Expression of Amyloidogenic Transthyretin Drives Hepatic Proteostasis Remodeling in an Induced Pluripotent Stem Cell Model of Systemic Amyloid Disease.
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ABSTRACT:
SUBMITTER: Giadone RM
PROVIDER: S-EPMC7419739 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The systemic amyloidoses are diverse disorders in which misfolded proteins are secreted by effector organs and deposited as proteotoxic aggregates at downstream tissues. Although well-described clinically, the contribution of synthesizing organs to amyloid disease pathogenesis is unknown. Here, we utilize hereditary transthyretin amyloidosis (ATTR amyloidosis) induced pluripotent stem cells (iPSCs) to define the contribution of HLCs to the proteotoxicity of secreted TTR. To this end, we generated isogenic, patient-specific iPSCs expressing either amyloidogenic or wild-type TTR. We subsequently differentiated these lines into HLCs and performed single cell RNA sequencing (scRNAseq) via the Fluidigm C1 platform in an effort to identify a destabilized TTR-derived disease signature. Upon doing so, we identified a number of hepatic proteostasis factors (e.g. the unfolded protein response, UPR, as well as known and novel chaperone genes) whose expression correlates with the production destabilized TTR production.
2020-07-01 | GSE153541 | GEO
Project description:Expression Of Amyloidogenic Transthyretin Drives Hepatic Proteostasis Remodeling In An Induced Pluripotent Stem Cell Model Of Systemic Amyloid Disease
| PRJNA643156 | ENA
Project description:Familial transthyretin amyloidosis (ATTR) is an autosomal-dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR, protein secreted from the liver aggregates and forms fibrils in target organs, chiefly the heart and peripheral nervous system, highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here, we describe the directed differentiation of ATTR patient-specific iPSCs into hepatocytes that produce mutant TTR, and the cardiomyocytes and neurons normally targeted in the disease. We demonstrate that iPSC-derived neuronal and cardiac cells display oxidative stress and an increased level of cell death when exposed to mutant TTR produced by the patient-matched iPSC-derived hepatocytes, recapitulating essential aspects of the disease in vitro. Furthermore, small molecule stabilizers of TTR show efficacy in this model, validating this iPSC-based, patient-specific in vitro system as a platform for testing therapeutic strategies.
| S-EPMC3841264 | biostudies-literature
Project description:Amyloid diseases are characterized by the deposition of proteins in the form of amyloid fibrils, in organs that eventually fail. The development of effective drug candidates follows from the understanding of the molecular processes that lead to protein aggregation. Here, we study amyloidogenic segments of transthyretin (TTR). TTR is a transporter of thyroxine and retinol in the blood and cerebrospinal fluid. When mutated and/or as a result of aging, TTR aggregates into amyloid fibrils that accumulate in organs such as the heart. Recently, we reported two amyloidogenic segments that drive amyloid aggregation. Here, we report the crystal structure of another six amyloidogenic segments of TTR. We found that the segments from the C-terminal region of TTR form in-register steric-zippers with highly-interdigitated, wet interfaces, whereas the ?-strand B from the N-terminal region of TTR forms an out-of-register assembly, previously associated with oligomeric formation. Our results contribute fundamental information for understanding the mechanism of aggregation of TTR.
| S-EPMC6032358 | biostudies-literature
Project description:The process of transthyretin (TTR) misfolding and aggregation, including amyloid formation, appears to cause a number of degenerative diseases. During amyloid formation, the native protein undergoes a tetramer-to-folded monomer transition, followed by local unfolding of the monomer to an assembly-competent amyloidogenic intermediate. Here we use NMR relaxation dispersion to probe conformational exchange at physiological pH between native monomeric TTR (the F87M/L110M variant) and a small population of a transiently formed amyloidogenic intermediate. The dispersion experiments show that a majority of the residues in the ?-sheet containing ?-strands D, A, G, and H undergo conformational fluctuations on microsecond-to-millisecond timescales. Exchange broadening is greatest for residues in the outer ?-strand H, which hydrogen bonds to ?-strand H' of a neighboring subunit in the tetramer, but the associated structural fluctuations propagate across the entire ?-sheet. Fluctuations in the other ?-sheet are limited to the outer ?-strand F, which packs against strand F' in the tetramer, while the B, C, and E ?-strands of this sheet remain stable. The structural changes were also investigated under more forcing amyloidogenic conditions (pH6.4-3.7), where ?-strand D and regions of the D-E and E-F loops were additionally destabilized, increasing the population of the amyloidogenic intermediate and accelerating amyloid formation. Strands B, C, and E appear to maintain native-like conformations in the partially unfolded, amyloidogenic state of wild-type TTR. In the case of the protective mutant T119M, the conformational fluctuations are suppressed under both physiological and mildly acidic conditions, indicating that the dynamic properties of TTR correlate well with its aggregation propensity.
| S-EPMC3594634 | biostudies-literature
Project description:Approximately 4% of black Americans carry a valine-to-isoleucine substitution (V122I) in the transthyretin protein, which has been associated with late-onset restrictive amyloid cardiomyopathy and increased risks of death and heart failure.We determined genotype status for the transthyretin gene (TTR) in 3856 black participants in the Atherosclerosis Risk in Communities study and assessed clinical profiles, mortality, and the risk of incident heart failure in V122I TTR variant carriers (124 participants [3%]) versus noncarriers (3732 participants). Cardiac structure and function and features suggestive of cardiac amyloidosis were assessed in participants who underwent echocardiography during visit 5 (2011 to 2013), when they were older than 65 years of age.After 21.5 years of follow-up, we did not detect a significant difference in mortality between carriers (41 deaths, 33%) and noncarriers (1382 deaths, 37%; age- and sex-stratified hazard ratio among carriers, 0.99; 95% confidence interval [CI], 0.73 to 1.36; P=0.97). The TTR variant was associated with an increased risk of incident heart failure (age- and sex-stratified hazard ratio, 1.47; 95% CI, 1.03 to 2.10; P=0.04). On echocardiography at visit 5, carriers (46 participants) had worse systolic and diastolic function, as well as a higher level of N-terminal pro-brain natriuretic peptide, than noncarriers (1194 participants), although carriers had a low prevalence (7%) of overt manifestations of amyloid cardiomyopathy.We did not detect a significant difference in mortality between V122I TTR allele carriers and noncarriers, a finding that contrasts with prior observations; however, the risk of heart failure was increased among carriers. The prevalence of overt cardiac abnormalities among V122I TTR carriers was low. (Funded by the National Heart, Lung, and Blood Institute and others.).
| S-EPMC4382209 | biostudies-literature
Project description:Transthyretin (TTR) is a thyroid hormone-binding protein which transports thyroxine from the bloodstream to the brain. The structural stability of TTR in tetrameric form is crucial for maintaining its original functions in blood or cerebrospinal fluid (CSF). The altered structure of TTR due to genetic mutations or its deposits due to aggregation could cause several deadly diseases such as cardiomyopathy and neuropathy in autonomic, motor, and sensory systems. The early diagnoses for hereditary amyloid TTR with cardiomyopathy (ATTR-CM) and wild-type amyloid TTR (ATTRwt) amyloidosis, which result from amyloid TTR (ATTR) deposition, are difficult to distinguish due to the close similarities of symptoms. Thus, many researchers investigated the role of ATTR as a biomarker, especially its potential for differential diagnosis due to its varying pathogenic involvement in hereditary ATTR-CM and ATTRwt amyloidosis. As a result, the detection of ATTR became valuable in the diagnosis and determination of the best course of treatment for ATTR amyloidoses. Assessing the extent of ATTR deposition and genetic analysis could help in determining disease progression, and thus survival rate could be improved following the determination of the appropriate course of treatment for the patient. Here, the perspectives of ATTR in various diseases were presented.
| S-EPMC6628571 | biostudies-literature
Project description:Many emerging cell-based therapies are based on pluripotent stem cells, though complete understanding of the properties of these cells is lacking. In these cells, much is still unknown about the cytoskeletal network, which governs the mechanoresponse. The objective of this study was to determine the cytoskeletal state in undifferentiated pluripotent stem cells and remodeling with differentiation. Mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs), as well as the original un-reprogrammed embryonic fibroblasts (MEFs), were evaluated for expression of cytoskeletal markers. We found that pluripotent stem cells overall have a less developed cytoskeleton compared to fibroblasts. Gene and protein expression of smooth muscle cell actin, vimentin, lamin A, and nestin were markedly lower for ESCs than MEFs. Whereas, iPSC samples were heterogeneous with most cells expressing patterns of cytoskeletal proteins similar to ESCs with a small subpopulation similar to MEFs. This indicates that dedifferentiation during reprogramming is associated with cytoskeletal remodeling to a less developed state. In differentiation studies, it was found that shear stress-mediated differentiation resulted in an increase in expression of cytoskeletal intermediate filaments in ESCs, but not in iPSC samples. In the embryoid body model of spontaneous differentiation of pluripotent stem cells, however, both ESCs and iPSCs had similar gene expression for cytoskeletal proteins during early differentiation. With further differentiation, however, gene levels were significantly higher for iPSCs compared to ESCs. These results indicate that reprogrammed iPSCs more readily reacquire cytoskeletal proteins compared to the ESCs that need to form the network de novo. The strategic selection of the parental phenotype is thus critical not only in the context of reprogramming but also the ultimate functionality of the iPSC-differentiated cell population. Overall, this increased characterization of the cytoskeleton in pluripotent stem cells will allow for the better understanding and design of stem cell-based therapies.
| S-EPMC4714815 | biostudies-literature
Project description:IntroductionTransthyretin amyloidosis (ATTR amyloidosis) is caused by the misfolding and deposition of the transthyretin (TTR) protein and results in progressive multi-organ dysfunction. TTR epitopes exposed by dissociation and misfolding are targets for immunotherapeutic antibodies. We developed and characterized antibodies that selectively bound to misfolded, non-native conformations of TTR.MethodsAntibody clones were generated by immunizing mice with an antigenic peptide comprising a cryptotope within the TTR sequence and screened for specific binding to non-native TTR conformations, suppression of in vitro TTR fibrillogenesis, promotion of antibody-dependent phagocytic uptake of mis-folded TTR and specific immunolabeling of ATTR amyloidosis patient-derived tissue.ResultsFour identified monoclonal antibodies were characterized. These antibodies selectively bound the target epitope on monomeric and non-native misfolded forms of TTR and strongly suppressed TTR fibril formation in vitro. These antibodies bound fluorescently tagged aggregated TTR, targeting it for phagocytic uptake by macrophage THP-1 cells, and amyloid-positive TTR deposits in heart tissue from patients with ATTR amyloidosis, but did not bind to other types of amyloid deposits or normal tissue.ConclusionsConformation-specific anti-TTR antibodies selectively bind amyloidogenic but not native TTR. These novel antibodies may be therapeutically useful in preventing deposition and promoting clearance of TTR amyloid and in diagnosing TTR amyloidosis.
| S-EPMC4898150 | biostudies-literature
Project description:The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single protein can generate different type of membrane curvatures using specific conformations for specific membrane structures and how EPR is a versatile tool well-suited to analyze subtle alterations in structures under such modifying conditions which otherwise would have been difficult using other biophysical tools.
| S-EPMC5461824 | biostudies-literature