Understanding the kinetic roles of the inducer heparin and of rod-like protofibrils during amyloid fibril formation by Tau protein

The aggregation of the natively disordered protein, Tau, to form lesions called neurofibrillary tangles is a characteristic feature of several neurodegenerative tauopathies. The polyanion, heparin, is commonly used as an inducer in studies of Tau aggregation in vitro, but there is surprisingly no comprehensive model describing, quantitatively, all aspects of the heparin-induced aggregation reaction. In this study, rate constants and extents of fibril formation by the four repeat domain of Tau (Tau4RD) have been reproducibly determined over a full range of heparin and protein concentrations. The kinetic role of heparin in the nucleation-dependent fibril formation reaction is shown to be limited to participation in the initial rate-limiting steps; a single heparin molecule binds two Tau4RD molecules, forming an aggregation-competent protein dimer, which then serves as a building block for further fibril growth. Importantly, the minimal kinetic model that is proposed can quantitatively account for the characteristic bell-shaped dependence of the aggregation kinetics on the stoichiometry of protein to heparin. Very importantly, this study also identifies for the first time short and thin, rod-like protofibrils that are populated transiently, early during the time course of fibril formation. The identification of these protofibrils as bona fide off-pathway species has implications for the development of therapies for tauopathies based on driving fibril formation as a means of protecting the cell from smaller, putatively toxic aggregates.     

Virus hunters: catching bugs in the field

Densely populated areas in rural China require constant vigilance and state-of-the-art technology to stop new pandemics in their tracks. Hurdles are not only scientific in some parts of the developing world.     

Degradation products analysis of an Fc fusion protein using LC/MS methods

The following analytical methods have been used to identify and quantify degradation products in an E. coli expressed human immunoglobulin G Fc fusion protein in both liquid and lyophilized forms: two-dimensional AEX/RP/MS, limited proteolysis followed by LC/MS, and tryptic digestion followed by LC/MS/MS. After aging in a potassium phosphate pH 7.0 buffer for 3 months at 29 °C, peptide map analysis revealed that asparagine N78 (N297 according to Edelman sequencing) of the CH2 domain was the most rapidly deamidated site in the molecule probably due to the lack of the N-linked glycan on this asparagine, but this deamidation can be prevented under properly formulated conditions. This is the first report on the rate of deamidation on N297 of an IgG molecule without glycosylation. The active protein portion of the Fc fusion protein contains two methionine residues that are potentially susceptible to oxidation. Limited proteolysis was employed to cleave the active protein portion and measure the amount of oxidation. LC/MS analysis identified that the liquid sample aged at 29 °C for 3 months produced 40% oxidation, while the control sample contained only 4% oxidation on the active protein. In contrast to the aged liquid sample, the aged lyophilized sample showed no increase of deamidation or oxidation after storage at 37 °C for 8 months.     

Site-specific ubiquitination exposes a linear motif to promote interferon-alpha receptor endocytosis

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCF^βTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.     

Purification and Characterization of Polygalacturonase-3 from Jamaica cherry (Muntingia calabura Linn)

Endo-polygalacturonase-3 (PG-3), the key enzyme of fruit ripening was purified to near homogeneity as judged by native PAGE from the fruit tissues of Jamaica cherry (Muntingia calabura) using ammonium sulphate fractionation, followed by anion-exchange, gel filtration and affinity chromatography. The molecular mass of the PG-3 enzyme was determined as 85 kD, by size exclusion chromatography. SDS-PAGE of PG-3 revealed two dissimilar bands of 62 and 21 kD as heterogenous subunits. The optimum pH of PG-3 was found to be 4.0. The enzyme had an optimum temperature of 40°C and was relatively stable at 50°C and 60°C. K_m for the substrate polygalacturonic acid was found to be 0.27%. The purified enzyme was a glycoprotein with 6.6 % carbohydrate content.     

Copsin,a Novel Peptide-based Fungal Antibiotic Interfering with the Peptidoglycan Synthesis

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/β-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.     

M. tuberculosis induces potent activation of IDO-1, but this is not essential for the immunological control of infection

Indoleamine 2,3-dioxygenesae-1 (IDO-1) catalyses the initial, rate-limiting step in tryptophan metabolism, thereby regulating tryptophan availability and the formation of downstream metabolites, including picolinic and quinolinic acid. We found that Mycobacterium tuberculosis infection induced marked upregulation of IDO-1 expression in both human and murine macrophages in vitro and in the lungs of mice following aerosol challenge with M. tuberculosis. The absence of IDO-1 in dendritic cells enhanced the activation of mycobacteria-specific T cells in vitro. Interestingly, IDO-1-deficiency during M. tuberculosis infection in mice was not associated with altered mycobacteria-specific T cell responses in vivo. The bacterial burden of infected organs, pulmonary inflammatory responses, and survival were also comparable in M. tuberculosis-infected IDO-1 deficient and wild type animals. Tryptophan is metabolised into either picolinic acid or quinolinic acid, but only picolinic acid inhibited the growth of M. tuberculosis in vitro. By contrast macrophages infected with pathogenic mycobacteria, produced quinolinic, rather than picolinic acid, which did not reduce M. tuberculosis growth in vitro. Therefore, although M. tuberculosis induces robust expression of IDO-1 and activation of tryptophan metabolism, IDO-1-deficiency fails to impact on the immune control and the outcome of the infection in the mouse model of tuberculosis.