Abstract
Perforin-2 (MPEG1) is an effector of the innate immune system that limits the proliferation and spread of medically relevant Gram-negative, -positive, and acid fast bacteria. We show here that a cullin-RING E3 ubiquitin ligase (CRL) complex containing cullin-1 and βTrCP monoubiquitylates Perforin-2 in response to pathogen associated molecular patterns such as LPS. Ubiquitylation triggers a rapid redistribution of Perforin-2 and is essential for its bactericidal activity. Enteric pathogens such as Yersinia pseudotuberculosis and enteropathogenic Escherichia coli disarm host cells by injecting cell cycle inhibiting factors (Cifs) into mammalian cells to deamidate the ubiquitin-like protein NEDD8. Because CRL activity is dependent upon NEDD8, Cif blocks ubiquitin dependent trafficking of Perforin-2 and thus, its bactericidal activity. Collectively, these studies further underscore the biological significance of Perforin-2 and elucidate critical molecular events that culminate in Perforin-2-dependent killing of both intracellular and extracellular, cell-adherent bacteria.
Highlights
As the largest class of ubiquitin ligases, cullin-RING E3 ubiquitin ligases (CRLs) regulate numerous cellular processes including signal transduction, gene expression, development, and cell cycling (Bosu and Kipreos, 2008; Metzger et al, 2012)
The presence of a membrane attack complex perforin (MACPF) domain within Perforin-2 suggests that it may have the ability to form lytic pores. This hypothesis is supported by the recovery of Perforin-2 from bacteria and images of pores with an average diameter of 100 Ain lipid membranes of mammalian cells expressing Perforin-2 and bacterial cell walls exposed to Perforin-2 (McCormack et al, 2015)
In this study we have shown that Perforin-2 is monoubiquitylated by a CRL complex in response to extracellular bacteria or pathogen associated molecular patterns (PAMPs) and that ubiquitylation is required for Perforin-2’s bactericidal activity against both extracellular, cell adherent, and intracellular bacteria
Summary
As the largest class of ubiquitin ligases, cullin-RING E3 ubiquitin ligases (CRLs) regulate numerous cellular processes including signal transduction, gene expression, development, and cell cycling (Bosu and Kipreos, 2008; Metzger et al, 2012). The placement of the substrate and E2 at opposite ends of the elongated cullin translates into a separation of ∼50 A (Wu et al, 2003; Hao et al, 2007; Merlet et al, 2009) This gap prohibits ubiquitylation of the substrate. This problem is solved by an additional E2 enzyme, such as UBC12, that conjugates NEDD8, an 8.6 kDa member of the ubiquitin family of proteins (UniProt entry Q15843, Pfam identifier PF00240), to a conserved lysine within the carboxy-terminal domain of the cullin (Petroski and Deshaies, 2005). CRL-dependent ubiquitylation of a protein substrate is itself dependent upon cullin neddylation (Morimoto et al, 2000; Ohh et al, 2002; Sakata et al, 2007)
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