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

Protein misassembly into aggregate structures, including cross-β-sheet amyloid fibrils, is linked to diseases characterized by the degeneration of post-mitotic tissue. While amyloid fibril deposition in the extracellular space certainly disrupts cellular and tissue architecture late in the course of amyloid diseases, strong genetic, pathological and pharmacologic evidence suggests that the process of amyloid fibril formation itself, known as amyloidogenesis, likely causes these maladies. It seems that the formation of oligomeric aggregates during the amyloidogenesis process causes the proteotoxicity and cytotoxicity characteristic of these disorders. Herein, we review what is known about the genetics, biochemistry and pathology of familial amyloidosis of Finnish type (FAF) or gelsolin amyloidosis. Briefly, autosomal dominant D187N or D187Y mutations compromise Ca(2+) binding in domain 2 of gelsolin, allowing domain 2 to sample unfolded conformations. When domain 2 is unfolded, gelsolin is subject to aberrant furin endoproteolysis as it passes through the Golgi on its way to the extracellular space. The resulting C-terminal 68 kDa fragment (C68) is susceptible to extracellular endoproteolytic events, possibly mediated by a matrix metalloprotease, affording 8 and 5 kDa amyloidogenic fragments of gelsolin. These amyloidogenic fragments deposit systemically, causing a variety of symptoms including corneal lattice dystrophy and neurodegeneration. The first murine model of the disease recapitulates the aberrant processing of mutant plasma gelsolin, amyloid deposition, and the degenerative phenotype. We use what we have learned from our biochemical studies, as well as insight from mouse and human pathology to propose therapeutic strategies that may halt the progression of FAF.     

Loss of a metal-binding site in gelsolin leads to familial amyloidosis-Finnish type.

Mutations in domain 2 (D2, residues 151-266) of the actin-binding protein gelsolin cause familial amyloidosis-Finnish type (FAF). These mutations, D187N or D187Y, lead to abnormal proteolysis of plasma gelsolin at residues 172-173 and a second hydrolysis at residue 243, resulting in an amyloidogenic fragment. Here we present the structure of human gelsolin D2 at 1.65 A and find that Asp 187 is part of a Cd2+ metal-binding site. Two Ca2+ ions are required for a conformational transition of gelsolin to its active form. Differential scanning calorimetry (DSC) and molecular dynamics (MD) simulations suggest that the Cd2+-binding site in D2 is one of these two Ca2+-binding sites and is essential to the stability of D2. Mutation of Asp 187 to Asn disrupts Ca2+ binding in D2, leading to instabilities upon Ca2+ activation. These instabilities make the domain a target for aberrant proteolysis, thereby enacting the first step in the cascade leading to FAF.     

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

Protein misassembly into aggregate structures, including cross-β-sheet amyloid fibrils, is linked to diseases characterized by the degeneration of post-mitotic tissue. While amyloid fibril deposition in the extracellular space certainly disrupts cellular and tissue architecture late in the course of amyloid diseases, strong genetic, pathological and pharmacologic evidence suggests that the process of amyloid fibril formation itself, known as amyloidogenesis, likely causes these maladies. It seems that the formation of oligomeric aggregates during the amyloidogenesis process causes the proteotoxicity and cytotoxicity characteristic of these disorders. Herein, we review what is known about the genetics, biochemistry and pathology of familial amyloidosis of Finnish type (FAF) or gelsolin amyloidosis. Briefly, autosomal dominant D187N or D187Y mutations compromise Ca²⁺ binding in domain 2 of gelsolin, allowing domain 2 to sample unfolded conformations. When domain 2 is unfolded, gelsolin is subject to aberrant furin endoproteolysis as it passes through the Golgi on its way to the extracellular space. The resulting C-terminal 68 kDa fragment (C68) is susceptible to extracellular endoproteolytic events, possibly mediated by a matrix metalloprotease, affording 8 and 5 kDa amyloidogenic fragments of gelsolin. These amyloidogenic fragments deposit systemically, causing a variety of symptoms including corneal lattice dystrophy and neurodegeneration. The first murine model of the disease recapitulates the aberrant processing of mutant plasma gelsolin, amyloid deposition, and the degenerative phenotype. We use what we have learned from our biochemical studies, as well as insight from mouse and human pathology to propose therapeutic strategies that may halt the progression of FAF.     

Functional consequences of amyloidosis mutation for gelsolin polypeptide -- analysis of gelsolin-actin interaction and gelsolin processing in gelsolin knock-out fibroblasts.

Gelsolin, an actin-modulating protein, derived from a single gene exists in intracellular and secreted forms. A point mutation at position 187 of both forms of gelsolin causes familial amyloidosis of the Finnish type (FAF). Here, we expressed both isoforms of the wild-type and FAF mutant gelsolin in mouse embryonic gelsolin-null fibroblasts. We demonstrate that the FAF mutation does not interfere with the normal actin-modulating function of intracellular gelsolin, and that aberrant processing of secreted FAF gelsolin to FAF amyloid precursor takes place in the gelsolin-negative background. These results suggest that, in patients with FAF, symptoms are caused by the accumulation in their tissues of amyloid derived from plasma gelsolin and are not due to functional differences in cytoplasmic gelsolin.     

Metalloendoprotease cleavage triggers gelsolin amyloidogenesis

Amyloid diseases like Alzheimer's disease and familial amyloidosis of Finnish type (FAF) stem from endoproteolytic cleavage of a precursor protein to generate amyloidogenic peptides that accumulate as amyloid deposits in a tissue-specific manner. FAF patients deposit both 8 and 5 kDa peptides derived from mutant (D187Y/N) plasma gelsolin in the extracellular matrix (ECM). The first of two aberrant sequential proteolytic events is executed by furin to yield a 68 kDa (C68) secreted fragment. We now identify the metalloprotease MT1-matrix metalloprotease (MMP), an integral membrane protein active in the ECM, as a protease that processes C68 to the amyloidogenic peptides. We further demonstrate that ECM components are capable of accelerating gelsolin amyloidogenesis. Proteolysis by MT1-MMP-like proteases proximal to the unique chemical environment of the ECM offers an explanation for the tissue-specific deposition observed in FAF and provides critical insight into new therapeutic strategies.     

Gelsolin domain 2 Ca2+ affinity determines susceptibility to furin proteolysis and familial amyloidosis of finnish type

Mutation of aspartic acid 187 to asparagine (D187N) or tyrosine (D187Y) in domain 2 of the actin-modulating protein gelsolin causes the neurodegenerative disease familial amyloidosis of Finnish type (FAF). These mutations render plasma gelsolin susceptible to aberrant proteolysis by furin in the trans-Golgi network, the initial proteolytic event in the formation of 71 and 53 residue fragments that assemble into amyloid fibrils. Ca(2+) binding stabilizes wild-type domain 2 gelsolin against denaturation and proteolysis, but the FAF variants are unable to bind and be stabilized by Ca(2+). Though the chain of events initiating FAF has been elucidated recently, uncertainty remains about the mechanistic details that allow the FAF variants to be processed. To test the hypothesis that impaired Ca(2+) binding in the D187 variants, but not other factors specific to residue 187, increases susceptibility to aberrant proteolysis and subsequent amyloidogenesis, we designed the gelsolin variant E209Q to remove a different Ca(2+) ligand from the same Ca(2+) site that is affected in the FAF variants. Here, we show that E209Q domain 2 does not bind Ca(2+) and is not stabilized against denaturation or furin proteolysis, analogous to the behavior exhibited by the FAF variants. Transfection of full-length E209Q into COS cells results in secretion of both the full-length and furin-processed fragments, as observed with D187N and D187Y. Mutation of the furin consensus sequence in D187N and E209Q gelsolin prevents cleavage during secretion, indicating that inhibition of proprotein convertases (furin) represents a viable therapeutic approach for the treatment of FAF. Mutations that diminish domain 2 Ca(2+) binding allow furin access to an otherwise protected cleavage site, initiating the proteolytic cascade that leads to gelsolin amyloidogenesis and FAF.     

Furin initiates gelsolin familial amyloidosis in the Golgi through a defect in Ca(2+) stabilization.

Hereditary familial amyloidosis of Finnish type (FAF) leading to amyloid in the peripheral and central nervous systems stems from deposition of a 71 residue fragment generated from the D187N/Y variants of plasma gelsolin by two sequential endoproteolytic events. We identify the protease accomplishing the first cleavage as furin, a proprotein convertase. Endoproteolysis of plasma gelsolin occurs in the trans-Golgi network due to the inability of the FAF variants to bind and be stabilized by Ca(2+). Secretion and processing of the FAF variants by furin can be uncoupled by blocking the convergence of the exocytic pathway transporting plasma gelsolin and the endocytic recycling of furin. We propose that coincidence of membrane trafficking pathways contributes to the development of proteolysis-initiated amyloid disease.     

Finnish type of familial amyloidosis: cosegregation of Asp187----Asn mutation of gelsolin with the disease in three large families.

Familial amyloidosis of Finnish type (FAF) is one of the familial amyloidotic polyneuropathy (FAP) syndromes, a group of inherited disorders characterized by extracellular accumulation of amyloid and by clinical symptoms and signs of polyneuropathy. FAF, an autosomal dominant trait, belongs to those rare monogenic disorders which occur with increased frequency in the Finnish population: only single FAF cases have been reported from other populations. In most types of FAP syndromes the accumulating protein is a transthyretin variant. However, recent evidence has suggested that the amyloid peptides in FAF are related to gelsolin, an actin modulating protein. The gelsolin fragments isolated from at least one patient with amyloidosis have been reported to have an amino acid substitution, with asparagine replacing aspartic acid at position 187 of the plasma gelsolin. In this study allele-specific oligonucleotides were used to analyze three large FAF families with multiple affected individuals as well as healthy family members. We found the corresponding G-A mutation in nucleotide 654 of the plasma gelsolin gene to cosegregate with the disease. The result was confirmed by sequencing and strongly suggests that the mutation has caused all the FAF cases of these families. Since the disease is clustered in restricted areas on the southern coast of Finland, this mutation most probably causes the majority, if not all, of FAF cases in Finland.     

The actin filament-severing domain of plasma gelsolin

Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.     

Molecular biology of gelsolin, a calcium-regulated actin filament severing protein

Gelsolin is a Ca2+-binding protein of mammalian leukocytes, platelets and other cells which has multiple and closely regulated powerful effects on actin. In the presence of micromolar Ca2+, gelsolin severs actin filaments, causing profound changes in the consistency of actin polymer networks. A variant of gelsolin containing a 25-amino acid extension at the NH2-terminus is present in plasma where it may be involved in the clearance of actin filaments released during tissue damage. Gelsolin has two sites which bind actin cooperatively. These sites have been localized using proteolytic cleavage and monoclonal antibody mapping techniques. The NH2-terminal half of the molecule contains a Ca2+-insensitive actin severing domain while the COOH-terminal half contains a Ca2+-sensitive actin binding domain which does not sever filaments. These data suggest that the NH2-terminal severing domain in intact gelsolin is influenced by the Ca2+-regulated COOH-terminal half of the molecule. The primary structure of gelsolin, deduced from human plasma gelsolin cDNA clones, supports the existence of actin binding domains and suggests that these may have arisen from a gene duplication event, and diverged subsequently to adopt their respective unique functions. The plasma and cytoplasmic forms of gelsolin are encoded by a single gene, and preliminary results indicate that separate mRNAs code for the two forms. Further application of molecular biological techniques will allow exploration into the structural basis for the multifunctionality of gelsolin, as well as the molecular basis for the genesis of the cytoplasmic and secreted forms of gelsolin.     

Gelsolin variant (Asn-187) in familial amyloidosis, Finnish type.

Familial amyloidosis, Finnish type (FAF), is an inherited form of systemic amyloidosis clinically characterized by cranial neuropathy and lattice corneal dystrophy. We have demonstrated that the protein subunit isolated from amyloid fibrils shows considerable sequence identity with gelsolin, an actin-binding protein. We have purified the amyloid subunit from a second case and further analysed different fractions from the previous one. Sequence analysis shows that, in both cases, the amyloid subunit starts at position 173 of the mature molecule; it has a heterogeneous N-terminus and contains one amino acid substitution, namely asparagine for aspartic acid, at position 15 (gelsolin residue 187), that is due to a guanine-to-adenine transversion corresponding to nucleotide-654 of human plasma gelsolin cDNA. The substitution maps in a fragment with actin-binding activity and is located in a repetitive motif highly conserved among species. Thus FAF is the first human disease known to be caused by an internal abnormal degradation of a gelsolin variant. We designate this variant of gelsolin-associated amyloidosis 'Agel Asn-187'.     

Purification and expression of gCap39. An intracellular and secreted Ca2(+)-dependent actin-binding protein enriched in mononuclear phagocytes

A protein of approximately 40 kDa was the major Ca2(+)-binding protein purified by Ca2(+)-dependent hydrophobic affinity chromatography from the cell lysates and conditioned media of RAW macrophages. Other Ca2(+)-binding proteins, including several annexins (calelectrins), S100-like proteins, and calmodulin, were less abundant and preferentially found in the cell lysates. Amino acid sequences of tryptic fragments from the purified 40-kDa protein revealed its identity to gCap39, an actin-binding protein encoded by a cDNA isolated on the basis of its homology with gelsolin. When an expression vector containing the gCap39 coding region was transfected into COS cells, high levels of gCap39 were found in both the cells and conditioned media, whereas annexins were only present in the cells. gCap39 could also be purified from human plasma where it appeared to be a minor component. No signal sequence was detected in the primary structure of gCap39 and the secreted and intracellular forms of gCap39 are of identical size, suggesting that unlike gelsolin, the mechanism of gCap39 secretion may not depend on a signal sequence. The high concentration of gCap39 in macrophages and its constitutive secretion as well as intracellular retention suggest that this protein may have a dual role in macrophage function, namely that of a Ca2(+)- and polyphosphoinositide-regulated intracellular modulator of the cytoskeleton as well as that of a secreted protein involved in the clearance of actin from the extracellular environment.     

Co-operative binding of Ca2+ ions to the regulatory binding sites of gelsolin.

The rate of association of actin with gelsolin was measured at various Ca2+ and ATP concentrations. The fraction of Ca2+-activated gelsolin was determined by quantitative evaluation of the association rates thereby assuming that Ca2+-binding gelsolin associates with actin and Ca2+-free gelsolin does not. A plot of the fraction of Ca2+-activated gelsolin vs. the free Ca2+ concentration revealed a sigmoidal shape suggesting that co-operative binding of Ca2+ ions is required for activation of gelsolin. A good fit of the experimental data by calculated binding curves was obtained if two Ca2+ ions were assumed to bind to actin in a highly co-operative manner. ATP decreased the rate of association of gelsolin with actin and bound to gelsolin at a low affinity (Kd = 32 microm for Ca2+-free and Kd = 400 microm for Ca2+-activated gelsolin). In contrast, a 1 : 1 gelsolin-actin complex was found to be activated for association with actin by a single Ca2+ ion in a non-co-operative manner.     

Monoclonal antibodies to the calcium-binding region peptide of human C-reactive protein alter its conformation.

Five mouse mAb were generated against a synthetic peptide corresponding to the proposed Ca(2+)-binding region of human C-reactive protein (CRP). The peptide consists of amino acids 134 to 148 and possesses a calmodulin Ca(2+)-binding sequence. The mAb reacted with a surface epitope(s) on native, intact CRP as well as the closely related pentraxin protein, serum amyloid P-component. Three of the 5 mAb inhibited the Ca(2+)-dependent phosphorylcholine-(PC) binding activity of CRP, but did not bind to the PC-binding region itself. Four of the five mAb also inhibited the recognition of an epitope in the PC-binding site of CRP. Four of the mAb partially, or completely, protected CRP from selective cleavage by pronase between residues 146 and 147. The findings suggest that the Ca(2+)-binding region is on the surface of CRP, has substantial flexibility, and is probably responsible for the allosteric effects of Ca2+ ions on CRP.     

1-Palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine, an oxidized phospholipid, accelerates Finnish type familial gelsolin amyloidosis in vitro

Finnish type familial amyloidosis (FAF) is a neurodegenerative disease, which involves the deposition of D187N or -Y mutant gelsolin fragments as amyloid in various tissues, accompanied by dermatologic, neurologic, and ophthalmologic disorders. Like the other amyloid diseases, FAF is associated with oxidative stress. The latter results in an extensive chemical modification of biomolecules, such as the formation of a myriad of phospholipids with oxidatively modified acyl chains containing various functional groups. Here we demonstrate that 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), a zwitterionic oxidized phospholipid bearing an aldehyde moiety at the end of its truncated sn-2 acyl chain, accelerates amyloidogenesis of FtG(179-194) (i.e., the core amyloidogenic segment of residues 179-194 of FAF gelsolin) as revealed by thioflavin T (ThT) fluorescence and electron microscopy. These techniques and Trp fluorescence show that the accelerated conversion of FtG(179-194) into amyloid fibrils consists of distinct consecutive phases. PoxnoPC at a close to critical micelle concentration (~22.5 μM) causes a maximal increase in ThT fluorescence and the K(app) for fibril formation. The rates of fibril elongation and nucleation were proportional to PoxnoPC concentration, while the rates of nucleation were different below and above the critical micelle concentration. Our data also suggest an initial rapid formation of a 1:1 complex by PoxnoPC and FtG(179-194). The latter could involve a transient Schiff base and reside at the membrane hydrocarbon-water interface in the proximity of the phosphocholine headgroup. Subsequently, these profibrils insert into a more hydrophobic milieu and undergo a slow structural transition and assemble into amyloid fibers. Different phases can be expected when proteins aggregate on the phospholipid membrane surfaces, underlying the importance of a detailed kinetic analysis to fully understand the effects of oxidized phospholipids on amyloidogenesis. This study represents the first comprehensive analysis of the kinetics and mechanisms of amyloid formation in the presence of an oxidized phospholipid.     

Regulation of neural differentiation by normal and mutant (G654A, amyloidogenic) gelsolin.

Gelsolin belongs to a family of proteins that modulate the structural dynamics of cytoskeletal actin. Gelsolin activity is required for the redistribution of actin occurring during membrane ruffling, cell crawling, and platelet activation. A point mutation (G654A) in the gelsolin gene causes a dominantly inherited systemic amyloidosis called familial amyloidosis of the Finnish type (FAF). This disease is characterized by a cranial neuropathy that cannot be explained solely by amyloid deposits. To address the question of whether gelsolin has a specific role in neural cell development, we transfected cDNA for wild type and G654A point-mutated gelsolin into a neural cell line, Paju, which can be induced to differentiate by treatment with phorbol 12-myristate 13-acetate. Overexpressed wild type gelsolin inhibited neural differentiation whereas mutated gelsolin did not, indicating that appropriate gelsolin activity is essential for neural sprouting. The G654A mutant gelsolin induced stabilization of F-actin and reduced the plasticity of neural development. This provides a novel etiopathogenetic mechanism for the neuronal dysfunction in FAF.     

Beyond genetic factors in familial amyloidotic polyneuropathy: protein glycation and the loss of fibrinogen's chaperone activity

Familial amyloidotic polyneuropathy (FAP) is a systemic conformational disease characterized by extracellular amyloid fibril formation from plasma transthyretin (TTR). This is a crippling, fatal disease for which liver transplantation is the only effective therapy. More than 80 TTR point mutations are associated with amyloidotic diseases and the most widely accepted disease model relates TTR tetramer instability with TTR point mutations. However, this model fails to explain two observations. First, native TTR also forms amyloid in systemic senile amyloidosis, a geriatric disease. Second, age at disease onset varies by decades for patients bearing the same mutation and some mutation carrier individuals are asymptomatic throughout their lives. Hence, mutations only accelerate the process and non-genetic factors must play a key role in the molecular mechanisms of disease. One of these factors is protein glycation, previously associated with conformational diseases like Alzheimer's and Parkinson's. The glycation hypothesis in FAP is supported by our previous discovery of methylglyoxal-derived glycation of amyloid fibrils in FAP patients. Here we show that plasma proteins are differentially glycated by methylglyoxal in FAP patients and that fibrinogen is the main glycation target. Moreover, we also found that fibrinogen interacts with TTR in plasma. Fibrinogen has chaperone activity which is compromised upon glycation by methylglyoxal. Hence, we propose that methylglyoxal glycation hampers the chaperone activity of fibrinogen, rendering TTR more prone to aggregation, amyloid formation and ultimately, disease.     

Isolation and characterization of cardiac amyloid in familial amyloid polyneuropathy type IV (Finnish): relation of the amyloid protein to variant gelsolin

Amyloid subunit protein was isolated from familial amyloid polyneuropathy type IV (Finnish type) cardiac tissue and purified to homogeneity. N-terminal amino acid sequence analysis shows that the amyloid protein is a fragment of the inner region of human gelsolin. When compared with the predicted sequence of human plasma gelsolin, the amyloid protein contains an asparagine-for-aspartic acid substitution at position 15 corresponding to residue 187 of the secreted protein. Antibodies raised against the amyloidogenic region of gelsolin specifically stained the amyloid deposited in tissues in familial amyloidosis type IV. The results show that the subunit amyloid protein in familial amyloid polyneuropathy type IV represents a unique type of amyloid derived from a variant (Asn-187) gelsolin molecule by limited proteolysis.     

Muscle is the major source of plasma gelsolin

Gelsolin, a Ca2+- and polyphosphoinositide-regulated actin-binding protein, is unique among vertebrate proteins in being both cytoplasmic and secreted. Plasma gelsolin, present at greater than 200 micrograms/ml in human plasma, may have a protective function by promoting the clearance of actin filaments released during tissue injury. Although there is evidence that smooth muscle tissues and HepG2 cells synthesize plasma gelsolin, the predominant secretory source is hitherto unknown. We report here that skeletal, cardiac, and smooth muscles have large amounts of plasma gelsolin mRNA and devote 0.5-3% of their biosynthetic activity to plasma gelsolin, whereas liver makes relatively little. Since skeletal muscle accounts for a large fraction of body mass and total protein synthesis, it is the major source of plasma gelsolin.     

A novel flagellar Ca2+-binding protein in trypanosomes

A 24-kDa protein of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is recognized by antisera from both humans and experimental animals infected with this organism. Near its C terminus are two regions that have sequence similarity with several Ca2+-binding proteins and that conform to the "E-F hand" Ca2+-binding structure. We expressed a cDNA encoding this protein in Escherichia coli and showed that both the recombinant protein and the 24-kDa native trypanosome protein do indeed bind Ca2+. The protein's low Ca2+-binding capacity (less than 2 mol of Ca2+/mol of protein) and high Ca2+-binding affinity (apparent Kd less than 50 microM Ca2+) are consistent with binding of Ca2+ via the E-F hand structures. Immunofluorescence assays using a mouse antiserum directed against the fusion protein localized the native protein to the trypanosome's flagellum. The protein's abundance, Ca2+-binding property, and flagellar localization suggest that it participates in molecular processes associated with the high motility of the parasite.