Microbial exposures and sex hormones exert potent effects on autoimmune diseases, many of which are more prevalent in women. We demonstrate that early-life microbial exposures determine sex hormone levels and modify progression to autoimmunity in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). Colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipient's microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and robust T1D protection. These effects were dependent on androgen receptor activity. Thus, the commensal microbial community alters sex hormone levels and regulates autoimmune disease fate in individuals with high genetic risk.

We have searched for a minimal interaction motif in protein that supports the aggregation into Alzheimer-like paired helical filaments. Digestion of the repeat domain with different proteases yields a GluC-induced fragment comprising 43 residues (termed PHF43), which represents the third repeat of plus some flanking residues. This fragment self assembles readily into thin filaments without a paired helical appearance, but these filaments are highly competent to nucleate bona fide PHFs from full-length . Probing the interactions of PHF43 with overlapping peptides derived from the full sequence yields a minimal hexapeptide interaction motif of 306 VQIVYK 311 at the beginning of the third internal repeat. This motif coincides with the highest predicted ␤-structure potential in . CD and Fourier transform infrared spectroscopy shows that PHF43 acquires pronounced ␤ structure in conditions of self assembly. Point mutations in the hexapeptide region by prolinescanning mutagenesis prevent the aggregation. The data indicate that PHF assembly is initiated by a short fragment containing the minimal interaction motif forming a local ␤ structure embedded in a largely random-coil protein.

The microtubule-associated protein tau is a natively unfolded protein in solution, yet it is able to polymerize into the ordered paired helical filaments (PHF) of Alzheimer's disease. In the splice isoforms lacking exon 10, this process is facilitated by the formation of ␤-structure around the hexapeptide motif PHF6 ( 306 VQIVYK 311 ) encoded by exon 11. We have investigated the structural requirements for PHF polymerization in the context of adult tau isoforms containing four repeats (including exon 10). In addition to the PHF6 motif there exists a related PHF6* motif ( 275 VQIINK 280 ) in the repeat encoded by the alternatively spliced exon 10. We show that this PHF6* motif also promotes aggregation by the formation of ␤-structure and that there is a cross-talk between the two hexapeptide motifs during PHF aggregation. We also show that two of the tau mutations found in hereditary frontotemporal dementias, ⌬K280 and P301L, have a much stronger tendency for PHF aggregation which correlates with their high propensity for ␤-structure around the hexapeptide motifs.

One of the hallmarks of Alzheimer's disease is the abnormal state of the microtubule-associated protein tau in neurons. It is both highly phosphorylated and aggregated into paired helical filaments, and it is commonly assumed that the hyperphosphorylation of tau causes its detachment from microtubules and promotes its assembly into PHFs. We have studied the relationship between the phosphorylation of tau by several kinases (MARK, PKA, MAPK, GSK3) and its assembly into PHFs. The proline-directed kinases MAPK and GSK3 are known to phosphorylate most Ser-Pro or Thr-Pro motifs in the regions flanking the repeat domain of tau: they induce the reaction with several antibodies diagnostic of Alzheimer PHFs, but this type of phosphorylation has only a weak effect on tau-microtubule interactions and on PHF assembly. By contrast, MARK and PKA phosphorylate several sites within the repeats (notably the KXGS motifs including Ser262, Ser324, and Ser356, plus Ser320); in addition PKA phosphorylates some sites in the flanking domains, notably Ser214. This type of phosphorylation strongly reduces tau's affinity for microtubules, and at the same time inhibits tau's assembly into PHFs. Thus, contrary to expectations, the phosphorylation that detaches tau from microtubules does not prime it for PHF assembly, but rather inhibits it. Likewise, although the phosphorylation sites on Ser-Pro or Thr-Pro motifs are the most prominent ones on Alzheimer PHFs (by antibody labeling), they are only weakly inhibitory to PHF assembly. This implies that the hyperphosphorylation of tau in Alzheimer's disease is not directly responsible for the pathological aggregation into PHFs; on the contrary, phosphorylation protects tau against aggregation.

The aggregation of the microtubule-associated tau protein and formation of "neurofibrillary tangles" is one of the hallmarks of Alzheimer disease. The mechanisms underlying the structural transition of innocuous, natively unfolded tau to neurotoxic forms and the detailed mechanisms of binding to microtubules are largely unknown. Here we report the high-resolution characterization of the repeat domain of soluble tau using multidimensional NMR spectroscopy. NMR secondary chemical shifts detect residual ␤-structure for 8 -10 residues at the beginning of repeats R2-R4. These regions correspond to sequence motifs known to form the core of the cross-␤-structure of tau-paired helical filaments. Chemical shift perturbation studies show that polyanions, which promote paired helical filament aggregation, as well as microtubules interact with tau through positive charges near the ends of the repeats and through the ␤-forming motifs at the beginning of repeats 2 and 3. The high degree of similarity between the binding of polyanions and microtubules supports the hypothesis that stable microtubules prevent paired helical filament formation by blocking the tau-polyanion interaction sites, which are crucial for paired helical filament formation.Alzheimer disease is characterized by abnormal protein deposits in the brain, such as amyloid plaques or neurofibrillary tangles, formed by fibrous assemblies of the A␤ peptide (1) or of the microtubule (MT) 1 -associated tau protein (2). These aggregates are thought to be toxic to neurons, either by causing some toxic signaling defect or by obstructing the cell interior. Therefore, one of the top priorities in Alzheimer research is to understand the reasons for the pathological aggregation and to find methods to prevent it. Although the structural principles governing A␤ aggregation are known in some detail, little is known for the tau protein.Tau is a microtubule-associated protein that regulates MT stability, neurite outgrowth, and other MT-dependent functions. The three or four repeats in the C-terminal half of the protein and the flanking proline-rich basic domains are known to be involved in MT binding (3). The affinity is regulated by phosphorylation particularly at KXGS-motifs in the repeats (4). Interestingly the same phosphorylation sites have an inhibitory influence on aggregation (5). Unbound tau can assemble into Alzheimer-like paired helical filaments (PHFs) whose polymerization can be enhanced by oxidation of SH groups and by polyanions (e.g. heparin, poly-Glu (6)). On the other hand, tau has a hydrophilic character, is highly soluble, and belongs to the class of natively unfolded proteins with no apparent ordered secondary structure detectable by far-UV CD or Fouriertransform infrared spectroscopy (7,8). Therefore, it is unclear why tau should aggregate in a specific manner and what structural principles could be responsible for this.Tau can aggregate as an intact protein, 352-441 residues in length (depending on isoform), so that all six tau isoforms are found in Alzheim...

The microtubule-associated protein tau stabilizes microtubules in its physiological role, whereas it forms insoluble aggregates (paired helical filaments) in Alzheimer's disease. Soluble tau is considered a natively unfolded protein whose residual folding and intramolecular interactions are largely undetermined. In this study, we have applied fluorescence resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) to examine the proximity and flexibility of tau domains and the global folding. FRET pairs spanning the tau molecule were created by inserting tryptophans (donor) and cysteines (labeled with IAEDANS as an acceptor) by site-directed mutagenesis. The observed FRET distances were significantly different from those expected for a random coil. Notably, the C-terminal end of tau folds over into the vicinity of the microtubule-binding repeat domain, the N-terminus remains outside the FRET distance of the repeat domain, yet both ends of the molecule approach one another. The interactions between the domains were obliterated by denaturation in GdnHCl. Paramagnetic spin-labels attached in various domains of tau were analyzed by EPR and exhibited a high mobility throughout. The data indicate that tau retains some global folding even in its "natively unfolded" state, combined with the high flexibility of the chain.

ObjectiveAntibiotic (AB) usage strongly affects microbial intestinal metabolism and thereby impacts human health. Understanding this process and the underlying mechanisms remains a major research goal. Accordingly, we conducted the first comparative omic investigation of gut microbial communities in faecal samples taken at multiple time points from an individual subjected to β-lactam therapy.MethodsThe total (16S rDNA) and active (16S rRNA) microbiota, metagenome, metatranscriptome (mRNAs), metametabolome (high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry) and metaproteome (ultra high performing liquid chromatography coupled to an Orbitrap MS2 instrument [UPLC-LTQ Orbitrap-MS/MS]) of a patient undergoing AB therapy for 14 days were evaluated.ResultsApparently oscillatory population dynamics were observed, with an early reduction in Gram-negative organisms (day 6) and an overall collapse in diversity and possible further colonisation by ‘presumptive’ naturally resistant bacteria (day 11), followed by the re-growth of Gram-positive species (day 14). During this process, the maximum imbalance in the active microbial fraction occurred later (day 14) than the greatest change in the total microbial fraction, which reached a minimum biodiversity and richness on day 11; additionally, major metabolic changes occurred at day 6. Gut bacteria respond to ABs early by activating systems to avoid the antimicrobial effects of the drugs, while ‘presumptively’ attenuating their overall energetic metabolic status and the capacity to transport and metabolise bile acid, cholesterol, hormones and vitamins; host–microbial interactions significantly improved after treatment cessation.ConclusionsThis proof-of-concept study provides an extensive description of gut microbiota responses to follow-up β-lactam therapy. The results demonstrate that ABs targeting specific pathogenic infections and diseases may alter gut microbial ecology and interactions with host metabolism at a much higher level than previously assumed.

Alzheimer's disease is characterized by two types of fibrous aggregates in the affected brains, the amyloid fibers (consisting of the A␤-peptide, generating the amyloid plaques), and paired helical filaments (PHFs; made up of tau protein, forming the neurofibrillary tangles). Hence, tau protein, a highly soluble protein that normally stabilizes microtubules, becomes aggregated into insoluble fibers that obstruct the cytoplasm of neurons and cause a loss of microtubule stability. We have developed recently a rapid assay for monitoring PHF assembly and show here that PHFs arise from a nucleated assembly mechanism. The PHF nucleus comprises about 8-14 tau monomers. A prerequisite for nucleation is the dimerization of tau because tau dimers act as effective building blocks. PHF assembly can be seeded by preformed filaments (made either in vitro or isolated from Alzheimer brain tissue). These results suggest that dimerization and nucleation are the rate-limiting steps for PHF formation in vivo.Alzheimer's disease, the most common age-relate dementia, is characterized by two pathological protein deposits in the brain, the amyloid plaques, consisting largely of amyloid fibers assembled from the A␤-peptide [a derivative of the membrane protein APP (amyloid precursor protein); reviewed in ref. 1] and the neurofibrillary tangles (NFT), which are bundles of paired helical filaments (PHFs) whose main constituent is the microtubule-associated protein tau (for reviews, see refs. 2 and 3). The uncontrolled precipitation of these aggregates is believed to be largely responsible for the neuronal degeneration, and the disease has been classified into several stages on the basis of the spreading of neurofibrillary deposits (4). It is therefore important to understand the factors underlying the abnormal aggregation of A␤ and tau. Both form filaments of Ϸ10 nm in width; in the case of A␤ they are smooth, while most tau filaments from Alzheimer brains show a characteristic ''paired helical'' structure, resembling two strands wound around one another, with a crossover periodicity of Ϸ80 nm and width varying between 10 and 20 nm (for review, see ref.The membrane-derived A␤-peptide is partly hydrophobic so that its tendency to aggregate is intuitively understandable (6, 7). By contrast, the cytosolic tau has a very hydrophilic character and is highly soluble (8, 9). Thus, it shows hardly any tendency to aggregate in physiological buffer conditions, and the formation of aggregates is very slow (days or weeks; ref. 10). Soluble tau protein contains very little secondary structure (␣-helix or ␤-sheet content Ͻ 5%), and the same holds for Alzheimer PHFs, in spite of their long-range periodicity (11). It therefore is not obvious why tau should aggregate in a specific manner and which structural principle could be responsible for this. Progress in understanding the mechanism has been correspondingly slow. Several factors supporting assembly have emerged in recent years. (i) The repeat domain in the C-terminal half of tau forms PHFs mo...