Membrane traffic in the secretory pathway

It is generally thought that microtubule-associated motors insure long-range movements of the secretory vesicles from the center of the cell to its periphery, while myosins insure short-range movements at the cell periphery. However, several of the myosins that have been reported during the last decade to be involved in the exocytic pathway are not processive, meaning that they do not have the ability to move cargos along actin polymers. We will review here the possible mechanisms by which these myosins could contribute to the traffic of secretory proteins from the Golgi complex to the plasma membrane.     

Na+ binding to meizothrombin desF1

Meizothrombin is the physiologically active intermediate generated by a single cleavage of prothrombin at R320 to separate the A and B chains. Recent evidence has suggested that meizothrombin, like thrombin, is a Na(+)-activated enzyme. In this study we present the first X-ray crystal structure of human meizothrombin desF1 solved in the presence of the active site inhibitor PPACK at 2.1 A resolution. The structure reveals a Na(+) binding site whose architecture is practically identical to that of human thrombin. Stopped-flow measurements of Na(+) binding to meizothrombin desF1 document a slow phase of fluorescence change with a k(obs) decreasing hyperbolically with increasing [Na(+)], consistent with the existence of three conformations in equilibrium, E*, E and E:Na(+), as for human thrombin. Evidence that meizothrombin exists in multiple conformations provides valuable new information for studies of the mechanism of prothrombin activation.     

Zinc binding to peptide analogs of the structural zinc site in alcohol dehydrogenase: Implications for an entatic state

Zinc binding to the peptide replica and analogs to residues 93–115 of horse liver alcohol dehydrogenase (ADH) was examined by competition of the peptides and the chromophoric chelator 4-(2- pyridylazo)resorcinol for zinc and X-ray absorption fine structure analysis of the zinc ligands. In the enzyme, zinc is coordinated by four Cys residues. In the peptide replica, zinc is bound to three Cys and one His residue. A four-Cys zinc coordination is observed only when His is removed, leading to increased zinc stability. ADH crystal structures reveal that the ε-amino group of the conserved residue Lys323 is within H-bond distance of the backbone amide oxygens of residues 103, 105 and 108, likely stabilizing the zinc coordination in the enzyme. The peptide data thus indicate structural strain and increased energy in the zinc-binding site in the protein, characteristic of an entatic state, implying a functional nature for this zinc site. Received 3 July 2008; received after revision 11 August 2008; accepted 1 September 2008     

Mutations in 15-hydroxyprostaglandin dehydrogenase cause primary hypertrophic osteoarthropathy

Digital clubbing, recognized by Hippocrates in the fifth century BC, is the outward hallmark of pulmonary hypertrophic osteoarthropathy, a clinical constellation that develops secondary to various acquired diseases, especially intrathoracic neoplasm. The pathogenesis of clubbing and hypertrophic osteoarthropathy has hitherto been poorly understood, but a clinically indistinguishable primary (idiopathic) form of hypertrophic osteoarthropathy (PHO) is recognized. This familial disorder can cause diagnostic confusion, as well as significant disability. By autozygosity methods, we mapped PHO to chromosome 4q33-q34 and identified mutations in HPGD, encoding 15-hydroxyprostaglandin dehydrogenase, the main enzyme of prostaglandin degradation. Homozygous individuals develop PHO secondary to chronically elevated prostaglandin E(2) levels. Heterozygous relatives also show milder biochemical and clinical manifestations. These findings not only suggest therapies for PHO, but also imply that clubbing secondary to other pathologies may be prostaglandin mediated. Testing for HPGD mutations and biochemical testing for HPGD deficiency in patients with unexplained clubbing might help to obviate extensive searches for occult pathology.     

What everybody should know about the rat genome and its online resources

It has been four years since the original publication of the draft sequence of the rat genome. Five groups are now working together to assemble, annotate and release an updated version of the rat genome. As the prevailing model for physiology, complex disease and pharmacological studies, there is an acute need for the rat's genomic resources to keep pace with the rat's prominence in the laboratory. In this commentary, we describe the current status of the rat genome sequence and the plans for its impending 'upgrade'. We then cover the key online resources providing access to the rat genome, including the new SNP views at Ensembl, the RefSeq and Genes databases at the US National Center for Biotechnology Information, Genome Browser at the University of California Santa Cruz and the disease portals for cardiovascular disease and obesity at the Rat Genome Database.     

Common variants near MC4R are associated with fat mass, weight and risk of obesity

To identify common variants influencing body mass index (BMI), we analyzed genome-wide association data from 16,876 individuals of European descent. After previously reported variants in FTO, the strongest association signal (rs17782313, P = 2.9 x 10(-6)) mapped 188 kb downstream of MC4R (melanocortin-4 receptor), mutations of which are the leading cause of monogenic severe childhood-onset obesity. We confirmed the BMI association in 60,352 adults (per-allele effect = 0.05 Z-score units; P = 2.8 x 10(-15)) and 5,988 children aged 7-11 (0.13 Z-score units; P = 1.5 x 10(-8)). In case-control analyses (n = 10,583), the odds for severe childhood obesity reached 1.30 (P = 8.0 x 10(-11)). Furthermore, we observed overtransmission of the risk allele to obese offspring in 660 families (P (pedigree disequilibrium test average; PDT-avg) = 2.4 x 10(-4)). The SNP location and patterns of phenotypic associations are consistent with effects mediated through altered MC4R function. Our findings establish that common variants near MC4R influence fat mass, weight and obesity risk at the population level and reinforce the need for large-scale data integration to identify variants influencing continuous biomedical traits.     

Gene silencing in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation

Epigenetic silencing in cancer cells is mediated by at least two distinct histone modifications, polycomb-based histone H3 lysine 27 trimethylation (H3K27triM) and H3K9 dimethylation. The relationship between DNA hypermethylation and these histone modifications is not completely understood. Using chromatin immunoprecipitation microarrays (ChIP-chip) in prostate cancer cells compared to normal prostate, we found that up to 5% of promoters (16% CpG islands and 84% non-CpG islands) were enriched with H3K27triM. These genes were silenced specifically in prostate cancer, and those CpG islands affected showed low levels of DNA methylation. Downregulation of the EZH2 histone methyltransferase restored expression of the H3K27triM target genes alone or in synergy with histone deacetylase inhibition, without affecting promoter DNA methylation, and with no effect on the expression of genes silenced by DNA hypermethylation. These data establish EZH2-mediated H3K27triM as a mechanism of tumor-suppressor gene silencing in cancer that is potentially independent of promoter DNA methylation.     

DNA deletions and clonal mutations drive premature aging in mitochondrial mutator mice

Mitochondrial DNA (mtDNA) mutations are thought to have a causal role in many age-related pathologies. Here we identify mtDNA deletions as a driving force behind the premature aging phenotype of mitochondrial mutator mice, and provide evidence for a homology-directed DNA repair mechanism in mitochondria that is directly linked to the formation of mtDNA deletions. In addition, our results demonstrate that the rate at which mtDNA mutations reach phenotypic expression differs markedly among tissues, which may be an important factor in determining the tolerance of a tissue to random mitochondrial mutagenesis.     

A cis-acting regulatory mutation causes premature hair graying and susceptibility to melanoma in the horse

In horses, graying with age is an autosomal dominant trait associated with a high incidence of melanoma and vitiligo-like depigmentation. Here we show that the Gray phenotype is caused by a 4.6-kb duplication in intron 6 of STX17 (syntaxin-17) that constitutes a cis-acting regulatory mutation. Both STX17 and the neighboring NR4A3 gene are overexpressed in melanomas from Gray horses. Gray horses carrying a loss-of-function mutation in ASIP (agouti signaling protein) had a higher incidence of melanoma, implying that increased melanocortin-1 receptor signaling promotes melanoma development in Gray horses. The Gray horse provides a notable example of how humans have cherry-picked mutations with favorable phenotypic effects in domestic animals.     

A Gata6-Wnt pathway required for epithelial stem cell development and airway regeneration

Epithelial organs, including the lung, are known to possess regenerative abilities through activation of endogenous stem cell populations, but the molecular pathways regulating stem cell expansion and regeneration are not well understood. Here we show that Gata6 regulates the temporal appearance and number of bronchioalveolar stem cells (BASCs) in the lung, its absence in Gata6-null lung epithelium leading to the precocious appearance of BASCs and concurrent loss in epithelial differentiation. This expansion of BASCs was the result of a pronounced increase in canonical Wnt signaling in lung epithelium upon loss of Gata6. Expression of the noncanonical Wnt receptor Fzd2 was downregulated in Gata6 mutants and increased Fzd2 or decreased beta-catenin expression rescued, in part, the lung epithelial defects in Gata6 mutants. During lung epithelial regeneration, canonical Wnt signaling was activated in the niche containing BASCs and forced activation of Wnt signaling led to a large increase in BASC numbers. Moreover, Gata6 was required for proper lung epithelial regeneration, and postnatal loss of Gata6 led to increased BASC expansion and decreased differentiation. Together, these data demonstrate that Gata6-regulated Wnt signaling controls the balance between progenitor expansion and epithelial differentiation required for both lung development and regeneration.