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Cell Mar 2020The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from...
The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from GSDMD-C-terminal domain. How the caspases recognize GSDMD and its connection with caspase activation are unknown. Here, we show site-specific caspase-4/11 autoprocessing, generating a p10 product, is required and sufficient for cleaving GSDMD and inducing pyroptosis. The p10-form autoprocessed caspase-4/11 binds the GSDMD-C domain with a high affinity. Structural comparison of autoprocessed and unprocessed capase-11 identifies a β sheet induced by the autoprocessing. In caspase-4/11-GSDMD-C complex crystal structures, the β sheet organizes a hydrophobic GSDMD-binding interface that is only possible for p10-form caspase-4/11. The binding promotes dimerization-mediated caspase activation, rendering a cleavage independently of the cleavage-site tetrapeptide sequence. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode. Our study reveals an unprecedented substrate-targeting mechanism for caspases. The hydrophobic interface suggests an additional space for developing inhibitors specific for pyroptotic caspases.
Topics: Animals; Caspase 1; Caspases, Initiator; Crystallography, X-Ray; HEK293 Cells; HeLa Cells; Humans; Hydrophobic and Hydrophilic Interactions; Inflammasomes; Intracellular Signaling Peptides and Proteins; Multiprotein Complexes; Phosphate-Binding Proteins; Protein Conformation, beta-Strand; Protein Domains; Protein Processing, Post-Translational; Proteolysis; Pyroptosis
PubMed: 32109412
DOI: 10.1016/j.cell.2020.02.002 -
Cells May 2022Following Alzheimer's, Parkinson's disease (PD) is the second-most common neurodegenerative disorder, sharing an unclear pathophysiology, a multifactorial profile, and... (Review)
Review
Following Alzheimer's, Parkinson's disease (PD) is the second-most common neurodegenerative disorder, sharing an unclear pathophysiology, a multifactorial profile, and massive social costs worldwide. Despite this, no disease-modifying therapy is available. PD is tightly associated with α-synuclein (α-Syn) deposits, which become organised into insoluble, amyloid fibrils. As a typical intrinsically disordered protein, α-Syn adopts a monomeric, random coil conformation in an aqueous solution, while its interaction with lipid membranes drives the transition of the molecule part into an α-helical structure. The central unstructured region of α-Syn is involved in fibril formation by converting to well-defined, β-sheet rich secondary structures. Presently, most therapeutic strategies against PD are focused on designing small molecules, peptides, and peptidomimetics that can directly target α-Syn and its aggregation pathway. Other approaches include gene silencing, cell transplantation, stimulation of intracellular clearance with autophagy promoters, and degradation pathways based on immunotherapy of amyloid fibrils. In the present review, we sum marise the current advances related to α-Syn aggregation/neurotoxicity. These findings present a valuable arsenal for the further development of efficient, nontoxic, and non-invasive therapeutic protocols for disease-modifying therapy that tackles disease onset and progression in the future.
Topics: Amyloid; Humans; Intrinsically Disordered Proteins; Parkinson Disease; Protein Conformation, beta-Strand; alpha-Synuclein
PubMed: 35681426
DOI: 10.3390/cells11111732 -
Methods in Enzymology 2019Chemical Shift-Rosetta (CS-Rosetta) is an automated method that employs NMR chemical shifts to model protein structures de novo. In this chapter, we introduce the...
Chemical Shift-Rosetta (CS-Rosetta) is an automated method that employs NMR chemical shifts to model protein structures de novo. In this chapter, we introduce the terminology and central concepts of CS-Rosetta. We describe the architecture and functionality of automatic NOESY assignment (AutoNOE) and structure determination protocols (Abrelax and RASREC) within the CS-Rosetta framework. We further demonstrate how CS-Rosetta can discriminate near-native structures against a large conformational search space using restraints obtained from NMR data, and/or sequence and structure homology. We highlight how CS-Rosetta can be combined with alternative automated approaches to (i) model oligomeric systems and (ii) create NMR-based structure determination pipeline. To show its practical applicability, we emphasize on the computational requirements and performance of CS-Rosetta for protein targets of varying molecular weight and complexity. Finally, we discuss the current Python interface, which enables easy execution of protocols for rapid and accurate high-resolution structure determination.
Topics: Algorithms; Binding Sites; Humans; Magnetic Resonance Imaging; Models, Molecular; Molecular Weight; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Folding; Protein Interaction Domains and Motifs; Proteins; Software; Structural Homology, Protein; Thermodynamics
PubMed: 30611429
DOI: 10.1016/bs.mie.2018.07.005 -
International Journal of Molecular... Oct 2021Insoluble protein aggregates with fibrillar morphology called amyloids and β-barrel proteins both share a β-sheet-rich structure. Correctly folded β-barrel proteins... (Review)
Review
Insoluble protein aggregates with fibrillar morphology called amyloids and β-barrel proteins both share a β-sheet-rich structure. Correctly folded β-barrel proteins can not only function in monomeric (dimeric) form, but also tend to interact with one another-followed, in several cases, by formation of higher order oligomers or even aggregates. In recent years, findings proving that β-barrel proteins can adopt cross-β amyloid folds have emerged. Different β-barrel proteins were shown to form amyloid fibrils in vitro. The formation of functional amyloids in vivo by β-barrel proteins for which the amyloid state is native was also discovered. In particular, several prokaryotic and eukaryotic proteins with β-barrel domains were demonstrated to form amyloids in vivo, where they participate in interspecies interactions and nutrient storage, respectively. According to recent observations, despite the variety of primary structures of amyloid-forming proteins, most of them can adopt a conformational state with the β-barrel topology. This state can be intermediate on the pathway of fibrillogenesis ("on-pathway state"), or can be formed as a result of an alternative assembly of partially unfolded monomers ("off-pathway state"). The β-barrel oligomers formed by amyloid proteins possess toxicity, and are likely to be involved in the development of amyloidoses, thus representing promising targets for potential therapy of these incurable diseases. Considering rapidly growing discoveries of the amyloid-forming β-barrels, we may suggest that their real number and diversity of functions are significantly higher than identified to date, and represent only "the tip of the iceberg". Here, we summarize the data on the amyloid-forming β-barrel proteins, their physicochemical properties, and their biological functions, and discuss probable means and consequences of the amyloidogenesis of these proteins, along with structural relationships between these two widespread types of β-folds.
Topics: Amyloid; Amyloid beta-Peptides; Amyloidogenic Proteins; Amyloidosis; Humans; Molecular Dynamics Simulation; Protein Aggregates; Protein Conformation, beta-Strand
PubMed: 34768745
DOI: 10.3390/ijms222111316 -
Journal of the American Chemical Society Mar 2019A family of proteases called caspases mediate apoptosis signaling in animals. We report a GFP-based fluorogenic protease reporter, dubbed "FlipGFP", by flipping a beta...
A family of proteases called caspases mediate apoptosis signaling in animals. We report a GFP-based fluorogenic protease reporter, dubbed "FlipGFP", by flipping a beta strand of the GFP. Upon protease activation and cleavage, the beta strand is restored, leading to reconstitution of the GFP and fluorescence. FlipGFP-based TEV protease reporter achieves 100-fold fluorescence change. A FlipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. FlipGFP also visualized apoptotic cells in the midgut of Drosophila. Thus, the FlipGFP-based caspase reporter will be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond caspases. The design strategy can be further applied to a red fluorescent protein for engineering a red fluorogenic protease reporter.
Topics: Animals; Apoptosis; Drosophila melanogaster; Genes, Reporter; Green Fluorescent Proteins; HEK293 Cells; HeLa Cells; Humans; Molecular Imaging; Peptide Hydrolases; Protein Conformation, beta-Strand
PubMed: 30821975
DOI: 10.1021/jacs.8b13042 -
Open Biology Nov 2022Amyloid diseases are linked to protein misfolding whereby the amyloidogenic protein undergoes a conformational change, aggregates and eventually forms amyloid fibrils.... (Review)
Review
Amyloid diseases are linked to protein misfolding whereby the amyloidogenic protein undergoes a conformational change, aggregates and eventually forms amyloid fibrils. While the amyloid fibrils and plaques are hallmarks of these diseases, they typically form late in the disease process and do not correlate with disease. Instead, there is growing evidence that smaller, soluble toxic oligomers form prior and appear to be early triggers of the molecular pathology underlying these diseases. Nearly 20 years ago, we proposed the α-sheet hypothesis after discovering that the early conformational changes observed during atomistic molecular dynamics simulations involve the formation of a non-standard protein structure, α-sheet. Furthermore, we proposed that toxic oligomers contain α-sheet structure and that preferentially targeting this structure could neutralize the toxicity, prevent further aggregation and serve as the basis for early detection of disease. Here, we present the origin of the α-sheet hypothesis and describe α-sheet structure and the corresponding mechanisms of conversion. We discuss experimental studies demonstrating that both mammalian and bacterial amyloid systems form α-sheet oligomers before converting to conventional β-sheet fibrils. Furthermore, we show that the process can be inhibited with designed α-sheet peptides complementary to the structure in the toxic oligomers.
Topics: Animals; Amyloid; Amyloidogenic Proteins; Protein Conformation, beta-Strand; Molecular Dynamics Simulation; Peptides; Mammals
PubMed: 36416010
DOI: 10.1098/rsob.220261 -
Trends in Biochemical Sciences Apr 2022Many neurodegenerative diseases, including Alzheimer's, originate from the conversion of proteins into pathogenic conformations. The microtubule-associated protein tau... (Review)
Review
Many neurodegenerative diseases, including Alzheimer's, originate from the conversion of proteins into pathogenic conformations. The microtubule-associated protein tau converts into β-sheet-rich amyloid conformations, which underlie pathology in over 25 related tauopathies. Structural studies of tau amyloid fibrils isolated from human tauopathy tissues have revealed that tau adopts diverse structural polymorphs, each linked to a different disease. Molecular chaperones play central roles in regulating tau function and amyloid assembly in disease. New data supports the model that chaperones selectively recognize different conformations of tau to limit the accumulation of proteotoxic species. The challenge now is to understand how chaperones influence disease processes across different tauopathies, which will help guide the development of novel conformation-specific diagnostic and therapeutic strategies.
Topics: Alzheimer Disease; Amyloid; Humans; Molecular Chaperones; Protein Conformation, beta-Strand; Tauopathies; tau Proteins
PubMed: 35045944
DOI: 10.1016/j.tibs.2021.12.009 -
Cold Spring Harbor Perspectives in... May 2019Polyphosphate (polyP), an extremely simple polyanion, has long been known to be involved in a variety of different cellular processes, ranging from stress resistance,... (Review)
Review
Polyphosphate (polyP), an extremely simple polyanion, has long been known to be involved in a variety of different cellular processes, ranging from stress resistance, biofilm formation, and virulence in bacteria to bone mineralization, blood clotting, and mammalian target of rapamycin (mTOR) signaling in mammalian organisms. Our laboratory recently discovered a completely unexpected role of polyP as a stabilizing scaffold for β-sheet-containing protein-folding intermediates. This realization led us to investigate the effects of polyP on amyloidogenic processes and the novel concept that polyP might play a role in neurodegenerative diseases. In this review, we will summarize recent results that show that polyP is a physiological modifier that accelerates amyloid fiber formation, alters fiber morphology, and protects cells against amyloid toxicity. We will review the current knowledge on the distribution, levels, and roles of polyP in the mammalian brain, and discuss potential mechanisms by which polyP might ameliorate amyloid toxicity.
Topics: Amyloid; Animals; Brain; Cell Survival; Humans; Polyphosphates; Protein Conformation, beta-Strand
PubMed: 30617049
DOI: 10.1101/cshperspect.a034041 -
ACS Chemical Neuroscience Oct 2022Abrupt aggregation of amyloid beta (Aβ) peptide is strongly associated with Alzheimer's disease. In this study, we used atomic force microscopy-infrared (AFM-IR)...
Abrupt aggregation of amyloid beta (Aβ) peptide is strongly associated with Alzheimer's disease. In this study, we used atomic force microscopy-infrared (AFM-IR) spectroscopy to characterize the secondary structure of Aβ oligomers, protofibrils and fibrils formed at the early (4 h), middle (24 h), and late (72 h) stages of protein aggregation. This innovative spectroscopic approach allows for label-free nanoscale structural characterization of individual protein aggregates. Using AFM-IR, we found that at the early stage of protein aggregation, oligomers with parallel β-sheet dominated. However, these species exhibited slower rates of fibril formation compared to the oligomers with antiparallel β-sheet, which first appeared in the middle stage. These antiparallel β-sheet oligomers rapidly propagated into fibrils that were simultaneously observed together with parallel β-sheet fibrils at the late stage of protein aggregation. Our findings showed that aggregation of Aβ is a complex process that yields several distinctly different aggregates with dissimilar toxicities.
Topics: Amyloid; Amyloid beta-Peptides; Microscopy, Atomic Force; Protein Aggregates; Protein Conformation, beta-Strand; Protein Structure, Secondary
PubMed: 36122250
DOI: 10.1021/acschemneuro.2c00180 -
The FEBS Journal Oct 2017The flavodoxin-like fold is a protein architecture that can be traced back to the universal ancestor of the three kingdoms of life. Many proteins share this α-β... (Review)
Review
The flavodoxin-like fold is a protein architecture that can be traced back to the universal ancestor of the three kingdoms of life. Many proteins share this α-β parallel topology and hence it is highly relevant to illuminate how they fold. Here, we review experiments and simulations concerning the folding of flavodoxins and CheY-like proteins, which share the flavodoxin-like fold. These polypeptides tend to temporarily misfold during unassisted folding to their functionally active forms. This susceptibility to frustration is caused by the more rapid formation of an α-helix compared to a β-sheet, particularly when a parallel β-sheet is involved. As a result, flavodoxin-like proteins form intermediates that are off-pathway to native protein and several of these species are molten globules (MGs). Experiments suggest that the off-pathway species are of helical nature and that flavodoxin-like proteins have a nonconserved transition state that determines the rate of productive folding. Folding of flavodoxin from Azotobacter vinelandii has been investigated extensively, enabling a schematic construction of its folding energy landscape. It is the only flavodoxin-like protein of which cotranslational folding has been probed. New insights that emphasize differences between in vivo and in vitro folding energy landscapes are emerging: the ribosome modulates MG formation in nascent apoflavodoxin and forces this polypeptide toward the native state.
Topics: Azotobacter vinelandii; Escherichia coli; Escherichia coli Proteins; Flavodoxin; Gene Expression; Methyl-Accepting Chemotaxis Proteins; Models, Molecular; Protein Biosynthesis; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Folding; Protein Isoforms; Thermodynamics
PubMed: 28380286
DOI: 10.1111/febs.14077