismatched base pair T/G in a hemi-methylated doublet pair. Because both bases in the mismatched pair are normal constituents of DNA, identifying the incorrect base is problematic. The only apparent distinction of the two is the methylation on the strand opposite the deamination event. Using available methylases we have produced hemi-methylated SV40 DNAs that are mismatched at a single T/G or A/C basepair in a sequence that mimics the lesion resulting from the deamination of a 5mCpG. Methylation at the adjacent cytosine results in the replacement of the T much more frequently than when no methylation is present in the heteroduplex. Cytosine methylation at sites farther removed from the mismatch is equally effective in replacing the incorrect T at the mismatch. Although methylation in vertebrates is almost exclusively on cytosine in the doublet CpG, methylation of cytosines in other doublets, as well as methylation of adenosine, also act as strand discrimination signals. Perhaps some of the excess methylation in vertebrate DNAs may serve to direct mismatch repair.

A pseudo-plaque method for infectious particle assay and clonal isolation of adeno-associated virus

A colorimetric method has been developed for the detection of adeno-associated virus (AAV) infectious centers in cell culture monolayers. Due to its non-cytopathic nature, AAV has not been amenable to the traditional plaque assay, involving an agar overlay and cellular stains. As a result, an alternate method was required. The pseudo-plaque assay is based on enzyme-catalyzed color development after a fixed cell monolayer is probed with anti-AAV monoclonal antibodies. In spite of chemical fixation, expected to damage the viral genomes and particles, infectious particles can be recovered and amplified for the propagation of viral clones.

Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-13C-glucose and 15N-glutamate as labeled precursors. This study suggests that uniformly 15N,13C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

A Simple Adaptable Blood-Brain Barrier Cell Model for Screening Matrix Metalloproteinase Inhibitor Functionality

The blood-brain barrier is a multicellular and basement membrane unit that regulates molecular transport between the blood and central nervous system. Many cerebral pathologies, such as acute stroke and chronic vascular dementia, result in a disrupted blood-brain barrier, increasing its permeability and allowing the entry of potentially neurotoxic molecules. The activation of matrix metalloproteinases mediates further blood-brain barrier damage. The inhibition of matrix metalloproteinases is a potential strategy for stroke therapy. As inhibitors are developed, efficient context-specific screening methods will be required. Models of the blood-brain barrier have been extensively used to study neuropathologies and the effect of various treatment options.Herein, we describe a co-culture model of the blood-brain barrier composed of brain microvascular endothelial cells and astrocytes grown on an artificial basement membrane-coated membrane insert. Our cell model forms a barrier and is a simple first approximation of blood-brain barrier integrity. As currently developed, the model may be applied to testing the effect of matrix metalloproteinases and matrix metalloproteinase inhibitors on blood-brain barrier physiology and pathophysiology. The model is a quick and effective evaluation tool for generating nonclinical data in a living cell system before proceeding to animal models.