Specific Chaperones and Regulatory Domains in Control of Amyloid Formation

Many proteins can form amyloid-like fibrils in vitro, but only about 30 amyloids are linked to disease, whereas some proteins form physiological amyloid-like assemblies. This raises questions of how the formation of toxic protein species during amyloidogenesis is prevented or contained in vivo. Intrinsic chaperoning or regulatory factors can control the aggregation in different protein systems, thereby preventing unwanted aggregation and enabling the biological use of amyloidogenic proteins. The molecular actions of these chaperones and regulators provide clues to the prevention of amyloid disease, as well as to the harnessing of amyloidogenic proteins in medicine and biotechnology.     

The chaperone domain BRICHOS prevents CNS toxicity of amyloid-β peptide in Drosophila melanogaster

Aggregation of the amyloid-β peptide (Aβ) into toxic oligomers and amyloid fibrils is linked to the development of Alzheimer’s disease (AD). Mutations of the BRICHOS chaperone domain are associated with amyloid disease and recent in vitro data show that BRICHOS efficiently delays Aβ42 oligomerization and fibril formation. We have generated transgenic Drosophila melanogaster flies that express the Aβ42 peptide and the BRICHOS domain in the central nervous system (CNS). Co-expression of Aβ42 and BRICHOS resulted in delayed Aβ42 aggregation and dramatic improvements of both lifespan and locomotor function compared with flies expressing Aβ42 alone. Moreover, BRICHOS increased the ratio of soluble:insoluble Aβ42 and bound to deposits of Aβ42 in the fly brain. Our results show that the BRICHOS domain efficiently reduces the neurotoxic effects of Aβ42, although significant Aβ42 aggregation is taking place. We propose that BRICHOS-based approaches should be explored with an aim towards the future prevention and treatment of AD.KEY WORDS: Amyloid, Alzheimer’s disease, Protein misfolding, Chaperone     

Sediment resuspension and transport patterns on a fringing reef flat,Molokai, Hawaii

Corals are known to flourish in various turbid environments around the world. The quantitative distinction between clear and turbid water in coral habitats is not well defined nor are the amount of sediment in suspension and rates of sedimentation used to evaluate the condition of reef environments well established. This study of sediment resuspension, transport, and resulting deposition on a fringing reef flat off Molokai, Hawaii, uses a year of time-series data from a small, instrumented tripod. It shows the importance of trade winds and ocean wave heights in controlling the movement of sediment. Sediment is typically resuspended daily and the dominant controls on the magnitude of events (10–25 mg/l) are the trade-wind-generated waves and currents and tidal elevation on the reef flat. The net flux of sediment on this reef is primarily along the reef flat in the direction of the prevailing trade winds (to the west), with a secondary direction of slightly offshore, towards a zone of low coral abundance.These results have application to reef studies and reef management in other areas in several ways. First, the observed resuspension and turbidity results from fine-grained terrigenous sediment that appears to be trapped and recycled on the reef flat. Thus corals are subjected to light attenuation by the same particles repeatedly, however small the amount. Secondly, the measurements show high temporal variability (from daily to seasonal scales) of sediment resuspension, indicating that single measurements are inadequate to accurately describe conditions on a reef flat.Communicated by: P.K. Swart     

Digestibility of amino acids in organically cultivated white-flowering faba bean and cake from cold-pressed rapeseed, linseed and hemp seed in growing pigs

The study aimed at determining the ileal apparent (IAD) and standardised ileal (SID) digestibility of crude protein (CP) and amino acids (AA) in organically cultivated white-flowering faba beans (Vicia faba), and cakes from hemp seed (Cannabis sativa), linseed (Linum usitatissimum) and rapeseed (Brassica napus). The experiment was designed as a four period cross-over trial with six castrated male Yorkshire pigs fitted with post valve T-caecum (PVTC) cannulas. The IAD and SID of CP for the feed ingredients ranged from 79.2-85.9% and were affected by dietary treatment, with significantly lower values in rapeseed cake. The IAD and SID of most AA in the feed ingredients were also significantly affected by dietary treatment, but without any consistent trend. However, the overall digestibilities were in general comparable with conventional protein feed ingredients. Thus, these alternative protein feed ingredients have the potential to be used to a greater extent when formulating organic pig diets.     

BRICHOS Domains Efficiently Delay Fibrillation of Amyloid β-Peptide

Amyloid diseases such as Alzheimer, Parkinson, and prion diseases are associated with a specific form of protein misfolding and aggregation into oligomers and fibrils rich in β-sheet structure. The BRICHOS domain consisting of ～100 residues is found in membrane proteins associated with degenerative and proliferative disease, including lung fibrosis (surfactant protein C precursor; pro-SP-C) and familial dementia (Bri2). We find that recombinant BRICHOS domains from Bri2 and pro-SP-C prevent fibril formation of amyloid β-peptides (Aβ(40) and Aβ(42)) far below the stoichiometric ratio. Kinetic experiments show that a main effect of BRICHOS is to prolong the lag time in a concentration-dependent, quantitative, and reproducible manner. An ongoing aggregation process is retarded if BRICHOS is added at any time during the lag phase, but it is too late to interfere at the end of the process. Results from circular dichroism and NMR spectroscopy, as well as analytical size exclusion chromatography, imply that Aβ is maintained as an unstructured monomer during the extended lag phase in the presence of BRICHOS. Electron microscopy shows that although the process is delayed, typical amyloid fibrils are eventually formed also when BRICHOS is present. Structural BRICHOS models display a conserved array of tyrosine rings on a five-stranded β-sheet, with inter-hydroxyl distances suited for hydrogen-bonding peptides in an extended β-conformation. Our data imply that the inhibitory mechanism is reliant on BRICHOS interfering with molecular events during the lag phase.     

The Brichos domain of prosurfactant protein C can hold and fold a transmembrane segment

Prosurfactant protein C (proSP‐C) is a 197‐residue integral membrane protein, in which the C‐terminal domain (CTC, positions 59–197) is localized in the endoplasmic reticulum (ER) lumen and contains a Brichos domain (positions 94–197). Mature SP‐C corresponds largely to the transmembrane (TM) region of proSP‐C. CTC binds to SP‐C, provided that it is in nonhelical conformation, and can prevent formation of intracellular amyloid‐like inclusions of proSP‐C that harbor mutations linked to interstitial lung disease (ILD). Herein it is shown that expression of proSP‐C (1–58), that is, the N‐terminal propeptide and the TM region, in HEK293 cells results in virtually no detectable protein, while coexpression of CTC in trans yields SDS‐soluble monomeric proSP‐C (1–58). Recombinant human (rh) CTC binds to cellulose‐bound peptides derived from the nonpolar TM region, but not the polar cytosolic part, of proSP‐C, and requires ≥5‐residues for maximal binding. Binding of rhCTC to a nonhelical peptide derived from SP‐C results in α‐helix formation provided that it contains a long TM segment. Finally, rhCTC and rhCTC Brichos domain shows very similar substrate specificities, but rhCTC^L188Q, a mutation linked to ILD is unable to bind all peptides analyzed. These data indicate that the Brichos domain of proSP‐C is a chaperone that induces α‐helix formation of an aggregation‐prone TM region.     

Liquid chromatography-tandem mass spectrometry method for routine measurement of oxalic acid in human plasma

A solid phase extraction (SPE)-LC-MSMS method for the routine determination of oxalic acid (OX) in plasma, a diagnostic marker of primary hyperoxaluria (PH), was developed and validated. The normal range of OX was found to be 3-11mumol/L (n=67), with no differences attributable to gender or age. The effect of pre-analytical factors on the in vitro production of OX was investigated, and plasma was found to be stable for 1-2h at room temperature, less after ingestion of vitamin C; the process was not completely stopped by preservation at either -20 or -70 degrees C.     

Conformational preferences of non-polar amino acid residues: an additional factor in amyloid formation

Amyloid consists of β-sheet polymers and is associated with disease and with functional assemblies. Amyloid-forming proteins differ widely in native structures and sequences. We describe here how conformational preferences of non-polar amino acid residues can affect amyloid formation. The most non-polar residues promote either β-strands (Val, Ile, Phe, and Cys, VIFC) or α-helices (Leu, Ala, and Met, LAM), while the most polar residues promote only α-helices. For 12 proteins associated with disease, the localizations of the amyloid core regions are known. Eleven of these contain segments that are biased for VIFC, but essentially lack segments that are biased for LAM. For the amyloid β-peptide associated with Alzheimer’s disease and an amyloidogenic fragment of the prion protein, observed effects of mutations support that VIFC bias favors formation of β-sheet aggregates and amyloid, while LAM bias prevents it. VIFC and LAM profiles combine information on secondary structure propensities and polarity, and add a simple criterion to the prediction of amyloidogenic regions.