The importin-alpha/nucleophosmin switch controls taspase1 protease function

Taspase1 is a threonine protease suspected to process (patho)biologically relevant nuclear and cytoplasmic substrates, such as the mixed lineage leukemia protein. However, neither the mechanisms regulating Taspase1's intracellular localization nor their functional consequences are known. Analysis of endogenous and ectopically expressed Taspase1 detected the protease predominantly in the nucleus accumulating at the nucleolus. Microinjection and ectopic expression studies identified an evolutionarily conserved bipartite nuclear import signal (NLS) (amino acids (197) KRNKRKLELA ERVDTDFMQLKKRR(220) ) interacting with importin-α. Notably, an NLS-mutated, import-deficient Taspase1 was biologically inactive. Although the NLS conferred nuclear transport already of the proenzyme, Taspase1's nucleolar localization required its autoproteolytic processing, triggering its interaction with the nucleolar shuttle protein nucleophosmin. In contrast, (auto)catalytically inactive Taspase1 mutants neither accumulated at the nucleolus nor bound nucleophosmin. Active nuclear import and interaction with nucleophosmin was found to be required for the formation of proteolytically active Taspase1 ensuring to efficiently process its nuclear targets. Intriguingly, coexpression of pathological nucleophosmin variants increased the amount of cytoplasmic Taspase1. Hence, Taspase1 appears to exploit the nuclear export activity of nucleophosmin to gain transient access to the cytoplasm required to also cleave its cytoplasmic substrates. Collectively, we here describe a hitherto unknown mechanism regulating the biological activity of this protease.     

Analysis of historic changes in regional ecosystem service provisioning using land use data

Land use change has a major impact on goods and services that our environment supplies for society. While detailed ecological or biophysical field studies are needed to quantify the exact amount of ecosystem service supply at local scales, such a monitoring might be unfeasible at the regional scale. Since field scale monitoring schemes for ecosystem services or ecosystem functioning are missing, proxy based indicators can help to assess the historic development of ecosystem services or ecosystem functioning at the regional scale. We show at the example of the historic development (1964–2004) in the district of Leipzig/Germany how land use/land cover data can be used to derive regional scale indicators for ecosystem functions. We focus thereby on two hypotheses: (1) the ecosystem functioning has degraded over time and (2) changes in land use configuration play an important role in this degradation. The study focuses on indicators for ecosystem functions related to (i) water purification by riparian buffer strips, (ii) pollination, (iii) food production and (iv) outdoor recreation. Each indicator builds on the analysis of land use configuration and land use composition information and is tested on sensitivity/robustness with respect to parameters which had to be estimated based on expert knowledge. We show that land use composition is an important aspect in our ecosystem service assessment. Although our study region is faced with a maximum land use change of 11% in the major land use classes between 1964 and 2004, we see a decrease of ecosystem function indicators up to 23%. The regional assessment shows an overall trend for degradation of ecosystem functioning from 1964 to 1984. This trend is reversed between 1984 and 1994 but the process slowed down until 2004 without reaching the level of 1964.     

The deubiquitinating enzyme Ubp1 affects sorting of the ATP-binding cassette-transporter Ste6 in the endocytic pathway

Deubiquitinating enzymes (Dubs) are potential regulators of ubiquitination-dependent processes. Here, we focus on a member of the yeast ubiquitin-specific processing protease (Ubp) family, the Ubp1 protein. We could show that Ubp1 exists in two forms: a longer membrane-anchored form (mUbp1) and a shorter soluble form (sUbp1) that seem to be independently expressed from the same gene. The membrane-associated mUbp1 variant could be localized to the endoplasmic reticulum (ER) membrane by sucrose density gradient centrifugation and by immunofluorescence microscopy. Overexpression of the soluble Ubp1 variant stabilizes the ATP-binding cassette-transporter Ste6, which is transported to the lysosome-like vacuole for degradation, and whose transport is regulated by ubiquitination. Ste6 stabilization was not the result of a general increase in deubiquitination activity, because overexpression of Ubp1 had no effect on the degradation of the ER-associated degradation substrate carboxypeptidase Y^* and most importantly on Ste6 ubiquitination itself. Also, overexpression of another yeast Dub, Ubp3, had no effect on Ste6 turnover. This suggests that the Ubp1 target is a component of the protein transport machinery. On Ubp1 overexpression, Ste6 accumulates at the cell surface, which is consistent with a role of Ubp1 at the internalization step of endocytosis or with enhanced recycling to the cell surface from an internal compartment.     

Ddx42p--a human DEAD box protein with RNA chaperone activities

The human gene ddx42 encodes a human DEAD box protein highly homologous to the p68 subfamily of RNA helicases. In HeLa cells, two ddx42 poly(A)⁺ RNA species were detected both encoding the nuclear localized 938 amino acid Ddx42p polypeptide. Ddx42p has been heterologously expressed and its biochemical properties characterized. It is an RNA binding protein, and ATP and ADP modulate its RNA binding affinity. Ddx42p is an NTPase with a preference for ATP, the hydrolysis of which is enhanced by various RNA substrates. It acts as a non-processive RNA helicase. Interestingly, RNA unwinding by Ddx42p is promoted in the presence of a single-strand (ss) binding protein (T4gp32). Ddx42p, particularly in the ADP-bound form (the state after ATP hydrolysis), also mediates efficient annealing of complementary RNA strands thereby displacing the ss binding protein. Ddx42p therefore represents the first example of a human DEAD box protein possessing RNA helicase, protein displacement and RNA annealing activities. The adenosine nucleotide cofactor bound to Ddx42p apparently acts as a switch that controls the two opposing activities: ATP triggers RNA strand separation, whereas ADP triggers annealing of complementary RNA strands.     

Run-Off Replication of Host-Adaptability Genes Is Associated with Gene Transfer Agents in the Genome of Mouse-Infecting Bartonella grahamii

The genus Bartonella comprises facultative intracellular bacteria adapted to mammals, including previously recognized and emerging human pathogens. We report the 2,341,328 bp genome sequence of Bartonella grahamii, one of the most prevalent Bartonella species in wild rodents. Comparative genomics revealed that rodent-associated Bartonella species have higher copy numbers of genes for putative host-adaptability factors than the related human-specific pathogens. Many of these gene clusters are located in a highly dynamic region of 461 kb. Using hybridization to a microarray designed for the B. grahamii genome, we observed a massive, putatively phage-derived run-off replication of this region. We also identified a novel gene transfer agent, which packages the bacterial genome, with an over-representation of the amplified DNA, in 14 kb pieces. This is the first observation associating the products of run-off replication with a gene transfer agent. Because of the high concentration of gene clusters for host-adaptation proteins in the amplified region, and since the genes encoding the gene transfer agent and the phage origin are well conserved in Bartonella, we hypothesize that these systems are driven by selection. We propose that the coupling of run-off replication with gene transfer agents promotes diversification and rapid spread of host-adaptability factors, facilitating host shifts in Bartonella.     

The evolutionary history of the white-rayed species of Melampodium (Asteraceae) involved multiple cycles of hybridization and polyploidization

? Premise of the study: Polyploidy plays an important role in race differentiation and eventually speciation. Underlying mechanisms include chromosomal and genomic changes facilitating reproductive isolation and/or stabilization of hybrids. A prerequisite for studying these processes is a sound knowledge on the origin of polyploids. A well-suited group for studying polyploid evolution consists of the three species of Melampodium ser. Leucantha (Asteraceae): M. argophyllum, M. cinereum, and M. leucanthum. ? Methods: The origin of polyploids was inferred using network and tree-based phylogenetic analyses of several plastid and nuclear DNA sequences and of fingerprint data (AFLP). Genome evolution was assessed via genome size measurements, karyotype analysis, and in situ hybridization of ribosomal DNA. ? Key results: Tetraploid cytotypes of the phylogenetically distinct M. cinereum and M. leucanthum had, compared to the diploid cytotypes, doubled genome sizes and no evidence of gross chromosomal rearrangements. Hexaploid M. argophyllum constituted a separate lineage with limited intermixing with the other species, except in analyses from nuclear ITS. Its genome size was lower than expected if M. cinereum and/or M. leucanthum were involved in its origin, and no chromosomal rearrangements were evident. ? Conclusions: Polyploids in M. cinereum and M. leucanthum are of recent autopolyploid origin in line with the lack of significant genomic changes. Hexaploid M. argophyllum also appears to be of autopolyploid origin against the previous hypothesis of an allopolyploid origin involving the other two species, but some gene flow with the other species in early phases of differentiation cannot be excluded.     

The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii

One of the most abundantly IFN-γ-induced protein families in different cell types is the 65-kDa guanylate-binding protein family that is recruited to the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to Toxoplasma gondii. The wild type protein exhibits low affinities to guanine nucleotides, self-assembles upon GTP binding, forming tetramers in the activated state, and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing multiparameter fluorescence image spectroscopy (MFIS) using a Homo-FRET assay shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase domain. The consistent results indicate that GTP binding, self-assembly, and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase domain regulates efficient recruitment to T. gondii in response to IFN-γ.     

Comparative metaproteomics and diversity analysis of human intestinal microbiota testifies for its temporal stability and expression of core functions

The human intestinal tract is colonized by microbial communities that show a subject-specific composition and a high-level temporal stability in healthy adults. To determine whether this is reflected at the functional level, we compared the faecal metaproteomes of healthy subjects over time using a novel high-throughput approach based on denaturing polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry. The developed robust metaproteomics workflow and identification pipeline was used to study the composition and temporal stability of the intestinal metaproteome using faecal samples collected from 3 healthy subjects over a period of six to twelve months. The same samples were also subjected to DNA extraction and analysed for their microbial composition and diversity using the Human Intestinal Tract Chip, a validated phylogenetic microarray. Using metagenome and single genome sequence data out of the thousands of mass spectra generated per sample, approximately 1,000 peptides per sample were identified. Our results indicate that the faecal metaproteome is subject-specific and stable during a one-year period. A stable common core of approximately 1,000 proteins could be recognized in each of the subjects, indicating a common functional core that is mainly involved in carbohydrate transport and degradation. Additionally, a variety of surface proteins could be identified, including potential microbes-host interacting components such as flagellins and pili. Altogether, we observed a highly comparable subject-specific clustering of the metaproteomic and phylogenetic profiles, indicating that the distinct microbial activity is reflected by the individual composition.     

Analysis of the fibroblast growth factor system reveals alterations in a mouse model of spinal muscular atrophy

The monogenetic disease Spinal Muscular Atrophy (SMA) is characterized by a progressive loss of motoneurons leading to muscle weakness and atrophy due to severe reduction of the Survival of Motoneuron (SMN) protein. Several models of SMA show deficits in neurite outgrowth and maintenance of neuromuscular junction (NMJ) structure. Survival of motoneurons, axonal outgrowth and formation of NMJ is controlled by neurotrophic factors such as the Fibroblast Growth Factor (FGF) system. Besides their classical role as extracellular ligands, some FGFs exert also intracellular functions controlling neuronal differentiation. We have previously shown that intracellular FGF-2 binds to SMN and regulates the number of a subtype of nuclear bodies which are reduced in SMA patients. In the light of these findings, we systematically analyzed the FGF-system comprising five canonical receptors and 22 ligands in a severe mouse model of SMA. In this study, we demonstrate widespread alterations of the FGF-system in both muscle and spinal cord. Importantly, FGF-receptor 1 is upregulated in spinal cord at a pre-symptomatic stage as well as in a mouse motoneuron-like cell-line NSC34 based model of SMA. Consistent with that, phosphorylations of FGFR-downstream targets Akt and ERK are increased. Moreover, ERK hyper-phosphorylation is functionally linked to FGFR-1 as revealed by receptor inhibition experiments. Our study shows that the FGF system is dysregulated at an early stage in SMA and may contribute to the SMA pathogenesis.