Coordinate Regulation of Ribosome and tRNA Biogenesis Controls Hypoxic Injury and Translation.

The translation machinery is composed of a myriad of proteins and RNAs whose levels must be coordinated to efficiently produce proteins without wasting energy or substrate. However, protein synthesis is clearly not always perfectly tuned to its environment, as disruption of translation machinery components can lengthen lifespan and stress survival. While much has been learned from bacteria and yeast about translational regulation, much less is known in metazoans.

Requirement for translocon-associated protein (TRAP) α in insulin biogenesis.

The mechanistic basis for the biogenesis of peptide hormones and growth factors is poorly understood. Here, we show that the conserved endoplasmic reticulum membrane translocon-associated protein α (TRAPα), also known as signal sequence receptor 1, plays a critical role in the biosynthesis of insulin. Genetic analysis in the nematode and biochemical studies in pancreatic β cells reveal that TRAPα deletion impairs preproinsulin translocation while unexpectedly disrupting distal steps in insulin biogenesis including proinsulin processing and secretion.

Ageing and hypoxia cause protein aggregation in mitochondria.

Aggregation of cytosolic proteins is a pathological finding in disease states, including ageing and neurodegenerative diseases. We have previously reported that hypoxia induces protein misfolding in Caenorhabditis elegans mitochondria, and electron micrographs suggested protein aggregates. Here, we seek to determine whether mitochondrial proteins actually aggregate after hypoxia and other cellular stresses.

N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3' Splice Site Function in Vivo.

Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3' splice site. In the nematode Caenorhabditis elegans, 3' splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking.

Chromoanasynthetic Genomic Rearrangement Identified in a N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Screen in Caenorhabditis elegans.

Chromoanasynthesis is a recently discovered phenomenon in humans with congenital diseases that is characterized by complex genomic rearrangements (CGRs) resulting from aberrant repair of catastrophic chromosomal damage. How these CGRs are induced is not known. Here, we describe the structure and function of dpDp667, a causative CGR that emerged from a Caenorhabditis elegans dauer suppressor screen in which animals were treated with the point mutagen N-ethyl-N-nitrosourea (ENU).

Longevity Genes Revealed by Integrative Analysis of Isoform-Specific daf-16/FoxO Mutants of Caenorhabditis elegans.

FoxO transcription factors promote longevity across taxa. How they do so is poorly understood. In the nematode Caenorhabditis elegans, the A- and F-isoforms of the FoxO transcription factor DAF-16 extend life span in the context of reduced DAF-2 insulin-like growth factor receptor (IGFR) signaling.

Assessing mucociliary transport of single particles in vivo shows variable speed and preference for the ventral trachea in newborn pigs.

Mucociliary transport (MCT) is an innate defense mechanism that removes particulates, noxious material, and microorganisms from the lung. Several airway diseases exhibit abnormal MCT, including asthma, chronic bronchitis, and cystic fibrosis. However, it remains uncertain whether MCT abnormalities contribute to the genesis of disease or whether they are secondary manifestations that may fuel disease progression.

Human cystic fibrosis airway epithelia have reduced Cl- conductance but not increased Na+ conductance.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function causes cystic fibrosis (CF) lung disease. CFTR is expressed in airway epithelia, but how CF alters electrolyte transport across airway epithelia has remained uncertain. Recent studies of a porcine model showed that in vivo, excised, and cultured CFTR(-/-) and CFTR(ΔF508/ΔF508) airway epithelia lacked anion conductance, and they did not hyperabsorb Na(+).

Acute regulation of tight junction ion selectivity in human airway epithelia.

Electrolyte transport through and between airway epithelial cells controls the quantity and composition of the overlying liquid. Many studies have shown acute regulation of transcellular ion transport in airway epithelia. However, whether ion transport through tight junctions can also be acutely regulated is poorly understood both in airway and other epithelia.

Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs.

Almost two decades after CFTR was identified as the gene responsible for cystic fibrosis (CF), we still lack answers to many questions about the pathogenesis of the disease, and it remains incurable. Mice with a disrupted CFTR gene have greatly facilitated CF studies, but the mutant mice do not develop the characteristic manifestations of human CF, including abnormalities of the pancreas, lung, intestine, liver, and other organs. Because pigs share many anatomical and physiological features with humans, we generated pigs with a targeted disruption of both CFTR alleles.

Basolateral chloride current in human airway epithelia.

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl(-) absorption.

Nedd4-2 isoforms differentially associate with ENaC and regulate its activity.

Mutations that disrupt a PY motif in epithelial Na(+) channel (ENaC) subunits increase surface expression of Na(+) channels in the collecting duct, resulting in greater Na(+) reabsorption. Nedd4 and Nedd4-2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. We recently reported that human Nedd4-2 (hNedd4-2) is expressed as many isoforms because of alternative promoter usage and/or variable splicing.

New insights into epithelial sodium channel function in the kidney: site of action, regulation by ubiquitin ligases, serum- and glucocorticoid-inducible kinase and proteolysis.

Purpose Of Review: The epithelial sodium channel (ENaC) sets the rate of Na+ reabsorption in the collecting duct. This review describes recent advances in our understanding of ENaC function.

Recent Findings: First, collecting duct-specific deletion of alphaENaC does not cause Na wasting in mice, suggesting that other regions can compensate.

Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains.

Mutations that disrupt a PY motif in epithelial Na+ channel (ENaC) subunits increase surface expression of Na+ channels in the collecting duct, resulting in greater Na+ reabsorption. Recently, Nedd4 and Nedd4-2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. To further understand the role of human Nedd4-2 (hNedd4-2), we cloned its cDNAs and determined its genomic organization using a bioinformatic approach.

Cycloheximide increases glucocorticoid-stimulated alpha -ENaC mRNA in collecting duct cells by p38 MAPK-dependent pathway.

Aldosterone and glucocorticoids (GCs) stimulate Na(+) reabsorption in the collecting ducts by increasing the activity of the epithelial Na(+) channel (ENaC). Our laboratory has used Madin-Darby canine kidney-C7 cells to demonstrate that this effect is associated with an increase in alpha-ENaC gene transcription (Mick VE, Itani OA, Loftus RW, Husted RF, Schmidt TJ, and Thomas CP, Mol Endocrinol 15: 575-588, 2001). Cycloheximide (CHX) superinduced the GC-stimulated alpha-ENaC expression in a dose-dependent manner, but had no effect on basal or aldosterone-stimulated alpha-ENaC expression, whereas anisomycin inhibited basal and corticosteroid-stimulated alpha-ENaC expression.

Glucocorticoids stimulate human sgk1 gene expression by activation of a GRE in its 5'-flanking region.

In lung and collecting duct epithelia, glucocorticoid (GC)-stimulated Na+ transport is preceded by an increase in the protein kinase sgk1, which in turn regulates the activity of the epithelial Na+ channel (ENaC). We investigated the mechanism for GC-regulated human sgk1 expression in lung and renal epithelia. sgk1 mRNA was increased in these epithelia by GCs, and this was inhibited by actinomycin D and superinduced by cycloheximide, consistent with a transcriptional effect that did not require protein synthesis.

Genomic organization of the 5' end of human beta-ENaC and preliminary characterization of its promoter.

The mRNA for the beta-subunit of the epithelial Na(+) channel (beta-ENaC) is regulated developmentally and, in some tissues, in response to corticosteroids. To understand the mechanisms of transcriptional regulation of the human beta-ENaC gene, we characterized the 5' end of the gene and its 5'-flanking regions. Adaptor-ligated human kidney and lung cDNA were amplified by 5' rapid amplification of cDNA ends, and transcription start sites of two 5' variant transcripts were determined by nuclease protection or primer extension assays.

Glucocorticoid-stimulated lung epithelial Na(+) transport is associated with regulated ENaC and sgk1 expression.

H441 cells, a bronchiolar epithelial cell line, develop a glucocorticoid-regulated amiloride-sensitive Na(+) transport pathway on permeable supports (R. Sayegh, S. D.