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INTRODUCTION. The structures of members of the Tcl1 oncoprotein family are being studied in order to understand their roles in lymphocyte biology and their development of lymphocytic diseases[1-3]. These ~15 kD proteins share 25–80% sequence identity between the members of the family. No sequence similarity was found with other human genes suggesting a unique cellular role(s). Family members share an uncommon tertiary structure of an eight-stranded beta barrel. Recently, the crystal structure of murine Tcl1 was determined[3]. The three structures were analyzed to reveal conserved features indicative of a potential binding site for an interacting protein.

Proteomic analysis of body fluids, including breast nipple aspirate fluid (NAF), holds promise to aid in early cancer detection. We conducted a prospective trial that collected NAF from women scheduled for diagnostic breast surgery to determine 1) the consistency of proteomic results, 2) protein masses associated with breast cancer, 3) subsets of women with a unique proteomic profile and 4) a breast cancer predictive model. NAF was collected preoperatively in 114 women and analyzed by SELDI-TOF mass spectrometry over a 3–50 kDa range using H4, NP and SAX ProteinChips. For all 3 chips, the same protein peaks were detected over 90% of the time in duplicate samples. The overall coefficient of variation was ≤ 0.17% for each chip for the internal standard and ≤ 0.29% for the unknown proteins. Seven candidate protein ion masses frequently expressed in NAF were identified. Three (5,200-H4, p=.04, 11,880-H4, p=.07 and 13,880 Da-SAX, p=.03) were differentially expressed in women with/without breast cancer. Protein expression differed between women with/without pathologic nipple discharge (PND), but the 5,200, 11,880 and 13,880 proteins remained associated with breast cancer even if PND samples were excluded. Subset analysis identified differences in expression between benign disease and DCIS and between DCIS and invasive cancer for the 5,200 and 33,400 Da proteins. The best cancer detection model included age, parity and the 11,880 Da protein, and excluded women with PND. 1) NAF proteomic analysis using SELDI-TOF is reproducible with the same sample set across different platforms, 2) differential proteomic expression exists between women/without breast cancer and 3) combining proteomic and clinical information that are available before surgery optimizes the prediction of which women have breast cancer. © 2004 Wiley-Liss, Inc.

The entire nef gene sequence of HIV-2, NIH-Z strain, has been cloned into the pJL6 expression vector and used for the synthesis of a 23-kDa protein in E. coli. The expressed protein is a fusion between the N-terminal 13 amino acids of the cII gene, 8 amino acids resulting from the ligation procedure, and the 180 amino acids that comprise the HIV-2 Nef sequence from the NIH-Z strain. The bacterially expressed Nef protein has been purified to apparent homogeneity on analytical scale (10-20 micrograms) by a combination of sequential detergent extraction, gel filtration, and reversed-phase high-performance chromatography. The expressed Nef protein is highly susceptible to proteolysis (chymotryptic-like activity) and this property accounts for the low yield obtained by gel filtration and RP-HPLC. Larger amounts (100 micrograms) of the purified Nef protein have been produced by a purification procedure that employs sequential detergent extraction, chromatography on Q-Sepharose in the presence of 7 M urea, and chromatography on hydroxylapatite, also in 7 M urea. The purified HIV-2 Nef protein has been used for the production of polyclonal and monoclonal antibodies. The milder method of purification should facilitate structure-function studies of the Nef protein and its role in the life cycle of HIV.

Significant improvement in the resolution and quality of the X-­ray diffraction of crystals of MTCP-1 protein was observed on post-crystallization soaking. The MTCP-1 crystals grown from 1.5 M ammonium sulfate diffracted to only 3.0 A resolution with some disorder in the diffraction. After post-crystallization soaking in a solution containing 2.0 M ammonium sulfate, the disorder was eliminated and diffraction extended to better than 2.0 A resolution. Both native and selenomethionine-enriched crystals demonstrated better diffraction after soaking for several months. This simple technique may be useful to improve the diffraction quality of protein crystals generally.

Members of the TCL1 family of oncogenes are abnormally expressed in mature T-cell leukemias and B-cell lymphomas. The proteins are involved in the coactivation of protein kinase B (Akt/PKB), a key intracellular kinase. The sequences and crystal structures of three Tcl1 proteins were analyzed in order to understand their interactions with Akt/PKB and the implications for lymphocyte malignancies. Tcl1 proteins are ~15 kD and share 25—80% amino acid sequence identity. The tertiary structures of mouse Tcl1, human Tcl1, and Mtcp1 are very similar. Analysis of the structures revealed conserved semi-planar surfaces that have characteristics of surfaces involved in protein-protein interactions. The Tcl1 proteins show differences in surface charge distribution and oligomeric state suggesting that they do not interact in the same way with Akt/PKB and other cellular protein(s).

The TCL-1 gene which is located on chromosome 14 plays a major role in human hematopoeitic malignancies and encodes a 14-kDa protein whose function has not been determined. The TCL-1 gene is expressed in pre-B cells, in immature thymocytes, and at low levels in activated T cells but not in peripheral mature B cells and in normal cells. The TCL-1 protein is similar in its primary structure to a protein encoded by the mature T cell proliferation gene (MTCP-1). The MTCP-1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T cell proliferative diseases. The TCL-1 and MTCP-1 genes appear to be members of a family of genes involved in lymphoid proliferation and T cell malignancies. Our laboratory has undertaken the study of the TCL-1 and MTCP-1 proteins to determine the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, both purified TCL-1 and MTCP-1 proteins in forms with and without a six His tag sequence. The recombinant proteins were purified by chromatography on a Ni–NTA resin followed by reverse-phase FPLC using a buffer system at pH 7.9 and a polymeric-based reverse-phase column. The MTCP-1 recombinant proteins display greater solubility, do not form disulfide linked dimers or oligomers, and elute at a lower isopropanol concentration than the corresponding TCL-1 proteins. The purified recombinant TCL-1 and MTCP-1 proteins have been characterized by N-terminal sequence analysis, time of flight mass spectrometry, and circular dichroism spectroscopy. Initial results have indicated that the MTCP-1 protein with the His tag removed is suitable for both NMR and X-ray crystallographic methods of structure determination.

Species variability of the lens protein ζ-crystallin was correlated with those of alcohol dehydrogenases of classes I and III and sorbitol dehydrogenase in the same protein family. The extent of overall variability, nature of residues conserved, and patterns of segment variability, all fall within the limits typical of the ‘variable’ group of medium-chain alcohol dehydrogenases. This shows that ζ-crystallin is subject to restrictions similar to those of classical liver alcohol dehydrogenase and therefore derived from a metabolically active enzyme like other enzyme crystalline. Special residues at the active site, however, differ substantially, including an apparent lack of a zinc-binding site. This is compatible with altered functional properties and makes the spread within this medium-chain dehydrogenase family resemble the wide spread within the short-chain dehydrogenases. Schematic plotting is useful for illustrating the differences between ‘variable’ and ‘constant’ enzymes.

Top of pageAbstract The use of AAV-mediated gene therapy appears to be an exciting approach for the treatment of inherited retinal diseases. However, there is at present, minimal information regarding the effect on the genome and proteome of the target cell following rAAV infection. Additional studies are critically needed to determine which genes and proteins are differentially expressed as the result of rAAV infection and as a result of the inclusion of a transgene. Using ARPE-19, a retinal pigment epithelial (RPE) cell line and recombinant AAV2/5 viruses, AAV2/5.EGFP, AAV2/5.EGFP-RO (reverse orientation), AAV2/5.EFEMP1wt and EFEMP1mut, as well as uninfected cells, we have examined the proteomes by 2D differential in-gel electrophoresis (DIGE) and the genomes by RNA transcript profiling. EGFP encodes the enhanced green flourescent protein and infection of RPE cells with this virus allowed the determination of the optimal multiplicity of infection (MOI) which was 1 |[times]| 106. The AAV2/5.EFEMP1wt and AAV2/5 EFEMP1mut encode epidermal growth factor containing fibrillin-like extracellular matrix protein wild-type and mutant R345W, where an arginine codon is substituted for a tryptophan codon. In patients with Malattia Leventinese (ML), an autosomal dominant macular degenerative disease, the EFEMP1 protein has been shown to accumulate within RPE cells and between the RPE and drusen. In vitro studies have shown that the EFEMP1wt protein is secreted while the EFEMP1mut protein is misfolded, poorly secreted and retained within cells. These results suggest that a misfolding and aberrant accumulation of EFEMP protein may underlie drusen formation and other abnormalities in the retinas of patients with ML and AMD. Initial analysis of the DIGE data shows the presence of both downregulated and upregulated proteins. Analysis of the microarray data shows the downregulation of a number of cell cycle regulator, DNA replication, kinase, kinesin-like (motor), and survivin [baculoviral IAP (apoptosis inhibitor protein) repeat-containing 5 and survivin-beta] genes for rAAV infected cells compared with uninfected RPE cells. Interestingly, there appears to be very little or no change in immune response, inflammatory response or signal transduction genes. Additional proteomic studies using DIGE and SILAC (stable isotope labeling with amino acids in cell culture) as well as continuing microarray studies are in progress to provide further understanding of the effect on the proteome and genome of these important gene therapy tools for the treatment of inherited retinal diseases.