No abstract

In search of metabolically regulated secreted proteins, we conducted a microarray study comparing gene expression in major metabolic tissues of fed and fasted ob/ob mice and C57BL/6 mice. The array used in this study included probes for ~4000 genes annotated as potential secreted proteins. Circulating macrophage inhibitory cytokine 1 (MIC-1)/growth differentiation factor 15 (GDF15) concentrations were increased in obese mice, rats, and humans in comparison to age-matched lean controls. Adeno-associated virus-mediated overexpression of GDF15 and recombinant GDF15 treatments reduced food intake and body weight and improved metabolic profiles in various metabolic disease models in mice, rats, and obese cynomolgus monkeys. Analysis of the GDF15 crystal structure suggested that the protein is not suitable for conventional Fc fusion at the carboxyl terminus of the protein. Thus, we used a structure-guided approach to design and successfully generate several Fc fusion molecules with extended half-life and potent efficacy. Furthermore, we discovered that GDF15 delayed gastric emptying, changed food preference, and activated area postrema neurons, confirming a role for GDF15 in the gut-brain axis responsible for the regulation of body energy intake. Our work provides evidence that GDF15 Fc fusion proteins could be potential therapeutic agents for the treatment of obesity and related comorbidities.

Protein disulfide isomerase (PDI), a very abundant protein in the endoplasmic reticulum, facilitates the formation and rearrangement of disulfide bonds using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F. (1994) J. Biol. Chem. 269, 30946-30952]. Each dithiol/disulfide active-site contains the thioredoxin consensus sequence CXXC. Four mutants of protein disulfide isomerase were constructed that have only a single active-site cysteine. Kinetic analysis of these mutants show that the first (more N-terminal) cysteine in either active site is essential for catalysis of oxidation and rearrangement during the refolding of reduced bovine pancreatic ribonuclease A (RNase). Mutant active sites with the sequence SGHC show no detectable activity for disulfide formation or rearrangement, even at concentrations of 25 microM. The second (more C-terminal) cysteine is not essential for catalysis of RNase disulfide rearrangements, but it is essential for catalysis of RNase oxidation, even in the presence of a glutathione redox buffer. Mutant active sites with the sequence CGHS show 12%-50% of the kcat activity of wild-type active sites during the rearrangement phase of RNase refolding but < 5% activity during the oxidation phase. In addition, mutants with the sequence CGHS accumulate significant levels of a covalent PDI-RNase complex during steady-state turnover while the wild-type enzyme and mutants with the sequence SGHC do not. Since both active-site cysteines are essential for catalysis of disulfide formation, the dominant mechanism for RNase oxidation may involve direct oxidation by the active-site PDI disulfide. Although it is not essential for catalysis of RNase rearrangements, the more C-terminal cysteine does contribute 2-8-fold to the rearrangement activity. A mechanism for substrate rearrangement is suggested in which the second active-site cysteine provides PDI with a way to "escape" from covalent intermediates that do not rearrange in a timely fashion. The second active-site cysteine may normally serve the wild-type enzyme as an internal clock that limits the time allowed for intramolecular substrate rearrangements.

Yeast methionine aminopeptidase I (MetAP I) is one of two enzymes in Saccharomyces cerevisiae that is responsible for cotranslational cleavage of initiator methionines. It has previously been classified as a Co2+ metalloprotease in all prokaryotic and eukaryotic forms studied. However, treatment of recombinant apo-MetAP I with 12.5 p M Zn2+ produces an enzyme that is as active as that reconstituted with 200 pM Co2+. In the presence of physiological concentrations of reduced glutathione (GSH), Co-MetAP I is inactive, while the activity of Zn-MetAP I is increased more than 1.7-fold over Zn-MetAP I assayed in the absence of GSH. Given that the in vivo concentration of Zn2+ is at least 1,000-fold higher than that of Co2+, and that Co2+ is insoluble in physiological concentrations of GSH, it is probable that yeast MetAP I is actually a Zn2+ metalloprotease. Furthermore, unless there are extraordinary conditions that insulate or sequester them from this reducing milieu, that have yet to be identified, there are not likely to be any cytoplasmic enzymes that use free Co2+.

During oxidative protein folding, efficient catalysis of disulfide rearrangements by protein-disulfide isomerase is found to involve an escape mechanism that prevents the enzyme from becoming trapped in covalent complexes with substrates that fail to rearrange in a timely fashion. Protein-disulfide isomerase mutants with only a single active-site cysteine catalyze slow disulfide rearrangements and become trapped in a covalent complex with substrate. Escape is mediated by the second, more carboxyl-terminal cysteine at the active site. A glutathione redox buffer increases the k cat for single-cysteine mutants by 20 -40-fold, but the presence of the second cysteine at the active site in the wild-type enzyme increases the k cat by over 200-fold. A model is developed in which kinetic scanning for disulfides of increasing reactivity is timed against an intramolecular clock provided by the second cysteine at the active site. This provides an alternative, more efficient mechanism for rearrangement involving the reduction and reoxidation of substrate disulfides.With no known sequence cues to designate which cysteines should be cross-linked in a native protein, the question arises as to how correct pairs of cysteines are selected during folding and how this selection is made quickly enough to be useful in the cell. The uncatalyzed formation of native disulfides during in vitro protein folding is generally not fast enough to support the folding rate that is found in the cell (1). To overcome this, catalysis of disulfide formation and rearrangement in eukaryotes is provided by protein-disulfide isomerase (PDI), 1 a 55-kDa protein of the endoplasmic reticulum (2).As a catalyst, PDI must deal with different mechanisms for directing disulfide formation. With some proteins, such as bovine pancreatic trypsin inhibitor, the specification of cysteine connectivity occurs early in folding and is directed by the formation of native-like structures that are interconverted by intramolecular disulfide rearrangements (3). With other proteins, such as ribonuclease A (RNase), the identification of which cysteines to connect occurs late in folding resulting in a large collection of intermediates with random disulfides and substantial non-native structure that must be rearranged to give native connectivity (4). PDI is capable of accelerating folding that proceeds by either of these mechanisms (5, 9).During the oxidative folding of reduced ribonuclease (RNase), PDI catalyzes the initial formation of substrate disulfides to yield a collection of inactive, RNase redox isomers that must be rearranged to the native disulfide pattern (6). PDI has two active sites that are housed in two, internally homologous thioredoxin domains, one near the amino terminus and the other near the carboxyl terminus (7). Both active sites contribute to PDI catalysis (8), but the two active sites are not equivalent (9). Each of the two active sites has two cysteine residues, found in the sequence WCGHCK (7). The two cysteines at each active site serve different functi...