Inhibiting Misfolding by Endogenous Amyloid Binding Proteins

Diverse proteins have been identified immunohistochemically to be associated with amyloid deposits (15). Some of these amyloid-associated proteins have been found in many diverse types of amyloid plaques and are known as universal components of amyloid, including apolipoprotein E (apoE), serum amyloid-P (SAP), and heparan sulfate proteoglycans (41-43). It has been hypothesized that some of these proteins and others known to interact with the protein before misfolding may serve as endogenous chaperones to stabilize the native folding and prevent protein aggregation (44-46).

Indeed, several reports have shown that some proteins (e.g., apoE, apolipoprotein J [apoJ], SAP, a-antichymotrypsin, laminin, transthyretin, albumin) can bind AP with high affinity and prevent amyloid formation in vitro (47-53). However, other studies have shown that these same proteins can also promote fibrillogenesis in vitro (54-56). These contradictory results have been explained by differences in the peptide concentration, assay sensitivity, solvents, or different peptide sources (45). Nevertheless, the relevance of these proteins in vivo is still unclear.

Transthyretin (TTR), a homotetramer with 127 amino acid residue in each chain, is synthesized in the liver and is found in blood plasma and cerebrospi-nal fluid (57). TTR is able to form amyloid which accumulate in different peripheral nerves of patients affected by familial amyloid polyneuropathy or in the heart of people affected by familial amyloid cardiomyopathy (58). It has been published that TTR is a major AP-binding protein in cerebrospinal fluid (59), and further studies demonstrated that TTR is able to prevent formation of AP fibrils in vitro, sequestering AP from cerebrospinal fluid by a stable complex formation (49).

Laminin is a four-arm glycoprotein component of the extracellular matrix. This protein is induced by brain injury, and it has been shown to accumulate in senile plaques of patient with AD (60). Thioflavin T-fluorescence assays and electronic microscopy studies shown that laminin inhibits the amyloid forma tion in a concentration-dependent manner (53). Experiments in rat hippocam-pal neurons show the ability of laminin-1 and laminin-2 to inhibit fibril formation and reduce AP toxicity (61). Also, it was demonstrated that amyloid fibrils are unstable in presence of laminin-1.

apoE is a homotetrameric, highly a-helical protein primarily known for its involvement in cholesterol metabolism (62). Contradictory results have been published for apoE's role in AP fibrillogenesis in vitro (47,54,55). In cell cultures, apoE produces the inhibition of AP aggregation (63), whereas in canine smooth muscle cells, the addition of apoE3 or apoE4 induces accumulation of AP-immunoreactive deposits (64). There are three alleles of ApoE in the human population. ApoE3 is the major allele and is considered the wild type. Considerable evidence demonstrates that the E4 allele of apoE is an important risk factor for AD, being present in 30 to 50% of patients who develop late-onset AD (65,66). By contrast, inheritance of the E2 allele appears to reduce the risk of developing AD (67). Amyloid precursor protein (APP) transgenic mice lacking apoE, develop diffuse, nonfibrillar AP deposits, but the amyloid burden is markedly reduced in old mice when compared with the transgenic complemented with murine apoE (68). Expression of apoE4 in the absence of mouse apoE increases hippocampal AP levels and amyloid burden (69). One hypothesis to explain the linkage between the E4 allele and late-onset AD is that apoE4 may form a less stable complex with AP than does apoE3 or apoE2, thereby rendering AP more prompt to aggregate or less sensitive to degradation by endogenous proteases.

Another protein implicated in controlling AP aggregation is apoJ, also known as clusterin because of its ability to elicit clustering of Sertoli cells (70). Overexpression of this protein has been observed in many pathological conditions involving injury and chronic inflammation of the brain. Also immunohis-tochemical studies show apoJ in association with amyloid plaques and vascular amyloid in AD cortex and hippocampus (71,72). Different data sources from in vitro studies demonstrates that apoJ plays an important role as a carrier for AP protein (73), forming a stable complex under physiological conditions. As a result of the complex formation, aggregation and polymerization is inhibited, and AP remains in the soluble form (48). In addition, resistance to neuronal death in apoJ-immunoreactive neurons suggests a neuroprotective role of clusterin (74). However, in vivo studies in APP transgenic mice show a significant reduction in fibrillar deposits when the apoJ gene is knocked out, arguing for an opposite effect as in vitro (75).

SAP is a calcium-dependent lectin, a normal plasma glycoprotein (76). It consists of five identical 25-kDa subunits of 204 amino acids. Each subunit has multiple antiparallel P-strands arranged in two sheets. It has been reported that SAP binds to DNA, chromatin, and glycosaminoglycans such as heparin, heparan, and dermatan sulfate, which are frequently associated with amyloid deposits (77). Purified SAP inhibits the fibrils formation of AP peptide, increasing its solubility in a dose-dependent manner (50). However, other studies provided evidence for an opposite effect of SAP in vitro (56). In addition, SAP has been shown to prevent the proteolysis of the AP fibrils when the protein is bound to the aggregates, contributing to their stabilization in the tissue (78). Experiments in SAP knockout animals demonstrated that the induction of reactive amyloidosis is retarded in mice with targeted deletion of the SAP gene

(79). These observations indicate that inhibition of SAP binding to amyloid fibrils might be an attractive therapeutic target for drugs to clear amyloidosis

(80). The drug CPHPC (R-1-[6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid) was developed to prevent the SAP binding to amyloid fibrils (80).Clinical trials with CPHPC show a decrease in circulating SAP, but its efficiency in amyloid clearance and reduction has not been demonstrated.

a-l-Antichymotrypsin (ACT) is a glial-derived serine protease inhibitor of cathepsin and chymotrypsin-like enzymes (81). ACT, a minor protein component of P-amyloid deposits, is able to inhibit AP aggregation into fibrils (82,83), but it is incapable of modulating the toxicity in primary rat hippocampal cell cultures (82). However, other in vitro studies have reported the enhancement of amyloid formation by ACT (55). The later conclusion is supported by in vivo experiments in transgenic animals that overexpresses the mutant APP and ACT showed that the protein promotes amyloid deposition (84). Further experiments indicated that depending on the ACT concentration, both inhibitory and promoter effects can be seen, and a molecular explanation was given based on the binding of AP to two different P-sheets of ACT (51).

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