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.

[1]  M. Satake,et al.  Cytotoxic and cytoskeletal effects of azaspiracid-1 on mammalian cell lines. , 2005, Toxicon : official journal of the International Society on Toxinology.

[2]  K. Nicolaou,et al.  Irreversible cytoskeletal disarrangement is independent of caspase activation during in vitro azaspiracid toxicity in human neuroblastoma cells. , 2007, Biochemical pharmacology.

[3]  J. Garin,et al.  AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture , 2004, The Journal of cell biology.

[4]  N. Kulagina,et al.  Azaspiracid-1 inhibits bioelectrical activity of spinal cord neuronal networks. , 2006, Toxicon : official journal of the International Society on Toxinology.

[5]  R. Dulbecco,et al.  Plaque production by the polyoma virus. , 1959, Virology.

[6]  M. Amagai.  A mystery of AHNAK/desmoyokin still goes on. , 2004, The Journal of investigative dermatology.

[7]  M. Twiner,et al.  Azaspiracid Shellfish Poisoning: A Review on the Chemistry, Ecology, and Toxicology with an Emphasis on Human Health Impacts , 2008, Marine drugs.

[8]  G. Egea,et al.  Association of Cdc42/N‐WASP/Arp2/3 Signaling Pathway with Golgi Membranes , 2004, Traffic.

[9]  K. Nicolaou,et al.  Cell growth inhibition and actin cytoskeleton disorganization induced by azaspiracid-1 structure-activity studies. , 2006, Chemical research in toxicology.

[10]  P. Hess,et al.  Discovery of new analogs of the marine biotoxin azaspiracid in blue mussels (Mytilus edulis) by ultra-performance liquid chromatography/tandem mass spectrometry. , 2008, Rapid communications in mass spectrometry : RCM.

[11]  M. Kessels,et al.  Actin Nucleation: Putting the Brakes on Arp2/3 , 2008, Current Biology.

[12]  Van Dolah Fm.  Marine algal toxins: origins, health effects, and their increased occurrence. , 2000 .

[13]  Anushya Muruganujan,et al.  PANTHER: a browsable database of gene products organized by biological function, using curated protein family and subfamily classification , 2003, Nucleic Acids Res..

[14]  G. Kroemer,et al.  The mitochondrion in apoptosis: how Pandora's box opens , 2001, Nature Reviews Molecular Cell Biology.

[15]  P. Bénit,et al.  AIF deficiency compromises oxidative phosphorylation , 2004, The EMBO journal.

[16]  Jody A. Vandergriff,et al.  Erratum: PANTHER: A browsable database of gene products organized by biological function, using curated protein family and subfamily classification (Nucleic Acids Research (2003) vol. 31 (334-341)) , 2003 .

[17]  A. Gross,et al.  Mitochondrial carriers and pores: Key regulators of the mitochondrial apoptotic program? , 2007, Apoptosis.

[18]  N. Vilariño.  Marine toxins and the cytoskeleton: azaspiracids , 2008, The FEBS journal.

[19]  M. Satake,et al.  Multiple organ damage caused by a new toxin azaspiracid, isolated from mussels produced in Ireland. , 2000, Toxicon : official journal of the International Society on Toxinology.

[20]  D. Dykxhoorn,et al.  Genetic Analysis of the Subunit Organization and Function of the Conserved Oligomeric Golgi (COG) Complex , 2005, Journal of Biological Chemistry.

[21]  M. Satake,et al.  Structures of Azaspiracid Analogs, Azaspiracid-4 and Azaspiracid-5, Causative Toxins of Azaspiracid Poisoning in Europe , 2001, Bioscience, biotechnology, and biochemistry.

[22]  O. Weisz,et al.  N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels. , 2007, American journal of physiology. Cell physiology.

[23]  K. Wirtz,et al.  "Wages of fear": transient threefold decrease in intracellular ATP level imposes apoptosis. , 2004, Biochimica et biophysica acta.

[24]  S. Lindquist,et al.  α-Synuclein Blocks ER-Golgi Traffic and Rab1 Rescues Neuron Loss in Parkinson's Models , 2006, Science.

[25]  Adam C. Martin,et al.  The Journal of Cell Biology , 2002 .

[26]  C. Delphin,et al.  Expression of the Giant Protein AHNAK (Desmoyokin) in Muscle and Lining Epithelial Cells , 2003, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[27]  K. Nicolaou,et al.  Effects of azaspiracid-1, a potent cytotoxic agent, on primary neuronal cultures. A structure-activity relationship study. , 2007, Journal of medicinal chemistry.

[28]  V. Pinelis,et al.  Changes in ATP content in cerebellar granule cells during hyperstimulation of glutamate receptors: Possible role of NO and nitrite ions , 2007, Bulletin of Experimental Biology and Medicine.

[29]  T. Smith,et al.  J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. , 1991, Biochemistry.

[30]  T D Pollard,et al.  Regulation of actin filament network formation through ARP2/3 complex: activation by a diverse array of proteins. , 2001, Annual review of biochemistry.

[31]  M. Vieytes,et al.  Azaspiracid-1, a potent, nonapoptotic new phycotoxin with several cell targets. , 2002, Cellular signalling.

[32]  T. Morita,et al.  Changes in the Balance between Caldesmon Regulated by p21-activated Kinases and the Arp2/3 Complex Govern Podosome Formation* , 2007, Journal of Biological Chemistry.

[33]  Y. Tsujimoto,et al.  Intracellular ATP levels determine cell death fate by apoptosis or necrosis. , 1997, Cancer research.

[34]  D. Lambright,et al.  Multivalent endosome targeting by homodimeric EEA1. , 2001, Molecular cell.

[35]  K. Nicolaou,et al.  The c-Jun-N-Terminal Kinase is Involved in the Neurotoxic Effect of Azaspiracid-1 , 2007, Cellular Physiology and Biochemistry.

[36]  T. Morita,et al.  Glucocorticoid Receptor-mediated Expression of Caldesmon Regulates Cell Migration via the Reorganization of the Actin Cytoskeleton* , 2008, Journal of Biological Chemistry.

[37]  F. Oosawa,et al.  Caldesmon Inhibits Arp2/3-mediated Actin Nucleation* , 2003, The Journal of Biological Chemistry.

[38]  Matthias Mann,et al.  Innovations: Functional and quantitative proteomics using SILAC , 2006, Nature Reviews Molecular Cell Biology.

[39]  P. Nicotera,et al.  Intracellular Adenosine Triphosphate (ATP) Concentration: A Switch in the Decision Between Apoptosis and Necrosis , 1997, The Journal of experimental medicine.

[40]  J. Silke,et al.  Two analogs of azaspiracid isolated from mussels, Mytilus edulis, involved in human intoxication in Ireland. , 1999, Natural toxins (Print).

[41]  A. Strasser,et al.  The BCL-2 protein family: opposing activities that mediate cell death , 2008, Nature Reviews Molecular Cell Biology.

[42]  J. Silke,et al.  AZASPIRACID, A NEW MARINE TOXIN HAVING UNIQUE SPIRO RING ASSEMBLIES, ISOLATED FROM IRISH MUSSELS, MYTILUS EDULIS , 1998 .