Amyloid Plaques Beyond Aβ: A Survey of the Diverse Modulators of Amyloid Aggregation

Aggregation of the amyloid-β (Aβ) peptide is strongly correlated with Alzheimer’s disease (AD). Recent research has improved our understanding of the kinetics of amyloid fibril assembly and revealed new details regarding different stages in plaque formation. Presently, interest is turning toward studying this process in a holistic context, focusing on cellular components which interact with the Aβ peptide at various junctures during aggregation, from monomer to cross-β amyloid fibrils. However, even in isolation, a multitude of factors including protein purity, pH, salt content, and agitation affect Aβ fibril formation and deposition, often producing complicated and conflicting results. The failure of numerous inhibitors in clinical trials for AD suggests that a detailed examination of the complex interactions that occur during plaque formation, including binding of carbohydrates, lipids, nucleic acids, and metal ions, is important for understanding the diversity of manifestations of the disease. Unraveling how a variety of key macromolecular modulators interact with the Aβ peptide and change its aggregation properties may provide opportunities for developing therapies. Since no protein acts in isolation, the interplay of these diverse molecules may differentiate disease onset, progression, and severity, and thus are worth careful consideration

Acute-Phase-HDL Remodeling by Heparan Sulfate Generates a Novel Lipoprotein with Exceptional Cholesterol Efflux Activity from Macrophages

During episodes of acute-inflammation high-density lipoproteins (HDL), the carrier of so-called good cholesterol, experiences a major change in apolipoprotein composition and becomes acute-phase HDL (AP-HDL). This altered, but physiologically important, HDL has an increased binding affinity for macrophages that is dependent on cell surface heparan sulfate (HS). While exploring the properties of AP-HDL∶HS interactions we discovered that HS caused significant remodeling of AP-HDL. The physical nature of this change in structure and its potential importance for cholesterol efflux from cholesterol-loaded macrophages was therefore investigated. In the presence of heparin, or HS, AP-HDL solutions at pH 5.2 became turbid within minutes. Analysis by centrifugation and gel electrophoresis indicated that AP-HDL was remodeled generating novel lipid poor particles composed only of apolipoprotein AI, which we designate β2. This remodeling is dependent on pH, glycosaminoglycan type, is promoted by Ca2+ and is independent of protease or lipase activity. Compared to HDL and AP-HDL, remodeled AP-HDL (S-HDL-SAA), containing β2 particles, demonstrated a 3-fold greater cholesterol efflux activity from cholesterol-loaded macrophage. Because the identified conditions causing this change in AP-HDL structure and function can exist physiologically at the surface of the macrophage, or in its endosomes, we postulate that AP-HDL contains latent functionalities that become apparent and active when it associates with macrophage cell surface/endosomal HS. In this way initial steps in the reverse cholesterol transport pathway are focused at sites of injury to mobilize cholesterol from macrophages that are actively participating in the phagocytosis of damaged membranes rich in cholesterol. The mechanism may also be of relevance to aspects of atherogenesis

Roles of the Amino Terminal Region and Repeat Region of the Plasmodium berghei Circumsporozoite Protein in Parasite Infectivity

The circumsporozoite protein (CSP) plays a key role in malaria sporozoite infection of both mosquito salivary glands and the vertebrate host. The conserved Regions I and II have been well studied but little is known about the immunogenic central repeat region and the N-terminal region of the protein. Rodent malaria Plasmodium berghei parasites, in which the endogenous CS gene has been replaced with the avian Plasmodium gallinaceum CS (PgCS) sequence, develop normally in the A. stephensi mosquito midgut but the sporozoites are not infectious. We therefore generated P. berghei transgenic parasites carrying the PgCS gene, in which the repeat region was replaced with the homologous region of P. berghei CS (PbCS). A further line, in which both the N-terminal region and repeat region were replaced with the homologous regions of PbCS, was also generated. Introduction of the PbCS repeat region alone, into the PgCS gene, did not rescue sporozoite species-specific infectivity. However, the introduction of both the PbCS repeat region and the N-terminal region into the PgCS gene completely rescued infectivity, in both the mosquito vector and the mammalian host. Immunofluorescence experiments and western blot analysis revealed correct localization and proteolytic processing of CSP in the chimeric parasites. The results demonstrate, in vivo, that the repeat region of P. berghei CSP, alone, is unable to mediate sporozoite infectivity in either the mosquito or the mammalian host, but suggest an important role for the N-terminal region in sporozoite host cell invasion

Establishment of a Transgenic Mouse Model Specifically Expressing Human Serum Amyloid A in Adipose Tissue

Obesity and obesity co-morbidities are associated with a low grade inflammation and elevated serum levels of acute phase proteins, including serum amyloid A (SAA). In the non-acute phase in humans, adipocytes are major producers of SAA but the function of adipocyte-derived SAA is unknown. To clarify the role of adipocyte-derived SAA, a transgenic mouse model expressing human SAA1 (hSAA) in adipocytes was established. hSAA expression was analysed using real-time PCR analysis. Male animals were challenged with a high fat (HF) diet. Plasma samples were subjected to fast protein liquid chromatography (FPLC) separation. hSAA, cholesterol and triglyceride content were measured in plasma and in FPLC fractions. Real-time PCR analysis confirmed an adipose tissue-specific hSAA gene expression. Moreover, the hSAA gene expression was not influenced by HF diet. However, hSAA plasma levels in HF fed animals (37.7±4.0 µg/mL, n = 7) were increased compared to those in normal chow fed animals (4.8±0.5 µg/mL, n = 10; p<0.001), and plasma levels in the two groups were in the same ranges as in obese and lean human subjects, respectively. In FPLC separated plasma samples, the concentration of hSAA peaked in high-density lipoprotein (HDL) containing fractions. In addition, cholesterol distribution over the different lipoprotein subfractions as assessed by FPLC analysis was similar within the two experimental groups. The established transgenic mouse model demonstrates that adipose tissue produced hSAA enters the circulation, resulting in elevated plasma levels of hSAA. This new model will enable further studies of metabolic effects of adipose tissue-derived SAA

Heparin Induces Harmless Fibril Formation in Amyloidogenic W7FW14F Apomyoglobin and Amyloid Aggregation in Wild-Type Protein In Vitro

Glycosaminoglycans (GAGs) are frequently associated with amyloid deposits in most amyloid diseases, and there is evidence to support their active role in amyloid fibril formation. The purpose of this study was to obtain structural insight into GAG-protein interactions and to better elucidate the molecular mechanism underlying the effect of GAGs on the amyloid aggregation process and on the related cytotoxicity. To this aim, using Fourier transform infrared and circular diochroism spectroscopy, electron microscopy and thioflavin fluorescence dye we examined the effect of heparin and other GAGs on the fibrillogenesis and cytotoxicity of aggregates formed by the amyloidogenic W7FW14 apomyoglobin mutant. Although this protein is unrelated to human disease, it is a suitable model for in vitro studies because it forms amyloid-like fibrils under physiological conditions of pH and temperature. Heparin strongly stimulated aggregation into amyloid fibrils, thereby abolishing the lag-phase normally detected following the kinetics of the process, and increasing the yield of fibrils. Moreover, the protein aggregates were harmless when assayed for cytotoxicity in vitro. Neutral or positive compounds did not affect the aggregation rate, and the early aggregates were highly cytotoxic. The surprising result that heparin induced amyloid fibril formation in wild-type apomyoglobin and in the partially folded intermediate state of the mutant, i.e., proteins that normally do not show any tendency to aggregate, suggested that the interaction of heparin with apomyoglobin is highly specific because of the presence, in protein turn regions, of consensus sequences consisting of alternating basic and non-basic residues that are capable of binding heparin molecules. Our data suggest that GAGs play a dual role in amyloidosis, namely, they promote beneficial fibril formation, but they also function as pathological chaperones by inducing amyloid aggregation

A Honey Bee Hexamerin, HEX 70a, Is Likely to Play an Intranuclear Role in Developing and Mature Ovarioles and Testioles

Insect hexamerins have long been known as storage proteins that are massively synthesized by the larval fat body and secreted into hemolymph. Following the larval-to-pupal molt, hexamerins are sequestered by the fat body via receptor-mediated endocytosis, broken up, and used as amino acid resources for metamorphosis. In the honey bee, the transcript and protein subunit of a hexamerin, HEX 70a, were also detected in ovaries and testes. Aiming to identify the subcellular localization of HEX 70a in the female and male gonads, we used a specific antibody in whole mount preparations of ovaries and testes for analysis by confocal laser-scanning microscopy. Intranuclear HEX 70a foci were evidenced in germ and somatic cells of ovarioles and testioles of pharate-adult workers and drones, suggesting a regulatory or structural role. Following injection of the thymidine analog EdU we observed co-labeling with HEX 70a in ovariole cell nuclei, inferring possible HEX 70a involvement in cell proliferation. Further support to this hypothesis came from an injection of anti-HEX 70a into newly ecdysed queen pupae where it had a negative effect on ovariole thickening. HEX 70a foci were also detected in ovarioles of egg laying queens, particularly in the nuclei of the highly polyploid nurse cells and in proliferating follicle cells. Additional roles for this storage protein are indicated by the detection of nuclear HEX 70a foci in post-meiotic spermatids and spermatozoa. Taken together, these results imply undescribed roles for HEX 70a in the developing gonads of the honey bee and raise the possibility that other hexamerins may also have tissue specific functions

A Computational Approach for Identifying the Chemical Factors Involved in the Glycosaminoglycans-Mediated Acceleration of Amyloid Fibril Formation

BACKGROUND: Amyloid fibril formation is the hallmark of many human diseases, including Alzheimer's disease, type II diabetes and amyloidosis. Amyloid fibrils deposit in the extracellular space and generally co-localize with the glycosaminoglycans (GAGs) of the basement membrane. GAGs have been shown to accelerate the formation of amyloid fibrils in vitro for a number of protein systems. The high number of data accumulated so far has created the grounds for the construction of a database on the effects of a number of GAGs on different proteins. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have constructed such a database and have used a computational approach that uses a combination of single parameter and multivariate analyses to identify the main chemical factors that determine the GAG-induced acceleration of amyloid formation. We show that the GAG accelerating effect is mainly governed by three parameters that account for three-fourths of the observed experimental variability: the GAG sulfation state, the solute molarity, and the ratio of protein and GAG molar concentrations. We then combined these three parameters into a single equation that predicts, with reasonable accuracy, the acceleration provided by a given GAG in a given condition. CONCLUSIONS/SIGNIFICANCE: In addition to shedding light on the chemical determinants of the protein∶GAG interaction and to providing a novel mathematical predictive tool, our findings highlight the possibility that GAGs may not have such an accelerating effect on protein aggregation under the conditions existing in the basement membrane, given the values of salt molarity and protein∶GAG molar ratio existing under such conditions

Implications of Heparan Sulfate and Heparanase in Amyloid Diseases

Amyloidosis refers to a group of diseases characterized by abnormal deposition of denatured endogenous proteins, termed amyloid, in the affected organs. Analysis of biopsy and autopsy tissues from patients revealed the presence of heparan sulfate proteoglycans (HSPGs) along with amyloid proteins in the deposits. For a long time, HSPGs were believed to occur in the deposits as an innocent bystander. Yet, the consistent presence of HSPGs in various deposits, regardless of the amyloid species, led to the hypothesis that these macromolecular glycoconjugates might play functional roles in the pathological process of amyloidosis. In vitro studies have revealed that HSPGs, or more precisely, the heparan sulfate (HS) side chains interact with amyloid peptides, thus promoting amyloid fibrillization. Although information on the mechanisms of HS participation in amyloid deposition is limited, recent studies involving a transgenic mouse model of Alzheimer’s disease point to an active role of HS in amyloid formation. Heparanase cleavage alters the molecular structure of HS, and thus modulates the functional roles of HS in homeostasis, as well as in diseases, including amyloidosis. The heparanase transgenic mice have provided models for unveiling the effects of heparanase, through cleavage of HS, in various amyloidosis conditions.</p