Mimicking kidney flow shear to investigate systemic amyloidosis - Project Summary Systemic amyloidosis (SA) is a group of serious disorders caused by misfolding of ~20 circulating proteins and their subsequent fibrillization and deposition in various organs. The most common forms of SA involve misfolding of immunoglobin light chain (LC), transthyretin (TTR), and leukocyte cell-derived chemotaxin-2 (LECT2), and aggregation of these proteins causes kidney and heart dysfunction. Some of the factors that contribute to the misfolding of these proteins are known or suspected, such as destabilizing mutations and loss of bound cofactors, but the underlying molecular mechanisms of SA remain undefined. This proposal pursues the hypothesis that laminar flow shear in the blood, in combination with known conditions that destabilize the affected proteins, is a major driver of protein misfolding in the circulatory system and development of SA. The principal innovation is the introduction of microfluidic devices that recapitulate the dimensions and highly branched structures of the human kidney vasculature, through which ~1 L of one’s blood passes each minute. These structures create shear stress conditions that are extensive and unique in the body. These devices will be used to subject LC, TTR, and LECT2 to the form of physio-mechanical stress that they experience in circulation, to induce specific local unfolding events that bring about their aggregation into amyloid fibrils. The project employs cryo-EM, fluorescence, and biophysical experiments to characterize the structures and properties of misfolded forms generated by the microfluidic devices, including liquid-liquid phase separated condensates and mature amyloid fibrils. Amyloid structures will be compared to those previously obtained from diseased human kidney and heart to test the hypothesis that flow shear reproduces the ex vivo structures. Finally, small binding domains that recognize misfolded forms of LC and TTR will be generated, and fluorescent and luminescent biosensors will be created by plugging these domains into the PI’s existing modular sensor designs. Biosensors will be tested using patient serum and urine. This aim addresses the critical need for an early diagnostic test that can be performed at point-of-care, to detect SA prior to the onset of organ damage.
