ABSTRACT
Variable domain (VL) gene segments exhibit variable tendencies to be associated with light chain amyloidosis (AL). While few of them are very frequent in AL and give rise to most of the amyloidogenic light chains compiled at the sequence databases, other are rarely found among the AL cases. To analyze to which extent these tendencies depend on folding stability and aggregation propensity of the germline VL protein, we characterized VL proteins encoded by four AL-associated germline gene segments and one not associated to AL. We found that the AL-associated germline rVL proteins differ widely in conformational stability and propensity to in vitro amyloid aggregation. While in vitro the amyloid formation kinetics of these proteins correlate well with their folding stabilities, the folding stability does not clearly correlate with their germline's frequencies in AL. We conclude that the association of the VL genes segments to amyloidosis is not determined solely by the folding stability and aggregation propensity of the germline VL protein. Other factors, such as the frequencies of destabilizing mutations and susceptibility to proteolysis, must play a role in determining the light chain amyloidogenicity.
Subject(s)
Amyloid/genetics , Amyloidosis/genetics , Immunoglobulin Variable Region/genetics , Protein Aggregation, Pathological/genetics , Amino Acid Sequence , Germ-Line Mutation , Humans , Microscopy, Electron, Transmission , Protein Domains , Protein Stability , Sequence Alignment , Spectrometry, FluorescenceABSTRACT
Tissue inhibitors of metalloproteases (TIMPs) are multifunctional proteins that inhibit matrix metalloproteases (MMPs). The latest described member of the family, TIMP-4, is expressed mainly in adipose tissue, with detectable levels in the brain and heart. Besides its high expression in fat, the role of this inhibitor in adipose tissue is unknown. In order to study the role of TIMP-4 during adipogenesis in vitro, 3T3-L1 cells were stably transfected with a TIMP-4 specific shRNA or a control shRNA. Unexpectedly, upon TIMP-4 knockdown, 3T3-L1 cells differentiated faster into mature adipocytes. To get better insight of TIMP-4's role in adipogenesis, microarray expression analyses were performed. Network enrichment analyses uncovered 25 significant upstream signaling pathways, among which the NFκB cascade was found. Previous works have shown that NFκB is a key regulator of adipogenesis. In accordance, we found that TIMP-4 knockdown decreased NFκB activity during adipogenesis. The present work suggests that TIMP-4 might act as a negative regulator of adipogenesis through NFκB cascade modulation.
