Light chain amyloidosis (AL) is a deadly disease characterized by the deposition of monoclonal immunoglobulin light chains as insoluble amyloid fibrils in different organs and tissues. Germ line λ VI has been closely related to this condition; moreover, the R24G mutation is present in 25% of the proteins of this germ line in AL patients. In this work, five small molecules were tested as inhibitors of the formation of amyloid fibrils from the 6aJL2-R24G protein. We have found by thioflavin T fluorescence and transmission electron microscopy that EGCG inhibits 6aJL2-R24G fibrillogenesis. Furthermore, using nuclear magnetic resonance spectroscopy, dynamic light scattering, and isothermal titration calorimetry, we have determined that the inhibition is due to binding to the protein in its native state, interacting mainly with aromatic residues.

Light chain amyloidosis is one of the most common systemic amyloidosis, characterized by the deposition of immunoglobulin light variable domain as insoluble amyloid fibrils in vital organs, leading to the death of patients. Germline λ6a is closely related with this disease and has been reported that 25% of proteins encoded by this germline have a change at position 24 where an Arg is replaced by a Gly (R24G). This germline variant reduces protein stability and increases the propensity to form amyloid fibrils. In this work, the crystal structure of 6aJL2-R24G has been determined to 2.0 Å resolution by molecular replacement. Crystal belongs to space group I2 1 2 1 2 1 (PDB ID 5JPJ) and there are two molecules in the asymmetric unit. This 6aJL2-R24G structure as several related in PDB (PDB entries: 5C9K, 2W0K, 5IR3 and 1PW3) presents by crystal packing the formation of an octameric assembly in a helicoidal arrangement, which has been proposed as an important early stage in amyloid fibril aggregation. However, other structures of other protein variants in PDB (PDB entries: 3B5G, 3BDX, 2W0L, 1CD0 and 2CD0) do not make the octameric assembly, regardless their capacity to form fibers in vitro or in vivo . The analysis presented here shows that the ability to form the octameric assembly in a helicoidal arrangement in crystallized light chain immunoglobulin proteins is not required for amyloid fibril formation in vitro . In addition, the fundamental role of partially folded states in the amyloid fibril formation in vitro , is not described in any crystallographic structure published or analyzed here, being those structures, in any case examples of proteins in their native states. Those partially folded states have been recently described by cryo-EM studies, showing the necessity of structural changes in the variants before the amyloid fiber formation process starts.

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.

caused by protein aggregates and soluble oligomers by disassembling these structures and recovering natively-folded proteins. The yeast protein disaggregase, Hsp104, disassembles disease-associated protein aggregates and soluble oligomers to suppress toxicity. Hsp104 has a yeast mitochondrial homologue, namely Hsp78. I targeted mitochondrial (mt) Hsp78 (mtHsp78) to the cytoplasm (cHsp78) and potentiated its activity via homologous mutations that nonselectively potentiated Hsp104. Three cHsp78 variants selectively rescue a-synuclein (aSyn), FUS, or TDP-43 toxicity in yeast. Three different mtHsp78 variants rescue aSyn toxicity in yeast without affecting cytoplasmic aSyn aggregation. We are exploring the mechanism of rescue for each of these variants. Additionally, we are interested in the possibility of targeting non-native disaggregases to other organelle such as the endoplasmic reticulum. The results further our understanding of the substrate and compartment specific demands of protein disaggregases in higher eukaryotes. How post-translational modifications alter the structures and interactions of proteins is of great interest for understanding proteomic changes during aging and disease. Oxidative modifications of the long-lived cysteine-rich lens g-crystallins are strongly associated with their aggregation into light-scattering structures that result in cataracts -the leading cause of age-related vision loss. How oxidation leads to aggregation is not well understood. Our previous computational and experimental work showed that formation of a particular non-native intramolecular disulfide bond in cataract-associated W42Q/R human gD-crystallin variants trapped a partially unfolded intermediate state prone to aggregation. Surprisingly, it also revealed that the wild-type protein was able to specifically promote aggregation of these variants without itself aggregating. The search for a biochemical mechanism behind this unprecedented ''inverse-prion'' interaction has now revealed that human gD-crystallin exhibits oxidoreductase activity. This activity depended on formation of a specific internal disulfide bond, which we mapped by LC/MS/MS and by comprehensive Cys mutagenesis. All-atom Monte-Carlo simulations with a statistical potential revealed conformational strain upon formation of this disulfide, which was confirmed by differential scanning flourometry. Disulfide exchange occurred among purified gD-crystallin molecules in solution. Both the Cys-oxidized (disulfide-bonded) wild-type protein and the destabilized (Trpoxidation mimicking) W42Q variant were highly soluble at physiological temperature and pH. When the two were mixed, however, the disulfide bond transferred from the WT to the mutant. Once oxidized, the mutant became aggregation-prone, its insolubilization helping drive the disulfide transfer. Destabilized or damaged gcrystallins may act as oxidation sinks in the lens, forming light-scattering aggregates as a consequence. There is evidence that human gD-crystallin's newly found oxidoreductase activity i...

Yeast prions are self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a a-helical arrangement, whereas others suggest a parallel in-register organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13