Toxoplasma gondii AP2IX-4 Regulates Gene Expression during Bradyzoite Development.

Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst that is imperceptible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immunocompromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, and yet the mechanisms underlying these alterations in gene expression are not well defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction-a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as the levels of many bradyzoite mRNAs dramatically increased beyond those seen under conditions of normal stress induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite. IMPORTANCEToxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite.

Opposing Transcriptional Mechanisms Regulate Toxoplasma Development.

The Toxoplasma biology that underlies human chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite stage. We investigated the roles of two alkaline-stress-induced ApiAP2 transcription factors, AP2IV-3 and AP2IX-9, in bradyzoite development. These factors were expressed in two overlapping waves during bradyzoite development, with AP2IX-9 increasing expression earlier than AP2IV-3, which peaked as AP2IX-9 expression was declining. Disruption of the AP2IX-9 gene enhanced, while deletion of AP2IV-3 gene decreased, tissue cyst formation, demonstrating that these factors have opposite functions in bradyzoite development. Conversely, conditional overexpression of FKBP-modified AP2IX-9 or AP2IV-3 with the small molecule Shield 1 had a reciprocal effect on tissue cyst formation, confirming the conclusions of the knockout experiments. The AP2IX-9 repressor and AP2IV-3 activator tissue cyst phenotypes were borne out in gene expression studies that determined that many of the same bradyzoite genes were regulated in an opposite manner by these transcription factors. A common gene target was the canonical bradyzoite marker BAG1, and mechanistic experiments determined that, like AP2IX-9, AP2IV-3 regulates a BAG1 promoter-luciferase reporter and specifically binds the BAG1 promoter in parasite chromatin. Altogether, these results suggest that the AP2IX-9 transcriptional repressor and the AP2IV-3 transcriptional activator likely compete to control bradyzoite gene expression, which may permit Toxoplasma to better adapt to different tissue environments and select a suitable host cell for long-term survival of the dormant tissue cyst. IMPORTANCEToxoplasma infections are lifelong because of the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control tissue cyst formation is still poorly understood. Significant changes in gene expression are associated with tissue cyst development, and ApiAP2 transcription factors are an important mechanism regulating this developmental transcriptome. However, the molecular composition of these ApiAP2 complexes and the operating principles of ApiAP2 mechanisms are not well defined. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway.

The role of the C-terminus of human α-synuclein: intra-disulfide bonds between the C-terminus and other regions stabilize non-fibrillar monomeric isomers.

Substantial evidence implicates that the aggregation of α-synuclein (αSyn) is a critical factor in the pathogenesis of Parkinson's disease. This study focuses on the role of αSyn C-terminus. We introduced two additional cysteine residues at positions 107 and 124 (A107C and A124C) to our previous construct. Five X-isomers of oxidative-folded mutation of α-synuclein with three disulfides were isolated and their secondary structures and aggregating features were analyzed. All isomers showed similar random coil structures as wild-type α-synuclein. However, these isomers did not form aggregates or fibrils, even with prolonged incubation, suggesting that the interactions between the C-terminal and N-terminal or central NAC region are important in maintaining the natively unfolded structure of αSyn and thus prevent αSyn from changing conformation, which is a critical step for fibrillation.

Characterization of the non-fibrillar α-synuclein oligomers.

Under certain in vitro conditions, α-Synuclein is an abundant 14 kDa presynaptic intrinsically disordered protein, involved in the pathogenesis of Parkinson's disease (PD) forms amyloid fibrils which resemble those found in Lewy bodies of PD patients. However, a substantial fraction of α-synuclein molecules (10-20 %) does not form fibrils during fibrillation and exists in a form of soluble oligomers. In this study, we examined these soluble oligomers by a variety of biophysical techniques including atomic force microscopy (AFM), circular dichroism, Fourier-transform infrared spectroscopy and thioflavin T fluorescence. We observed that the fibrillation kinetics is affected by the variation in salt and protein concentrations. Although both high salt and high protein concentrations noticeably accelerated α-synuclein fibrillation, the amount of non-fibrillar oligomers is independent of the salt content. The oligomers formed at low salt concentration adopt more ß-sheet structure and are smaller in size than those formed at high salt concentration. AFM analysis shows that the low salt oligomers represent a mixture of small oligomers and some amorphous aggregates, whereas oligomers formed at high salt concentrations are noticeably larger, more homogenous, and are mostly spherical in shape. All the late stage non-fibrillar oligomers do not form fibrils even when seeded with pre-formed fibrils, are characterized by negligible rates of dissociation, likely due to their intertwined structure, and are able to disrupt the integrity of the biological membrane. These findings suggest that these soluble oligomers are important players in the multi-pathway aggregation of α-synuclein and should be taken into account in studies on the molecular mechanisms of this protein fibrillation.

At low concentrations, 3,4-dihydroxyphenylacetic acid (DOPAC) binds non-covalently to alpha-synuclein and prevents its fibrillation.

Several studies have shown that catecholamines can inhibit the fibrillation of alpha-synuclein (alpha-Syn), a small presynaptic protein whose aggregation is believed to be a critical step in the etiology of Parkinson's disease and several other neurodegenerative disorders. However, the mechanism of this inhibition is uncertain. We show here that substoichiometric concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), a normal product of the metabolism of dopamine, can inhibit the fibrillation of alpha-Syn, due to non-covalent binding of DOPAC to alpha-Syn monomer. Intriguingly, the presence of alpha-Syn accelerates the spontaneous oxidation of DOPAC, and the oxidized form of DOPAC (the quinone) is responsible for the fibrillation inhibition. In addition, the presence of DOPAC leads to the oxidation of the methionine residues of alpha-Syn, probably due to the H(2)O(2) production as a by-product of DOPAC oxidation. The lack of fibrillation results from the formation of stable oligomers, which are very similar to those observed transiently at early stages of the alpha-Syn fibrillation. A possible explanation for this phenomenon is that DOPAC stabilizes the normally transient oligomers and prevents them from subsequent fibril formation. The analysis of the alpha-Syn Y39W variant suggests that DOPAC binds non-covalently to the same N-terminal region of alpha-Syn as lipid vesicles, probably in the vicinity of residue 39. In contrast to the compounds with 1,2-dihydroxyphenyl groups (DOPAC and catechol), their 1,4-dihydroxyphenyl isomers (hydroquinone and homogentisic acid) are able to modify alpha-Syn covalently, probably due to the less steric hindrance in the Michael addition.

Smoking and Parkinson's disease: does nicotine affect alpha-synuclein fibrillation?

alpha-synuclein is a small presynaptic protein (14,460 D) that is abundantly distributed in the brain. Although, its function is unknown, the aggregated form of alpha-synuclein is a pathological hallmark of several neurodegenerative diseases, including Parkinson's disease (PD). Epidemiological studies have shown that smoking can lessen the incidence of Parkinson's disease, indicating that smoke may contain chemicals that are neuro-protective. The fibrillation of alpha-synuclein was studied in relation to five different compounds found in cigarette smoke: anabasine, cotinine, hydroquinone, nicotine and nornicotine. Thioflavin T assays, gel electrophoresis, size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) and atomic force microscopy (AFM) were utilized to monitor the rate of alpha-synuclein fibrillation and the inhibitory effects of the cigarette smoke components. We show that nicotine and hydroquinone inhibit alpha-synuclein fibril formation in a concentration-dependent manner, with nicotine being more effective. The SEC-HPLC data show that nicotine and hydroquinone stabilize soluble oligomers. The morphology of the oligomers stabilized by nicotine was evaluated by AFM, which showed the presence of three stable oligomers with an average height of 16 nm, 10 nm and 4 nm. Comparable results were obtained for the effect of the cigarette smoke components on the A53T mutant fibrillation. These results show that nicotine and hydroquinone inhibit alpha-synuclein fibrillation and stabilize soluble oligomeric forms. This information can be used to understand the molecular mechanism of the nicotine and hydroquinone action to develop therapeutic solutions for PD.

Structural characteristics of alpha-synuclein oligomers stabilized by the flavonoid baicalein.

The flavonoid baicalein inhibits fibrillation of alpha-synuclein, which is a major component of Lewy bodies in Parkinson's disease. It has been known that baicalein induces the formation of alpha-synuclein oligomers and consequently prevents their fibrillation. In order to evaluate the structural properties of baicalein-stabilized oligomers, we purified oligomer species by HPLC and examined their stability and structure by CD, Fourier transform infrared spectroscopy, size exclusion chromatography HPLC, small-angle X-ray scattering, and atomic force microscopy. Baicalein-stabilized oligomers are beta-sheet-enriched according to CD and Fourier transform infrared spectroscopy analyses. They did not form fibrils even after very prolonged incubation. From small-angle X-ray scattering data and atomic force microscopy images, the oligomers were characterized as quite compact globular species. Oligomers were extremely stable, with a GdmCl C(m)=3.3 M. This high stability explains the previously observed inhibition properties of baicalein against alpha-synuclein fibrillation. These baicalein-stabilized oligomers, added to the solution of aggregating alpha-synuclein, were able to noticeably inhibit its fibrillation. After prolonged coincubation, short fibrils were formed, suggesting an effective interaction of oligomers with monomeric alpha-synuclein. Membrane permeability tests suggested that the baicalein-stabilized oligomers had a mild effect on the integrity of the membrane surface. This effect was rather similar to that of the monomeric protein, suggesting that targeted stabilization of certain alpha-synuclein oligomers might offer a potential strategy for the development of novel Parkinson's disease therapies.

Role of different regions of alpha-synuclein in the assembly of fibrils.

Elucidating the details of the assembly of amyloid fibrils is a key step to understanding the mechanism of amyloid deposition diseases including Parkinson's disease. Although several models have been proposed, based on analyses of polypeptides and short peptides, a detailed understanding of the structure and mechanism of alpha-synuclein fibrillation remains elusive. In this study, we used trypsin and endoproteinase GluC to digest intact alpha-synuclein fibrils and to analyze the detailed morphology of the resultant fibrils/remnants. We also created three mutants of alpha-synuclein, in which the N-terminal and C-terminal regions were removed, both individually and in combination, and investigated the detailed morphology of the fibrils from these mutants. Our results indicate that the assembly of mature alpha-synuclein fibrils is hierarchical: protofilaments --> protofibrils --> mature fibrils. There is a core region of approximately 70 amino acids, from residues approximately 32 to 102, which comprises the beta-rich core of the protofilaments and fibrils. In contrast, the two terminal regions show no evidence of participating in the assembly of the protofilament core but play a key role in the interactions between the protofilaments, which is necessary for the fibril maturation.

Fibrillation of human insulin A and B chains.

Human insulin, which consists of disulfide cross-linked A and B polypeptide chains, readily forms amyloid fibrils under slightly destabilizing conditions. We examined whether the isolated A and B chain peptides of human insulin would form fibrils at neutral and acidic pH. Although insulin exhibits a pH-dependent lag phase in fibrillation, the A chain formed fibrils without a lag at both pHs. In contrast, the B chain exhibited complex concentration-dependent fibrillation behavior at acidic pH. At higher concentrations, e.g., >0.2 mg/mL, the B chains preferentially and rapidly formed stable protofilaments rather than mature fibrils upon incubation at 37 degrees C. Surprisingly, these protofilaments did not convert into mature fibrils. At lower B chain concentrations, however, mature fibrils were formed. The explanation for the concentration dependence of B chain fibrillation is as follows. The B chains exist as soluble oligomers at acidic pH, have a beta-sheet rich conformation as determined by CD, and bind ANS strongly, and these oligomers rapidly form dead-end protofilaments. However, under conditions in which the B chain monomer is present, such as low B chain concentration (<0.2 mg/mL) or in the presence of low concentrations of GuHCl, which dissociates the soluble oligomers, mature fibrils were formed. Thus, both A and B chain peptides can form amyloid fibrils, and both are likely to be involved in the interactions leading to the fibrillation of intact insulin.

Characterization of oligomers during alpha-synuclein aggregation using intrinsic tryptophan fluorescence.

The aggregation of the presynaptic protein alpha-synuclein is associated with Parkinson's disease (PD). The details of the mechanism of aggregation, as well as the cytotoxic species, are currently not well understood. alpha-Synuclein has four tyrosine and no tryptophan residues. We introduced a tyrosine to tryptophan mutation at position 39 to create an intrinsic fluorescence probe and allow additional characterization of the aggregation process. Y39W alpha-synuclein had similar fibrillation kinetics (2-fold slower), pH-induced conformational changes, and fibril morphology to wild-type alpha-synuclein. In addition to intrinsic Trp fluorescence, acrylamide quenching, fluorescence anisotropy, ANS binding, dynamic light scattering, and FTIR were employed to monitor the kinetics of aggregation. These biophysical probes revealed the significant population of two classes of oligomeric intermediates, one formed during the lag period of fibrillation and the other present at the completion of fibrillation. As expected for a natively unfolded protein, Trp 39 was highly solvent-exposed in the monomer and is solvent-exposed in the two oligomeric intermediates; however, it is partially, but not fully, buried in the fibrils. These observations demonstrate the utility of Trp fluorescence labeled alpha-synuclein and demonstrate the existence of an oligomeric intermediate that exists as a transient reservoir of alpha-synuclein for fibrillation.

Independent heterologous fibrillation of insulin and its B-chain peptide.

Insulin is very prone to form amyloid fibrils under slightly destabilizing conditions, and the B-chain region plays a critical role in the fibrillation. We show here that the isolated B-chain peptide of bovine insulin also forms fibrils at both acidic and neutral pH. When a mixture of insulin and the B-chain peptide was incubated at either acidic or neutral pH, the formation of fibrils was clearly separated into two phases, with the faster phase corresponding to the formation of homogeneous fibrils from the B-chain and the slower phase corresponding to homogeneous fibrillation of insulin. To further investigate the interaction (or lack thereof) between the two polypeptides, we examined the effects of cross-seeding. The results indicate that seeds of B-chain fibrils accelerate the fibrillation of insulin at pH 1.6 and inhibit the fibrillation at pH 7.5, but seeds of insulin fibrils have little effect on the fibrillation of the B-chain. We conclude that at pH 7.5 simultaneous independent homologous fibrillation occurs, but at low pH, heterologous fibrillation takes place, and with B-chain seeding of insulin, a unique conformation of fibrils is formed. Our results demonstrate that in the co-aggregation of closely related peptides each peptide species may undergo concurrent homogeneous or heterologous polymerization and that fibrils of one species may or may not seed fibrillation of the other. The results demonstrate the significant "species" barrier in amyloid fibril formation between fibrillation induced by different fibrils. A model for the fibrillation of the heterogeneous system of insulin and B-chain insulin is proposed.

Characterization of oligomeric intermediates in alpha-synuclein fibrillation: FRET studies of Y125W/Y133F/Y136F alpha-synuclein.

The aggregation of alpha-synuclein is believed to be a critical step in the etiology of Parkinson's disease. A variety of biophysical techniques were used to investigate the aggregation and fibrillation of alpha-synuclein in which one of the four intrinsic Tyr residues was replaced by Trp, and two others by Phe, in order to permit fluorescence resonance energy transfer (FRET) between residues 39 (Tyr) and 125 (Trp). The mutant Y125W/Y133F/Y136F alpha-synuclein (one Tyr, one Trp) showed fibrillation kinetics similar to that of the wild-type, as did the Y125F/Y133F/Y136F (one Tyr, no Trp) and Y39F/Y125W/Y133F/Y136F (no Tyr, one Trp) mutants. Time-dependent changes in FRET, Fourier transform infrared, Trp fluorescence, dynamic light-scattering and other probes, indicate the existence of a transient oligomer, whose population reaches a maximum at the end of the lag time. This oligomer, in which the alpha-synuclein is in a partially folded conformation, is subsequently converted into fibrils, and has physical properties that are distinct from those of the monomer and fibrils. In addition, another population of soluble oligomers was observed to coexist with fibrils at completion of the reaction. The average distance between Tyr39 and Trp125 decreases from 24.9A in the monomer to 21.9A in the early oligomer and 18.8A in the late oligomer. Trp125 remains solvent-exposed in both the oligomers and fibrils, indicating that the C-terminal domain is not part of the fibril core. No FRET was observed in the fibrils, due to quenching of Tyr39 fluorescence in the fibril core. Thus, aggregation of alpha-synuclein involves multiple oligomeric intermediates and competing pathways.

Conformation of beta 2-microglobulin amyloid fibrils analyzed by reduction of the disulfide bond.

Beta2-microglobulin (beta2-m), a major component of dialysis-related amyloid fibrils, has an intrachain disulfide bond buried inside the native structure. We examined the conformation of beta2-m amyloid fibrils by analyzing the reactivity of the disulfide bond to a reducing reagent, dithiothreitol. Although the disulfide bond in the native structure was highly protected from reduction, the disulfide bonds in the amyloid fibrils prepared at pH 2.5 were progressively reduced at pH 8.5 by 50 mm dithiothreitol. Because beta2-m amyloid fibrils prepared under acidic conditions have been known to depolymerize at a neutral pH, we examined the relation between depolymerization and reduction of the disulfide bond. The results indicate that the disulfide bonds in the amyloid fibrils were protected from reduction, and the reduction occurred during depolymerization. On the other hand, the disulfide bonds of immature filaments, the thin and flexible filaments prepared under conditions of high salt at pH 2.5, were reduced at pH 8.5 more readily than those of amyloid fibrils, suggesting that the disulfide bonds are exposed to the solvent. Taken together, the disulfide bond once exposed to the solvent upon acid denaturation may be progressively buried in the interior of the amyloid fibrils during its formation.

Aggregation of beta(2)-glycoprotein I induced by sodium lauryl sulfate and lysophospholipids.

beta(2)-Glycoprotein I (beta(2)-GPI) is a plasma protein that binds to negatively charged substances such as DNA, heparin, and anionic phospholipids. The interaction of beta(2)-GPI with anionic phospholipids is intriguing in the context of the autoimmune disease antiphospholipid syndrome. To extend understanding of the binding mechanism to phospholipids, the interactions of beta(2)-GPI with amphiphiles, i.e., sodium lauryl sulfate and lysophospholipids, were examined. These amphiphiles induced the aggregation of beta(2)-GPI below the critical micelle concentration, indicating that the interaction of beta(2)-GPI with monodispersed amphiphiles is unstable, resulting in the formation of large aggregates. However, highly soluble monocaproylphosphatidic acid did not induce aggregation, suggesting that the hydrophobicity of the acyl chain is also an important factor for aggregate formation in addition to negative charges in the headgroup. A series of experiments using deletion mutants and a peptide showed that the fifth domain of beta(2)-GPI (domain V) is responsible for formation of aggregates observed for intact full-length beta(2)-GPI. In addition, the flexible loop (F307-C326) in the C-terminal of domain V, which consists of hydrophobic and positively charged residues, was identified as the important region for aggregation. These results indicate that beta(2)-GPI binds to the amphiphiles through the flexible loop of domain V, resulting in formation of large aggregates where both electrostatic and hydrophobic interactions are involved.