Garrett DuBois

ALL-1 Is a Histone Methyltransferase that Assembles a Supercomplex of Proteins Involved in Transcriptional Regulation.

ALL-1 is a member of the human trithorax/Polycomb gene family and is also involved in acute leukemia. ALL-1 is present within a stable, very large multiprotein supercomplex composed of > or =29 proteins. The majority of the latter are components of the human transcription complexes TFIID (including TBP), SWI/SNF, NuRD, hSNF2H, and Sin3A. Other components are involved in RNA processing or in histone methylation. The complex remodels, acetylates, deacetylates, and methylates nucleosomes and/or free histones. The complexs H3-K4 methylation activity is conferred by the ALL-1 SET domain. Chromatin immunoprecipitations show that ALL-1 and other complex components examined are bound at the promoter of an active ALL-1-dependent Hox a9 gene. In parallel, H3-K4 is methylated, and histones H3 and H4 are acetylated at this promoter.

Cell-cell fusion and internalization of the CNS-based, HIV-1 co-receptor, APJ.

APJ, a member of the human G protein-coupled seven-transmembrane receptor family, has been shown to serve as a coreceptor for the entry of human immunodeficiency virus type I (HIV-1) and simian immunodeficiency virus (SIV), and it is dramatically expressed in central nervous system (CNS)-based cells. In this study, expression of APJ tagged with the green fluorescent protein (GFP) and a fluorescent peptide, 5-carboxyfluorescein (5-CF) conjugated Apelin-13, were utilized for studying receptor internalization and recycling, in stably expressing indicator cells, human neurons, primary CNS microvascular endothelial cells (MVECs), and astrocytes. Fusion of the C-terminus of APJ to the N-terminus of GFP did not alter receptor ligand binding and functions, including signaling and internalization. Using 293 cells stably expressing APJ-GFP, we demonstrated that rapid internalization of the APJ receptor was induced by stimulation with Apelin-36 and Apelin-13, in a dose-dependent manner. Furthermore, investigations showed that the internalized APJ was colocalized with transferrin receptors, suggesting that the internalization of APJ induced by Apelin is likely to be via clathrin-coated pits. Interestingly, we found that the internalized APJ molecules were recycled to the cell surface within 60 min after removal of Apelin-13, but most of the internalized APJ still remained in the cytoplasm, even 2 h after washout of Apelin-36. The intact cytoplasmic C-terminal domain was found to be required for ligand-induced APJ internalization. Human neurons were dramatically stained by the APJ-binding fluorescent peptides. Primary human fetal astrocytes were less strongly labeled with 5-CF-Apelin-13, and in primary human CNS MVECs only weak distribution of green fluorescence specific for APJ in the cytoplasm was observed. Apelin-36 blocked cell membrane fusion mostly due to steric interference, with only a very modest effect on receptor internalization. The CNS represents a unique reservoir site for HIV-1. As such, molecular therapeutics and small molecular inhibitors of HIV-1 entry via this unique CNS receptor are now able to be rationally designed.

Heterogeneity of nef proteins in cells infected with human immunodeficiency virus type 1

Human T-lymphocytic cell line H9 infected with the HTLV-IIIB isolate of human immunodeficiency virus type 1 (HIV-1) synthesizes two forms of the Nef protein (p25 and p27) that differ both in molecular weight and charge. Different subpopulations of viruses were isolated from the HTLV-IIIB stock which induce expression of only p25 or p27. Cells infected with HIV-1 derived from the HXB3 clone of the HTLV-IIIB isolate made only the p25 species, whereas the 8E5/LAV cell line which harbors a single defective LAV provirus produces only the p27 species. These findings are consistent with the notion that the HTLV-IIIB isolate consists of at least two distinct variants with different nef genes, one specifying p25 and the other encoding p27. After a considerable number of passages in culture, H9 cells chronically infected with the HTLV-IIIB isolate produced high levels of p25 and lower levels of p27. Passages in culture appear to select for a subpopulation of virus variants that specify high levels of p25 Nef expression.

Structure of murine Tcl1 at 2.5 A resolution and implications for the TCL oncogene family.

Tcl1 and Mtcp1, members of the Tcl1 family, are implicated in T-cell prolymphocytic leukemia. The crystal structure of a dimer of murine Tcl1 has been determined at 2.5 A resolution with an R factor of 0.225. Murine Tcl1, human Tcl1 and Mtcp1 share very similar subunit structures, with RMS differences of 0.6 and 1.4 A for C(alpha) atoms, respectively, while the sequences share 50 and 36% identity, respectively. These structures fold into an eight-stranded beta-barrel of unique topology and high internal symmetry of 1.1-1.3 A for the two halves of human and murine Tcl1 and 1.7 A for Mtcp1, despite the low 12-13% sequence identity. The molecular surfaces of all three structures showed a common planar region which is likely to be involved in protein-protein interactions.

Effects of different post‐crystallization soaking conditions on the diffraction of Mtcp1 crystals

The crystal structure of human Mtcp1 was determined at 2 A resolution after the X-ray diffraction limit was improved by post-crystallization soaking in 2.0 M ammonium sulfate for 1-5 months. The effects of varying the ammonium sulfate concentration and addition of polyethylene glycol to the soaking solution were examined in order to understand the phenomenon and to reduce the soaking time. Soaking the crystal for one week in a solution of 1.5 M ammonium sulfate and 2% PEG 3400 gave the desired improvement in diffraction quality. Therefore, different soaking conditions should be explored when crystals show disordered and low-resolution diffraction.