David T. McPherson

Elastic protein-based polymers in soft tissue augmentation and generation

Five elastic protein-based polymers, designed as variations of polymer I, (GVGVP)251, elicited different responses when injected as subcutaneous implants in the guinea pig, a preclinical test used to evaluate materials for soft tissue augmentation and specifically for correction of urinary incontinence. All six polymers, prepared using recombinant DNA technology, expressed at good levels using transformed E. coli fermentation. These E. coli-produced polymers were purified for the first time to the exacting levels required for use as biomaterials where a large quantity could disperse into the tissues in a few days. Time periods of 2 and 4 weeks were used. Polymer I functioned as a bulking agent around which a fine fibrous capsule formed. Inclusion of (GVGVAP)8, a chemoattractant toward monocytes and elastin-synthesizing fibroblasts in the sequence of polymer I, resulted in an appropriate tissue response of invasion of macrophages. Inclusion of lysine residues, for lysyl oxidase cross-linking, suggested a possible remodeling of the implant toward fibers. Most promising however, when the cell attachment sequence, GRGDSP, was added to polymer I, the implant elicited tissue generation with a normal complement of collagen and elastic fibers, spindle-shaped histiocytes and angiogenesis. If this response is retained over time, the desired soft tissue augmentation and generation will have been achieved. Our working hypothesis is that on formation of elastin, with a half-life of the order of 70 years, a long lasting soft tissue augmentation would result rather than scar tissue as occurs with Contigen, the currently approved injectable implant for soft tissue augmentation.

Hyper expression of an environmentally friendly synthetic polymer gene

SummaryBiodegradable polymers offer an environmentally friendly alternative to petroleum-based polymers. Applications of protein based polymers include the use of these compounds in the fields of medicine, molecular-based energy conversions, the manufacture of unique fibers, coatings and biodegradable plastics. We report here expression of a synthetic gene G-(VPGVG) 119-VPGV coding for the EG-120mer (elastomer) in E. coli. Polymer expression is observed in uninduced cells grown in terrific broth in polyacrylamide gels negatively stained with CuCl2. Electron micrographs reveal formation of inclusion bodies in uninduced cells occupying upto 80–90% of the cell volume under optimal growth conditions. To the best of our knowledge this report represents the first demonstration of hyper expression of a synthetic gene (with no natural analog) in E. coli.

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.

Purification, crystallization and preliminary X-ray diffraction of a complex between IL-10 and soluble IL-10R1

A complex between interleukin-10 and the extracellular domain of its high-affinity receptor (sIL-10R1) has been crystallized from polyethylene glycol solutions. Crystals suitable for diffraction analysis required the modification of the NXS/T glycosylation sites on sIL-10R1 by site-directed mutagenesis and inclusion of the detergent cyclohexyl-methyl-beta-D-maltopyranoside in the crystallization experiments. The crystals belong to space group P3(2)12 or its enantimorph P3(1)12, with unit-cell parameters a = b = 46.23, c = 307.78 A, alpha = beta = 90, gamma = 120 degrees, and diffract X-rays to approximately 2.9 A. The IL-10 dimer is positioned on a crystallographic twofold, resulting in one IL-10 chain and one sIL-10R1 chain in the asymmetric unit, which corresponds to a solvent content of approximately 44%.

Expression of the recombinant human immunoglobulin J chain in Escherichia coli

Selective transport of polymeric (p) immunoglobulins (Ig) of IgA and IgM isotypes into external secretions by pIg receptor-mediated mechanism depends on the incorporation of joining (J) chain into the polymers. Until now, availability of a free J chain for immunological and biophysical studies has been limited to preparations of denatured J chain forms with moderate yield. Here we report that a recombinant J chain (rJ) can be over-expressed as a soluble fusion protein with thioredoxin using a modified vector pET32 in Escherichia coli. An intact J chain was released by digestion with IgA1 protease from Neisseria gonorrhoeae and isolated in a good yield with immunological and biochemical properties similar to those of J chain obtained by chemical cleavage from pIgA.

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.

Engineering protein-based machines to emulate key steps of metabolism (biological energy conversion)

Metabolism is the conversion of available energy sources to those energy forms required for sustaining and propagating living organisms; this is simply biological energy conversion. Proteins are the machines of metabolism; they are the engines of motility and the other machines that interconvert energy forms not involving motion. Accordingly, metabolic engineering becomes the use of natural protein-based machines for the good of society. In addition, metabolic engineering can utilize the principles, whereby proteins function, to design new protein-based machines to fulfill roles for society that proteins have never been called upon throughout evolution to fulfill. This article presents arguments for a universal mechanism whereby proteins perform their diverse energy conversions; it begins with background information, and then asserts a set of five axioms for protein folding, assembly, and function and for protein engineering. The key process is the hydrophobic folding and assembly transition exhibited by properly balanced amphiphilic protein sequences. The fundamental molecular process is the competition for hydration between hydrophobic and polar, e.g., charged, residues. This competition determines Tt, the onset temperature for the hydrophobic folding and assembly transition, Nhh, the numbers of waters of hydrophobic hydration, and the pKa of ionizable functions. Reported acid-base titrations and pH dependence of microwave dielectric relaxation data simultaneously demonstrate the interdependence of Tt, Nhh and the pKa using a series of microbially prepared protein-based poly(30mers) with one glutamic acid residue per 30mer and with an increasing number of more hydrophobic phenylalanine residues replacing valine residues. Also, reduction of nicotinamides and flavins is shown to lower Tt, i.e., to increase hydrophobicity. Furthermore, the argument is presented, and related to an extended Henderson-Hasselbalch equation, wherein reduction of nicotinamides represents an increase in hydrophobicity and resulting hydrophobic-induced pKa shifts become the basis for understanding a primary energy conversion (proton transport) process of mitochondria. Copyright 1998 John Wiley & Sons, Inc.

Key amino acid residues in the melanocortin-4 receptor for nonpeptide THIQ specific binding and signaling.

Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and glucose homeostasis. Synthetic nonpeptide compound N- (3R)-1 4-tetrahydroisoquinolinium-3-ylcarbonyl-(1R)-1-(4-chlorobenzyl)-2-4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl-2-oxoethylamine (THIQ) is a potent agonist at MC4R but not at hMC2R. In this study, we utilized two approaches (chimeric receptor and site-directed mutagenesis) to narrow down the key amino acid residues of MC4R responsible for THIQ binding and signaling. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TMs) of the human MC4R (hMC4R) with the homologous regions of hMC2R were constructed. Our results indicate that the cassette substitutions of these TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter THIQ binding affinity and potency except the substitution of the hMC4R TM3, suggesting that the conserved amino acid residues in these TMs of the hMC4R are main potential candidates for THIQ binding and signaling while non conserved residues in TM3 of MC4R may also be involved. Nineteen MC4R mutants were then created, including 13 conserved amino acid residues and 6 non-conserved amino acid residues. Our results indicate that seven conserved residue [E100 (TM2), D122 (TM3), D126 (TM3), F254 (TM6), W258 (TM6), F261 (TM6), H264 (TM6)] are important for THIQ binding and three non-conserved residues [N123 (TM3), I129 (TM3) and S131 (TM3)] are involved in THIQ selectivity. In conclusion, our results suggest that THIQ utilize both conserved and non-conserved amino acid residues for binding and signaling at hMC4R and non conserved residues may be responsible for MC4R selectivity.

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-β 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.

Structural insight into the role of the human melanocortin 3 receptor cysteine residues on receptor function.

Melanocortin-3 receptor (MC3R), expressed in the hypothalamus and limbic systems of the brain, as well as by peripheral sites, plays an important role in the regulation of energy homeostasis and other physiological functions. Past work shows that MC3R-deficiency resulted in fat mass increase, feeding efficiency increase, hyperleptinemia and mild hyperinsulinemia in mice and human. MC3R belongs to G-protein coupled receptor (GPCR) family and many studies indicate that some cysteine residues in GPCR play key roles in maintaining receptor tertiary structure and function. In this study, we examined the role of cysteine residues in MC3R on receptor function. Human MC3R (hMC3R) has eighteen cysteine residues where they are located in the extracellular loops (ELs), the transmembrane domains (TMs) and the intracellular loops (ILs). We replaced these cysteines with serine and expressed these receptors in HEK-293 cells which lack endogenous MC3R. Our results indicate that five cysteines in eighteen of the hMC3R are important for hMC3R function. Mutations, C305S, C311S, and C313S in EL3, resulted in significant decrease in receptor expression and receptor function while two other mutations C115S and C162S in TM3 significantly decreased NDP-MSH binding affinity and potency. These results suggest that extracellular cysteine residue 305, 311 and 313 are crucial for receptor expression and the transmembrane cysteine residue, C115 and 162 are important for ligand binding and signaling. These findings provide important insights into the importance of cysteine residues of hMC3R on receptor tertiary structure and function.