Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
1.
Chembiochem ; 24(12): e202300320, 2023 06 15.
Article in English | MEDLINE | ID: mdl-37186077

ABSTRACT

There is considerable interest in drug discovery targeting the aggregation of α-synuclein (αSyn) since this molecular process is closely associated with Parkinson's disease. However, inhibiting αSyn aggregation remains a major challenge because of its highly dynamic nature which makes it difficult to form a stable binding complex with a drug molecule. Here, by exploiting Random non-standard Peptides Integrated Discovery (RaPID) system, we identified a macrocyclic peptide, BD1, that could interact with immobilized αSyn and inhibit the formation of fibrils. Furthermore, improving the solubility of BD1 suppresses the co-aggregation with αSyn fibrils while it kinetically inhibits more effectively without change in their morphology. We also revealed the molecular mechanism of kinetic inhibition, where peptides bind to fibril ends of αSyn, thereby preventing further growth of fibrils. These results suggest that our approach for generating non-standard macrocyclic peptides is a promising approach for developing potential therapeutics against neurodegeneration.


Subject(s)
Parkinson Disease , alpha-Synuclein , Humans , alpha-Synuclein/metabolism , Amyloid/chemistry , Parkinson Disease/drug therapy , Parkinson Disease/metabolism , Peptides/pharmacology , Kinetics
2.
Cell Chem Biol ; 29(4): 544-554.e4, 2022 04 21.
Article in English | MEDLINE | ID: mdl-34936860

ABSTRACT

The Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator contains a four-protein subcomplex called the deubiquitinating enzyme (DUB) module that removes ubiquitin from histone H2B-K120. The human DUB module contains the catalytic subunit ubiquitin-specific protease 22 (USP22), which is overexpressed in a number of cancers that are resistant to available therapies. We screened a massive combinatorial library of cyclic peptides and identified potent inhibitors of USP22. The top hit was highly specific for USP22 compared with a panel of 44 other human DUBs. Cells treated with peptide had increased levels of H2B monoubiquitination, demonstrating the ability of the cyclic peptides to enter human cells and inhibit H2B deubiquitination. These macrocycle inhibitors are, to our knowledge, the first reported inhibitors of USP22/SAGA DUB module and show promise for development.


Subject(s)
Histones , Ubiquitin , Histones/metabolism , Humans , Peptides, Cyclic/pharmacology , Transcription Factors/metabolism , Ubiquitin Thiolesterase , Ubiquitination
3.
Front Neurosci ; 15: 623097, 2021.
Article in English | MEDLINE | ID: mdl-33716651

ABSTRACT

There is great interest in drug discovery programs targeted at the aggregation of the 42-residue form of the amyloid ß peptide (Aß42), since this molecular process is closely associated with Alzheimer's disease. The use of bicyclic peptides may offer novel opportunities for the effective modification of Aß42 aggregation and the inhibition of its cytotoxicity, as these compounds combine the molecular recognition ability of antibodies with a relatively small size of about 2 kD. Here, to pursue this approach, we rationally designed a panel of six bicyclic peptides targeting various epitopes along the sequence of Aß42 to scan its most amyloidogenic region (residues 13-42). Our kinetic analysis and structural studies revealed that at sub-stoichiometric concentrations the designed bicyclic peptides induce a delay in the condensation of Aß42 and the subsequent transition to a fibrillar state, while at higher concentrations they inhibit such transition. We thus suggest that designed bicyclic peptides can be employed to inhibit amyloid formation by redirecting the aggregation process toward amorphous assemblies.

4.
Sci Rep ; 10(1): 15280, 2020 09 17.
Article in English | MEDLINE | ID: mdl-32943652

ABSTRACT

Bicyclic peptides have great therapeutic potential since they can bridge the gap between small molecules and antibodies by combining a low molecular weight of about 2 kDa with an antibody-like binding specificity. Here we apply a recently developed in silico rational design strategy to produce a bicyclic peptide to target the C-terminal region (residues 31-42) of the 42-residue form of the amyloid ß peptide (Aß42), a protein fragment whose aggregation into amyloid plaques is linked with Alzheimer's disease. We show that this bicyclic peptide is able to remodel the aggregation process of Aß42 in vitro and to reduce its associated toxicity in vivo in a C. elegans worm model expressing Aß42. These results provide an initial example of a computational approach to design bicyclic peptides to target specific epitopes on disordered proteins.


Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Caenorhabditis elegans/metabolism , Protein Aggregation, Pathological/metabolism , Amyloid/metabolism , Animals , Disease Models, Animal , Peptide Fragments , Plaque, Amyloid/metabolism
5.
Biochim Biophys Acta Biomembr ; 1860(3): 757-766, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29273335

ABSTRACT

Amyloid fibrillation causes serious neurodegenerative diseases and amyloidosis; however, the detailed mechanisms by which the structural states of precursor proteins in a lipid membrane-associated environment contribute to amyloidogenesis still remains to be elucidated. We examined the relationship between structural states of intrinsically-disordered wild-type and mutant α-synuclein (αSN) and amyloidogenesis on two-types of model membranes. Highly-unstructured wild-type αSN (αSNWT) and a C-terminally-truncated mutant lacking negative charges (αSN103) formed amyloid fibrils on both types of membranes, the model membrane mimicking presynaptic vesicles (Mimic membrane) and the model membrane of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC membrane). Unstructured αSNWT and αSN103 both bound to Mimic membranes in a helical conformation with similar binding affinity. Promotion and then inhibition of amyloidogenesis of αSNWT were observed as the concentration of Mimic lipids increased. We explain this by the two-state binding model: at lower lipid concentrations, binding of αSNWT to membranes enhances amyloidogenicity by increasing the local concentration of membrane-bound αSN and so promoting amyloid nucleation; at higher lipid concentrations, membrane-bound αSNWT is actually in a sense diluted by increasing the number of model membranes, which blocks amyloid fibrillation due to an insufficient bound population for productive nucleation. Meanwhile, αSN103 formed amyloid fibrils over the whole concentration of Mimic lipids used here without inhibition, revealing the importance of helical structures for binding affinity and negatively charged unstructured C-terminal region for modulating amyloidogenesis. We propose that membrane binding-induced initial conformations of αSN, its overall charge states, and the population of membrane-bound αSN are key determinants of amyloidogenesis on membranes.


Subject(s)
Amyloid/biosynthesis , Unilamellar Liposomes , alpha-Synuclein/chemistry , Dose-Response Relationship, Drug , Dynamic Light Scattering , Humans , Membrane Lipids/chemistry , Models, Chemical , Nuclear Magnetic Resonance, Biomolecular , Phosphatidylcholines/chemistry , Phosphatidylethanolamines/chemistry , Phosphatidylserines/chemistry , Protein Binding , Protein Conformation , Sequence Deletion , alpha-Synuclein/genetics
6.
Phys Chem Chem Phys ; 19(24): 16257-16266, 2017 Jun 21.
Article in English | MEDLINE | ID: mdl-28608875

ABSTRACT

We herein report the mechanism of amyloid formation of amyloid-ß (Aß) peptides on small (SUV) and large unilamellar vesicles (LUVs), which consist of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids. Although Aß1-42 formed fibrils on SUVs at all POPC concentrations used, the lag time, elongation rate, maximum thioflavin T intensity, and fibrillar morphology were distinct, indicating polymorphic amyloid formation. LUVs, at low POPC concentrations, did not markedly affect fibrillation kinetics; however, increases in POPC concentrations suppressed amyloid formation. No significant differences in the thermal stabilities of Aß1-42 fibrils formed with and without vesicles were observed, although fibrils formed on SUVs showed some differences with dilution. SUVs markedly promoted Aß1-40 fibrillation by condensing Aß1-40, whereas no effects of LUVs on amyloidogenesis were detected. Salts greatly increased Aß1-40 amyloidogenicity on vesicles. We proposed comprehensive models for vesicle size-dependent Aß amyloidogenesis. Inhomogeneous packing defects in SUVs may induce distinct nucleation in the polymorphisms of amyloids and decreasing local concentrations of Aß with higher amounts of LUVs inhibits amyloid formation. We also pointed out that C-terminal hydrophobicity of Aß is important for amyloidogenesis on membranes.


Subject(s)
Amyloid beta-Peptides/metabolism , Peptide Fragments/metabolism , Unilamellar Liposomes/metabolism , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Peptides/chemistry , Calorimetry , Circular Dichroism , Humans , Kinetics , Microscopy, Atomic Force , Peptide Fragments/chemistry , Phosphatidylcholines/chemistry , Unilamellar Liposomes/chemistry
7.
Langmuir ; 32(8): 2010-22, 2016 Mar 01.
Article in English | MEDLINE | ID: mdl-26824789

ABSTRACT

Despite extensive studies on the folding and function of cytochrome c, the mechanisms underlying its aggregation remain largely unknown. We herein examined the aggregation behavior of the physiologically relevant two types of cytochrome c, metal-bound cytochrome c, and its fragment with high amyloidogenicity as predicted in alcohol/water mixtures. Although the aggregation propensity of holo cytochrome c was low due to high solubility, markedly unfolded apo cytochrome c, lacking the heme prosthetic group, strongly promoted the propensity for amorphous aggregation with increases in hydrophobicity. Silver-bound apo cytochrome c increased the capacity of fibrillar aggregation (i.e., protofibrils or immature fibrils) due to subtle structural changes of apo cytochrome c by strong binding of silver. However, mature amyloid fibrils were not detected for any of the cytochrome c variants or its fragment, even with extensive ultrasonication, which is a powerful amyloid inducer. These results revealed the intrinsically low amyloidogenicity of cytochrome c, which is beneficial for its homeostasis and function by facilitating the folding and minimizing irreversible amyloid formation. We propose that intrinsically low amyloidogenicity of cytochrome c is attributed to the low metastability of supersaturation. The phase diagram constructed based on solubility and aggregate type is useful for a comprehensive understanding of protein aggregation. Furthermore, amorphous aggregation, which is also viewed as a generic property of proteins, and amyloid fibrillation can be distinguished from each other by the metastability of supersaturation.

8.
Biochim Biophys Acta ; 1854(3): 209-17, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25528988

ABSTRACT

Ultrasonication can be used to break the supersaturation of α-synuclein, a protein associated with Parkinson's disease, at pH7.4 above the critical concentration of fibrillation, thereby inducing the formation of amyloid fibrils. We speculated that ultrasonication could also be used to depolymerize preformed fibrils below the critical concentration. However, extensive ultrasonic irradiation transformed preformed fibrils into amorphous aggregates even above the critical concentration. Exposing preformed fibrils to the hydrophobic air-water interface of cavitation bubbles may have destabilized the fibrils and stabilized amorphous aggregates. Upon extensive ultrasonic irradiation, the accompanying decomposition of chemical structures was suggested when monitored by analytical ultracentrifugation. Amorphous aggregates produced by extensive ultrasonication showed higher cytotoxicity, suggesting that, although ultrasonication might be a useful approach for inactivating amyloid fibrils, potential cytotoxicity of amorphous aggregates should be considered.


Subject(s)
Amyloid/chemical synthesis , Amyloid/radiation effects , Sonication/methods , alpha-Synuclein/chemistry , alpha-Synuclein/radiation effects , Amyloid/administration & dosage , Animals , Cell Survival/drug effects , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , High-Energy Shock Waves , PC12 Cells , Protein Aggregates , Proteolysis , Radiation Dosage , Rats , alpha-Synuclein/administration & dosage
9.
Angew Chem Int Ed Engl ; 53(30): 7799-804, 2014 Jul 21.
Article in English | MEDLINE | ID: mdl-24920162

ABSTRACT

Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α-Synuclein fibrils cold-denatured to monomers at 0-20 °C and heat-denatured at 60-110 °C. Meanwhile, the fibrils of ß2-microglobulin, Alzheimer's Aß1-40/Aß1-42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α-synuclein fibrils. We propose that although cold-denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid structures may explain amyloid-specific cold denaturation arising from the unfavorable burial of charged side-chains in fibril cores.


Subject(s)
Amyloid/chemistry , alpha-Synuclein/chemistry , Cold Temperature , Protein Denaturation , Protein Folding
10.
Proc Natl Acad Sci U S A ; 111(18): 6654-9, 2014 May 06.
Article in English | MEDLINE | ID: mdl-24753579

ABSTRACT

Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. Although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. Furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. Here, with ß2-microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (ITC). The spontaneous fibrillation after a lag phase was accompanied by exothermic heat. The thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. We also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. These results indicate that ITC will become a promising approach for clarifying comprehensively the thermodynamics of protein folding and misfolding.


Subject(s)
Amyloid/chemistry , beta 2-Microglobulin/chemistry , Amyloid/ultrastructure , Amyloidosis/metabolism , Calorimetry , Hot Temperature , Humans , Microscopy, Atomic Force , Protein Folding , Protein Stability , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Solutions , Thermodynamics , beta 2-Microglobulin/ultrastructure
SELECTION OF CITATIONS
SEARCH DETAIL
...