Andrew P. Shaw

Systematic study of protein sumoylation: Development of a site‐specific predictor of SUMOsp 2.0

Protein sumoylation is an important reversible post‐translational modification on proteins, and orchestrates a variety of cellular processes. Recently, computational prediction of sumoylation sites has attracted much attention for its cost‐efficiency and power in genomic data mining. In this work, we developed SUMOsp 2.0, an accurate computing program with an improved group‐based phosphorylation scoring algorithm. Our analysis demonstrated that SUMOsp 2.0 has greater prediction accuracy than SUMOsp 1.0 and other existing tools, with a sensitivity of 88.17% and a specificity of 92.69% under the medium threshold. Previously, several large‐scale experiments have identified a list of potential sumoylated substrates in Saccharomyces cerevisiae and Homo sapiens; however, the exact sumoylation sites in most of these proteins remain elusive. We have predicted potential sumoylation sites in these proteins using SUMOsp 2.0, which provides a great resource for researchers and an outline for further mechanistic studies of sumoylation in cellular plasticity and dynamics. The online service and local packages of SUMOsp 2.0 are freely available at: http://sumosp.biocuckoo.org/.

Phospho-regulation of HsCdc14A by polo-like kinase 1 is essential for mitotic progression

Chromosome segregation in mitosis is orchestrated by dynamic interactions between spindle microtubules and centromeres, which in turn are governed by protein kinase- and phosphatase-signaling cascades. Previous studies showed that overexpression of human phosphatase HsCdc14A, an antagonist of cyclin-dependent kinase 1, affects several aspects of cell division. However, the molecular mechanism underlying HsCdc14A regulation in mitosis has remained elusive. Here we show that HsCdc14A activity is regulated by an auto-inhibitory mechanism via its intra-molecular association. Our biochemical study demonstrated that Polo-like kinase 1 (PLK1) interacts with and phosphorylates HsCdc14A. This phosphorylation partially releases the auto-inhibition of HsCdc14A judged by its phosphatase activity in vitro. To examine the functional relevance of such phospho-regulation of HsCdc14A in vivo, a phospho-mimicking mutant of HsCdc14A was expressed in HeLa cells. Importantly, overexpression of the phospho-mimicking mutants caused aberrant chromosome alignment with a prometaphase delay, suggesting the temporal regulation of HsCdc14A activity is critical for orchestrating mitotic events. Given the fact that HsCdc14A forms an intra-molecular association and PLK1-mediated phospho-regulation promotes HsCdc14A phosphatase activity, we propose that PLK1-HsCdc14A interaction provides a temporal regulation of HsCdc14A in chromosome segregation during mitosis.

Relationship between the Host Cell Mitochondria and the Parasitophorous Vacuole in Cells Infected with Encephalitozoon Microsporidia

Abstract Encephalitozoon microsporidia proliferate and differentiate within a parasitophorous vacuole. Using the fluorescent probe, calcein, and the mitochondrial probe, MitoTracker-CMXRos, a vital method was developed that confirmed ultrastructural reports that the host cell mitochondria frequently lie in immediate proximity to the parasitophorous vacuole. Morphometry failed to demonstrate any infection-induced increase in host cell mitochondria as there was no correlation between the mitochondrial volume and the extent of infection as judged by the parasitophorous vacuole volume. The total ATP concentration of infected cells did not differ from that of uninfected cells in spite of the increased metabolic demands of the infection. Treatment with 10−6 M albendazole, more than ten times the antiparasitic IC50 dose, and demecolcine had no subjective effect on the proximity of mitochondria to the parasitophorous vacuole membrane when studied by either transmission electron microscopy or by confocal microscopy even though these drug concentrations affected microtubule structure. Thus, once the association between mitochondria and the parasitophorous vacuole has been established, host cell microtubule integrity is probably not required for its maintenance. It is unlikely that the antimicrosporidial action of albendazole involves physically uncoupling developing parasite stages from host cell organelle metabolic support.

Infection by Microsporidia Disrupts the Host Cell Cycle

Abstract Microsporidia of the genus Encephalitozoon infect mammalian cells and have become a source of morbidity and mortality in immunocompromised humans. Encephalitozoon microsporidia develop and mature within parasitophorous vacuoles, enlarging the vacuole over time until it eventually occupies most of the cytoplasm of the host cell. The ability of the host cell to accommodate such a large burden for several days suggests that the parasite subverts normal host cell processes to ensure optimal environmental conditions for its growth and development. Since this environment would be threatened if cell division of the host cell occurred, we have formulated the hypothesis that infection with Encephalitozoon microsporidia induces an arrest in the cell cycle of the host cell. In support of this hypothesis, we have found that mitotic index and DNA duplication are reduced in infected cells as compared to uninfected cells. The number of host cell nuclei in S phase is increased. The levels of cyclin D1 and the percentage of cells in G1 are reduced; however, the levels of cyclin B1 are elevated even though the percentage of cells in G2/M is decreased. These results suggest that host cells infected with Encephalitozoon microsporidia are blocked at multiple points in the cell cycle.

Apical Spore Phagocytosis Is Not a Significant Route of Infection of Differentiated Enterocytes by Encephalitozoon intestinalis

ABSTRACT Encephalitozoon intestinalis is a microsporidian species that infects the intestinal mucosal epithelium, primarily in immunodeficient individuals. The present study employed undifferentiated and differentiated human colonic carcinoma cell lines to determine if this parasite species infected polarized epithelial cells by spore phagocytosis or by impalement with the deployed spore polar tube. Apical surface spore attachment differed between cell lines such that SW480>HT-29>Caco-2>HCT-8, with attachment being greater to undifferentiated Caco-2 cells than differentiated cells and greater to partially differentiated HCT-8 cells than differentiated HCT-8 cells. Attachment was inhibited by chondroitin sulfate A, suggesting that it was mediated by host cell sulfated glycoaminoglycans. Infection rates 3 days postinfection paralleled spore attachment in the various cell lines. The undifferentiated cell line SW480 and undifferentiated Caco-2 and HCT-8 cells exhibited modest spore phagocytosis while the more differentiated cell line HT29 and differentiated Caco-2 and HCT-8 cells did not. All cell lines were impaled by the polar tubes of germinating spores. When normalized to the number of spores attached to the apical membrane, such impalement was greatest in the more differentiated Caco-2 and HCT-8 cells. The host cell apical surface influenced parasite spore germination, as in populations of large undifferentiated Caco-2 cells to which >3 spores had attached, the frequency distribution of the percentages of spores germinated per cell was bimodal, indicating that the surface of some cells favored germination, while others did not. This study suggests that phagocytosis is not a biologically significant mode of infection in differentiated intestinal epithelial cells.

Role of P Glycoprotein in the Course and Treatment of Encephalitozoon Microsporidiosis

ABSTRACT Encephalitozoon microsporidia are obligate intracellular protozoan parasites that proliferate and differentiate within a parasitophorous vacuole inside host cells that are usually epithelial in nature. Isolates of the three species of theEncephalitozoon microsporidia, E. cuniculi,E. hellem, and E. intestinalis, were obtained from AIDS patients and cultured in green monkey (E6) kidney cells. Anti-P-glycoprotein (anti-Pgp) and anti-multidrug resistance-associated protein (anti-MRP) monoclonal antibodies were used to probe for multidrug resistance (MDR) pump epitopes and verapamil- or cyclosporin A- and probenecid-modulated intracellular calcein fluorescence were used to assess the expression of Pgp and MRP respectively in uninfected and infected cells. Pgp, but not MRP, was detected immunocytochemically and by verapamil- and cyclosporin A-potentiated intracellular fluorescence in both host cells and parasite developing stages. When an in vitro infection assay was employed, verapamil and cyclosporin A acted as chemosensitizing agents for the antiparasitic drug albendazole. These observations suggest that inhibiting host cell and perhaps parasite MDR pumps may increase the efficacy of antiparasitic agents in these and other microsporidia species.