Addition of an oligoglutamate domain to bone morphogenic protein 2 confers binding to hydroxyapatite materials and induces osteoblastic signaling.

Nonautologous bone grafts have limited osteoinductive potential and thus there is substantial interest in reconstituting these graft materials with osteogenic factors such as bone morphogenic protein 2 (BMP2). However, one limitation of this approach is that BMP2 is typically weakly bound to the graft, which can lead to side effects associated with BMP2 dissemination. In the current study we added a hydroxyapatite (HA)-binding domain onto BMP2 to increase coupling to the graft surface. A sequence consisting of eight glutamate residues (E8) was inserted into the C-terminus of BMP2, and the recombinant protein (rBMP2-E8) was expressed in E. coli. Compared with rBMP2, rBMP2-E8 displayed markedly enhanced binding to HA disks and was better retained on the disks following exposure to vigorous wash steps. Furthermore, rBMP2-E8 was purified using a heparin column, and evaluated for its capacity to stimulate osteoblastic cell signaling. Treatment of SAOS2 cells with rBMP2-E8 induced SMAD 1/5 activation, confirming that the protein retains activity. Collectively these results suggest that the E8 domain serves as an effective tool for improving rBMP2 coupling to graft materials. The increased retention of rBMP2-E8 on the graft surface is expected to prolong BMP2's osteoinductive activity within the graft site, while simultaneously reducing off-target effects.

Lipid complex of apolipoprotein A-I mimetic peptide 4F is a novel platform for paraoxonase-1 binding and enhancing its activity and stability.

High density lipoprotein (HDL) associated paraoxonase-1 (PON1) is crucial for the anti-oxidant, anti-inflammatory, and anti-atherogenic properties of HDL. Discoidal apolipoprotein (apo)A-I:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) complex has been shown to be the most effective in binding PON1, stabilizing it, and enhancing its lactonase and inhibitory activity of low density lipoprotein oxidation. Based on our earlier study demonstrating that apoA-I mimetic peptide 4F forms discoidal complex with 1,2-dimyristoyl-sn-glycero-3-phosphocholine, we hypothesized that lipid complexes of 4F would be able to bind PON1 and enhance its activity and stability. To test our hypothesis, we have expressed and purified a recombinant PON1 (rPON1) and studied its interaction with 4F:POPC complex. Our studies show significant increase, compared to the control, in the paraoxonase activity and stability of rPON1 in the presence of 4F:POPC complex. We propose that 4F:POPC complex is a novel platform for PON1 binding, increasing its stability, and enhancing its enzyme activity. We propose a structural model for the 4F:POPC:PON1 ternary complex that is consistent with our results and published observations.

Sidedness of interfacial arginine residues and anti-atherogenicity of apolipoprotein A-I mimetic peptides.

To test the hypothesis that sidedness of interfacial arginine (Arg) in apoA-I mimetic peptides, similar to that observed in apoA-I (Bashtovyy, D. et al. 2011. Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function. J. Lipid Res. 52: 435-450.), may be important for biological activity, we compared properties of 4F and analogs, [K4,¹5>R]4F and [K9,¹³>R]4F, with Lys>Arg substitutions on the right and left side, respectively, of the 4F amphipathic helix. Intraperitoneal administration of these peptides into female apoE null mice (n = 13 in each group) reduced en face lesions significantly compared with controls; 4F and [K4,¹5>R]4F were equally effective whereas [K9,¹³>R]4F was less effective. Turnover experiments indicated that [K4,¹5>R]4F reached the highest, whereas [K9,¹³>R]4F had the lowest, plasma peak levels with a similar half life as the [K4,¹5>R]4F analog. The half life of 4F was two times longer than the other two peptides. The order in their abilities to associate with HDL in human plasma, generation of apoA-I particles with pre-ß mobility from isolated HDL, lipid associating ability, and sensitivity of lipid complexes to trypsin digestion was: 4F>[K4,¹5,>R]4F>[K9,¹³>R]4F. These studies support our hypothesis that the sidedness of interfacial Arg residues in the polar face of apoA-I mimetics results in differential biological properties.

A rev1-vpu polymorphism unique to HIV-1 subtype A and C strains impairs envelope glycoprotein expression from rev-vpu-env cassettes and reduces virion infectivity in pseudotyping assays.

Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev-vpu-env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1-vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1-vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01_A/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev-vpu-env cassettes since this increases Env expression irrespective of the presence of the polymorphism.

Effect of NaCl on the exothermic and endothermic components of the inverse temperature transition of a model elastin-like polymer.

TMDSC data have been employed to observe the effect of NaCl on the inverse temperature transition of the model elastin-like polymer (GVGVP)251. NaCl causes a decrease in Tt and an increase in DeltaH. The increase in enthalpy appears both in the enthalpy related with the folding of the polymer and in the contribution associated with disruption of the structured water of hydrophobic hydration. It has been suggested that the presence of NaCl may cause a better formation of water structures surrounding the apolar polymer chains.

Micro-RNA-like effects of complete intronic sequences.

MicroRNAs (miRNAs) have been suggested as suppressors of numerous target genes in human cells. In this report, we present gene chip array data indicating that in the absence of miRNA sequences, complete human introns are similarly capable of coordinating expression of large numbers of gene products at spatially diverse sites within the genome. The expression of selected intronic sequences (6a, 14b and 23) derived from the cystic fibrosis transmembrane conductance regulator (CFTR) gene caused extensive and specific transcriptional changes in epithelial cells (HeLa) that do not normally express this gene product. Each intron initiated a distinctive pattern of gene transcription. Affected genes such as FOXF1, sucrase-isomaltase, collagen, interferon, complement and thrombospondin 1 have previously been linked to CFTR function or are known to contribute to the related processes of epithelial differentiation and repair. A possible regulatory function of this nature has not been demonstrated previously for non-coding sequences within eukaryotic DNA. The results are consistent with the observation that splicesomal introns are found only in eukaryotic organisms and that the number of introns increases with phylogenetic complexity.

Designer gene therapy using an Escherichia coli purine nucleoside phosphorylase/prodrug system.

Activation of prodrugs by Escherichia coli purine nucleoside phosphorylase (PNP) provides a method for selectively killing tumor cells expressing a transfected PNP gene. This gene therapy approach requires matching a prodrug and a known enzymatic activity present only in tumor cells. The specificity of the method relies on avoiding prodrug cleavage by enzymes already present in the host cells or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave new prodrug substrates more efficiently than does the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(6-deoxy-alpha-L-talofuranosyl)-6-methylpurine with a kcat/Km over 100 times greater than for native E. coli PNP. In a xenograft tumor experiment, this compound caused regression of tumors expressing the M64V PNP gene.

The function of hydrophobic residues in the catalytic cleft of Streptococcus pneumoniae hyaluronate lyase. Kinetic characterization of mutant enzyme forms.

Streptococcus pneumoniae hyaluronate lyase is a surface antigen of this Gram-positive human bacterial pathogen. The primary function of this enzyme is the degradation of hyaluronan, which is a major component of the extracellular matrix of the tissues of vertebrates and of some bacteria. The enzyme degrades its substrate through a beta-elimination process called proton acceptance and donation. The inherent part of this degradation is a processive mode of action of the enzyme degrading hyaluronan into unsaturated disaccharide hyaluronic acid blocks from the reducing to the nonreducing end of the polymer following the initial random endolytic binding to the substrate. The final degradation product is the unsaturated disaccharide hyaluronic acid. The residues of the enzyme that are involved in various aspects of such degradation were identified based on the three-dimensional structures of the native enzyme and its complexes with hyaluronan substrates of various lengths. The catalytic residues were identified to be Asn(349), His(399), and Tyr(408). The residues responsible for the release of the product of the reaction were identified as Glu(388), Asp(398), and Thr(400), and they were termed negative patch. The hydrophobic residues Trp(291), Trp(292), and Phe(343) were found to be responsible for the precise positioning of the substrate for enzyme catalysis and named hydrophobic patch. The comparison of the specific activities and kinetic properties of the wild type and the mutant enzymes involving the hydrophobic patch residues W292A, F343V, W291A/W292A, W292A/F343V, and W291A/W292A/F343V allowed for the characterization of every mutant and for the correlation of the activity and kinetic properties of the enzyme with its structure as well as the mechanism of catalysis.