Intermolecular interactions between the SH3 domain and the proline-rich TH region of Bruton’s tyrosine kinase

AbstractThe SH3 domain of Bruton’s tyrosine kinase (Btk) is preceded by the Tec homology (TH) region containing proline-rich sequences. We have studied a protein fragment containing both the Btk SH3 domain and the proline-rich sequences of the TH region (PRR-SH3). Intermolecular NMR cross-relaxation measurements, gel permeation chromatography profiles, titrations with proline-rich peptides, and 15N NMR relaxation measurements are all consistent with a monomer–dimer equilibrium with a dissociation constant on the order of 60 μM. The intermolecular interactions do, at least in part, involve proline-rich sequences in the TH region. This behavior of Btk PRR-SH3 may have implications for the functional action of Btk

Recombinant amyloid beta-peptide production by coexpression with an affibody ligand.

BACKGROUND: Oligomeric and fibrillar aggregates of the amyloid beta-peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). The characterization of Abeta assemblies is essential for the elucidation of the mechanisms of Abeta neurotoxicity, but requires large quantities of pure peptide. Here we describe a novel approach to the recombinant production of Abeta. The method is based on the coexpression of the affibody protein ZAbeta3, a selected affinity ligand derived from the Z domain three-helix bundle scaffold. ZAbeta3 binds to the amyloidogenic central and C-terminal part of Abeta with nanomolar affinity and consequently inhibits aggregation. RESULTS: Coexpression of ZAbeta3 affords the overexpression of both major Abeta isoforms, Abeta(1-40) and Abeta(1-42), yielding 4 or 3 mg, respectively, of pure 15N-labeled peptide per liter of culture. The method does not rely on a protein-fusion or -tag and thus does not require a cleavage reaction. The purified peptides were characterized by NMR, circular dichroism, SDS-PAGE and size exclusion chromatography, and their aggregation propensities were assessed by thioflavin T fluorescence and electron microscopy. The data coincide with those reported previously for monomeric, largely unstructured Abeta. ZAbeta3 coexpression moreover permits the recombinant production of Abeta(1-42) carrying the Arctic (E22G) mutation, which causes early onset familial AD. Abeta(1-42)E22G is obtained in predominantly monomeric form and suitable, e.g., for NMR studies. CONCLUSION: The coexpression of an engineered aggregation-inhibiting binding protein offers a novel route to the recombinant production of amyloidogenic Abeta peptides that can be advantageously employed to study the molecular basis of AD. The presented expression system is the first for which expression and purification of the aggregation-prone Arctic variant (E22G) of Abeta(1-42) is reported.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Protofibrillar and Fibrillar Amyloid-β Binding Proteins in Cerebrospinal Fluid

Aggregation and deposition of misfolded amyloid-β (Aβ) peptide in the brain is central to Alzheimer’s disease (AD). Oligomeric, protofibrillar, and fibrillar forms of Aβ are believed to be neurotoxic and cause neurodegeneration in AD, but the toxicity mechanisms are not well understood and may involve Aβ-interacting molecular partners. In a previous study, we identified potential Aβ₄₂ protofibrillar-binding proteins in serum and cerebrospinal fluid (CSF) using an engineered version of Aβ₄₂ (Aβ₄₂CC) that forms protofibrils, but not fibrils. Here we studied binding of proteins to Aβ₄₂ fibrils in AD and non-AD CSF and compared these with protofibrillar Aβ₄₂CC-binding partners. Aβ₄₂ fibrils sequestered 2.4-fold more proteins than Aβ₄₂CC protofibrils. Proteins with selective binding to fibrillar aggregates with low nanomolar affinity were identified. We also found that protofibrillar and fibrillar Aβ-binding proteins represent distinct functional categories. Aβ₄₂CC protofibrils triggered interactions with proteins involved in catalytic activities, like transferases and oxidoreductases, while Aβ₄₂ fibrils were more likely involved in binding to proteoglycans, growth factors and neuron-associated proteins, e.g., neurexin-1, -2, and -3. Interestingly, 10 brain-enriched proteins were identified among the fibril-binding proteins, while protofibril-extracted proteins had more general expression patterns. Both types of Aβ aggregates bound several extracellular proteins. Additionally, we list a set of CSF proteins that might have potential to discriminate between AD and non-AD CSF samples. The results may be of relevance both for biomarker studies and for studies of Aβ-related toxicity mechanisms

A Potential Role for Drosophila Mucins in Development and Physiology

Vital vertebrate organs are protected from the external environment by a barrier that to a large extent consists of mucins. These proteins are characterized by poorly conserved repeated sequences that are rich in prolines and potentially glycosylated threonines and serines (PTS). We have now used the characteristics of the PTS repeat domain to identify Drosophila mucins in a simple bioinformatics approach. Searching the predicted protein database for proteins with at least 4 repeats and a high ST content, more than 30 mucin-like proteins were identified, ranging from 300–23000 amino acids in length. We find that Drosophila mucins are present at all stages of the fly life cycle, and that their transcripts localize to selective organs analogous to sites of vertebrate mucin expression. The results could allow for addressing basic questions about human mucin-related diseases in this model system. Additionally, many of the mucins are expressed in selective tissues during embryogenesis, thus revealing new potential functions for mucins as apical matrix components during organ morphogenesis

Sequestration of the Aβ Peptide Prevents Toxicity and Promotes Degradation In Vivo

An engineered protein prevents aggregation of the Aβ peptide and facilitates clearance of Aβ from the brain in a fruit fly model of Alzheimer's disease

Allosteric Modulators of Steroid Hormone Receptors : Structural Dynamics and Gene Regulation

Peer reviewedPublisher PD

Temporary Electrostatic Impairment of DNA Recognition: Light‐Driven DNA Binding of Peptide Dimers

This is the peer reviewed version of the following article: Jiménez‐Balsa, A. , Pazos, E. , Martínez‐Albardonedo, B. , Mascareñas, J. L. and Vázquez, M. E. (2012), Temporary Electrostatic Impairment of DNA Recognition: Light‐Driven DNA Binding of Peptide Dimers. Angew. Chem. Int. Ed., 51: 8825-8829, which has been published in final form at https://doi.org/10.1002/anie.201201627. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsAppending negatively charged Glu8 tails to a peptide dimer derived from the GCN4 transcription factor leads to an effective suppression of its DNA binding. The specific DNA recognition can be restored by irradiation with UV light by using a photolabile linker between the acidic tail and the DNA binding peptideConsolider Ingenio. Grant Numbers: SAF2010‐20822‐C02, CTQ2009‐14431/BQU, CSD2007‐00006 Xunta de Galicia. Grant Numbers: INCITE09 209 084PR, PGIDIT08CSA‐047209PR, GRC2010/12 MINECOS

The amyloid fibril-forming properties of the amphibian antimicrobial peptide uperin 3.5

The amphibian skin is a vast resource for bioactive peptides, which form the basis of the animals' innate immune system. Key components of the secretions of the cutaneous glands are antimicrobial peptides (AMPs), which exert their cytotoxic effects often as a result of membrane disruption. It is becoming increasingly evident that there is a link between the mechanism of action of AMPs and amyloidogenic peptides and proteins. In this work, we demonstrate that the broad-spectrum amphibian AMP uperin 3.5, which has a random-coil structure in solution but adopts an α-helical structure in membrane-like environments, forms amyloid fibrils rapidly in solution at neutral pH. These fibrils are cytotoxic to model neuronal cells in a similar fashion to those formed by the proteins implicated in neurodegenerative diseases. The addition of small quantities of 2,2,2-trifluoroethanol accelerates fibril formation by uperin 3.5, and is correlated with a structural stabilisation induced by this co-solvent. Uperin 3.5 fibril formation and the associated cellular toxicity are inhibited by the polyphenol (-)-epigallocatechin-3-gallate (EGCG). Furthermore, EGCG rapidly dissociates fully formed uperin 3.5 fibrils. Ion mobility-mass spectrometry reveals that uperin 3.5 adopts various oligomeric states in solution. Combined, these observations imply that the mechanism of membrane permeability by uperin 3.5 is related to its fibril-forming properties.Antonio N. Calabrese, Yanqin Liu, Tianfang Wang, Ian F. Musgrave, Tara L. Pukala, Rico F. Tabor, Lisandra L. Martin, John A. Carver, John H. Bowi

Rejuvenating conventional dendritic cells and T follicular helper cell formation after vaccination.

Germinal centres (GCs) are T follicular helper cell (Tfh)-dependent structures that form in response to vaccination, producing long-lived antibody secreting plasma cells and memory B cells that protect against subsequent infection. With advancing age the GC and Tfh cell response declines, resulting in impaired humoral immunity. We sought to discover what underpins the poor Tfh cell response in ageing and whether it is possible to correct it. Here, we demonstrate that older people and aged mice have impaired Tfh cell differentiation upon vaccination. This deficit is preceded by poor activation of conventional dendritic cells type 2 (cDC2) due to reduced type 1 interferon signalling. Importantly, the Tfh and cDC2 cell response can be boosted in aged mice by treatment with a TLR7 agonist. This demonstrates that age-associated defects in the cDC2 and Tfh cell response are not irreversible and can be enhanced to improve vaccine responses in older individuals