Interplay between conformational selection and zymogen activation.

Trypsin-like proteases are synthesized as zymogens and activated through a mechanism that folds the active site for efficient binding and catalysis. Ligand binding to the active site is therefore a valuable source of information on the changes that accompany zymogen activation. Using the physiologically relevant transition of the clotting zymogen prothrombin to the mature protease thrombin, we show that the mechanism of ligand recognition follows selection within a pre-existing ensemble of conformations with the active site accessible (E) or inaccessible (E*) to binding.

Induced Fit Is a Special Case of Conformational Selection.

Conformational selection (CS) and induced fit (IF) are two widely used interpretations of binding of a ligand to biological macromolecules. Both mechanisms envision a two-step reaction in which a conformational transition either precedes (CS) or follows (IF) the binding step. Under pseudo-first-order conditions where the ligand is in excess compared to the macromolecule, both mechanisms produce two relaxations.

Kinetic dissection of the pre-existing conformational equilibrium in the trypsin fold.

Structural biology has recently documented the conformational plasticity of the trypsin fold for both the protease and zymogen in terms of a pre-existing equilibrium between closed (E*) and open (E) forms of the active site region. How such plasticity is manifested in solution and affects ligand recognition by the protease and zymogen is poorly understood in quantitative terms. Here we dissect the E*-E equilibrium with stopped-flow kinetics in the presence of excess ligand or macromolecule.

Immunoproteasome Activation During Early Antiviral Response in Mouse Pancreatic β-cells: New Insights into Auto-antigen Generation in Type I Diabetes?

Type 1 diabetes results from autoimmune destruction of the insulin producing pancreatic β-cells. The immunoproteasome, a version of the proteasome that collaborates with the 11S/PA28 activator to generate immunogenic peptides for presentation by MHC class I molecules, has long been implicated in the onset of the disease, but little is known about immunoproteasome function and regulation in pancreatic β-cells. Interesting insight into these issues comes from a recent analysis of the immunoproteasome expressed in pancreatic β-cells during early antiviral defenses mediated by interferon β (IFNβ), a type I IFN implicated in the induction of the diabetic state in human and animal models.

Reduction in ATP levels triggers immunoproteasome activation by the 11S (PA28) regulator during early antiviral response mediated by IFNβ in mouse pancreatic β-cells.

Autoimmune destruction of insulin producing pancreatic β-cells is the hallmark of type I diabetes. One of the key molecules implicated in the disease onset is the immunoproteasome, a protease with multiple proteolytic sites that collaborates with the constitutive 19S and the inducible 11S (PA28) activators to produce immunogenic peptides for presentation by MHC class I molecules. Despite its importance, little is known about the function and regulation of the immunoproteasome in pancreatic β-cells.