Expression, purification, and characterization of human malonyl-CoA decarboxylase.

The recombinant human malonyl-CoA decarboxylase (hMCD) was overexpressed in Escherichia coli with and without the first 39 N-terminal amino acids via a cleavable MBP-fusion construct. Proteolytic digestion using genenase I to remove the MBP-fusion tag was optimized for both the full length and truncated hMCD. The apo-hMCD enzymes were solubilized and purified to homogeneity. Steady-state kinetic characterization showed similar kinetic parameters for the MBP-fused and apo-hMCD enzymes with an apparent Km value of approximately 330-520 microM and a turnover rate kcat of 13-28s(-1). For the apo-hMCD enzymes, the N-terminal truncated hMCD was well tolerated over a broad pH range (pH 4-10); whereas the full-length hMCD appeared to be stable only at pH >/= 8.5. Our results showed that the N-terminal region of hMCD has no effect on the catalytic activity of the enzyme but plays a role in the folding process and conformation stability of hMCD.

SP500263, a novel SERM, blocks osteoclastogenesis in a human bone cell model: role of IL-6 and GM-CSF.

Bone metabolism requires tightly coupled activities exhibited by two unique cell populations, the bone-resorbing osteoclasts and the bone-forming osteoblasts. Imbalance in the function of these two cell types can result in osteoporosis, a condition characterized by loss in bone integrity and of bone mass. We developed a human bone cell culture model that allows the in vitro study of bone formation and osteoclastogenesis and employed this bone model for the screening and pharmacological analyses of protein and small molecule therapeutics. The cytokines, interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF), play an intricate role in osteoclastogenesis in this system. Neutralizing antibodies to IL-6 and GM-CSF decreased the formation of osteoclast-like cells. SP500263, an early lead compound from a novel class of selective estrogen receptor modulators (SERMs), was more efficacious than estrogen and comparable to raloxifene in blocking cytokine production and formation of osteoclast-like cells. Our research demonstrates the usefulness of the in vitro co-culture model in the dissection of molecular events relevant to bone metabolism and provides greater insight into a potential novel role for cytokines in bone resorption. Furthermore, representatives of the SP500263 family of SERMs may be effective as therapeutics for the treatment of osteoporosis.

In vitro high-throughput screening assay for modulators of transcription.

We developed a 96-well microtiter-plate high-throughput screening (HTS) assay for the detection of modulators of transcription. This HTS assay consists of three steps: (1) the in vitro transcription reaction; (2) modification and hybridization of RNA products; and (3) washing and quantification. During the first step, a DNA template containing the promoter of interest upstream of a cassette lacking guanosine residues in one of its strands (G-less cassette) is incubated with nuclear extract and the necessary cofactors/activators and substrates. During the second step, the in vitro synthesized transcripts are digested with RNase T1 and hybridized to two DNA oligonucleotides. One oligonucleotide is biotinylated for trapping of the RNA products to a streptavidin-coated plate, and the other is europium-labeled for detection by time-resolved fluorescence. We show that this assay is highly reproducible and robust, yielding results comparable to those obtained by standard methodologies employing radioactive nucleotide incorporation and gel electrophoresis while offering a very significant advantage in terms of throughput (>2000 assay points per operator per day). We demonstrate the usefulness of the assay for the discovery of small molecule inhibitors of transcription, and applications of this approach for the high-throughput discovery of transcriptional modulators are discussed.

Late temporal gene expression from the human cytomegalovirus pp28US (UL99) promoter when integrated into the host cell chromosome.

Toward understanding the temporal regulation of human cytomegalovirus (HCMV) late genes, we studied the regulation of the late gene promoter (pp28US, UL99) when outside the context of the viral genome and its response to the immediate early (IE) proteins. Expression of the luciferase reporter gene, regulated by the pp28US promoter, was synchronous with that of the endogenous viral pp28 gene, independently of whether the reporter was episomal or integrated into the glioblastoma cell line U373MG. Cotransfection of the reporter with expression vectors for each of the three major IE genes, IE72, IE86 and IE55, indicated that only IE86 transactivated the pp28US promoter. However, the magnitude of the promoter activation upon HCMV infection suggested that additional factors are also required for higher promoter activity. The promoter activation was specific to HCMV, as herpes simplex virus type 1 infection did not induce luciferase expression.

Human papillomavirus type 6: classification of clinical isolates and functional analysis of E2 proteins.

Human papillomaviruses (HPVs) cause a variety of clinical manifestations, including the most prevalent viral sexually transmitted disease, genital warts. HPV-6 is found in a greater number of genital warts than any other HPV. To increase our understanding of the structural and functional relationships between HPV-6 isolates and to provide information for epidemiological studies, the sequences of the E2, E6 and E7 coding regions of HPV-6 genomes in clinical samples were determined. This sequence analysis was performed on isolates originally designated HPV-6a on the basis of analysis of patterns generated by restriction enzyme digestion. It was found that the designation of subtype on the basis of restriction enzyme digestion correlated poorly with the designation of subtype on the basis of sequence comparison; in fact, the clinical isolates were clearly categorized into HPV-6a and HPV-6b groups, with the previously described HPV-6vc being a member of the HPV-6a group. It was also found that the HPV-6a E2 protein is a much less potent activator of transcription than the HPV-16 E2 protein, generalizing our previous results with the HPV-6b E2 protein to this second HPV-6 E2 protein. These studies indicate that the amino acid differences observed between these natural variants of the HPV-6 E2 protein do not affect its function.