Alzheimer's disease as an autoimmune disorder of innate immunity endogenously modulated by tryptophan metabolites.

Introduction: Alzheimer's disease (AD) is characterized by neurotoxic immuno-inflammation concomitant with cytotoxic oligomerization of amyloid beta (Aß) and tau, culminating in concurrent, interdependent immunopathic and proteopathic pathogeneses. Methods: We performed a comprehensive series of in silico, in vitro, and in vivo studies explicitly evaluating the atomistic-molecular mechanisms of cytokine-mediated and Aß-mediated neurotoxicities in AD.  Next, 471 new chemical entities were designed and synthesized to probe the pathways identified by these molecular mechanism studies and to provide prototypic starting points in the development of small-molecule therapeutics for AD. Results: In response to various stimuli (e.g., infection, trauma, ischemia, air pollution, depression), Aß is released as an early responder immunopeptide triggering an innate immunity cascade in which Aß exhibits both immunomodulatory and antimicrobial properties (whether bacteria are present, or not), resulting in a misdirected attack upon "self" neurons, arising from analogous electronegative surface topologies between neurons and bacteria, and rendering them similarly susceptible to membrane-penetrating attack by antimicrobial peptides (AMPs) such as Aß. After this self-attack, the resulting necrotic (but not apoptotic) neuronal breakdown products diffuse to adjacent neurons eliciting further release of Aß, leading to a chronic self-perpetuating autoimmune cycle.  AD thus emerges as a brain-centric autoimmune disorder of innate immunity. Based upon the hypothesis that autoimmune processes are susceptible to endogenous regulatory processes, a subsequent comprehensive screening program of 1137 small molecules normally present in human brain identified tryptophan metabolism as a regulator of brain innate immunity and a source of potential endogenous anti-AD molecules capable of chemical modification into multi-site therapeutic modulators targeting AD's complex immunopathic-proteopathic pathogenesis. Discussion:  Conceptualizing AD as an autoimmune disease, identifying endogenous regulators of this autoimmunity, and designing small molecule drug-like analogues of these endogenous regulators represents a novel therapeutic approach for AD.

N-terminal helix-cap in α-helix 2 modulates ß-state misfolding in rabbit and hamster prion proteins.

Susceptibility of a particular species to prion disease is affected by small differences in the sequence of PrP and correlates with the propensity of its PrP to assume the ß-state. A helix-cap motif in the ß2-α2-loop of native α-helical rabbit PrP, a resistant species, contains sequence differences that influence intra- and interspecies transmission. To determine the effect the helix-cap motif on ß-state refolding propensity, we mutated S170N, S174N, and S170N/S174N of the rabbit PrP helix-cap to resemble that of hamster PrP and conversely, N170S, N174S, and N170S/N174S of hamster PrP to resemble the helix-cap of rabbit PrP. High-resolution crystal structures (1.45-1.6 Å) revealed that these mutations ablate hydrogen-bonding interactions within the helix-cap motif in rabbit PrP(C). They also alter the ß-state-misfolding propensity of PrP; the serine mutations in hamster PrP decrease the propensity up to 35%, whereas the asparagine mutations in rabbit PrP increase it up to 42%. Rapid dilution of rabbit and hamster into ß-state buffer conditions causes quick conversion to ß-state monomers. Kinetic monitoring using size-exclusion chromatography showed that the monomer population decreases exponentially mirrored by an increase in an octameric species. The monomer-octamer transition rates are faster for hamster than for rabbit PrP. The N170S/N174S mutant of hamster PrP has a smaller octamer component at the endpoint compared to the wild-type, whereas the kinetics of octamer formation in mutant and wild-type rabbit PrP are comparable. These findings demonstrate that the sequence of the ß2-α2 helix-cap affects refolding to the ß-state and subsequently, may influence susceptibility to prion disease.

Relative and regional stabilities of the hamster, mouse, rabbit, and bovine prion proteins toward urea unfolding assessed by nuclear magnetic resonance and circular dichroism spectroscopies.

The residue-specific urea-induced unfolding patterns of recombinant prion proteins from different species (bovine, rabbit, mouse, and Syrian hamster) were monitored using high-resolution (1)H nuclear magnetic resonance (NMR) spectroscopy. Protein constructs of different lengths, and with and without a His tag attached at the N-terminus, were studied. The various species showed different overall sensitivities toward urea denaturation with stabilities in the following order: hamster ≤ mouse < rabbit < bovine protein. This order is in agreement with recent circular dichroism (CD) spectroscopic measurements for several species [Khan, M. Q. (2010) Proc. Natl. Acad. Sci. U.S.A.107, 19808-19813] and for the bovine protein presented herein. The [urea](1/2) values determined by CD spectroscopy parallel those of the most stable residues observed by NMR spectroscopy. Neither the longer constructs containing an additional hydrophobic region nor the His tag influenced the stability of the structured domain of the constructs studied. The effect of the S174N mutation in rabbit PrP(C) was also investigated. The rank order of the regional stabilities within each protein remained the same for all species. In particular, the residues in the ß-sheet region in all four species were more sensitive to urea-induced unfolding than residues in the α2 and α3 helical regions. These observations indicate that the regional specific unfolding pattern is the same for the four mammalian prion proteins studied but militate against the idea that PrP(Sc) formation is linked with the global stability of PrP(C).

Prion disease susceptibility is affected by beta-structure folding propensity and local side-chain interactions in PrP.

Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological ß-structured state with prion infectivity (PrP(Sc)). Exposure to PrP(Sc) from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic ß-structured state, which exists in a monomer-octamer equilibrium. It has been controversial whether ß-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a ß-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the ß-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease.

The real-time monitoring of the thermal unfolding of tetramethylrhodamine-labeled actin.

Modification of actin at Cys (374) with tetramethylrhodamine maleimide (TMR-actin) has been used for visualization of actin filaments and to produce high-resolution crystal structures of actin. We show that TMR-actin exhibits a 21% decrease in absorbance at 557 nm upon thermal unfolding, likely due to the movement of TMR to a more hydrophobic environment upon rapid unfolding and protein aggregation. We took advantage of this property to test models of actin protein unfolding. A transition temperature ( T m) of 60.2 +/- 0.2 degrees C for Ca (2+).ATP.TMR-actin was determined using A 557 and agreed with our own determinations employing different techniques and previous work with unlabeled actin. Our data show that the dependence of TMR-actin thermal stability on the bound nucleotide and cations follows a trend of Ca (2+).ATP > Mg (2+).ATP > Ca (2+).ADP > Mg (2+).ADP. The activation energies and frequency factors for the thermal unfolding of TMR-actin determined with two methods were in good agreement with those previously determined for unlabeled actin. We observed a biphasic trend in the T m of TMR-actin with increasing nucleotide concentrations, supporting a two-pathway model for actin protein unfolding where one pathway dominates at different concentrations of nucleotide. Additionally, TMR-actin bound by DNase I or gelsolin segment-1 exhibited elevated transition temperatures.

Purification and characterization of a nonpolymerizing long-pitch actin dimer.

Atomic resolution structures of filamentous actin have not been obtained owing to the self-association of actin under crystallization conditions. Obtaining short filamentous actin complexes of defined lengths is therefore a highly desirable goal. Here we report the production and isolation of a long-pitch actin dimer employing chemical crosslinking between wild-type actin and Q41C/C374A mutant actin. The Q41C/C374A mutant actin possessed altered polymerization properties, with a 2-fold reduction in the rate of elongation and an increased critical concentration relative to wild-type actin. The Q41C/C374A mutant actin also displayed an increase in the IC50 for DNase I, a pointed-end actin-binding protein. The long-pitch dimer was bound by DNase I to prevent polymerization and purified. It was found that each actin dimer is bound by 2 DNase I molecules, 1 likely bound to each of the actin protomers. The long-pitch dimer bound by DNase I did not form short F actin structures, as assessed by the binding of rhodamine-phalloidin.