Showing papers by "David A. Pearce published in 2000"

A Common Motif within the Negative Regulatory Regions of Multiple Factors Inhibits Their Transcriptional Synergy

Abstract: DNA regulatory elements frequently harbor multiple recognition sites for several transcriptional activators. The response mounted from such compound response elements is often more pronounced than the simple sum of effects observed at single binding sites. The determinants of such transcriptional synergy and its control, however, are poorly understood. Through a genetic approach, we have uncovered a novel protein motif that limits the transcriptional synergy of multiple DNA-binding regulators. Disruption of these conserved synergy control motifs (SC motifs) selectively increases activity at compound, but not single, response elements. Although isolated SC motifs do not regulate transcription when tethered to DNA, their transfer to an activator lacking them is sufficient to impose limits on synergy. Mechanistic analysis of the two SC motifs found in the glucocorticoid receptor N-terminal region reveals that they function irrespective of the arrangement of the receptor binding sites or their distance from the transcription start site. Proper function, however, requires the receptor's ligand-binding domain and an engaged dimer interface. Notably, the motifs are not functional in yeast and do not alter the effect of p160 coactivators, suggesting that they require other nonconserved components to operate. Many activators across multiple classes harbor seemingly unrelated negative regulatory regions. The presence of SC motifs within them, however, suggests a common function and identifies SC motifs as critical elements of a general mechanism to modulate higher-order interactions among transcriptional regulators.

Batten disease: evaluation of CLN3 mutations on protein localization and function

Abstract: Juvenile neuronal ceroid lipofuscinosis (JNCL), Batten disease, is an autosomal recessive lysosomal storage disease associated with mutations in CLN3 .C LN3 has no known homology to other proteins and a function has not yet been described. The predominant mutation in CLN3 is a 1.02 kb genomic deletion that accounts for nearly 85% of the disease alleles. In this mutation, truncation of the protein by a premature stop codon results in the classical phenotype. Additional missense and nonsense mutations have been described. Some missense substitutions result in a protracted phenotype, with delays in the onset of classical clinical features, whereas others lead to classical JNCL. In this study, we examined the effect of naturally occurring point mutations on the intracellular localization of CLN3 and their ability to complement the CLN3deficient yeast, btn1-∆. We also examined a putative farnesylation motif thought to be involved in CLN3 trafficking. All of the point mutations, like wild-type CLN3, were highly associated with lysosomeassociated membrane protein II in non-neuronal cells and with synaptophysin in neuronal cell lines. In the yeast functional assay, point mutations correlating with a mild phenotype also demonstrated CLN3 activity, whereas the mutations associated with severe disease failed to restore CLN3 function completely. CLN3 with a mutation in the farnesylation motif trafficked normally but was functionally impaired. These data suggest that these clinically relevant point mutations, causative of Batten disease, do not affect protein trafficking but rather exert their effects by impairing protein function.

Human immunodeficiency virus type 1 Vpr contains two leucine-rich helices that mediate glucocorticoid receptor coactivation independently of its effects on G(2) cell cycle arrest.

Abstract: Human immunodeficiency virus type 1 (HIV-1) Vpr participates in nuclear targeting of the viral preintegration complex in nondividing cells and induces G 2 cell cycle arrest in proliferating cells, which creates an intracellular milieu favorable for viral replication. Vpr also activates the transcription of several promoters and enhancers by a poorly understood mechanism. Vpr enhances glucocorticoid receptor (GR) signaling and may mediate the effects of steroids on HIV replication. More specifically, recombinant Vpr can potentiate virion production from U937 cells, downregulate NF-κB induction, and enhance programmed cell death, all effects also mediated by glucocorticoids. Vpr has been proposed to act as a GR coactivator, although other studies suggest that these enhancing effects are merely a consequence of G 2 cell cycle arrest. We now demonstrate that Vpr functions as a GR coactivator and that this activity is independent of cell cycle arrest. In addition, we show that the Vpr-induced coactivation requires an intact glucocorticoid response element, that it is dependent on the presence of hormone and the corresponding receptor, and that it is mediated by the two highly conserved leucine-rich domains within Vpr that resemble the GR coactivator signature motif.

The yeast model for batten disease: mutations in BTN1, BTN2, and HSP30 alter pH homeostasis.

Abstract: Juvenile neuronal ceroid-lipofuscinoses, or Batten disease, is an autosomal progressive neurodegenerative disease in children, with an incidence as high as one in 12,500 live births and with about 440,000 carriers in the United States (1, 7). Diagnosis is often based on visual defects, behavioral changes, and seizures. Progression is characterized by a decline in mental abilities, increased severity of untreatable seizures, blindness, loss of motor skills, and premature death. The CLN3 gene, positionally cloned in 1995, was shown to be responsible for Batten disease, with most diseased individuals harboring a major deletion of the gene (11). However, the function of the CLN3 protein and the molecular basis for the disease remain elusive. Batten disease is characterized by the accumulation of autofluorescent hydrophobic material in the lysosomes of neurons and, to a lesser extent, in other cell types (9, 14). Furthermore, protein sequencing and immunological studies have revealed that subunit c of mitochondrial ATP synthase is the major component of the lysosomal storage material (8, 20). Although initially located in the mitochondria, mitochondrial ATP synthase subunit c accumulates in lysosomes of NCL cells, whereas the degradation of another mitochondrial inner membrane protein, cytochrome oxidase subunit IV, is unaffected, with no lysosomal accumulation (5, 15). CLN3 localizes in the lysosome (12, 13), suggesting that accumulation of mitochondrial ATP synthase subunit c in the lysosome is not due to defective mitochodria but rather to a defect within the lysosome. Genes encoding predicted proteins with high sequence similarity to the Cln3 protein have been identified in mouse, dog, rabbit, Caenorhabditis elegans, and the yeast Saccharomyces cerevisiae (18, 28; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"U92812","term_id":"2388632","term_text":"U92812"}}U92812 and 249335 and Swiss-Prot accession no. {"type":"entrez-protein","attrs":{"text":"O29611","term_id":"74570216","term_text":"O29611"}}O29611). We previously reported that the corresponding yeast gene, BTN1, encodes a nonessential protein that is 39% identical and 59% similar to human Cln3p (21). Deletion of BTN1 had no effect on mitochondrial function or the degradation of mitochondrial ATP synthase subunit c. We further demonstrated that yeast strains lacking Btn1p were resistant to d-(−)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP) and that this phenotype was complemented by expression of human Cln3p, indicating that yeast Btn1p and human Cln3p have the same function (22). Furthermore, the degree of ANP resistance was correlated with point mutations identified in CLN3 that are associated with less severe forms of Batten disease, further establishing the functional equivalence of Btn1p and CLN3 (22). The resistance of btn1-Δ yeast strains to ANP was caused by an apparent decrease in the pH of the growth media brought about by an elevated ability to acidify growth medium through an increased activity of the plasma membrane H+-ATPase (23). This increased activity is caused by a response to an imbalance in pH homeostasis within the cell, produced from an abnormally acidic vacuolar pH in btn1-Δ strains (23). DNA microarray analysis revealed that expression of only two genes, HSP30 and BTN2, is abnormally increased as btn1-Δ strains grow, apparently returning the plasma membrane H+-ATPase activity and vacuolar pH to normal. Furthermore, like CLN3, Btn1p has been localized to the lysosome, called the vacuole in yeast (4, 23). We report here that deletion of either HSP30 (hsp30-Δ) or BTN2 (btn2-Δ) does not result in ANP resistance, as vacuolar pH is unaffected by these mutations. However, the vacuolar H+-ATPase activity is nevertheless increased in hsp30-Δ or btn2-Δ, particularly btn2-Δ. Furthermore, btn1-Δ, hsp30-Δ, or btn2-Δ causes poor growth at low pH in the presence of sorbic acid. This new phenotype for the Batten disease yeast model, btn1-Δ, and for hsp30-Δ and btn2-Δ, whose gene products exhibit increased expression in btn1-Δ strains, provides more evidence that deletion of BTN1 affects the ability of a yeast cell to regulate intracellular pH.

Plasma Membrane Calcium Pump Isoform 1 Gene Expression Is Repressed by Corticosterone and Stress in Rat Hippocampus

Abstract: Glucocorticoids (GCs) are critical to learning and memory, in large part because of their actions in the hippocampus. Chronic high levels of GCs have profound effects on hippocampal structure and function and can even result in irreversible neurodegeneration. Hippocampal GC actions are mediated by intracellular receptors that modulate the transcription of specific target genes. In a screen for genes repressed by GCs in rat hippocampus, we identified plasma membrane calcium pump isoform 1 (PMCA1), a plasma membrane calcium ATPase. In Northern blots, PMCA1 was repressed ∼33% after a high, but not a low dose of the GC, corticosterone (B), suggesting glucocorticoid (but not mineralocorticoid) receptor-mediated repression. Furthermore, in situ hybridization demonstrated that B significantly downregulated PMCA1 mRNA in all brain regions examined. Repression of PMCA1 was also observed in cultured hippocampal neurons, but only when the cells were in the differentiated state. Stress also repressed PMCA1 expression in hippocampus of adrenal-intact animals, and a clear inverse correlation between B level and PMCA1 mRNA could be discerned. However, other non-B-dependent factors appeared to be involved in the response of PMCA1 to stress because, unlike exogenous B, cold stress did not repress PMCA1 in brain regions other than hippocampus. Moreover, in the presence of constant B (B-replaced, adrenalectomized animals), cold stress led to increased hippocampal PMCA1 expression. These observations suggest that repression of PMCA1 represents one molecular mechanism by which corticosteroids regulate Ca2+ homeostasis and hence influence neuronal activity. Moreover, other stress-related neurohumoral factors appear to counter the repressive effects of B. Defects in the balance between GC-mediated and non-GC-mediated effects on PMCA1 expression may have adverse effects on neuronal function and ultimately result in irreversible neuronal damage.

Localization and processing of CLN3, the protein associated to Batten disease: where is it and what does it do?

Abstract: Although the CLN3 gene for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995, the function of the corresponding protein still remains elusive. A key to understanding the pathology of this devastating disease will be to elucidate the function of CLN3 at the molecular level. CLN3 has proven difficult to study, as it is predicted to be a membrane protein, and is of apparently low abundance in cells. Different groups have reported differing subcellular localization of CLN3. The purpose of this review is to critically examine the various cell biological approaches undertaken to localize CLN3 and to piece together a potential function for CLN3 in neuronal cells. The most likely conclusion of this is that CLN3 is a lysosomal/endosomal protein that is trafficked through the endoplasmic reticulum (ER) and Golgi. Furthermore, studies are required to confirm whether CLN3 has a potential role in the recycling of synaptic vesicles through the endosome/lysosome.

Transmission electron microscope analysis of virus-like particles in the freshwater lakes of Signy Island, Antarctica

Abstract: Water samples from a range of fresh-water Antarctic lakes on Signy Island (South Orkney Islands: 60°45′S, 45 °38′W) were examined for the presence of virus-like particles (VLPs) during the 1998/1999 field season. It was discovered that VLPs were ubiquitous, morphologically diverse and abundant, with high concentrations ranging from 4.9 × 106 ml−1 to 3.1 × 107 ml−1. Likely hosts include bacteria, cyanobacteria and eukaryotic algae. In addition, an unusually large virus morphotype was observed with a head diameter 370 × 330 nm and a tail 1.3 μm long.

A method to study bacterioplankton community structure in Antarctic lakes

Abstract: A method to study bacterioplankton community structure in Antarctic freshwater lake samples is described. Small samples (between 300 and 1000 ml) taken in remote field locations were used for crude DNA extraction, followed by PCR amplification of 16S rRNA gene fragments using group-specific primers. The amplification products of the PCR reaction were then separated using denaturing gradient gel electrophoresis to produce a profile of the bacterioplankton community. Whilst the technique is only semi-quantitative, it readily differentiated communities from lakes of different trophic status and from vertical profiles within different lake types. The method offers a sensitive tool for screening and monitoring Antarctic freshwater environments as a precursor and adjunct to more detailed studies.