The 20S proteasome isolated from Alzheimer's disease brain shows post-translational modifications but unchanged proteolytic activity

Chronic neurodegenerative diseases are characterized by the accumulation of aggregated protein species, and functional impairment of the ubiquitin proteasome system has been hypothesized to contribute to neuronal cell loss. Decreased proteolytic activity of the 20S proteasome has been shown postmortem in crude brain lysates from Alzheimer's disease (AD) patients. In the present study, we demonstrate, however, that catalytic activity of the 20S proteasome increases during chromatographic purification from AD brains as compared with age-matched controls. By two-dimensional difference gel electrophoresis we detected pI shifts in several proteasome subunits in AD samples pointing to differential post-translational modifications. Moreover, we identified N-terminal acetylation and dephosphorylation of subunit alpha7 in AD by tandem mass spectrometry. Thus, reduced peptidase activity in AD brain extracts is not an intrinsic property of the 20S proteasome, but may be resulting from the presence of endogenous inhibitory proteins or substrates. Post-translational modifications of non-catalytic subunits in situ may contribute to the trend towards enhanced hydrolytic activity of the isolated 20S proteasome after removal of the endogenous inhibitors.     

Assessment of local genotoxic effects of formaldehyde in humans measured by the micronucleus test with exfoliated buccal mucosa cells

Volunteers (10 women, 11 men) were exposed to formaldehyde (FA) vapors for 4h per day over a period of 10 working days under strictly controlled conditions. Exposure varied randomly each day from constant 0.15 ppm up to 0.5 ppm with four peaks of 1.0 ppm for 15 min each (13.5 ppm h cumulative exposure over 10 working days). FA was masked on four days by co-exposure to ethyl acetate. During exposure, subjects had to perform bicycle exercises (about 80 W) three times for 15 min. Buccal smears were prepared 1 week before the start of the study (control 1), at the start of the study before the first exposure (control 2), at the end of the exposure period of 10 days and 7, 14 and 21 days thereafter. Two thousand cells per data point were analyzed for the presence of micronuclei (MN) and the frequency of MN per 1000 cells was determined on slides coded by an independent quality-assurance unit. No significant increase in the frequency of MN was measured at any time point after the end of the exposure. Twenty-one days after the end of the exposure MN frequencies were significantly lower in comparison with control 1. This study, which was performed under GLP-like conditions, clearly indicates that FA does not induce MN in buccal mucosa cells after peak exposures up to 1 ppm and a cumulative exposure of 13.5 ppm h over 2 weeks.     

Characterization of the preprotein and amino acid transporter gene family in Arabidopsis

Seventeen loci encode proteins of the preprotein and amino acid transporter family in Arabidopsis (Arabidopsis thaliana). Some of these genes have arisen from recent duplications and are not in annotated duplicated regions of the Arabidopsis genome. In comparison to a number of other eukaryotic organisms, this family of proteins has greatly expanded in plants, with 24 loci in rice (Oryza sativa). Most of the Arabidopsis and rice genes are orthologous, indicating expansion of this family before monocot and dicot divergence. In vitro protein uptake assays, in vivo green fluorescent protein tagging, and immunological analyses of selected proteins determined either mitochondrial or plastidic localization for 10 and six proteins, respectively. The protein encoded by At5g24650 is targeted to both mitochondria and chloroplasts and, to our knowledge, is the first membrane protein reported to be targeted to mitochondria and chloroplasts. Three genes encoded translocase of the inner mitochondrial membrane (TIM)17-like proteins, three TIM23-like proteins, and three outer envelope protein16-like proteins in Arabidopsis. The identity of Arabidopsis TIM22-like proteins is most likely a protein encoded by At3g10110/At1g18320, based on phylogenetic analysis, subcellular localization, and complementation of a yeast (Saccharomyces cerevisiae) mutant and coexpression analysis. The lack of a preprotein and amino acid transporter domain in some proteins, localization in mitochondria, plastids, or both, variation in gene structure, and the differences in expression profiles indicate that the function of this family has diverged in plants beyond roles in protein translocation.     

The alternative complex III from Rhodothermus marinus - a prototype of a new family of quinol:electron acceptor oxidoreductases

The biochemical and genetic search for a bc(1) complex in Rhodothermus marinus was always fruitless; however, a functional equivalent, i.e. having quinol:cytochrome c oxidoreductase activity was characterized. Now, with the sequencing of R. marinus genome, it was possible to assign the N-terminal sequences of several proteins of this complex to its coding genes. The alternative complex III from R. marinus has the same genomic organization of the so-called MFIcc complexes, proposed to be oxidoreductases of the respiratory and photosynthetic electron transfer chains. In this report, we establish undoubtedly the existence of an alternative complex III, a functional substitute of the bc(1) complex, by its identification at both the biochemical and genomic level.     

Urea denaturation of alpha-hemolysin pore inserted in planar lipid bilayer detected by single nanopore recording: loss of structural asymmetry

The aim of this work is to study pore protein denaturation inside a lipid bilayer and to probe current asymmetry as a function of the channel conformation. We describe the urea denaturation of alpha-hemolysin channel and the channel formation of alpha-hemolysin monomer incubated with urea prior to insertion into a lipid bilayer. Analysis of single-channel recordings of current traces reveals a sigmoid curve of current intensity as a function of urea concentration. The normalized current asymmetry at 29+/-4% is observed between 0 and 3.56M concentrations and vanishes abruptly down to 0 concentration exceeds 4M. The loss of current asymmetry through alpha-hemolysin is due to the denaturation of the channel's cap. We also show that the alpha-hemolysin pore inserted into a lipid bilayer is much more resistant to urea denaturation than the alpha-hemolysin monomer in solution: The pore remains in the lipid bilayer up to 7.2M urea. The pore formation is possible up to 4.66M urea when protein monomers were previously incubated in urea.     

Hypoxia-inducible factor mediates hypoxic and tumor necrosis factor alpha-induced increases in tumor necrosis factor-alpha converting enzyme/ADAM17 expression by synovial cells

Chronic hypoxia and inflammatory cytokines are hallmarks of inflammatory joint diseases like rheumatoid arthritis (RA), suggesting a link between this microenvironment and central pathological events. Because TACE/ADAM17 is the predominant protease catalyzing the release of tumor necrosis factor alpha (TNFalpha), a cytokine that triggers a cascade of events leading to RA, we examined the regulation of this metalloprotease in response to hypoxia and TNFalpha itself. We report that low oxygen concentrations and TNFalpha enhance TACE mRNA levels in synovial cells through direct binding of hypoxia-inducible factor-1 (HIF-1) to the 5' promoter region. This is associated with elevated TACE activity as shown by the increase in TNFalpha shedding rate. By the use of HIF-1-deficient cells and by obliterating NF-kappaB activation, it was determined that the hypoxic TACE response is mediated by HIF-1 signaling, whereas the regulation by TNFalpha also requires NF-kappaB activation. As a support for the in vivo relevance of the HIF-1 axis for TACE regulation, immunohistological analysis of TACE and HIF-1 expression in RA synovium indicates that TACE is up-regulated in both fibroblast- and macrophage-like synovial cells where it localizes with elevated expression of both HIF-1 and TNFalpha. These findings suggest a mechanism by which TACE is increased in RA-affected joints. They also provide novel mechanistic clues on the influence of the hypoxic and inflammatory microenvironment on joint diseases.     

Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here, we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. Mrp4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea.     

Low TCR avidity and lack of tumor cell recognition in CD8<Superscript>+</Superscript> T cells primed with the CEA-analogue CAP1-6D peptide

The use of “altered peptide ligands” (APL), epitopes designed for exerting increased immunogenicity as compared with native determinants, represents nowadays one of the most utilized strategies for overcoming immune tolerance to self-antigens and boosting anti-tumor T cell-mediated immune responses. However, the actual ability of APL-primed T cells to cross-recognize natural epitopes expressed by tumor cells remains a crucial concern. In the present study, we show that CAP1-6D, a superagonist analogue of a carcinoembriyonic antigen (CEA)-derived HLA-A*0201-restricted epitope widely used in clinical setting, reproducibly promotes the generation of low-affinity CD8⁺ T cells lacking the ability to recognized CEA-expressing colorectal carcinoma (CRC) cells. Short-term T cell cultures, obtained by priming peripheral blood mononuclear cells from HLA-A*0201⁺ healthy donors or CRC patients with CAP1-6D, were indeed found to heterogeneously cross-react with saturating concentrations of the native peptide CAP1, but to fail constantly lysing or recognizing through IFN- γ release CEA⁺CRC cells. Characterization of anti-CAP1-6D T cell avidity, gained through peptide titration, CD8-dependency assay, and staining with mutated tetramers (D227K/T228A), revealed that anti-CAP1-6D T cells exerted a differential interaction with the two CEA epitopes, i.e., displaying high affinity/CD8-independency toward the APL and low affinity/CD8-dependency toward the native CAP1 peptide. Our data demonstrate that the efficient detection of self-antigen expressed by tumors could be a feature of high avidity CD8-independent T cells, and underline the need for extensive analysis of tumor cross-recognition prior to any clinical usage of APL as anti-cancer vaccines.