Biomedical subjects

Gordon D Strachan

Publications and source records attributed to Gordon D Strachan.

6 recordsLinked to original sources

PubMed2005

E2F1 induces cell death, calpain activation, and MDMX degradation in a transcription independent manner implicating a novel role for E2F1 in neuronal loss in SIV encephalitis.

The E2F1 transcription factor can initiate proliferation or apoptosis, the latter by both transcription-dependent and -independent mechanisms. Recently, an E2F1 mutant lacking the DNA binding domain, E2F1(180-437), has been implicated in degradation of MDMX and MDM2 proteins via lysosomal proteases. MDM proteins block p53 dependent apoptosis by directly inhibiting p53 stability and function. Here we demonstrate E2F1(180-437) induces death in HEK293 cells independent of E2F1 transcriptional activation and p53 stabilization. E2F1(180-437) elevates the activity of the calcium-activated protease, calpain, which is required for E2F1 induced proteolysis of MDMX and E2F1 induced cell loss. To determine if E2F1 could be activating proteolysis via calpains in neurodegeneration, we examined MDMX immunofluorescence in simian immunodeficiency virus encephalitis (SIVE). We found a reciprocal relationship between E2F1 and MDMX staining: in SIVE where E2F1 immunostaining is increased, MDMX is decreased, while in controls where E2F1 immunostaining is low, MDMX is high. Together these experiments support a new function for E2F1 in the activation of calpain proteases and suggest a role for this pathway in SIVE.

Animals↗

PubMed2005

Expression of the fetal Alz-50 clone 1 protein induces apoptotic cell death.

The fetal Alz-50 clone 1 (FAC1) protein exhibits altered expression patterns in neurodegenerative disease. Though it has been shown to bind DNA in a site-specific, phosphorylation-dependent manner, its cellular function remains unknown. Here, we demonstrate that overexpression of FAC1 in PT67 fibroblasts induces nuclear condensation and cleavage of caspase 3 to its active form indicating induction of apoptosis. The amino-terminal domain of FAC1 is necessary and sufficient to induce both nuclear condensation and activation of caspase 3. Disruption of FAC1 interaction with a known binding partner, kelch-like ECH-associated protein 1 (Keap1), enhances activation of caspase 3. Keap1 is known to block activation of the antioxidant response gene products by direct interaction with the transcriptional activator, Nrf2. Disruption of the Keap1:Nrf2 interaction enhances FAC1 induction of apoptosis. These findings suggest a role for FAC1 in apoptosis following release of Nrf2 from Keap1 in response to oxidative stress.

Antigens, Nuclear↗

PubMed2005

E2F4 expression patterns in SIV encephalitis.

The E2F1 transcriptional regulator has been shown to exhibit altered expression and localization in HIVE and SIVE. However, other E2F family members are expressed in mature neurons and participate in neuronal differentiation. In an in vitro model of neuronal differentiation, E2F4 protein levels have been shown to increase. Further reduction in E2F4 leads to loss of neurites in this model. Neuritic damage and loss are also seen in progression of HIVE and SIVE. To determine if changes in E2F4 may contribute to altered neuronal morphology and survival, we assessed E2F4 immunostaining in caudate and mid-frontal cortex from SIVE macaques and non-encephalitic controls. We found that E2F4 was expressed in neurons and localized to nuclei in both SIVE and non-encephalitic controls. Quantification of E2F4 fluorescence intensity indicated that there was an overall decrease in E2F4 in caudate of SIVE macaques as compared to non-encephalitic controls, which correlated with a decrease in the neuronal phenotypic marker, MAP2. In contrast, we observed a slight increase in E2F4 in mid-frontal cortex of SIVE despite a significant decrease in MAP2. When E2F4 is normalized to MAP2, we found an increase in E2F4 fluorescence intensity per MAP2 in SIVE mid-frontal cortex. These findings suggest changes in E2F4 may be contributing to altered neuronal morphology or survival in SIVE.

Animals↗

PubMed2005

Chemokine- and neurotrophic factor-induced changes in E2F1 localization and phosphorylation of the retinoblastoma susceptibility gene product (pRb) occur by distinct mechanisms in murine cortical cultures.

The retinoblastoma susceptibility gene product (pRb) and E2F1 have been found to exhibit altered localization and increased staining in several neurodegenerative diseases. We have observed similar localization in primary murine cortical cultures treated with neurotrophic factors (NTF) or chemokines. In untreated cultures, E2F1 exhibited minimal immunostaining using the KH95 antibody, which recognizes the pRb interaction domain. In primary E16 murine cortical cultures, NTF- or chemokine-treated neurons, KH95 E2F1 staining was increased in the cytoplasm. However, an antibody recognizing the amino-terminus of E2F1 (KH20) stained the cytoplasm of both untreated and treated neurons. Taken together these results suggest that the change seen in E2F1 using the KH95 antibody is due to antigen unmasking of a carboxy-terminal epitope in response to NTF and chemokines. When we assessed staining for the hyperphosphorylated, inactive form of pRb (ppRb) in untreated cultures, ppRb was predominantly cytoplasmic. In response to NTF or chemokine treatment, staining for ppRb was observed predominantly in nuclei of neurons indicating a change in subcellular distribution. Immunoblot analysis demonstrated increased levels of ppRb in response to NTF and chemokines. Inhibitors of translation, nuclear export, and phoshpatidylinositol-3-kinase blocked NTF- and chemokine-induced nuclear ppRb localization while having no effect on E2F1 staining. Instead increased cytoplasmic KH95 E2F1 staining was dependent on cytoskeletal destabilization which did not influence ppRb localization. These findings demonstrate that alterations in ppRb distribution and E2F1 antigen availability by NTF and chemokines occur by distinct mechanisms suggesting that E2F1 function may be independent of pRb regulation in post-mitotic neurons.

Animals↗

PubMed2004

Fetal Alz-50 clone 1 interacts with the human orthologue of the Kelch-like Ech-associated protein.

The fetal Alz-50 reactive clone 1 (FAC1) protein exhibits altered expression and subcellular localization during neuronal development and neurodegenerative diseases such as Alzheimer's disease. Using the yeast two-hybrid screen, the human orthologue of Keap1 (hKeap1) was identified as a FAC1 interacting protein. Keap1 is an important regulator of the oxidative stress response pathway through its interaction with the Nrf family of transcription factors. An interaction between full-length FAC1 and hKeap1 proteins has been demonstrated, and the FAC1 binding domain of hKeap1 has been identified as the Kelch repeats. In addition, FAC1 colocalizes with endogenous Keap1 within the cytoplasm of PT67 cells. Exogenously introduced eGFP:hKeap1 fusion protein redistributed FAC1 to colocalize with eGFP:hKeap1 in perinuclear, spherical structures. The interaction between FAC1 and hKeap1 is reduced by competition with the Nrf2 protein. However, competition by Nrf2 for hKeap1 is reduced by diethylmaleate (DEM), a known disrupter of the Nrf2:Keap1 interaction. DEM does not affect the ability of FAC1 to bind hKeap1 in our assay. These results suggest that hKeap1 regulates FAC1 in addition to its known role in control of Nrf2. Furthermore, the observed competition between FAC1 and Nrf2 for binding hKeap1 indicates that the interplay between these three proteins has important implications for neuronal response to oxidative stress.

Actins↗

PubMed2003

The E2F-1 transcription factor is negatively regulated by its interaction with the MDMX protein.

Several proteins with important roles in oncogenesis have been shown to regulate the function of the E2F-1 transcription factor, which is known to activate the expression of genes required for proliferation and apoptosis. Here we identify the MDMX oncoprotein as an E2F-1-binding factor, from a yeast-two hybrid screen using a portion of the E2F-1 protein as "bait." We demonstrate that the region within MDMX needed for the E2F-1:MDMX interaction is located in the central part of the protein, C-terminal of the p53-binding domain. The region within E2F-1 needed for this association is adjacent to the DNA binding domain. Further, when expressed in vivo or in vitro the MDMX protein migrates as two isoforms on SDS-PAGE, the faster migrating isoform having the stronger affinity for the E2F-1 proteins. It appears that this interaction reduces the ability of E2F-1 to bind DNA. Expression of MDMX along with E2F-1 and Dp-1 in Saos2 cells reduces the ability of E2F-1 to bind to its consensus DNA sequence, without altering E2F-1 protein levels. These data indicate that the MDMX protein is capable of associating with E2F-1 and negatively regulating its DNA binding ability.

Animals↗