Biomedical subjects

Miranda Thomas

Publications and source records attributed to Miranda Thomas.

9 recordsLinked to original sources

PubMed2007

Cleavage of MAGI-1, a tight junction PDZ protein, by caspases is an important step for cell-cell detachment in apoptosis.

MAGI-1, a member of the MAGUK family of proteins, is shown to be rapidly cleaved during Fas-induced apoptosis in mouse 3T3 A31 cells, and in UV irradiation- and staurosporine-induced apoptosis in HaCaT cells. This generates a 97 kDa N-terminal fragment that dissociates from the cell membrane; a process that is largely prevented in the presence of the caspase inhibitor Z-VAD-fmk. In addition, we show that in vitro translated radiolabelled MAGI-1 is efficiently cleaved into 97 kDa and 68 kDa fragments by caspases-3 and -7 at physiological concentrations and mutating the MAGI-1 Asp(761) to Ala completely abolished the caspase-induced cleavage. Moreover, in HaCaT cells overexpressing the MAGI-1 Asp(761)Ala mutant the disruption of cell-cell contacts was delayed during apoptosis, whereas other caspase-dependent processes such as nuclear condensation were not affected, suggesting that cell detachment is parallel to them. Thus, MAGI-1 cleavage appears to be an important step in the disassembly of cell-cell contacts during apoptosis.

3T3 Cells↗

PubMed2005

The hScrib/Dlg apico-basal control complex is differentially targeted by HPV-16 and HPV-18 E6 proteins.

The E6 proteins of the high-risk Human papillomaviruses (HPV) types have a well-documented ability to target certain cellular proteins for ubiquitin-mediated degradation via the proteasome. Previous studies have shown that E6 proteins interact differently with different target proteins, and that the viral proteins, depending upon the target, may recruit diverse cellular ubiquitin-protein ligases. In this study, we have examined the abilities of E6 proteins from HPV-16 and HPV-18 to interact with and induce the degradation of two PDZ domain-containing targets, Dlg and hScrib. We have also mapped the binding site of E6 on hScrib and shown that the interaction of E6 with hScrib is distinct from its interactions with other PDZ domain-containing targets. This is reflected in the efficiency with which the two viral E6 proteins can inhibit hScrib's suppression of cell transformation.Dlg and hScrib have complementary activities in the control of epithelial cell polarity and the fact that both are targeted by high-risk HPV E6 proteins underlines their importance. Our finding that they are each targeted differently by HPV-16 and HPV-18 E 6 s suggests that the two viruses are subjected to somewhat different constraints and provides a possible explanation for the apparent redundancy in targeting both parts of this important control mechanism.

Adaptor Proteins, Signal Transducing↗

PubMed2005

In vitro assays of substrate degradation induced by high-risk HPV E6 oncoproteins.

The high-risk mucosal human papillomavirus E6 proteins were the first viral proteins that were shown to use the ubiquitin proteasome pathway for the inactivation of their cellular target proteins. The first substrate to be identified was the p53 tumor suppressor protein, and since then many other substrates for E6-induced degradation have been described. All of these require the presence of high-risk mucosal E6 together with the E1, E2, and E3 enzymes of the ubiquitin pathway. This activity of E6, although complex, is nonetheless amenable to in vitro analysis. Many different protocols have been described over the years for performing these assays. In this chapter we describe the most easily used and robust procedure that is routinely used in our laboratory.

Animals↗

PubMed2005

hDLG/SAP97, a member of the MAGUK protein family, is a novel caspase target during cell-cell detachment in apoptosis.

Cell-cell detachment is one of the hallmarks of apoptosis. To date, several transmembrane and plaque proteins from tight and adherent junctions have been characterised as caspase targets during apoptosis. Human discs large protein (hDLG)/SAP97 is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins, localised at the adherent junctions of epithelial and endothelial cells, that is required for adherens junction assembly and differentiation. Here, hDLG is shown to be a caspase target during UV irradiation and staurosporine (STS)-induced apoptosis in HaCaT and CaCo-2 cells. Immunohistological data show a rapid loss of hDLG localisation at the sites of cell-cell contacts, preceding actual cell-cell detachment. In vitro experiments revealed cleavages at multiple sites located in the N-terminal half of the protein by caspase-3 only. Using Ala scanning mutagenesis, one cleavage site with an unusual recognition sequence for the executioner caspases (QSVD427/N) was identified. These data suggest that caspase-mediated cleavage of hDLG, and other MAGUKs, and their removal from sites of cell-cell contacts is an early step in the disruption of adherens junctions and dismantling of cell-cell contacts during apoptosis.

Adaptor Proteins, Signal Transducing↗

PubMed2004

HPV E6 specifically targets different cellular pools of its PDZ domain-containing tumour suppressor substrates for proteasome-mediated degradation.

The high-risk HPV E6 proteins have been shown to direct the degradation of a variety of cellular proteins that contain PDZ domains. Although some of these proteins are involved in regulating processes of cell growth and polarity in Drosophila, little is known about their function in higher eukaryotic epithelial cells. In HPV-containing cells derived from cervical tumours, we find that the patterns of expression of the E6 targets hDlg (discs large), hScrib (Scribble), and MUPP1 are consistent with their being substrates for E6-induced degradation. It is also clear that, in the case of hDlg, E6 is specifically targeting nuclear pools of the protein rather than membrane-bound forms. We have also analysed the activity of a subset of E6 target proteins in the suppression of oncogene-induced cell transformation. Interestingly, Dlg, MAGI-1 and MUPP1 efficiently suppressed cell transformation, while MAGI-2 and MAGI-3 were ineffective in this assay. These results suggest that in the context of HPV-induced transformation Dlg, MAGI-1 and MUPP1 can function as tumour suppressors.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Activity of the human papillomavirus E6 PDZ-binding motif correlates with an enhanced morphological transformation of immortalized human keratinocytes.

Human papillomavirus E6 oncoproteins induce the proteasomal degradation of several multi-PDZ (PSD95/Dlg/ZO-1) domain-containing proteins such as the human homologue of Drosophila discs large. Binding to PDZ domain-containing proteins is mediated by a PDZ-binding motif contained within the C-terminus of E6. The ability of E6 proteins to induce degradation of PDZ domain-containing proteins correlates with their oncogenic potential. Here we examined the biological effect of this region of the human papillomavirus type 18 E6 oncoprotein on keratinocyte morphology. Our results show that in simian virus 40-immortalized human keratinocytes, stable expression of E6 correlated with the induction of an exaggerated mesenchymal-like morphology and actin cytoskeleton disorganization compared with parental cells. The altered phenotype was accentuated in cells expressing an E6 protein containing a mutation (Arg153Leu) within a protein kinase A recognition motif that abrogates protein kinase A's negative regulation of the activity of the PDZ-binding domain. The E6-induced changes indicated an epithelial-mesenchymal transition and were supported by the finding that E6-expressing cells contained vimentin. Changes to the epithelial phenotype of cells expressing a mutant E6 protein (Thr156Glu) that is unable to degrade discs large was significantly less marked, although they did show evidence of epithelial-mesenchymal transition. These observations imply that the activity of the E6 PDZ-binding motif contributes only to a part of the transition. Further analysis of the E6 cell lines showed a decrease in adherens junction and desmosome formation. Cells expressing a functional PDZ-binding motif showed the greatest disruption of intercellular junction formation, but this did not correlate with a decrease in total cellular levels of the individual components of adhesion junctions. This suggests that the activity of the PDZ-binding motif may have influenced either the assembly or integrity of functional adhesion complexes. An E6-mediated decrease in peripheral membrane levels of PDZ proteins like discs large could be the basis for the enhanced morphological transformation of immortalized keratinocytes.

Actins↗

PubMed2003

Viruses and the 26S proteasome: hacking into destruction.

The discovery that the human papillomavirus E6 oncoprotein could direct the ubiquitination and degradation of the p53 tumour suppressor at the 26S proteasome was the beginning of a new view on virus-host interactions. A decade later, a plethora of viral proteins have been shown to direct host-cell proteins for proteolytic degradation. These activities are required for various aspects of the virus life-cycle from entry, through replication and enhanced cell survival, to viral release. As with oncogenes and cell-cycle control, the study of apparently simple viruses has provided a wealth of information on the function of a whole class of cellular proteins whose function is arguably as important as that of the kinases: the ubiquitin-protein ligases.

Adenoviridae↗

PubMed2002

Chimaeric HPV E6 proteins allow dissection of the proteolytic pathways regulating different E6 cellular target proteins.

The ability of HPV E6 oncoproteins to induce the degradation of PDZ domain-containing MAGUK proteins correlates with their malignant potential. We previously showed that the HPV-6 E6 protein, when provided with the PDZ-binding domain from HPV-18 E6, acquires the ability to bind the Discs Large (Dlg) tumour suppressor and target it for degradation. Based on this finding we have extended this analysis to E6 proteins from a variety of different papillomavirus types. Cloning a PDZ-binding sequence onto the C-terminus of E6 proteins derived from low-risk mucosal, and low and high-risk cutaneous papillomavirus types, enables them to bind Dlg and a second MAGUK family member, MAGI-1. This renders the mucosally-derived low-risk chimaeric HPV E6 proteins capable of targeting Dlg for degradation, but they are unable to induce significant levels of degradation of MAGI-1. In contrast, none of the E6 proteins derived from cutaneous papillomavirus types induce significant degradation of either MAGI-1 or Dlg when provided with a PDZ-binding domain. These results demonstrate significant differences, both between mucosal and cutaneous HPV E6 proteins and in the pathways required for Dlg and MAGI-1 degradation.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Oncogenic human papillomavirus E6 proteins target the MAGI-2 and MAGI-3 proteins for degradation.

The E6 proteins from the high-risk human papillomavirus (HPV) types have previously been shown to target a number of PDZ domain-containing proteins for proteasome-mediated degradation. These include the hDlg tumour suppressor and the MAGI-1 protein. In this study we show that high-risk HPV E6 proteins also target the related MAGI-2 and MAGI-3 proteins for degradation. Moreover, we show that the interaction is specific to one PDZ domain, and that co-expression of this domain can protect each of the full-length MAGI proteins from E6-mediated degradation. These data provide clear indicators for the potential design of compounds that could specifically inhibit the interaction of oncogenic HPV E6 proteins with an important class of target proteins.

Activin Receptors, Type II↗