HLA class I and II antigens are partially co-clustered in the plasma membrane of human lymphoblastoid cells

Major histocompatibility complex (MHC) class II molecules displayed clustered patterns at the surfaces of T (HUT-102B2) and B (JY) lymphoma cells characterized by interreceptor distances in the micrometer range as detected by scanning force microscopy of immunogold-labeled antigens. Electron microscopy revealed that a fraction of the MHC class II molecules was also heteroclustered with MHC class I antigens at the same hierarchical level as described by the scanning force microscopy data, after specifically and sequentially labeling the antigens with 30- and 15-nm immunogold beads. On JY cells the estimated fraction of co-clustered HLA II was 0.61, whereas that of the HLA I was 0.24. Clusterization of the antigens was detected by the deviation of their spatial distribution from the Poissonian distribution representing the random case. Fluorescence resonance energy transfer measurements also confirmed partial co-clustering of the HLA class I and II molecules at another hierarchical level characterized by the 2- to 10-nm Förster distance range and providing fine details of the molecular organization of receptors. The larger-scale topological organization of the MHC class I and II antigens may reflect underlying membrane lipid domains and may fulfill significant functions in cell-to-cell contacts and signal transduction.

Relationship between donor age and the replicative lifespan of human cells in culture: A reevaluation

Normal human diploid fibroblasts have a finite replicative lifespan in vitro, which has been postulated to be a cellular manifestation of aging in vivo. Several studies have shown an inverse relationship between donor age and fibroblast culture replicative lifespan; however, in all cases, the correlation was weak, and, with few exceptions, the health status of the donors was unknown. We have determined the replicative lifespans of 124 skin fibroblast cell lines established from donors of different ages as part of the Baltimore Longitudinal Study of Aging. All of the donors were medically examined and were declared “healthy,” according to Baltimore Longitudinal Study of Aging protocols, at the time the biopsies were taken. Both long- and short-lived cell lines were observed in all age groups, but no significant correlation between the proliferative potential of the cell lines and donor age was found. A comparison of multiple cell lines established from the same donors at different ages also failed to reveal any significant trends between proliferative potential and donor age. The rate of [3H]thymidine incorporation and the initial rates of growth during the first few subcultivations were examined in a subset of cell lines and were found to be significantly greater in fetal lines than in postnatal lines. Cell lines established from adults did not vary significantly either in initial growth rate or in [3H]thymidine incorporation. These results clearly indicate that, if health status and biopsy conditions are controlled, the replicative lifespan of fibroblasts in culture does not correlate with donor age.

Stable expression in yeast of the mature form of human telomerase RNA depends on its association with the box H/ACA small nucleolar RNP proteins Cbf5p, Nhp2p and Nop10p

Telomerase is a ribonucleoprotein (RNP) particle required for the replication of telomeres. The RNA component, termed hTR, of human telomerase contains a domain structurally and functionally related to box H/ACA small nucleolar RNAs (snoRNAs). Furthermore, hTR is known to be associated with two core components of H/ACA snoRNPs, hGar1p and Dyskerin (the human counterpart of yeast Cbf5p). To assess the functional importance of the association of hTR with H/ACA snoRNP core proteins, we have attempted to express hTR in a genetically tractable system, Saccharomyces cerevisiae. Both mature non-polyadenylated and polyadenylated forms of hTR accumulate in yeast. The former is associated with all yeast H/ACA snoRNP core proteins, unlike TLC1 RNA, the endogenous RNA component of yeast telomerase. We show that the presence of the H/ACA snoRNP proteins Cbf5p, Nhp2p and Nop10p, but not Gar1p, is required for the accumulation of mature non-polyadenylated hTR in yeast, while accumulation of TLC1 RNA is not affected by the absence of any of these proteins. Our results demonstrate that yeast telomerase is unrelated to H/ACA snoRNPs. In addition, they show that the accumulation in yeast of the mature RNA component of human telomerase depends on its association with three of the four core H/ACA snoRNP proteins. It is likely that this is the case in human cells as well.

Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-Å resolution. The α/β structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3γ (HNF-3γ), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3γ and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the β subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein

IL-18 can be considered a proinflammatory cytokine mediating disease as well as an immunostimulatory cytokine that is important for host defense against infection and cancer. The high-affinity, constitutively expressed, and circulating IL-18 binding protein (IL-18BP), which competes with cell surface receptors for IL-18 and neutralizes IL-18 activity, may act as a natural antiinflammatory as well as immunosuppressive molecule. In the present studies, the IL-18 precursor caspase-1 cleavage site was changed to a factor Xa site, and, after expression in Escherichia coli, mature IL-18 was generated by factor Xa cleavage. Mature IL-18 generated by factor Xa cleavage was fully active. Single point mutations in the mature IL-18 peptide were made, and the biological activities of the wild-type (WT) IL-18 were compared with those of the mutants. Mutants E42A and K89A exhibited 2-fold increased activity compared with WT IL-18. A double mutant, E42A plus K89A, exhibited 4-fold greater activity. Unexpectedly, IL-18BP failed to neutralize the double mutant E42A plus K89A compared with WT IL-18. The K89A mutant was intermediate in being neutralized by IL-18BP, whereas neutralization of the E42A mutant was comparable to that in the WT IL-18. The identification of E42 and K89 in the mature IL-18 peptide is consistent with previous modeling studies of IL-18 binding to IL-18BP and explains the unusually high affinity of IL-18BP for IL-18.

Approaches to enhancing the retroviral transduction of human synoviocytes

This report concerns a clinical trial for rheumatoid arthritis (RA), approved by the US National Institutes of Health and the Food and Drug Administration. An amphotropic retrovirus (MFG-IRAP) was used ex vivo to transfer a cDNA encoding human interleukin-1 receptor antagonist (IL-1Ra) to synovium. The protocol required the transduced cells to secrete at least 30 ng IL-1Ra/106 cells per 48 h before reimplantation. Here we have evaluated various protocols for their efficiency in transducing cultures of human rheumatoid synoviocytes. The most reliably efficient methods used high titer retrovirus (approximately 108 infectious particles/ml). Transduction efficiency was increased further by exposing the cells to virus under flow-through conditions. The use of dioctadecylamidoglycylspermine (DOGS) as a polycation instead of Polybrene (hexadimethrine bromide) provided an additional small increment in efficiency. Under normal conditions of static transduction, standard titer, clinical grade retrovirus (approximately 5 × 105 infectious particles/ml) failed to achieve the expression levels required by the clinical trial. However, the shortfall could be remedied by increasing the time of transduction under static conditions, transducing under flow-through conditions, or transducing during centrifugation.

Activation of the JC virus Tat-responsive transcriptional control element by association of the Tat protein of human immunodeficiency virus 1 with cellular protein Purα

JC virus is activated to replicate in glial cells of many AIDS patients with neurological disorders. In human glial cells, the human immunodeficiency virus 1 (HIV-1) Tat protein activates the major late promoter of JC virus through a Tat-responsive DNA element, termed upTAR, which is a recognition site for cellular Purα, a sequence-specific single-stranded DNA binding protein implicated in cell cycle control of DNA replication and transcription. Tat interacts with two leucine-rich repeats in Purα to form a complex that can be immunoprecipitated from cell extracts. Tat enhances the ability of purified glutathione S-transferase-Purα (GST-Purα) to bind the upTAR element. Tat acts synergistically with Purα, in a cell-cycle-dependent manner, to activate transcription at an upTAR element placed upstream of a heterologous promoter. Since Purα is ubiquitously expressed in human cells and since PUR elements are located near many promoters and origins of replication, the Tat-Purα interaction may be implicated in effects of HIV-1 throughout the full range of HIV-1-infected cells.

Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells

The effects of Escherichia coli heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [3H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [3H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.

The crystal structure of human glycosylation-inhibiting factor is a trimeric barrel with three 6-stranded beta-sheets.

Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis. The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method. The structure was refined to an R factor of 0.168 at 1.9 angstrom resolution. The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded beta-sheets on the inside and six alpha-helices on the outside. There is a 5-angstrom-diameter "hole" through the middle of the barrel. The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor. Each subunit has a new class of alpha + beta sandwich structure consisting of two beta-alpha-beta motifs. These beta-alpha-beta motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different.

A mean field model of ligand-protein interactions: implications for the structural assessment of human immunodeficiency virus type 1 protease complexes and receptor-specific binding.

We propose a general mean field model of ligand-protein interactions to determine the thermodynamic equilibrium of a system at finite temperature. The method is employed in structural assessments of two human immuno-deficiency virus type 1 protease complexes where the gross effects of protein flexibility are incorporated by utilizing a data base of crystal structures. Analysis of the energy spectra for these complexes has revealed that structural and thermo-dynamic aspects of molecular recognition can be rationalized on the basis of the extent of frustration in the binding energy landscape. In particular, the relationship between receptor-specific binding of these ligands to human immunodeficiency virus type 1 protease and a minimal frustration principle is analyzed.

Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands.

We have used an in vitro selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) to identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A randomized RNA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presence of Rev. Those sequences with the ability to photocrosslink to Rev were partitioned from the rest of the RNA pool, amplified, and used for the next round of selection. Rounds of photocrosslinking selection were alternated with rounds of selection for RNA sequences with high affinity to Rev. This iterative, dual-selection method yielded RNA molecules with subnanomolar dissociation constants and high efficiency photocrosslinking to Rev. Some of the RNA molecules isolated by this procedure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. In vitro selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule.

Specific mutations in the ligand binding domain selectively abolish the silencing function of human thyroid hormone receptor beta.

Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (V174A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/D177, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription.

The 94- to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich liposomes is identical to macrosialin, the mouse homologue of human CD68.

We have previously reported the partial purification of a 94- to 97-kDa plasma membrane protein from mouse peritoneal macrophages that binds oxidatively modified low density lipoprotein (OxLDL) and phosphatidylserine-rich liposomes. We have now identified that protein as macrosialin, a previously cloned macrophage-restricted membrane protein in the lysosomal-associated membrane protein family (mouse homologue of human CD68). Early in the course of purification of the 94- to 97-kDa protein, a new OxLDL-binding band at 190-200 kDa appeared and copurified with the 94- to 97-kDa protein. The HPLC pattern of tryptic peptides from this higher molecular mass ligand-binding band closely matched that derived from the 94- to 97-kDa band. Specifically, the same three macrosialin-derived tryptic peptides (9, 9, and 15 residues) were present in the purified 94- to 97-kDa band and in the 190- to 200-kDa band and antisera raised against peptide sequences in macrosialin recognized both bands. An antiserum against macrosialin precipitated most of the 94- to 97-kDa OxLDL-binding material. We conclude that the binding of OxLDL to mouse macrophage membranes is in part attributable to macrosialin. Our previous studies show that OxLDL competes with oxidized red blood cells and with apoptotic thymocytes for binding to mouse peritoneal macrophages. Whether macrosialin plays a role in recognition of OxLDL and oxidatively damaged cells by intact macrophages remains uncertain.

SPC3, a synthetic peptide derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120, inhibits HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms.

The third variable region (V3 loop) of gp120, the HIV-1 surface envelope glycoprotein, plays a key role in HIV-1 infection and pathogenesis. Recently, we reported that a synthetic multibranched peptide (SPC3) containing eight V3-loop consensus motifs (GPGRAF) inhibited HIV-1 infection in both CD4+ and CD4- susceptible cells. In the present study, we investigated the mechanisms of action of SPC3 in these cell types--i.e., CD4+ lymphocytes and CD4- epithelial cells expressing galactosylceramide (GalCer), an alternative receptor for HIV-1 gp120. We found that SPC3 was a potent inhibitor of HIV-1 infection in CD4+ lymphocytes when added 1 h after initial exposure of the cells to HIV-1, whereas it had no inhibitory effect when present only before and/or during the incubation with HIV-1. These data suggested that SPC3 did not inhibit the binding of HIV-1 to CD4+ lymphocytes but interfered with a post-binding step necessary for virus entry. In agreement with this hypothesis, SPC3 treatment after HIV-1 exposure dramatically reduced the number of infected cells without altering gp120-CD4 interaction or viral gene expression. In contrast, SPC3 blocked HIV-1 entry into CD4-/GalCer+ human colon epithelial cells when present in competition with HIV-1 but had no effect when added after infection. Accordingly, SPC3 was found to inhibit the binding of gp120 to the GalCer receptor. Thus, the data suggest that SPC3 affects HIV-1 infection by two distinct mechanisms: (i) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4-/GalCer+ cells and (ii) post-binding inhibition of HIV-1 entry into CD4+ lymphocytes.

Mutagenesis of the putative alpha-helical domain of the Vpr protein of human immunodeficiency virus type 1: effect on stability and virion incorporation.

vpr is one of the auxiliary genes of human immunodeficiency virus type 1 (HIV-1) and is conserved in the related HIV-2/simian immunodeficiency virus lentiviruses. The unique feature of Vpr is that it is the only nonstructural protein incorporated into the virus particle. Secondary structural analysis predicted an amphipathic alpha-helical domain in the amino terminus of Vpr (residues 17-34) which contains five acidic and four leucine residues. To evaluate the role of specific residues of the helical domain for virion incorporation, mutagenesis of this domain was carried out. Substitution of proline for any of the individual acidic residues (Asp-17 and Glu-21, -24, -25, and -29) eliminated the virion incorporation of Vpr and also altered the stability of Vpr in cells. Conservative replacement of glutamic residues of the helical domain with aspartic residues resulted in Vpr characteristic of wild type both in stability and virion incorporation, as did substitution of glutamine for the acidic residues. In contrast, replacement of leucine residues of the helical domain (residues 20, 22, 23, and 26) by alanine eliminated virion incorporation function of Vpr. These data indicate that acidic and hydrophobic residues and the helical structure in this region are critical for the stability of Vpr and its efficient incorporation into virus-like particles.