Increased nuclear envelope permeability and Pep4p-dependent degradation of nucleoporins during hydrogen peroxide-induced cell death

The death of yeast treated with hydrogen peroxide (H(2)O(2)) shares a number of morphological and biochemical features with mammalian apoptosis. In this study, we report that the permeability of yeast nuclear envelopes (NE) increased during H(2)O(2)-induced cell death. Similar phenomena have been observed during apoptosis in mammalian tissue culture cells. Increased NE permeability in yeast was temporally correlated with an increase in the production of reactive-oxygen species (ROS). Later, after ROS levels began to decline and viability was lost, specific nuclear pore complex (NPC) proteins (nucleoporins) were degraded. Although caspases are responsible for the degradation of mammalian nucleoporins during apoptosis, the deletion of the metacaspase gene YCA1 had no effect on the stability of yeast nucleoporins. Instead, Pep4p, a vacuolar cathepsin D homolog, was responsible for the proteolysis of nucleoporins. Coincident with nucleoporin degradation, a Pep4p-EGFP reporter migrated out of the vacuole in H(2)O(2)-treated cells. We conclude that increases in ROS and NPC permeability occur relatively early during H(2)O(2)-induced cell death. Later, Pep4p migrates out of vacuoles and degrades nucleoporins after the cells are effectively dead.     

Synthetic lethality of PARP inhibition in BRCA-network disrupted tumor cells is associated with interferon pathway activation and enhanced by interferon-γ

Tumor suppressor genes BRCA1 and BRCA2 function in a complex gene network that regulates homologous recombination and DNA double-strand break repair. Disruption of the BRCA-network through gene mutation, deletion, or RNAi-mediated silencing can sensitize cells to small molecule inhibitors of poly (ADP-ribose) polymerase (PARPi). Here, we demonstrate that BRCA-network disruption in the presence of PARPi leads to the selective induction and enhancement of interferon pathway and apoptotic gene expression in cultured tumor cells. In addition, we report PARPi cytotoxicity in BRCA1-deficient tumor cells is enhanced >10-fold when combined with interferon-γ. These findings establish a link between synthetic lethality of PARPi in BRCA-network disrupted cells and interferon pathway activation triggered by genetic instability.     

Structure of a classical MHC class I molecule that binds "non-classical" ligands

Chicken YF1 genes share a close sequence relationship with classical MHC class I loci but map outside of the core MHC region. To obtain insights into their function, we determined the structure of the YF1*7.1/β₂-microgloblin complex by X-ray crystallography at 1.3 Å resolution. It exhibits the architecture typical of classical MHC class I molecules but possesses a hydrophobic binding groove that contains a non-peptidic ligand. This finding prompted us to reconstitute YF1*7.1 also with various self-lipids. Seven additional YF1*7.1 structures were solved, but only polyethyleneglycol molecules could be modeled into the electron density within the binding groove. However, an assessment of YF1*7.1 by native isoelectric focusing indicated that the molecules were also able to bind nonself-lipids. The ability of YF1*7.1 to interact with hydrophobic ligands is unprecedented among classical MHC class I proteins and might aid the chicken immune system to recognize a diverse ligand repertoire with a minimal number of MHC class I molecules.     

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

Background

F₁F_O ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.

Methods

We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.

Results

We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F₁F_O ATP synthases.

Conclusions

Our system now allows performing previously impossible structural and functional studies on A. aeolicus F₁F_O ATP synthase.

General significance

More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.     

Active Surveillance of Hansen's Disease (Leprosy): Importance for Case Finding among Extra-domiciliary Contacts

Hansen''s disease (leprosy) remains an important health problem in Brazil, where 34,894 new cases were diagnosed in 2010, corresponding to 15.3% of the world''s new cases detected in that year. The purpose of this study was to use home visits as a tool for surveillance of Hansen''s disease in a hyperendemic area in Brazil. A total of 258 residences were visited with 719 individuals examined. Of these, 82 individuals had had a previous history of Hansen''s disease, 209 were their household contacts and 428 lived in neighboring residences. Fifteen new Hansen''s disease cases were confirmed, yielding a detection rate of 2.0% of people examined. There was no difference in the detection rate between household and neighbor contacts (p = 0.615). The two groups had the same background in relation to education (p = 0.510), household income (p = 0.582), and the number of people living in the residence (p = 0.188). Spatial analysis showed clustering of newly diagnosed cases and association with residential coordinates of previously diagnosed multibacillary cases. Active case finding is an important tool for Hansen''s disease control in hyperendemic areas, enabling earlier diagnosis, treatment, decrease in disability from Hansen''s disease and potentially less spread of Mycobacterium leprae.     

Analysis of protein fragmentation inhibition by an MMP-inhibitor in an in vivo model of heart failure using automated chromatography

Proteomic strategies have continued to demonstrate value in studying disease by exploiting new technologies that can develop significant numbers of measurements from single samples. However, using complex samples such as tissues or blood has continued to be problematic due to the presence of major interfering substances. In this study, a process is described that uses denaturing peptide extraction from whole tissue and automated chromatography in order to allow subsequent analysis of more than 1000 tissue-derived peptides per sample. The process was employed to identify cardiac proteins that were spared degradation by administration of a heart-protecting matrix metalloproteinase (MMP) inhibitor (compound SC-621) following experimental myocardial infarction (MI). HPLC peptide fingerprints were developed from rat heart left ventricles and the resultant integrated peak data was compared across experimental animals. Surprisingly, although protein fragmentation was generally increased in MI hearts, the effect of the MMP inhibitor was only observed on a few species. The results from this study demonstrated that whole-tissue sample enrichment and peptide analysis using HPLC could be linked in order to study the effects of new compounds on a disease state. The system is flexible and amenable to improvements such as incorporating detection by mass spectrometry.     

Antinociceptive effects of tetrahydrophthalimides and related compounds

This paper describes the antinociceptive effects of tetrahydrophthalimides and related compounds in mice. Twenty compounds were obtained by the reaction of cis-1,2,3,6-tetrahydrophthalic anhydride with appropriate amines, dehydration, and addition to the imidic double bond. They were analyzed in the writhing test at 10 mg/kg given intraperitoneally. The most active compound 2-benzyl-5-morpholin-4-yl-hexahydroisoindole-1,3-dione (19) was studied on formalin, capsaicin, glutamate and hot plate models. The antinociceptive activity demonstrated by some studied compounds is promising, and some of them were more active than acetylsalicylic acid and paracetamol used as reference drugs in writhing tests in mice. Compound 19 was about 5-fold more potent than the reference drugs, being also effective by oral route and against the inflammatory response in the formalin test. The results suggest that compound 19 could be used as a model to obtain new and more potent antinociceptive agents. It exhibits an interesting antinociceptive profile, and does not interact with opioid systems.     

γ-Herpesvirus reactivation differentially stimulates epitope-specific CD8 T cell responses

The γ-herpesviruses are characterized by their ability to establish lifelong latency. Subsequent immune suppression leads to viral reactivation from latency and the onset of a variety of pathologies, including lymphoproliferative disease and cancers. CD8 T cells play a key role in preventing reactivation of latent virus. Therefore, to develop effective therapeutic immune strategies, it is essential to understand the maintenance of CD8 T cell responses during latency. Because the γ-herpesviruses are highly species-specific and mice cannot be infected with the human pathogens, EBV or Kaposi's sarcoma-associated herpesvirus, we have used a natural rodent γ-herpesvirus experimental infection model, γ-herpesvirus-68. In this report, we show that during long-term latent infection, naive CD8 T cells are recruited into the ongoing immune response in an epitope-specific manner. When virus reactivation is induced in vivo, the recruitment of CD8 T cells for some, but not all, epitopes is enhanced. The variation in recruitment is not due to differences in epitope presentation. We also show that CD8 T cells that are newly stimulated during reactivation are functionally impaired compared with acutely stimulated cells in terms of cytokine production. Thus, our results demonstrate unexpected complexity in the response of CD8 T cells specific for different viral epitopes that were stimulated during acute infection, quiescent latency, and reactivation.     

Camptosemin, a tetrameric lectin of Camptosema ellipticum: structural and functional analysis

Lectins have been classified into a structurally diverse group of proteins that bind carbohydrates and glycoconjugates with high specificity. They are extremely useful molecules in the characterization of saccharides, as drug delivery mediators, and even as cellular surface makers. In this study, we present camptosemin, a new lectin from Camptosema ellipticum. It was characterized as an N-acetyl-d-galactosamine-binding homo-tetrameric lectin, with a molecular weight around 26 kDa/monomers. The monomers were stable over a wide range of pH values and exhibited pH-dependent oligomerization. Camptosemin promoted adhesion of breast cancer cells and hemagglutination, and both activities were inhibited by its binding of sugar. The stability and unfolding/folding behavior of this lectin was characterized using fluorescence and far-UV circular dichroism spectroscopies. The results indicate that chemical unfolding of camptosemin proceeds as a two-state monomer-tetramer process. In addition, small-angle X-ray scattering shows that camptosemin behaves as a soluble and stable homo-tetramer molecule in solution.