Zasp/Cypher internal ZM-motif containing fragments are sufficient to co-localize with alpha-actinin--analysis of patient mutations

Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) has an important role in maintaining Z-disc stability in striated and cardiac muscle. ZASP/Cypher interacts through its PDZ domain with the major Z-disc actin cross-linker, alpha-actinin. ZASP/Cypher also has a conserved sequence called the ZM-motif, and it is found in two alternatively spliced exons 4 and 6. We have shown earlier that the ZM-motif containing internal regions of two related proteins ALP and CLP36 interact with alpha-actinin rod region, and that the ZM-motif is important in targeting ALP to the alpha-actinin containing structures in cell. Here, we show that the ZASP/Cypher internal fragments containing either ZM exon 4 or 6 co-localized with alpha-actinin in cultured myoblasts and nonmuscle cells. Fragments of 130 residues around the ZM-consensus were sufficient for localization, which is similar to our previous results of ALP. Moreover, ZASP/Cypher protein interacted directly with the alpha-actinin rod and competed with ALP in binding to the rod. During the inhibition of stress fiber assembly ZASP/Cypher and alpha-actinin co-localization could be partially disturbed, suggesting that ZASP/Cypher is bound to alpha-actinin mainly when alpha-actinin is localizing in stress fibers. Many point mutations found in cardiomyopathy patients are located in the internal region of ZASP/Cypher. However, we found no evidence that human patient mutations in the internal domain would affect the ZASP/Cypher co-localization with alpha-actinin, or that the mutations would destabilize the ZASP/Cypher protein.     

Cortinarius sect. Brunnei (Basidiomycota, Agaricales) in North Europe

The section Brunnei was extensively studied based on material from North Europe. To stabilise the nomenclature we studied the relevant types of taxa included in this section. Phylogenetic relationships and species limits were investigated using rDNA ITS sequences and the results were compared with the morphological data. We recognised 11 species: Cortinarius brunneus, C. clarobrunneus comb. nov., C. coleoptera, C. ectypus, C. gentilis, C. glandicolor (neotypified), C. pseudorubricosus, and four species described as new C. caesiobrunneus, C. albogaudis, C. carabus, and C. cicindela. They are described here and their taxonomy, ecology, distribution, and relationships are discussed. In addition, a key to species of the section Brunnei is provided. A total of 77 new sequences of 11 species are published including nine type sequences. Also the taxonomic assignments of sequences in the public databases belonging to the section Brunnei are revised.     

Parvovirus Induced Alterations in Nuclear Architecture and Dynamics

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analyzis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications.     

Validating quantitative fatty acid signature analysis to estimate diets of spectacled and Steller’s eiders (Somateria fischeri and Polysticta stelleri)

Fatty acid (FA) signature analysis has been used to study foraging ecology and food webs in marine ecosystems. This powerful method provides information about diets over an extended time period (e.g., 2–4 weeks), rather than just the most recent meal as with most traditional approaches. Using consumer FA signatures, along with a comprehensive database of diet FA signatures, and accounting for consumer FA metabolism, it is possible to estimate the proportions of diet items in the consumer’s diet using quantitative FA signature analysis (QFASA). However, before applying QFASA to free-ranging populations, ideally, controlled feeding studies are performed to determine FA deposition and turnover characteristics. We conducted feeding experiments to validate QFASA in captive spectacled eiders (Somateria fischeri) and Steller’s eiders (Polysticta stelleri) as a minimally invasive method for studying the diets of these threatened species. We determined FA deposition in eider adipose tissue relative to long-term diet, and developed calibration coefficients (CCs) to account for eider lipid metabolism. Using these CCs with subsequent diet trials, QFASA accurately indicated diet and diet switches. QFASA estimates also indicated that turnover of dietary FAs was not complete by 21 or 29 days, and confirmed that diets could be estimated over an extended period of >29 days. Thus, our understanding of diet can be backtracked to more than a month in captive feeding eiders. We conclude that applying QFASA techniques to eiders and other birds in the wild has the potential to provide valuable information about their diets at various life history stages.     

Pripper: prediction of caspase cleavage sites from whole proteomes

Background  

Caspases are a family of proteases that have central functions in programmed cell death (apoptosis) and inflammation. Caspases mediate their effects through aspartate-specific cleavage of their target proteins, and at present almost 400 caspase substrates are known. There are several methods developed to predict caspase cleavage sites from individual proteins, but currently none of them can be used to predict caspase cleavage sites from multiple proteins or entire proteomes, or to use several classifiers in combination. The possibility to create a database from predicted caspase cleavage products for the whole genome could significantly aid in identifying novel caspase targets from tandem mass spectrometry based proteomic experiments.     

Local and average gloss from flat-faced sodium chloride tablets

The purpose of this study was to detect local gloss and surface structure changes of sodium chloride tablets. The changes in surface structure were reflected by gloss variation, which was measured using a diffractive optical element-based gloss-meter (DOG). By scanning a surface area, we constructed a 2-dimensional gloss map that characterized the tablet’s surface structure. The gloss variation results were compared with scanning electron microscopy (SEM) images and average surface roughness values that were measured by conventional diamond stylus profilometry. The profilometry data showed a decrease in tablet surface roughness as a function of compression force. In general, a smoother surface contributes to higher average gloss values. The average gloss values for this material, in contrast, showed a decrease as a function of the compression force. The sequence of particle fragmentation and deformation together with crack formation in sodium chloride particles resulted in a loss of gloss for single sodium chloride particles at the tablet surfaces, which could be detected by the DOG. These results were supported by the SEM images. The results show that detailed information regarding tablets’ surface structure changes can be obtained by detection of local gloss variation and average gloss. Published: January 13, 2006     

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations.The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.     

Probing pH-dependent functional elements in proteins: modification of carboxylic acid pairs in Trichoderma reesei cellobiohydrolase Cel6A

Two carboxylic acid side chains can, depending on their geometry and environment, share a proton in a hydrogen bond and form a carboxyl-carboxylate pair. In the Trichoderma reesei cellobiohydrolase Cel6A structure, five carboxyl-carboxylate pairs are observed. One of these pairs (D175-D221) is involved in catalysis, and three other pairs are found in, or close to the two surface loops covering the active site tunnel of the catalytic domain. To stabilize Cel6A at alkaline pH values, where deprotonation of the carboxylic acids leads to repulsion of their side chains, we designed two mutant enzymes. In the first mutant, one carboxyl-carboxylate pair (E107-E399) was replaced by a corresponding amide-carboxylate pair (Q107-E399), and in the second mutant, all three carboxyl-carboxylate pairs (E107-E399, D170-E184, and D366-D419) were mutated in a similar manner. The unfolding studies using both intrinsic tryptophan fluorescence and far-ultraviolet circular dichroism spectroscopy at different pH values demonstrate that the unfolding temperature (T(m)) of both mutants has changed, resulting in destabilization of the mutant enzymes at acidic pH and stabilization at alkaline pH. The effect of stabilization seems additive, as a Cel6A triple mutant is the most stable enzyme variant. This increased stability is also reflected in the 2- or 4-fold increased half-life of the two mutants at alkaline pH, while the catalytic rate on cellotetraose (at t = 0) has not changed. Increased operational stability at alkaline pH was also observed on insoluble cellulosic substrates. Local conformational changes are suggested to take place in the active site loops of Cel6A wild-type enzyme at elevated pHs (pH 7), affecting to the end-product spectrum on insoluble cellulose. The triple mutant does not show such pH-dependent behavior. Overall, our results demonstrate that carboxyl-carboxylate pair engineering is a useful tool to alter pH-dependent protein behavior.     

A novel specific role for I kappa B kinase complex-associated protein in cytosolic stress signaling

We demonstrate here a novel role for the I kappa B kinase complex-associated protein (IKAP) in the regulation of activation of the mammalian stress response via the c-Jun N-terminal kinase (JNK)-signaling pathway. We cloned IKAP as a JNK-associating protein using the Ras recruitment yeast two-hybrid system. IKAP efficiently and specifically enhanced JNK activation induced by ectopic expression of MEKK1 and ASK1, upstream activators of JNK. Importantly, IKAP also enhanced JNK activation induced by ultraviolet light irradiation as well as treatments with tumor necrosis factor or epidermal growth factor. The JNK association site in IKAP was mapped to the C-terminal part of IKAP. Interestingly, this region is deleted from IKAP expressed in the autonomous nervous system of the patients affected by familial dysautonomia. Ectopic expression of this C-terminal fragment of IKAP was sufficient to support JNK activation. Taken together, our data demonstrate a novel role for IKAP in the regulation of the JNK-mediated stress signaling. Additionally, our results point to a role of JNK signaling in familial dysautonomia and, thus, further support the involvement of JNK signaling in the development, survival, and degeneration of the sensory and autonomic nervous system.