Biochemical and aggregation analysis of Bence Jones proteins from different light chain diseases

Deposition of immunoglobulin light chains is a result of clonal proliferation of monoclonal plasma cells that secrete free immunoglobulin light chains, also called Bence Jones proteins (Bence Jones proteins). These Bence Jones proteins are present in circulation in large amounts and excreted in urine in various light chain diseases such as light chain amyloidosis (AL), light chain deposition disease (LCDD) and multiple myeloma (MM). BJP from patients with AL, LCDD and MM were purified from their urine and studies were performed to determine their secondary structure, thermodynamic stability and aggregate formation kinetics. Our results show that LCDD and MM proteins have the lowest free energy of folding while all proteins show similar melting temperatures. Incubation of the BJP at their melting temperature produced morphologically different aggregates: amyloid fibrils from the AL proteins, amorphous aggregates from the LCDD proteins and large spherical species from the MM proteins. The aggregates formed under in vitro conditions suggested that the various proteins derived from patients with different light chain diseases might follow different aggregation pathways

Regulatory Light Chain Mutants Linked to Heart Disease Modify the Cardiac Myosin Lever Arm

Myosin is the chemomechanical energy transducer in striated heart muscle. The myosin cross-bridge applies impulsive force to actin while consuming ATP chemical energy to propel myosin thick filaments relative to actin thin filaments in the fiber. Transduction begins with ATP hydrolysis in the cross-bridge driving rotary movement of a lever arm converting torque into linear displacement. Myosin regulatory light chain (RLC) binds to the lever arm and modifies its ability to translate actin. Gene sequencing implicated several RLC mutations in heart disease, and three of them are investigated here using photoactivatable GFP-tagged RLC (RLC-PAGFP) exchanged into permeabilized papillary muscle fibers. A single-lever arm probe orientation is detected in the crowded environment of the muscle fiber by using RLC-PAGFP with dipole orientation deduced from the three-spatial dimension fluorescence emission pattern of the single molecule. Symmetry and selection rules locate dipoles in their half-sarcomere, identify those at the minimal free energy, and specify active dipole contraction intermediates. Experiments were performed in a microfluidic chamber designed for isometric contraction, total internal reflection fluorescence detection, and two-photon excitation second harmonic generation to evaluate sarcomere length. The RLC-PAGFP reports apparently discretized lever arm orientation intermediates in active isometric fibers that on average produce the stall force. Disease-linked mutants introduced into RLC move intermediate occupancy further down the free energy gradient, implying lever arms rotate more to reach stall force because mutant RLC increases lever arm shear strain. A lower free energy intermediate occupancy involves a lower energy conversion efficiency in the fiber relating a specific myosin function modification to the disease-implicated mutant

Domain:domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure

The major heat shock protein (Hsp) chaperones Hsp70 and Hsp90 both bind the co-chaperone Hop (Hsp70/Hsp90 organizing protein), which coordinates Hsp actions in folding protein substrates. Hop contains three tetratricopeptide repeat (TPR) domains that have binding sites for the conserved EEVD C termini of Hsp70 and Hsp90. Crystallographic studies have shown that EEVD interacts with positively charged amino acids in Hop TPR-binding pockets (called carboxylate clamps), and point mutations of these carboxylate clamp positions can disrupt Hsp binding. In this report, we use circular dichroism to assess the effects of point mutations and Hsp70/Hsp90 peptide binding on Hop conformation. Our results show that Hop global conformation is destabilized by single point mutations in carboxylate clamp positions at pH 5, while the structure of individual TPR domains is unaffected. Binding of peptides corresponding to the C termini of Hsp70 and Hsp90 alters the global conformation of wild-type Hop, whereas peptide binding does not alter conformation of individual TPR domains. These results provide biophysical evidence that Hop-binding pockets are directly involved with domain:domain interactions, both influencing Hop global conformation and Hsp binding, and contributing to proper coordination of Hsp70 and Hsp90 interactions with protein substrates

Mutations in specific structural regions of immunoglobulin light chains are associated with free light chain levels in patients with AL amyloidosis.

BACKGROUND: The amyloidoses are protein misfolding diseases characterized by the deposition of amyloid that leads to cell death and tissue degeneration. In immunoglobulin light chain amyloidosis (AL), each patient has a unique monoclonal immunoglobulin light chain (LC) that forms amyloid deposits. Somatic mutations in AL LCs make these proteins less thermodynamically stable than their non-amyloidogenic counterparts, leading to misfolding and ultimately the formation of amyloid fibrils. We hypothesize that location rather than number of non-conservative mutations determines the amyloidogenicity of light chains. METHODOLOGY/PRINCIPAL FINDINGS: We performed sequence alignments on the variable domain of 50 kappa and 91 lambda AL light chains and calculated the number of non-conservative mutations over total number of patients for each secondary structure element in order to identify regions that accumulate non-conservative mutations. Among patients with AL, the levels of circulating immunoglobulin free light chain varies greatly, but even patients with very low levels can have very advanced amyloid deposition. CONCLUSIONS: Our results show that in specific secondary structure elements, there are significant differences in the number of non-conservative mutations between normal and AL sequences. AL sequences from patients with different levels of secreted light chain have distinct differences in the location of non-conservative mutations, suggesting that for patients with very low levels of light chains and advanced amyloid deposition, the location of non-conservative mutations rather than the amount of free light chain in circulation may determine the amyloidogenic propensity of light chains

"Teriam feito alguma coisa...". Uma análise sobre as discussões morais no acesso à condição de ativista militar em casos de mortes violentas (Córdoba, Argentina)

Las figuras de familiares -especialmente de las madres- han venido ganando legitimidad en los últimos años en Argentina para reclamar por sus víctimas, ocupando también un lugar central en las aproximaciones de las ciencias sociales. Este artículo pretende analizar aquellas contiendas morales que restringen el acceso a la condición de "activista familiar" en los sectores populares, en la medida en que han sido escasamente problematizadas por la academia. El texto se inscribe en una investigación etnográfica y comparativa que desarrollo desde 2007, y que aborda redes de relaciones familiares, sociales y políticas vinculadas a muertes violentas en sectores populares de Córdoba (Argentina). A través del recorrido etnográfico se propone señalar, por un lado, el modo en que la apelación a la figura de la madre para legitimar su intervención política tiende a reforzar las acusaciones morales que pretende trascender. Y por otro, reflexionar sobre las exclusiones de aquellas muertes que quedan aparentemente inscriptas fuera de los "contextos políticos".The figure of the relative (especially the mother) has recently been gaining legitimacy in Argentina during the process of claiming justice for victims of violent deaths. It has also played a vital role in the social sciences. This article aims to analyze the moral strife that limits the access to the status of "family activist" in popular sectors, one that has been scarcely problematized in academia. The text is part of a comparative ethnographic research that has been developed since 2007 that deals with family, social and political relations linked to violent deaths in popular neighborhoods of Córdoba (Argentina). The ethnographic journey, on one hand points outs how the appealing to figure of the mother in order to legitimize her political intervention tends to reinforce the moral accusations that it seeks to transcend. On the other hand, it seeks to reflect on the exclusion of deaths that are thought to be circumscribed outside "political contexts".As figuras familiares -especialmente a das mães- vêm ganhando legitimidade nos últimos anos na Argentina para reivindicar por suas vítimas, ocupando também um lugar central nas aproximações das ciências sociais. Este artigo pretende analisar aquelas discussões morais que restringem o acesso à condição de "ativista militar" nos setores populares, na medida em que têm sido escassamente problematizadas pela academia. O texto se inscreve numa pesquisa etnográfica e comparativa desenvolvida desde 2007 e que aborda redes de relações familiares, sociais e políticas vinculadas a mortes violentas em setores populares de Córdoba (Argentina). Por meio do percorrido etnográfico, propõe-se indicar, por um lado, o modo em que a apelação à figura da mãe pode legitimar sua intervenção política tende a reforçar as acusações morais que pretende transcender. Por outro lado, refletir sobre as exclusões daquelas mortes que ficam aparentemente inscritas fora dos "contextos políticos".Fil: Bermúdez, Natalia Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Antropología de Córdoba. Universidad Nacional de Córdoba. Facultad de Filosofía y Humanidades. Instituto de Antropología de Córdoba; Argentin