The Co-Chaperone Hch1 Regulates Hsp90 Function Differently than Its Homologue Aha1 and Confers Sensitivity to Yeast to the Hsp90 Inhibitor NVP-AUY922

Hsp90 is a dimeric ATPase responsible for the activation or maturation of a specific set of substrate proteins termed ‘clients’. This molecular chaperone acts in the context of a structurally dynamic and highly regulated cycle involving ATP, co-chaperone proteins and clients. Co-chaperone proteins regulate conformational transitions that may be impaired in mutant forms of Hsp90. We report here that the in vivo impairment of commonly studied Hsp90 variants harbouring the G313S or A587T mutation are exacerbated by the co-chaperone Hch1p. Deletion of HCH1, but not AHA1, mitigates the temperature sensitive phenotype and high sensitivity to Hsp90 inhibitor drugs observed in Saccharomyces cerevisiae that express either of these two Hsp90 variants. Moreover, the deletion of HCH1 results in high resistance to Hsp90 inhibitors in yeast that express wildtype Hsp90. Conversely, the overexpression of Hch1p greatly increases sensitivity to Hsp90 inhibition in yeast expressing wildtype Hsp90. We conclude that despite the similarity between these two co-chaperones, Hch1p and Aha1p regulate Hsp90 function in distinct ways and likely independent of their roles as ATPase stimulators. We further conclude that Hch1p plays a critical role in regulating Hsp90 inhibitor drug sensitivity in yeast.     

Modeling Future Conservation of Hawaiian Honeycreepers by Mosquito Management and Translocation of Disease-Tolerant Amakihi

Avian malaria is an important cause of the decline of endemic Hawaiian honeycreepers. Because of the complexity of this disease system we used a computer model of avian malaria in forest birds to evaluate how two proposed conservation strategies: 1) reduction of habitat for mosquito larvae and 2) establishment of a low-elevation, malaria-tolerant honeycreeper (Hawaii Amakihi) to mid-elevation forests would affect native Hawaiian honeycreeper populations. We evaluated these approaches in mid-elevation forests, where malaria transmission is seasonal and control strategies are more likely to work. Our model suggests the potential benefit of larval habitat reduction depends on the level of malaria transmission, abundance of larval cavities, and the ability to substantially reduce these cavities. Permanent reduction in larval habitat of >80% may be needed to control abundance of infectious mosquitoes and benefit bird populations. Establishment of malaria-tolerant Amakihi in mid-elevation forests increases Amakihi abundance, creates a larger disease reservoir, and increases the abundance of infectious mosquitoes which may negatively impact other honeycreepers. For mid-elevation sites where bird populations are severely affected by avian malaria, malaria-tolerant Amakihi had little impact on other honeycreepers. Both management strategies may benefit native Hawaiian honeycreepers, but benefits depend on specific forest characteristics, the amount of reduction in larval habitat that can be achieved, and how malaria transmission is affected by temperature.     

Regulation of the membrane-localized prostaglandin E synthases mPGES-1 and mPGES-2 in cardiac myocytes and fibroblasts

The proinflammatory mediator cyclooxygenase (COX)-2 and its product PGE(2) are induced in the ischemic heart, contributing to inflammatory cell infiltration, fibroblast proliferation, and cardiac hypertrophy. PGE(2) synthesis coupled to COX-2 involves two membrane-localized PGE synthases, mPGES-1 and mPGES-2; however, it is not clear how these synthases are regulated in cardiac myocytes and fibroblasts. To study this, we used primary cultures of neonatal ventricular myocytes (VM) and fibroblasts (VF) treated with IL-1beta for 24 h. To test for involvement of MAPKs in IL-1beta regulation of mPGES-1 and-2, cells were pretreated with the pharmacological inhibitors of p42/44 MAPK, p38 MAPK, and c-Jun kinase (JNK). mRNA was analyzed by RT-PCR. Protein was analyzed by densitometry of Western blots. mPGES-1 was undetectable in untreated VF but induced by IL-1beta; inhibition of either p42/44 MAPK or JNK, but not p38 MAPK, was almost completely inhibitory. In VM, inhibition of the three MAPKs reduced IL-1beta-stimulated mPGES-1 protein by 70-90%. mPGES-2 was constitutively synthesized in both VM and VF and was not regulated by IL-1beta or MAPKs. Confocal microscopy revealed colocalization of both mPGES-1 and mPGES-2 with COX-2 in the perinuclear area of both VF and VM. Finally, PGE(2) production was higher in VM than VF. Our data show that 1) mPGES-1 is induced in both VF and VM, 2) regulation of mPGES-1 by MAPK family members is different in the two cell types, 3) mPGES-2 is constitutively synthesized in both VM and VF and is not regulated, and 4) mPGES-1 and mPGES-2 are colocalized with COX-2 in both cells. Thus differences in activity of mPGES-1 and COX-2 or coupling of COX-2 with mPGES-1 may contribute to differences in PGE(2) production by myocytes and fibroblasts.     

Conditions used with a continuous cultivation system to screen for d-hydantoinase-producing microorganisms

The continuous cultivation technique has been used to screen for microorganisms producing d-hydantoinase, a biocatalyst involved in the production of optically active amino acids. Pseudomonas putida strain DSM 84 was used as a model hydantoinase producer to establish selective culture conditions through the addition of various pyrimidines, dihydropyrimidines, hydantoins and 5-monosubstituted hydantoins. Thymine induced more activity than all cyclic amides tested. Addition of thymine as a non-metabolised inducer at a concentration of 0.05 g l^–1 in a continuous culture of P. putida stimulated hydantoinase production up to 80 times the basal level. Using continuous culture conditions established with the model strain, a different strain of P. putida having hydantoinase activity was isolated from commercial mixed cultures of microorganisms. DNA fingerprinting revealed that this new isolate was distinct from strain DSM 84. When used as a probe, the d-hydantoinase gene of strain DSM 84 hybridized with the DNA of the new P. putida isolate.     

Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.     

Larval habitat for the avian malaria vector Culex quinquefasciatus (Diptera: Culicidae) in altered mid‐elevation mesic‐dry forests in Hawai'i

Effective management of avian malaria (Plasmodium relictum) in Hawai'i's endemic honeycreepers (Drepanidinae) requires the identification and subsequent reduction or treatment of larval habitat for the mosquito vector, Culex quinquefasciatus (Diptera: Culicidae). We conducted ground surveys, treehole surveys, and helicopter aerial surveys from 2001–2003 to identify all potential larval mosquito habitat within two 100+ ha mesic‐dry forest study sites in Hawai'i Volcanoes National Park, Hawai'i; ‘Ainahou Ranch and Mauna Loa Strip Road. At ‘Ainahou Ranch, anthropogenic sites (43%) were more likely to contain mosquitoes than naturally occurring (8%) sites. Larvae of Cx. quinquefasciatus were predominately found in anthropogenic sites while Aedes albopictus larvae occurred less frequently in both anthropogenic sites and naturally‐occurring sites. Additionally, moderate‐size (≈20–22,000 liters) anthropogenic potential larval habitat had >50% probability of mosquito presence compared to larger‐ and smaller‐volume habitat (<50%). Less than 20% of trees surveyed at ‘Ainahou Ranch had treeholes and few mosquito larvae were detected. Aerial surveys at ‘Ainahou Ranch detected 56% (95% CI: 42–68%) of the potential larval habitat identified in ground surveys. At Mauna Loa Strip Road, Cx. quinquefasciatus larvae were only found in the rock holes of small intermittent stream drainages that made up 20% (5 of 25) of the total potential larval habitat. The volume of the potential larval habitat did not influence the probability of mosquito occurrence at Mauna Loa Strip Road. Our results suggest that Cx. quinquefasciatus abundance, and subsequently avian malaria, may be controlled by larval habitat reduction in the mesic‐dry landscapes of Hawai'i where anthropogenic sources predominate.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.