Structure and evolution of vertebrate aldehyde oxidases: from gene duplication to gene suppression

Aldehyde oxidases (AOXs) and xanthine dehydrogenases (XDHs) belong to the family of molybdo-flavoenzymes. Although AOXs are not identifiable in fungi, these enzymes are represented in certain protists and the majority of plants and vertebrates. The physiological functions and substrates of AOXs are unknown. Nevertheless, AOXs are major drug metabolizing enzymes, oxidizing a wide range of aromatic aldehydes and heterocyclic compounds of medical/toxicological importance. Using genome sequencing data, we predict the structures of AOX genes and pseudogenes, reconstructing their evolution. Fishes are the most primitive organisms with an AOX gene (AOXα), originating from the duplication of an ancestral XDH. Further evolution of fishes resulted in the duplication of AOXα into AOXβ and successive pseudogenization of AOXα. AOXβ is maintained in amphibians and it is the likely precursors of reptilian, avian, and mammalian AOX1. Amphibian AOXγ is a duplication of AOXβ and the likely ancestor of reptilian and avian AOX2, which, in turn, gave rise to mammalian AOX3L1. Subsequent gene duplications generated the two mammalian genes, AOX3 and AOX4. The evolution of mammalian AOX genes is dominated by pseudogenization and deletion events. Our analysis is relevant from a structural point of view, as it provides information on the residues characterizing the three domains of each mammalian AOX isoenzyme. We cloned the cDNAs encoding the AOX proteins of guinea pig and cynomolgus monkeys, two unique species as to the evolution of this enzyme family. We identify chimeric RNAs from the human AOX3 and AOX3L1 pseudogenes with potential to encode a novel microRNA.     

ING1 and ING2: multifaceted tumor suppressor genes

Inhibitor of Growth 1 (ING1) was identified and characterized as a “candidate” tumor suppressor gene in 1996. Subsequently, four more genes, also characterized as “candidate” tumor suppressor genes, were identified by homology search: ING2, ING3, ING4, and ING5. The ING proteins are characterized by a high homology in their C-terminal domain, which contains a Nuclear Localization Sequence and a Plant HomeoDomain (PHD), which has a high affinity to Histone 3 tri-methylated on lysine 4 (H3K4Me3). The ING proteins have been involved in the control of cell growth, senescence, apoptosis, chromatin remodeling, and DNA repair. Within the ING family, ING1 and ING2 form a subgroup since they are evolutionarily and functionally close. In yeast, only one gene, Pho23, is related to ING1 and ING2 and possesses also a PHD. Recently, the ING1 and ING2 tumor suppressor status has been fully established since several studies have described the loss of ING1 and ING2 protein expression in human tumors and both ING1 and ING2 knockout mice were reported to have spontaneously developed tumors, B cell lymphomas, and soft tissue sarcomas, respectively. In this review, we will describe for the first time what is known about the ING1 and ING2 genes, proteins, their regulations in both human and mice, and their status in human tumors. Furthermore, we explore the current knowledge about identified functions involving ING1 and ING2 in tumor suppression pathways especially in the control of cell cycle and in genome stability.     

Evolution of the Cdk-activator Speedy/RINGO in vertebrates

Successful completion of the cell cycle relies on the precise activation and inactivation of cyclin-dependent kinases (Cdks) whose activity is mainly regulated by binding to cyclins. Recently, a new family of Cdk regulators termed Speedy/RINGO has been discovered, which can bind and activate Cdks but shares no apparent amino acid sequence homology with cyclins. All Speedy proteins share a conserved domain of approximately 140 amino acids called “Speedy Box”, which is essential for Cdk binding. Speedy/RINGO proteins display an important role in oocyte maturation in Xenopus. Interestingly, a common feature of all Speedy genes is their predominant expression in testis suggesting that meiotic functions may be the most important physiological feature of Speedy genes. Speedy homologs have been reported in mammals and can be traced back to the most primitive clade of chordates (Ciona intestinalis). Here, we investigated the evolution of the Speedy genes and have identified a number of new Speedy/RINGO proteins. Through extensive analysis of numerous species, we discovered diverse evolutionary histories: the number of Speedy genes varies considerably among species, with evidence of substantial gains and losses. Despite the interspecies variation, Speedy is conserved among most species examined. Our results provide a complete picture of the Speedy gene family and its evolution.     

Aspartate 338 contributes to the cationic specificity and to driver-amino acid coupling in the insect cotransporter KAAT1

To investigate the peculiar ionic specificity of KAAT1, an Na⁺- and K⁺-coupled amino acid cotransporter from Lepidoptera, a detailed analysis of membrane topology predictions was performed, together with sequence comparison with strictly Na⁺-dependent mammalian cotransporters from the same family. The analysis identified aspartate 338, a residue present also in the other cotransporter accepting K⁺ (CAATCH1), but absent in most mammalian transporters that have, instead, an asparagine in the corresponding position. Mutation of D338 in KAAT1 led either to non-functional transporters (D338G, D338C), or to an altered ionic selectivity (D338E, D338N), observable in uptake experiments and in electrophysiological properties. In particular, in D338E, the transport activity, while persisting in the presence of Na⁺, appeared to be completely abolished in the presence of K⁺. D338E also showed uncoupling between transport-associated current and uptake. The opposite mutation in the -aminobutyric acid transporter rGAT-1 (N327D) resulted in complete loss of function. In conclusion, aspartate 338 in KAAT1 appears to be important in allowing K⁺, in addition to Na⁺, to drive the transport mechanism, although other residues in different parts of the protein may also play a role in the complete determination of ionic selectivity.Received 23 September 2003; received after revision 11 November 2003; accepted 25 November 2003     

The FHIT gene product: tumor suppressor and genome “caretaker”

The FHIT gene at FRA3B is one of the earliest and most frequently altered genes in the majority of human cancers. It was recently discovered that the FHIT gene is not the most fragile locus in epithelial cells, the cell of origin for most Fhit-negative cancers, eroding support for past claims that deletions at this locus are simply passenger events that are carried along in expanding cancer clones, due to extreme vulnerability to DNA damage rather than to loss of FHIT function. Indeed, recent reports have reconfirmed FHIT as a tumor suppressor gene with roles in apoptosis and prevention of the epithelial–mesenchymal transition. Other recent works have identified a novel role for the FHIT gene product, Fhit, as a genome “caretaker.” Loss of this caretaker function leads to nucleotide imbalance, spontaneous replication stress, and DNA breaks. Because Fhit loss-induced DNA damage is “checkpoint blind,” cells accumulate further DNA damage during subsequent cell cycles, accruing global genome instability that could facilitate oncogenic mutation acquisition and expedite clonal expansion. Loss of Fhit activity therefore induces a mutator phenotype. Evidence for FHIT as a mutator gene is discussed in light of these recent investigations of Fhit loss and subsequent genome instability.     

Current understanding of ZIP and ZnT zinc transporters in human health and diseases

Zinc transporters, the Zrt-, Irt-like protein (ZIP) family and the Zn transporter (ZnT) family transporters, are found in all aspects of life. Increasing evidence has clarified the molecular mechanism, in which both transporters play critical roles in cellular and physiological functions via mobilizing zinc across the cellular membrane. In the last decade, mutations in ZIP and ZnT transporter genes have been shown to be implicated in a number of inherited human diseases. Moreover, dysregulation of expression and activity of both transporters has been suggested to be involved in the pathogenesis and progression of chronic diseases including cancer, immunological impairment, and neurodegenerative diseases, although comprehensive understanding is far from complete. The diverse phenotypes of diseases related to ZIP and ZnT transporters reflect the multifarious biological functions of both transporters. The present review summarizes the current understanding of ZIP and ZnT transporter functions from the standpoint of human health and diseases. The study of zinc transporters is currently of great clinical interest.     

Potentiation of antifungal activity of sesquiterpene dialdehydes againstCandida albicans and two other fungi

The antifungal activity of two drimane sesquiterpene dialdehydes, polygodial (1) and warburganal (2), alone and in combination with several other substances, was examined against three fungi,Candida albicans, Saccharomyces cerevisiae andPityrosporum ovale employing a broth dilution method. Anethole significantly synergized the activity of the two sesquiterpenoids againstC. albicans andS. cerevisiae however, it had only an, additive effect againstP. ovale. By contrast, two antioxidants, ascorbic acid (vitamin C) and BHA (butylated hydroxyanisole), noticeably enhanced the activity of the sesquiterpenoids againstP. ovale, but had no, effect againstC. albicans andS. cerevisiae.     

Gene structure of the murine 2'-5'-oligoadenylate synthetase family

The 2'-5'-oligoadenylate synthetases (OASs) are members of a family of interferon-induced proteins playing an important role in the antiviral effect of interferons as well as being involved in apoptosis and control of cellular growth. Based on sequence data from the murine BAC clone (RP23-39M18), and a number of EST and IMAGE clones and the Celera Mouse database, we identified twelve Oas genes in the mouse genome, all localized to the chromosome 5F region. In contrast to the single OAS1 gene found in humans, we identified eight closely linked Oas1 genes in the murine genome, together with the genes of Oas2 and Oas3. Compared to the single OASL gene found in humans, two genes of OAS-like proteins, Oasl1 and Oasl2, were identified. All the putative genes seem to be transcribed. The exon/intron structures of the murine Oas genes were found to be identical to those of the human genes.     

Interaction of aging-associated signaling cascades: Inhibition of NF-κB signaling by longevity factors FoxOs and SIRT1

Research on aging in model organisms has revealed different molecular mechanisms involved in the regulation of the lifespan. Studies on Saccharomyces cerevisiae have highlighted the role of the Sir2 family of genes, human Sirtuin homologs, as the longevity factors. In Caenorhabditis elegans, the daf-16 gene, a mammalian homolog of FoxO genes, was shown to function as a longevity gene. A wide array of studies has provided evidence for a role of the activation of innate immunity during aging process in mammals. This process has been called inflamm-aging. The master regulator of innate immunity is the NF-κB system. In this review, we focus on the several interactions of aging-associated signaling cascades regulated either by Sirtuins and FoxOs or NF-κB signaling pathways. We provide evidence that signaling via the longevity factors of FoxOs and SIRT1 can inhibit NF-κB signaling and simultaneously protect against inflamm-aging process. Received 4 October 2007; received after revision 7 November 2007; accepted 9 November 2007     

Molecular functions and cellular roles of the ChlR1 (DDX11) helicase defective in the rare cohesinopathy Warsaw breakage syndrome

In 2010, a new recessive cohesinopathy disorder, designated Warsaw breakage syndrome (WABS), was described. The individual with WABS displayed microcephaly, pre- and postnatal growth retardation, and abnormal skin pigmentation. Cytogenetic analysis revealed mitomycin C (MMC)-induced chromosomal breakage; however, an additional sister chromatid cohesion defect was also observed. WABS is genetically linked to bi-allelic mutations in the ChlR1/DDX11 gene which encodes a protein of the conserved family of Iron–Sulfur (Fe–S) cluster DNA helicases. Mutations in the budding yeast ortholog of ChlR1, known as Chl1, were known to cause sister chromatid cohesion defects, indicating a conserved function of the gene. In 2012, three affected siblings were identified with similar symptoms to the original WABS case, and found to have a homozygous mutation in the conserved Fe–S domain of ChlR1, confirming the genetic linkage. Significantly, the clinically relevant mutations perturbed ChlR1 DNA unwinding activity. In addition to its genetic importance in human disease, ChlR1 is implicated in papillomavirus genome maintenance and cancer. Although its precise functions in genome homeostasis are still not well understood, ongoing molecular studies of ChlR1 suggest the helicase plays a critically important role in cellular replication and/or DNA repair.     

Antitumoral effects of 9-cis retinoic acid in adrenocortical cancer

The currently available medical treatment options of adrenocortical cancer (ACC) are limited. In our previous meta-analysis of adrenocortical tumor genomics data, ACC was associated with reduced retinoic acid production and retinoid X receptor-mediated signaling. Our objective has been to study the potential antitumoral effects of 9-cis retinoic acid (9-cisRA) on the ACC cell line NCI-H295R and in a xenograft model. Cell proliferation, hormone secretion, and gene expression have been studied in the NCI-H295R cell line. A complex bioinformatics approach involving pathway and network analysis has been performed. Selected genes have been validated by real-time qRT-PCR. Athymic nude mice xenografted with NCI-H295R have been used in a pilot in vivo xenograft model. 9-cisRA significantly decreased cell viability and steroid hormone secretion in a concentration- and time-dependent manner in the NCI-H295R cell line. Four major molecular pathways have been identified by the analysis of gene expression data. Ten genes have been successfully validated involved in: (1) steroid hormone secretion (HSD3B1, HSD3B2), (2) retinoic acid signaling (ABCA1, ABCG1, HMGCR), (3) cell-cycle damage (GADD45A, CCNE2, UHRF1), and the (4) immune response (MAP2K6, IL1R2). 9-cisRA appears to directly regulate the cell cycle by network analysis. 9-cisRA also reduced tumor growth in the in vivo xenograft model. In conclusion, 9-cisRA might represent a promising new candidate in the treatment of hormone-secreting adrenal tumors and adrenocortical cancer.     

The ZIC gene family encodes multi-functional proteins essential for patterning and morphogenesis

The zinc finger of the cerebellum gene (ZIC) discovered in Drosophila melanogaster (odd-paired) has five homologs in Xenopus, chicken, mice, and humans, and seven in zebrafish. This pattern of gene copy expansion is accompanied by a divergence in gene and protein structure, suggesting that Zic family members share some, but not all, functions. ZIC genes are implicated in neuroectodermal development and neural crest cell induction. All share conserved regions encoding zinc finger domains, however their heterogeneity and specification remain unexplained. In this review, the evolution, structure, and expression patterns of the ZIC homologs are described; specific functions attributable to individual family members are supported. A review of data from functional studies in Xenopus and murine models suggest that ZIC genes encode multifunctional proteins operating in a context-specific manner to drive critical events during embryogenesis. The identification of ZIC mutations in congenital syndromes highlights the relevance of these genes in human development.     

Mitochondrial DNA replication and disease: insights from DNA polymerase γ mutations

DNA polymerase γ (pol γ), encoded by POLG, is responsible for replicating human mitochondrial DNA. About 150 mutations in the human POLG have been identified in patients with mitochondrial diseases such as Alpers syndrome, progressive external ophthalmoplegia, and ataxia-neuropathy syndromes. Because many of the mutations are described in single citations with no genotypic family history, it is important to ascertain which mutations cause or contribute to mitochondrial disease. The vast majority of data about POLG mutations has been generated from biochemical characterizations of recombinant pol γ. However, recently, the study of mitochondrial dysfunction in Saccharomyces cerevisiae and mouse models provides important in vivo evidence for the role of POLG mutations in disease. Also, the published 3D-structure of the human pol γ assists in explaining some of the biochemical and genetic properties of the mutants. This review summarizes the current evidence that identifies and explains disease-causing POLG mutations.     

Ribosomal gene numbers in Microseridinae (Compositae: Cichorieae)

Summary 8 species of the subtribe Microseridinae contain between 1100 and 3400 genes for 25 and 18 S ribosomal RNA. The gene numbers seem to evolve by discrete steps. Their trend follows a general reduction in genome size during the evolution of the annual species ofMicroseris, but numbers remain high in one of them and inAgoseris grandiflora. 2 species ofPyrrhopappus differ by a duplication of the ribosomal gene numbers; 5 S ribosomal RNA genes in 4 species are repeated roughly 10,000 times.We thank Miss S. Werner, Miss A. Roth and Miss U. Krehan for help with some of the experiments. This paper is part of a project supported by grant Ba 536/1-5 from the Deutsche Forschungsgemeinschaft.     

Genetic determinants of neuronal vulnerability to apoptosis

Apoptosis is a common mode of cell death that contributes to neuronal loss associated with neurodegeneration. Single-nucleotide polymorphisms (SNPs) in chromosomal DNA are contributing factors dictating natural susceptibility of humans to disease. Here, the most common SNPs affecting neuronal vulnerability to apoptosis are reviewed in the context of neurological disorders. Polymorphic variants in genes encoding apoptotic proteins, either from the extrinsic (FAS, TNF-α, CASP8) or the intrinsic (BAX, BCL2, CASP3, CASP9) pathways could be highly valuable in the diagnosis of neurodegenerative diseases and stroke. Interestingly, the Arg72Pro SNP in TP53, the gene encoding tumor suppressor p53, was recently revealed a biomarker of poor prognosis in stroke due to its ability to modulate neuronal apoptotic death. Search for new SNPs responsible for genetic variability to apoptosis will ensure the implementation of novel diagnostic and prognostic tools, as well as therapeutic strategies against neurological diseases.