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Background: Immunoglobulin light chain (LC) amyloidosis is a life-threatening disease complicated by vast numbers of patient-specific mutations. We explored 14 patient-derived and engineered proteins related to j1-family germline genes IGKVLD-33-01 and IGKVLD-39-01. Methods: Hydrogen-deuterium exchange mass spectrometry analysis of conformational dynamics in recombinant LCs and their fragments was integrated with studies of thermal stability, proteolytic susceptibility, amyloid formation and amyloidogenic sequence propensity. The results were mapped on the structures of native and fibrillary proteins. Results: Proteins from two j1 subfamilies showed unexpected differences. Compared to their germline counterparts, amyloid LC related to IGKVLD-33-01 was less stable and formed amyloid faster, whereas amyloid LC related to IGKVLD-39-01 had similar stability and formed amyloid slower, suggesting different major factors influencing amyloidogenesis. In 33-01-related amyloid LC, these factors involved destabilization of the native structure and probable stabilization of amyloid. The atypical behavior of 39-01-related amyloid LC stemmed from increased dynamics/exposure of amyloidogenic segments in bC0V and bEV that could initiate aggregation and decreased dynamics/exposure near the Cys23-Cys88 disulfide. Conclusions: The results suggest distinct amyloidogenic pathways for closely related LCs and point to the complementarity-defining regions CDR1 and CDR3, linked via the conserved internal disulfide, as key factors in amyloid formation. [ABSTRACT FROM AUTHOR]

The amyloidogenic transthyretin (TTR) variant, V122I, occurs in 4% of the African American population and frequently presents as a restricted cardiomyopathy. While heterozygosity for TTR V122I predominates, several compound heterozygous cases have been previously described. Herein, we detail features of ATTRv amyloidosis associated with novel compound heterozygous TTR mutation, T60I/V122I and provide evidence supporting the amyloidogenecity of T60I. A 63-year-old African American female presented with atrial fibrillation, congestive heart failure, autonomic and peripheral neuropathy. In vitro studies of TTR T60I and V122I were undertaken to compare the biophysical properties of the proteins. Congophilic deposits in a rectal biopsy were immunohistochemically positive for TTR. Serum screening by isoelectric focussing revealed two TTR variants in the absence of wild-type protein. DNA sequencing identified compound heterozygous TTR gene mutations, c.239C > T and c.424G > A. Adipose amyloid deposits were composed of both T60I and V122I. While kinetic stabilities of T60I and V122I variants were similar, distinct thermodynamic stabilities and amyloid growth kinetics were observed. This report provides clinical and experimental results supporting the amyloidogenic nature of a novel TTR T60I variant. In vitro data indicate that the destabilising effect of individual T60I and V122I variants appears to be additive rather than synergistic. [ABSTRACT FROM AUTHOR]

The thermal stability of full-length light chain (LC) and corresponding variable-joining [V.sub.L] and constant [C.sub.L] fragments are compared to assess the role of [C.sub.L] in fibrillogenesis. The full-length LCs are more likely to initiate aggregation instead of their fragments, during unfolding and provide a template for the [V.sub.L] deposition.

The circulating protein transthyretin (TTR) can unfold, oligomerize, and form highly structured amyloid fibrils that are deposited in tissues, causing organ damage and disease. This pathogenic process is caused by a heritable TTR point mutation in cases of familial TTR-related amyloidosis or wild-type TTR in cases of age-associated amyloidosis (previously called senile systemic amyloidosis). The TTR amyloid cascade is hypothesized to begin with the dissociation of the TTR native tetrameric structure into folded but unstable monomeric TTR subunits. Unfolding of monomeric TTR initiates an oligomerization process leading to aggregation and fibril formation. Numerous proteostatic mechanisms for regulating the TTR amyloid cascade exist. Extracellular chaperones provide an innate defense against misfolded proteins. Clusterin (CLU), a plasma protein, has the capacity to recognize exposed hydrophobic regions of misfolded proteins, shielding them from aggregation. We have previously demonstrated that CLU is associated with the amyloid fibrils in cardiac tissues from patients with TTR amyloidosis. In this study, we have used tetrameric and monomeric TTR structural variants to determine the ability of CLU to inhibit TTR amyloid fibril formation. Using circular dichroism spectroscopy, we determined that CLU preferentially stabilizes monomeric TTR and generates increasingly stable conformations under acid stress. Moreover, studies using surface plasmon resonance showed a direct interaction of CLU with high-molecular weight TTR oligomers. The interactions of CLU with monomeric and aggregated TTR proceed in a cooperative manner in the presence of diflunisal, a small molecule drug used to stabilize TTR tetramers. [ABSTRACT FROM AUTHOR]

Calbindin-D28K is a biologically important protein required for normal neural function and for the transport of calcium in epithelial cells of the intestine and kidney. We have used fluorescence and circular dichroism (CD) spectroscopy to characterize the effects of calcium binding on the structure and stability of calbindin. Ca2+ titration monitored by fluorescence spectroscopy reveals the presence of two classes of calcium-binding sites with association constants ∼107.5 and ∼108.9M−1. CD spectra in the far-UV spectral range show minor changes upon Ca2+ titration, implying that the secondary structure of calbindin-D28K is not greatly affected. On the basis of the CD spectra in the near-UV spectral range, we conclude that the tertiary structure is more sensitive to Ca2+ addition. The most significant change occurs between pCa 7.0 and pCa 8.0. The variations in the protein thermostability are correlated with those in the near-UV CD spectra. The enthalpy changes upon heat denaturation of calbindin in the apo-state are characteristic of proteins containing several weakly interacting domains with similar thermodynamical properties. Thus, calcium binding by calbindin-D28K largely affects the local structure around the aromatic residues and the thermal stability of the protein; the changes in the secondary structure are insignificant. [Copyright &y& Elsevier]