A fresh colony was inoculated into tryptic soy broth containing 0

A fresh colony was inoculated into tryptic soy broth containing 0.5% yeast extract (TSBY) (Difco Laboratories, Detroit, MI, USA) for GnRH Associated Peptide (GAP) (1-13), human 16 h and then renewed with fresh TSBY broth for another 3 h incubation at 37C. is mostly regulated by TLR-myeloid differentiation factor 88 (MyD88) signaling (2, 3). Group A (GAS) infection causes various diseases ranging from mild pharyngitis and impetigo to severe necrotizing fasciitis and streptococcal toxic shock syndrome (STSS) (4). In STSS, the excessive production of various cytokines is thought to be responsible for severe systemic effects, and serum levels of TNF- Rabbit polyclonal to POLR3B and IL-6 show the highest correlation with disease severity (5, 6). Thioredoxin-interacting protein (Txnip), a vitamin D3-upregulated protein in 1,25-dihydroxyvitamin D3 (1,25[OH]2D3)-treated HL-60 cells (7), acts as an endogenous inhibitor of the antioxidant thioredoxin (Trx), which is involved in a wide variety of cellular processes including the response to oxidative stress, cancer development, metabolic diseases, and inflammatory processes (8C13). GnRH Associated Peptide (GAP) (1-13), human The reduction of Txnip protein facilitates tumor progression, whereas the overexpression of Txnip results in the inhibition of metastasis or further triggers cells undergoing apoptosis (9, 14, 15). In addition to the pro-apoptotic role of Txnip under stress, it also plays a crucial role in the induction of reactive oxygen species (ROS)-mediated NLRP3 inflammasomes whereby initiating inflammatory responses (12, 15, 16). As a member of the alpha-arrestin protein family, Txnip comprises a PXXP sequence for the binding of SH3 domain-containing proteins such as Trx and a PPXY sequence for the recognition of WW domain-containing proteins such as the E3 ubiquitin ligase Itch (17, 18). Itch belongs to the Nedd4-like family of E3 ubiquitin ligases and has been reported to specifically mediate the transfer of ubiquitin from E2 ubiquitin-conjugating enzymes to Txnip followed by the triggering of proteasomal degradation (18). In addition, AMP-dependent protein kinase (AMPK) has been demonstrated to phosphorylate Txnip, causing its rapid degradation during energy stress (19). Reports indicate that the TNF–stimulated reduction of Txnip effectively causes Trx-mediated p65 denitrosylation, which results in the increased DNA binding activities of NF-B (20). Consistent with this, exacerbated endotoxic shock occurs along with overactivated NF-B and excessive nitric oxide (NO) induction in Txnip-deficient mice during lipopolysaccharide (LPS) stimulation (21). Therefore, the stability GnRH Associated Peptide (GAP) (1-13), human of Txnip has certain pathophysiological impacts on inflammatory diseases. Txnip is a vital regulator of NF-B activation; however, little is known about its stability in controlling inflammation during bacterial infection. In this study, we investigated TLR2/NADPH oxidase-initiated HECT E3 ubiquitin ligase-dependent Txnip degradation for cytokine induction during GAS infection. Materials and Methods Bacteria GAS strain NZ131 (type M49) was a gift from Dr. D. R. Martin (New Zealand Communicable Disease Center, Porirua). GAS strain A20 (type M1) and (S2-1790) was kindly provided by Dr. C. F. Lin (Taipei Medical University, Taiwan). A fresh colony was inoculated into tryptic soy broth containing 0.5% yeast extract (TSBY) (Difco Laboratories, Detroit, MI, USA) for 16 h and then renewed with fresh TSBY broth for another 3 h incubation at 37C. The bacterial density was determined by measuring the absorbance at 600 nm with a spectrophotometer (Beckman Instruments, Somerset, NJ, USA) and plating serial dilutions of the samples on TSBY agar for counting CFU after incubation overnight at 37C. For the preparation of heat-killed GAS, suspended bacteria were treated at 100C for 30 min. Cell Cultures and Reagents RAW264.7 macrophage cells and THP-1 monocytic cells kindly provided by Dr. C. F. Lin (Taipei Medical University, Taiwan) were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), respectively. Murine BMDMs were isolated from wild-type, 0.05. Results GAS Infection Triggers Txnip Degradation mutant strain.