Pink1 protects cortical neurons from thapsigargin-induced oxidative stress and neuronal apoptosis.

Apoptosis mediates the precise and programmed natural death of neurons and is a physiologically important process in neurogenesis during maturation of the central nervous system. However, premature apoptosis and/or an aberration in apoptosis regulation are implicated in the pathogenesis of neurodegeneration. Thus, it is important to identify neuronal pathways/factors controlling apoptosis. Pink1 [phosphatase and tensin homologue (PTEN)-induced kinase 1] is a ubiquitously expressed gene and has been reported to have a physiological role in mitochondrial maintenance, suppressing mitochondrial oxidative stress, fission and autophagy. However, how Pink1 is involved in neuronal survival against oxidative stress remains not well understood. In the present paper, we demonstrate that thapsigargin, a specific irreversible inhibitor of endoplasmic reticulum (ER) calcium-ATPase, could lead to dramatic oxidative stress and neuronal apoptosis by ectopic calcium entry. Importantly, the neuronal toxicity of thapsigargin inhibits antioxidant gene Pink1 expression. Although Pink1 knockdown enhances the neuronal apoptosis by thapsigargin, its overexpression restores it. Our findings have established the neuronal protective role of Pink1 against oxidative stress and afford rationale for developing new strategy to the therapy of neurodegenerative diseases.

Cloning and expression of an alpha-amylase gene from Phanerochaete chrysosporium.

Based on the genomic sequence and cDNA library screening, the cDNA sequence encoding an alpha-amylase was cloned from the filamentous white-rot fungus Phanerochaete chrysosporium and designated as pcamy1. Alignment results showed that the predicted protein has up to 43% amino acid homology to the known alpha-amylases in other organisms and is close to those from some filamentous fungi. Under nitrogen-starvation condition, the transcription of pcamy1 was accordingly upregulated or downregulated when soluble starch or glucose is sole carbon source. Addition of oxygen to nitrogen-limited media led to pcamy1 transcription and removal of glucose metabolic repression. The result indicated that the pcamy1 transcript was not only regulated by nutrients such as the carbon source but also by the cultivation environment, such as oxygen. This coordinate-regulatory model is likely common in P. chrysosporium. The expressed product of this gene in Escherichia coli could hydrolyze soluble starch, and its enzymatic activity was determined. As far as we know, this is the first report about cloning and expression study on the alpha-amylase in P. chrysosporium.

Molecular cloning and characterization of a peroxiredoxin from Phanerochaete chrysosporium.

Peroxiredoxins (Prxs) are a ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Here, we report on the cloning and functional characterization of a cDNA designated PcPrx-1, encoding peroxiredoxin from the white-rot fungus Phanerochaete chrysosporium. The full-length PcPrx-1 cDNA (932 bp) contains an open reading frame of 200 amino acid residues with a molecular mass of 22.1 kDa. The deduced primary structure of PcPrx-1 polypeptide shows a high level of sequence identity to other recently identified 2-cys peroxiredoxins. The recombinant PcPrx-1 protein was expressed as a histidine fusion protein in Escherichia coli and purified with a Ni2+-column. The purified protein was shown to have a protective effect against plasmid DNA cleavage by reactive oxygen species. The PcPrx-1 protein displays the ability to remove H2O2 in a ferrithiocyanate system. The results of this study suggest that PcPrx-1 may play a protective role against oxidative stress in P. chrysosporium.