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1.
J Am Chem Soc ; 134(33): 13535-7, 2012 Aug 22.
Article in English | MEDLINE | ID: mdl-22857257

ABSTRACT

Mammalian Nod2 is an intracellular protein that is implicated in the innate immune response to the bacterial cell wall and is associated with the development of Crohn's disease, Blau syndrome, and gastrointestinal cancers. Nod2 is required for an immune response to muramyl dipeptide (MDP), an immunostimulatory fragment of bacterial cell wall, but it is not known whether MDP binds directly to Nod2. We report the expression and purification of human Nod2 from insect cells. Using novel MDP self-assembled monolayers (SAMs), we provide the first biochemical evidence for a direct, high-affinity interaction between Nod2 and MDP.


Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/immunology , Bacteria/immunology , Cell Wall/immunology , Immunity, Innate , Nod2 Signaling Adaptor Protein/immunology , Animals , Cell Line , Cloning, Molecular , HEK293 Cells , Humans , Insecta/cytology , Nod2 Signaling Adaptor Protein/genetics , Nod2 Signaling Adaptor Protein/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification
2.
Biochemistry ; 48(23): 5291-302, 2009 Jun 16.
Article in English | MEDLINE | ID: mdl-19405474

ABSTRACT

Adenylosuccinate lyase (ASL), a catalyst of key reactions in purine biosynthesis, is normally a homotetramer in which three subunits contribute to each of four active sites. Human ASL deficiency is an inherited metabolic disease associated with autism and mental retardation. We have characterized five disease-associated ASL mutants: R194C and K246E are located at subunit interfaces, L311V is in the central helical region away from the active site, and R396C and R396H are at the entrance to the active site. The V(max) (at 25 degrees C) for R194C is comparable to that of WT, while those of L311V, R396C, R396H, and K246E are considerably reduced and affinity for adenylosuccinate is retained. The mutant enzymes have decreased positive cooperativity as compared to WT. K246E exists mainly as dimer or monomer, accounting for its negligible activity, whereas the other mutant enzymes are similar to WT in the predominance of tetramer. At 37 degrees C, the specific activity of WT and these mutant enzymes slowly decreases 30-40% with time and reaches a limiting specific activity without changing significantly the amount of tetramer. Mutant R194C is unique in being rapidly inactivated at the harsher temperature of 60 degrees C, indicating that it is the least stable enzyme in vitro. Conformational changes in the mutant enzymes are evident from protein fluorescence intensity at 25 degrees C and after incubation at 37 degrees C, which correlates with the loss of enzymatic activity. Thus, these disease-associated single mutations can yield enzyme with reduced activity either by affecting the active site or by perturbing the enzyme's structure and/or native conformation which are required for catalytic function.


Subject(s)
Adenylosuccinate Lyase/chemistry , Adenylosuccinate Lyase/genetics , Autistic Disorder/enzymology , Intellectual Disability/enzymology , Mutation , Adenylosuccinate Lyase/metabolism , Autistic Disorder/metabolism , Circular Dichroism , Crystallography, X-Ray , Humans , Intellectual Disability/metabolism , Kinetics , Models, Molecular , Molecular Weight , Mutagenesis, Site-Directed , Protein Structure, Secondary , Spectrometry, Fluorescence , Temperature
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