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Khaled, M., Rönnbäck, I., Ilag, L. L., Gräslund, A., Strodel, B. & Österlund, N. (2023). A Hairpin Motif in the Amyloid-& beta Peptide Is Important for Formation of Disease-Related Oligomers. Journal of the American Chemical Society, 145(33), 18340-18354
Open this publication in new window or tab >>A Hairpin Motif in the Amyloid-& beta Peptide Is Important for Formation of Disease-Related Oligomers
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2023 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 145, no 33, p. 18340-18354Article in journal (Refereed) Published
Abstract [en]

The amyloid-& beta;(A & beta;) peptide is associated with the developmentof Alzheimer's disease and is known to form highly neurotoxicprefibrillar oligomeric aggregates, which are difficult to study dueto their transient, low-abundance, and heterogeneous nature. To obtainhigh-resolution information about oligomer structure and dynamicsas well as relative populations of assembly states, we here employa combination of native ion mobility mass spectrometry and moleculardynamics simulations. We find that the formation of A & beta; oligomersis dependent on the presence of a specific & beta;-hairpin motif inthe peptide sequence. Oligomers initially grow spherically but startto form extended linear aggregates at oligomeric states larger thanthose of the tetramer. The population of the extended oligomers couldbe notably increased by introducing an intramolecular disulfide bond,which prearranges the peptide in the hairpin conformation, therebypromoting oligomeric structures but preventing conversion into maturefibrils. Conversely, truncating one of the & beta;-strand-formingsegments of A & beta; decreased the hairpin propensity of the peptideand thus decreased the oligomer population, removed the formationof extended oligomers entirely, and decreased the aggregation propensityof the peptide. We thus propose that the observed extended oligomerstate is related to the formation of an antiparallel sheet state,which then nucleates into the amyloid state. These studies provideincreased mechanistic understanding of the earliest steps in A & beta;aggregation and suggest that inhibition of A & beta; folding into thehairpin conformation could be a viable strategy for reducing the amountof toxic oligomers.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-221322 (URN)10.1021/jacs.3c03980 (DOI)001044984100001 ()37555670 (PubMedID)2-s2.0-85168362360 (Scopus ID)
Available from: 2023-09-19 Created: 2023-09-19 Last updated: 2023-09-19Bibliographically approved
Österlund, N. (2023). Capturing transient peptide assemblies associated with Alzheimer's disease: Native mass spectrometry studies of amyloid-β oligomerization. (Doctoral dissertation). Stockholm: Department of Biochemistry and Biophysics, Stockholm University
Open this publication in new window or tab >>Capturing transient peptide assemblies associated with Alzheimer's disease: Native mass spectrometry studies of amyloid-β oligomerization
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Correct folding of proteins is essential for maintaining a functional living cell. Misfolding and aggregation of proteins, where non-native intermolecular interactions form large and highly ordered amyloid aggregates with low free energy, is hence associated with multiple diseases. One example is Alzheimer’s disease (AD) where the Amyloid-β (Aβ) peptide aggregates into amyloid fibrils, which deposit as neuritic plaques in the brains of AD patients. Nucleation of amyloid fibrils takes place via formation of smaller pre-nucleation clusters, so-called oligomers, which are considered to be especially toxic and are therefore potentially important in AD pathology. Detailed mechanistic molecular knowledge of Aβ aggregation is important for design of AD treatments that target these processes. The oligomeric species are however challenging to study experimentally due to their low abundance and high polydispersity.  

Aβ oligomers are in this thesis studied under controlled in vitro conditions using bottom-up biophysics. Highly pure recombinant Aβ peptides are studied, primarily using native ion-mobility mass spectrometry, to monitor the spontaneous formation of oligomers in aqueous solution. Mass spectrometry is capable of resolving individual oligomeric states, while ion mobility provides low-resolution structure information. This is complemented with other biophysical techniques, as well as theoretical modeling. The oligomers are also studied upon modulating intrinsic factors, such as peptide length and sequence, or extrinsic factors, such as the chemical environment. Interactions with two important biological interaction partners are studied: chaperone proteins and cell membranes.  

We show how Aβ oligomers assemble, and form extended structures which may be linked to continued growth into amyloid fibrils. We also show how different amyloid chaperone proteins interact with growing aggregates, which modifies and delays the aggregation process. These interactions are shown to depend on specific sequence-motifs in the chaperones and client peptides. Membrane-mimicking micelles are on the other hand able to stabilize globular compact forms of the Aβ oligomers and to inhibit the formation of extended structures which nucleate into amyloid fibrils. This may contribute to enrichment of toxic species in vivo. Interactions with membrane-mimicking systems are shown to be highly dependent on both the Aβ peptide isoform and the properties of the membrane environment, such as headgroup charges. It is also demonstrated how addition of a designed small peptide construct can inhibit formation of Aβ oligomers in the membrane environment. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2023. p. 74
Keywords
Protein aggregation, amyloid-β, neurodegeneration, Alzheimer’s disease, chaperones, DnaJ, biomembranes, native mass spectrometry
National Category
Biophysics Biochemistry and Molecular Biology Analytical Chemistry
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-212567 (URN)978-91-8014-130-7 (ISBN)978-91-8014-131-4 (ISBN)
Public defence
2023-01-27, Magnélisalen (AR-L208), Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2023-01-02 Created: 2022-12-08 Last updated: 2022-12-21Bibliographically approved
Liu, C., Henning-Knechtel, A., Österlund, N., Wu, J., Wang, G., Gräslund, R. A., . . . Luo, J. (2023). Oligomer Dynamics of LL-37 Truncated Fragments Probed by α-Hemolysin Pore and Molecular Simulations. Small, 19(37), Article ID 2206232.
Open this publication in new window or tab >>Oligomer Dynamics of LL-37 Truncated Fragments Probed by α-Hemolysin Pore and Molecular Simulations
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2023 (English)In: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 19, no 37, article id 2206232Article in journal (Refereed) Published
Abstract [en]

Oligomerization of antimicrobial peptides (AMPs) is critical in their effects on pathogens. LL-37 and its truncated fragments are widely investigated regarding their structures, antimicrobial activities, and application, such as developing new antibiotics. Due to the small size and weak intermolecular interactions of LL-37 fragments, it is still elusive to establish the relationship between oligomeric states and antimicrobial activities. Here, an α-hemolysin nanopore, mass spectrometry (MS), and molecular dynamic (MD) simulations are used to characterize the oligomeric states of two LL-37 fragments. Nanopore studies provide evidence of trapping events related to the oligomer formation and provide further details on their stabilities, which are confirmed by MS and MD simulations. Furthermore, simulation results reveal the molecular basis of oligomer dynamics and states of LL-37 fragments. This work provides unique insights into the relationship between the oligomer dynamics of AMPs and their antimicrobial activities at the single-molecule level. The study demonstrates how integrating methods allows deciphering single molecule level understanding from nanopore sensing approaches. 

Keywords
dynamics, LL-37 fragments, nanopores, oligomers
National Category
Biophysics
Identifiers
urn:nbn:se:su:diva-220214 (URN)10.1002/smll.202206232 (DOI)000986112000001 ()37170734 (PubMedID)2-s2.0-85159128314 (Scopus ID)
Available from: 2023-08-25 Created: 2023-08-25 Last updated: 2023-10-12Bibliographically approved
Österlund, N., Frankel, R., Carlsson, A., Thacker, D., Karlsson, M., Matus, V., . . . Linse, S. (2023). The C-terminal domain of the antiamyloid chaperone DNAJB6 binds to amyloid-β peptide fibrils and inhibits secondary nucleation. Journal of Biological Chemistry, 299(11), Article ID 105317.
Open this publication in new window or tab >>The C-terminal domain of the antiamyloid chaperone DNAJB6 binds to amyloid-β peptide fibrils and inhibits secondary nucleation
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2023 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 299, no 11, article id 105317Article in journal (Refereed) Published
Abstract [en]

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Here, we studied the role of its C-terminal domain (CTD) using constructs comprising either the entire CTD or the first two or all four of the CTD β-strands grafted onto a scaffold protein. Each construct was expressed as WT and as a variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first β-strand. We investigated the stability, oligomerization, antiamyloid activity, and affinity for amyloid-β (Aβ42) species using optical spectroscopy, native mass spectrometry, chemical crosslinking, and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers, and tetramers of low affinity. Kinetic analyses showed a shift in inhibition mechanism. Whereas full-length DNAJB6 activity is dependent on the serine and threonine residues and efficiently inhibits primary and secondary nucleation, all CTD constructs inhibit secondary nucleation only, independently of the serine and threonine residues, although their dimerization and thermal stabilities are reduced by alanine substitution. While the full-length DNAJB6 inhibition of primary nucleation is related to its propensity to form coaggregates with Aβ, the CTD constructs instead bind to Aβ42 fibrils, which affects the nucleation events at the fibril surface. The retardation of secondary nucleation by DNAJB6 can thus be ascribed to the first two β-strands of its CTD, whereas the inhibition of primary nucleation is dependent on the entire protein or regions outside the CTD.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-224307 (URN)10.1016/j.jbc.2023.105317 (DOI)001101315100001 ()37797698 (PubMedID)2-s2.0-85175246202 (Scopus ID)
Available from: 2023-12-06 Created: 2023-12-06 Last updated: 2023-12-06Bibliographically approved
Kaldmäe, M., Vosselman, T., Zhong, X., Lama, D., Chen, G., Saluri, M., . . . Landreh, M. (2022). A “spindle and thread” mechanism unblocks p53 translation by modulating N-terminal disorder. Structure, 30(5), 733-742, e1-e7
Open this publication in new window or tab >>A “spindle and thread” mechanism unblocks p53 translation by modulating N-terminal disorder
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2022 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 30, no 5, p. 733-742, e1-e7Article in journal (Refereed) Published
Abstract [en]

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of “life on the edge of solubility.” Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT domain. We conclude that interactions with NT help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT. In summary, we demonstrate that inducing co-translational folding via a molecular “spindle and thread” mechanism unblocks protein translation in vitro.

Keywords
protein translation, tumor suppressor, protein folding, intrinsically disordered proteins
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-206250 (URN)10.1016/j.str.2022.02.013 (DOI)000799220200009 ()35290795 (PubMedID)2-s2.0-85129416955 (Scopus ID)
Available from: 2022-06-13 Created: 2022-06-13 Last updated: 2022-06-13Bibliographically approved
Österlund, N., Wärmländer, S. K. T. & Gräslund, A. (2022). Cell-Penetrating Peptides with Unexpected Anti-Amyloid Properties. Pharmaceutics, 14(4), Article ID 823.
Open this publication in new window or tab >>Cell-Penetrating Peptides with Unexpected Anti-Amyloid Properties
2022 (English)In: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 14, no 4, article id 823Article, review/survey (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) with sequences derived originally from a prion protein (PrP) have been shown to exhibit both anti-prion and anti-amyloid properties particularly against prion proteins and the amyloid-β (Aβ) peptide active in Alzheimer’s disease. These disease-modifying properties are so far observed in cell cultures and in vitro. The CPP sequences are composed of a hydrophobic signal sequence followed by a highly positively charged hexapeptide segment. The original signal sequence of the prion protein can be changed to the signal sequence of the NCAM1 protein without losing the anti-prion activity. Although the detailed molecular mechanisms of these CPP peptides are not fully understood, they do form amyloid aggregates by themselves, and molecular interactions between the CPPs and PrP/Aβ can be observed in vitro using various spectroscopic techniques. These initial intermolecular interactions appear to re-direct the aggregation pathways for prion/amyloid formation to less cell-toxic molecular structures (i.e., co-aggregates), which likely is why the disease-inducing PrP/Aβ aggregation is counteracted in vivo.

Keywords
protein aggregation, secretion signal peptide, peptide engineering, drug design
National Category
Neurosciences Biological Sciences
Identifiers
urn:nbn:se:su:diva-204559 (URN)10.3390/pharmaceutics14040823 (DOI)000785290300001 ()35456657 (PubMedID)2-s2.0-85128454673 (Scopus ID)
Available from: 2022-05-10 Created: 2022-05-10 Last updated: 2022-05-10Bibliographically approved
Wu, J., Österlund, N., Wang, H., Sternke-Hoffmann, R., Pupart, H., Ilag, L. L., . . . Luo, J. (2022). Identifying the role of co-aggregation of Alzheimer's amyloid-beta with amorphous protein aggregates of non-amyloid proteins. Cell Reports Physical Science, 3(9), Article ID 101028.
Open this publication in new window or tab >>Identifying the role of co-aggregation of Alzheimer's amyloid-beta with amorphous protein aggregates of non-amyloid proteins
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2022 (English)In: Cell Reports Physical Science, E-ISSN 2666-3864, Vol. 3, no 9, article id 101028Article in journal (Refereed) Published
Abstract [en]

Protein homeostasis collapse typically leads to protein aggregation into amyloid fibrils and diffuse amorphous aggregates, which both occur in Alzheimer’s and other neurodegenerative diseases, but their relationship remains to be clarified. Here we examine the interactions between the amorphously aggregated non-chaperone proteins (albumin, β-lactoglobulin, and superoxide dismutase 1) and Alzheimer’s amyloid-β (Aβ) peptides. Amorphous aggregates suppress the primary nucleation and elongation of Aβ fibrillation and modulate Aβ toxicity. The higher inhibitory efficiency of intermediately sized molten globular aggregates (20–300 nm) on Aβ fibrillation is hypothesized to be due to the higher amount of exposed hydrophobic residues and higher free energy. The formed co-aggregates are off-pathway species that favor formation of the amorphous end state instead of fibrillar amyloid structures normally formed by Aβ. Our findings expand our knowledge of how the native and aggregated cellular proteins modulate Aβ aggregation at the molecular and mesoscopic level and point out the major conclusions.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:su:diva-210647 (URN)10.1016/j.xcrp.2022.101028 (DOI)000865445200009 ()2-s2.0-85138140111 (Scopus ID)
Available from: 2022-10-25 Created: 2022-10-25 Last updated: 2022-10-25Bibliographically approved
Österlund, N., Vosselman, T., Leppert, A., Gräslund, A., Jörnvall, H., Ilag, L. L., . . . Landreh, M. (2022). Mass Spectrometry and Machine Learning Reveal Determinants of Client Recognition by Antiamyloid Chaperones. Molecular & Cellular Proteomics, 21(10), Article ID 100413.
Open this publication in new window or tab >>Mass Spectrometry and Machine Learning Reveal Determinants of Client Recognition by Antiamyloid Chaperones
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2022 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 21, no 10, article id 100413Article in journal (Refereed) Published
Abstract [en]

The assembly of proteins and peptides into amyloid fibrils is causally linked to serious disorders such as Alzheimer’s disease. Multiple proteins have been shown to prevent amyloid formation in vitro and in vivo, ranging from highly specific chaperone–client pairs to completely nonspecific binding of aggregation-prone peptides. The underlying interactions remain elusive. Here, we turn to the machine learning–based structure prediction algorithm AlphaFold2 to obtain models for the nonspecific interactions of β-lactoglobulin, transthyretin, or thioredoxin 80 with the model amyloid peptide amyloid β and the highly specific complex between the BRICHOS chaperone domain of C-terminal region of lung surfactant protein C and its polyvaline target. Using a combination of native mass spectrometry (MS) and ion mobility MS, we show that nonspecific chaperoning is driven predominantly by hydrophobic interactions of amyloid β with hydrophobic surfaces in β-lactoglobulin, transthyretin, and thioredoxin 80, and in part regulated by oligomer stability. For C-terminal region of lung surfactant protein C, native MS and hydrogen–deuterium exchange MS reveal that a disordered region recognizes the polyvaline target by forming a complementary β-strand. Hence, we show that AlphaFold2 and MS can yield atomistic models of hard-to-capture protein interactions that reveal different chaperoning mechanisms based on separate ligand properties and may provide possible clues for specific therapeutic intervention.

Keywords
structural proteomics, machine learning, protein misfolding, molecular chaperones
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:su:diva-211847 (URN)10.1016/j.mcpro.2022.100413 (DOI)000877820400002 ()36115577 (PubMedID)2-s2.0-85140395227 (Scopus ID)
Available from: 2022-11-28 Created: 2022-11-28 Last updated: 2023-09-04Bibliographically approved
Sahin, C., Østerlund, E. C., Österlund, N., Costeira-Paulo, J., Nedergaard Pedersen, J., Christiansen, G., . . . Otzen, D. E. (2022). Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization. Journal of the American Chemical Society, 144(27), 11949-11954
Open this publication in new window or tab >>Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization
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2022 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 144, no 27, p. 11949-11954Article in journal (Refereed) Published
Abstract [en]

α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinson’s disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation. 

National Category
Chemical Sciences Biological Sciences
Identifiers
urn:nbn:se:su:diva-208098 (URN)10.1021/jacs.2c03607 (DOI)000819232700001 ()35749730 (PubMedID)2-s2.0-85134426743 (Scopus ID)
Available from: 2022-08-19 Created: 2022-08-19 Last updated: 2022-09-28Bibliographically approved
Abramsson, M. L., Sahin, C., Hopper, J. T. S., Branca, R. M. M., Danielsson, J., Xu, M., . . . Landreh, M. (2021). Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry. JACS Au, 1(12), 2385-2393
Open this publication in new window or tab >>Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry
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2021 (English)In: JACS Au, E-ISSN 2691-3704, Vol. 1, no 12, p. 2385-2393Article in journal (Refereed) Published
Abstract [en]

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

Keywords
protein folding, gas-phase conformations, ion mobility mass spectrometry
National Category
Chemical Sciences Biological Sciences
Identifiers
urn:nbn:se:su:diva-202027 (URN)10.1021/jacsau.1c00458 (DOI)000746335000003 ()34977906 (PubMedID)
Available from: 2022-02-11 Created: 2022-02-11 Last updated: 2022-07-27Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0003-0905-7911

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