Loss of function mutations in EIF2AK3, encoding the Pancreatic Endoplasmic Reticulum (ER) Kinase, PERK, are associated with dysfunction of the endocrine pancreas and diabetes. However to date it has not been possible to uncouple the long term developmental effects of PERK deficiency from sensitization to physiological levels of ER unfolded protein stress upon interruption of PERK-modulation of protein synthesis rates. Here we report that a selective PERK inhibitor acutely deregulates protein synthesis in freshly isolated islets of Langerhans, across a range of glucose concentrations. Acute loss of the PERK-mediated strand of the unfolded protein response leads to rapid accumulation of misfolded pro-insulin in cultured beta cells and is associated with a kinetic defect in pro-insulin processing. These in vitro observations uncouple the latent role of PERK in beta cell development from the regulation of unfolded protein flux through the ER and attest to the importance of the latter in beta cell proteostasis.
Coexistence of ribbon and helical fibrils originating from hIAPP20-29 revealed by quantitative nanomechanical atomic force microscopy
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 7 years ago
Uncontrolled misfolding of proteins leading to the formation of amyloid deposits is associated with more than 40 types of diseases, such as neurodegenerative diseases and type-2 diabetes. These irreversible amyloid fibrils typically assemble in distinct stages. Transitions among the various intermediate stages are the subject of many studies but are not yet fully elucidated. Here, we combine high-resolution atomic force microscopy and quantitative nanomechanical mapping to determine the self-assembled structures of the decapeptide hIAPP(20-29), which is considered to be the fibrillating core fragment of the human islet amyloid polypeptide (hIAPP) involved in type-2 diabetes. We successfully follow the evolution of hIAPP(20-29) nanostructures over time, calculate the average thickening speed of small ribbon-like structures, and provide evidence of the coexistence of ribbon and helical fibrils, highlighting a key step within the self-assembly model. In addition, the mutations of individual side chains of wide-type hIAPP(20-29) shift this balance and destabilize the helical fibrils sufficiently relative to the twisted ribbons to lead to their complete elimination. We combine atomic force microscopy structures, mechanical properties, and solid-state NMR structural information to build a molecular model containing β sheets in cross-β motifs as the basis of self-assembled amyloids.
IAPP, a 37 amino-acid peptide hormone belonging to the calcitonin family, is an intrinsically disordered protein that is coexpressed and cosecreted along with insulin by pancreatic islet β-cells in response to meals. IAPP plays a physiological role in glucose regulation; however, in certain species, IAPP can aggregate and this process is linked to β-cell death and Type II Diabetes. Using replica exchange molecular dynamics with extensive sampling (16 replicas per sequence and 600 ns per replica), we investigate the structure of the monomeric state of two species of aggregating peptides (human and cat IAPP) and two species of non-aggregating peptides (pig and rat IAPP). Our simulations reveal that the pig and rat conformations are very similar, and consist of helix-coil and helix-hairpin conformations. The aggregating sequences, on the other hand, populate the same helix-coil and helix-hairpin conformations as the non-aggregating sequence, but, in addition, populate a hairpin structure. Our exhaustive simulations, coupled with available peptide-activity data, leads us to a structure-activity relationship (SAR) in which we propose that the functional role of IAPP is carried out by the helix-coil conformation, a structure common to both aggregating and non-aggregating species. The pathological role of this peptide may have multiple origins, including the interaction of the helical elements with membranes. Nonetheless, our simulations suggest that the hairpin structure, only observed in the aggregating species, might be linked to the pathological role of this peptide, either as a direct precursor to amyloid fibrils, or as part of a cylindrin type of toxic oligomer. We further propose that the helix-hairpin fold is also a possible aggregation prone conformation that would lead normally non-aggregating variants of IAPP to form fibrils under conditions where an external perturbation is applied. The SAR relationship is used to suggest the rational design of therapeutics for treating diabetes.
Amylin (islet amyloid polypeptide) and amyloid-beta (Aβ) protein, which are deposited within pancreatic islets of diabetics and brains of Alzheimer’s patients respectively, share many biophysical and physiological properties. Emerging evidence indicates that the amylin receptor is a putative target receptor for the actions of human amylin and Aβ in the brain. The amylin receptor consists of the calcitonin receptor dimerized with a receptor activity-modifying protein and is widely distributed within central nervous system. Both amylin and Aβ directly activate this G protein-coupled receptor and trigger multiple common intracellular signal transduction pathways that can culminate in apoptotic cell death. Moreover, amylin receptor antagonists can block both the biological and neurotoxic effects of human amylin and Aβ. Amylin receptors thus appear to be involved in the pathophysiology of Alzheimer’s disease and diabetes, and could serve as a molecular link between the two conditions that are associated epidemiologically.
Zinc is essential for the activities of pancreatic beta-cells, especially insulin storage and secretion. Insulin secretion leads to co-release of zinc which contributes to the paracrine communication in the pancreatic islets. Zinc-transporting proteins (zinc-regulated transporter, iron-regulated transporter-like proteins [ZIPs] and zinc transporters [ZnTs]) and metal-buffering proteins (metallothioneins, MTs) tightly regulate intracellular zinc homeostasis. The present study investigated how modulation of cellular zinc availability affects beta-cell function using INS-1E cells.
Peptide mediated gain-of-toxic function is central to pathology in Alzheimer’s, Parkinson’s and diabetes. In each system, self-assembly into oligomers is observed and can also result in poration of artificial membranes. Structural requirements for poration and the relationship of structure to cytotoxicity is unaddressed. Here we focus on islet amyloid polypeptide (IAPP) mediated loss-of-insulin secreting cells in patients with diabetes. Newly developed methods enable structure-function enquiry to focus on intracellular oligomers composed of hundreds of IAPP. The key insights are that porating oligomers are internally dynamic, grow in discrete steps and are not canonical amyloid. Moreover, two classes of poration occur; an IAPP-specific ligand establishes that only one is cytotoxic. Toxic rescue occurs by stabilising non-toxic poration without displacing IAPP from mitochondria. These insights illuminate cytotoxic mechanism in diabetes and also provide a generalisable approach for enquiry applicable to other partially ordered protein assemblies.
Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique b-strand structure distinct from the conventional amyloid b-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.
- Proceedings of the National Academy of Sciences of the United States of America
- Published almost 7 years ago
Amyloid formation is implicated in more than 20 human diseases, yet the mechanism by which fibrils form is not well understood. We use 2D infrared spectroscopy and isotope labeling to monitor the kinetics of fibril formation by human islet amyloid polypeptide (hIAPP or amylin) that is associated with type 2 diabetes. We find that an oligomeric intermediate forms during the lag phase with parallel β-sheet structure in a region that is ultimately a partially disordered loop in the fibril. We confirm the presence of this intermediate, using a set of homologous macrocyclic peptides designed to recognize β-sheets. Mutations and molecular dynamics simulations indicate that the intermediate is on pathway. Disrupting the oligomeric β-sheet to form the partially disordered loop of the fibrils creates a free energy barrier that is the origin of the lag phase during aggregation. These results help rationalize a wide range of previous fragment and mutation studies including mutations in other species that prevent the formation of amyloid plaques.
TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas the ΔN isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes that promote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ΔN isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the β cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.
The deposition of fibrillated human islet β-cell peptide islet amyloid polypeptide (hIAPP) into amyloid plaques is characteristic of the pathogenesis of islet cell death during type 2 diabetes. We investigated the effects of the neuroendocrine secretory proteins 7B2 and proSAAS on hIAPP fibrillation in vitro and on cytotoxicity. In vitro, 21-kDa 7B2 and proSAAS blocked hIAPP fibrillation. Structure-function studies showed that a central region within 21-kDa 7B2 is important in this effect and revealed the importance of the N-terminal region of proSAAS. Both chaperones blocked the cytotoxic effects of exogenous hIAPP on Rin5f cells; 7B2 generated by overexpression was also effective. ProSAAS and 7B2 may perform a chaperone role as secretory anti-aggregants in normal islet cell function and in type 2 diabetes.