A bipartite NLS motif mediates the nuclear import of Drosophila moesin.

The ERM protein family, which consists of three closely related proteins in vertebrates, ezrin, radixin, and moesin (ERM), is an ancient and important group of cytoplasmic actin-binding and organizing proteins. With their FERM domain, ERMs bind various transmembrane proteins and anchor them to the actin cortex through their C-terminal F-actin binding domain, thus they are major regulators of actin dynamics in the cell. ERMs participate in many fundamental cellular processes, such as phagocytosis, microvilli formation, T-cell activation and tumor metastasis. We have previously shown that, besides its cytoplasmic activities, the single ERM protein of Drosophila melanogaster, moesin, is also present in the cell nucleus, where it participates in gene expression and mRNA export. Here we study the mechanism by which moesin enters the nucleus. We show that the nuclear import of moesin is an NLS-mediated, active process. The nuclear localization sequence of the moesin protein is an evolutionarily highly conserved, conventional bipartite motif located on the surface of the FERM domain. Our experiments also reveal that the nuclear import of moesin does not require PIP2 binding or protein activation, and occurs in monomeric form. We propose, that the balance between the phosphorylated and non-phosphorylated protein pools determines the degree of nuclear import of moesin.

A comparative analysis of fruit fly and human glutamate dehydrogenases in Drosophila melanogaster sperm development.

Glutamate dehydrogenases are enzymes that take part in both amino acid and energy metabolism. Their role is clear in many biological processes, from neuronal function to cancer development. The putative testis-specific Drosophila glutamate dehydrogenase, Bb8, is required for male fertility and the development of mitochondrial derivatives in spermatids. Testis-specific genes are less conserved and could gain new functions, thus raising a question whether Bb8 has retained its original enzymatic activity. We show that while Bb8 displays glutamate dehydrogenase activity, there are significant functional differences between the housekeeping Gdh and the testis-specific Bb8. Both human GLUD1 and GLUD2 can rescue the bb8 ms mutant phenotype, with superior performance by GLUD2. We also tested the role of three conserved amino acids observed in both Bb8 and GLUD2 in Gdh mutants, which showed their importance in the glutamate dehydrogenase function. The findings of our study indicate that Drosophila Bb8 and human GLUD2 could be novel examples of convergent molecular evolution. Furthermore, we investigated the importance of glutamate levels in mitochondrial homeostasis during spermatogenesis by ectopic expression of the mitochondrial glutamate transporter Aralar1, which caused mitochondrial abnormalities in fly spermatids. The data presented in our study offer evidence supporting the significant involvement of glutamate metabolism in sperm development.

Biofilm formation initiating rotifer-specific biopolymer and its predicted components.

The rotifer-specific biopolymer, namely Rotimer, is a recently discovered group of the biomolecule family. Rotimer has an active role in the biofilm formation initiated by rotifers (e.g., Euchlanis dilatata or Adineta vaga) or in the female-male sexual interaction of monogononts. To understand the Ca2+- and polarity-dependent formation of this multifunctional viscoelastic material, it is essential to explore its molecular composition. The investigation of the rotifer-enhanced biofilm and Rotimer-inductor conglomerate (RIC) formation yielded several protein candidates to predict the Rotimer-specific main components. The exudate of E. dilatata males was primarily applied from different biopolimer-containing samples (biofilm or RIC). The advantage of males over females lies in their degenerated digestive system and simple anatomy. Thus, their exudate is less contaminated with food and endosymbiont elements. The sequenced and annotated genome and transcriptome of this species opened the way for identifying Rotimer proteins by mass spectrometry. The predicted rotifer-biopolymer forming components are SCO-spondins and 14-3-3 protein. The characteristics of Rotimer are similar to Reissner's fiber, which is found in the central nervous system of vertebrates and is mainly formed from SCO-spondins. This molecular information serves as a starting point for its interdisciplinary investigation and application in biotechnology, biomedicine, or neurodegeneration-related drug development.

Evolutionary mode for the functional preservation of fast-evolving Drosophila telomere capping proteins.

DNA end protection is fundamental for the long-term preservation of the genome. In vertebrates the Shelterin protein complex protects telomeric DNA ends, thereby contributing to the maintenance of genome integrity. In the Drosophila genus, this function is thought to be performed by the Terminin complex, an assembly of fast-evolving subunits. Considering that DNA end protection is fundamental for successful genome replication, the accelerated evolution of Terminin subunits is counterintuitive, as conservation is supposed to maintain the assembly and concerted function of the interacting partners. This problem extends over Drosophila telomere biology and provides insight into the evolution of protein assemblies. In order to learn more about the mechanistic details of this phenomenon we have investigated the intra- and interspecies assemblies of Verrocchio and Modigliani, two Terminin subunits using in vitro assays. Based on our results and on homology-based three-dimensional models for Ver and Moi, we conclude that both proteins contain Ob-fold and contribute to the ssDNA binding of the Terminin complex. We propose that the preservation of Ver function is achieved by conservation of specific amino acids responsible for folding or localized in interacting surfaces. We also provide here the first evidence on Moi DNA binding.

TERT promoter alterations could provide a solution for Peto's paradox in rodents.

Cancer is a genetic disease caused by changes in gene expression resulting from somatic mutations and epigenetic changes. Although the probability of mutations is proportional with cell number and replication cycles, large bodied species do not develop cancer more frequently than smaller ones. This notion is known as Peto's paradox, and assumes stronger tumor suppression in larger animals. One of the possible tumor suppressor mechanisms involved could be replicative senescence caused by telomere shortening in the absence of telomerase activity. We analysed telomerase promoter activity and transcription factor binding in mammals to identify the key element of telomerase gene inactivation. We found that the GABPA transcription factor plays a key role in TERT regulation in somatic cells of small rodents, but its binding site is absent in larger beavers. Protein binding and reporter gene assays verify different use of this site in different species. The presence or absence of the GABPA TF site in TERT promoters of rodents correlates with TERT promoter activity; thus it could determine whether replicative senescence plays a tumor suppressor role in these species, which could be in direct relation with body mass. The GABPA TF binding sites that contribute to TERT activity in somatic cells of rodents are analogous to those mutated in human tumors, which activate telomerase by a non-ALT mechanism.

Alternative linker histone permits fast paced nuclear divisions in early Drosophila embryo.

In most animals, the start of embryogenesis requires specific histones. In Drosophila linker histone variant BigH1 is present in early embryos. To uncover the specific role of this alternative linker histone at early embryogenesis, we established fly lines in which domains of BigH1 have been replaced partially or completely with that of H1. Analysis of the resulting Drosophila lines revealed that at normal temperature somatic H1 can substitute the alternative linker histone, but at low temperature the globular and C-terminal domains of BigH1 are essential for embryogenesis. In the presence of BigH1 nucleosome stability increases and core histone incorporation into nucleosomes is more rapid, while nucleosome spacing is unchanged. Chromatin formation in the presence of BigH1 permits the fast-paced nuclear divisions of the early embryo. We propose a model which explains how this specific linker histone ensures the rapid nucleosome reassembly required during quick replication cycles at the start of embryogenesis.

Cross-Species Interaction between Rapidly Evolving Telomere-Specific Drosophila Proteins.

Telomere integrity in Drosophila melanogaster is maintained by a putative multisubunit complex called terminin that is believed to act in analogy to the mammalian shelterin complex in protecting chromosome ends from being recognized as sites of DNA damage. The five proteins supposed to form the terminin complex are HP1-ORC associated protein, HP1-HOAP interacting protein, Verrocchio, Drosophila Telomere Loss/Modigliani and Heterochromatic Protein 1. Four of these proteins evolve rapidly within the Drosophila genus. The accelerated evolution of terminin components may indicate the involvement of these proteins in the process by which new species arise, as the resulting divergence of terminin proteins might prevent hybrid formation, thus driving speciation. However, terminin is not an experimentally proven entity, and no biochemical studies have been performed to investigate its assembly and action in detail. Motivated by these facts in order to initiate biochemical studies on terminin function, we attempted to reconstitute terminin by co-expressing its subunits in bacteria and investigated the possible role of the fast-evolving parts of terminin components in complex assembly. Our results suggest formation of stable subcomplexes of terminin, but not of the whole complex in vitro. We found that the accelerated evolution is restricted to definable regions of terminin components, and that the divergence of D. melanogaster Drosophila Telomere Loss and D. yakuba Verrocchio proteins does not preclude their stable interaction.

mRNA levels of related Abcb genes change opposite to each other upon histone deacetylase inhibition in drug-resistant rat hepatoma cells.

The multidrug-resistant phenotype of tumor cells is acquired via an increased capability of drug efflux by ABC transporters and causes serious problems in cancer treatment. With the aim to uncover whether changes induced by epigenetic mechanisms in the expression level of drug transporter genes correlates with changes in the drug resistance phenotypes of resistant cells, we studied the expression of drug transporters in rat hepatoma cell lines. We found that of the three major rat ABC transporter genes Abcb1a, Abcb1b and Abcc1 the activity of only Abcb1b increased significantly in colchicine-selected, drug-resistant cells. Increased transporter expression in drug-resistant cells results primarily from transcriptional activation. A change in histone modification at the regulatory regions of the chromosomally adjacent Abcb1a and Abcb1b genes differentially affects the levels of corresponding mRNAs. Transcriptional up- and down-regulation accompany an increase in acetylation levels of histone H3 lysine 9 at the promoter regions of Abcb1b and Abcb1a, respectively. Drug efflux activity, however, does not follow tightly the transcriptional activity of drug transporter genes in hepatoma cells. Our results point out the need for careful analysis of cause-and-effect relationships between changes in histone modification, drug transporter expression and drug resistance phenotypes.