| Staphylococcu.s aureus is a common human pathogen that causes a wide range of infections and diseases,including fasciitis,pneumonia,endocarditis,septicemia,osteomyelitis,and toxic shock syndrome.In order to destroy the host innate immune system,lots of immune evasion proteins were secreted by S.aureus to inhibit neutrophils recruitment and activation.S.aureus(NCTC 8325 strain)secretes 14 staphylococcal superantigen-like(SSLs)proteins,the SSL proteins were originally named as staphylococcal exotoxin-like proteins(Sets).Sets were latterly renamed as staphylococcal superantigen-like(SSLs)due to their sequence and structural similarity to superantigen.However,S.aureus contains many different strains and the naming of SSLs/Sets among strains is confusing.The 14 ssl genes in NCTC 8325 strain have been well identified,while only 12 ssl/set genes were found in Mu50 strain.SSL proteins have multiple functions by interacting with different host targets.For example,SSL7 binds complement C5 to inhibit complement system.SSL10 disrupts the IgG1-mediated phagocytosis by interacting with IgG1.SSL11 inhibits neutrophil extravasation by blocking the interaction of PSGL-1 with P-selectin.SSL3 and SSL4 inhibit the expression of Toll-like receptor 2 on the surface of dendritic cells,macrophages and neutrophils.In addition,SSL10 not only binds to IgG1 but also interacts with prothrombin and Factor Xa to inhibit blood coagulation system.To get insight into the functions of SSL proteins,more SSLs targets need to be identified.Set11 is one of the SSL proteins in Mu50 strain,of which structure and function remain unknown.In part 1,to identify and characterize Set11 and its possible targets proteins from host blood/serum,we tried to solve the crystal structure of Set11 and identify its host targets by CNBr-pull down assays.To obtain high quality cryatals,a number of Set11 truncations were expressed,purified and crystallized.Unfortunately,we failed to get better crystals with the X-ray diffraction resolution higher than 3.6 A.We performed the bio informatics analysis of SSLs/Sets proteins in Mu50 strain and renamed all the proteins as SSLs.We found Set11 has 90%sequence identity to SSL7 from NCTC 8325 strain,indicating Setll is a SSL7 ortholog.In addition,the SSL7 in ATCC 12598 strain was reported to bind complement C5 to inhibit complement system.To investigate if Set11 also binds C5,we made the homology model of Set11 and the Set11-C5 complex model based on SSL7(PDB ID:1V10)and SSL7-C5(PDB ID:3KLS)structure,respectively.Structural analysis and sequence alignment reveal that the residues on SSL7 involved in C5 binding are highly conserved in Set11,indicating C5 as the potential target for Set11 in Mu50 strain.To identify new targets for Setll,we cloned,expressed and purified Set11 and performed CNBr-pull down combined mass spectrum assays using human blood and serum.CNBr-Pull down experimental results show that human blood exists 5 Setll targets and they respectively are fibronectin 1 isoform 3 preproprotein,complement C5,complement C3 precursor,serum albumin,and alpha-1-microglobulin precursor identified by mass spectrum.The potential target proteins for Set11 indicate new functions of Set11.E3 ubiquitin ligase TRAIP is the abbreviation of tumor necrosis factor(TNF)receptor-associated factors interacting protein.TRAIP was reported to interact with tumor necrosis factor(TNF)receptor-assoc iated factors and the two tumor suppressors CYLD and SYK to inhibit the NF-κB activation and apoptosis.Recently,TRAIP was identified as an RING type E3 ubiquitin ligase that interacts with E2 enzyme UBE2U through RING domian and played an important role in ubiquitylation.However,the ubiquitylation substrates and the molecular mechnism of RING domian-mediated TRIAP-UBE2U interaction remians unknown.In part 2,we coloned,expressed,purified and crystallized TRAIP RING Domain and its truncated fragments from mouse,zebrafish,african clawed frog and fruit flies.Unlucky,no crystal was observed up to now. |
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