Identification of two hydrophilins that contribute to the desiccation and freezing tolerance of yeast (Saccharomyces cerevisiae) cells

Hydrophilins are a group of proteins that are present in all organisms and that have been defined as being highly hydrophilic and rich in glycine. They are assumed to play important roles in cellular dehydration tolerance. There are 12 genes in the yeast Saccharomyces cerevisiae that encode hydrophilins and most of these genes are stress responsive. However, the functional role of yeast hydrophilins, especially in desiccation and freezing tolerance, is largely unknown. Here, we selected six candidate hydrophilins for further analysis. All six proteins were predicted to be intrinsically disordered, i.e. to have no stable structure in solution. The contribution of these proteins to the desiccation and freezing tolerance of yeast was investigated in the respective knock-out strains. Only the disruption of the genes YJL144W and YMR175W (SIP18) resulted in significantly reduced desiccation tolerance, while none of the strains was affected in its freezing tolerance under our experimental conditions. Complementation experiments showed that yeast cells overexpressing these two genes were both more desiccation and freezing tolerant, confirming the role of these two hydrophilins in yeast dehydration stress tolerance.     

Cold stability of intrinsically disordered proteins

Contrary to globular proteins, intrinsically disordered proteins (IDPs) lack a folded structure and they do not lose solubility at elevated temperatures. Although this should also be true at low temperatures, cold stability of IDPs has not been addressed in any scientific work so far. As direct characterization of cold-denaturation is difficult, we approached the problem through a freezing-induced loss-of-function model of globular-disordered functional protein pairs (m-calpain-calpastatin, tubulin-Map2c, Hsp90-ERD14). Our results affirm that in contrast with globular proteins IDPs are resistant to cold treatment. The theoretical and functional aspects of this observation are discussed.     

Hsp105 reduces the protein aggregation and cytotoxicity by expanded-polyglutamine proteins through the induction of Hsp70

Hsp105α and Hsp105β are major heat shock proteins in mammalian cells and belong to the HSP105/110 family. Hsp105α is expressed constitutively in the cytoplasm of cells, while Hsp105β, an alternatively spliced form of Hsp105α, is expressed specifically in the nucleus of cells during mild heat shock. Here, we show that not only Hsp105β but also Hsp105α accumulated in the nucleus of cells following the expression of enhanced green fluorescent protein with a pathological length polyQ tract (EGFP-polyQ97) and suppressed the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Mutants of Hsp105α and Hsp105β with changes in the nuclear localization signal sequences, which localized exclusively in the cytoplasm with or without the expression of EGFP-polyQ97, did not suppress the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Furthermore, Hsp70 was induced by the co-expression of Hsp105α and EGFP-polyQ97, and the knockdown of Hsp70 reduced the inhibitory effect of Hsp105α and Hsp105β on the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. These observations suggested that Hsp105α and Hsp105β suppressed the expanded polyQ tract-induced protein aggregation and apoptosis through the induction of Hsp70.     

Anti-apoptotic protein TCTP controls the stability of the tumor suppressor p53

In this study, we identified p53 as a novel TCTP-interacting protein using TCTP as bait. Also, we determined the critical binding sites between TCTP and p53. To elucidate the functional consequence of the interaction, we developed the overexpression and inhibition system of TCTP and p53 expression. Overexpression of TCTP in lung carcinoma cells reversed p53 mediated apoptosis and inhibition of TCTP expression by small interfering RNA increased apoptosis of lung carcinoma cells. Moreover, it was observed that TCTP overexpression promotes degradation of p53. These results clearly indicate that the interaction between TCTP and p53 prevents apoptosis by destabilizing p53. Thus, TCTP acts as a negative regulator of apoptosis in lung cancer.

Structured summary

MINT-8057107, MINT-8057116: p53 (uniprotkb:P04637) physically interacts (MI:0915) with TCTP (uniprotkb:P13693) by anti bait coimmunoprecipitation (MI:0006)MINT-8057141: TCTP (uniprotkb:P13693) physically interacts (MI:0915) with p53 (uniprotkb:P04637) by two hybrid pooling approach (MI:0398)MINT-8057126: p53 (uniprotkb:P04637) physically interacts (MI:0915) with TCTP (uniprotkb:P13693) by anti tag coimmunoprecipitation (MI:0007) MINT-8057160: TCTP (uniprotkb:P13693) physically interacts (MI:0915) with p53 (uniprotkb:P04637) by two hybrid (MI:0018)     

Interaction of two intrinsically disordered plant stress proteins (COR15A and COR15B) with lipid membranes in the dry state

COR15A and COR15B form a tandem repeat of highly homologous genes in Arabidopsis thaliana. Both genes are highly cold induced and the encoded proteins belong to the Pfam LEA_4 group (group 3) of the late embryogenesis abundant (LEA) proteins. Both proteins were predicted to be intrinsically disordered in solution. Only COR15A has previously been characterized and it was shown to be localized in the soluble stroma fraction of chloroplasts. Ectopic expression of COR15A in Arabidopsis resulted in increased freezing tolerance of both chloroplasts after freezing and thawing of intact leaves and of isolated protoplasts frozen and thawed in vitro. In the present study we have generated recombinant mature COR15A and COR15B for a comparative study of their structure and possible function as membrane protectants. CD spectroscopy showed that both proteins are predominantly unstructured in solution and mainly α-helical after drying. Both proteins showed similar effects on the thermotropic phase behavior of dry liposomes. A decrease in the gel to liquid-crystalline phase transition temperature depended on both the unsaturation of the fatty acyl chains and lipid headgroup structure. FTIR spectroscopy indicated no strong interactions between the proteins and the lipid phosphate and carbonyl groups, but significant interactions with the galactose headgroup of the chloroplast lipid monogalactosyldiacylglycerol. These findings were rationalized by modeling the secondary structure of COR15A and COR15B. Helical wheel projection indicated the presence of amphipathic α-helices in both proteins. The helices lacked a clear separation of positive and negative charges on the hydrophilic face, but contained several hydroxylated amino acids.     

Compaction properties of an intrinsically disordered protein: Sic1 and its kinase-inhibitor domain

IDPs in their unbound state can transiently acquire secondary and tertiary structure. Describing such intrinsic structure is important to understand the transition between free and bound state, leading to supramolecular complexes with physiological interactors. IDP structure is highly dynamic and, therefore, difficult to study by conventional techniques. This work focuses on conformational analysis of the KID fragment of the Sic1 protein, an IDP with a key regulatory role in the cell-cycle of Saccharomyces cerevisiae. FT-IR spectroscopy, ESI-MS, and IM measurements are used to capture dynamic and short-lived conformational states, probing both secondary and tertiary protein structure. The results indicate that the isolated Sic1 KID retains dynamic helical structure and populates collapsed states of different compactness. A metastable, highly compact species is detected. Comparison between the fragment and the full-length protein suggests that chain length is crucial to the stabilization of compact states of this IDP. The two proteins are compared by a length-independent compaction index.     

Over-expression of mitochondrial heat shock protein 70 suppresses programmed cell death in rice

In this study, we identified and functionally characterized the mitochondrial heat shock protein 70 (mtHsp70). Over-expression of mtHsp70 suppressed heat- and H₂O₂-induced programmed cell death (PCD) in rice protoplasts, as reflected by higher cell viability, decreased DNA laddering and chromatin condensation. Mitochondrial membrane potential (Δψ_m) after heat shock was destroyed gradually in protoplasts, but mtHsp70 over-expression showed higher Δψ_m relative to the vector control cells, and partially inhibited cytochrome c release from mitochondria to cytosol. Heat treatment also significantly increased reactive oxygen species (ROS) generation, a phenomenon not observed in protoplasts over-expressing mtHsp70. Together, these results suggest that mtHsp70 may suppress PCD in rice protoplasts by maintaining mitochondrial Δψ_m and inhibiting the amplification of ROS.     

Histone H3K4me3 binding is required for the DNA repair and apoptotic activities of ING1 tumor suppressor

Inhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair, and apoptosis. Mutations within the ING1 gene and altered expression levels of ING1 are found in multiple human cancers. Here, we show that both DNA repair and apoptotic activities of ING1 require the interaction of the C-terminal plant homeodomain (PHD) finger with histone H3 trimethylated at Lys4 (H3K4me3). The ING1 PHD finger recognizes methylated H3K4 but not other histone modifications as revealed by the peptide microarrays. The molecular mechanism of the histone recognition is elucidated based on a 2.1 Å-resolution crystal structure of the PHD-H3K4me3 complex. The K4me3 occupies a deep hydrophobic pocket formed by the conserved Y212 and W235 residues that make cation-π contacts with the trimethylammonium group. Both aromatic residues are essential in the H3K4me3 recognition, as substitution of these residues with Ala disrupts the interaction. Unlike the wild-type ING1, the W235A mutant, overexpressed in the stable clones of melanoma cells or in HT1080 cells, was unable to stimulate DNA repair after UV irradiation or promote DNA-damage-induced apoptosis, indicating that H3K4me3 binding is necessary for these biological functions of ING1. Furthermore, N216S, V218I, and G221V mutations, found in human malignances, impair the ability of ING1 to associate with H3K4me3 or to induce nucleotide repair and cell death, linking the tumorigenic activity of ING1 with epigenetic regulation. Together, our findings reveal the critical role of the H3K4me3 interaction in mediating cellular responses to genotoxic stresses and offer new insight into the molecular mechanism underlying the tumor suppressive activity of ING1.     

Fructose-bisphosphate aldolase and enolase from Echinococcus granulosus: genes, expression patterns and protein interactions of two potential moonlighting proteins

Glycolytic enzymes, such as fructose-bisphosphate aldolase (FBA) and enolase, have been described as complex multifunctional proteins that may perform non-glycolytic moonlighting functions, but little is known about such functions, especially in parasites. We have carried out in silico genomic searches in order to identify FBA and enolase coding sequences in Echinococcus granulosus, the causative agent of cystic hydatid disease. Four FBA genes and 3 enolase genes were found, and their sequences and exon-intron structures were characterized and compared to those of their orthologs in Echinococcus multilocularis, the causative agent of alveolar hydatid disease. To gather evidence of possible non-glycolytic functions, the expression profile of FBA and enolase isoforms detected in the E. granulosus pathogenic larval form (hydatid cyst) (EgFBA1 and EgEno1) was assessed. Using specific antibodies, EgFBA1 and EgEno1 were detected in protoscolex and germinal layer cells, as expected, but they were also found in the hydatid fluid, which contains parasite's excretory-secretory (ES) products. Besides, both proteins were found in protoscolex tegument and in vitro ES products, further suggesting possible non-glycolytic functions in the host-parasite interface. EgFBA1 modeled 3D structure predicted a F-actin binding site, and the ability of EgFBA1 to bind actin was confirmed experimentally, which was taken as an additional evidence of FBA multifunctionality in E. granulosus. Overall, our results represent the first experimental evidences of alternative functions performed by glycolytic enzymes in E. granulosus and provide relevant information for the understanding of their roles in host-parasite interplay.     

An abundant LEA protein in the anhydrobiotic midge,PvLEA4, acts as a molecular shield by limiting growth of aggregating protein particles

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A sesquiterpene lactone, costunolide, interacts with microtubule protein and inhibits the growth of MCF-7 cells

Costunolide is an active sesquiterpene lactone of medicinal herbs with anti-inflammatory and potential anti-cancer activity. Nevertheless, the pharmacological pathways of costunolide have not yet been fully elucidated. In this study we showed that costunolide exerts a dose-dependent antiproliferative activity in the human breast cancer MCF-7 cells. In addition, light microscopy observations indicated that costunolide affected nuclear organization and reorganized microtubule architecture. The antiproliferative and antimicrotubular effects of costunolide were not influenced by paclitaxel, well-known microtubule-stabilizing anticancer agent. The microtubule-interacting activity of costunolide was confirmed by in vitro studies on purified microtubular protein. In fact, costunolide demonstrated polymerizing ability, by inducing the formation of well organized microtubule polymers. Our data suggest an interaction of costunolide with microtubules, which may represent a new intracellular target for this drug.     

Histidine-rich protein Hpn from Helicobacter pylori forms amyloid-like fibrils in vitro and inhibits the proliferation of gastric epithelial AGS cells

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms oligomers in physiologically relevant conditions. In this present study, Hpn oligomers were found to develop amyloid-like fibrils as confirmed by negative stain transition electron microscopy, thioflavin T and Congo red binding assays. The amyloid-like fibrils of Hpn inhibit the proliferation of gastric epithelial AGS cells through cell cycle arrest in the G2/M phase, which may be closely related to the disruption of mitochondrial bioenergetics as reflected by the significant depletion of intracellular ATP levels and the mitochondrial membrane potential. The collective data presented here shed some light on the pathologic mechanisms of H. pylori infections.     

Modulation of cytochrome P450 gene expression in primary hepatocytes on various artificial extracellular matrices

We studied effect of artificial extracellular matrices (ECMs), such as collagen I, poly (N-p-vinylbenzyl-4-O-β-d-galactopyranosyl-d-gluconamide)(PVLA) and E-cadherin–IgG Fc (E-cad-Fc) on hepatic metabolism to identify the mechanism of in vivo hepatocellular functional and metabolic integrity. mRNA expression of liver function marker, cytochrome P450 (CYP) and transporter genes in hepatocytes were compared among used ECMs using real-time RT-PCR. mRNA expressions of Cyp2c29 and Cyp2d22 among CYP genes in hepatocytes on PVLA were recovered after 3 days due to enhanced liver-specific function by the spheroid formation of hepatocytes whereas mRNA expressions of CYP genes in hepatocytes on collagen and E-cad-Fc drastically decreased with time. mRNA expressions of the Cyp2c29 and Cyp2d22 in hepatocytes on PVLA were more recovered in the presence of epidermal growth factor (EGF) due to the more and bigger spheroid formation of hepatocytes. Multidrug resistance-associated protein 2 (Mrp2) protein was accumulated at intracellular lumen as similar to bile duct in hepatocyte spheroid formed on PVLA, indicating that spheroid formation of hepatocytes is very important for maintaining liver functions.