Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.

Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic ß cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration.

Harmonizing Experimental Data with Modeling to Predict Membrane Protein Insertion in Yeast.

Membrane proteins must adopt their proper topologies within biological membranes, but achieving the correct topology is compromised by the presence of marginally hydrophobic transmembrane helices (TMHs). In this study, we report on a new model membrane protein in yeast that harbors two TMHs fused to an unstable nucleotide-binding domain. Because the second helix (TMH2) in this reporter has an unfavorable predicted free energy of insertion, we employed established methods to generate variants that alter TMH2 insertion free energy. We first found that altering TMH2 did not significantly affect the extent of protein degradation by the cellular quality control machinery. Next, we correlated predicted insertion free energies from a knowledge-based energy scale with the measured apparent free energies of TMH2 insertion. Although the predicted and apparent insertion energies showed a similar trend, the predicted free-energy changes spanned an unanticipated narrow range. By instead using a physics-based model, we obtained a broader range of free energies that agreed considerably better with the magnitude of the experimentally derived values. Nevertheless, some variants still inserted better in yeast than predicted from energy-based scales. Therefore, molecular dynamics simulations were performed and indicated that the corresponding mutations induced conformational changes within TMH2, which altered the number of stabilizing hydrogen bonds. Together, our results offer insight into the ability of the cellular quality control machinery to recognize conformationally distinct misfolded topomers, provide a model to assess TMH insertion in vivo, and indicate that TMH insertion energy scales may be limited depending on the specific protein and the mutation present.

A shift to organismal stress resistance in programmed cell death mutants.

Animals have many ways of protecting themselves against stress; for example, they can induce animal-wide, stress-protective pathways and they can kill damaged cells via apoptosis. We have discovered an unexpected regulatory relationship between these two types of stress responses. We find that C. elegans mutations blocking the normal course of programmed cell death and clearance confer animal-wide resistance to a specific set of environmental stressors; namely, ER, heat and osmotic stress. Remarkably, this pattern of stress resistance is induced by mutations that affect cell death in different ways, including ced-3 (cell death defective) mutations, which block programmed cell death, ced-1 and ced-2 mutations, which prevent the engulfment of dying cells, and progranulin (pgrn-1) mutations, which accelerate the clearance of apoptotic cells. Stress resistance conferred by ced and pgrn-1 mutations is not additive and these mutants share altered patterns of gene expression, suggesting that they may act within the same pathway to achieve stress resistance. Together, our findings demonstrate that programmed cell death effectors influence the degree to which C. elegans tolerates environmental stress. While the mechanism is not entirely clear, it is intriguing that animals lacking the ability to efficiently and correctly remove dying cells should switch to a more global animal-wide system of stress resistance.

Hsp70 targets a cytoplasmic quality control substrate to the San1p ubiquitin ligase.

Accumulation of misfolded proteins in cellular compartments can result in stress-induced cell death. In the endoplasmic reticulum (ER), ER-associated degradation clears aberrant proteins from the secretory pathway. In the cytoplasm and nucleus, this job is left to the cytoplasmic quality control (CytoQC) machinery. Both processes utilize chaperones and the ubiquitin-proteasome system to aid in protein elimination. Previous studies in yeast have drawn comparisons between these processes using data from structurally and topologically different substrates. We sought to draw a direct comparison between ERAD and CytoQC by studying the elimination of a single misfolded domain that, depending on its residence, is disposed by either of these pathways. The truncated, second nucleotide binding domain (NBD2*) from a yeast ERAD substrate, Ste6p*, resides at the cytoplasmic face of the ER. We show that a soluble form of NBD2* is cytoplasmic and unlike wild-type NBD2 is targeted for proteasome-mediated degradation. In contrast to Ste6p*, which employs the ER-localized Doa10p ubiquitin ligase, NBD2* is ubiquitinated by a nuclear E3 ligase San1p, a factor that is also required for its degradation. Although the yeast cytoplasmic Hsp70 chaperone, Ssa1p, has been thought to facilitate the nuclear import or to maintain the solubility of most CytoQC substrates, we discovered that Ssa1p facilitates the interaction between San1p and NBD2*, demonstrating that chaperones can aid in substrate recognition and San1p-dependent protein degradation. These results emphasize the diverse action of molecular chaperones during CytoQC.

Transmembrane helix hydrophobicity is an energetic barrier during the retrotranslocation of integral membrane ERAD substrates.

Integral membrane proteins fold inefficiently and are susceptible to turnover via the endoplasmic reticulum-associated degradation (ERAD) pathway. During ERAD, misfolded proteins are recognized by molecular chaperones, polyubiquitinated, and retrotranslocated to the cytoplasm for proteasomal degradation. Although many aspects of this pathway are defined, how transmembrane helices (TMHs) are removed from the membrane and into the cytoplasm before degradation is poorly understood. In this study, we asked whether the hydrophobic character of a TMH acts as an energetic barrier to retrotranslocation. To this end, we designed a dual-pass model ERAD substrate, Chimera A*, which contains the cytoplasmic misfolded domain from a characterized ERAD substrate, Sterile 6* (Ste6p*). We found that the degradation requirements for Chimera A* and Ste6p* are similar, but Chimera A* was retrotranslocated more efficiently than Ste6p* in an in vitro assay in which retrotranslocation can be quantified. We then constructed a series of Chimera A* variants containing synthetic TMHs with a range of ΔG values for membrane insertion. TMH hydrophobicity correlated inversely with retrotranslocation efficiency, and in all cases, retrotranslocation remained Cdc48p dependent. These findings provide insight into the energetic restrictions on the retrotranslocation reaction, as well as a new computational approach to predict retrotranslocation efficiency.

Serine 421 regulates mutant huntingtin toxicity and clearance in mice.

Huntington's disease (HD) is a progressive, adult-onset neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the N-terminal region of the protein huntingtin (HTT). There are no cures or disease-modifying therapies for HD. HTT has a highly conserved Akt phosphorylation site at serine 421, and prior work in HD models found that phosphorylation at S421 (S421-P) diminishes the toxicity of mutant HTT (mHTT) fragments in neuronal cultures. However, whether S421-P affects the toxicity of mHTT in vivo remains unknown. In this work, we used murine models to investigate the role of S421-P in HTT-induced neurodegeneration. Specifically, we mutated the human mHTT gene within a BAC to express either an aspartic acid or an alanine at position 421, mimicking tonic phosphorylation (mHTT-S421D mice) or preventing phosphorylation (mHTT-S421A mice), respectively. Mimicking HTT phosphorylation strongly ameliorated mHTT-induced behavioral dysfunction and striatal neurodegeneration, whereas neuronal dysfunction persisted when S421 phosphorylation was blocked. We found that S421 phosphorylation mitigates neurodegeneration by increasing proteasome-dependent turnover of mHTT and reducing the presence of a toxic mHTT conformer. These data indicate that S421 is a potent modifier of mHTT toxicity and offer in vivo validation for S421 as a therapeutic target in HD.