Lavatelli Francesca, Imperlini Esther, Orrù Stefania, Rognoni Paola, Sarnataro Daniela, Palladini Giuseppina, Malpasso Giuseppe, Soriano Maria Eugenia, Di Fonzo Andrea, Valentini Veronica, Gnecchi Massimiliano, Perlini Stefano, Salvatore Francesco, Merlini Giampaolo
*Amyloidosis Research and Treatment Center, Department of Molecular Medicine, **Department of Internal Medicine, Department of Cardiothoracic and Vascular Sciences, Laboratory of Experimental Cardiology for Cell and Molecular Therapy, University of Pavia, and Clinical Chemistry Laboratory, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, Pavia, Italy; Centro di Ricerca di Ingegneria Genetica (CEINGE)-Biotecnologie Avanzate, Naples, Italy; Department of Movement Sciences, Parthenope University of Naples, Naples, Italy; Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Naples, Italy; Department of Biology, University of Padua, Padua, Italy; and Department of Medicine, University of Cape Town, Cape Town, South Africa.
*Amyloidosis Research and Treatment Center, Department of Molecular Medicine, **Department of Internal Medicine, Department of Cardiothoracic and Vascular Sciences, Laboratory of Experimental Cardiology for Cell and Molecular Therapy, University of Pavia, and Clinical Chemistry Laboratory, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, Pavia, Italy; Centro di Ricerca di Ingegneria Genetica (CEINGE)-Biotecnologie Avanzate, Naples, Italy; Department of Movement Sciences, Parthenope University of Naples, Naples, Italy; Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Naples, Italy; Department of Biology, University of Padua, Padua, Italy; and Department of Medicine, University of Cape Town, Cape Town, South Africa
FASEB J. 2015 Nov;29(11):4614-28. doi: 10.1096/fj.15-272179. Epub 2015 Jul 28.
In immunoglobulin (Ig) light-chain (LC) (AL) amyloidosis, AL deposition translates into life-threatening cardiomyopathy. Clinical and experimental evidence indicates that soluble cardiotoxic LCs are themselves harmful for cells, by which they are internalized. Hypothesizing that interaction of soluble cardiotoxic LCs with cellular proteins contributes to damage, we characterized their interactome in cardiac cells. LCs were purified from patients with AL amyloidosis cardiomyopathy or multiple myeloma without amyloidosis (the nonamyloidogenic/noncardiotoxic LCs served as controls) and employed at concentrations in the range observed in AL patients' sera. A functional proteomic approach, based on direct and inverse coimmunoprecipitation and mass spectrometry, allowed identifying LC-protein complexes. Findings were validated by colocalization, fluorescence lifetime imaging microscopy (FLIM)-fluorescence resonance energy transfer (FRET), and ultrastructural studies, using human primary cardiac fibroblasts (hCFs) and stem cell-derived cardiomyocytes. Amyloidogenic cardiotoxic LCs interact in vitro with specific intracellular proteins involved in viability and metabolism. Imaging confirmed that, especially in hCFs, cardiotoxic LCs (not controls) colocalize with mitochondria and spatially associate with selected interactors: mitochondrial optic atrophy 1-like protein and peroxisomal acyl-coenzyme A oxidase 1 (FLIM-FRET efficiencies 11 and 6%, respectively). Cardiotoxic LC-treated hCFs display mitochondrial ultrastructural changes, supporting mitochondrial involvement. We show that cardiotoxic LCs establish nonphysiologic protein-protein contacts in human cardiac cells, offering new clues on the pathogenesis of AL cardiomyopathy.
在免疫球蛋白（Ig）轻链（LC）（AL）淀粉样变性中，AL沉积会导致危及生命的心肌病。临床和实验证据表明，可溶性心脏毒性轻链本身对细胞有害，细胞会将它们内化。假设可溶性心脏毒性轻链与细胞蛋白的相互作用会导致损伤，我们对它们在心脏细胞中的相互作用组进行了表征。从患有AL淀粉样变性心肌病的患者或无淀粉样变性的多发性骨髓瘤患者（非淀粉样变性/非心脏毒性轻链用作对照）中纯化轻链，并以在AL患者血清中观察到的浓度使用。一种基于直接和反向共免疫沉淀及质谱的功能蛋白质组学方法，能够鉴定轻链-蛋白质复合物。使用人原代心脏成纤维细胞（hCFs）和干细胞衍生的心肌细胞，通过共定位、荧光寿命成像显微镜（FLIM）-荧光共振能量转移（FRET）和超微结构研究对结果进行了验证。淀粉样变性心脏毒性轻链在体外与参与细胞活力和代谢的特定细胞内蛋白质相互作用。成像证实，尤其是在hCFs中，心脏毒性轻链（而非对照）与线粒体共定位，并在空间上与选定的相互作用蛋白相关联：线粒体视神经萎缩1样蛋白和过氧化物酶体酰基辅酶A氧化酶1（FLIM-FRET效率分别为11%和6%）。经心脏毒性轻链处理的hCFs显示出线粒体超微结构变化，支持线粒体参与其中。我们表明，心脏毒性轻链在人心脏细胞中建立了非生理性的蛋白质-蛋白质接触，为AL心肌病的发病机制提供了新线索。
