核蛋白SP110a特性之研究

Abstract

Tuberculosis (TB) is one of the most important infectious diseases in the world. According to the epidemiological statistics, one-third of the world population is estimated to be infected with Mycobacterium tuberculosis (MTB) which causes approximatedly 8 million new cases of tuberculosis globally each year and results in about 2 million TB-related deaths annually. The majority of individuals infected with Mycobacterium tuberculosis remains asymptomatic and noninfectious, and only 10% will progress to active tuberculosis. There are many factors involved in the risk of individual infection and development of TB, and these include the interaction of the host body and it's pathogens, stress, malnutrition, concomitant infections (for example HIV) and senescence. Evidence indicates that the susceptibility of host to MTB is associated with host immunity and genetic variation. Until now, the relationship between host resistance to TB and genetic variation has remained unclear. In previous studies, a genetic locus on mouse chromosome 1, named sst1 (supersusceptibility to tuberculosis 1), controlling the host resistance and susceptibility to tuberculosis was mapped. Furthermore, a gene, Ipr1 (intracellular pathogen resistance 1), within the sst1 locus was identified. Ipr1 is upregulated in macrophages resistant to MTB but not in the sst1 susceptible microphages. Therefore, Ipr1 gene may be participated in innate immunity in mouse models of MTB infection and in the cellular regulatory mechanism of disease onset. The SP110 nuclear body protein (SP110) is the closest human homologue of the mouse IPR1 protein. SP110 protein family has 3 major isoforms: SP110a, SP110b and SP110c. The alternative splicing makes different isoforms of SP110; however, the differences of roles and functions among these isoforms are still unclear. Thus, in this study, we investigated the characteristics and functional differences among the isoforms of SP110 proteins. For this purpose, we constructed the eGFP-SP110 expression vector and co-transfected with four lentiviral structural gene into HEK293T cell line to generate lentiviral particles containing our target genes. Then, we used dual-promoter lentiviral system to transduce human THP1 cell line with the lentiviral particles to generate a stable clone, in which the expression of eGFP-SP110a was regulated by the Tet-on system. After treatment with doxycycline, the expression of eGFP-SP110a protein was induced, and the confocal images showed that eGFP-SP110a protein was mainly localized in the nucleus. In the previous studies, we found that the expression of eGFP-SP110b/c protein was increased after treatment with IFN, probably through the increase of protein stability. Thus, we examined whether SP110a protein has the same characteristics as SP110b/c proteins and found that the phenomena of SP110a were the same with the latter. In addition, by coimmunoprecipitation, we found that some proteins binding with SP110a/b/c protein were pulled down with an anti-eGFP antibody, including OAS1 (2, 5-oligoadenylate synthetase 1). The proteins interacting with eGFP-SP110a/b/c will be further characterized by proteomics approaches. We will identify the proteins interacting with SP110a by LC-MS/MS analysis and compare the protein complex profiles with that of SP110b/c.