Anna Kaplan

Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins

A hallmark of many neurodegenerative diseases is accumulation of misfolded proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving protein disulfide isomerase (PDI), a protein chaperone that catalyzes isomerization, reduction, and oxidation of disulfides. Through a small-molecule-screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated-ER-membranes and trigger apoptotic cell death, via mitochondrial outer membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon1 and Aβ peptides processed from the amyloid precursor protein. This pro-apoptotic function of PDI provides a new mechanism linking protein misfolding and apoptotic cell death.

Protein Folding Drives Disulfide Formation

PDI catalyzes the oxidative folding of disulfide-containing proteins. However, the sequence of reactions leading to a natively folded and oxidized protein remains unknown. Here we demonstrate a technique that enables independent measurements of disulfide formation and protein folding. We find that non-native disulfides are formed early in the folding pathway and can trigger misfolding. In contrast, a PDI domain favors native disulfides by catalyzing oxidation at a late stage of folding. We propose a model for cotranslational oxidative folding wherein PDI acts as a placeholder that is relieved by the pairing of cysteines caused by substrate folding. This general mechanism can explain how PDI catalyzes oxidative folding in a variety of structurally unrelated substrates.

Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis

Apoptosis is known as programmed cell death. Some non-apoptotic cell death is increasingly recognized as genetically controlled, or ‘regulated’. However, the full extent and diversity of these alternative cell death mechanisms remains uncharted. Here, we surveyed the landscape of pharmacologically-accessible cell death mechanisms. Of 56 caspase-independent lethal compounds, modulatory profiling revealed ten inducing three types of regulated non-apoptotic cell death. Lead optimization of one of the ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis occurs when the lipid repair enzyme GPX4 is inhibited. We found that FIN56 promotes degradation of GPX4. We performed chemoproteomics to reveal that FIN56 also binds to and activates squalene synthase, an enzyme involved in the cholesterol synthesis, in a manner independent of GPX4 degradation. These discoveries reveal that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes.

Therapeutic approaches to preventing cell death in Huntington disease

Neurodegenerative diseases affect the lives of millions of patients and their families. Due to the complexity of these diseases and our limited understanding of their pathogenesis, the design of therapeutic agents that can effectively treat these diseases has been challenging. Huntington disease (HD) is one of several neurological disorders with few therapeutic options. HD, like numerous other neurodegenerative diseases, involves extensive neuronal cell loss. One potential strategy to combat HD and other neurodegenerative disorders is to intervene in the execution of neuronal cell death. Inhibiting neuronal cell death pathways may slow the development of neurodegeneration. However, discovering small molecule inhibitors of neuronal cell death remains a significant challenge. Here, we review candidate therapeutic targets controlling cell death mechanisms that have been the focus of research in HD, as well as an emerging strategy that has been applied to developing small molecule inhibitors-fragment-based drug discovery (FBDD). FBDD has been successfully used in both industry and academia to identify selective and potent small molecule inhibitors, with a focus on challenging proteins that are not amenable to traditional high-throughput screening approaches. FBDD has been used to generate potent leads, pre-clinical candidates, and has led to the development of an FDA approved drug. This approach can be valuable for identifying modulators of cell-death-regulating proteins; such compounds may prove to be the key to halting the progression of HD and other neurodegenerative disorders.

Small molecule-induced oxidation of protein disulfide isomerase is neuroprotective

Significance Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum. It is up-regulated in mouse models of, and brains of patients with, neurological protein folding diseases. Irreversible inhibition of PDI activity by the small molecule 16F16 results in protection in cell and organotypic brain slice culture models of Huntington disease. Here, we identified lead optimized compound (LOC)14 as a nanomolar, reversible inhibitor of PDI that protects PC12 cells and medium spiny neurons from the toxic mutant huntingtin protein. LOC14 has improved potency compared with 16F16 and displays favorable pharmaceutical properties, making it a suitable compound to evaluate the therapeutic potential of inhibiting PDI in multiple disease models. Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum that is up-regulated in mouse models of, and brains of patients with, neurodegenerative diseases involving protein misfolding. PDI’s role in these diseases, however, is not fully understood. Here, we report the discovery of a reversible, neuroprotective lead optimized compound (LOC)14, that acts as a modulator of PDI. LOC14 was identified using a high-throughput screen of ∼10,000 lead-optimized compounds for potent rescue of viability of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a Kd of 62 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using 2D heteronuclear single quantum correlation NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site and to observe that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures. LOC14 exhibited high stability in mouse liver microsomes and blood plasma, low intrinsic microsome clearance, and low plasma-protein binding. These results suggest that LOC14 is a promising lead compound to evaluate the potential therapeutic effects of modulating PDI in animal models of disease.

Structural Elucidation of a Small Molecule Inhibitor of Protein Disulfide Isomerase

Compound libraries provide a starting point for multiple biological investigations, but the structural integrity of compounds is rarely assessed experimentally until a late stage in the research process. Here, we describe the discovery of a neuroprotective small molecule that was originally incorrectly annotated with a chemical structure. We elucidated the correct structure of the active compound using analytical chemistry, revealing it to be the natural product securinine. We show that securinine is protective in a cell model of Huntington disease and identify the binding site of securinine to its target, protein disulfide isomerase using NMR chemical shift perturbation studies. We show that securinine displays favorable pharmaceutical properties, making it a promising compound for in vivo studies in neurodegenerative disease models. In addition to finding this unexpected activity of securinine, this study provides a systematic roadmap to those who encounter compounds with incorrect structural annotation in the course of screening campaigns.

Voices from the Peace Corps: Fifty Years of Kentucky Volunteers

As reflected in its cover photograph of a Peace Corps volunteer (PCV) walking hand-in-hand with a young Malawi girl named Memory, Voices from the Peace Corps: Fifty Years of Kentucky Volunteers centers on oral histories. It offers no oral history theory or analysis of the construction of the narratives but uses the stories to illuminate both the enjoyable and the unpleasant aspects of the PCV experience. When long sections of the interviews are quoted, the excerpts are printed in a different font from the rest of the text to highlight their status as personal narrative. Shorter quotations remain in the same typeface as the rest of the sentence or paragraph. However, the authors do not include any indication (ellipses, brackets) of the editing that polished the quotations or any pauses or filler words from the original interviews.

Unraveling the Mechanisms of Oxidative Folding using Single Molecule Force Spectroscopy

Disulfide bonds are formed as posttranslational modifications in a third of human proteins. The biological significance of disulfide formation is further underscored by its critical importance in numerous pathological processes including bacterial infection, viral assembly and protein misfolding disease. In eukaryotic cells, protein synthesis takes place in the cytosol where thioredoxin (TRX) prevents the formation of disulfides. Oxidative folding occurs mainly in the endoplasmic reticulum (ER) and is catalyzed by the thioredoxin-like oxidase Protein Disulfide Isomerase (PDI). Previous studies have suggested the engagement of PDI with unfolded substrates during ongoing ER translocation. However, the precise involvement of PDI during protein folding has remained elusive. Here we present a kinetic model for PDI activity during catalyzed oxidative folding. We introduce a method enabling, for the first time, independent kinetic measurements of folding and disulfide formation in a single protein substrate. Direct manipulation of the substrate enables initiation of oxidative folding from a well-defined extended state resembling the in vivo scenario. Our data indicate that structural folding is rate-limiting during catalyzed disulfide formation. Resolution of an intermediate enzyme-substrate complex appears necessary for folding to complete. Based on these observations, we propose the spontaneous rate of enzyme release as determinant of catalytic activity. We validate this hypothesis by showing that replacement of a single atom in TRX enables catalysis of disulfide formation, with PDI-like efficiency. Our findings reveal a universal catalytic mechanism common to disulfide reductases and oxidases, and can explain the functional diversification of a ubiquitous family of enzymes. Furthermore, the method presented here opens the possibility for highly specific functional screens for inhibitors of oxidative folding.