Amyloidogenesis of SARS-CoV-2 Spike Protein (2022, Nyström & Hammarström)

SNT Gatchaman

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Amyloidogenesis of SARS-CoV-2 Spike Protein
Sofie Nyström, Per Hammarström

Abstract
SARS-CoV-2 infection is associated with a surprising number of morbidities. Uncanny similarities with amyloid-disease associated blood coagulation and fibrinolytic disturbances together with neurologic and cardiac problems led us to investigate the amyloidogenicity of the SARS-CoV-2 spike protein (S-protein).

Amyloid fibril assays of peptide library mixtures and theoretical predictions identified seven amyloidogenic sequences within the S-protein. All seven peptides in isolation formed aggregates during incubation at 37 °C. Three 20-amino acid long synthetic spike peptides (sequence 192-211, 601-620, 1166-1185) fulfilled three amyloid fibril criteria: nucleation dependent polymerization kinetics by ThT, Congo red positivity, and ultrastructural fibrillar morphology.

Full-length folded S-protein did not form amyloid fibrils, but amyloid-like fibrils with evident branching were formed during 24 h of S-protein coincubation with the protease neutrophil elastase (NE) in vitro. NE efficiently cleaved S-protein, rendering exposure of amyloidogenic segments and accumulation of the amyloidogenic peptide 194-203, part of the most amyloidogenic synthetic spike peptide.

NE is overexpressed at inflamed sites of viral infection. Our data propose a molecular mechanism for potential amyloidogenesis of SARS-CoV-2 S-protein in humans facilitated by endoproteolysis. The prospective of S-protein amyloidogenesis in COVID-19 disease associated pathogenesis can be important in understanding the disease and long COVID-19.

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SNT Gatchaman

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We obtained a 316 peptide pool library (divided into two subpools) from a peptide scan through the entire SARS-CoV-2 S-protein ... we generated 20-AA peptides from the full-length SARS-CoV-2 S-protein ... aimed to address the most amyloidogenic sequences and used the WALTZ ... prediction algorithm to identify such segments
Seven amyloidogenic sequences distributed over the entire S-protein were identified and named according to the starting position in the S-protein. ... All but one of the predicted sequences are in β-sheet conformation in the SARS-CoV-2 Spike cryo-EM structure in its closed state.
Solubilized peptides were monitored for in vitro amyloid fibril formation kinetics using ThT, Congo red birefringence (CR), and negative stain transmission electron microscopy (TEM).
Spike192, Spike601, and Spike1166 fulfilled all our amyloid criteria: sigmoidal ThT kinetics, Congophilicity, and fibrillar ultrastructure. Spike192 formed exceptionally well-ordered fibrils comparable to a mix of all peptides
 

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SARS-CoV-2 S-protein is fairly stable (Tm > 50 °C) and would not readily denature spontaneously. Furthermore, such a large protein with complex folding will not easily misfold into an amyloid state. However, proteolysis is an obvious candidate mechanism. ... Endoproteolysis of precursor proteins is a well-known molecular initiation mechanism in several amyloidoses
SARS-CoV-2 S-protein is proteolyzed during infection by host furin-like enzymes and by serine proteases such as the transmembrane protease, serine 2 (TMPRSS2), at the cell surface and is further proteolyzed during inflammation.
Neutrophils act by phagocytosis of opsonized pathogens and by extracellular release of enzymes such as neutrophil elastase (NE). NE is a serine protease coupled to obstructive lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease, and alpha-1-antitrypsin deficiency.
in silico proteolytic cleavage by [neutrophil elastase] using Expasy Peptide cutter. One of the resulting peptides, Spike194−213, closely matched Spike192, only frame shifted by two amino acids.
subjected full-length SARS-CoV-2 S-protein to NE cleavage in vitro ... we discovered amyloid-like fibril formation upon proteolytic cleavage using TEM. Neither NE nor SARS-CoV-2 S-protein incubated alone formed fibrils. Fibrils were found only after co-incubation of the two proteins. ... fibrils showed unusual morphology with evident branching, suggesting involvement of proteolytically nicked S-protein within the fibril, rendering nodes for branching of different amyloidogenic sequences
 

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... performed LC-MS/MS analysis of peptides formed after 1 min and 6 h of digestion at 2:1 excess of NE

... identified 98 NE cleavage peptides from the S-protein structure and classified these into three groups: (i) formed after 1 min, (ii) formed after 1 min and still present after 6 h, and (iii) only present after 6 h
Several peptides only formed after 6 h of incubation. Strikingly, the peptide from segment 194−203 (FKNIDGYFKI, included in Spike192) was part of this group and was highly abundant after 6 h. Three peptides containing segments from our seven spike peptides were formed as free peptides still present after 6 h of co-incubation, two were digested early and disappeared, and two were likely still present in the parent nicked S-protein. Hence, the observed formed branched fibrils are likely composed of a mix of fibrils initiated by an amyloidogenic peptide seed recruiting nicked S-protein for elongation and branching.
We performed fibrillation experiments on the short fragment 194−203. The peptide was less amyloidogenic than Spike192. Formed fibrils of Spike194−203 were however amyloidogenic by our three criteria, ThT kinetics, Congo red birefringence, and fibrillar ultrastructure.
worth noting that the Spike194−203 peptide lacks one amino acid in the predicted amyloidogenic sequence. To test the significance of this amino acid deletion in the peptide, we performed a simple in silico mutation experiment where we substituted the final tyrosine in the amyloidogenic segment of Spike192 with a glycine to mimic its deletion. The in silico substitution Y204G abolished the amyloid prediction

Fibril “shaving” is known for several amyloidogenic proteins and peptides. It is possible that Y204 is cleaved after S-protein aggregation.
 

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We performed a thrombin induced fibrinogen to fibrin conversion followed by plasminogen tPA assay in the presence and absence of Spike peptide fibrils. The addition of 10 μg/mL amyloid fibrils formed from a mix of the seven spike peptides during fibrin formation decreased the fibrinolysis.
addition of 2% fibrils of Spike192 and 194−203 increased persistent plasmin indigestible fibrin. As expected, the more amyloidogenic Spike192 induced more plasmin resistant fibrin clots than did Spike194−203.
 

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... tested two fluorescent analogues of positron emission tomography (PET) amyloid tracers, CN-PiB and DF-9, known to bind to neurological Aβ amyloid and cardiac AL, AA, and ATTR amyloid and found strong binding with concomitant fluorescence response toward Spike192 fibrils in vitro. As a translational strategy, PET imaging may hence serve as an option for clinical studies to complement liquid biopsies to assess amyloid microclots.
We found that all common coronaviruses infecting humans contain amyloidogenic sequences. Nonetheless, the magnitude of diverse COVID-19 symptoms was not previously reported. The segment 194− 213 is unique for SARS-CoV-2 which, in combination with acute inflammation and neutrophil recruitment known to be more prevalent in COVID-19 compared to other viral infections, could explain the putative COVID-19 associated amyloid formation.