Search

EP-4735891-A1 - USES OF AEROLYSIN NANOPORES

EP4735891A1EP 4735891 A1EP4735891 A1EP 4735891A1EP-4735891-A1

Abstract

The invention relates to use of an aerolysin nanopore for the discrimination of different protein isoforms bearing different post-translational modifications (PTMs) and to methods for detecting adverse medical conditions associated with the presence of peptides, polypeptides or proteins bearing particular PTMs.

Inventors

  • CAO, Chan
  • DAL PERARO, Matteo
  • LASHUEL, HILAL

Assignees

  • ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE (EPFL)

Dates

Publication Date
20260506
Application Date
20240628

Claims (13)

  1. Claims 1. Use of an aerolysin nanopore for the discrimination of at least two isoforms of a peptide, polypeptide, or protein, wherein the at least two isoforms differ from each other at least in that they bear different post-translational modifications at the same position of the peptide, polypeptide, or protein.
  2. 2. Use according to claim 1 wherein at least one of the said post-translational modifications is a phosphorylation, a nitration or an oxidation, preferably a nitration or an oxidation.
  3. 3. Use according to claim 1 or 2, wherein the said peptide, polypeptide or protein is α-synuclein or a fragment thereof, preferably the C-terminal domain of α -synuclein, more preferably the residues 124 to 140 of α-synuclein.
  4. 4. Use according to claim 3, wherein the peptide, polypeptide or protein bears multiple PTMs at multiple residues, wherein the PTMs are selected from oxidation, nitration, and mixtures thereof.
  5. 5. A method of discriminating between at least two isoforms of a peptide, polypeptide, or protein, wherein the at least two isoforms differ from each other at least in that they bear different post-translational modifications at the same position of the peptide, polypeptide, or protein, the method comprising: a. optionally contacting one or more peptide, polypeptide or protein isoform(s) with a protease such as to cleave one or more fragment(s) comprising one or more amino acids of interest; b. placing a sample comprising the peptide, polypeptide or protein isoform(s) or the fragment(s) thereof obtained in step a) in a first compartment of a chamber separated into a first and a second compartments by a membrane holding an aerolysin nanopore, wherein the aerolysin nanopore connects both compartments of the chamber; c. triggering translocation of the peptide, polypeptide or protein isoform(s) or of the fragment(s) thereof obtained in step a) through the aerolysin nanopore by applying a potential, preferably a voltage potential, across the aerolysin nanopore; d. measuring at least one characteristic indicative of the type(s), number and position(s) of any post-translational modification(s) of the peptide, polypeptide or protein isoform(s) or of the fragment(s) thereof obtained in step a) in the aerolysin nanopore, wherein said characteristic is preferably selected from dwell time, averaged relative current, relative current variation, skewness of relative current, kurtosis of relative current, minimum of relative current, maximum of relative current, peak to peak variation, integrated current and combinations thereof; and e. identifying the type(s), number and position(s) of any post-translational modification of the peptide, polypeptide or protein isoform(s) or of the fragment(s) thereof obtained in step a) present in the sample by comparing the at least one characteristic measured in step d) with characteristics previously measured for specific peptide, polypeptide or protein isoforms or fragments thereof for which the type(s), number and position(s) of post-translational modification(s) were known.
  6. 6. A method of detecting in a subject a disease or disorder associated with the presence of one or more PTM(s) of an aberrant type on one or more amino acid(s) of interest in a peptide, polypeptide or protein instead of one or more PTM(s) of a healthy type on the same amino acid(s), the method comprising: a. optionally contacting the peptide, polypeptide or protein from the subject with a protease such as to cleave one or more fragment(s) comprising one or more amino acids of interest; b. placing a sample comprising the peptide, polypeptide or protein from the subject or the fragment(s) thereof obtained in step a) in a first compartment of a chamber separated into a first and a second compartments by a membrane holding an aerolysin nanopore, wherein the aerolysin nanopore connects both compartments of the chamber; c. triggering translocation of the peptide, polypeptide or protein or of the fragment(s) thereof obtained in step a) through the aerolysin nanopore by applying a potential, preferably a voltage potential, across the aerolysin nanopore; d. measuring at least one characteristic indicative of the type(s), number and position(s) of any post-translational modification(s) of the peptide, polypeptide or protein or of the fragment(s) thereof obtained in step a) in the aerolysin nanopore, wherein said characteristic is preferably selected from dwell time, averaged relative current, relative current variation, skewness of relative current, kurtosis of relative current, minimum of relative current, maximum of relative current, peak to peak variation, integrated current and combinations thereof; and e. identifying the type(s), number and position(s) of any post-translational modification(s) of the peptide, polypeptide or protein or of the fragment(s) thereof obtained in step a) by comparing the at least one characteristic measured in step d) with characteristics previously measured for specific peptides, polypeptides or proteins or fragments thereof for which the type(s), number and position(s) of post-translational modification(s) was known.
  7. 7. The method of claim 5 or 6, wherein the peptide, polypeptide or protein is α- synuclein or a fragment thereof.
  8. 8. The method of claim 7, wherein the α-synuclein fragment is a C-terminal fragment of α-synuclein, preferably comprising the residues 124 to 140 of α -synuclein.
  9. 9. The method of any one of claims 5 to 8, wherein step e. is performed using a deep learning algorithm trained with a database comprising characteristics previously measured for specific isoforms for which the type(s), number and position(s) of post-translational modification(s) was known.
  10. 10.The method according to any one of claims 6 to 9, wherein the disorder associated with the presence of a PTM of an aberrant type on one or more amino acid(s) of interest in a peptide, polypeptide or protein instead of a PTM(s) of a healthy type on the same amino acid is a neurodegenerative disease, preferably a synucleinopathy, more preferably selected from Alzheimer disease and Parkinson diseases.
  11. 11.The use according to any one of claims 1 to 4 or the method according to any one of claims 5 to 10, wherein the peptide, polypeptide or protein or the α- synuclein or fragment thereof is in unfolded form, and wherein the aerolysin nanopore is in folded form.
  12. 12. The use according to any one of claims 1 to 4 or the method according to any one of claims 5 to 11, wherein the peptide, polypeptide, or protein or α- synuclein or the fragment thereof is provided in a biological sample, preferably plasma or a sample comprising red blood cells, preferably wherein haemoglobin proteins have been removed.
  13. 13.The use according to any one of claims 1 to 4 or the method according to any one of claims 5 to 12, wherein the peptide, polypeptide, or protein or α- synuclein or the fragment thereof is provided in a biological sample diluted with a biologically compatible aqueous solvent at a biological sample to solvent ratio of 1:10 to 1:50. The use according to any one of claims 1 to 4 or the method according to any one of claims 5 to 13, wherein the aerolysin nanopore comprises a monomer consisting of a mutant polypeptide selected from a. a polypeptide comprising a first amino acid substitution selected from the group consisting of K238A, K238S, K238G and K238D of the amino acid sequence of any one of SEQ ID NO:1, 2, 4, 6, 7, 8, 10, 12, 14 or 15 or a variant or fragment thereof; and b. a polypeptide comprising a first amino acid substitution selected from the group consisting of V238A, V238S, V238G and V238D of the amino acid sequence of any one of SEQ ID NO:3 or 11 or a variant or fragment thereof. The use or the method according to claim 14, wherein the aerolysin monomer further comprises at least one of - a second amino acid substitution selected from the group consisting of K242W and K242A, preferably K242A when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 6, 7, 8, 10, 12, 14 and 15 of variants or fragments thereof or a second amino acid substitution selected from the group consisting of N242W and N242A, preferably N242A, when the sequence is selected from any one of SEQ ID NO:3 or 11 or a variant or fragment thereof or a second amino acid substitution selected from the group consisting of S242W and S242A, preferably S242A, when the sequence is selected from any one of SEQ ID NO:5 or 13 or a variant or fragment thereof. - a third amino acid substitution selected from the group consisting of R282A, R282S, R282G, R282D, R282W, preferably selected from the group consisting of R282A and R282S, most preferably R282S when the sequence is selected from any one of SEQ ID NO:1,2, 4, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a third amino acid substitution selected from P282A, P282S, P282G, P282D, P282W, preferably selected from the group consisting of P282A and P282S, most preferably P282S when the sequence is selected from SEQ ID NO:3 and 11 or a variant or fragment thereof; - a fourth amino acid substitution selected from the group consisting of D216A, D216S, D216G, D216D and D216W, preferably selected from the group consisting of D216A and D216S, most preferably D216S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 7, 8, 10, 12 and 15 or a variant or fragment thereof or a fourth amino acid substitution selected from selected from the group consisting of V216A, V216S, V216G, V216D and V216W, preferably selected from the group consisting of V216A and V216S, most preferably V216S when the sequence is selected from SEQ ID NO: 3, 6, 11 or 14 or a variant or fragment thereof or a fourth amino acid substitution selected from selected from the group consisting of G216A and G216S, most preferably V216S when the sequence is selected from SEQ ID NO: 5 and 13; - a fifth amino acid substitution selected from the group consisting of D222A, D222S, D222G and D222D, preferably selected from the group consisting of D222A and D222S, most preferably D222S when the sequence is selected from any one of SEQ ID NO:1,2, 4, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a fifth amino acid substitution selected the group consisting of K222A, K222S, K222G and K222D, preferably selected from the group consisting of K222A and K222S, most preferably K222S when the sequence is selected from SEQ ID NO:3 or 11 or a variant or fragment thereof or a fifth amino acid substitution selected from the group consisting of N222A, N222S, N222G and N222D, preferably selected from the group consisting of N222A and N222S, most preferably N222S when the sequence is selected from any one of SEQ ID NO: 5 and 13; - a sixth amino acid substitution selected from the group consisting of E258A, E258S, E258G, E258D and E258W, preferably selected from the group consisting of E258A and E258S, most preferably E258S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a sixth amino acid substitution selected from the group consisting of I258A, I258S, I258G, I258D and I258W, preferably selected from the group consisting of I258A and I258S, most preferably I258S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof; - a seventh amino acid substitution selected from the group consisting of E254A, E254S, E254G, E254D and E254W, preferably selected from the group consisting of E254A and E254S, most preferably E254S when the sequence is selected from any one of SEQ ID NO:1,2, 4, 5, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a seventh amino acid substitution selected from the group consisting of L254A, L254S, L254G, L254D and L254W, preferably selected from the group consisting of L254A and L254S, most preferably L254S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof; - an eighth amino acid substitution selected from the group consisting of K244A, K244S, K244G, K244D and K244W, preferably selected from the group consisting of K244A and K244S, most preferably K244S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 5, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or an eighth amino acid substitution selected from the group consisting of F244A, F244S, K244G, F244D and F244W, preferably selected from the group consisting of F244A and F244S, most preferably F244S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof; - a ninth amino acid substitution selected from the group consisting of E252A, E252S, E252G, E252D and E252W, preferably selected from the group consisting of E252A and E252S, most preferably E252S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a ninth amino acid substitution selected from the group consisting of T252A, T252S, T252G, T252D and T252W, preferably selected from the group consisting of T252A and T252S, most preferably T252S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof or a ninth amino acid substitution selected from the group consisting of V252A, V252S, V252G, V252D and V252W, preferably selected from the group consisting of V252A and V252S, most preferably V252S when the sequence is selected from any one of SEQ ID NO: 5 and 13 or a variant or fragment thereof; and - a tenth amino acid substitution selected from the group consisting of K246S, K246A K246G, K246D and K246W, preferably selected from the group consisting of K246A and K246S, most preferably K246S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 5, 7, 8, 10, 12, 13, and 15 or a variant or fragment thereof or a tenth amino acid substitution selected from the group consisting of W246S, W246A W246G, W246D and W246W, preferably selected from the group consisting of W246A and W246S, most preferably W246S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof or a tenth amino acid substitution selected from the group consisting of Q246S, Q246A Q246G, Q246D and Q246W, preferably selected from the group consisting of Q246A and Q246S, most preferably Q246S when the sequence is selected from any one of SEQ ID NO: 6 and 14 or a variant or fragment thereof.

Description

Uses of aerolysin nanopores Technical Field [0001] The invention relates to use of an aerolysin nanopore for the discrimination of different protein isoforms bearing different post- translational modifications (PTMs) and to methods for detecting adverse medical conditions associated with the presence of peptides, polypeptides or proteins bearing particular PTMs. Background Art [0002] Using biological nanopores to sequence biopolymers, particularly nucleic acids, was proposed many years ago. Recent advances in enzyme-based control of DNA translocation and in DNA nucleotide resolution using mutated biological pores have satisfied the needs for a functional DNA sequencing biological device. [0003] Nanopore sensing is an approach that relies on the exploitation of individual binding or interaction events between to-be-analysed molecules and pore-forming macromolecules. Nanopore sensors can be created by placing nanometric-scaled pore peptide structures in an insulating membrane and measuring voltage-driven ionic transport through the pore in the presence of peptides, polypeptides or proteins. The identity of a peptide, polypeptide or protein can be ascertained through its peculiar electric signature, particularly the duration and extent of current block and the variance of current levels. Two of the essential components of nanopore sensing are (1) the control of analyte movement through the pore and (2) the discrimination of nucleotides as the analyte is moved through the pore. [0004] Pore-forming proteins are produced by a variety of organisms and are often involved in defence or attack mechanisms. One notable feature is that they are produced as soluble proteins that subsequently oligomerize and convert into a transmembrane pore in the target membrane. The most extensively characterized pore-forming proteins are the bacterial pore-forming toxins (PFTs), which, depending on the secondary structure elements that cross the bilayer, have been classified as α- or β-PFTs. [0005] Aerolysin, produced by Aeromonas species, is the founding member of a large superfamily that spans all of the kingdoms of life. Although it was the first β-PFT for which the X-ray structure of the soluble form was solved, the structure of the pore has remained elusive for long time. Aerolysin forms a heptameric beta-barrel in biological membranes. It is secreted as a monomer that binds to the outer membrane of susceptible cells. Upon binding, the monomers oligomerize to form a water-filled transmembrane channel that facilitates uncontrolled permeation of water, ions, and small organic molecules. Rapid discharge of vital molecules, such as ATP, dissipation of the membrane potential and ionic gradients, and irreversible osmotic swelling leading to rupture or lysis of the cell wall, frequently causing death of the host cell. This pore-forming property has been identified as a major mechanism by which protein toxins cause damage to cells. [0006] Aerolysin is the most promising biological nanopore for identifying the subtle differences between detected molecules, because of its unique structure profile. Cao C. et al. (Nat Nanotechnol. 2016 Apr 25. doi: 10.1038/nnano.2016.66) demonstrated the ability of aerolysin nanopore to resolve at high resolution individual short oligonucleotides that are 2 to 10 bases long without any extra chemicals or modifications, useful for single-molecule analysis of oligonucleotides. [0007] Additionally, Cao C. et al. (Nature Communications volume 9, Article number: 2823, 2018) described nanopore experimental results and molecular simulations based on an aerolysin structural model to map the sensing spots for ssDNA translocation. Computational and experimental results revealed two critical sensing spots (R220, K238) generating two constriction points along the pore lumen. Taking advantage of the sensing spots, all four nucleobases, cytosine methylation and oxidation of guanine can be clearly identified in a mixture sample. [0008] Beyond sequencing of biological polymers, there is a need for methods that make it possible to identify protein post-translational modifications. Protein post-translational modifications (PTMs) play crucial roles in biology and have emerged as reliable biomarkers for several diseases. However, only a handful of techniques are available to accurately measure their levels, capture their complexity at a single molecule level and characterize their multifaceted roles in health and disease. [0009] The standard methods currently available for the detection of PTMs are mass spectrometry (MS) and enzyme linked antibody-based assays. Recently great progress has been made towards developing and improving MS methods and immunoassays to map, detect and quantify modified proteins in biological samples. For instance, proximity extension assays and single molecule array have shown a high sensitivity for detecting PTMs. However, these techniques have fundamental drawbacks in terms of detection of the