Browsing by Subject "nucleolus"

Bisphosphate 3'-nucleotidase (Bpnt1) is a member of a family of small molecule phosphatases whose activities depend on divalent cations and are inhibited by lithium. While the enzymes share many commonalities, they have distinct and non-­overlapping substrate pools. Of the seven mammalian members, two enzymes, gPAPP and Bpnt1, hydrolyze the same small molecule 3'-phosphoadenosine 5'-phosphate (PAP) but act in separate subcellular compartments, the Golgi apparatus and cytoplasm respectively. Hydrolysis of PAP, which is a metabolite of the inorganic sulfate incorporation pathway, is highly conserved throughout evolution from bacteria to yeast to humans. Evidence in multiple species has shown that inhibiting PAP hydrolysis leads to cellular toxicity as a result of its accumulation and also that these effects can be ameliorated by modulating the rate of its production. However, despite the abundant evidence of its importance

from studies in lower eukaryotes, the role of the cytoplasmic PAP phosphatase, Bpnt1, in more complicated mammalian physiological remains poorly understood. Here we report for the first time the generation and characterization of mice deficient for Bpnt1. Bpnt1 null mice do not exhibit skeletal defects, but instead develop severe liver

pathologies and deficiencies in intestinal iron absorption. Loss of Bpnt1 leads to tissue-specific elevations of the substrate PAP. To test the hypothesis that a toxic cellular accumulation of PAP accounts for the observed phenotypes, we generated a double mutant mouse that concomitantly down regulates bisphosphorylated nucleotide synthesis in the context of Bpnt1 deficiency. Remarkably, double mutants do not display any detectable physiological defects seen in Bpnt1 null mice. In addition, we have identified and characterized a novel substrate of 3'­nucleotidases, 3'-phosphoadenosine 5'-diphosphate (PAPP) that co-accumulates with PAPS and PAP and might play a role in mediating certain aspects of the physiological defects of Bpnt1 null mice. Overall, our study defines a role for Bpnt1 in mammalian physiology and provides mechanistic insights into the importance of cytoplasmic 3'-­

nucleotide hydrolysis to normal cellular function.

The relatively simple innate immune system of Caenorhabditis elegans and the number of traits that facilitate genetic and genomic analysis using this organism have nurtured rapid advances into the understanding of C. elegans innate immunity during the last few years. However, traditional methods of isolating and mapping C. elegans mutants exhibiting aberrant immune responses to pathogen infection are often labor intensive and time consuming. Therefore, a simple and rapid means of isolating and mapping C. elegans immune mutants will increase the number of mutants that can be studied. Salmonella enterica, as well as other bacterial pathogens, has been described to cause a significant distension of the C. elegans intestinal lumen, which correlates with death of the nematode. C. elegans mutants which exhibit a weakened immune response would therefore be expected to develop intestinal distension at an earlier time point than wild type. Likewise, mutants which exhibit an enhanced immune response would be expected to develop intestinal distension at a later time point than wild type. Taking advantage of this correlation, we designed a novel approach to isolating C. elegans mutants which exhibit aberrant immune responses to the bacterial pathogen, S. enterica. Furthermore, we validated and optimized the use of Amplifluor®, a high-throughput genotyping system, for use in C. elegans single nucleotide polymorphism (SNP) mapping.

To date, the only known negative regulators of innate immunity in C. elegans are dependent on the FOXO transcription factor, DAF-16 and regulate lifespan in addition to immunity. Therefore, we focused our efforts on identifying additional negative regulators of innate immunity by screening for mutants which display a reduced accumulation of S. enterica at a time point when wild-type nematodes are packed with bacteria. In a genetic screen for C. elegans mutants which display reduced accumulation of S. enterica/GFP, we identified a mutation in nol-6, a nucleolar protein containing a nucleolar RNA-associated protein (Nrap) domain which is conserved across eukaryotic organisms. nol-6 is implicated in ribosomal RNA (rRNA) processing during the early stages of ribosome biogenesis. We show that knockdown of nol-6 as well as other nucleolar genes leads to a reduction of pathogen accumulation and enhanced resistance to killing by pathogen. In addition, we demonstrate that enhanced resistance is dependent on p53/cep-1. Furthermore, microarray analysis shows a significant enrichment of upregulated genes that have previously been shown to be dependent on p53/cep-1 for induction following ultraviolet radiation. These results represent the first evidence that C. elegans innate immunity is regulated by the nucleolus through a p53/cep-1-dependent mechanism.