Qualitative and quantitative multiplexed proteomic analysis of complex yeast protein fractions that modulate the assembly of the yeast prion Sup35p

Background

The aggregation of the baker''s yeast prion Sup35p is at the origin of the transmissible [PSI⁺] trait. We and others have shown that molecular chaperones modulate Sup35p aggregation. However, other protein classes might be involved in [PSI⁺] formation.

Results

We designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI⁺] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI⁺] and [psi⁻] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel-free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes.

Conclusion

Our functional proteomic study provides the first inventory list of over 300 proteins that directly or indirectly affect Sup35p aggregation and [PSI⁺] formation. Our results highlight the complexity of the cellular changes accompanying [PSI⁺] formation and pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified here, susceptible of affecting Sup35p assembly.     

α-Synuclein expression selectively affects tumorigenesis in mice modeling Parkinson's disease

Alpha Synuclein (α-Syn) is a protein implicated in mechanisms of neuronal degeneration in Parkinson's disease (PD). α-Syn is primarily a neuronal protein, however, its expression is found in various tumors including ovarian, colorectal and melanoma tumors. It has been hypothesized that neurodegeneration may share common mechanisms with oncogenesis. We tested whether α-Syn expression affects tumorigenesis of three types of tumors. Specifically, B16 melanoma, E0771 mammary gland adenocarcinoma and D122 Lewis lung carcinoma. For this aim, we utilized transgenic mice expression the human A53T α-Syn form. We found that the in vivo growth of B16 and E0771 but not D122 was enhanced in the A53T α-Syn mice. The effect on tumorigenesis was not detected in age-matched APP/PS1 mice, modeling Alzheimer's disease (AD), suggesting a specific effect for α-Syn-dependent neurodegeneration. Importantly, transgenic α-Syn expression was detected within the three tumor types. We further show uptake of exogenously added, purified α-Syn, by the cultured tumor cells. In accord, with the affected tumorigenesis in the young A53T α-Syn mice, over-expression of α-Syn in cultured B16 and E0771 cells enhanced proliferation, however, had no effect on the proliferation of D122 cells. Based on these results, we suggest that certain forms of α-Syn may selectively accelerate cellular mechanisms leading to cancer.     

NF-kappaB/Egr-1/Gadd45 are sequentially activated upon UVB irradiation to mediate epidermal cell death

Chronic sun exposure can lead to severe skin disorders such as carcinogenesis. The cell death process triggered by ultraviolet B (UVB) irradiation is crucial because it protects the surrounding tissue from the emergence and the accumulation of cells that bear the risk of becoming transformed. Here, we show that repression of NF-kappaB and Egr-1 expression drastically inhibits UVB-mediated cell death. Furthermore, we demonstrate that Egr-1 is induced upon UVB irradiation through NF-kappaB activation and the binding of p65/RelA within the Egr-1 promoter. We show that Egr-1 contributes to the regulation of the Gadd45a and Gadd45b genes, which are involved in the control of cell cycle, DNA repair and apoptosis, by direct binding to their promoter. Our study demonstrates for the first time a signaling cascade involving sequential activation of NF-kappaB, Egr-1 and Gadd45 to induce UVB-mediated cell death. Failure in the induction of each protagonist of this pathway alters the UVB-mediated cell death process. Therefore, impairment of the cascade could be at the onset of skin carcinogenesis mediated by genotoxic stress.     

Cross-regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor

A complex programme of regulation governs gene expression during development of the morphologically and biochemically complex eubacterial genus Streptomyces. Earlier work has suggested a model in which 'higher level' pleiotropic regulators activate 'pathway-specific' regulators located within chromosomal gene clusters encoding biosynthesis of individual antibiotics. We used mutational analysis and adventitious overexpression of key Streptomyces coelicolor regulators to investigate functional interactions among them. We report here that cluster-situated regulators (CSRs) thought to be pathway-specific can also control other antibiotic biosynthetic gene clusters, and thus have pleiotropic actions. Surprisingly, we also find that CSRs exhibit growth-phase-dependent control over afsR2/afsS, a 'higher level' pleiotropic regulatory locus not located within any of the chromosomal gene clusters it targets, and further demonstrate that cross-regulation by CSRs is modulated globally and differentially during the S. coelicolor growth cycle by the RNaseIII homologue AbsB. Our results, which reveal a network of functional interactions among regulators that govern production of antibiotics and other secondary metabolites in S. coelicolor, suggest that revision of the currently prevalent view of higher-level versus pathway-specific regulation of secondary metabolism in Streptomyces species is warranted.     

Action of multiple base excision repair enzymes on the 2'-deoxyribonolactone

Free radical attack on the sugar-phosphate backbone generates oxidized apurinic/apyrimidinic (AP) residues in DNA. 2'-deoxyribonolactone (dL) is a C1'-oxidized AP site damage generated by UV and gamma-irradiation, and certain anticancer drugs. If not repaired dL produces G-->A transitions in Escherichia coli. In the base excision repair (BER) pathway, AP endonucleases are the major enzymes responsible for 5'-incision of the regular AP site (dR) and dL. DNA glycosylases with associated AP lyase activity can also efficiently cleave regular AP sites. Here, we report that dL is a substrate for AP endonucleases but not for DNA glycosylases/AP lyases. The kinetic parameters of the dL-incision were similar to those of the dR. DNA glycosylases such as E. coli formamidopyrimidine-DNA glycosylase, mismatch-specific uracil-DNA glycosylase, and human alkylpurine-DNA N-glycosylase bind strongly to dL without cleaving it. We show that dL cross-links with the human proteins 8-oxoguanine-DNA (hOGG1) and thymine glycol-DNA glycosylases (hNth1), and dR cross-links with Nth and hNth1. These results suggest that dL and dR induced genotoxicity might be strengthened by BER pathway in vivo.     

Functional Differences Among Benthic Macroinvertebrate Communities in Reference Streams of Same Order in a Given Biogeographic Area

Ecological theory (Southwood, 1977. Journal of Animal Ecology 46: 337–365, 1988. Oikos 52: 3–18; Townsend, 1989. Journal of the North American Benthological Society 8: 36–50; Townsend & Hildrew, 1994. Freshwater Biology 31: 265–275) considers that spatio-temporal variations in habitats act as evolutionary forces on organisms, selecting for traits that maximize fitness. As a consequence, communities developed under same environmental conditions should present the same combination of species bio-ecological characteristics. The objective was to verify, using data from the same biogeographical zone, (1) if there was a unique suite of bio-ecological profiles for reference macrobenthic communities of `comparable' rivers or (2) if the distribution of bio-ecological traits within reference communities was significantly influenced by geology. The variability of 22 bio-ecological traits in 12 different sites was tested to evaluate the potential influence of geological substrate on biological and ecological features of reference stream communities. Observed patterns suggested that communities displayed highly stable bio-ecological profiles among sites (within a given biogeographical zone) whatever the substrate was, even if communities on clayey substrate exhibited slightly different bio/ecological characteristics than on others geologies. Nevertheless, the functional structure of macrobenthic communities in reference sites was quite stable in this biogeographical area. This study was restricted to the selected stream types and its results may not directly be transferred to other biogeographical areas and stream types. However, the perspective of a unique functional reference for streams of the same order in a given biogeographical area, improve functional comparison between observed vs. reference communities. This could simplify and objectively define the ecological status of a given site.     

Influence of chain length and polymer concentration on the gelation of (amidated) low-methoxyl pectin induced by calcium

The gelation of low-methoxyl pectin (LMP) induced by addition of Ca2+ was studied by measuring the storage modulus as a function of temperature during cooling. Samples with different molar masses were prepared by mechanical degradation. The effect of the molar mass and the pectin concentration on the gelation properties was investigated. The effect of partial amidation was studied by comparing LMP and partially amidated LMP with the same molar mass and degree of methylation. The results are compared to those from a model developed for Ca2+-induced pectin gelation, and good agreement is found except at low concentrations and low molar masses where the gels are weaker than predicted. At low concentrations intrachain bonding weakens the gel, while the presence of small pectin chains weakens the gel because it neutralizes binding sites on larger chains.     

The endoproteolytic processing of alphavbeta5 integrin is involved in cytoskeleton remodelling and cell migration

We previously showed that the post-translational cleavage of alphav subunit is essential for integrin-dependent signalling and cell adhesion. Here, we report that blocking alphav subunit cleavage by expression of alpha1-PDX, a convertase inhibitor, modified the capacity of cells to change shape, via a remodelling of the actin cytoskeleton upon cell attachment. These changes are associated with cell scattering and with a dramatic increase in cell migration to vitronectin. The alphav subunit cleavage is thus essential for integrin function and has a considerable impact on integrin-dependent events, especially those leading to cell migration.