A Novel Mechanism for SUMO System Control: Regulated Ulp1 Nucleolar Sequestration

The small ubiquitin-related modifiers (SUMOs) are evolutionarily conserved polypeptides that are covalently conjugated to protein targets to modulate their subcellular localization, half-life, or activity. Steady-state SUMO conjugation levels increase in response to many different types of environmental stresses, but how the SUMO system is regulated in response to these insults is not well understood. Here, we characterize a novel mode of SUMO system control: in response to elevated alcohol levels, the Saccharomyces cerevisiae SUMO protease Ulp1 is disengaged from its usual location at the nuclear pore complex (NPC) and sequestered in the nucleolus. We further show that the Ulp1 region previously demonstrated to interact with the karyopherins Kap95 and Kap60 (amino acids 150 to 340) is necessary and sufficient for nucleolar targeting and that enforced sequestration of Ulp1 in the nucleolus significantly increases steady-state SUMO conjugate levels, even in the absence of alcohol. We have thus characterized a novel mechanism of SUMO system control in which the balance between SUMO-conjugating and -deconjugating activities at the NPC is altered in response to stress via relocalization of a SUMO-deconjugating enzyme.The small ubiquitin-related modifiers (SUMOs) are a family of evolutionarily conserved polypeptides that are conjugated to protein targets via the concerted action of SUMO-specific E1 (activation), E2 (conjugation), and E3 (ligase) enzymes to effect changes in subcellular localization, half-life, or target activity. A family of SUMO-specific proteases act to remove the modifier from conjugates (8, 20). The SUMO system has been implicated in a variety of critical cellular functions, such as DNA repair and replication, RNA metabolism, and stress responses (8, 16, 20). Importantly, the SUMO system is highly dynamic and the SUMO pathway enzymes appear to work together to precisely control SUMO conjugate levels in the cell (8, 16, 20). However, how the SUMO system itself is regulated is poorly understood.Localization of the SUMO pathway enzymes may play an important role in SUMO system function (21). For example, the budding yeast SUMO protease Ulp1 is tethered to the nuclear face of the nuclear pore complex (NPC) via an unconventional interaction with the karyopherin Kap121 and the heterodimeric Kap95/Kap60 complex (12, 13, 23). However, this SUMO protease is not maintained exclusively at the NPC but appears to be mobile, effecting desumoylation at diverse subcellular locations: e.g., during mitosis, Saccharomyces cerevisiae Ulp1 is recruited to the septin ring to desumoylate septins (15), Schizosaccharomyces pombe Ulp1 localization is regulated throughout the cell cycle (31), and a mammalian Ulp1 homolog, SENP2, is shuttled between the nucleus and the cytoplasm (7). Consistent with these observations, SUMO conjugate levels are significantly altered in yeast strains expressing mislocalized Ulp1 (13, 37).Dramatic changes in SUMO conjugate populations have been noted in response to many different types of stresses in yeasts, mammals, and plants (9, 17, 27, 32, 38). For example, in S. cerevisiae, significantly increased steady-state SUMO conjugate levels are observed in response to elevated concentrations of ethanol (38). To better understand how the SUMO system is regulated in response to stress, we utilized alcohol as a model of a physiologically relevant stressor in yeast. Here, we demonstrate that alcohol stress results in a rapid, reversible nucleolar sequestration of Ulp1 and that enforced localization of Ulp1 in the nucleolus leads to a dramatic increase in steady-state SUMO conjugate levels. This is the first demonstration of regulated modulation of the intracellular localization of a SUMO enzyme in response to stress and thus represents a novel mechanism for SUMO system control.     

Synthesis and pharmacological evaluation of novel 1,3,8- and 1,3,7,8-substituted xanthines as adenosine receptor antagonists

A number of novel xanthines bearing a variety of substituents at positions 1, 3, 7 and 8 were prepared and evaluated for their binding affinity to the human adenosine receptor A₁, A_2A, A_2B and A₃ subtypes. Several of the 1,3,8- and 1,3,7,8-substituted xanthines showed moderate-to-high affinity at human A_2B and A₁ receptors, with the most active compound (14q) having a pK_i of 7.57 nM for hA_2B receptors and a selectivity over hA_2A receptors of 8.1-fold and hA₁ receptors of 3.7-fold.     

A new series of flavones,thioflavones, and flavanones as selective monoamine oxidase-B inhibitors

A new series of synthetic flavones, thioflavones, and flavanones has been synthesized and evaluated as potential inhibitors of monoamine oxidase isoforms (MAO-A and -B). The most active series is the flavanone one with higher selective inhibitory activity against MAO-B. Some of these flavanones (mainly the most effective) have been separated and tested as single enantiomers. In order to investigate the MAOs recognition of the most active and selective compounds, a molecular modeling study has been performed using available Protein Data Bank (PDB) structures as receptor models for docking experiments.     

A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus

The functional expression of heterologous genes using standard bacterial expression hosts such as Escherichia coli is often limited, e.g. by incorrect folding, assembly or targeting of recombinant proteins. Consequently, alternative bacterial expression systems have to be developed to provide novel strategies for protein synthesis exceeding the repertoire of the standard expression host E. coli.Here, we report on the construction of a novel expression system that combines the high processivity of T7 RNA polymerase with the unique physiological properties of the facultative photosynthetic bacterium Rhodobacter capsulatus. This system basically consists of a recombinant R. capsulatus T7 expression strain (R. capsulatus B10S-T7) harboring the respective polymerase gene under control of a fructose inducible promoter. In addition, a set of different broad-host-range vectors (pRho) was constructed allowing T7 RNA polymerase dependent and independent target gene expression in R. capsulatus and other Gram-negative bacteria. The expression efficiency of the novel system was studied in R. capsulatus and E. coli using the yellow fluorescent protein (YFP) as model protein. Expression levels were comparable in both expression hosts and yielded up to 80 mg/l YFP in phototrophically grown R. capsulatus cultures. This result clearly indicates that the novel R. capsulatus-based expression system is well suited for the high-level expression of soluble proteins.     

Molecular ecology of fungal entomopathogens: molecular genetic tools and their applications in population and fate studies

The power of molecular genetic techniques to address ecological research questions has opened a distinct interdisciplinary research area collectively referred to as molecular ecology. Molecular ecology combines aspects of diverse research fields like population and evolutionary genetics, as well as biodiversity, conservation biology, behavioural ecology, or species-habitat interactions. Molecular techniques detect specific DNA sequence characteristics that are used as genetic markers to discriminate individuals or taxonomic groups, for instance in analyses of population and community structures, for elucidation of phylogenetic relationships, or for the characterization and monitoring of specific strains in the environment. Here, we summarize the PCR-based molecular techniques used in molecular ecological research on fungal entomopathogens and discuss novel techniques that may have relevance to the studies of entomopathogenic fungi in the future. We discuss the flow chart of the molecular ecology approaches and we highlight some of the critical steps involved. There are still many unresolved questions in the understanding of the ecology of fungal entomopathogens. These include population characteristics and relations of genotypes and habitats as well as host-pathogen interactions. Molecular tools can provide substantial support for ecological research and offer insight into this far inaccessible systems. Application of molecular ecology approaches will stimulate and accelerate new research in the field of entomophathogen ecology.     

Characterisation of Zea mays L. plastidial transglutaminase: interactions with thylakoid membrane proteins

Chloroplast transglutaminase (chlTGase) activity is considered to play a significant role in response to a light stimulus and photo‐adaptation of plants, but its precise function in the chloroplast is unclear. The characterisation, at the proteomic level, of the chlTGase interaction with thylakoid proteins and demonstration of its association with photosystem II (PSII) protein complexes was accomplished with experiments using maize thylakoid protein extracts. By means of a specific antibody designed against the C‐terminal sequence of the maize TGase gene product, different chlTGase forms were immunodetected in thylakoid membrane extracts from three different stages of maize chloroplast differentiation. These bands co‐localised with those of lhcb 1, 2 and 3 antenna proteins. The most significant, a 58 kDa form present in mature chloroplasts, was characterised using biochemical and proteomic approaches. Sequential fractionation of thylakoid proteins from light‐induced mature chloroplasts showed that the 58 kDa form was associated with the thylakoid membrane, behaving as a soluble or peripheral membrane protein. Two‐dimensional gel electrophoresis discriminated, for the first time, the 58‐kDa band in two different forms, probably corresponding to the two different TGase cDNAs previously cloned. Electrophoretic separation of thylakoid proteins in native gels, followed by LC‐MS mass spectrometry identification of protein complexes indicated that maize chlTGase forms part of a specific PSII protein complex, which includes LHCII, ATPase and pSbS proteins. The results are discussed in relation to the interaction between these proteins and the suggested role of the enzyme in thylakoid membrane organisation and photoprotection.     

Rock weathering creates oases of life in a High Arctic desert

During primary colonization of rock substrates by plants, mineral weathering is strongly accelerated under plant roots, but little is known on how it affects soil ecosystem development before plant establishment. Here we show that rock mineral weathering mediated by chemolithoautotrophic bacteria is associated to plant community formation in sites recently released by permanent glacier ice cover in the Midtre Lovénbreen glacier moraine (78°53′N), Svalbard. Increased soil fertility fosters growth of prokaryotes and plants at the boundary between sites of intense bacterial mediated chemolithotrophic iron‐sulfur oxidation and pH decrease, and the common moraine substrate where carbon and nitrogen are fixed by cyanobacteria. Microbial iron oxidizing activity determines acidity and corresponding fertility gradients, where water retention, cation exchange capacity and nutrient availability are increased. This fertilization is enabled by abundant mineral nutrients and reduced forms of iron and sulfur in pyrite minerals within a conglomerate type of moraine rock. Such an interaction between microorganisms and moraine minerals determines a peculiar, not yet described model for soil genesis and plant ecosystem formation with potential past and present analogues in other harsh environments with similar geochemical settings.     

Changes in mouse whole saliva soluble proteome induced by tannin-enriched diet

Background  

Previous studies suggested that dietary tannin ingestion may induce changes in mouse salivary proteins in addition to the primarily studied proline-rich proteins (PRPs). The aim of the present study was to determine the protein expression changes induced by condensed tannin intake on the fraction of mouse whole salivary proteins that are unable to form insoluble tannin-protein complexes. Two-dimensional polyacrylamide gel electrophoresis protein separation was used, followed by protein identification by mass spectrometry.     

Analysis of changes in tyrosine and serine phosphorylation of red cell membrane proteins induced by P. falciparum growth

Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti‐phosphotyrosine and anti‐phosphoserine antibodies following 2‐DE in conjunction with double channel laser‐induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans‐membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.