Functional Analysis of the Yeast Glc7-Binding Protein Reg1 Identifies a Protein Phosphatase Type 1-Binding Motif as Essential for Repression of ADH2 Expression

In Saccharomyces cerevisiae, the protein phosphatase type 1 (PP1)-binding protein Reg1 is required to maintain complete repression of ADH2 expression during growth on glucose. Surprisingly, however, mutant forms of the yeast PP1 homologue Glc7, which are unable to repress expression of another glucose-regulated gene, SUC2, fully repressed ADH2. Constitutive ADH2 expression in reg1 mutant cells did require Snf1 protein kinase activity like constitutive SUC2 expression and was inhibited by unregulated cyclic AMP-dependent protein kinase activity like ADH2 expression in derepressed cells. To further elucidate the functional role of Reg1 in repressing ADH2 expression, deletions scanning the entire length of the protein were analyzed. Only the central region of the protein containing the putative PP1-binding sequence RHIHF was found to be indispensable for repression. Introduction of the I466M F468A substitutions into this sequence rendered Reg1 almost nonfunctional. Deletion of the central region or the double substitution prevented Reg1 from significantly interacting with Glc7 in two-hybrid analyses. Previous experimental evidence had indicated that Reg1 might target Glc7 to nuclear substrates such as the Snf1 kinase complex. Subcellular localization of a fully functional Reg1-green fluorescent protein fusion, however, indicated that Reg1 is cytoplasmic and excluded from the nucleus independently of the carbon source. When the level of Adr1 was modestly elevated, ADH2 expression was no longer fully repressed in glc7 mutant cells, providing the first direct evidence that Glc7 can repress ADH2 expression. These results suggest that the Reg1-Glc7 phosphatase is a cytoplasmic component of the machinery responsible for returning Snf1 kinase activity to its basal level and reestablishing glucose repression. This implies that the activated form of the Snf1 kinase complex must cycle between the nucleus and the cytoplasm.     

Distinct Phenotypic Differences Associated with Differential Amplification of Receptor Tyrosine Kinase Genes at 4q12 in Glioblastoma

Gene amplification at chromosome 4q12 is a common alteration in human high grade gliomas including glioblastoma, a CNS tumour with consistently poor prognosis. This locus harbours the known oncogenes encoding the receptor tyrosine kinases PDGFRA, KIT, and VEGFR2. These receptors are potential targets for novel therapeutic intervention in these diseases, with expression noted in tumour cells and/or associated vasculature. Despite this, a detailed assessment of their relative contributions to different high grade glioma histologies and the underlying heterogeneity within glioblastoma has been lacking. We studied 342 primary high grade gliomas for individual gene amplification using specific FISH probes, as well as receptor expression in the tumour and endothelial cells by immunohistochemistry, and correlated our findings with specific tumour cell morphological types and patterns of vasculature. We identified amplicons which encompassed PDGFRA only, PDGFRA/KIT, and PDGFRA/KIT/VEGFR2, with distinct phenotypic correlates. Within glioblastoma specimens, PDGFRA amplification alone was linked to oligodendroglial, small cell and sarcomatous tumour cell morphologies, and rare MGMT promoter methylation. A younger age at diagnosis and better clinical outcome in glioblastoma patients is only seen when PDGFRA and KIT are co-amplified. IDH1 mutation was only found when all three genes are amplified; this is a subgroup which also harbours extensive MGMT promoter methylation. Whilst PDGFRA amplification was tightly linked to tumour expression of the receptor, this was not the case for KIT or VEGFR2. Thus we have identified differential patterns of gene amplification and expression of RTKs at the 4q12 locus to be associated with specific phenotypes which may reflect their distinct underlying mechanisms.     

CELFish ways to modulate mRNA decay

The CELF family of RNA-binding proteins regulates many steps of mRNA metabolism. Although their best characterized function is in pre-mRNA splice site choice, CELF family members are also powerful modulators of mRNA decay. In this review we focus on the different modes of regulation that CELF proteins employ to mediate mRNA decay by binding to GU-rich elements. After starting with an overview of the importance of CELF proteins during development and disease pathogenesis, we then review the mRNA networks and cellular pathways these proteins regulate and the mechanisms by which they influence mRNA decay. Finally, we discuss how CELF protein activity is modulated during development and in response to cellular signals. We conclude by highlighting the priorities for new experiments in this field. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

Drug-Encoded Biomarkers for Monitoring Biological Therapies

Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers.     

The Vertebrate Tooth Row: Is It Initiated by a Single Organizing Tooth?

Teeth are one of the most fascinating innovations of vertebrates. Their diversity of shape, size, location, and number in vertebrates is astonishing. If the molecular mechanisms underlying the morphogenesis of individual teeth are now relatively well understood, thanks to the detailed experimental work that has been performed in model organisms (mainly mouse and zebrafish), the mechanisms that control the organization of the dentition are still a mystery. Mammals display simplified dentitions when compared to other vertebrates with only a single tooth row positioned in the anterior part of the mouth, whereas other vertebrates exhibit tooth rows in many locations. As proposed 60 years ago, tooth rows can be formed sequentially from an initiator tooth. Recent results in zebrafish have now largely confirmed this hypothesis. Here this observation is generalized upon and it is suggested that in most vertebrates tooth rows could form sequentially from a single initiator tooth.     

A Population Accounting Approach to Assess Tourism Contributions to Conservation of IUCN-Redlisted Mammal Species

Over 1,000 mammal species are red-listed by IUCN, as Critically Endangered, Endangered or Vulnerable. Conservation of many threatened mammal species, even inside protected areas, depends on costly active day-to-day defence against poaching, bushmeat hunting, invasive species and habitat encroachment. Many parks agencies worldwide now rely heavily on tourism for routine operational funding: >50% in some cases. This puts rare mammals at a new risk, from downturns in tourism driven by external socioeconomic factors. Using the survival of individual animals as a metric or currency of successful conservation, we calculate here what proportions of remaining populations of IUCN-redlisted mammal species are currently supported by funds from tourism. This proportion is ≥5% for over half of the species where relevant data exist, ≥15% for one fifth, and up to 66% in a few cases. Many of these species, especially the most endangered, survive only in one single remaining subpopulation. These proportions are not correlated either with global population sizes or recognition as wildlife tourism icons. Most of the more heavily tourism-dependent species, however, are medium sized (>7.5 kg) or larger. Historically, biological concern over the growth of tourism in protected areas has centered on direct disturbance to wildlife. These results show that conservation of threatened mammal species has become reliant on revenue from tourism to a previously unsuspected degree. On the one hand, this provides new opportunities for conservation funding; but on the other, dependence on such an uncertain source of funding is a new, large and growing threat to red-listed species.     

Self-association of the Lentivirus protein, Nef

Background

The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization.

Results

By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIV_MAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface.

Conclusions

We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.     

The origin of EFNB1 mutations in craniofrontonasal syndrome: frequent somatic mosaicism and explanation of the paucity of carrier males

Craniofrontonasal syndrome (CFNS) is an X-linked disorder that exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis, and additional minor malformations, but males are usually mildly affected with hypertelorism only. Despite this, males appear underrepresented in CFNS pedigrees, with carrier males encountered infrequently compared with affected females. To investigate these unusual genetic features of CFNS, we exploited the recent discovery of causative mutations in the EFNB1 gene, which encodes ephrin-B1, to survey the molecular alterations in 59 families (39 newly investigated and 20 published elsewhere). We identified the first complete deletions of EFNB1, catalogued 27 novel intragenic mutations, and used Pyrosequencing and analysis of nearby polymorphic alleles to quantify mosaic cases and to determine the parental origin of verified germline mutations. Somatic mosaicism was demonstrated in 6 of 53 informative families, and, of 17 germline mutations in individuals for whom the parental origin of mutation could be demonstrated, 15 arose from the father. We conclude that the major factor accounting for the relative scarcity of carrier males is the bias toward mutations in the paternal germline (which present as affected female offspring) combined with reduced reproductive fitness in affected females. Postzygotic mutations also contribute to the female preponderance, whereas true nonpenetrance in males who are hemizygous for an EFNB1 mutation appears unusual. These results highlight the importance of considering possible origins of mutation in the counseling of families with CFNS and provide a generally applicable approach to the combined analysis of mosaic and germline mutations.