Conformational Differences in the Light Chain Constant Domain of Immunoglobulin G and Free Light Chain May Influence Proteolysis in AL Amyloidosis.

Immunoglobulin light chain amyloidosis (AL) is a life-threatening disease caused by the deposition of light chain (LC) and its fragments containing variable (V) and portions of constant (C) domains. AL patients feature either monoclonal free LCs (FLCs) circulating as covalent and noncovalent homodimers, or monoclonal immunoglobulin (Ig) wherein the LC and heavy chain (HC) form disulfide-linked heterodimers, or both. The role of full-length Ig in AL amyloidosis is unclear as prior studies focused on FLC or V domain.

Role of complementarity-determining regions 1 and 3 in pathologic amyloid formation by human immunoglobulin κ1 light chains.

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 κ1-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.

Role of Complementarity-Determining Regions 1 and 3 in Pathologic Amyloid Formation by Human Immunoglobulin κ1 Light Chains.

Immunoglobulin light chain (LC) amyloidosis is a life-threatening disease whose understanding and treatment is complicated by vast numbers of patient-specific mutations. To address molecular origins of the disease, we explored 14 patient-derived and engineered proteins related to κ1-family germline genes IGKVLD-33*01 and IGKVLD-39*01. Hydrogen-deuterium exchange mass spectrometry analysis of local conformational dynamics in full-length recombinant LCs and their fragments was integrated with studies of thermal stability, proteolytic susceptibility, amyloid formation, and amyloidogenic sequence propensities using spectroscopic, electron microscopic and bioinformatics tools.

A Conservative Point Mutation in a Dynamic Antigen-binding Loop of Human Immunoglobulin λ6 Light Chain Promotes Pathologic Amyloid Formation.

Immunoglobulin light chain (LC) amyloidosis (AL) is a life-threatening human disease wherein free mono-clonal LCs deposit in vital organs. To determine what makes some LCs amyloidogenic, we explored patient-based amyloidogenic and non-amyloidogenic recombinant LCs from the λ6 subtype prevalent in AL. Hydrogen-deuterium exchange mass spectrometry, structural stability, proteolysis, and amyloid growth studies revealed that the antigen-binding CDR1 loop is the least protected part in the variable domain of λ6 LC, particularly in the AL variant.

In vitro co-expression of human amyloidogenic immunoglobulin light and heavy chain proteins: a relevant cell-based model of AL amyloidosis.

Immunoglobulin (Ig) light chain (LC) amyloidosis (AL) is characterized by the overproduction and tissue deposition of monoclonal LC in various organs and tissues. The plasma circulating monoclonal LC is believed to be the precursor of the deposited protein and in vitro studies aimed at understanding AL pathobiology have mainly focused on LC and its variable domain. While 33% of patients have free circulating monoclonal LC, ∼40% feature LC complexed to heavy chain (HC) forming a monoclonal intact Ig; the significance of free vs.

Cooperative stabilization of transthyretin by clusterin and diflunisal.

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.

Solution structure and backbone dynamics of human liver fatty acid binding protein: fatty acid binding revisited.

Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the promiscuous binding and transport properties of L-FABP, we investigated structure and dynamics of human L-FABP with and without bound ligands by means of heteronuclear NMR.

The critical role of the constant region in thermal stability and aggregation of amyloidogenic immunoglobulin light chain.

Light chain (LC) amyloidosis (AL) is a fatal disease in which immunoglobulin LC deposit as fibrils. Although the LC amyloid-forming propensity is attributed primarily to the variable region, fibrils also contain full-length LC comprised of variable-joining (V(L)) and constant (C(L)) regions. To assess the role of C(L) in fibrillogenesis, we compared the thermal stability of full-length LC and corresponding V(L) and C(L) fragments.

The modulation of transthyretin tetramer stability by cysteine 10 adducts and the drug diflunisal. Direct analysis by fluorescence-detected analytical ultracentrifugation.

Transthyretin (TTR) is normally a stable plasma protein. However, in cases of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril and the factors that promote this process are largely undetermined.

Expression, purification, and in vitro cysteine-10 modification of native sequence recombinant human transthyretin.

Transthyretin (TTR) is a serum protein that is also a prominent component of deposits in two different types of systemic amyloid disease, senile systemic and familial TTR amyloidoses. Studies of recombinant TTR (rTTR) have provided many insights into the relationship between protein structure and amyloidogenicity. Yet, there is no existing recombinant system that results in high yield production of a protein that is identical in primary structure to human TTR.

Photophysics of ANS. I. Protein-ANS complexes: Intestinal fatty acid binding protein and single-trp mutants.

We continue investigations into the physical chemistry of intestinal fatty acid binding protein, I-FABP, and its interaction with ANS and other ligands [cf references [Kirk, W., E. Kurian, and F.

Photophysics of ANS. III: Circular dichroism of ANS and anilinonaphthalene in I-FABP.

We investigate the circular dichroism of the I-FABP system with the ligands ANS (1,8-anilinonaphthalene sulfonate) and AnN (anilinonaphthalene) as previously reported in our earlier publications in the series (referred to as I and II here) on ANS photophysics. We employ our semi-empirical calculated spectral functions (from II) to compute the actual CD spectra, without any additional assumptions or data except what we have previously presented with respect to binding geometry (in I). The common mechanisms fail to produce the observed spectra.

Interactions between fatty acids and alpha-synuclein.

alpha-Synuclein (alphaS) is an amyloidogenic neuronal protein associated with several neurodegenerative disorders. Although unstructured in solution, alphaS forms alpha-helices in the presence of negatively charged lipid surfaces. Moreover, alphaS was shown to interact with FAs in a manner that promotes protein aggregation.

Changes in structure and stability of calbindin-D(28K) upon calcium binding.

Calbindin-D(28K) 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. Ca(2+) titration monitored by fluorescence spectroscopy reveals the presence of two classes of calcium-binding sites with association constants approximately 10(7.

The effects of hexanol on Galpha(i) subunits of heterotrimeric G proteins.

Unlabelled: Alcohols and other anesthetics interfere with the function of a variety of systems regulated by guanosine triphosphate (GTP)-binding proteins (G proteins). We examined the effect of hexanol on the activity of the alpha subunit (Galpha(i1)) of heterotrimeric G proteins. The GTP hydrolysis activity of recombinant Galpha(i1) was 0.

The metal-binding properties of DREAM: evidence for calcium-mediated changes in DREAM structure.

DREAM, an EF-hand protein, associates with and modulates the activity of presenilins and Kv4 potassium channels in neural and cardiac tissues and represses prodynorphin and c-fos gene expression by binding to DNA response elements in these genes. Information concerning the metal-binding properties of DREAM and the consequences of metal binding on protein structure are important in understanding how this protein functions in cells. We now show that DREAM binds 1 mol of calcium/mol of protein with relatively high affinity and another 3 mol of calcium with lower affinity.