New insight into the molecular control of bacterial functional amyloids.

New insight into the molecular control of bacterial functional amyloids. Front. Cell. Infect. Microbiol. 5:33. doi: 10.3389/fcimb.2015.00033 New insight into the molecular control of bacterial functional amyloid

The transcriptional regulator GalR self-assembles to form highly regular tubular structures

The Gal repressor regulates transport and metabolism of D-galactose in Escherichia coli and can mediate DNA loop formation by forming a bridge between adjacent or distant sites. GalR forms insoluble aggregates at lower salt concentrations in vitro, which can be solubilized at higher salt concentrations. Here, we investigate the assembly and disassembly of GalR aggregates. We find that a sharp transition from aggregates to soluble species occurs between 200 and 400 mM NaCl, incompatible with a simple salting-in effect. The aggregates are highly ordered rod-like structures, highlighting a remarkable ability for organized self-assembly. Mutant studies reveal that aggregation is dependent on two separate interfaces of GalR. The highly ordered structures dissociate to smaller aggregates in the presence of D-galactose. We propose that these self-assembled structures may constitute galactose-tolerant polymers for chromosome compaction in stationary phase cells, in effect linking self-assembly with regulatory function

Oleuropein derivatives from olive fruit extracts reduce α-synuclein fibrillation and oligomer toxicity

Aggregation of α-synuclein (αSN) is implicated in neuronal degeneration in Parkinson's disease and has prompted searches for natural compounds inhibiting αSN aggregation and reducing its tendency to form toxic oligomers. Oil from the olive tree (Olea europaea L.) represents the main source of fat in the Mediterranean diet and contains variable levels of phenolic compounds, many structurally related to the compound oleuropein. Here, using αSN aggregation, fibrillation, size-exclusion chromatography–multiangle light scattering (SEC-MALS)-based assays, and toxicity assays, we systematically screened the fruit extracts of 15 different olive varieties to identify compounds that can inhibit αSN aggregation and oligomer toxicity and also have antioxidant activity. Polyphenol composition differed markedly among varieties. The variety with the most effective antioxidant and aggregation activities, Koroneiki, combined strong inhibition of αSN fibril nucleation and elongation with strong disaggregation activity on preformed fibrils and prevented the formation of toxic αSN oligomers. Fractionation of the Koroneiki extract identified oleuropein aglycone, hydroxyl oleuropein aglycone, and oleuropein as key compounds responsible for the differences in inhibition across the extracts. These phenolic compounds inhibited αSN amyloidogenesis by directing αSN monomers into small αSN oligomers with lower toxicity, thereby suppressing the subsequent fibril growth phase. Our results highlight the molecular consequences of differences in the level of effective phenolic compounds in different olive varieties, insights that have implications for long-term human health

Skp is a multivalent chaperone of outer membrane proteins

The trimeric chaperone Skp sequesters outer-membrane proteins (OMPs) within a hydrophobic cage, thereby preventing their aggregation during transport across the periplasm in Gram-negative bacteria. Here, we studied the interaction between Escherichia coli Skp and five OMPs of varying size. Investigations of the kinetics of OMP folding revealed that higher Skp/OMP ratios are required to prevent the folding of 16-stranded OMPs compared with their 8-stranded counterparts. Ion mobility spectrometry–mass spectrometry (IMS–MS) data, computer modeling and molecular dynamics simulations provided evidence that 10- to 16-stranded OMPs are encapsulated within an expanded Skp substrate cage. For OMPs that cannot be fully accommodated in the expanded cavity, sequestration is achieved by binding of an additional Skp trimer. The results suggest a new mechanism for Skp chaperone activity involving the coordination of multiple copies of Skp in protecting a single substrate from aggregation

An Evolutionary Trade-Off between Protein Turnover Rate and Protein Aggregation Favors a Higher Aggregation Propensity in Fast Degrading Proteins

We previously showed the existence of selective pressure against protein aggregation by the enrichment of aggregation-opposing ‘gatekeeper’ residues at strategic places along the sequence of proteins. Here we analyzed the relationship between protein lifetime and protein aggregation by combining experimentally determined turnover rates, expression data, structural data and chaperone interaction data on a set of more than 500 proteins. We find that selective pressure on protein sequences against aggregation is not homogeneous but that short-living proteins on average have a higher aggregation propensity and fewer chaperone interactions than long-living proteins. We also find that short-living proteins are more often associated to deposition diseases. These findings suggest that the efficient degradation of high-turnover proteins is sufficient to preclude aggregation, but also that factors that inhibit proteasomal activity, such as physiological ageing, will primarily affect the aggregation of short-living proteins

Amyloid Formation by the Pro-Inflammatory S100A8/A9 Proteins in the Ageing Prostate

BACKGROUND: The conversion of soluble peptides and proteins into polymeric amyloid structures is a hallmark of many age-related degenerative disorders, including Alzheimer's disease, type II diabetes and a variety of systemic amyloidoses. We report here that amyloid formation is linked to another major age-related phenomenon--prostate tissue remodelling in middle-aged and elderly men. METHODOLOGY/PRINCIPAL FINDINGS: By using multidisciplinary analysis of corpora amylacea inclusions in prostate glands of patients diagnosed with prostate cancer we have revealed that their major components are the amyloid forms of S100A8 and S100A9 proteins associated with numerous inflammatory conditions and types of cancer. In prostate protease rich environment the amyloids are stabilized by dystrophic calcification and lateral thickening. We have demonstrated that material closely resembling CA can be produced from S100A8/A9 in vitro under native and acidic conditions and shows the characters of amyloids. This process is facilitated by calcium or zinc, both of which are abundant in ex vivo inclusions. These observations were supported by computational analysis of the S100A8/A9 calcium-dependent aggregation propensity profiles. We found DNA and proteins from Escherichia coli in CA bodies, suggesting that their formation is likely to be associated with bacterial infection. CA inclusions were also accompanied by the activation of macrophages and by an increase in the concentration of S100A8/A9 in the surrounding tissues, indicating inflammatory reactions. CONCLUSIONS/SIGNIFICANCE: These findings, taken together, suggest a link between bacterial infection, inflammation and amyloid deposition of pro-inflammatory proteins S100A8/A9 in the prostate gland, such that a self-perpetuating cycle can be triggered and may increase the risk of malignancy in the ageing prostate. The results provide strong support for the prediction that the generic ability of polypeptide chains to convert into amyloids could lead to their involvement in an increasing number of otherwise apparently unrelated diseases, particularly those associated with ageing.Original Publication:Kiran Yanamandra, Oleg Alexeyev, Vladimir Zamotin, Vaibhav Srivastava, Andrei Shchukarev, Ann-Christin Brorsson, Gian Gaetano Tartaglia, Thomas Vogl, Rakez Kayed, Gunnar Wingsle, Jan Olsson, Christopher M Dobson, Anders Bergh, Fredrik Elgh and Ludmilla A Morozova-Roche, Amyloid formation by the pro-inflammatory S100A8/A9 proteins in the ageing prostate., 2009, PloS one, (4), 5, e5562.http://dx.doi.org/10.1371/journal.pone.000556

Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins

Outer membrane protein folding from an energy landscape perspective

The cell envelope is essential for the survival of Gram-negative bacteria. This specialised membrane is densely packed with outer membrane proteins (OMPs), which perform a variety of functions. How OMPs fold into this crowded environment remains an open question. Here, we review current knowledge about OFMP folding mechanisms in vitro and discuss how the need to fold to a stable native state has shaped their folding energy landscapes. We also highlight the role of chaperones and the β-barrel assembly machinery (BAM) in assisting OMP folding in vivo and discuss proposed mechanisms by which this fascinating machinery may catalyse OMP folding

Latent analysis of unmodified biomolecules and their complexes in solution with attomole detection sensitivity

The study of biomolecular interactions is central to an understanding of function, malfunction and therapeutic modulation of biological systems, yet often involves a compromise between sensitivity and accuracy. Many conventional analytical steps and the procedures required to facilitate sensitive detection, such as the incorporation of chemical labels, are prone to perturb the complexes under observation. Here we present a 'latent' analysis approach that uses chemical and microfluidic tools to reveal, through highly sensitive detection of a labelled system, the behaviour of the physiologically relevant unlabelled system. We implement this strategy in a native microfluidic diffusional sizing platform, allowing us to achieve detection sensitivity at the attomole level, determine the hydrodynamic radii of biomolecules that vary by over three orders of magnitude in molecular weight, and study heterogeneous mixtures. We illustrate these key advantages by characterizing a complex of an antibody domain in the solution phase and under physiologically relevant conditions.We would like to thank the ERC, BBSRC, Wellcome Trust, Newman Foundation, Winston Churchill Foundation, and Elan Pharmaceuticals for financial support. E.D.G was supported by the MRC (G1002272)