ABSTRACT
Histone deacetylase (HDAC) 8 is a zinc ion dependent enzyme involved in removing the acetyl group from the core histones and other proteins which belong to Class I HDACs. It was reported that nitric oxide (NO) is a key regulator of HDAC function and S-nitrosylation of HDAC2 induces chromatin remodelling in neurons. This work reports the successful recombinant expression of human HDAC8 in Escherichia coli with two plasmids and the purification and S-nitrosylation in vitro. It was found that HDAC8 can be S-nitrosylated by the NO donor S-nitrosoglutathione (GSNO) in vitro, and the activity of HDAC8 was significantly inhibited when incubated with GSNO and S-nitrosocysteine in a time- and dosage-dependent manner, but sodium nitroprusside (SNP), and dithiothreitol cannot reverse this inhibition. These observations support and extend the concept that NO may regulate HDAC8 function by S-nitrosylation.
Subject(s)
Histone Deacetylases/isolation & purification , Histone Deacetylases/metabolism , Nitric Oxide/metabolism , Recombinant Proteins/metabolism , Repressor Proteins/isolation & purification , Repressor Proteins/metabolism , S-Nitrosoglutathione/metabolism , Blotting, Western , Cysteine/analogs & derivatives , Cysteine/chemical synthesis , Cysteine/metabolism , DNA Primers/genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Humans , In Vitro Techniques , Plasmids/genetics , S-Nitrosoglutathione/chemical synthesis , S-Nitrosothiols/chemical synthesis , S-Nitrosothiols/metabolismABSTRACT
The three-dimensional structure of an acidic phospholipase A(2) purified from the venom of Deinagkistrodon acutus (Agkistrodon acutus) was determined in a new crystal form by molecular replacement at 0.28 nm resolution with a crystallographic R factor of 21.9% (R-free=25.7%) and reasonable stereochemistry. Being similar to the previous reported crystal form, a significant conformational adaptation of segment 14-23 at the dimer interface was observed. This segment was related to the "interface recognition site" (IRS). It was found that a positively charged residue at position 34 seems to be a common feature for most of hemolytic PLA(2)s belonging to group II. Structural comparison between the two crystal forms showed that NaCl had significant effects on the crystal packing, thus leading to dramatic changes of the unit cell parameters. In the new crystal form, MPD (2-methyl-2, 4-pentanediol) molecules exist in the hydrophobic channel of the enzyme.
Subject(s)
Agkistrodon , Crotalid Venoms/enzymology , Phospholipases A/chemistry , Amino Acid Sequence , Animals , Calcium/chemistry , Crotalid Venoms/chemistry , Crystallization , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Phospholipases A/metabolism , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
The thermal stability of IL-1 beta in aqueous solution as a function of temperature (5-60 degrees C), pH (2-9), buffer (acetate, citrate, tris, and phosphate), and cyroprotectants (sugars, HSA) was investigated in this study. The analytical methodologies included RP-HPLC, SEC, ELISA, IEF-PAGE, SDS-PAGE, and bioassay. The degradation and inactivation of IL-1 beta at or above 39 degrees C were attributed to autoxidation of the two cysteine residues in the denatured protein, followed by hydrophobic/covalent aggregation and precipitation. At or below 30 degrees C, IEF- and SDS-PAGE results suggest a possible deamidation reaction. The difference in mechanism of degradation precludes the prediction of formulation shelf life from accelerated temperature data. Nonetheless, the good stability observed at 5 degrees C suggests that a solution formulation may be feasible for IL-1 beta.