Browsing by Subject "proteasome"
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Item Persistent “hijacking” of brain proteasomes in HIV-associated dementia(2008-11-05) Trung Phuoc Nguyen; Benjamin B. Gelman, MD, PhD; Miles W. Cloyd, PhD; Dennis L. Kolson, MD, PhD; David J. McAdoo, PhD; Claudio A. Soto, PhDHuman immunodeficiency virus-1 (HIV) infection of the central nervous system results in a syndrome of neuropsychological impairment, motor deficits, and behavioral changes diagnosed as HIV-associated dementia. Findings of increased ubiquitin-stained deposits and high molecular weight ubiquitin-protein conjugates in brains of HIV-positive subjects suggest impaired protein turnover by the ubiquitin-proteasome system analogous to neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. HIV infection of the brain and the consequent inflammatory response was hypothesized to alter the ubiquitin-proteasome system, leading to altered brain protein turnover and neuronal function. Investigations into the ubiquitin-proteasome system in HIV-infected brains were performed using the resources of the Texas NeuroAIDS Research Center and the National NeuroAIDS Tissue Consortium, including clinical data and frontal cortex brain tissue from 153 autopsy cases. Assessment of frontal cortex proteasome proteolytic activity revealed an abnormal catalytic profile that was more severe in those with HIV-associated neuropsychological impairment or HIV encephalitis. Proteasome subunit composition analysis by immunoblotting showed increases in immunoproteasome subunits LMP7 and PA28 alpha and decreases in constitutive proteasome regulatory subunit Rpn2 in the frontal cortex of HIV-positive subjects that correlated with the abnormal proteasome proteinase profile and were associated with neuropsychological impairment and HIV encephalitis. Immunoproteasome increases correlated with lower performance on neurocognitive tests specific for frontal lobe functioning domains, providing indications of regional specificity. Immunoproteasome increases also correlated with increases in frontal cortex tissue HIV loads. Immunoproteasomes were localized by immunofluorescence to the perikarya and distal processes of neurons, as well as to oligodendrocytes, astrocytes, and microglia. The potential consequence of immunoproteasomes in neurons was investigated with the analysis of synaptic proteins. Decreases in synaptophysin correlated with immunoproteasome increases, indicating the potential for synaptic protein alterations associated with immunoproteasomes. Analysis of isolated nerve endings, or synaptosomes, revealed immunoproteasome increases in synapses of HIV-positive subjects that correlated with increases in 14-3-3 zeta and decreases in synapsin 1. These findings suggest “hijacking” of constitutive proteasomes by immunoproteasomes with the persistent inflammatory response in HIV-infected brains. Ubiquitin-mediated protein turnover by constitutive proteasomes is consequently impaired, resulting in the dysregulation of neuronal and synaptic protein composition that leads to neuronal dysfunction.Item West Nile virus versus the host cell: Identification of host factors that modulate infection(2008-08-27) Felicia Gilfoy Santa Maria Guerra; Peter W. Mason; Robert Davey; Nigel Bourne; Gregg Milligan; Frank Scholle; Don M. EstesThere are two competing aspects of virus-host interactions: (i) those that enhance virus transmission and (ii) those that reduce or prevent viral replication and transmission. Each of these types of interactions is critical and both must be investigated to fully understand viral pathogenesis. Cells encode a variety of molecules which are critical for recognizing viral infections. One such molecule, the dsRNA sensor PKR, has been shown to be important for IFN-beta induction. Therefore, to determine whether PKR was involved in the recognition of WNV infection, cells lacking PKR were infected with WNV and assayed for IFN production. Interestingly, PKR-null cells demonstrated dramatically lower levels of WNV-induced IFN compared to wild type cells. Additionally, chemical inhibition of PKR activity or post-translational gene silencing of PKR expression severely impaired WNV-induced IFN production, suggesting that PKR is critical for the induction of IFN following WNV infection. Further analysis suggested that PKR may be important for the activation of NF-kappaB, suggesting a possible mechanism of IFN-beta induction. Consistent with cell line data, PKR was shown to be critical for WNV-induced IFN production in primary mouse bone marrow-derived dendritic cells.\r\nThe recognition of WNV is an important aspect of controlling infection; however, it is only one side of the story. Host factors which WNV utilizes to facilitate its infection and replication are also key to understanding viral pathogenesis. The presence or absence of specific factors may control the level of viral replication, host tropisms and, ultimately, viral pathogenesis. To identify host co-factors that are essential for WNV infection and/or replication, small interfering RNAs (siRNAs) were used to systematically knockdown host gene products and levels of WNV infection and/or replication was assayed in the absence of these factors. A siRNA library screen identified ten cellular proteins which are essential for WNV infection and/or replication. Two of these genes encoded subunits of the proteasome. Chemical inhibitors of proteasome activity confirmed that the proteasome is critical for efficient WNV replication.