Kari E. Fladmark

Ultrarapid caspase-3 dependent apoptosis induction by serine/threonine phosphatase inhibitors

The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induced apoptosis with unprecedented rapidity, more than 50% of primary hepatocytes showing extensive surface budding and shrinkage of cytoplasm and nucleoplasm within 2 min. The apoptosis was retarded by the general caspase inhibitor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodularin into nonhepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells started to undergo budding typical of apoptosis within 0.5–3 min after injection. This was accompanied by cytoplasmic and nuclear shrinkage and externalization of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosis. Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 deficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and Z-VAD.fmk dependent apoptotic response to nodularin. Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apoptosis was preceded by increased phosphorylation of several proteins, including myosin light chain. The protein phosphorylation occurred even in the presence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase-inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation.

Sensitive detection of apoptogenic toxins in suspension cultures of rat and salmon hepatocytes

A number of algal toxins were tested for the ability to induce apoptosis (regulated cell death) in primary hepatocytes from salmon and rat. The tested toxins included the liver targeting substances microcystin-LR and nodularin, substances associated with the diarrhetic shellfish poison complex (okadaic acid, dinophysistoxin-1 and pectenotoxin-1) and calyculin A. All toxins induced apoptosis in both salmon and rat hepatocytes in less than 2 h. The apoptotic changes were evident both by electron and light microscopy and were counteracted by the caspase inhibitor ZVAD-fmk and by the Ca2+/calmodulin dependent kinase II inhibitor KN-93. The salmon hepatocytes were 10-20-fold more sensitive to okadaic acid and dinophysistoxin-1 (EC50=20 nM) than rat hepatocytes and other mammalian cell lines tested. An assay was devised using hepatocyte apoptosis as parameter for detection of algal toxins. This assay was at least as sensitive as HPLC determination for okadaic acid in mussel extracts. It also detected algal toxins which do not inhibit protein phosphatases, like pectenotoxin-1. Subapoptotic concentrations of the toxins inhibited hepatocyte aggregation. Using this parameter, less than 200 pg okadaic acid could be detected. In conclusion, salmon hepatocytes in suspension culture provide a rapid and sensitive system for detection of a broad range of apoptogenic toxins.

Apoptosis induced by microinjection of cytochrome c is caspase-dependent and is inhibited by Bcl-2.

Microinjection of cytochrome c induced apoptosis in all the cell types we tested (IPC-81, Swiss 3T3, Clone 8 fibroblasts, NRK, H295, Y1, HEK 293). The apoptotic phenotype induced by injected cytochrome c was characterized by externalization of phosphatidyl serine, cell detachment from substratum and from neighbor cells, and had the classic ultrastructural features of membrane budding, chromatin condensation and cell shrinkage. Depending on the cell type and concentration of cytochrome c, the induction of apoptosis was remarkably rapid. The development of apoptosis was prevented by the caspase inhibitor Z-VAD.fmk. Four of the cell types (Clone 8, Swiss 3T3, NRK, Y1) were transfected with bcl-2 and these all showed a markedly decreased sensitivity towards injected cytochrome c. Our data suggest that extramitochondrial cytochrome c is a general apoptogen in cells with a functioning caspase system. They also indicate that, in preventing apoptosis, Bcl-2 acts not only at the level of regulation of cytochrome c release from mitochondria, but can also interfere with caspase activation induced by cytochrome c microinjected directly into the cytoplasm.

CaM-kinaseII-dependent commitment to microcystin- induced apoptosis is coupled to cell budding, but not to shrinkage or chromatin hypercondensation

The protein phosphatase inhibitor microcystin-LR (MC) induced hepatocyte apoptosis mediated by the calcium-calmodulin-dependent multifunctional protein kinase II (CaMKII). CaMKII antagonists were added at various times after MC to define for how long the cells depended on CaMKII activity to be committed to execute the various parameters of death. Shrinkage and nonpolarized budding were reversible and not coupled to commitment. A critical commitment step was observed 15–20 min after MC (0.5 μM) addition. After this, CaMKII inhibitors no longer protected against polarized budding, DNA fragmentation, lost protein synthesis capability, and cell disruption. Commitment to chromatin hypercondensation occurred 40 min after MC addition. In conclusion, irreversible death commitment was coupled to polarized budding, but not to shrinkage or chromatin condensation. Antioxidant prevented chromatin condensation when given after the CaMKII-dependent commitment point, suggesting that CaMKII had mediated the accumulation of a second messenger of reactive oxygen species nature.

Redox control of protein degradation

Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies.

An ultrasensitive competitive binding assay for the detection of toxins affecting protein phosphatases

An ultrasensitive assay is described for microcystin-LR and other substances (microcystins, nodularin, okadaic acid, calyculin A, tautomycin) which block the active site of protein phosphatases (PP) 1 and 2A. The assay is based on competition between the unknown sample and [125I]microcystin-YR for binding to the catalytic subunit of PP2A. The PP2A-bound [125I]microcystin-YR was stable (half-time of dissociation = 1.8 h), allowing non-bound [125I]microcystin-YR to be removed by Sephadex G-50 size-exclusion chromatography. Compared to current assays based on inhibition of protein phosphatase activity the present assay was more robust against interference (from fluoride, ATP, histone, and casein), and had an even better sensitivity. The detection limit was below 50 pM (2.5 fmol) for nodularin and microcystin-LR, and below 200 pM (10 fmol) for okadaic acid. The method was used successfully to detect extremely low concentrations of either microcystin or nodularin in drinking water or seawater, and okadaic acid in shellfish extract.

Proteomic response of human neuroblastoma cells to azaspiracid-1.

Azaspiracid-1 is a novel algal toxin, which causes an instantaneous rise of intracellular messengers, and an irreversible disarrangement of the actin cytoskeleton. Little is known regarding the molecular mechanisms that are involved in azaspiracid-1 toxicity. This study investigated global changes in protein expression by stable-isotope labelling with amino acids in culture and mass spectrometry, following exposure of human neuroblastoma cells to azaspiracid-1. The most highly upregulated proteins were involved in cellular energy metabolism, followed by cytoskeleton regulating proteins. The majority of downregulated proteins were involved in transcription, translation and protein modification. In addition, two proteins, component of oligomeric Golgi complex 5 and ras-related protein RAB1, which are involved in the maintenance of the Golgi complex and vesicle transport, respectively, were downregulated. Electron microscopy revealed a disruption of the Golgi complex by azaspiracid-1, and an accumulation of vesicles. In this study, the differential protein expression was examined prior to changes of the cytoskeleton structure in order to capture the primary effects of azaspiracid-1, however the observed changes were of unexpected complexity. Azaspiracid-1 caused a pronounced, but temporary depletion of ATP, which may be the reason for the observed complexity of cellular changes.

The N-terminal acetyltransferase Naa10 is essential for zebrafish development

The Naa10 (Nα acetyltransferase 10) N-terminal acetyltransferase is implicated in cancer and developmental syndromes in humans. We show that its enzymatic activity is conserved in zebrafish, and that Naa10 depletion leads to developmental abnormalities.

Identification of Dynamic Changes in Proteins Associated with the Cellular Cytoskeleton after Exposure to Okadaic Acid

Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100–500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment.

Increased interaction between DJ-1 and the Mi-2/ nucleosome remodelling and deacetylase complex during cellular stress

DJ‐1 was originally identified to be an oncogenic product, but has later been shown to be highly multifunctional. DJ‐1 plays a role in oxidative stress response and transcriptional regulation, and loss of its function leads to an early onset of Parkinsonism. To further understand the mechanisms behind DJ‐1s role in cell survival and death, we investigated alternations in endogenous DJ‐1 protein–protein interaction in apoptotic cells exposed to the phosphatase inhibitor okadaic acid. By combining cellular stable isotopic labelling of amino acids in cell culture, sub‐cellular fractionation, co‐immunoprecipitation, and MS, we identified a novel group of DJ‐1 interaction partners that increased their association to DJ‐1 in okadaic acid‐exposed cells. These proteins were integral components of the Mi‐2/nucleosome remodelling and deacetylase (NuRD) complex. Knockdown of DJ‐1 and MTA2, a core component of the NuRD complex, had a similar and pro‐apoptotic effect on the transcriptional‐ and p53‐dependent cell death induced by daunorubicin. On the other hand, MTA2 knockdown had no significant effect on the progression of p53‐independent okadaic acid‐induced apoptosis. Our data suggest that the increased DJ‐1/NuRD interaction is a general anti‐stress response regulated by okadaic acid‐induced modifications of DJ‐1. The observed interaction between DJ‐1 and the NuRD complex may give new clues to how DJ‐1 can protect cells from p53‐dependent cell death.