Amyloid-β, tau, and α-synuclein disorders
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Sengupta and Kayed, Progress in Neurobiology, 2022,04
Fig. 1. Aβ, tau and α-Syn proteinopathies. (A) Overlapping protein pathologies of Aβ, tau and α-Syn in several neurodegenerative diseases. (B) Molecular cross-talks among the trio depicting Aβ/tau, α-Syn/tau and Aβ/α-Syn interactions and their plausible convergence into a combined effect. Each interacting protein pair has direct and indirect effects culminating into impaired cellular processes. Blue continuous line represents direct interaction and purple dotted line represents indirect interaction.
Fig. 2. Mixed brain protein pathologies in several neurodegenerative diseases. The three most widely studied protein pathologies of Aβ, tau and α-Syn interact mechanistically in AD and related disorders where they often co-exist. Studies show that interacting networks of co-pathologies affect neuron/glia brain cell biology leading to several cellular dysfunctions such as proteostasis, synaptic, mitochondrial, and vascular dysfunctions and inflammation.
Käufer et al., eBioMedicine, 2022.04
Background “Neurological symptoms such as cognitive decline and depression contribute substantially to post-COVID-19 syndrome, defined as lasting symptoms several weeks after initial SARS-CoV-2 infection. The pathogenesis is still elusive, which hampers appropriate treatment. Neuroinflammatory responses and neurodegenerative processes may occur in absence of overt neuroinvasion.”
Findings “Viral protein in the nasal cavity led to pronounced microglia activation in the olfactory bulb beyond viral clearance. Cortical but not hippocampal neurons accumulated hyperphosphorylated tau and alpha-synuclein, in the absence of overt inflammation and neurodegeneration. Importantly, not all brain regions were affected, which is in line with selective vulnerability.”
Interpretation Thus, despite the absence of virus in brain, neurons develop signatures of proteinopathies that may contribute to progressive neuronal dysfunction.”
Introduction “In progressive neurodegenerative diseases, alpha-synuclein and tau are upregulated as part of the neuronal stress response, leading to intracellular accumulation, post-translational modification/truncation, loss or gain of toxic function, and perpetuating toxicity to the neuron. These processes are hallmarks of synucleinopathies (e.g. Parkinson’s disease) or tauopathies (e.g. Alzheimers disease, progressive supranuclear palsy). In fact, neurodegenerative disorders display protein pathology in the olfactory bulb, typically associated with deficits in olfactory perception in the very early phase of these diseases. Such pathology can spread prion-like across neurons, or even from the gut nervous system to the brain. Therefore, even subtle local alterations can present a starting point to more widespread progressive pathology. Here we present evidence that upon intranasal inoculation of SARS-CoV-2 in hamsters, neurodegenerative processes are initiated from microglia activation in the olfactory bulb to accumulation of hyper-phosphorylated tau and alpha-synuclein protein in cortical neurons.”
Figure 1 b. Viral S2 protein was stained in olfactory turbinates of hamsters following SARS-CoV-2 or mock infection and infection burden was semiquantitatively scored (no virus, low, medium and high degree of virus). c. Immunofluorescent images from nasal tissue stained against IBA1 (myeloid cells), viral S2 protein and DAPI (cell nuclei) from 3 days post infection (mock and SARS-CoV-2 infection). Note the high number of Iba1-positive cells in infected noses compared to the low number of Iba1-positive myeloid cells in the mock infected tissue (hollow arrowheads in Iba-1 (red) images). Also a vast amount of viral S2 protein in different parts of the nasal tissue can be observed (white arrow heads in viral S2 protein (green) images). m. Iba1-positive cells (i.e. microglia) in the olfactory bulb from mock and SARS-CoV-2 infected hamsters. While cells from mock infected animals appear to be resting. i.e. they have small cell bodies and fine protrusions (hollow arrowheads), cells from SARS-CoV-2 infected animals appear activated (white arrowheads), i.e. they have enlarged cell bodies and processes, on both 3 and 14 dpi.
Brain Inflammation and Intracellular α-Synuclein Aggregates in Macaques after SARS-CoV-2 Infection
Philippens et al., Viruses, 2022.03
“SARS-CoV-2 causes acute respiratory disease, but many patients also experience neurological complications. Neuropathological changes with pronounced neuroinflammation have been described in individuals after lethal COVID-19, as well as in the CSF of hospitalized patients with neurological complications. To assess whether neuropathological changes can occur after a SARS-CoV-2 infection, leading to mild-to-moderate disease, we investigated the brains of four rhesus and four cynomolgus macaques after pulmonary disease and without overt clinical symptoms. Postmortem analysis demonstrated the infiltration of T-cells and activated microglia in the parenchyma of all infected animals, even in the absence of viral antigen or RNA. Moreover, intracellular α-synuclein aggregates were found in the brains of both macaque species. The heterogeneity of these manifestations in the brains indicates the virus’ neuropathological potential and should be considered a warning for long-term health risks, following SARS-CoV-2 infection.”
Figure 3. Overview of CNS effects of SARS-CoV-2 infection detected in a macaque brain in this study. The presence of viral RNA was investigated in multiple regions of the brain, as indicated by the numbers. Viral RNA-positive regions in cynomolgus macaque C3 are indicated by a yellow background. Brain areas with infiltrated T-cells (CD3+) and activated microglia (Mamu-DR+) are shown in light blue (low expression) and dark blue (moderate expression). Brain areas with Lewy bodies (α-synuclein+) are depicted in orange. Blue and orange areas comprise of the combined observations from all animals. The horizontal dotted line indicates the border between the dorsal and ventral parts of the brain.
Figure 4. SARS-CoV-2 causes brain inflammation and Lewy body formation in brains of macaques. The immunohistochemistry of macaque brain tissues (20×). Arrows indicate the presence of some clear positive cells for the immunohistochemical staining. First and second row: CD3+ T-cells. In infected animals, T-cells were found in the pituitary gland (A), perivascular (B,C), and in the brain parenchyma (D). Third row: Mamu-DR+-activated microglia cells. Activated microglia cells are shown in the pituitary gland of R3 (E). Amoeboid microglia cells are shown in the olfactory bulb of C3 (F). Bottom panel: α-Synuclein positive staining was found in the ventral midbrain in all SARS-CoV-2-infected rhesus macaques. α-Synuclein accumulations were found in the ventral midbrain region, next to the caudate nucleus (G). A positive control of the α-synuclein staining of a brain slice from a 22-year-old cynomolgus monkey, showing signs of parkinsonism from the brain bank of the BPRC, is shown (H).
Kell et al., Biochemical Journal, 2022.02
“Post-acute sequelae of COVID (PASC), usually referred to as ‘Long COVID’ (a phenotype of COVID-19), is a relatively frequent consequence of SARS-CoV-2 infection, in which symptoms such as breathlessness, fatigue, ‘brain fog’, tissue damage, inflammation, and coagulopathies (dysfunctions of the blood coagulation system) persist long after the initial infection. It bears similarities to other post-viral syndromes, and to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Many regulatory health bodies still do not recognize this syndrome as a separate disease entity, and refer to it under the broad terminology of ‘COVID’, although its demographics are quite different from those of acute COVID-19. A few years ago, we discovered that fibrinogen in blood can clot into an anomalous ‘amyloid’ form of fibrin that (like other β-rich amyloids and prions) is relatively resistant to proteolysis (fibrinolysis). The result, as is strongly manifested in platelet-poor plasma (PPP) of individuals with Long COVID, is extensive fibrin amyloid microclots that can persist, can entrap other proteins, and that may lead to the production of various autoantibodies. These microclots are more-or-less easily measured in PPP with the stain thioflavin T and a simple fluorescence microscope. Although the symptoms of Long COVID are multifarious, we here argue that the ability of these fibrin amyloid microclots (fibrinaloids) to block up capillaries, and thus to limit the passage of red blood cells and hence O2 exchange, can actually underpin the majority of these symptoms. Consistent with this, in a preliminary report, it has been shown that suitable and closely monitored ‘triple’ anticoagulant therapy that leads to the removal of the microclots also removes the other symptoms. Fibrin amyloid microclots represent a novel and potentially important target for both the understanding and treatment of Long COVID and related disorders.”
“Conclusions: Here we have argued, and focus on the fact, that Long COVID is characterized by the presence of persistent fibrin amyloid microclots that might block capillaries and inhibit the transport of O2 to tissues, entrapping numerous inflammatory molecules, including those that prevent clot breakdown (as we have indeed recently shown) (Figure 10).
In addition to microclot formation, significant platelet dysfunction and a systemic endotheliitis drive systemic cellular hypoxia. These pathologies can explain most, if not all, of the lingering symptoms to which individuals with long COVID refer. We have noted that amyloid microclots, platelet hyperactivation and endothelial dysfunction, might form a suitable set of foci for the clinical treatment of the symptoms of long COVID. Therefore, if fibrinaloid microclots are largely responsible for the symptoms of Long COVID, their removal is to be seen as paramount for relieving these symptoms and allowing the body to repair itself.”
Figure 7. A simplified diagram to explain microclot formation that might either be resolved via fibrinolytic processes after acute COVID-19 or, in some patients, result in a failed fibrinolytic process.
Figure 10. Some of the sequelae of fibrinaloid microclot formation in the symptomology of Long COVID. Many others, such as a role for auto-antibodies, are not shown.
Amyloid and Hydrogel Formation of a Peptide Sequence from a Coronavirus Spike Protein
Castelletto and Hamley, American Chemical Society Nano, 2022.01
“We demonstrate that a conserved coronavirus spike protein peptide forms amyloid structures, differing from the native helical conformation and not predicted by amyloid aggregation algorithms. We investigate the conformation and aggregation of peptide RSAIEDLLFDKV, which is a sequence common to many animal and human coronavirus spike proteins. This sequence is part of a native α-helical S2 glycoprotein domain, close to and partly spanning the fusion sequence. This peptide aggregates into β-sheet amyloid nanotape structures close to the calculated pI = 4.2, but forms disordered monomers at high and low pH. The β-sheet conformation revealed by FTIR and circular dichroism (CD) spectroscopy leads to peptide nanotape structures, imaged using transmission electron microscopy (TEM) and probed by small-angle X-ray scattering (SAXS). The nanotapes comprise arginine-coated bilayers. A Congo red dye UV–vis assay is used to probe the aggregation of the peptide into amyloid structures, which enabled the determination of a critical aggregation concentration (CAC). This peptide also forms hydrogels under precisely defined conditions of pH and concentration, the rheological properties of which were probed. The observation of amyloid formation by a coronavirus spike has relevance to the stability of the spike protein conformation (or its destabilization via pH change), and the peptide may have potential utility as a functional material. Hydrogels formed by coronavirus peptides may also be of future interest in the development of slow-release systems, among other applications.”
Figure 1. Structure of the porcine deltacoronavirus spike protein, obtained from high resolution cryo-EM. (3,4) The sequence RSAIEDLLFNKV is highlighted (in red); this is the closest sequence to RSAIEDLLFDKV for which a pdb file could be obtained. The spike has a trimeric structure. Top, side and top views; bottom, enlargement of RSAIEDLLFNKV region with residue numbers for the A chain. This clearly lies in a surface coil sequence.
Effect of an amyloidogenic SARS-COV-2 protein fragment on α-synuclein monomers and fibrils
Jana et al., bioRxiv, 2022.02
“Using molecular dynamic simulations we study whether amyloidogenic regions in viral proteins can initiate and modulate formation of α-synuclein aggregates, thought to be the disease-causing agent in Parkinson’s Disease. As an example we choose the nine-residue fragment SFYVYSRVK (SK9), located on the C-terminal of the Envelope protein of SARS-COV-2. We probe how the presence of SK9 affects the conformational ensemble of α-synuclein monomers and the stability of two resolved fibril polymorphs. We find that the viral protein fragment SK9 may alter α-synuclein amyloid formation by shifting the ensemble toward aggregation-prone and preferentially rod-like fibril seeding conformations. However, SK9 has only little effect of the stability of pre-existing or newly-formed fibrils.”
Figure 6: Representative final configurations extracted from simulations starting from (a) the experimentally determined rod-like α-synuclein fibril model (PDB-ID: 6CU7) and (c) the extended model. Corresponding final snapshots extracted from simulations in the presence of SK9-segment are shown in (b), and (d). N- and C-terminus are represented by blue and red spheres, respectively. Only residues 38-97 are shown for the extended model configurations in (c) and(d). The time evolution of the RMSD in the simulation of these systems is shown in (e), and residue-wise RMSF in (f). We calculate RMSD and RMSF again only for the experimentally resolved region 38-97, i.e., ignoring the disordered and unresolved parts of the fibril models, considering all backbone atoms.
MSH3 Homology and Potential Recombination Link to SARS-CoV-2 Furin Cleavage Site
Ambati et al., Frontiers in Virology, 2022.02
“Among numerous point mutation differences between the SARS-CoV-2 and the bat RaTG13 coronavirus, only the 12-nucleotide furin cleavage site (FCS) exceeds 3 nucleotides. A BLAST search revealed that a 19 nucleotide portion of the SARS.Cov2 genome encompassing the furing cleavage site is a 100% complementary match to a codon-optimized proprietary sequence that is the reverse complement of the human mutS homolog (MSH3). The reverse complement sequence present in SARS-CoV-2 may occur randomly but other possibilities must be considered. Recombination in an intermediate host is an unlikely explanation. Single stranded RNA viruses such as SARS-CoV-2 utilize negative strand RNA templates in infected cells, which might lead through copy choice recombination with a negative sense SARS-CoV-2 RNA to the integration of the MSH3 negative strand, including the FCS, into the viral genome. In any case, the presence of the 19-nucleotide long RNA sequence including the FCS with 100% identity to the reverse complement of the MSH3 mRNA is highly unusual and requires further investigations.”
Figure 1. The origin of the furin sequence in SARS-CoV-2. Comparison of the protein sequences at the S1/S2 junction in SARS-CoV, RaTG13, & SARS-CoV-2 demonstrating the presence of the furin cleavage site (FCS) PRRA only in SARS-CoV-2. Based on a BLAST search of the 12-nucleotide stretch coding for the FCS PRRA, a 19-nucleotide long identical sequence was identified in the patented (US 958 7003) sequence Seq ID11652. SEQ ID11652 is transcribed to a MSH3 mRNA that appears to be codon optimized for humans. This 19-nucleotide sequence including 12 nucleotides coding for the FCS PRRA, present in the human MSH3 gene might have been introduced into the SARS-CoV-2 genome by the illustrated copy choice recombination mechanism in SARS-CoV-2 infected human cells overexpressing the MSH3 gene.
Amyloidogenesis of SARS-CoV-2 Spike Protein
Nyström and Hammarström, bioRxiv, 2021.12
“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 191-210, 599-618, 1165-1184) 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 hours of S-protein co-incubation with the protease neutrophil elastase (NE) in vitro. NE efficiently cleaved S-protein rendering exposure of amyloidogenic segments and accumulation of the peptide 193-202, part of the most amyloidogenic synthetic Spike peptide. NE is overexpressed at inflamed sites of viral infection and at vaccine injection sites. Our data propose a molecular mechanism for amyloidogenesis of SARS-CoV-2 S-protein in humans facilitated by endoproteolysis. The potential implications of S-protein amyloidogenesis in COVID-19 disease associated pathogenesis and consequences following S-protein based vaccines should be addressed in understanding the disease, long COVID-19, and vaccine side effects.”
Figure 1 A. The structure of one protomer of the trimeric SARS-CoV-2 S-protein in its closed state, PDB code: 6VXX [8] with the predicted full sequence of the amyloidogenic peptides highlighted in the same colors as the predictions in Table 1. B. Conformation of peptides within the folded S-protein in comparison with Alpha Fold 2 models of the synthetic peptides (Table 1).
Figure 3. Amyloid fibrillation of 7 mixed SARS-CoV-2 S-peptides (total concentration 0.1 mg/ml). A. Fibril formation kinetics monitored by ThT fluorescence. B. Fibrillar structures by negative stain TEM. C. Fibrillar structures by negative stain TEM of Spike191 resembling the mix.
Interactions between SARS-CoV-2 N-Protein and α-Synuclein Accelerate Amyloid Formation
Semerdzhiev et al., ACS Chemical Neuroscience, 2021.12
“First cases that point at a correlation between SARS-CoV-2 infections and the development of Parkinson’s disease (PD) have been reported. Currently, it is unclear if there is also a direct causal link between these diseases. To obtain first insights into a possible molecular relation between viral infections and the aggregation of α-synuclein protein into amyloid fibrils characteristic for PD, we investigated the effect of the presence of SARS-CoV-2 proteins on α-synuclein aggregation. We show, in test tube experiments, that SARS-CoV-2 spike protein (S-protein) has no effect on α-synuclein aggregation, while SARS-CoV-2 nucleocapsid protein (N-protein) considerably speeds up the aggregation process. We observe the formation of multiprotein complexes and eventually amyloid fibrils. Microinjection of N-protein in SH-SY5Y cells disturbed the α-synuclein proteostasis and increased cell death. Our results point toward direct interactions between the N-protein of SARS-CoV-2 and α-synuclein as molecular basis for the observed correlation between SARS-CoV-2 infections and Parkinsonism.”
“We have identified a SARS-CoV-2 protein that induces the aggregation of αS in the test tube. In the initial interaction between the SARS-CoV-2 N-protein and αS, multiprotein complexes are formed. In the presence of N-protein, the onset of αS aggregation into amyloid fibrils is strongly accelerated, indicating that N-protein facilitates the formation of a critical nucleus for aggregation. Fibril formation is not only faster but it also proceeds in an unusual two-step process. In cells, the presence of N-protein changes the distribution of αS over different conformations that likely represent different functions at already short time scales. Disturbance of αS proteostasis might be a first step toward nucleation of fibrils. Our results point toward a direct interaction between the N-protein of SARS-CoV-2 and αS as a molecular basis for the observed relations between virus infections and Parkinsonism. The observed molecular interactions thus suggest that SARS-CoV-2 infections may have long-term implications and that caution is required in considering N-protein as an alternative target in vaccination strategies.”
SARS-CoV-2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro
Jiang and Mei, Viruses, 2021.10
“Severe acute respiratory syndrome coronavirus 2 (SARS–CoV–2) has led to the coronavirus disease 2019 (COVID–19) pandemic, severely affecting public health and the global economy. Adaptive immunity plays a crucial role in fighting against SARS–CoV–2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID–19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS–CoV–2 impedes adaptive immunity remains unclear. Here, by using an in vitro cell line, we report that the SARS–CoV–2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full-length spike-based vaccines.”
Fig. 1. Effect of severe acute respiratory syndrome coronavirus 2 (SARS–CoV–2) nuclear-localized proteins on DNA damage repair. (A) Subcellular distribution of the SARS–CoV–2 proteins. Immunofluorescence was performed at 24 h after transfection of the plasmid expressing the viral proteins into HEK293T cells. Scale bar: 10 µm. (B) Schematic of the EJ5-GFP reporter used to monitor non-homologous end joining (NHEJ).
Ryu et al., bioRxiv, 2021.10
“SARS-CoV-2 spike induces structurally abnormal blood clots and thromboinflammation neutralized by a fibrin-targeting antibody.”
“Blood clots are a central feature of coronavirus disease-2019 (COVID-19) and can culminate in pulmonary embolism, stroke, and sudden death. However, it is not known how abnormal blood clots form in COVID-19 or why they occur even in asymptomatic and convalescent patients. Here we report that the Spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the blood coagulation factor fibrinogen and induces structurally abnormal blood clots with heightened proinflammatory activity. SARS-CoV-2 Spike virions enhanced fibrin-mediated microglia activation and induced fibrinogen-dependent lung pathology. COVID-19 patients had fibrin autoantibodies that persisted long after acute infection. Monoclonal antibody 5B8, targeting the cryptic inflammatory fibrin epitope, inhibited thromboinflammation. Our results reveal a procoagulant role for the SARS-CoV-2 Spike and propose fibrin-targeting interventions as a treatment for thromboinflammation in COVID-19.”
Fig. 1. Characterization of the interaction between SARS-CoV-2 Spike and fibrinogen.
(A) Turbidity assay of fibrin polymerization in healthy human donor plasma in the presence or absence of Spike protein. Data are representative of four independent experiments with similar results. (B) SEM images of fibrin clots in healthy human donor plasma or in the presence of Spike protein. Scale bar, 1 µm. Topographic visualization of fibrin fiber surface in SEM images. Quantification of fibrin fiber radius and intersection density. Data are from three independent experiments (mean ± s.e.m.). ****P < 0.0001, ***P < 0.001 (multiple testing Holm procedure and two-tailed Mann-Whitney test). (C) ELISA of the binding of recombinant SARS-CoV-2 Spike protein (Spike) to fibrinogen or fibrin presented as absorbance at 450 nm (A450), plus the dissociation constants (Kd). Representative binding curvefits are shown from two independent experiments performed in duplicates (mean ± s.e.m.). (D). Immunoprecipitation of fibrinogen with His-tagged recombinant trimeric SARS-CoV-2 Spike protein produced in CHO cells (i) or monomeric SARS-CoV-2 Spike produced in E.coli (ii) blotted with anti-spike, anti-His or anti-fibrinogen. Representative immunoblots from three independent experiments are shown. (E) Peptide array mapping with immobilized peptides of fibrinogen chains Aα, Bβ, and γ blotted with Spike protein. Heatmap of signal intensity showing binding sites (red-orange) indicated by their aa sequences on chains Bβ (β119-129) and γ (γ163-181 and γ364-395). Color key indicates fluorescence intensities signal values from low (white) to high (red). Crystal structure of fibrinogen (PDB:3GHG) showing the three mapped peptides β119-129, γ163-181 and γ364-395 (red). Structural proximity of the γ163-181 and γ364-395 peptides indicating a 3D conformational epitope (inset). (F) Immunoblot of fibrin degradation after 0, 1, 2, 4 and 6 h of plasmin digestion. Data are from five (timepoints 0, 2 and 4 h) or three (timepoints 1 and 6 h) independent experiments (mean ± s.e.m.). Representative immunoblot is shown. (G) Quantification of ROS production detected with dihydroethidum in unstimulated BMDMs or stimulated for 24 h with fibrin in the presence of Spike protein. Data are from three independent experiments (mean ± s.e.m.). *P < 0.05; **P < 0.01; ***P < 0.001 (one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test).
Laudicella et al., SN Comprehensive Clinical Medicine, 2021.09
“Introduction: Large-scale worldwide COVID-19 vaccination programs are being rapidly deployed, and high-risk patients with comorbidity are now receiving the first doses of the vaccine. Physicians should be, therefore, aware of new pitfalls associated with the current pandemic vaccination program, also in the case of [18F]Florbetaben PET/CT.
Case Presentation: We described the first image of [18F]Florbetaben PET/CT in the evaluation of a 70-year-old male with suspicious Alzheimer disease and unclear history of heart disease. We detailed the diagnostic imaging PET/CT workup with different findings.
Conclusion: In this case, [18F]Florbetaben PET/CT can demonstrate potential beta-amyloid immune-reactivity and deposition associated with the current COVID-19 pandemic vaccination programs.”
Fig. 1 [18F]Florbetaben PET/CT: MIP (A), PET (axial-B, coronal-G), CT (axial-C, coronal-E), PET/CT (axial-D, coronal-F) images demonstrated ill-defined uptake in the right arm’s subcutaneous tissues (SUVmax 5.6; white-arrows) and next to a possible right-axillar lymph node (SUVmax 4.75; yellow-arrows) evident on low-dose CT scan without breathing control (red arrows). Reprinted with permission from Nuclear Medicine Unit, Fondazione Istituto G. Giglio, Cefalù (Palermo), Italy.
Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes
Bhardwaj et al., bioRxiv, 2021.06
Abstract: The phenomenon of protein aggregation is associated with a wide range of human diseases. Our knowledge on the aggregation behaviour of viral proteins, however, is still rather limited. Here, we investigated this behaviour in the the SARS-CoV and SARS-CoV-2 proteomes. An initial analysis using a panel of sequence-based predictors suggested the presence of multiple aggregation-prone regions in these proteomes, and revealed an enhanced aggregation propensity in some SARS-CoV-2 proteins. We then studied the in vitro aggregation of predicted aggregation-prone SARS-CoV-2 proteins, including the signal sequence peptide and fusion peptide 1 of the spike protein, a peptide from the NSP6 protein (NSP6-p), the ORF10 protein, and the NSP11 protein. Our results show that these peptides and proteins form aggregates via a nucleation-dependent mechanism. Moreover, we demonstrated that the aggregates of NSP11 are toxic to mammalian cell cultures. These findings provide evidence about the aggregation of proteins in the SARS-CoV-2 proteome.
Significance: The aggregation of proteins is linked with human disease in a variety of ways. In the case of viral infections, one could expect that the aberrant aggregation of viral proteins may damage the host cells, and also that viral particles may trigger the misfolding and aggregation of host proteins, resulting in damage to the host organism. Here we investigate the aggregation propensity of SARS-CoV-2 proteins and show that many of them can form aggregates that are potentially cytotoxic. In perspective, these results suggest that a better understanding of the effects of viruses on the human protein homeostasis system could help future therapeutic efforts.
Fig. 2. Amyloidogenic propensity analysis of the SARS-CoV and SARS-CoV-2 proteomes.
(a,b) Mean predicted percentage amyloidogenic propensity (PPAP) calculated using the mean of percentage of aggregation prone regions obtained from four servers (MetAmyl, AGGRESCAN, FoldAmyloid, FISH Amyloid) for SARS-CoV-2 (a) and SARS-CoV (b). (c-e) Average profile value for protein obtained from FoldAmyloid analysis of proteins against protein length for structural proteins (c), accessory proteins (d), and non-structural proteins (e). The analysis was done at default settings in the FoldAmyloid server (threshold: 21.4, represented by the red-colored short-dashed line, and scale, i.e. the expected number of contacts within 8 Å).
Dr. Malone discusses the SARS-CoV-2 vs. vaccine spike proteins, 2021.07
Be aware of SARS-CoV-2 spike protein: There is more than meets the eye
Theoharides & Conti, Journal of Biological Regulators & Homeostatic Agents, 2021.06
“The COVID-19 pandemic necessitated the rapid production of vaccines aimed at the production of neutralizing antibodies against the COVID-19 spike protein required for the corona virus binding to target cells. The best well-known vaccines have utilized either mRNA or an adenovirus vector to direct human cells to produce the spike protein against which the body produces mostly neutralizing antibodies. However, recent reports have raised some skepticism as to the biologic actions of the spike protein and the types of antibodies produced. One paper reported that certain antibodies in the blood of infected patients appear to change the shape of the spike protein so as to make it more likely to bind to cells, while other papers showed that the spike protein by itself (without being part of the corona virus) can damage endothelial cells and disrupt the blood-brain barrier. These findings may be even more relevant to the pathogenesis of long-COVID syndrome that may affect as many as 50% of those infected with SARS-CoV-2. In COVID-19, a response to oxidative stress is required by increasing anti-oxidant enzymes. In this regard, it is known that polyphenols are natural anti-oxidants with multiple health effects. Hence, there are even more reasons to intervene with the use of anti-oxidant compounds, such as luteolin, in addition to available vaccines and anti-inflammatory drugs to prevent the harmful actions of the spike protein.”
Fig. 1. Diagrammatic representation of how luteolin and methoxyluteolin could block SARS-CoV-2 Spike protein from stimulating microglia. The biologic action of SARS-CoV-2 Spike protein could be via different steps (red rectangles): (1) Spike protein binding to its ACE2 receptor; (2) Activation of serine proteinases responsible for “priming” the Spike protein for entry into the cells; (3) Viral replication within the nucleus; (4) Activation of TLR7/8 found in the endosomes by single- stranded RNA viruses like SARS-CoV-2; (5) Production of proinflammatory cytokines. Luteolin and methoxyluteolin could protect against SARS-CoV-2 Spike protein-associated damage by interfering (green line) at practically all steps
SARS, ACE-2, and spike proteins, Dr. Jessica Rose, 2021.04
Nuovo et al., Annals of Diagnostic Pathology, 2021.04
“Neurologic complications of symptomatic COVID-19 are common. Brain tissues from 13 autopsies of people who died of COVID-19 were examined. Cultured endothelial and neuronal cells were incubated with and wild type mice were injected IV with different spike subunits. In situ analyses were used to detect SARS-CoV-2 proteins and the host response. In 13/13 brains from fatal COVID-19, pseudovirions (spike, envelope, and membrane proteins without viral RNA) were present in the endothelia of microvessels ranging from 0 to 14 positive cells/200× field (mean 4.3). The pseudovirions strongly co-localized with caspase-3, ACE2, IL6, TNFα, and C5b-9. The surrounding neurons demonstrated increased NMDAR2 and neuronal NOS plus decreased MFSD2a and SHIP1 proteins. Tail vein injection of the full length S1 spike subunit in mice led to neurologic signs (increased thirst, stressed behavior) not evident in those injected with the S2 subunit. The S1 subunit localized to the endothelia of microvessels in the mice brain and showed co-localization with caspase-3, ACE2, IL6, TNFα, and C5b-9. The surrounding neurons showed increased neuronal NOS and decreased MFSD2a. It is concluded that ACE2+ endothelial damage is a central part of SARS-CoV-2 pathology and may be induced by the spike protein alone. Thus, the diagnostic pathologist can use either hematoxylin and eosin stain or immunohistochemistry for caspase 3 and ACE2 to document the endothelial cell damage of COVID-19.”
Idrees and Kumar, Biochemical and Biophysical Research Communications, 2021.05
Highlights
SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) binds to heparin and heparin binding proteins.
Heparin binding accelerates the aggregation of the pathological amyloid proteins present in the brain.
SARS-CoV-2 S1 RBD binds to a number of aggregation-prone, heparin binding proteins including Aβ, α-synuclein, tau, prion, and TDP-43 RRM.
Heparin-binding site on the S1 might assist the binding of amyloid proteins to the viral surface and thus could leads to neurodegeneration in brain.
Abstract: “The post-infection of COVID-19 includes a myriad of neurologic symptoms including neurodegeneration. Protein aggregation in brain can be considered as one of the important reasons behind the neurodegeneration. SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) binds to heparin and heparin binding proteins. Moreover, heparin binding accelerates the aggregation of the pathological amyloid proteins present in the brain. In this paper, we have shown that the SARS-CoV-2 S1 RBD binds to a number of aggregation-prone, heparin binding proteins including Aβ, α-synuclein, tau, prion, and TDP-43 RRM. These interactions suggests that the heparin-binding site on the S1 protein might assist the binding of amyloid proteins to the viral surface and thus could initiate aggregation of these proteins and finally leads to neurodegeneration in brain.”
Results: “The protein-protein docking suggests that the binding affinity of SARS-CoV-2 S1 towards the selected proteins is favourable with higher docking energy scores. On the basis of docking scores, the increasing affinity of proteins towards S1 is arranged as: Prion > Aβ > Tau > RRM > α-Syn (Table 1). Interestingly, interaction of heparin with S1 protein is also strong with the docking score of -282.57, much higher than all the proteins studied except prion protein.” …
“Based on docking scores, the interaction between protein and heparin is arranged in order: S1-heparin > Prion-heparin > RRM-heparin > Aβ-heparin > Tau-heparin > α-Syn-heparin (Table 2). Also, the docking score of FGF2-heparin (-220.74) is less than all of these proteins, indicating that the neurodegeneration causing proteins and SARS-CoV-2 S1 protein binds more strongly to heparin.”
Conclusion: “In summary, the findings reported here support the hypothesis that the SARS-CoV-2 spike protein can interact with heparin binding amyloid forming proteins. Our results indicate stable binding of the S1 protein to these aggregation-prone proteins which might initiates aggregation of brain protein and accelerate neurodegeneration.”
Fig. 1. SARS-CoV-2 spike protein S1 RBD domain interactions to amyloid forming HBPs. (A) Docking model of the interaction of heparin-binding domains of spike protein, S1 (green) and Aβ (brown). (B) Detailed molecular interactions between S1 (chain E) and Aβ (chain A) residues deduced by PDBsum. (C) Docking model showing the interaction of S1 (green) to Prion (yellow). (D) Molecular interactions of S1 (chain E) to prion protein (chain A). (E) Surface diagram of S1 (green)-α-Syn complex (red) model and (F) residual interactions of this complex. (G) Model of S1(green)-tau complex structure and (H) the molecular interactions between the tau (chain A) and the heparin-binding domain of spike protein S1 (chain E). (I) Docking model showing the interaction of S1 (green) with the RRM of TDP-43 (blue), and (J) detailed molecular interactions between spike protein S1 (chain E) and RRM (chain A). Key interactions between residues are shown as dotted lines. The key interactions are color coded as: salt bridges (red), disulfide bonds (yellow), hydrogen bonds (blue), and non-bonded contacts (orange). The number of lines indicates the potential number of bonds. For non-bonded contacts, the width of the striped line indicates the number of potential contacts.
SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE 2
Lei et al., Circulation Research, 2021.03, 📰
“SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection relies on the binding of S protein (Spike glycoprotein) to ACE (angiotensin-converting enzyme) 2 in the host cells. Vascular endothelium can be infected by SARS-CoV-2,1 which triggers mitochondrial reactive oxygen species production and glycolytic shift.2 Paradoxically, ACE2 is protective in the cardiovascular system, and SARS-CoV-1 S protein promotes lung injury by decreasing the level of ACE2 in the infected lungs.3 In the current study, we show that S protein alone can damage vascular endothelial cells (ECs) by downregulating ACE2 and consequently inhibiting mitochondrial function.”
Figure. SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) Spike protein exacerbates endothelial cell (EC) function via ACE (angiotensin-converting enzyme) 2 downregulation and mitochondrial impairment.
Mycroft-West et al., bioRxiv, 2020.04
Abstract: “Many pathogens take advantage of the dependence of the host on the interaction of hundreds of extracellular proteins with the glycosaminoglycans heparan sulphate to regulate homeostasis and use heparan sulphate as a means to adhere and gain access to cells. Moreover, mucosal epithelia such as that of the respiratory tract are protected by a layer of mucin polysaccharides, which are usually sulphated. Consequently, the polydisperse, natural products of heparan sulphate and the allied polysaccharide, heparin have been found to be involved and prevent infection by a range of viruses including S-associated coronavirus strain HSR1. Here we use surface plasmon resonance and circular dichroism to measure the interaction between the SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) and heparin. The data demonstrate an interaction between the recombinant surface receptor binding domain and the polysaccharide. This has implications for the rapid development of a first-line therapeutic by repurposing heparin and for next-generation, tailor-made, GAG-based antivirals.”
Discussion and Conclusion: “Studying SARS-CoV-2 Spike protein structure and behaviour in solution is a vital step for the development of effective therapeutics against SARS-CoV-2. Here, the ability of the SARS-CoV-2 S1 RBD to bind pharmaceutical heparin has been studied using spectroscopic techniques in concert with molecular modelling. The data show that SARS-CoV-2 S1 RBD binds to heparin and that upon binding, a significant structural change is induced. Moreover, moieties of basic amino acid residues, known to constitute heparin binding domains, are solvent accessible on the SARS-CoV-2 S1 RBD surface and form a continuous patch that is suitable for heparin binding.”
Figure 2. The structural change of the SARS-CoV-2 S1 RBD observed in the presence of heparin by circular dichroism (CD) spectroscopy.
(A) CD spectra of spike 1 RBD alone (black) or with heparin (red) in phosphate buffered saline pH 7.4. Theoretical sum of spike 1 RBD alone and heparin (control) if no interaction was observed (dotted red). (B) Δ secondary structure (%) of (A). A 1.5% increase in helix and 2.1% decrease in antiparallel secondary structural features were calculated (BestSel) for the observed spectrum compared to that of the theoretical, summative spectrum of the SARS-CoV-2 S1 RBD in the presence of heparin.
Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein
Walls et al., Cell, 2020.03
“The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.”
Figure 3. Cryo-EM Structures of the SARS-CoV-2 S Glycoprotein
(A) Closed SARS-CoV-2 S trimer unsharpened cryo-EM map. (B and C) Two orthogonal views from the side (B) and top (C) of the atomic model of the closed SARS-CoV-2 S trimer. (D) Partially open SARS-CoV-2 S trimer unsharpened cryo-EM map (one SB domain is open). (E-F) Two orthogonal views from the side (E) and top (F) of the atomic model of the closed SARS-CoV-2 S trimer. The glycans were omitted for clarity. See also Figures S1 and S2.
Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor
Lan et al., Nature, 2020.03
“A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from December 2019 that quickly spread nationwide and to other countries around the world. Here, to better understand the initial step of infection at an atomic level, we determined the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor. Structural analysis identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses. The epitopes of two SARS-CoV antibodies that target the RBD are also analysed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.”
Fig. 1: Overall structure of SARS-CoV-2 RBD bound to ACE2.
a, Overall topology of the SARS-CoV-2 spike monomer. FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; IC, intracellular domain; NTD, N-terminal domain; SD1, subdomain 1; SD2, subdomain 2; TM, transmembrane region. b, Sequence and secondary structures of SARS-CoV-2 RBD. The RBM sequence is shown in red. c, Overall structure of the SARS-CoV-2 RBD bound to ACE2. ACE2 is shown in green. The SARS-CoV-2 RBD core is shown in cyan and RBM in red. Disulfide bonds in the SARS-CoV-2 RBD are shown as sticks and indicated by arrows. The N-terminal helix of ACE2 responsible for binding is labelled.