Hereditary Amyloidosis: Insights Into a Fibrinogen A Variant Protein.

Amyloidosis are a group of diseases in which soluble proteins aggregate and deposit in fibrillar conformation extracellularly in tissues. The effectiveness of therapeutic strategies depends on the specific protein involved, being crucial to accurately determine its nature. Moreover, following the diagnosis, the search for the mutation within relatives allows the clinical advice. Here we report the precise diagnosis and explored the possible reasons of the structural pathogenicity for a renal amyloidosis related to a fibrinogen Aα-chain variant. Whole-exome sequencing and GATK calling pipeline were leveraged to characterize the protein variant present in a patient with kidney failure. Bioinformatics strategies were applied to suggest potential explanations of the variants aggregation. Our pipeline allowed the identification of a single-point variant of fibrinogen Aα-chain, which opened the possibility of curative transplantation. In silico structural analysis suggested that the pathogenicity of the variant may be attributed to a heightened susceptibility to yield a peptide prone to deposit as an oligomer with a ß-sheet structure. Exploiting the comprehensive coverage of whole-genome sequencing, we managed to fill a vacant stage in the diagnosis of hereditary amyloidosis and to stimulate the advancement in biomedicine.

Interactions of variants of human apolipoprotein A-I with biopolymeric model matrices. Effect of collagen and heparin.

BACKGROUND: The extracellular matrix (ECM) is a complex tridimensional scaffold that actively participates in physiological and pathological events. The objective of this study was to test whether structural proteins of the ECM and glycosaminoglycans (GAGs) may favor the retention of human apolipoprotein A-I (apoA-I) variants associated with amyloidosis and atherosclerosis. METHODS: Biopolymeric matrices containing collagen type I (Col, a main macromolecular component of the ECM) with or without heparin (Hep, a model of GAGs) were constructed and characterized, and used to compare the binding of apoA-I having the native sequence (Wt) or Arg173Pro, a natural variant inducing cardiac amyloidosis. Protein binding was observed by fluorescence microscopy and unbound proteins quantified by a colorimetric assay. RESULTS: Both, Wt and Arg173Pro bound to the scaffolds containing Col, but the presence of Hep diminished the binding efficiency. Col-Hep matrices retained Arg173Pro more than the Wt. The retained protein was only partially removed from the matrices with saline solutions, indicating that electrostatic interactions may occur but are not the main driving force. Using in addition thermodynamic molecular simulations and size exclusion chromatography approaches, we suggest that the binding of apoA-I variants to the biopolymeric matrices is driven by many low affinity interactions. CONCLUSIONS: Under this scenario Col-Hep scaffolds contribute to the binding of Arg173Pro, as a cooperative platform which could modify the native protein conformation affecting protein folding. GENERAL SIGNIFICANCE: We show that the composition of the ECM is key to the protein retention, and well characterized biosynthetic matrices offer an invaluable in vitro model to mimic the hallmark of pathologies with interstitial infiltration such as cardiac amyloidosis.

Evolutionary and structural constraints influencing apolipoprotein A-I amyloid behavior.

Apolipoprotein A-I (apoA-I) has a key function in the reverse cholesterol transport. However, aggregation of apoA-I single point mutants can lead to hereditary amyloid pathology. Although several studies have tackled the biophysical and structural consequences introduced by these mutations, there is little information addressing the relationship between the evolutionary and structural features that contribute to the amyloid behavior of apoA-I. We combined evolutionary studies, in silico mutagenesis and molecular dynamics (MD) simulations to provide a comprehensive analysis of the conservation and pathogenic role of the aggregation-prone regions (APRs) present in apoA-I. Sequence analysis demonstrated that among the four amyloidogenic regions described for human apoA-I, only two (APR1 and APR4) are evolutionary conserved across different species of Sarcopterygii. Moreover, stability analysis carried out with the FoldX engine showed that APR1 contributes to the marginal stability of apoA-I. Structural properties of full-length apoA-I models suggest that aggregation is avoided by placing APRs into highly packed and rigid portions of its native fold. Compared to silent variants extracted from the gnomAD database, the thermodynamic and pathogenic impact of amyloid mutations showed evidence of a higher destabilizing effect. MD simulations of the amyloid variant G26R evidenced the partial unfolding of the alpha-helix bundle with the concomitant exposure of APR1 to the solvent, suggesting an insight into the early steps involved in its aggregation. Our findings highlight APR1 as a relevant component for apoA-I structural integrity and emphasize a destabilizing effect of amyloid variants that leads to the exposure of this region.

Understanding the role of apolipoproteinA-I in atherosclerosis. Post-translational modifications synergize dysfunction?

BACKGROUND: The identification of dysfunctional human apolipoprotein A-I (apoA-I) in atherosclerotic plaques suggests that protein structure and function may be hampered under a chronic pro inflammatory scenario. Moreover, the fact that natural mutants of this protein elicit severe cardiovascular diseases (CVD) strongly indicates that the native folding could shift due to the mutation, yielding a structure more prone to misfold or misfunction. To understand the events that determine the failure of apoA-I structural flexibility to fulfill its protective role, we took advantage of the study of a natural variant with a deletion of the residue lysine 107 (K107del) associated with atherosclerosis. METHODS: Biophysical approaches, such as electrophoresis, fluorescence and spectroscopy were used to characterize proteins structure and function, either in native conformation or under oxidation or intramolecular crosslinking. RESULTS: K107del structure was more flexible than the protein with the native sequence (Wt) but interactions with artificial membranes were preserved. Instead, structural restrictions by intramolecular crosslinking impaired the Wt and K107del lipid solubilization function. In addition, controlled oxidation decreased the yield of the native dimer conformation for both variants. CONCLUSIONS: We conclude that even though mutations may alter protein structure and spatial arrangement, the highly flexible conformation compensates the mild shift from the native folding. Instead, post translational apoA-I modifications (probably chronic and progressive) are required to raise a protein conformation with significant loss of function and increased aggregation tendency. GENERAL SIGNIFICANCE: The results learnt from this variant strength a close association between amyloidosis and atherosclerosis.

Dataset of the construction and characterization of stable biological nanoparticles.

This article shows the dataset of clearance assays and the reconstitution of stable biological nano-complexes using both detergent-assisted and spontaneous solubilization of phospholipids by the recombinant purified apolipoprotein A-I (apoA-I). Protein was intra-chain crosslinked in order to introduce steric constrains. Then, native and crosslinked protein function was evaluated by a data collection of dimiristoyl phosphatidyl choline (DMPC) micellization curves. Additionally, resulting particles from spontaneous or detergent-assisted lipid solubilization were characterized by transmission electron microscopy (TEM), size exclusion chromatography (SEC), and native polyacrylamide gel electrophoresis (PAGE). Here we set up an experimental design that may help study protein structure based on its function, since interaction with biological membranes and lipids is an intrinsic activity attributed to many proteins in circulation. In addition, by t-test analysis of collected-data, we examined the formation of lipoprotein particles by native and intra-chain crosslinked proteins under different conditions like temperature and time incubation. Thus, data shown here strengthen the usefulness of an easy, rapid, accessible and inexpensive approach to test protein flexibility related to its function.

Data regarding the sensibility to proteolysis of a natural apolipoprotein A-I mutant.

The article shows dataset of the proteolysis of a natural variant of apolipoprotein A-I (apoA-I) with a substitution of a leucine by and arginine in position 60 (L60R), in comparison with the protein with the native sequence (Wt). This information demonstrates the potential of in vitro partial proteolysis experiments as it may be applicable to different approaches in the biophysical field. We have analyzed by different electrophoresis techniques apoA-I variants, quantified the degree of proteolysis after staining and compared the proteolysis efficiency with the computed cleavage patterns. The data shown here clearly strengthen the usefulness of this approach to test protein flexibility, as it may be attained with enzymes which are not expected to modify in vivo this protein but have a well-known digestion pattern. In addition it is appropriate for evaluating protein catabolism, as it is exemplified here by the evidence with metalloproteinase 12 (MMP-12), which is a physiological protease that may elicit the pro-inflammatory processing of this variant within the lesions. We support the work "Structural analysis of a natural apolipoprotein A-I variant (L60R) associated with amyloidosis" (Gaddi, et al., 2020), gaining insights on protein folding from a characterization by proteolysis analysis [1].

Structural analysis of a natural apolipoprotein A-I variant (L60R) associated with amyloidosis.

The reason that determines the pathological deposition of human apolipoprotein A-I variants inducing organ failure has been under research since the early description of natural mutations in patients. To shed light into the events associated with protein aggregation, we studied the structural perturbations that may occur in the natural variant that shows a substitution of a Leucine by an Arginine in position 60 (L60R). Circular dichroism, intrinsic fluorescence measurements, and proteolysis analysis indicated that L60R was more unstable, more sensitive to cleavage and the N-terminus was more disorganized than the protein with the native sequence (Wt). A higher tendency to aggregate was also detected when L60R was incubated at physiological pH. In addition, the small structural rearrangement observed for the freshly folded variant led to the release of tumor necrosis factor-α and interleukin-1ß from a model of macrophages. However, the mutant preserved both its dimeric conformation and its lipid-binding capacity. Our results strongly suggest that the chronic disease may be a consequence of the native conformation loss which elicits the release of protein conformations that could be either cytotoxic or precursors of amyloid conformations.

Fibrillar conformation of an apolipoprotein A-I variant involved in amyloidosis and atherosclerosis.

BACKGROUND: Different protein conformations may be involved in the development of clinical manifestations associated with human amyloidosis. Although a fibrillar conformation is usually the signature of damage in the tissues of patients, it is not clear whether this species is per se the cause or the consequence of the disease. Hereditary amyloidosis due to variants of apolipoprotein A-I (apoA-I) with a substitution of a single amino acid is characterized by the presence of fibrillar protein within the lesions. Thus mutations result in increased protein aggregation. Here we set up to characterize the folding of a natural variant with a mutation leading to a deletion at position 107 (apoA-I Lys107-0). Patients carrying this variant show amyloidosis and severe atherosclerosis. METHODS: We oxidized this variant under controlled concentrations of hydrogen peroxide and analyzed the structure obtained after 30-day incubation by fluorescence, circular dichroism and microscopy approaches. Neutrophils activation was characterized by confocal microscopy. RESULTS: We obtained a high yield of well-defined stable fibrillar structures of apoA-I Lys107-0. In an in vitro neutrophils system, we were able to detect the induction of Neutrophils Extracellular Traps (NETs) when we incubated with oxidized apoA-I variants. This effect was exacerbated by the fibrillar structure of oxidized Lys 107-0. CONCLUSIONS: We conclude that a pro-inflammatory microenvironment could result in the formation of aggregation-prone species, which, in addition may induce a positive feed-back in the activation of an inflammatory response. GENERAL SIGNIFICANCE: These events may explain a close association between amyloidosis due to apoA-I Lys107-0 and atherosclerosis.

Human apolipoprotein A-I Gly26Arg stimulation of inflammatory responses via NF-kB activation: Potential roles in amyloidosis?

The cascade of molecular events leading to Human apolipoprotein A-I (apoA-I) amyloidosis is not completely understood, not even the pathways that determine clinical manifestations associated to systemic protein deposition in organs such as liver, kidney and heart. About twenty natural variants of apoA-I were described as inducing amyloidosis, but the mechanisms driving their aggregation and deposition are still unclear. We previously identified that the mutant Gly26Arg but not Lys107-0 induced the release of cytokines and reactive oxygen species from cultured RAW 264.7 murine macrophages, suggesting that part of the pathogenic pathway could elicit of an inflammatory signal. In this work we gained deep insight into this mechanism and determined that Gly26Arg induced a specific pro-inflammatory cascade involving activation of NF-κB and its translocation into the nucleus. These findings suggest that some but not all apoA-I natural variants might promote a pro-oxidant microenvironment which could in turn result in oxidative processing of the variants into a misfolded conformation.

Amyloidogenic propensity of a natural variant of human apolipoprotein A-I: stability and interaction with ligands.

A number of naturally occurring mutations of human apolipoprotein A-I (apoA-I) have been associated with hereditary amyloidoses. The molecular mechanisms involved in amyloid-associated pathology remain largely unknown. Here we examined the effects of the Arg173Pro point mutation in apoA-I on the structure, stability, and aggregation propensity, as well as on the ability to bind to putative ligands. Our results indicate that the mutation induces a drastic loss of stability, and a lower efficiency to bind to phospholipid vesicles at physiological pH, which could determine the observed higher tendency to aggregate as pro-amyloidogenic complexes. Incubation under acidic conditions does not seem to induce significant desestabilization or aggregation tendency, neither does it contribute to the binding of the mutant to sodium dodecyl sulfate. While the binding to this detergent is higher for the mutant as compared to wt apoA-I, the interaction of the Arg173Pro variant with heparin depends on pH, being lower at pH 5.0 and higher than wt under physiological pH conditions. We suggest that binding to ligands as heparin or other glycosaminoglycans could be key events tuning the fine details of the interaction of apoA-I variants with the micro-environment, and probably eliciting the toxicity of these variants in hereditary amyloidoses.

Human apolipoprotein A-I natural variants: molecular mechanisms underlying amyloidogenic propensity.

Human apolipoprotein A-I (apoA-I)-derived amyloidosis can present with either wild-type (Wt) protein deposits in atherosclerotic plaques or as a hereditary form in which apoA-I variants deposit causing multiple organ failure. More than 15 single amino acid replacement amyloidogenic apoA-I variants have been described, but the molecular mechanisms involved in amyloid-associated pathology remain largely unknown. Here, we have investigated by fluorescence and biochemical approaches the stabilities and propensities to aggregate of two disease-associated apoA-I variants, apoA-IGly26Arg, associated with polyneuropathy and kidney dysfunction, and apoA-ILys107-0, implicated in amyloidosis in severe atherosclerosis. Results showed that both variants share common structural properties including decreased stability compared to Wt apoA-I and a more flexible structure that gives rise to formation of partially folded states. Interestingly, however, distinct features appear to determine their pathogenic mechanisms. ApoA-ILys107-0 has an increased propensity to aggregate at physiological pH and in a pro-inflammatory microenvironment than Wt apoA-I, whereas apoA-IGly26Arg elicited macrophage activation, thus stimulating local chronic inflammation. Our results strongly suggest that some natural mutations in apoA-I variants elicit protein tendency to aggregate, but in addition the specific interaction of different variants with macrophages may contribute to cellular stress and toxicity in hereditary amyloidosis.

Human apolipoprotein A-I-derived amyloid: its association with atherosclerosis.

Amyloidoses constitute a group of diseases in which soluble proteins aggregate and deposit extracellularly in tissues. Nonhereditary apolipoprotein A-I (apoA-I) amyloid is characterized by deposits of nonvariant protein in atherosclerotic arteries. Despite being common, little is known about the pathogenesis and significance of apoA-I deposition. In this work we investigated by fluorescence and biochemical approaches the impact of a cellular microenvironment associated with chronic inflammation on the folding and pro-amyloidogenic processing of apoA-I. Results showed that mildly acidic pH promotes misfolding, aggregation, and increased binding of apoA-I to extracellular matrix elements, thus favoring protein deposition as amyloid like-complexes. In addition, activated neutrophils and oxidative/proteolytic cleavage of the protein give rise to pro amyloidogenic products. We conclude that, even though apoA-I is not inherently amyloidogenic, it may produce non hereditary amyloidosis as a consequence of the pro-inflammatory microenvironment associated to atherogenesis.

Changes in cucumber hypocotyl cell wall dynamics caused by Azospirillum brasilense inoculation.

We previously reported that Azospirillum brasilense induced a more elastic cell wall and a higher apoplastic water fraction in both wheat coleoptile and flag leaf. These biophysical characteristics could permit increased growth. Knowledge of the biochemical effects the bacteria could elicit in plant cell walls and how these responses change plant physiology is still scarce. The objective of this work was to analyze whether A. brasilense Sp245 inoculation affected elongation and extensibility of growing cucumber (Cucumis sativus) hypocotyls and ionically bound cell wall peroxidase activities. Hypocotyl tip and basal segments were excised from A. brasilense Sp245-inoculated cucumber seedlings growing in darkness under hydroponic conditions. Elongation, cell wall extensibility, cell wall peroxidase activities against ferulic acid and guaiacol and NADH oxidase activities were analyzed. Azospirillum-inoculated cucumber seedlings grew bigger than non-inoculated ones. Dynamic cell wall differences were detected between inoculated and non-inoculated hypocotyls. They included greater acid-induced cell wall extension and in vivo elongation when incubated in distilled water. Although there was no difference between treatments in either region of the hypocotyl NADH oxidase and ferulic acid peroxidase activities were lower in both regions in inoculated seedlings. These lesser activities could be delaying the stiffening of cell wall in inoculated seedlings. These results showed that the cell wall is a target for A. brasilense growth promotion.