Volume 390, Issue 2

Knockout mice are the gold standard to probe for the role of a specific protease within the interacting network of proteases, substrates, and inhibitors. This proteolytic network, or protease web, determines cell signaling and organ homeostasis. Therefore, protease deficiency or inhibition is intrinsically tied to alterations within this network, always leading to new molecular phenotypes, which define susceptibility of an organ to disease. Furthermore, recent hints, mainly from research on matrix metalloproteinases, about the impact of the protease web on inter-organ signaling molecules suggest the existence of a proteolytic internet of communicating local organ- or molecular polymorphism-specific networks, thereby defining homeostasis and disease susceptibility in the whole organism.

Enzymes of the peptidylarginine deiminase (PAD) family catalyze the posttranslational deimination of polypeptide-bound arginine residues. Here, we report the selection of peptide substrates by PAD-4, an isoform thought to be involved in the pathogenesis of rheumatoid arthritis. First, we investigated whether PAD-4-mediated deimination is influenced by the nature of amino acid residues flanking the targeted arginine. Using two peptide substrates, residues in positions -2, -1, +1, and +2 relative to the central arginine targeted by PAD-4 were systematically replaced by all natural l -amino acids except cysteine. Each peptide was treated with recombinant human PAD-4 and deimination was analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In all four flanking positions, amino acids which positively or negatively influenced deimination were identified. We next designed peptides with expected high or low deimination rates and determined their K m and k cat values. These peptides showed PAD-4 substrate behavior as predicted, demonstrating that residues flanking the targeted arginine are important for deimination. Further truncation of peptide substrates suggested additional effects on deimination by residues outside the -2 to +2 region. Finally, we observed that a methylated lysine residue flanking the targeted arginine influences PAD-4-mediated deimination, also suggesting that posttranslational modifications can affect substrate efficiency.

Organisms from insects to mammals respond to heavy metal load (copper, zinc, cadmium, and mercury) by activating the metal-responsive transcription factor 1 (MTF-1). MTF-1 binds to short DNA sequence motifs, termed metal response elements, and boosts transcription of a number of genes, notably those for metallothioneins. In Drosophila , MTF-1 somewhat counter-intuitively also activates transcription of a copper importer gene ( Ctr1B ) in response to copper starvation. Here, we report that mutant flies lacking Ctr1B are extremely sensitive to cadmium and mercury treatment, but can be rescued by excess copper in the food. We thus propose that copper, by competing for binding sites on cellular proteins, alleviates the toxic effects of mercury and cadmium. Such a scenario also explains a seemingly fortuitous metal response, namely, that cadmium and mercury strongly induce the expression of a Ctr1B reporter gene. Thus, the transcription enhancer/promoter region of the Ctr1B copper importer gene is subject to three modes of regulation. All of them depend on MTF-1 and all make biological sense, namely, (i) induction by copper starvation, (ii) repression by copper abundance, and (iii), as shown here, induction by cadmium or mercury at normal copper supply.

HIV infection is characterized by progressive immunodeficiency: HIV-infected peripheral blood mononuclear cells (PBMCs) cannot properly react to stimulation with allo-antigens and mitogens. In this study, we examined interferon-γ (IFN-γ)-mediated pathways and the proliferative response of mitogen-stimulated HIV-infected PBMCs in vitro . PBMCs of 30 HIV-infected patients were stimulated with the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM). Mitogen stimulation induced expression of IFN-γ, GTP cyclohydrolase I (GCH-I), and indoleamine (2,3)-dioxygenase (IDO) resulting in enhanced neopterin formation and tryptophan degradation by HIV-infected and control PBMCs. IFN-γ concentrations correlated with neopterin levels and tryptophan degradation. Proliferative responses to PHA and PWM cytokine were lower in HIV patients, with IFN-γ formation predicting proliferative responses. Higher mRNA expression of IFN-γ, GCH-I and IDO after 6 h was related to better proliferative responses in HIV-infected PBMCs. In conclusion, induction of IFN-γ and subsequent enzymes appears to importantly influence the proliferative response of HIV-infected PBMCs in vitro , suggesting a prominent role of the cytokine in the development of immunodeficiency.

We recently identified a group of proteins which are dysregulated in renal cell carcinoma (RCC). In this study, we performed bioinformatics and pathway analysis of these proteins. Proteins were mapped to gene ontology biological processes. The upregulated proteins tend to cluster in processes, such as cancer initiation and progression. In addition, we identified a number of pathways that are significantly enriched in RCC. Some of these are ‘common’ pathways which are dysregulated in many cancers, but we also identified a number of pathways which were not previously linked to RCC. In addition to their potential prognostic values, many of these pathways have a potential as therapeutic targets for RCC. To verify our findings, we compared our proteins to a pool of datasets from published reports. Although there were only a minimal number of common proteins, there was a significant overlap between the identified pathways in the two groups. Moreover, out of 16 individually discovered genes identified by a literature search, 10 were found to be related to our dysregulated pathways. We also verified the upregulation of the mammalian target of rapamycin signaling pathway in RCC by immunohistochemistry. Finally, we highlight the potential clinical applications of pathway analysis in kidney cancer.

Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein uniquely expressed in glioblastoma, but not in normal brain tissues. To develop targeted therapies for brain tumors, we selected RNA aptamers against the histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for protein expression and purification. Representative aptamer E21 has a dissociation constant ( K d ) of 33×10 -9   m , and exhibits high affinity and specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However, selected aptamers cannot bind the same protein expressed from eukaryotic cells because glycosylation, a post-translational modification present only in eukaryotic systems, significantly alters the structure of the target protein. By transfecting EGFRvIII aptamers into cells, we find that membrane-bound, glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis is increased. We postulate that transfected aptamers can interact with newly synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of mature EGFRvIII reaching the cell surface. Our work establishes the feasibility of disrupting protein post-translational modifications in situ with aptamers. This finding is useful for elucidating the function of proteins of interest with various modifications, as well as dissecting signal transduction pathways.

In this study, we analyzed the influence of proteoglycans on the interaction between human high molecular weight kininogen (HK) and the cell surface. We found that D5-related peptide inhibits HK-biotin cellular uptake. Confocal microscopy showed that HK colocalizes with heparan sulfate proteoglycan (HSPG) at the cell surface. When biotin-HK is incubated with rabbit aorta endothelial cells (RAECs) and CHO-K1 cells, it is internalized into acidic intracellular vesicles, whereas when incubated with CHO-745 cells, which express reduced levels of glycosaminoglycans, HK is not internalized. To further verify the hypothesis that HSPG-dependent mechanisms are involved in HK uptake and proteolytic processing in lysosomes, we tested chloroquine, which blocks Alexa 488-HK colocalization with Lyso Tracker in acidic endosomal vesicles. The process of HK internalization was blocked by low temperatures, methyl-β-cyclodextrin, FCCP and 2-deoxy- d -glucose, implying that HK uptake into acidic vesicles is energy-dependent and most likely involves binding to HSPG structures localized in cholesterol-rich domains present in the plasma membrane. Kinin generation at the cell surface was much higher in tumorigenic cells (CHO-K1) when compared to endothelial cells (RAECs). The present data indicate that the process of HK endocytosis involving HSPG is a novel additional mechanism which may control kinin generation at the cell surface.

Pygopus, a very important component of the Wnt signaling transcriptional complex, has multiple functions in both Wnt-dependent and -independent pathways. Human Pygopus2 (Pygo2) is expressed in many cancers and plays an important role in tumor growth. In the present study, we generated human carcinoma (HeLa) cell lines stably expressing Pygo2, which counteracts vinblastine-induced apoptosis. The anti-apoptotic function was determined by DNA fragmentation, sub-G1 appearance, loss of mitochondrial membrane potential (Δ ψ m) and the activation of caspase-9 and caspase-3. In addition, we found that Pygo2 effectively blocks vinblastine-induced c-Jun and AP-1 activation, maintains the anti-apoptotic protein Bcl-2 in an unphosphorylated state, and thus can render cells resistant to apoptosis. However, Pygo2 does not alter the vinblastine-induced cell cycle changes. Here, we describe an anti-apoptotic activity exerted by Pygo2 through blocking activation of the JNK/AP-1 signaling pathway induced by vinblastine.

Cathepsin L-like endopeptidases of the papain family are synthesized as proenzymes. N-terminal proregions are essential for folding and latency of the enzyme unit. While selectivity has been reported for the inhibitory function of papain-family propeptides, there is no systematic investigation of the selectivity of their chaperone-like function to date. The chaperone-like cross-reactivity between the cathepsins S, K, and L were thoroughly quantified in trans- experiments, i.e., with isolated propeptides and mature enzymes, and compared to the inhibitory cross-reactivity. The three endopeptidases have been chosen due to only minimal evolutionary distance and nearly identical X-ray structures of their zymogenes. The intramolecular chaperone function of the proregion was found to be more selective than the inhibitory activity and significant differences were found between the selectivity profiles, underlining the assumption that the inhibitory and the chaperone-like propeptide functions are autonomous. Considering the data published on the intramolecular chaperone-like propeptide function within other protease classes as well, our data suggest that intrinsically structured propeptides are more selective than intrinsically unstructured propeptides, i.e., those adopting tertiary structure elements only in complex with their maternal enzyme.

Validation of drug targets and subsequent preclinical studies are usually carried out on animal disease models, with mouse being the most commonly used. However, results from mouse models cannot always be directly related to human disease. Major discrepancies between the properties of murine and human variants were observed during the evaluation of compounds targeting cathepsins S and K. It is important, therefore, to know whether similar differences exist between murine and human cathepsin B. Thus, both enzymes were expressed and biochemically characterized. The enzymes exhibited similar biochemical properties, indicating that cathepsin B transgenic mouse models could be useful for studying its role in human pathologies.

Chemiluminescence assays are used widely for the detection of nitric oxide (NO)-derived species in biological fluids and tissues. Here, we demonstrate that these assays can be interfaced with mass-sensitive detectors for parallel determination of isotopic abundance. Results obtained with tri-iodide and ascorbic acid-based reductive assays indicate that mass spectrometric detection enables NO isotope-tracing experiments to be carried out to a limit of detectability of a few picomoles, a sensitivity similar to that of standard gas phase chemiluminescence methods. The advantage afforded by mass spectrometric detection is demonstrated using the murine macrophage cell line J774, which is shown here to reduce 15 NO 3 - to 15 NO 2 - under anoxic conditions. The particular combination of an analytical and cellular system described here may hold promise for future characterization of the enzymatic pathways contributing to mammalian nitrate reductase activity, without background interference from 14 NO 2 - derived from other sources.