CJ-023423 Among the listed GSN variants
Among the listed GSN variants, the D187N protein is the best biochemically and biophysically characterized. >20 years of in vitro and in vivo studies led to a consensus on the pathological mechanism underlying Finnish AGel type, although this model has been questioned by recent findings . According to this model, aspartic CJ-023423 187 is part of a cluster of residues in the G2 domain of GSN, able to chelate a calcium ion . Its N or Y substitution compromises calcium binding [, , , ], leading to the exposure of an otherwise buried sequence, which is recognized by the furin protease . In the Golgi, this intracellular enzyme cleaves GSN producing a C-terminal 68 kDa fragment (C68). C68 is later exported to the extracellular space where it is further processed by matrix metalloproteases, eventually producing 5 and 8 kDa highly amyloidogenic peptides . These fragments rapidly aggregate and deposit in different tissues and organs [17,18]. In stark contrast to the extensive biochemical knowledge available on the D187N mutant, its crystal structure has never been obtained, limiting the mechanistic understanding of GSN instability and aberrant proteolysis. This study aims at characterizing the crystal structure of the isolated G2 domain (Fig. 1) of the D187N protein (D187NG2) by exploiting a recently-developed nanobody (Nb) targeting GSN . Different Nbs able to bind mutated GSN and to detect or prevent its aggregation, have been developed and tested [, , , ]. Among them, Nb11 proved to be the most efficient one. Studies performed in vitro and in vivo demonstrated that Nb11 binds G2 domain of GSN with high affinity, irrespective of calcium, and protects the mutated domain from furin proteolysis, thus skipping the first event of the aberrant proteolytic cascade (19–22). Inspired by the recent use of Nbs as a unique tool for structural biological studies , we employed Nb11 to increase the stability of D187NG2. The successful co-crystallization of D187NG2 in complex with Nb11 (D187NG2:Nb11) showed that the nanobody protects D187NG2 from furin-induced proteolysis, stabilizing the G2 C-terminal linker. Such stabilization is achieved allosterically since the Nb11 binding site locates far from the furin cleavage site. We complemented the structural results cross-referencing molecular dynamics (MD) simulations insights with thermal denaturation studies and furin proteolysis assays. These studies were extended to other mutations causing AGel, such as G167R and N184K. In the absence of cellular or animal models recapitulating G167R and N184K-related AGel as well as the toxicity of the WT or mutated G2 domains, we decided to employ the invertebrate nematode Caenorhabditis elegans as “biosensor”, able to recognize proteins which exert in vivo a biologically relevant effect [, , , ]. This approach takes advantage of the ability of the pharynx of worms, fundamental for their feeding and survival, to be inhibited when it meets molecules acting as chemical stressors . This nematode-based method has been widely applied to recognize the toxicity of different amyloidogenic proteins in vivo, demonstrating that singular molecular mechanisms underlie their proteotoxic activity [, , , ,29]. The protein folding, oligomerization propensity and the exposure of hydrophobic residues on the outside of the protein are relevant for the toxic action of β-amyloid (Aβ) and HIV-matrix protein p17 [25,27,29]. Instead, amyloidogenic cardiotoxic light chains are recognized as stressors by C. elegans thanks to their ability to interact with metal ions and continuously generate reactive oxygen species [24,26]. Our findings indicate that C. elegans efficiently recognizes the proteotoxic potential of the G2 domains and can discriminate between different level of toxicity. Furthermore, the stabilizing effects induced by Nb11 on G2 translated into an effective protection in vivo. These observations point to the use of this nematode-based model as a valuable tool for investigating the mechanisms underlying AGel.