Abstract
MGA_0676 has been characterized as a Mycoplasma gallisepticum nuclease that can induce apoptosis of chicken cells. However, the mechanism by which MGA_0676 induces apoptosis has remained unclear. In this study, we evaluated MGA_0676-induced apoptosis and internalization in immortalized chicken embryo fibroblasts (DF-1) and cancer cell lines. The internalization of MGA_0676 was proven through caveolin-mediated endocytosis by blocking the endocytosis with specific inhibitors or with siRNA. We identified the Thif domain of NEDD8-activating enzyme E1 regulatory subunit (NAE) in DF-1 as the target region interacting with the SNC domain of MGA_0676. The interaction between the Thif and SNC domains was observed co-located in the perinuclear and nuclear of DF-1. We found that the interaction between NAE and MGA_0676 increased the ability of apoptosis and accelerated the process of cullin neddylation in DF-1 cells, in turn activating NF-κB. This resulted in the observed aggregation of NF-κB in the nuclei of DF-1 cells. Moreover, the apoptosis induced by MGA_0676 decreased significantly when NF-κB was inhibited by siRNA or BAY 11-7082 or when NAE was silenced by siRNA. Overall, our results demonstrate that MGA_0676 is internalized through caveolin-mediated endocytosis, interacts with SNC-dependent Thif to accelerate the process of cullin neddylation and activates NF-κB in DF-1 cells, ultimately playing a key role in apoptosis in chicken cells. Our results indicate MGA_0676 constitutes a critical etiological virulence factor of the respiratory disease caused by M. gallisepticum. This study also opens a venue to investigate MGA_0676 as a potential candidate as pro-apoptotic drug in cancer studies.
Highlights
Microbes have been reported to mediate pathogenicity in host cells by utilizing their virulence proteins (Fitzgerald et al, 2006; Humphrys et al, 2013; Krachler and Orth, 2013)
We found that neddylation specific inhibitor MLN4924 blocked the activation of NF-κB induced by MGA_0676 (Figure 7B), for further confirming this results, we observed the translocation of Rela in DF-1 cells by confocal microscopy, from the Figure 7C, we found that MGA_0676 lost its ability of inducing NF-κB translocated into nucleus when the DF-1 was pretreated with MLN4924
Endocytosis is involved in cell signaling generally mediated by clathrin or caveolin (Pascual-Lucas et al, 2014; Haucke, 2015; Garcia et al, 2017)
Summary
Microbes have been reported to mediate pathogenicity in host cells by utilizing their virulence proteins (Fitzgerald et al, 2006; Humphrys et al, 2013; Krachler and Orth, 2013). Some bacteria possess more than one kind of nucleases, which are vital to virulence and contribute to a wide variety of cellular biological processes, such as inducing host cell death, repairing DNA damage, and evading immune clearance from host tissues (Rottem, 2003; Amundsen et al, 2008; Delaney et al, 2012; Derré-Bobillot et al, 2013; Shields et al, 2013). Due to their extremely limited biosynthetic capabilities, Mycoplasma adopts a parasitic lifestyle in order to obtain their nutritional needs from host cells (Chung et al, 2010; Fan et al, 2010; Großhennig et al, 2013). It is worthwhile to examine the biological properties and mechanisms of mycoplasmal membrane-associated nucleases
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