Destabilization of Ca²⁺-free gelsolin may not be responsible for proteolysis in Familial Amyloidosis of Finnish Type

Abstract

Mutations at position 187 in secreted gelsolin enable aberrant proteolysis at the 172-173 and 243-244 amide bonds, affording the 71-residue amyloidogenic peptide deposited in Familial Amyloidosis of Finnish Type (FAF). Thermodynamic comparisons of two different domain 2 constructs were carried out to study possible effects of the mutations on proteolytic susceptibility. In the construct we consider to be most representative of domain 2 in the context of the full-length protein (134 -266), the D187N FAF variant is slightly destabilized relative to wild type (WT) under the conditions of urea denaturation, but exhibits a T m identical to WT. The D187Y variant is less stable to intermediate urea concentrations and exhibits a T m that is estimated to be Ϸ5°C lower than WT (pH 7.4, Ca 2؉ -free). Although the thermodynamic data indicate that the FAF mutations may slightly destabilize domain 2, these changes are probably not sufficient to shift the native to denatured state equilibrium enough to enable the proteolysis leading to FAF. Biophysical data indicate that these two FAF variants may have different native state structures and possibly different pathways of amyloidosis.A myloid diseases are associated with abnormal fibrillar protein deposits (1-7) from the self-assembly of misfolded proteins or peptides (3,(8)(9)(10)(11). Both amyloid and intermediates of assembly have been implicated as causative agents in these diseases (12)(13)(14)(15). Deposition of a fragment of mutated human plasma gelsolin (173-243) putatively causes Familial Amyloidosis of Finnish Type (FAF), a disease characterized by lattice corneal dystrophy, progressive cranial neuropathy, and skin elasticity complications (16)(17)(18).Gelsolin is an actin-binding protein that nucleates, caps, and severs actin filaments. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and Ca 2ϩ ions regulate gelsolin's dissociation from and binding to actin, respectively (19)(20)(21)(22). Human gelsolin is expressed as an 81-kDa protein in cytoplasm and 84-kDa protein in plasma (23). The secreted protein differs by a signal sequence required for export and a 25-residue N-terminal extension. The cysteines at positions 188 and 201 form a disulfide bond in the plasma, but remain reduced in the cytoplasmic form (24).The mutation of Asp-187 in domain 2 to either an Asn or a Tyr allows aberrant proteolysis at the 172-173 amide bond during secretion (25). A subsequent proteolytic cleavage generates the 173-243 amyloidogenic fragment.Sequence comparisons and limited proteolysis defined gelsolin to be composed of six domains (26-28). The crystal structure of horse plasma gelsolin, highly homologous to human, in the absence of Ca 2ϩ confirmed this architecture and revealed a fold common to each domain ( Figs. 1 and 2; ref. 29). The fold consists of a central five-or six-stranded ␤-sheet sandwiched between a long ␣-helix parallel to the ␤-strands and a short ␣-helix almost perpendicular to the strands (Fig. 2). The definition of domain 2 based on sequence homology and proteolysis ...

Destabilization of apoprotein is insufficient to explain Cu,Zn-superoxide dismutase-linked ALS pathogenesis

“…Pathogenic mutations at metal coordinating residues have also been reported for the calciumbinding protein gelsolin, which causes familial amyloidosisFinnish type (6,26). These mutations are located at Ca 2ϩ coordinating residues and, similar to MBR ALS mutations in SOD1, do not destabilize the apoprotein but disrupt metal binding, leading to the generation of amyloidogenic peptides via aberrant proteolysis.…”

Gelsolin amyloidosis: genetics, biochemistry, pathology and possible strategies for therapeutic intervention

“…This mutation eliminates one of the four calcium binding ligands in the second domain of plasma gelsolin, substantially compromising calcium binding. This allows domain 2 of mutant plasma gelsolin to sample unfolded conformations, during which it can be aberrantly proteolytically processed, first in the Golgi and later outside the cell, to form 8 and 5 kDa amyloidogenic fragments that deposit systemically (Figure 1) (Chen et al, 2001, Huff et al, 2003b, Page et al, 2004, Ratnaswamy et al, 2001, Page et al, 2005, Huff et al, 2003a). One copy of the mutant allele appears to result in complete penetrance of FAF.…”

“…These partially folded states have aggregation-prone sequences that are exposed and have a high propensity to aggregate because of the high β-sheet propensity of the exposed residues, their hydrophobicity and/or their lack of charge (Chiti et al, 2003, Fernandez-Escamilla et al, 2004). In addition, point mutations can also destabilize normally folded proteins leading to a higher concentration of partially denatured protease-sensitive states, affording a higher concentration of an amyloidogenic protein fragment, as in the case of gelsolin amyloidosis—the subject of this review (Chen et al, 2001, Page et al, 2005, Kangas et al, 2002, Huff et al, 2003a, Huff et al, 2003b, Page et al, 2004, Ratnaswamy et al, 2001). Finally, point mutations in either a precursor protein endoprotease substrate or in the endoprotease itself that processes an intrinsically disordered protein can afford a higher concentration of an amyloidogenic fragment or generate alternative polypeptide fragments with increased amyloidogenic potential, as in the case of Alzheimer’s disease where mutations in APP or γ-secretase lead to more aggregation-prone Aβ 1–42 relative to Aβ 1–40 (Tanzi and Bertram, 2005, Selkoe, 2003).…”

Gelsolin Domain 2 Ca2+ Affinity Determines Susceptibility to Furin Proteolysis and Familial Amyloidosis of Finnish Type

“…14,18 The Quickchange method (Stratagene) was used to introduce the E209Q mutation into these constructs and to introduce the R172A mutation into full-length D187N and E209Q.…”

“…thermodynamic stabilities between the wild-type and FAF-variant proteins. 18 Zapun et al established that wild-type gelsolin domain 2 contains a Ca 2þ binding site and suggested that, if D187 contributes to Ca 2þ binding, the FAF mutations could eliminate a potential source of stabilization. 19 Subsequent studies demonstrated that D187 is a ligand for Ca 2þ binding; wild-type domain 2 binds Ca 2þ with a K d of 650 nM, whereas binding is undetectable at micromolar concentrations for D187N and D187Y domain 2.…”