Christopher Gerner

Executioner caspase-3 and caspase-7 are functionally distinct proteases

Members of the caspase family of cysteine proteases play central roles in coordinating the stereotypical events that occur during apoptosis. Because the major executioner caspases, caspase-3 and caspase-7, exhibit almost indistinguishable activity toward certain synthetic peptide substrates, this has led to the widespread view that these proteases occupy functionally redundant roles within the cell death machinery. However, the distinct phenotypes of mice deficient in either of these caspases, as well as mice deficient in both, is at odds with this view. These distinct phenotypes could be related to differences in the relative expression levels of caspase-3 and caspase-7 in vivo, or due to more fundamental differences between these proteases in terms of their ability to cleave natural substrates. Here we show that caspase-3 and caspase-7 exhibit differential activity toward multiple substrate proteins, including Bid, XIAP, gelsolin, caspase-6, and cochaperone p23. Caspase-3 was found to be generally more promiscuous than caspase-7 and appears to be the major executioner caspase during the demolition phase of apoptosis. Our observations provide a molecular basis for the different phenotypes seen in mice lacking either caspase and indicate that these proteases occupy nonredundant roles within the cell death machinery.

The Fas-induced apoptosis analyzed by high throughput proteome analysis.

The fate of cytosolic proteins was studied during Fas-induced cell death of Jurkat T-lymphocytes by proteome analysis. Among 1000 spots resolved in two-dimensional gels, comparison of control versus apoptotic cells revealed that the signal intensity of 19 spots decreased or even disappeared, whereas 38 novel spots emerged. These proteins were further analyzed with respect tode novo protein synthesis, phosphorylation status, and intracellular localization by metabolic labeling and analysis of subcellular protein fractions in combination with two-dimensional Western blots and mass spectrometry analysis of tryptic digests. We found that e.g. hsp27, hsp70B, calmodulin, and H-ras synthesis was induced upon Fas signaling. 34 proteins were affected by dephosphorylation (e.g. endoplasmin) and phosphorylation (e.g. hsc70, hsp57, and hsp90). Nuclear annexin IV translocated to the cytosol, whereas decreasing cytosolic TCP-1α became detectable in the nucleus. In addition, degradation of 12 proteins was observed; among them myosin heavy chain was identified as a novel caspase target. Fas-induced proteome alterations were compared with those of other cell death inducers, indicating specific physiological characteristics of different cell death mechanisms, consequent to as well as independent of caspase activation. Characteristic proteome alterations of apoptotic cells at early time points were found reminiscent of those of malignant cells in vivo.

Serum Amyloid A in Uremic HDL Promotes Inflammation

Uremia impairs the atheroprotective properties of HDL, but the mechanisms underlying why this occurs are unknown. Here, we observed that HDL isolated from healthy individuals inhibited the production of inflammatory cytokines by peripheral monocytes stimulated with a Toll-like receptor 2 agonist. In contrast, HDL isolated from the majority of patients with ESRD did not show this anti-inflammatory property; many HDL samples even promoted the production of inflammatory cytokines. To investigate this difference, we used shotgun proteomics to identify 49 HDL-associated proteins in a uremia-specific pattern. Proteins enriched in HDL from patients with ESRD (ESRD-HDL) included surfactant protein B (SP-B), apolipoprotein C-II, serum amyloid A (SAA), and α-1-microglobulin/bikunin precursor. In addition, we detected some ESRD-enriched proteins in earlier stages of CKD. We did not detect a difference in oxidation status between HDL isolated from uremic and healthy patients. Regarding function of these uremia-specific proteins, only SAA mimicked ESRD-HDL by promoting inflammatory cytokine production. Furthermore, SAA levels in ESRD-HDL inversely correlated with its anti-inflammatory potency. In conclusion, HDL has anti-inflammatory activities that are defective in uremic patients as a result of specific changes in its molecular composition. These data suggest a potential link between the high levels of inflammation and cardiovascular mortality in uremia.

Cell death and autophagy: Cytokines, drugs, and nutritional factors

Cells may use multiple pathways to commit suicide. In certain contexts, dying cells generate large amounts of autophagic vacuoles and clear large proportions of their cytoplasm, before they finally die, as exemplified by the treatment of human mammary carcinoma cells with the anti-estrogen tamoxifen (TAM, < or = 1 microM). Protein analysis during autophagic cell death revealed distinct proteins of the nuclear fraction including GST-pi and some proteasomal subunit constituents to be affected during autophagic cell death. Depending on the functional status of caspase-3, MCF-7 cells may switch between autophagic and apoptotic features of cell death [Fazi, B., Bursch, W., Fimia, G.M., Nardacci R., Piacentini, M., Di Sano, F., Piredda, L., 2008. Fenretinide induces autophagic cell death in caspase-defective breast cancer cells. Autophagy 4(4), 435-441]. Furthermore, the self-destruction of MCF-7 cells was found to be completed by phagocytosis of cell residues [Petrovski, G., Zahuczky, G., Katona, K., Vereb, G., Martinet, W., Nemes, Z., Bursch, W., Fésüs, L., 2007. Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes. Cell Death Diff. 14 (6), 1117-1128]. Autophagy also constitutes a cells strategy of defense upon cell damage by eliminating damaged bulk proteins/organelles. This biological condition may be exemplified by the treatment of MCF-7 cells with a necrogenic TAM-dose (10 microM), resulting in the lysis of almost all cells within 24h. However, a transient (1h) challenge of MCF-7 cells with the same dose allowed the recovery of cells involving autophagy. Enrichment of chaperones in the insoluble cytoplasmic protein fraction indicated the formation of aggresomes, a potential trigger for autophagy. In a further experimental model HL60 cells were treated with TAM, causing dose-dependent distinct responses: 1-5 microM TAM, autophagy predominant; 7-9 microM, apoptosis predominant; 15 microM, necrosis. These phenomena might be attributed to the degree of cell damage caused by tamoxifen, either by generating ROS, increasing membrane fluidity or forming DNA-adducts. Finally, autophagy constitutes a cells major adaptive (survival) strategy in response to metabolic challenges such as glucose or amino acid deprivation, or starvation in general. Notably, the role of autophagy appears not to be restricted to nutrient recycling in order to maintain energy supply of cells and to adapt cell(organ) size to given physiological needs. For instance, using a newly established hepatoma cell line HCC-1.2, amino acid and glucose deprivation revealed a pro-apoptotic activity, additive to TGF-beta1. The pro-apoptotic action of glucose deprivation was antagonized by 2-deoxyglucose, possibly by stabilizing the mitochondrial membrane involving the action of hexokinase II. These observations suggest that signaling cascades steering autophagy appear to provide links to those regulating cell number. Taken together, our data exemplify that a given cell may flexibly respond to type and degree of (micro)environmental changes or cell death stimuli; a cells response may shift gradually from the elimination of damaged proteins by autophagy and the recovery to autophagic or apoptotic pathways of cell death, the failure of which eventually may result in necrosis.

Concomitant Determination of Absolute Values of Cellular Protein Amounts, Synthesis Rates, and Turnover Rates by Quantitative Proteome Profiling

Two-dimensional gel electrophoresis of protein fractions isolated from 35S-radiolabeled cells provides qualitative information on intracellular amounts, 35S incorporation rates, protein modifications, and subcellular localizations of up to thousands of individual proteins. In this study we extended proteome profiling to provide quantitative data on synthesis rates of individual proteins. We combined fluorescence detection of radiolabeled proteins with SYPRO ruby™ staining and subsequent autoradiography of the same gels, thereby quantifying protein amounts and 35S incorporation. To calibrate calculation of absolute synthesis rates, we determined the amount and autoradiograph intensity of radiolabeled haptoglobin secreted by interleukin-6 pretreated HepG2 cells. This allowed us to obtain a standard calibration value for 35S incorporation per autoradiograph intensity unit. This value was used to measure protein synthesis rates during time course experiments of heat-shocked U937 cells. We measured the increasing amounts of hsp70 and calculated it by integration of the determined hsp70 synthesis rates over time. Similar results were obtained by both methods, validating our standardization procedure. Based on the assumption that the synthesis rate of proteins in a steady state of cell metabolism would essentially compensate protein degradation, we calculated biological half-lives of proteins from protein amounts and synthesis rates determined from two-dimensional gels. Calculated protein half-lives were found close to those determined by pulse-chase experiments, thus validating this new method. In conclusion, we devised a method to assess quantitative proteome profiles covering determination of individual amounts, synthesis, and turnover rates of proteins.

Use of conventional and -omics based methods for health claims of dietary antioxidants: a critical overview.

This article describes the principles and limitations of methods used to investigate reactive oxygen species (ROS) protective properties of dietary constituents and is aimed at providing a better understanding of the requirements for science based health claims of antioxidant (AO) effects of foods. A number of currently used biochemical measurements aimed of determining the total antioxidant capacity and oxidised lipids and proteins are carried out under unphysiological conditions and are prone to artefact formation. Probably the most reliable approaches are measurements of isoprostanes as a parameter of lipid peroxidation and determination of oxidative DNA damage. Also the design of the experimental models has a strong impact on the reliability of AO studies: the common strategy is the identification of AO by in vitro screening with cell lines. This approach is based on the assumption that protection towards ROS is due to scavenging, but recent findings indicate that activation of transcription factors which regulate genes involved in antioxidant defence plays a key role in the mode of action of AO. These processes are not adequately represented in cell lines. Another shortcoming of in vitro experiments is that AO are metabolised in vivo and that most cell lines are lacking enzymes which catalyse these reactions. Compounds with large molecular configurations (chlorophylls, anthocyans and polyphenolics) are potent AO in vitro, but weak or no effects were observed in animal/human studies with realistic doses as they are poorly absorbed. The development of -omics approaches will improve the scientific basis for health claims. The evaluation of results from microarray and proteomics studies shows that it is not possible to establish a general signature of alterations of transcription and protein patterns by AO. However, it was shown that alterations of gene expression and protein levels caused by experimentally induced oxidative stress and ROS related diseases can be normalised by dietary AO.

Cell characterization by proteome profiling applied to primary hepatocytes and hepatocyte cell lines Hep-G2 and Hep-3B.

Hepatocytes are known to express a large number of characteristic proteins. Transformed and cultured hepatocytes only partially maintain functional cell differentiation characteristics, which can be assessed by proteome profiling. Here, we applied 2D-PAGE analysis in addition to shotgun proteomics to assess the functional cell state of primary human hepatocytes (PHH), HepG2 and Hep3B cells. Out of a total of 1995 proteins identified in the cytoplasm of these cells, we filtered 107 proteins which are characteristic for hepatocytes. A total of 104 of those were identified in primary human hepatocytes, 20 in HepG2, and only 6 in Hep3B. Forty-six out of 72 proteins identified in the secretome of PHH, 55 out of 139 in HepG2, and only 24 out of 72 in Hep3B were plasma proteins characteristic for hepatocytes. Beside other biomarker candidates presently identified, 11 proteins of the HepG2 secretome have been described previously as biomarkers for hepatocellular carcinoma. Because of indications that epithelial to mesenchymal transition (EMT) may have occurred in the cultured hepatoma cells, we included the analysis of fibroblasts representative for mesenchymal cells. Hep3B, but not HepG2, secreted five proteins including follistatin-related protein 1 which are characteristic for mesenchymal cells and may be marker proteins for EMT. Our data demonstrate that HepG2 show more features characteristic for hepatocytes than Hep3B, while Hep3B express more mesenchymal proteins indicative for EMT. Proteome profiling thus proved to enable comprehensive assessment of functional cell states and cell differentiation states of cultured hepatocytes and enabled the identification of numerous biomarkers for hepatocellular carcinoma and EMT.

Local complement activation triggers neutrophil recruitment to the site of thrombus formation in acute myocardial infarction

Atherosclerotic plaque rupture with subsequent mural thrombus formation is considered the main event compromising epicardial flow in acute myocardial infarction (AMI). However, the precise mechanisms underlying acute coronary occlusion are unknown. We compared the proteomic profiles of systemic plasma and plasma derived from the site of thrombus formation of patients with AMI by two-dimensional gel electrophoresis and ELISA. We identified a local activation of the complement system, with selective accumulation of the complement activator C-reactive protein (CRP) and the downstream complement effectors C3a and C5a. CRP in coronary thrombus co-localised with C1q and C3 immunoreactivities, suggesting classical complement activation. In vitro, coronary thrombus derived plasma enhanced neutrophil chemotaxis in a C5a dependent fashion. In vivo, neutrophil accumulation at the site of thrombus formation paralleled the time delay from symptom onset to first balloon inflation or aspiration, and was correlated with C5a and enzymatic infarct size. We present the first direct evidence for localised complement activation in acute coronary thrombi. Our data indicate that local complement effectors amplify the vascular occlusion process in AMI by enhanced neutrophil recruitment.

Proteome analysis of nuclear matrix proteins during apoptotic chromatin condensation

The nuclear matrix (NM) is considered a proteinaceous scaffold spatially organizing the interphase nucleus, the integrity of which is affected during apoptosis. Caspase-mediated degradation of NM proteins, such as nuclear lamins, precedes apoptotic chromatin condensation (ACC). Nevertheless, other NM proteins remain unaffected, which most likely maintain a remaining nuclear structure devoid of chromatin. We, therefore, screened various types of apoptotic cells for changes of the nuclear matrix proteome during the process of apoptotic ACC. Expectedly, we observed fundamental alterations of known chromatin-associated proteins, comprising both degradation and translocation to the cytosol. Importantly, a consistent set of abundant NM proteins, some (e.g. hNMP 200) of which displaying structural features, remained unaffected during apoptosis and might therefore represent constituents of an elementary scaffold. In addition, proteins involved in DNA replication and DNA repair were found accumulated in the NM fraction before cells became irreversibly committed to ACC, a time point characterized in detail by inhibitor studies with orthovanadate. In general, protein alterations of a consistent set of NM proteins (67 of which were identified), were reproducibly detectable in Fas-induced Jurkat cells, in UV-light treated U937 cells and also in staurosporine-treated HeLa cells. Our data indicate that substantial alterations of proteins linking chromatin to an elementary nuclear protein scaffold might play an intriguing role for the process of ACC.

Proteome maps of the main human peripheral blood constituents.

Clinical proteome analysis will almost inevitably be confronted with blood constituents. Purified plasma, serum, cell or tissue samples may easily be contaminated with some other constituents, affecting the final proteome analysis result. To recognize proteins which are potentially indicative for the presence of major blood constituents, we purified T cells, monocytes, neutrophils, erythrocytes, platelets and plasma and performed comparative proteome profiling employing 2D-PAGE in addition to shotgun proteomics. By mass analysis, 594 different proteins were identified in the 2D gels. Six of the 594 proteins displayed a highly specific expression pattern. A total of 1774 proteins were identified by shotgun proteomics, including 50 proteins with highly specific expression patterns. Indeed, proteins specific for each of the constituents were successfully identified. All protein lists including mass spectrometry details and expression specificity are freely available via the PRIDE database and the CPL/MUW database. The present protein maps of each of the constituents may serve as references for comparative analyses and will aid the interpretation of proteome profiles of clinical samples.