Huntingtin Inclusions Trigger Cellular Quiescence, Deactivate Apoptosis, and Lead to Delayed Necrosis.

Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.

Interactions outside the proteinase-binding loop contribute significantly to the inhibition of activated coagulation factor XII by its canonical inhibitor from corn.

Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (Ki = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a Ki of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20-45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (Ki = 11.7 ± 1.2 µm) and activated factor XI (Ki = 94 ± 11 µm). Full-length CHFI inhibited trypsin with a Ki of 1.3 ± 0.2 nm and activated factor XI with a Ki of 5.4 ± 0.2 µm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.