An Interaction Network Predicted from Public Data as a Discovery Tool: Application to the Hsp90 Molecular Chaperone Machine

Understanding the functions of proteins requires information about their protein-protein interactions (PPI). The collective effort of the scientific community generates far more data on any given protein than individual experimental approaches. The latter are often too limited to reveal an interactome comprehensively. We developed a workflow for parallel mining of all major PPI databases, containing data from several model organisms, and to integrate data from the literature for a protein of interest. We applied this novel approach to build the PPI network of the human Hsp90 molecular chaperone machine (Hsp90Int) for which previous efforts have yielded limited and poorly overlapping sets of interactors. We demonstrate the power of the Hsp90Int database as a discovery tool by validating the prediction that the Hsp90 co-chaperone Aha1 is involved in nucleocytoplasmic transport. Thus, we both describe how to build a custom database and introduce a powerful new resource for the scientific community

The Hsp70-Hsp90 co-chaperone Hop/Stip1 shifts the proteostatic balance from folding towards degradation.

Hop/Stip1/Sti1 is thought to be essential as a co-chaperone to facilitate substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Despite this proposed key function for protein folding and maturation, it is not essential in a number of eukaryotes and bacteria lack an ortholog. We set out to identify and to characterize its eukaryote-specific function. Human cell lines and the budding yeast with deletions of the Hop/Sti1 gene display reduced proteasome activity due to inefficient capping of the core particle with regulatory particles. Unexpectedly, knock-out cells are more proficient at preventing protein aggregation and at promoting protein refolding. Without the restraint by Hop, a more efficient folding activity of the prokaryote-like Hsp70-Hsp90 complex, which can also be demonstrated in vitro, compensates for the proteasomal defect and ensures the proteostatic equilibrium. Thus, cells may act on the level and/or activity of Hop to shift the proteostatic balance between folding and degradation

IL28B gene polymorphism rs12979860, but not rs8099917, contributes to the occurrence of chronic HCV infection in uruguayan patients

Background: Host single-nucleotide polymorphisms (SNPs) near the interleukin 28B (IL28B) locus are associated with sustained virological response to antiviral therapy and with spontaneous Hepatitis C Virus (HCV) clearance. Prevalence of these SNPs varies depending on ethnicity. The impact of IL28B SNPs in HCV-infected patients is currently unknown in Uruguay. Therefore, the aim of this study was to evaluate and compare the distribution of polymorphisms in the IL28B gene (rs12979860 and rs8099917) among HCV-infected patients and healthy individuals in Uruguay and thus assess their possible association with the establishment of HCV infection. Methods: DNA was recovered from 92 non-infected individuals and 78 HCV-infected patients and SNPs were determined by RFLP and allelic discrimination by real-time PCR. Results: The distribution of rs12979860 genotypes for the infected population was 29.5%-CC, 47.4%-CT and 23.1%-TT and for the control group 45.7,% 42.4% and 11.9,% respectively. Prevalence in both infected and uninfected individuals is similar to that reported in other countries with admixed populations. The distribution of rs8099917 genotypes for the infected population was 57.7%-TT, 27.2%-TG and 14.1%-GG and for the control group 60.9,% 33.7% and 5.4,% respectively. The comparison of rs12979860 genotype distribution between the two populations evidenced a higher prevalence of the favourable genotype (CC) in the uninfected control group (p < 0.05). Additionally, results generated using logistic regression analysis show that individuals carrying rs12979860-TT or CT genotypes have a higher likelihood of developing chronic hepatitis upon infection with HCV, when compared to CC carriers, considering rs8099917 genotype as constant. Conclusion: Patients with HCV infection have a statistically significant lower prevalence of the favourable rs12979860 genotype when compared to uninfected individuals; therefore we can establish that only IL28B rs12979860-CT and TT genotypes seem to contribute to the occurrence of chronic HCV infection in the cohort of Uruguayan population studied. Considering that a trend towards a higher frequency of "good" response genotypes was observed in responder patients, we believe that IL28B rs12979860 genotyping could be a useful tool for predicting different therapies outcome, including in the DAA era

Impairment of exogenous lactate clearance in experimental hyperdynamic septic shock is not related to total liver hypoperfusion

Introduction: Although the prognostic value of persistent hyperlactatemia in septic shock is unequivocal, its physiological determinants are controversial. Particularly, the role of impaired hepatic clearance has been underestimated and is only considered relevant in patients with liver ischemia or cirrhosis. Our objectives were to establish whether endotoxemia impairs whole body net lactate clearance, and to explore a potential role for total liver hypoperfusion during the early phase of septic shock. Methods: After anesthesia, 12 sheep were subjected to hemodynamic/perfusion monitoring including hepatic and portal catheterization, and a hepatic ultrasound flow probe. After stabilization (point A), sheep were alternatively assigned to lipopolysaccharide (LPS) (5 mcg/kg bolus followed by 4 mcg/kg/h) or sham for a three-hour study period. After 60 minutes of shock, animals were fluid resuscitated to normalize mean arterial pressure. Repeated series of measurements were performed immediately after fluid resuscitation (point B), and one (point C) and two hours later (point D). Monitoring included systemic and regional hemodynamics, blood gases and lactate measurements, and ex-vivo hepatic mitochondrial respiration at point D. Parallel exogenous lactate and sorbitol clearances were performed at points B and D. Both groups included an intravenous bolus followed by serial blood sampling to draw a curve using the least squares method. Results: Significant hyperlactatemia was already present in LPS as compared to sham animals at point B (4.7 (3.1 to 6.7) versus 1.8 (1.5 to 3.7) mmol/L), increasing to 10.2 (7.8 to 12.3) mmol/L at point D. A significant increase in portal and hepatic lactate levels in LPS animals was also observed. No within-group difference in hepatic DO2, VO2 or O2 extraction, total hepatic blood flow (point D: 915 (773 to 1,046) versus 655 (593 to 1,175) ml/min), mitochondrial respiration, liver enzymes or sorbitol clearance was found. However, there was a highly significant decrease in lactate clearance in LPS animals (point B: 46 (30 to 180) versus 1,212 (743 to 2,116) ml/min, P <0.01; point D: 113 (65 to 322) versus 944 (363 to 1,235) ml/min, P <0.01). Conclusions: Endotoxemia induces an early and severe impairment in lactate clearance that is not related to total liver hypoperfusion

Microbiology and Nitrogen Cycle in the Benthic Sediments of a Glacial Oligotrophic Deep Andean Lake as Analog of Ancient Martian Lake-Beds

Potential benthic habitats of early Mars lakes, probably oligotrophic, could range from hydrothermal to cold sediments. Dynamic processes in the water column (such as turbidity or UV penetration) as well as in the benthic bed (temperature gradients, turbation, or sedimentation rate) contribute to supply nutrients to a potential microbial ecosystem. High altitude, oligotrophic, and deep Andean lakes with active deglaciation processes and recent or past volcanic activity are natural models to assess the feasibility of life in other planetary lake/ocean environments and to develop technology for their exploration. We sampled the benthic sediments (down to 269 m depth) of the oligotrophic lake Laguna Negra (Central Andes, Chile) to investigate its ecosystem through geochemical, biomarker profiling, and molecular ecology studies. The chemistry of the benthic water was similar to the rest of the water column, except for variable amounts of ammonium (up to 2.8 ppm) and nitrate (up to 0.13 ppm). A life detector chip with a 300-antibody microarray revealed the presence of biomass in the form of exopolysaccharides and other microbial markers associated to several phylogenetic groups and potential microaerobic and anaerobic metabolisms such as nitrate reduction. DNA analyses showed that 27% of the Archaea sequences corresponded to a group of ammonia-oxidizing archaea (AOA) similar (97%) to Nitrosopumilus spp. and Nitrosoarchaeum spp. (Thaumarchaeota), and 4% of Bacteria sequences to nitrite-oxidizing bacteria from the Nitrospira genus, suggesting a coupling between ammonia and nitrite oxidation. Mesocosm experiments with the specific AOA inhibitor 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) demonstrated an AOA-associated ammonia oxidation activity with the simultaneous accumulation of nitrate and sulfate. The results showed a rich benthic microbial community dominated by microaerobic and anaerobic metabolisms thriving under aphotic, low temperature (4°C), and relatively high pressure, that might be a suitable terrestrial analog of other planetary settings

Detection of changes in gene regulatory patterns, elicited by perturbations of the Hsp90 molecular chaperone complex, by visualizing multiple experiments with an animation

<p>Abstract</p> <p>Background</p> <p>To make sense out of gene expression profiles, such analyses must be pushed beyond the mere listing of affected genes. For example, if a group of genes persistently display similar changes in expression levels under particular experimental conditions, and the proteins encoded by these genes interact and function in the same cellular compartments, this could be taken as very strong indicators for co-regulated protein complexes. One of the key requirements is having appropriate tools to detect such regulatory patterns.</p> <p>Results</p> <p>We have analyzed the global adaptations in gene expression patterns in the budding yeast when the Hsp90 molecular chaperone complex is perturbed either pharmacologically or genetically. We integrated these results with publicly accessible expression, protein-protein interaction and intracellular localization data. But most importantly, all experimental conditions were simultaneously and dynamically visualized with an animation. This critically facilitated the detection of patterns of gene expression changes that suggested underlying regulatory networks that a standard analysis by pairwise comparison and clustering could not have revealed.</p> <p>Conclusions</p> <p>The results of the animation-assisted detection of changes in gene regulatory patterns make predictions about the potential roles of Hsp90 and its co-chaperone p23 in regulating whole sets of genes. The simultaneous dynamic visualization of microarray experiments, represented in networks built by integrating one's own experimental with publicly accessible data, represents a powerful discovery tool that allows the generation of new interpretations and hypotheses.</p

Dung removal increases under higher dung beetle functional diversity regardless of grazing intensification

Dung removal by macrofauna such as dung beetles is an important process for nutrient cycling in pasturelands. Intensification of farming practices generally reduces species and functional diversity of terrestrial invertebrates, which may negatively affect ecosystem services. Here, we investigate the effects of cattle-grazing intensification on dung removal by dung beetles in field experiments replicated in 38 pastures around the world. Within each study site, we measured dung removal in pastures managed with low- and high-intensity regimes to assess between-regime differences in dung beetle diversity and dung removal, whilst also considering climate and regional variations. The impacts of intensification were heterogeneous, either diminishing or increasing dung beetle species richness, functional diversity, and dung removal rates. The effects of beetle diversity on dung removal were more variable across sites than within sites. Dung removal increased with species richness across sites, while functional diversity consistently enhanced dung removal within sites, independently of cattle grazing intensity or climate. Our findings indicate that, despite intensified cattle stocking rates, ecosystem services related to decomposition and nutrient cycling can be maintained when a functionally diverse dung beetle community inhabits the human-modified landscape

The PREDICTS database: a global database of how local terrestrial biodiversity responds to human impacts

Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species’ threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project – and avert – future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local-scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups – including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems – www.predicts.org.uk). We make site-level summary data available alongside this article. The full database will be publicly available in 2015

Characterization of Toxoplasma gondii Hsp90 and co-chaperones and their involment during parasite development

Toxoplasma gondii es una parásito intracelular obligado y un patógeno humano de importancia. Dos formas replicativas son la característica principal del ciclo asexual de éste parásito: uno de replicación rápida (el taquizoito) y otro de división lenta (los bradizoitos enquistados). El proceso que permite la interconversión entre taquizoitos y bradizoitos es central para la patogenicidad y duración de la infección. Todavía no están claramente comprendidos los mecanismos que regulan este proceso de diferenciación. La inducción del desarrollo del estadío bradizoito in vitro se asocia con varios tipos de estresores, estímulos que son conocidos también como inductores de la expresión de proteínas de choque térmico (HSPs). Las HSPs son un enorme grupo de proteínas que se asocian en forma transiente con intermediarios de plegado de proteínas nacientes o también con proteínas desnaturalizadas, con el objeto de evitar la agregación. In vivo, la proteína Hsp90 tiene un rol fundamental en la maduración de componentes de numerosas vías de transducción de señales. El objetivo de esta tesis fue identificar factores asociados con la diferenciación del parásito. Nuestros estudios se centraron en el análisis del perfil de expresión y la regulación durante el desarrollo de la proteína Hsp90 de T. gondii en tres tipos de parásitos, la cepa RH ΔUPRT, la cepa cistogénica ME49 y un clon derivado de esta última, PK todas útiles para estudios de diferenciación in vitro. Nuestros resultados muestran que el transcripto y nivel proteico de Hsp90 se ven incrementados ante condiciones de estrés y de diferenciación del parásito a bradizoíto. Estudios de microscopía confocal e inmunofluorescencia revelaron que la proteína Hsp90 está presente en el citoplasma de taquizoitos y en el citoplasma y núcleo de bradizoitos maduros, lo cual, sugiere una correlación entre la localización subcelular y los dos estadios asexuales de desarrollo. Para caracterizar el papel de la proteína Hsp90 en la diferenciación a bradizoíto, se estudiaron mutantes de T. gondii incapaces de desarrollar a bradizoitos. Bajo condiciones de diferenciación a bradizoitos éstos parásitos mostraron un patrón de localización celular equivalente al observado en taquizoitos. Se utilizó una droga capaz de unirse e inhibir la función de Hsp90, la geldanamicina, la cual, bloqueó la conversión de taquizoito a bradizoíto y también el camino inverso. Estos resultados sugieren un papel crucial para la proteína Hsp90 durante el cambio de estadío. Se sabe, a partir de estudios realizados sobre la maduración de los receptores de esteroides, que la proteína Hsp90 no actúa sola, sino, que lo hace en asociación con variadas co-chaperonas (Hip, Hsp40, Hop, P23). Estas proteínas se unen a la proteína Hsp90 y se organizan en sub-complejos discretos, cada uno conteniendo un grupo diferencial de co-chaperonas. En el genoma de T. gondii pudimos encontrar varias co-chaperonas pertenecientes a la extensivamente estudiada, “maquinaria” chaperona de las proteínas Hsp90/Hsp70. Se clonaron las proteínas Hip y P23, las cuales, una vez expresadas nos permitieron obtener anticuerpos policlonales que las reconozcan. Por ensayos de co-inmunoprecipitación se pudo determinar que Hip y P23 interactúan con la proteína Hsp90. Ambas co-chaperonas colocalizan intracelularmente con Hsp90 en el estadío taquizoito. En el estadío bradizoito solamente P23 colocaliza con Hsp90, manteniéndose Hip excluida del núcleo. También se clonaron y caracterizaron preliminarmente las co-chaperonas Hop y Hsp40.Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Two replicative forms characterize the asexual cycle of the protozoan parasite Toxoplasma gondii: rapidly growing tachyzoites and slowly dividing encysted bradyzoites. This process of tachyzoite–bradyzoite interconversion is central to the pathogenesis and longevity of infection. The mechanisms that regulate the transition between these two stages are not clearly understood. However, stress inducers that also activate heat shock protein expression can trigger formation of bradyzoites in vitro. A large and diverse group of proteins, known as chaperones, transiently associate with protein folding intermediates or proteins needing to be refolded after stress, to prevent aggregation, but in many cases the specific functions of individual chaperones are no still clear. In vivo, Hsp90 (heat shock protein 90) plays a key role in the maturation of components of signal transduction pathways. In order to identify molecular factors associated with parasite differentiation, in this thesis it was studied the association of the T. gondii Hsp90 with the modulation of parasite differentiation and response to stress stimuli using RH ΔUPRT parasites and the cystogenic strain ME49, as well as, a clone derivative of that strain, PK. Our results show that Hsp90 transcript and protein levels increase under stress or bradyzoite differentiation conditions. Moreover, fluorescence microscopy studies revealed that Hsp90 is present in the cytosol of tachyzoites and both in the nucleus and cytosol of mature bradyzoites, suggesting a correlation between its subcellular organization and these two developmental stages. To further characterize the role for Hsp90 in bradyzoite differentiation, T. gondii tachyzoite mutants that are defective in differentiation showed the same staining pattern as tachyzoites under differentiation conditions. In addition, geldanamycin, a benzoquinone ansamycin antibiotic capable of binding and disrupting the function of Hsp90, blocked conversion both from the tachyzoite to bradyzoite and the bradyzoite to tachyzoite stage, suggesting an critical role for this protein in the regulation of stage interconversion. These results thus suggest that Hsp90 may play a role in stage switch. It is known from studies on steroid hormone receptor maturation that Hsp90 does not act alone but in association 4 4 with several different co-chaperones. These co-chaperones bind to Hsp90 and organize it into discrete subcomplexes, each containing a different set of cochaperones. In T. gondii genome we could find several co-chaperones members of the extensively studied Hsp90/Hsp70 based chaperone machinery. Hip and P23 proteins were cloned, sequenced and expressed in order to obtain specific polyclonal antibodies against them. Furthermore, co-immunoprecipitation assays show that Hip and P23 interact with Hsp90 but not between them, suggesting that Hip belongs to early/intermediate Hsp90 complexes (also interacts with Hsp70), and P23 to mature complexes. Both co-chaperones colocalize with Hsp90 at tachyzoite stage. While, at bradyzoite stage only P23 colocalize with Hsp90, Hip is located mainly out of nucleus. Also we cloned and made preliminary characterization of Hop and Hsp40 co-chaperones.Fil: Echeverría, Pablo C.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Nuclear Import of the Glucocorticoid Receptor-hsp90 Complex through the Nuclear Pore Complex Is Mediated by Its Interaction with Nup62 and Importin β ▿

Glucocorticoid receptor (GR) is cytoplasmic in the absence of ligand and localizes to the nucleus after steroid binding. Previous evidence demonstrated that the hsp90-based heterocomplex bound to GR is required for the efficient retrotransport of the receptor to the nuclear compartment. We examined the putative association of GR and its associated chaperone heterocomplex with structures of the nuclear pore. We found that importin β and the integral nuclear pore glycoprotein Nup62 interact with hsp90, hsp70, p23, and the TPR domain proteins FKBP52 and PP5. Nup62 and GR were able to interact in a more efficient manner when chaperoned by the hsp90-based heterocomplex. Interestingly, the binding of hsp70 and p23 to Nup62 does not require the presence of hsp90, whereas the association of FKBP52 and PP5 is hsp90 dependent, as indicated by the results of experiments where the hsp90 function was disrupted with radicicol. The ability of both FKBP52 and PP5 to interact with Nup62 was abrogated in cells overexpressing the TPR peptide. Importantly, GR cross-linked to the hsp90 heterocomplex was able to translocate to the nucleus in digitonin-permeabilized cells treated with steroid, suggesting that GR could pass through the pore in its untransformed state