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

The Effect of Iron Limitation on the Transcriptome and Proteome of Pseudomonas fluorescens Pf-5

One of the most important micronutrients for bacterial growth is iron, whose bioavailability in soil is limited. Consequently, rhizospheric bacteria such as Pseudomonas fluorescens employ a range of mechanisms to acquire or compete for iron. We investigated the transcriptomic and proteomic effects of iron limitation on P. fluorescens Pf-5 by employing microarray and iTRAQ techniques, respectively. Analysis of this data revealed that genes encoding functions related to iron homeostasis, including pyoverdine and enantio-pyochelin biosynthesis, a number of TonB-dependent receptor systems, as well as some inner-membrane transporters, were significantly up-regulated in response to iron limitation. Transcription of a ribosomal protein L36-encoding gene was also highly up-regulated during iron limitation. Certain genes or proteins involved in biosynthesis of secondary metabolites such as 2,4-diacetylphloroglucinol (DAPG), orfamide A and pyrrolnitrin, as well as a chitinase, were over-expressed under iron-limited conditions. In contrast, we observed that expression of genes involved in hydrogen cyanide production and flagellar biosynthesis were down-regulated in an iron-depleted culture medium. Phenotypic tests revealed that Pf-5 had reduced swarming motility on semi-solid agar in response to iron limitation. Comparison of the transcriptomic data with the proteomic data suggested that iron acquisition is regulated at both the transcriptional and post-transcriptional levels

A β-Hairpin-Binding Protein for Three Different Disease-Related Amyloidogenic Proteins

Amyloidogenic proteins share a propensity to convert to the b-structure-rich amyloid state that is associated with the progression of several protein-misfolding disorders. Here we show that a single engineered b-hairpin-binding protein, the bwrapin AS10, binds monomers of three different amyloidogenic proteins, that is, amyloid-b peptide, a-synuclein, and islet amyloid polypeptide, with sub-micromolar affinity. AS10 binding inhibits the aggregation and toxicity of all three proteins. The results demonstrate common conformational preferences and related binding sites in a subset of the amyloidogenic proteins. These commonalities enable the generation of multispecific monomer-binding agents

Contact between the β1 and β2 Segments of α-Synuclein that Inhibits Amyloid Formation

Conversion of the intrinsically disordered protein α-synuclein (α-syn) into amyloid aggregates is a key process in Parkinson’s disease. The sequence region 35–59 contains β-strand segments β1 and β2 of α-syn amyloid fibril models and most disease-related mutations. β1 and β2 frequently engage in transient interactions in monomeric α-syn. The consequences of β1–β2 contacts are evaluated by disulfide engineering, biophysical techniques, and cell viability assays. The double-cysteine mutant α-synCC, with a disulfide linking β1 and β2, is aggregation-incompetent and inhibits aggregation and toxicity of wild-type α-syn. We show that α-syn delays the aggregation of amyloid-β peptide and islet amyloid polypeptide involved in Alzheimer’s disease and type 2 diabetes, an effect enhanced in the α-synCC mutant. Tertiary interactions in the β1–β2 region of α-syn interfere with the nucleation of amyloid formation, suggesting promotion of such interactions as a potential therapeutic approach

Autoproteolysis accelerated by conformational strain - a novel biochemical mechanism

Natural fragmentation of polypeptide chains by autoproteolysis occurs in a number of protein families. It is a vital step in the maturation of several enzymes and in the formation of membrane-associated mucins that constitute a part of the protective mucus barrier lining epithelial cells. These reactions follow similar routes involving an initial N-O or N-S acyl shift starting with a nucleophilic attack by a hydroxyl or thiol group on a carbonyl carbon followed by resolution of the ester intermediate. Previous studies indicate that distortion of the scissile peptide bond may play a role in autoproteolysis. Our structural, biochemical and molecular dynamics studies of the autoproteolyzed SEA domains from human membrane-bound mucin MUC1 and human orphan receptor GPR116 confirmed this by revealing a novel biochemical mechanism where the folding free energy accelerates cleavage by imposing conformational strain in the precursor structure. This mechanism may well be general for autoproteolysis. The structure of the cleaved MUC1 SEA domain was determined using NMR spectroscopy. It consists of four alpha-helices packed against the concave surface of a four-stranded anti-parallel beta-sheet. There are no disordered loops. The site of autoproteolysis is a conserved GSVVV sequence located at the ends of beta-sheets 2 and 3 where the resulting N- and C-terminal residues become integrated parts of these sheets after cleavage. The structure does not reveal any charge-relay system or oxyanion hole as would be expected if catalysis proceeded by way of transition state stabilization. The surface of the domain contains two hydrophobic patches that may serve as sites of interaction with other proteins, giving it a potential function in the regulation of the protective mucus layer. Combined studies of autoproteolysis and adoption of native fold show that these mechanisms proceed with the same rate and that the autoproteolysis has a global effect on structure. Studies of the stability and cleavage kinetics were performed by destabilizing core mutations or addition of denaturing co-solvents. Analysis revealed that ~7 kcal mol-1 of conformational free energy is partitioned as strain in the precursor. The results corroborate a mechanism where the autoproteolysis is accelerated by the concerted action of a conserved serine residue and strain imposed on the precursor structure upon folding, that is, the catalytic mechanism is substrate destabilization. The autoproteolysis of SEA is pH dependent. This is in line with a proposed mechanism with an initial N-O acyl shift, involving transient protonation of the amide nitrogen, and subsequent hydroxyl-mediated hydrolysis of the resulting ester. The mechanistic link between strain and cleavage kinetics is that strain induces a pyramidal conformation of the amide nitrogen which results in an increase of the pKa and thereby an acceleration of the N-O acyl shift. Furthermore we propose a water hydronium as proton donor in this step. This explains the absence of conserved acid-base functionality within the SEA structure