Comparative analysis of the cytotoxicity of homopolymeric amino acids.

Many human proteins have homopolymeric amino acid (HPAA) tracts, although the physiological significance or cellular effects of their presence is poorly understood. We previously reported that 20 kinds of HPAAs show characteristic intracellular localization and that among those, hydrophobic HPAAs aggregate strongly and form high molecular weight proteins when expressed in cultured cells. In this study, we investigated the cytotoxicity of 20 kinds of HPAAs. HPAA tracts of approximately 30 residues fused to the C-terminus of YFP were expressed in COS-7 cells. Cells expressing homopolymeric-Cys, -Ile, -Leu, and -Val showed low viability in Trypan Blue assay. Caspase-3 activity, which is usually upregulated in dying cells, was determined by measuring the cleavage of the peptide substrate Ac-DEVD-MCA and by detecting the cleaved active form of the caspase-3 by Western blotting. The activity of caspase-3 was drastically elevated in cells expressing those HPAAs which showed low viability in Trypan Blue assay. Interestingly, it was found that there is a correlation between the hydrophobicity of a single amino acid and the cytotoxicity of the corresponding HPAA as a homopolymer. These results indicate that the hydrophobicity of HPAAs may cause cytotoxicity.

[1]  C. Dobson,et al.  Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases , 2002, Nature.

[2]  K. Sakamaki,et al.  Polyglutamine aggregates stimulate ER stress signals and caspase-12 activation. , 2002, Human molecular genetics.

[3]  K. Moulder,et al.  Generation of Neuronal Intranuclear Inclusions by Polyglutamine-GFP: Analysis of Inclusion Clearance and Toxicity as a Function of Polyglutamine Length , 1999, The Journal of Neuroscience.

[4]  C. Ross,et al.  A repeat expansion in the gene encoding junctophilin-3 is associated with Huntington disease–like 2 , 2001, Nature Genetics.

[5]  C. Ross,et al.  Expansion explosion: new clues to the pathogenesis of repeat expansion neurodegenerative diseases. , 2001, Trends in Molecular Medicine.

[6]  J. Castilla,et al.  Caspase‐12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein , 2003, The EMBO journal.

[7]  M. MacDonald,et al.  Amyloid Formation by Mutant Huntingtin: Threshold, Progressivity and Recruitment of Normal Polyglutamine Proteins , 1998, Somatic cell and molecular genetics.

[8]  Carl W. Cotman,et al.  Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis , 2003, Science.

[9]  K. Fischbeck,et al.  Toxic Proteins in Neurodegenerative Disease , 2002, Science.

[10]  Christine Van Broeckhoven,et al.  Pathogenesis of polyglutamine disorders: aggregation revisited. , 2003, Human molecular genetics.

[11]  A. Goldberg,et al.  Cellular Defenses against Unfolded Proteins A Cell Biologist Thinks about Neurodegenerative Diseases , 2001, Neuron.

[12]  John Q Trojanowski,et al.  ‘Unfolding’ pathways in neurodegenerative disease , 2003, Trends in Neurosciences.

[13]  H. Zoghbi,et al.  Glutamine repeats and neurodegeneration. , 2000, Annual review of neuroscience.

[14]  Johan T den Dunnen,et al.  Strong aggregation and increased toxicity of polyleucine over polyglutamine stretches in mammalian cells. , 2002, Human molecular genetics.

[15]  R. Kopito,et al.  Impairment of the ubiquitin-proteasome system by protein aggregation. , 2001, Science.

[16]  Lucia Y Brown,et al.  Alanine tracts: the expanding story of human illness and trinucleotide repeats. , 2004, Trends in genetics : TIG.

[17]  R. Guigó,et al.  Comparative analysis of amino acid repeats in rodents and humans. , 2004, Genome research.

[18]  L. Davis,et al.  Intranuclear inclusions in oculopharyngeal muscular dystrophy contain poly(A) binding protein 2 , 2000, Annals of neurology.

[19]  R. Houghten,et al.  Polyalanine-based peptides as models for self-associated beta-pleated-sheet complexes. , 1997, Biochemistry.

[20]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[21]  A. Munnich,et al.  Polyalanine expansions in human. , 2004, Human molecular genetics.

[22]  Junying Yuan,et al.  Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-β , 2000, Nature.

[23]  Christopher A Ross,et al.  Polyglutamine Pathogenesis Emergence of Unifying Mechanisms for Huntington's Disease and Related Disorders , 2002, Neuron.

[24]  D. Rubinsztein,et al.  Intracellular green fluorescent protein-polyalanine aggregates are associated with cell death. , 2000, The Biochemical journal.

[25]  Mark Turmaine,et al.  Formation of Neuronal Intranuclear Inclusions Underlies the Neurological Dysfunction in Mice Transgenic for the HD Mutation , 1997, Cell.

[26]  Y. Kino,et al.  Intracellular Localization of Homopolymeric Amino Acid-containing Proteins Expressed in Mammalian Cells* , 2004, Journal of Biological Chemistry.

[27]  S. W. Davies,et al.  Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. , 1997, Science.

[28]  Hans Lehrach,et al.  Huntingtin-Encoded Polyglutamine Expansions Form Amyloid-like Protein Aggregates In Vitro and In Vivo , 1997, Cell.

[29]  B. Brais,et al.  PABP2 polyalanine tract expansion causes intranuclear inclusions in oculopharyngeal muscular dystrophy , 2000, Annals of neurology.

[30]  Kiyoshi Inoue,et al.  ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. , 2002, Genes & development.

[31]  E. Slee,et al.  Serial killers: ordering caspase activation events in apoptosis , 1999, Cell Death and Differentiation.