Stress proteins located in the cytosol or endoplasmic reticulum (ER) maintain cell homeostasis and afford tolerance to severe insults. In neurodegenerative diseases, several chaperones ameliorate the accumulation of misfolded proteins triggered by oxidative or nitrosative stress, or of mutated gene products. Although severe ER stress can induce apoptosis, the ER withstands relatively mild insults through the expression of stress proteins or chaperones such as glucose-regulated protein (GRP) and protein-disulphide isomerase (PDI), which assist in the maturation and transport of unfolded secretory proteins. PDI catalyses thiol-disulphide exchange, thus facilitating disulphide bond formation and rearrangement reactions. PDI has two domains that function as independent active sites with homology to the small, redox-active protein thioredoxin. During neurodegenerative disorders and cerebral ischaemia, the accumulation of immature and denatured proteins results in ER dysfunction, but the upregulation of PDI represents an adaptive response to protect neuronal cells. Here we show, in brains manifesting sporadic Parkinson's or Alzheimer's disease, that PDI is S-nitrosylated, a reaction transferring a nitric oxide (NO) group to a critical cysteine thiol to affect protein function. NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leads to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. S-nitrosylation also abrogates PDI-mediated attenuation of neuronal cell death triggered by ER stress, misfolded proteins or proteasome inhibition. Thus, PDI prevents neurotoxicity associated with ER stress and protein misfolding, but NO blocks this protective effect in neurodegenerative disorders through the S-nitrosylation of PDI.
Many hereditary and sporadic neurodegenerative disorders are characterized by the accumulation of aberrant proteins. In sporadic Parkinson's disease, representing the most prevalent movement disorder, oxidative and nitrosative stress are believed to contribute to disease pathogenesis, but the exact molecular basis for protein aggregation remains unclear. In the case of autosomal recessive-juvenile Parkinsonism, mutation in the E3 ubiquitin ligase protein parkin is linked to death of dopaminergic neurons. Here we show both in vitro and in vivo that nitrosative stress leads to S-nitrosylation of wild-type parkin and, initially, to a dramatic increase followed by a decrease in the E3 ligase-ubiquitin-proteasome degradative pathway. The initial increase in parkin's E3 ubiquitin ligase activity leads to autoubiquitination of parkin and subsequent inhibition of its activity, which would impair ubiquitination and clearance of parkin substrates. These findings may thus provide a molecular link between free radical toxicity and protein accumulation in sporadic Parkinson's disease
A major obstacle to improving prognoses in ovarian cancer is the lack of effective screening methods for early detection. Circulating microRNAs (miRNAs) have been recognized as promising biomarkers that could lead to clinical applications. Here, to develop an optimal detection method, we use microarrays to obtain comprehensive miRNA profiles from 4046 serum samples, including 428 patients with ovarian tumors. A diagnostic model based on expression levels of ten miRNAs is constructed in the discovery set. Validation in an independent cohort reveals that the model is very accurate (sensitivity, 0.99; specificity, 1.00), and the diagnostic accuracy is maintained even in early-stage ovarian cancers. Furthermore, we construct two additional models, each using 9–10 serum miRNAs, aimed at discriminating ovarian cancers from the other types of solid tumors or benign ovarian tumors. Our findings provide robust evidence that the serum miRNA profile represents a promising diagnostic biomarker for ovarian cancer.
We isolated and identified a stress protein that is upregulated in response to hypoxia in primary-cultured glial cells. Protein-disulfide isomerase (PDI) was up-regulated not only by hypoxia in glia in vitro, but also by transient forebrain ischemia in rats in vivo. To determine whether newly synthesized PDI is involved in tolerance to ischemic stress, we carried out two procedures to induce PDI gene expression in human neuroblastoma SK-N-MC cells, as well as intrahippocampal injection following electroporation of an expression vector capable of overexpressing PDI in rats. Overexpression of this gene resulted in attenuation of the loss of cell viability induced by hypoxia in neuroblastoma SK-N-MC cells and a reduction in the number of DNAfragmented cells in the CA1 area of the hippocampus in brain ischemic rats, respectively. These findings suggest that up-regulated PDI may play a critical role in resistance to ischemic damage, and that the elevation of levels of this protein in the brain may have beneficial effects against brain stroke.Two distinct phenomena are observed in the CA1 subfield of the hippocampus after transient forebrain ischemia in rodents: the death of neurons, which is referred to as "delayed neuronal death," and the proliferation of glial cells, which is termed "gliosis" (1). Neurons are thus thought to be fragile and very sensitive to such stress, and, as a consequence, apoptotic cell death occurs (2, 3). Several studies have shown that caspases are involved in this neuronal apoptosis triggered by brain ischemia. Transgenic mice expressing dominant-negative mutants of caspase-1 and caspase-1-deficient mice show a reduced neuronal cell death induced by ischemic brain injury (4 -6). Caspase inhibitors such as Z-VAD-fmk, 1 YVAD-fmk, and DEVD-fmk also significantly reduce the neuronal cell death induced by ischemia (7-9). Furthermore, overexpression of Bcl-2 in transgenic mice as well as of neuronal apoptosis inhibitory protein, a member of the inhibitor of apoptosis protein family, by injection of adenovirus expression vectors has a protective effect against the neuronal cell death induced by focal cerebral or transient forebrain ischemia (10, 11). These results suggest that caspases are involved in ischemia-induced neuronal death in an anti-apoptotic protein-dependent manner.On the other hand, glial cells show tolerance to ischemic stress, and their numbers are increased in areas originally containing neurons. Due to their abundance and ability to sustain environmental perturbations, glia play a critical role in maintaining neuronal function under both physiological and pathological conditions (12). Glial cells subjected to hypoxia express stress proteins such as a 70-kDa heat shock protein (HSP70), a 78-kDa glucose-regulated protein (GRP78), a 150-kDa oxygen-regulated protein (ORP150), a 36-kDa putative RNA-binding protein (RA301), and a 70-kDa putative vesicle transport-related protein (RA410) (13-16). Hori et al. (17) have reported that the inhibition of protein synthesis during early reoxy...
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