Unfolding and Aggregation Pathways of Variable Domains from Immunoglobulin Light Chains.

Light chain amyloidosis is the most common form of systemic amyloidosis. This disease is caused by the formation and deposition of amyloid fibers made from immunoglobulin light chains. Environmental conditions such as pH and temperature can affect protein structure and induce the development of these fibers. Several studies have shed light on the native state, stability, dynamics, and final amyloid state of these proteins; however, the initiation process and the fibril formation pathway remain poorly understood structurally and kinetically. To study this, we analyzed the unfolding and aggregation process of the 6aJL2 protein under acidic conditions, with temperature changes, and upon mutation, using biophysical and computational techniques. Our results suggest that the differences in amyloidogenicity displayed by 6aJL2 under these conditions are caused by traversing different aggregation pathways, including unfolded intermediates and the formation of oligomers.

Site-Specific Interactions with Copper Promote Amyloid Fibril Formation for λ6aJL2-R24G.

Light-chain amyloidosis (AL) is one of the most common systemic amyloidoses, and it is characterized by the deposition of immunoglobulin light chain (LC) variable domains as insoluble amyloid fibers in vital organs and tissues. The recombinant protein 6aJL2-R24G contains λ6a and JL2 germline genes and also contains the Arg24 by Gly substitution. This mutation is present in 25% of all amyloid-associated λ6 LC cases, reduces protein stability, and increases the propensity to form amyloid fibers. In this study, it was found that the interaction of 6aJL2-R24G with Cu(II) decreases the thermal stability of the protein and accelerates the amyloid fibril formation, as observed by fluorescence spectroscopy. Isothermal calorimetry titration showed that Cu(II) binds to the protein with micromolar affinity. His99 may be one of the main Cu(II) interaction sites, as observed by nuclear magnetic resonance spectroscopy. The binding of Cu(II) to His99 induces larger fluctuations of the CDR1 and loop C″, as shown by molecular dynamics simulations. Thus, Cu(II) binding may be inducing the loss of interactions between CDR3 and CDR1, making the protein less stable and more prone to form amyloid fibers. This study provides insights into the mechanism of metal-induced aggregation of the 6aJL2-R24G protein and sheds light on the bio-inorganic understanding of AL disease.

Different Dynamics in 6aJL2 Proteins Associated with AL Amyloidosis, a Conformational Disease.

Light-chain amyloidosis (AL) is the most common systemic amyloidosis and is caused by the deposition of mainly insoluble immunoglobulin light chain amyloid fibrils in multiple organs, causing organ failure and eventually death. The germ-line λ6a has been implicated in AL, where a single point mutant at amino acid 24 (6aJL2-R24G) has been observed in around 25% of patient samples. Structural analysis has shown only subtle differences between both proteins; nevertheless, 6aJL2-R24G is more prone to form amyloid fibrils. To improve our understanding of the role of protein flexibility in amyloid fibril formation, we have used a combination of solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations to complement the structural insight with dynamic knowledge. Fast timescale dynamics (ps-ns) were equivalent for both proteins, but suggested exchange events for some residues. Even though most of the intermediate dynamics (µs-ms) occurred at a similar region for both proteins, the specific characteristics are very different. A minor population detected in the dispersion experiments could be associated with the formation of an off-pathway intermediate that protects from fiber formation more efficiently in the germ-line protein. Moreover, we found that the hydrogen bond patterns for both proteins are similar, but the lifetime for the mutant is significantly reduced; as a consequence, there is a decrease in the stability of the tertiary structure that extends throughout the protein and leads to an increase in the propensity to form amyloid fibers.

A family 13 thioesterase isolated from an activated sludge metagenome: Insights into aromatic compounds metabolism.

Activated sludge is produced during the treatment of sewage and industrial wastewaters. Its diverse chemical composition allows growth of a large collection of microbial phylotypes with very different physiologic and metabolic profiles. Thus, activated sludge is considered as an excellent environment to discover novel enzymes through functional metagenomics, especially activities related with degradation of environmental pollutants. Metagenomic DNA was isolated and purified from an activated sludge sample. Metagenomic libraries were subsequently constructed in Escherichia coli. Using tributyrin hydrolysis, a screening by functional analysis was conducted and a clone that showed esterase activity was isolated. Blastx analysis of the sequence of the cloned DNA revealed, among others, an ORF that encodes a putative thioesterase with 47-64% identity to GenBank CDS reported genes, similar to those in the hotdog fold thioesterase superfamily. On the basis of its amino acid similarity and its homology-modelled structure we deduced that this gene encodes an enzyme (ThYest_ar) that belongs to family TE13, with a preference for aryl-CoA substrates and a novel catalytic residue constellation. Plasmid retransformation in E. coli confirmed the clone's phenotype, and functional complementation of a paaI E. coli mutant showed preference for phenylacetate over chlorobenzene as a carbon source. This work suggests a role for TE13 family thioesterases in swimming and degradation approaches for phenyl acetic acid. Proteins 2017; 85:1222-1237. © 2017 Wiley Periodicals, Inc.