THP-1 sh2 cells were transduced with computer virus containing HA-NEDD4 for 48?h and determined with 10?g/ml blasticidin (Invivogen, ant-bl-05) for stable clones
THP-1 sh2 cells were transduced with computer virus containing HA-NEDD4 for 48?h and determined with 10?g/ml blasticidin (Invivogen, ant-bl-05) for stable clones. Immunofluorescence Cells were fixed with 4% (v:v) paraformaldehyde in PBS, pH 7.4 for 10?min at room heat (RT). increased autophagy. Thus, NEDD4 participates in killing of intracellular bacterial pathogens TCS PIM-1 1 via autophagy by sustaining the stability of BECN1. (Mtb) and (Lm).4-6 Dysregulated autophagy has also been implicated in pathological development, emphasizing its critical involvement in maintaining homeostasis at the cellular and organismic level.3,7 The ULK1 and BECN1 complexes are 2 key players in the formation of autophagosomes. The ULK1 complex, which plays pivotal functions in autophagy initiation, consists of the serine/threonine kinase ULK1, ATG13, RB1CC1 and ATG101.8,9 ULK1 is activated by AMPK-mediated phosphorylation or loss of MTOR-mediated phosphorylation.10-12 After activation, ULK1 phosphorylates downstream targets to initiate autophagy. One such target is the BECN1 complex, comprising BECN1, PIK3R4/VPS15, PIK3C3/VPS34 and ATG14.13,14 Both abundances and activities of ULK1 and BECN1 are extensively regulated by ubiquitination. TRAF6 mediates K63 ubiquitination of ULK1 through AMBRA1, which increases stability and function of ULK1.15 TRAF6 also mediates K63 ubiquitination of BECN1, facilitating TLR4-triggered autophagy.15 In contrast, the deubiquitinase TNFAIP3/A20 reduces TLR4-triggered autophagy by decreasing K63 ubiquitination of BECN1.16 RNF216 also regulates K48 ubiquitination of BECN1, resulting in proteasomal degradation of BECN1 and decreased autophagy.17 Recently CUL3-KLHL20 was shown to ubiquitinate ULK1, leading to the proteasomal degradation of ULK1.18 NEDD4 (neuronal precursor cell expressed, developmentally downregulated 4) is a HECT (homologous E6-AP carboxyl?terminus) type E3 ubiquitin ligase, and is comprised of one N-terminal C2 domain name, 3 or 4 4 WW domains and 1 C-terminal HECT domain name.19,20 The C2 domain mediates its intracellular localization by interacting with phospholipids and proteins. The WW domain name, named for the presence of 2 conserved tryptophan residues, is responsible for substrate acknowledgement by binding to a PPxY motif. The HECT domain TCS PIM-1 1 name confers E3 catalytic activity.19,20 Numerous studies have exhibited that NEDD4 facilitates budding of various viruses by ubiquitinating viral matrix proteins via PPxY motifs. These include vesicular stomatitis computer virus,21 Marburg computer virus,22 Ebola computer virus,23,24 Rous sarcoma computer virus,25 and human immunodeficiency computer virus (HIV). 26 NEDD4 also ubiquitinates capsid protein VI of adenovirus in a PPxY-dependent manner and facilitates its intracellular targeting and effective contamination.27 Moreover, NEDD4 increases influenza virus contamination by decreasing the large quantity of the antiviral protein IFITM3 via ubiquitination.28 While the role of NEDD4 in viral infections is well documented, its function in bacterial infections remains elusive. As intracellular pathogens, Lm and Mtb have developed specific strategies to subvert defenses inside host cells.29,30 The common mechanism underlying intracellular survival shared by Lm and Mtb is the egression into the cytosol.30 For Lm, listeriolysin O (LLO) is essential for its rapid escape into the cytosol and for subsequent autophagy activation.31,32 Unlike Lm, Mtb induces phagosomal escape at later time points of contamination via the ESX-1 secretion system,33,34 which is encoded in region of difference 1 (RD1). ESX-1 is also required for sequestering Mtb within autophagosomes. 35 BCG (BCG) lacks the RD1 region and hence remains in the phagosomes within macrophages.34,36 The capacity of LLO to facilitate egression has been harnessed Rabbit Polyclonal to NXF3 for the generation of a recombinant BCG vaccine strain, BCG (rBCG).37 Although it perturbs the phagosomal membrane, this vaccine candidate remains in the phagosomes; however, bacterial components including proteins, glycolipids and double-stranded DNA are released into the cytosol, thereby inducing autophagy and inflammasome activation.38 Here we investigated the role of NEDD4 in bacterial TCS PIM-1 1 infections with Mtb, BCG, rBCG and Lm. We show that NEDD4 contributes to killing of intracellular Mtb, rBCG and Lm knockdown (KD) induced elevated K48-linkage ubiquitination of endogenous BECN1. BECN1 stability was increased by NEDD4 via K6 and K27 ubiquitination during autophagy induction, thereby promoting autophagy activation. Collectively, our data reveal a novel regulation of autophagy in which TCS PIM-1 1 NEDD4 increases BECN1 stability via ubiquitination and hence promotes autophagy, thereby contributing to killing of membrane-perturbing intracellular bacterial pathogens. Results NEDD4 contributes to efficient control of bacteria perturbing the phagosomal membrane To investigate the role of NEDD4 in bacterial infection, stable KD THP-1 and HeLa cell lines were established with lentivirus-delivered shRNAs (Fig.?S1A-D). shRNA #2 (sh2) was selected for further experiments due to its strong KD efficiency. THP-1 cells expressing scrambled shRNA or sh2 were employed for in vitro contamination with different intracellular bacteria. For Mtb contamination, the intracellular bacterial weight in KD TCS PIM-1 1 cells was significantly higher than in control cells expressing scrambled shRNA at 48?h post-infection (p.i.) (Fig.?1A). However, the intracellular growth of attenuated strain Mtb KD and control cells (Fig.?1B). For both Mtb.