Foldability of a Natural De Novo Evolved Protein.

The de novo evolution of protein-coding genes from noncoding DNA is emerging as a source of molecular innovation in biology. Studies of random sequence libraries, however, suggest that young de novo proteins will not fold into compact, specific structures typical of native globular proteins. Here we show that Bsc4, a functional, natural de novo protein encoded by a gene that evolved recently from noncoding DNA in the yeast S. cerevisiae, folds to a partially specific three-dimensional structure. Bsc4 forms soluble, compact oligomers with high ß sheet content and a hydrophobic core, and undergoes cooperative, reversible denaturation. Bsc4 lacks a specific quaternary state, however, existing instead as a continuous distribution of oligomer sizes, and binds dyes indicative of amyloid oligomers or molten globules. The combination of native-like and non-native-like properties suggests a rudimentary fold that could potentially act as a functional intermediate in the emergence of new folded proteins de novo.

High-resolution identification of human adiponectin oligomers and regulation by pioglitazone in type 2 diabetic patients.

Adiponectin is an adipokine with insulin-sensitizing, anti-inflammatory, and cardiac protective actions. It homo-oligomerizes into trimers, hexamers, and higher molecular weight (HMW) species, which are not fully characterized. We describe high-resolution separation of adiponectin oligomers under native conditions in polyacrylamide gel coupled with methods for producing standards to provide facile and accurate identification of the oligomers. Using these procedures, adiponectin trimers in human and rodent plasma were found to migrate as two distinct populations. Distributions of these two populations are linearly proportional in plasma from type 2 diabetic patients before (R(2)=0.903, P<0.001) and after (R(2)=0.960, P<0.0001) 12weeks of treatment with pioglitazone as well as from control subjects (R(2)=0.891, P<0.0001). In addition, HMW adiponectin could be separated into three distinct oligomers: nonamer (9mer), dodecamer (12mer), and the previously characterized octadecamer (18mer). Plasma concentrations of all oligomers increased on pioglitazone treatment, with the largest fold increase being observed in 9mers and 12mers compared with baseline. Increasing concentrations of adiponectin during oligomerization in vitro led to a disproportionate increase in 18mers. The difference between in vivo and in vitro observations suggests that higher total adiponectin protein concentration contributes to pioglitazone's ability to enhance HMW adiponectin levels, but additional factors likely affect oligomer assembly or turnover independently.

Extracellular conversion of adiponectin hexamers into trimers.

Adiponectin is an adipocyte-secreted hormone that exists as trimers, hexamers and larger species collectively referred to as HMW (high-molecular-weight) adiponectin. Whether hexamers or HMW adiponectin serve as precursors for trimers outside the circulation is currently unknown. Here, we demonstrate that adiponectin trimers can be generated from larger oligomers secreted from primary rat adipose cells or differentiated 3T3-L1 adipocytes. Purified hexameric, but not HMW, adiponectin converted into trimers in conditioned media separated from 3T3-L1 adipocytes or, more efficiently, when enclosed in the dialysis membrane in the presence of adipocytes. Several lines of evidence indicate that the conversion is mediated by an extracellular redox system. First, N-terminal epitope-tagged hexamers converted into trimers without proteolytic removal of the tag. Secondly, appearance of trimers was associated with conversion of disulfide-bonded dimers into monomers. Thirdly, thiol-reactive agents inhibited conversion into trimers. Consistent with a redox-based mechanism, purified hexamers reductively converted into trimers in defined glutathione redox buffer with reduction potential typically found in the extracellular environment while the HMW adiponectin remained stable. In addition, conversion of hexamers into trimers was enhanced by NADPH, but not by NADP+. Collectively, these data strongly suggest the presence of an extracellular redox system capable of converting adiponectin oligomers.