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Background: Amyloidosis is a major long-term complication of chronic disease; however, whether it represents one of the complications of post-myocardial infarction (MI) is yet to be fully understood. Methods: Using wild-type and knocked-out MI mouse models and characterizing in vitro the exosomal communication between bone marrow-derived macrophages and activated mesenchymal stromal cells (MSC) isolated after MI, we investigated the mechanism behind Serum Amyloid A 3 (SAA3) protein overproduction in injured hearts. Results: Here, we show that amyloidosis occurs after MI and that amyloid fibers are composed of macrophage-derived SAA3 monomers. SAA3 overproduction in macrophages is triggered by exosomal communication from a subset of activated MSC, which, in response to MI, acquire the expression of a platelet aggregation-inducing type I transmembrane glycoprotein named Podoplanin (PDPN). Cardiac MSC PDPN+ communicate with and activate macrophages through their extracellular vesicles or exosomes. Specifically, MSC PDPN+ derived exosomes (MSC PDPN+ Exosomes) are enriched in SAA3 and exosomal SAA3 protein engages with Toll-like receptor 2 (TRL2) on macrophages, triggering an overproduction and impaired clearance of SAA3 proteins, resulting in aggregation of SAA3 monomers as rigid amyloid deposits in the extracellular space. The onset of amyloid fibers deposition alongside extra-cellular-matrix (ECM) proteins in the ischemic heart exacerbates the rigidity and stiffness of the scar, hindering the contractility of viable myocardium and overall impairing organ function. Using SAA3 and TLR2 deficient mouse models, we show that SAA3 delivered by MSC PDPN+ exosomes promotes post-MI amyloidosis. Inhibition of SAA3 aggregation via administration of a retro-inverso D-peptide, specifically designed to bind SAA3 monomers, prevents the deposition of SAA3 amyloid fibrils, positively modulates the scar formation, and improves heart function post-MI. Conclusion: Overall, our findings provide mechanistic insights into post-MI amyloidosis and suggest that SAA3 may be an attractive target for effective scar reversal after ischemic injury and a potential target in multiple diseases characterized by a similar pattern of inflammation and amyloid deposition. NOVELTY AND SIGNIFICANCE: What is known? Accumulation of rigid amyloid structures in the left ventricular wall impairs ventricle contractility.After myocardial infarction cardiac Mesenchymal Stromal Cells (MSC) acquire Podoplanin (PDPN) to better interact with immune cells.Amyloid structures can accumulate in the heart after chronic inflammatory conditions. What information does this article contribute? Whether accumulation of cumbersome amyloid structures in the ischemic scar impairs left ventricle contractility, and scar reversal after myocardial infarction (MI) has never been investigated.The pathophysiological relevance of PDPN acquirement by MSC and the functional role of their secreted exosomes in the context of post-MI cardiac remodeling has not been investigated.Amyloid structures are present in the scar after ischemia and are composed of macrophage-derived Serum Amyloid A (SAA) 3 monomers, although mechanisms of SAA3 overproduction is not established. SUMMARY OF NOVELTY AND SIGNIFICANCE: Here, we report that amyloidosis, a secondary phenomenon of an already preexisting and prolonged chronic inflammatory condition, occurs after MI and that amyloid structures are composed of macrophage-derived SAA3 monomers. Frequently studied cardiac amyloidosis are caused by aggregation of immunoglobulin light chains, transthyretin, fibrinogen, and apolipoprotein in a healthy heart as a consequence of systemic chronic inflammation leading to congestive heart failure with various types of arrhythmias and tissue stiffness. Although chronic MI is considered a systemic inflammatory condition, studies regarding the possible accumulation of amyloidogenic proteins after MI and the mechanisms involved in that process are yet to be reported. Here, we show that SAA3 overproduction in macrophages is triggered in a Toll-like Receptor 2 (TLR2)-p38MAP Kinase-dependent manner by exosomal communication from a subset of activated MSC, which, in response to MI, express a platelet aggregation-inducing type I transmembrane glycoprotein named Podoplanin. We provide the full mechanism of this phenomenon in murine models and confirm SAA3 amyloidosis in failing human heart samples. Moreover, we developed a retro-inverso D-peptide therapeutic approach, "DRI-R5S," specifically designed to bind SAA3 monomers and prevent post-MI aggregation and deposition of SAA3 amyloid fibrils without interfering with the innate immune response.
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Resistance to available antibiotics poses a growing challenge to modern medicine, as this often disallows infections to be controlled. This problem can only be alleviated by the development of new drugs. Nisin, a natural lantibiotic with broad antimicrobial activity, has shown promise as a potential candidate for combating antibiotic-resistant bacteria. However, nisin is poorly soluble and barely stable at physiological pH, which despite attempts to address these issues through mutant design has restricted its use as an antibacterial drug. Therefore, gaining a deeper understanding of the antimicrobial effectiveness, which relies in part on its ability to form pores, is crucial for finding innovative ways to manage infections caused by resistant bacteria. Using large-scale molecular dynamics simulations, we find that the bacterial membrane-specific lipid II increases the stability of pores formed by nisin and that the interplay of nisin and lipid II reduces the overall integrity of bacterial membranes by changing the local thickness and viscosity.
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Simulación de Dinámica Molecular , Nisina , Uridina Difosfato Ácido N-Acetilmurámico , Antibacterianos/farmacología , Antibacterianos/química , Membrana Celular/efectos de los fármacos , Membrana Celular/química , Membrana Celular/metabolismo , Nisina/química , Nisina/farmacología , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/química , Uridina Difosfato Ácido N-Acetilmurámico/metabolismoRESUMEN
Parkinson's disease is accompanied by the presence of amyloids in the brain that are formed of α-synuclein chains. The correlation between COVID-19 and the onset of Parkinson's disease led to the idea that amyloidogenic segments in SARS-COV-2 proteins can induce aggregation of α-synuclein. Using molecular dynamic simulations, we show that the fragment FKNIDGYFKI of the spike protein, which is unique for SARS-COV-2, preferentially shifts the ensemble of α-synuclein monomer toward rod-like fibril seeding conformations and, at the same time, differentially stabilizes this polymorph over the competing twister-like structure. Our results are compared with earlier work relying on a different protein fragment that is not specific for SARS-COV-2.
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Enfermedad de Parkinson , Glicoproteína de la Espiga del Coronavirus , alfa-Sinucleína , Humanos , alfa-Sinucleína/química , Amiloide/química , Enfermedad de Parkinson/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
Parkinson's Disease is accompanied by presence of amyloids in the brain formed of α-synuclein chains. Correlation between COVID-19 and the onset of Parkinson's disease let to the idea that amyloidogenic segments in SARS-COV-2 proteins can induce aggregation of α-synuclein. Using molecular dynamic simulations, we show that the fragment FKNIDGYFKI of the spike protein, which is unique for SARS-COV-2, shifts preferentially the ensemble of α-synuclein monomer towards rod-like fibril seeding conformations, and at the same time stabilizes differentially this polymorph over the competing twister-like structure. Our results are compared with earlier work relying on a different protein fragment that is not specific for SARS-COV-2.
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COVID-19 can lead to the onset of type-II diabetes, which is associated with the aggregation of islet amyloid polypeptides, also called amylin. Using molecular dynamics simulations, we investigate how the equilibrium between amylin monomers in its functional form and fibrils associated with diabetes is altered in the presence of SARS-COV-2 protein fragments. For this purpose, we study the interaction between the fragment SFYVYSRVK of the envelope protein or the fragment FKNIDGYFKI of the spike protein with the monomer and two amylin fibril models. Our results are compared with earlier work studying such interactions for the two different proteins.
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Covid-19 can lead to the onset of type-II diabetes which is associated with aggregation of islet amyloid polypeptides, also called amylin. Using molecular dynamics simulations, we investigate how the equilibrium, between amylin monomers in its functional form and fibrils associated with diabetes, is altered in presence of SARS-COV-2 protein fragments. For this purpose, we study the interaction between the fragment SFYVYSRVK of the Envelope protein or the fragment FKNIDGYFKI of the Spike protein with the monomer and two amylin fibril models. Our results are compared with earlier work studying such interactions for two different proteins.
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Simulations of protein folding and protein association happen on timescales that are orders of magnitude larger than what can typically be covered in all-atom molecular dynamics simulations. Use of low-resolution models alleviates this problem but may reduce the accuracy of the simulations. We introduce a replica-exchange-based multiscale sampling technique that combines the faster sampling in coarse-grained simulations with the potentially higher accuracy of all-atom simulations. After testing the efficiency of our Resolution Exchange with Tunneling (ResET) in simulations of the Trp-cage protein, an often used model to evaluate sampling techniques in protein simulations, we use our approach to compare the landscape of wild-type and A2T mutant Aß_{1-42} peptides. Our results suggest a mechanism by that the mutation of a small hydrophobic alanine (A) into a bulky polar threonine (T) may interfere with the self-assembly of Aß fibrils.
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Simulación de Dinámica Molecular , Proteínas , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/química , Pliegue de Proteína , Proteínas/químicaRESUMEN
Prion diseases are characterized by the conversion of prion proteins from a PrPC fold into a disease-causing PrPSC form that is self-replicating. A possible agent to trigger this conversion is polyadenosine RNA, but both mechanism and pathways of the conversion are poorly understood. Using coarse-grained molecular dynamic simulations we study the time evolution of PrPC over 600 µs. We find that both the D178N mutation and interacting with polyadenosine RNA reduce the helicity of the protein and encourage formation of segments with strand-like motifs. We conjecture that these transient ß-strands nucleate the conversion of the protein to the scrapie conformation PrPSC.
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Priones , Animales , Humanos , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Proteínas Priónicas/genética , Priones/genética , Conformación Proteica en Lámina beta , ARNRESUMEN
Aggregates of α-synuclein are thought to be the disease-causing agent in Parkinson's disease. Various case studies have hinted at a correlation between COVID-19 and the onset of Parkinson's disease. For this reason, we use molecular dynamics simulations to study whether amyloidogenic regions in SARS-COV-2 proteins can initiate and modulate aggregation of α-synuclein. 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 a small effect on the stability of pre-existing or newly formed fibrils. A potential mechanism and key residues for potential virus-induced amyloid formation are described.
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Proteínas Amiloidogénicas , Proteínas de la Envoltura de Coronavirus , Enfermedad de Parkinson , Fragmentos de Péptidos , alfa-Sinucleína , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/metabolismo , COVID-19/virología , Proteínas de la Envoltura de Coronavirus/química , Proteínas de la Envoltura de Coronavirus/metabolismo , Humanos , Enfermedad de Parkinson/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , SARS-CoV-2/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMEN
Overexpression of serum amyloid A (SAA) can lead to a form of amyloidosis where the fibrils are made of SAA fragments, most often SAA1-76. Using Replica Exchange with Tunneling, we study the conversion of a SAA1-76 chain between the folded conformation and a fibril conformation. We find that the basins in the free energy landscape corresponding to the two motifs are separated by barriers of only about 2-3 k B T. Crucial for the assembly into the fibril structure is the salt bridge 26E-34K that provides a scaffold for forming the fibril conformation.
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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.
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Deposition of human serum amyloid A (SAA) amyloids in blood vessels, causing inflammation, thrombosis, and eventually organ damage, is commonly seen as a consequence of certain cancers and inflammatory diseases and may also be a risk after SARS-COV-2 infections. Several attempts have been made to develop peptide-based drugs that inhibit or at least slow down SAA amyloidosis. We use extensive all-atom molecular dynamic simulations to compare three of these drug candidates for their ability to destabilize SAA fibrils and to propose for the best candidate, the N-terminal sequence SAA1-5, a mechanism for inhibition. As the lifetime of peptide drugs can be increased by replacing l-amino acids with their mirror d-amino acids, we have also studied corresponding d-peptides. We find that DRI-SAA1-5, formed of d-amino acids with the sequence of the peptide reversed, has similar inhibitory properties compared to the original l-peptide and therefore may be a promising candidate for drugs targeting SAA amyloidosis.
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A marker for the severeness and disease progress of COVID-19 is overexpression of serum amyloid A (SAA) to levels that in other diseases are associated with a risk for SAA amyloidosis. To understand whether SAA amyloidosis could also be a long-term risk of SARS-CoV-2 infections, we have used long all-atom molecular dynamic simulations to study the effect of a SARS-CoV-2 protein segment on SAA amyloid formation. Sampling over 40 µs, we find that the presence of the nine-residue segment SK9, located at the C-terminus of the envelope protein, increases the propensity for SAA fibril formation by three mechanisms: it reduces the stability of the lipid-transporting hexamer shifting the equilibrium toward monomers, it increases the frequency of aggregation-prone configurations in the resulting chains, and it raises the stability of SAA fibrils. Our results therefore suggest that SAA amyloidosis and related pathologies may be a long-term risk of SARS-CoV-2 infections.
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Amiloidosis , COVID-19 , Amiloide , Humanos , SARS-CoV-2 , Proteína Amiloide A SéricaRESUMEN
A marker for the severeness and disease progress of COVID-19 is overexpression of serum amyloid A (SAA) to levels that in other diseases are associated with a risk for SAA amyloidosis. In order to understand whether SAA amyloidosis could also be a long-term risk of SARS-COV-2 infections we have used long all-atom molecular dynamic simulations to study the effect of a SARS-COV-2 protein segment on SAA amyloid formation. Sampling over 40 µs we find that presence of the nine-residue segment SK9, located at the C-terminus of the Envelope protein, increases the propensity for SAA fibril formation by three mechanisms: it reduces the stability of the lipid-transporting hexamer shifting the equilibrium toward monomers, it increases the frequency of aggregation-prone configurations in the resulting chains, and it raises the stability of SAA fibrils. Our results therefore suggest that SAA amyloidosis and related pathologies may be a long-term risk of SARS-COV-2 infections.
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While Alzheimer's disease is correlated with the presence of Aß fibrils in patient brains, the more likely agents are their precursors, soluble oligomers that may form pores or otherwise distort cell membranes. Using all-atom molecular dynamics simulation, we study how the presence of fatty acids such as lauric acid changes the stability of pore-forming oligomers built from three-stranded Aß42 chains. Such a change would alter the distribution of amyloids in the fatty acid-rich brain environment and therefore could explain the lower polymorphism observed in Aß fibrils derived from brains of patients with Alzheimer's disease. We find that lauric acid stabilizes both ring-like and barrel-shaped models, with the effect being stronger for barrel-like models than for ring-like oligomers.
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In systemic amyloidosis, serum amyloid A (SAA) fibril deposits cause widespread damages to tissues and organs that eventually may lead to death. A therapeutically intervention therefore has either to dissolve these fibrils or inhibit their formation. However, only recently has the human SAA fibril structure been resolved at a resolution that is sufficient for development of drug candidates. Here, we use molecular dynamic simulations to probe the factors that modulate the stability of this fibril model. Our simulations suggest that fibril formation starts with the stacking of two misfolded monomers into metastable dimers, with the stacking depending on the N-terminal amyloidogenic regions of different chains forming anchors. The resulting dimers pack in a second step into a 2-fold two-layer tetramer that is stable enough to nucleate fibril formation. The stability of the initial dimers is enhanced under acidic conditions by a strong salt bridge and side-chain hydrogen bond network in the C-terminal cavity (residues 23-51) but is not affected by the presence of the disordered C-terminal tail.
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Amiloidosis , Proteína Amiloide A Sérica , Amiloide , Humanos , Simulación de Dinámica MolecularRESUMEN
The growing bacterial resistance to available antibiotics makes it necessary to look for new drug candidates. An example is the lanthionine-containing nisin, which has a broad spectrum of antimicrobial activity. While nisin is widely utilized as a food preservative, its poor solubility and low stability at physiological pH hinder its use as an antibiotic. As the solubility of nisin is controlled by the residues of the hinge region, we have performed molecular dynamics simulations of various mutants and studied their effects on nisin's solubility. These simulations are complicated by the presence of two uncommon residues (dehydroalanine and dehydrobutyrine) in the peptide. The primary goal of the present study is to derive rules for designing new mutants that will be more soluble at physiological pH and, therefore, may serve as a basis for the future antibiotic design. Another aim of our study is to evaluate whether existing force fields can model the solubility of these amino acids accurately in order to motivate further developments of force fields to account for solubility information.
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Motivated by the role that amylin aggregates play in type-II diabetes, we compare the stability of regular amylin fibrils with the stability of fibrils where l-amino acid chains are replaced by d-retro inverso (DRI) amylin, that is, peptides where the sequence of amino acids is reversed, and at the same time, the l-amino acids are replaced by their mirror images. Our molecular dynamics simulations show that despite leading to only a marginal difference in the fibril structure and stability, aggregating DRI-amylin peptides have different patterns of contacts and hydrogen bonding. Because of these differences, DRI-amylin, when interacting with regular (l) amylin, alters the elongation process and lowers the stability of hybrid amylin fibrils. Our results not only suggest the potential use of DRI-amylin as an inhibitor of amylin fibril formation but also point to the possibility of using the insertion of DRI proteins in l-assemblies as a way to probe the role of certain kinds of hydrogen bonds in supramolecular assemblies or aggregates.
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Polipéptido Amiloide de los Islotes Pancreáticos/química , Simulación de Dinámica Molecular , Humanos , Estabilidad ProteicaRESUMEN
Lymphotactin (Ltn) exists under physiological conditions in an equilibrium between two interconverting structures with distinct biological functions. Using replica-exchange-with-tunneling, we study the conversion between the 2-folds. Unlike previously proposed, we find that the fold switching does not require unfolding of lymphotactin but proceeds through a series of intermediates that remain partially structured. This process relies on two bifurcated hydrogen bonds that connect the ß2 and ß3 strands and ease the transition between the hydrogen bond pattern by which the central three-stranded ß-sheet in the two forms differs.
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Linfocinas , Sialoglicoproteínas , Hidrógeno , Enlace de Hidrógeno , Pliegue de ProteínaRESUMEN
Various diseases cause overexpression of the serum amyloid A (SAA) protein, which in some cases, but not in all cases, leads to amyloidosis as a secondary disease. Response to the overexpression involves dissociation of the SAA hexamer and subsequent cleavage of the released monomers, most commonly yielding fragments SAA1-76 of the full-sized SAA1-104. We report results from molecular dynamic simulations that probe the role of this cleavage for downregulating the activity and concentration of SAA. We propose a mechanism that relies on two elements. First, the probability to assemble into hexamers is lower for the fragments than it is for the full-sized protein. Second, unlike other fragments, SAA1-76 can switch between two distinct configurations. The first kind is easy to proteolyse (allowing a fast reduction of the SAA concentration) but prone to aggregation, whereas the situation is opposite for the second kind. If the time scale for amyloid formation is longer than the one for proteolysis, the aggregation-prone species dominates. However, if environmental conditions such as low pH increases the risk of amyloid formation, the ensemble shifts toward the more protected form. We speculate that SAA amyloidosis is a failure of this switching mechanism leading to accumulation of the aggregation-prone species and subsequent amyloid formation.