Cutaneous biomarkers of therapeutic efficacy in early treatment of hereditary ATTR amyloid polyneuropathy with tafamidis.

BACKGROUND: ATTR (ATTRv) amyloidosis neuropathy is characterized by progressive sensorimotor and autonomic nerve degeneration secondary to amyloid deposition caused by a misfolded transthyretin protein (TTR). Small nerve fiber neuropathy is an early clinical manifestation of this disease resulting from the dysfunction of the Aδ and C small nerve fibers. Tafamidis, a selective TTR stabilizer, has proven its efficacy in the earlier stages of hATTR. OBJECTIVES: To evaluate the clinical course and utility of cutaneous pathological biomarkers in patients with ATTR amyloidosis treated with tafamidis compared to control patients. METHODS: Forty patients diagnosed with early stages of ATTRv amyloidosis (polyneuropathy disability [PND] scores 0-II) underwent small and large nerve fiber neurological evaluations, and annual skin biopsies for intraepidermal nerve fiber density (IENFD) and amyloid deposition index (ADI) estimation. Thirty patients were allocated to receive tafamidis, and 10 patients served as controls. Tafamidis pharmacokinetics analysis was performed in patients who received the treatment. RESULTS: At baseline, 12% of patients in stage PND 0 and 28% in PND I displayed small nerve fiber denervation in the distal thigh, whereas 23% and 38%, respectively, in the distal leg. Similarly, 72% and 84% had amyloid deposition in the distal thigh and 56% and 69% in the distal leg. Following 1 year of treatment, the tafamidis group showed significant clinical improvement compared to the control group, revealed by the following mean differences (1) -9.3 versus -4 points (p = <.00) in the patient's neuropathy total symptom score 6 (NTSS-6) questionnaire, (2) -2.5 versus +2.8 points (p = <.00) in the Utah Early Neuropathy Score (UENS), and (3) +1.2°C versus -0.6 (p = .01) in cold detection thresholds. Among the patients who received tafamidis, 65% had stable or increased IENFD in their distal thigh and 27% in the distal leg. In contrast, all patients in the control group underwent denervation. The ADI either decreased or remained constant in 31% of the biopsies in the distal thigh and in 24% of the biopsies in the distal leg of the tafamidis-treated patients, whereas it rose across all the biopsies in the control group. At the 4-year follow-up, the tafamidis group continued to display less denervation in the distal thigh (mean difference [MD] of -3.0 vs. -9.3 fibers/mm) and the distal leg (mean difference [MD] -4.9 vs. -8.6 fibers/mm). ADI in tafamidis-treated patients was also lower in the distal thigh (10 vs. 30 amyloid/mm2) and the distal leg (23 vs. 40 amyloid/mm2) compared to control patients. Plasma tafamidis concentrations were higher in patients with IENFD improvement and in patients with reduced amyloid deposition. Patients without amyloid deposition in the distal leg at baseline displayed delayed disease progression at 4 years. CONCLUSIONS: Cutaneous IENFD and amyloid deposition assessments in the skin of the distal thigh and distal leg are valuable biomarkers for early diagnosis of ATTR amyloidosis and for measuring the progression of small nerve fiber neuropathy. Early treatment with tafamidis slows the clinical progression of the disease, skin denervation, and amyloid deposition in the skin. Higher plasma concentrations of tafamidis are associated with better disease outcomes, suggesting that increasing the drug dose could achieve better plasma concentrations and response rates. This study describes the longest small nerve fiber neuropathy therapeutic trial with tafamidis and is the first to report small fiber symptoms, function, and structural assessments as outcomes.

Activation of XBP1s attenuates disease severity in models of proteotoxic Charcot-Marie-Tooth type 1B.

Mutations in myelin protein zero (MPZ) are generally associated with Charcot-Marie-Tooth type 1B (CMT1B) disease, one of the most common forms of demyelinating neuropathy. Pathogenesis of some MPZ mutants, such as S63del and R98C, involves the misfolding and retention of MPZ in the endoplasmic reticulum (ER) of myelinating Schwann cells. To cope with proteotoxic ER-stress, Schwann cells mount an unfolded protein response (UPR) characterized by activation of the PERK, ATF6 and IRE1α/XBP1 pathways. Previous results showed that targeting the PERK UPR pathway mitigates neuropathy in mouse models of CMT1B; however, the contributions of other UPR pathways in disease pathogenesis remains poorly understood. Here, we probe the importance of the IRE1α/XBP1 signalling during normal myelination and in CMT1B. In response to ER stress, IRE1α is activated to stimulate the non-canonical splicing of Xbp1 mRNA to generate spliced Xbp1 (Xbp1s). This results in the increased expression of the adaptive transcription factor XBP1s, which regulates the expression of genes involved in diverse pathways including ER proteostasis. We generated mouse models where Xbp1 is deleted specifically in Schwann cells, preventing XBP1s activation in these cells. We observed that Xbp1 is dispensable for normal developmental myelination, myelin maintenance and remyelination after injury. However, Xbp1 deletion dramatically worsens the hypomyelination and the electrophysiological and locomotor parameters observed in young and adult CMT1B neuropathic animals. RNAseq analysis suggested that XBP1s exerts its adaptive function in CMT1B mouse models in large part via the induction of ER proteostasis genes. Accordingly, the exacerbation of the neuropathy in Xbp1 deficient mice was accompanied by upregulation of ER-stress pathways and of IRE1-mediated RIDD signaling in Schwann cells, suggesting that the activation of XBP1s via IRE1 plays a critical role in limiting mutant protein toxicity and that this toxicity cannot be compensated by other stress responses. Schwann cell specific overexpression of XBP1s partially re-established Schwann cell proteostasis and attenuated CMT1B severity in both the S63del and R98C mouse models. In addition, the selective, pharmacologic activation of IRE1α/XBP1 signaling ameliorated myelination in S63del dorsal root ganglia explants. Collectively, these data show that XBP1 has an essential adaptive role in different models of proteotoxic CMT1B neuropathy and suggest that activation of the IRE1α/XBP1 pathway may represent a therapeutic avenue in CMT1B and possibly for other neuropathies characterized by UPR activation.

Biochemistry and Protein Interactions of the CYREN Microprotein.

Over the past decade, advances in genomics have identified thousands of additional protein-coding small open reading frames (smORFs) missed by traditional gene finding approaches. These smORFs encode peptides and small proteins, commonly termed micropeptides or microproteins. Several of these newly discovered microproteins have biological functions and operate through interactions with proteins and protein complexes within the cell. CYREN1 is a characterized microprotein that regulates double-strand break repair in mammalian cells through interaction with Ku70/80 heterodimer. Ku70/80 binds to and stabilizes double-strand breaks and recruits the machinery needed for nonhomologous end join repair. In this study, we examined the biochemical properties of CYREN1 to better understand and explain its cellular protein interactions. Our findings support that CYREN1 is an intrinsically disordered microprotein and this disordered structure allows it to enriches several proteins, including a newly discovered interaction with SF3B1 via a distinct short linear motif (SLiMs) on CYREN1. Since many microproteins are predicted to be disordered, CYREN1 is an exemplar of how microproteins interact with other proteins and reveals an unknown scaffolding function of this microprotein that may link NHEJ and splicing.

Divergent Proteome Reactivity Influences Arm-Selective Activation of the Unfolded Protein Response by Pharmacological Endoplasmic Reticulum Proteostasis Regulators.

Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the unfolded protein response (UPR) has proven useful for ameliorating proteostasis deficiencies in cellular and mouse models of numerous etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino-p-cresol substructure affording a quinone methide, which then covalently modifies a subset of endoplasmic reticulum (ER) protein disulfide isomerases (PDIs). Another compound identified in this screen, AA132, also contains a 2-amino-p-cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent modification of proteins including multiple PDIs. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. However, the extent of PDI labeling by AA147 approaches a plateau more rapidly than PDI labeling by AA132. These observations together suggest that AA132 can access a larger pool of proteins for covalent modification, possibly because its activated form is less susceptible to quenching than activated AA147. In other words, the lower reactivity of activated AA132 allows it to persist longer and modify more PDIs in the cellular environment. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and enables selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

Pharmacological stabilization of the native state of full-length immunoglobulin light chains to treat light chain amyloidosis.

Immunoglobulin light chain amyloidosis (AL) is a cancer of plasma cells that secrete unstable full-length immunoglobulin light chains. These light chains misfold and aggregate, often with aberrant endoproteolysis, leading to organ toxicity. AL is currently treated by pharmacological elimination of the clonal plasma cells. Since it remains difficult to completely kill these cells in the majority of patients, we seek a complementary drug that inhibits light chain aggregation, which should diminish organ toxicity. We discovered a small-molecule binding site on full-length immunoglobulin light chains by structurally characterizing hit stabilizers emerging from a high-throughput screen seeking small molecules that protect full-length light chains from conformational excursion-linked endoproteolysis. The x-ray crystallographic characterization of 7 structurally distinct hit native-state stabilizers provided a structure-based blueprint, reviewed herein, to design more potent stabilizers. This approach enabled us to transform hits with micromolar affinity into stabilizers with nanomolar dissociation constants that potently prevent light chain aggregation.

PERK signaling promotes mitochondrial elongation by remodeling membrane phosphatidic acid.

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are linked in the onset and pathogenesis of numerous diseases. This has led to considerable interest in defining the mechanisms responsible for regulating mitochondria during ER stress. The PERK signaling arm of the unfolded protein response (UPR) has emerged as a prominent ER stress-responsive signaling pathway that regulates diverse aspects of mitochondrial biology. Here, we show that PERK activity promotes adaptive remodeling of mitochondrial membrane phosphatidic acid (PA) to induce protective mitochondrial elongation during acute ER stress. We find that PERK activity is required for ER stress-dependent increases in both cellular PA and YME1L-dependent degradation of the intramitochondrial PA transporter PRELID1. These two processes lead to the accumulation of PA on the outer mitochondrial membrane where it can induce mitochondrial elongation by inhibiting mitochondrial fission. Our results establish a new role for PERK in the adaptive remodeling of mitochondrial phospholipids and demonstrate that PERK-dependent PA regulation adapts organellar shape in response to ER stress.

Design and Rationale the SCAN-MP (Screening for Cardiac Amyloidosis With Nuclear Imaging in Minority Populations) Study.

Background Transthyretin amyloid cardiomyopathy (ATTR-CM) is an important cause of heart failure in older individuals. Misfolding and deposition of transthyretin or prealbumin protein causes ATTR-CM in the context of a normal (wild-type) or variant TTR sequence. Variant ATTR-CM is most commonly caused by the substitution of valine for isoleucine at position 122 in transthyretin (Val122Ile or pV142I, almost exclusively observed in individuals of West African ancestry), demonstrated in 3.4% of self-identified Black individuals in the United States with an estimated 1.5 million carriers. Despite the large number of known pV142I carriers, the proportion of older Black patients with heart failure attributable to ATTR-CM remains unknown. Methods To address this knowledge gap, the SCAN-MP (Screening for Cardiac Amyloidosis with Nuclear Imaging in Minority Populations) study was funded by the National Institutes of Health/National Heart, Lung, and Blood Institute (R01HL139671) to enroll a targeted population of self-identified, community-dwelling Black or Caribbean Hispanic patients (many of whom are of West African ancestry) >60 years of age with heart failure and identify ATTR-CM by noninvasive nuclear imaging. The principal objective of SCAN-MP is to determine the prevalence of ATTR-CM in this population. Secondary objectives will explore TTR genotype, demographics, progression of variant versus wild-type ATTR-CM, and biochemical mechanisms of transthyretin amyloid fibril formation. Conclusions The SCAN-MP study is the largest, prospective study of cardiac amyloidosis in Black and Hispanic individuals. Both wild-type and variant ATTR-CM are now treatable with the US Food and Drug-approved drug tafamidis. The insights gained from SCAN-MP are likely to improve those at risk for or afflicted with ATTR-CM. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03812172.

Proteostasis and lysosomal quality control deficits in Alzheimer's disease neurons.

The role of proteostasis and organelle homeostasis dysfunction in human aging and Alzheimer's disease (AD) remains unclear. Analyzing proteome-wide changes in human donor fibroblasts and their corresponding transdifferentiated neurons (tNeurons), we find aging and AD synergistically impair multiple proteostasis pathways, most notably lysosomal quality control (LQC). In particular, we show that ESCRT-mediated lysosomal repair defects are associated with both sporadic and PSEN1 familial AD. Aging- and AD-linked defects are detected in fibroblasts but highly exacerbated in tNeurons, leading to enhanced neuronal vulnerability, unrepaired lysosomal damage, inflammatory factor secretion and cytotoxicity. Surprisingly, tNeurons from aged and AD donors spontaneously develop amyloid-ß inclusions co-localizing with LQC markers, LAMP1/2-positive lysosomes and proteostasis factors; we observe similar inclusions in brain tissue from AD patients and APP-transgenic mice. Importantly, compounds enhancing lysosomal function broadly ameliorate these AD-associated pathologies. Our findings establish cell-autonomous LQC dysfunction in neurons as a central vulnerability in aging and AD pathogenesis.

A Comprehensive Enumeration of the Human Proteostasis Network. 2. Components of the Autophagy-Lysosome Pathway.

The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Establishing and maintaining proteostasis is the province of the proteostasis network, approximately 2,700 components that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental entity in biology that is essential for cellular health and has direct relevance to many diseases of protein conformation. However, it is not well defined or annotated, which hinders its functional characterization in health and disease. In this series of manuscripts, we aim to operationally define the human proteostasis network by providing a comprehensive, annotated list of its components. We provided in a previous manuscript a list of chaperones and folding enzymes as well as the components that make up the machineries for protein synthesis, protein trafficking into and out of organelles, and organelle-specific degradation pathways. Here, we provide a curated list of 838 unique high-confidence components of the autophagy-lysosome pathway, one of the two major protein degradation systems in human cells.

Tafamidis concentration required for transthyretin stabilisation in cerebrospinal fluid.

BACKGROUND: Hereditary transthyretin (TTR) amyloidosis (ATTRv) initially presents as a polyneuropathy and/or a cardiomyopathy. Central nervous system (CNS) pathology in ATTRv amyloidosis, including focal neurological episodes, dementia, cerebrovascular bleeding, and seizures, appears around a decade later. Wild-type (WT) TTR amyloidosis (ATTRwt) causes a cardiomyopathy. CNS pathology risk likely also increases in these patients as cardiomyopathy progresses. Herein, we study tafamidis-mediated TTR kinetic stabilisation in cerebrospinal fluid (CSF). METHODS: Varying tafamidis concentrations (50-1000 nM) were added to CSF from healthy donors or ATTRv patients, and TTR stabilisation was measured via the decrease in dissociation rate. RESULTS: Tafamidis meglumine (Vyndaqel) can be dosed at 20 or 80 mg QD. The latter dose is bioequivalent to a 61 mg QD dose of tafamidis free acid (Vyndamax). The tafamidis CSF concentration in ATTRv patients on 20 mg Vyndaqel is ∼125 nM. By linear extrapolation, we expect a CSF concentration of ∼500 nM at the higher dose. When tafamidis is added to healthy donor CSF at 125 or 500 nM, the WT TTR dissociation rate decreases by 42% or 87%, respectively. CONCLUSIONS: Tafamidis stabilises TTR in CSF to what is likely a clinically meaningful extent at CSF concentrations achieved by the normal tafamidis dosing regimen.

Capturing the conversion of the pathogenic alpha-1-antitrypsin fold by ATF6 enhanced proteostasis.

Genetic variation in alpha-1 antitrypsin (AAT) causes AAT deficiency (AATD) through liver aggregation-associated gain-of-toxic pathology and/or insufficient AAT activity in the lung manifesting as chronic obstructive pulmonary disease (COPD). Here, we utilize 71 AATD-associated variants as input through Gaussian process (GP)-based machine learning to study the correction of AAT folding and function at a residue-by-residue level by pharmacological activation of the ATF6 arm of the unfolded protein response (UPR). We show that ATF6 activators increase AAT neutrophil elastase (NE) inhibitory activity, while reducing polymer accumulation for the majority of AATD variants, including the prominent Z variant. GP-based profiling of the residue-by-residue response to ATF6 activators captures an unexpected role of the "gate" area in managing AAT-specific activity. Our work establishes a new spatial covariant (SCV) understanding of the convertible state of the protein fold in response to genetic perturbation and active environmental management by proteostasis enhancement for precision medicine.

Divergent Proteome Reactivity Influences Arm-Selective Activation of Pharmacological Endoplasmic Reticulum Proteostasis Regulators.

Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the Unfolded Protein Response (UPR) has proven useful for ameliorating proteostasis deficiencies in a variety of etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino- p -cresol substructure affording a quinone methide, which then covalently modifies a subset of ER protein disulfide isomerases (PDIs). Intriguingly, another compound identified in this screen, AA132, also contains a 2-amino- p -cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent PDI modification. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. Paradoxically, activated AA132 reacts slower with PDIs, indicating it is less reactive than activated AA147. This suggests that the higher labeling of PDIs observed with activated AA132 can be attributed to its lower reactivity, which allows this activated compound to persist longer in the cellular environment prior to quenching by endogenous nucleophiles. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and allows for selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

Characterising diflunisal as a transthyretin kinetic stabilizer at relevant concentrations in human plasma using subunit exchange.

Transthyretin (TTR) dissociation is the rate limiting step for both aggregation and subunit exchange. Kinetic stabilisers, small molecules that bind to the native tetrameric structure of TTR, slow TTR dissociation and inhibit aggregation. One such stabiliser is the non-steroidal anti-inflammatory drug (NSAID), diflunisal, which has been repurposed to treat TTR polyneuropathy. Previously, we compared the efficacy of diflunisal, tafamidis, tolcapone, and AG10 as kinetic stabilisers for transthyretin. However, we could not meaningfully compare diflunisal because we were unsure of its plasma concentration after long-term oral dosing. Herein, we report the diflunisal plasma concentrations measured by extraction, reversed phase HPLC separation, and fluorescence detection after long-term 250 mg BID oral dosing in two groups: a placebo-controlled diflunisal clinical trial group and an open-label Japanese polyneuropathy treatment cohort. The measured mean diflunisal plasma concentration from both groups was 282.2 µM ± 143.7 µM (mean ± standard deviation). Thus, quantification of TTR kinetic stabilisation using subunit exchange was carried out at 100, 200, 300, and 400 µM diflunisal concentrations, all observed in patients after 250 mg BID oral dosing. A 250 µM diflunisal plasma concentration reduced the wild-type TTR dissociation rate in plasma by 95%, which is sufficient to stop transthyretin aggregation, consistent with the clinical efficacy of diflunisal for ameliorating transthyretin polyneuropathy.

Tafamidis polyneuropathy amelioration requires modest increases in transthyretin stability even though increases in plasma native TTR and decreases in non-native TTR do not predict response.

BACKGROUND: TTR aggregation causes hereditary transthyretin (TTR) polyneuropathy (ATTRv-PN) in individuals with destabilised TTR variants. ATTRv-PN can be treated with ligands that bind TTR and prevent aggregation. One such ligand, tafamidis, is widely approved to treat ATTRv-PN. We explore how TTR stabilisation markers relate to clinical efficacy in 210 ATTRv-PN patients taking tafamidis. METHODS: TTR concentration in patient plasma was measured before and after tafamidis treatment using assays for native or combined native + non-native TTR. TTR tetramer dissociation kinetics, which are slowed by tafamidis binding, were also measured. RESULTS: Native TTR levels increased by 56.8% while combined native + non-native TTR levels increased by 3.1% after 24 months of tafamidis treatment, implying that non-native TTR decreased. Accordingly, the fraction of native TTR increased from 0.54 to 0.71 with tafamidis administration. Changes in native and non-native TTR levels were uncorrelated with clinical response to tafamidis. TTR tetramer dissociation generally slowed to an extent consistent with ∼40% of TTR being tafamidis-bound. Male non-responders had a lower extent of binding. CONCLUSIONS: Native and non-native TTR concentration changes cannot be used as surrogate measures for therapeutic efficacy. Also, successful tafamidis therapy requires only moderate TTR stabilisation. Male patients may benefit from higher tafamidis doses.

Analysis of lumbar spine stenosis specimens for identification of amyloid.

BACKGROUND: Lumbar spinal stenosis (LSS) is a common reason for spine surgery in which ligamentum flavum is resected. Transthyretin (TTR) amyloid is an often unrecognized and potentially modifiable mechanism for LSS that can also cause TTR cardiac amyloidosis. Accordingly, older adult patients undergoing lumbar spine (LS) surgery were evaluated for amyloid and if present, the precursor protein, as well as comprehensive characterization of the clinical phenotype. METHODS: A prospective, cohort study in 2 academic medical centers enrolled 47 subjects (age 69 ± 7 years, 53% male) undergoing clinically indicated LS decompression. The presence of amyloid was evaluated by Congo Red staining and in those with amyloid, precursor protein was determined by laser capture microdissection coupled to mass spectrometry (LCM-MS). The phenotype was assessed by disease-specific questionnaires (Swiss Spinal Stenosis Questionnaire and Kansas City Cardiomyopathy Questionnaire) and the 36-question short-form health survey, as well as biochemical measures (TTR, retinol-binding protein, and TTR stability). Cardiac testing included technetium-99m-pyrophosphate scintigraphy, electrocardiograms, echocardiograms, and cardiac biomarkers as well as measures of functional capacity. RESULTS: Amyloid was detected in 16 samples (34% of participants) and was more common in those aged ≥ 75 years of age (66.7%) compared with those <75 years (22.3%, p < 0.05). LCM-MS demonstrated TTR as the precursor protein in 62.5% of participants with amyloid while 37.5% had an indeterminant type of amyloid. Demographic, clinical, quality-of-life measures, electrocardiographic, echocardiographic, and biochemical measures did not differ between those with and without amyloid. Among those with TTR amyloid (n = 10), one subject had cardiac involvement by scintigraphy. CONCLUSIONS: Amyloid is detected in more than a third of older adults undergoing LSS. Amyloid is more common with advancing age and is particularly common in those >75 years old. No demographic, clinical, biochemical, or cardiac parameter distinguished those with and without amyloid. In more than half of subjects with LS amyloid, the precursor protein was TTR indicating the importance of pathological assessment.

ATF6 Activation Reduces Amyloidogenic Transthyretin Secretion through Increased Interactions with Endoplasmic Reticulum Proteostasis Factors.

The extracellular aggregation of destabilized transthyretin (TTR) variants is implicated in the onset and pathogenesis of familial TTR-related amyloid diseases. One strategy to reduce the toxic, extracellular aggregation of TTR is to decrease the population of aggregation-prone proteins secreted from mammalian cells. The stress-independent activation of the unfolded protein response (UPR)-associated transcription factor ATF6 preferentially decreases the secretion and subsequent aggregation of destabilized, aggregation-prone TTR variants. However, the mechanism of this reduced secretion was previously undefined. Here, we implement a mass-spectrometry-based interactomics approach to identify endoplasmic reticulum (ER) proteostasis factors involved in ATF6-dependent reductions in destabilized TTR secretion. We show that ATF6 activation reduces amyloidogenic TTR secretion and subsequent aggregation through a mechanism involving ER retention that is mediated by increased interactions with ATF6-regulated ER proteostasis factors including BiP and PDIA4. Intriguingly, the PDIA4-dependent retention of TTR is independent of both the single TTR cysteine residue and the redox activity of PDIA4, indicating that PDIA4 retains destabilized TTR in the ER through a redox-independent mechanism. Our results define a mechanistic basis to explain the ATF6 activation-dependent reduction in destabilized, amyloidogenic TTR secretion that could be therapeutically accessed to improve treatments of TTR-related amyloid diseases.

Pharmacological activation of ATF6 remodels the proteostasis network to rescue pathogenic GABAA receptors.

BACKGROUND: Genetic variants in the subunits of the gamma-aminobutyric acid type A (GABAA) receptors are implicated in the onset of multiple pathologic conditions including genetic epilepsy. Previous work showed that pathogenic GABAA subunits promote misfolding and inefficient assembly of the GABAA receptors, limiting receptor expression and activity at the plasma membrane. However, GABAA receptors containing variant subunits can retain activity, indicating that enhancing the folding, assembly, and trafficking of these variant receptors offers a potential opportunity to mitigate pathology associated with genetic epilepsy. RESULTS: Here, we demonstrate that pharmacologically enhancing endoplasmic reticulum (ER) proteostasis using small molecule activators of the ATF6 (Activating Transcription Factor 6) signaling arm of the unfolded protein response (UPR) increases the assembly, trafficking, and surface expression of variant GABAA receptors. These improvements are attributed to ATF6-dependent remodeling of the ER proteostasis environment, which increases protein levels of pro-folding ER proteostasis factors including the ER chaperone BiP (Immunoglobulin Binding Protein) and trafficking receptors, such as LMAN1 (Lectin Mannose-Binding 1) and enhances their interactions with GABAA receptors. Importantly, we further show that pharmacologic ATF6 activators increase the activity of GABAA receptors at the cell surface, revealing the potential for this strategy to restore receptor activity to levels that could mitigate disease pathogenesis. CONCLUSIONS: These results indicate that pharmacologic ATF6 activators offer an opportunity to restore GABAA receptor activity in diseases including genetic epilepsy and point to the potential for similar pharmacologic enhancement of ER proteostasis to improve trafficking of other disease-associated variant ion channels implicated in etiologically-diverse diseases.

Amyloidogenic immunoglobulin light chain kinetic stabilizers comprising a simple urea linker module reveal a novel binding sub-site.

In immunoglobulin light chain (LC) amyloidosis, the misfolding, or misfolding and misassembly of LC a protein or fragments thereof resulting from aberrant endoproteolysis, causes organ damage to patients. A small molecule "kinetic stabilizer" drug could slow or stop these processes and improve prognosis. We previously identified coumarin-based kinetic stabilizers of LCs that can be divided into four components, including a "linker module" and "distal substructure". Our prior studies focused on characterizing carbamate, hydantoin, and spirocyclic urea linker modules, which bind in a solvent-exposed site at the VL-VL domain interface of the LC dimer. Here, we report structure-activity relationship data on 7-diethylamino coumarin-based kinetic stabilizers. This substructure occupies the previously characterized "anchor cavity" and the "aromatic slit". The potencies of amide and urea linker modules terminating in a variety of distal substructures attached at the 3-position of this coumarin ring were assessed. Surprisingly, crystallographic data on a 7-diethylamino coumarin-based kinetic stabilizer reveals that the urea linker module and distal substructure attached at the 3-position bind a solvent-exposed region of the full-length LC dimer distinct from previously characterized sites. Our results further elaborate the small-molecule binding surface of LCs that could be occupied by potent and selective LC kinetic stabilizers.

Inverse Drug Discovery identifies weak electrophiles affording protein conjugates.

Traditional biochemical target-based and phenotypic cell-based screening approaches to drug discovery have produced the current covalent and non-covalent pharmacopoeia. Strategies to expand the druggable proteome include Inverse Drug Discovery, which involves incubating one weak organic electrophile at a time with the proteins of a living cell to identify the conjugates formed. An alkyne substructure in each organic electrophile enables affinity chromatography-mass spectrometry, which produces a list of proteins that each distinct compound reacts with. Herein, we review Inverse Drug Discovery in the context of organic compounds of intermediate complexity harboring Sulfur(VI)-fluoride exchange (SuFEx) electrophiles used to expand the cellular proteins that can be targeted covalently.

Small molecule protein binding to correct cellular folding or stabilize the native state against misfolding and aggregation.

Protein misfolding diseases are caused by the difficulty of a protein to attain or stably maintain its native three-dimensional structure. In 2011, the first small molecule that specifically binds to the folded state of a protein was approved by a regulatory agency to treat a protein misfolding disease (tafamidis, transthyretin amyloidosis). Subsequently, folded state binders for three additional pathologies were approved. All of these molecules bind specifically to and stabilize the native state of a misfolding-prone protein and either correct cellular folding or stabilize the native state against misfolding and aggregation. We will use these four case studies to explain how protein folding coupled to small molecule binding is a promising approach to treat a variety of human maladies.