Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis

In vivo, collagen I, the major structural protein in human body, is found assembled into fibrils. In the present work, we study a high concentrated collagen sample in its soluble, fibrillar, and denatured states using one and two dimensional {¹H}-¹³C solid-state NMR spectroscopy. We interpret ¹³C chemical shift variations in terms of dihedral angle conformation changes. Our data show that fibrillogenesis increases the side chain and backbone structural complexity. Nevertheless, only three to five rotameric equilibria are found for each amino acid residue, indicating a relatively low structural heterogeneity of collagen upon fibrillogenesis. Using side chain statistical data, we calculate equilibrium constants for a great number of amino acid residues. Moreover, based on a ¹³C quantitative spectrum, we estimate the percentage of residues implicated in each equilibrium. Our data indicate that fibril formation greatly affects hydroxyproline and proline prolyl pucker ring conformation. Finally, we discuss the implication of these structural data and propose a model in which the attractive force of fibrillogenesis comes from a structural reorganization of 10 to 15% of the amino acids. These results allow us to further understand the self-assembling process and fibrillar structure of collagen.     

A hIAPP-derived all-d-amino-acid inhibits hIAPP fibrillation efficiently at membrane surface by targeting α-helical oligomeric intermediates

A variety of peptides and peptide derivatives have been constructed using the “β-sheet core segment” of amyloid proteins as inhibitors of amyloidogenic fibrillation. A novel all-d-amino-acid from hIAPP β-sheet core segment (hIAPP 22–27) is demonstrated to inhibit hIAPP fibril formation efficiently both at the phospholipid membrane and in bulk solution. The inhibitor terminates hIAPP aggregation to the α-helical oligomeric intermediates at the membrane surface, whereas it stops the aggregation at the stage of β-sheet oligomeric intermediates in bulk solution. This is the first evidence that the inhibition mechanism of the inhibitor at membrane surface is significantly different from that in bulk solution.     

In-situ spectroscopic investigation of ultrasonic assisted unfolding and aggregation of insulin

It is well-known that fibrillogenesis of proteins can be influenced by diverse external parameters, such as temperature, pressure, agitation or chemical agents. The present preliminary study suggests that ultrasonic excitation at moderate intensities has a significant influence on the unfolding and aggregation behaviour of insulin. Irradiation with an average sound intensity of even as low as 70 mW/cm² leads to a lowering of the unfolding and aggregation temperature up to 7 K. The effect could be explained by an increase of the aggregation kinetics due to ultrasonically induced acoustic micro-streaming in the insulin solution that most probably enhances the aggregation rate. The clear and remarkable effect at relatively low sound intensities offers interesting options for further applications of ultrasound in biophysics and biochemistry. On the other hand, a process that causes a change of kinetics equivalent to 7 K also gives a warning signal concerning the safety of those medical ultrasonic devices that work in this intensity range.     

The inhibitory effects of Escherichia coli maltose binding protein on β-amyloid aggregation and cytotoxicity

The aggregation of β-amyloid (Aβ) peptide from its monomeric to its fibrillar form importantly contributes to the development of Alzheimer’s disease. Here, we investigated the effects of Escherichia coli maltose binding protein (MBP), which has been previously used as a fusion protein, on Aβ42 fibrillization, in order to improve understanding of the self-assembly process and the cytotoxic mechanism of Aβ42. MBP, at a sub-stoichiometric ratio with respect to Aβ42, was found to have chaperone-like inhibitory effects on β-sheet fibril formation, due to the accumulation of Aβ42 aggregates by sequestration of active Aβ42 species as Aβ42-MBP complexes. Furthermore, MBP increased the lag time of Aβ42 polymerization, decreased the growth rate of fibril extension, and suppressed Aβ42 mediated toxicity in human neuroblastoma SH-SY5Y cells. It appears that MBP decreases the active concentration of Aβ42 by sequestering it as Aβ42-MBP complex, and that this sequestration suppresses ongoing nucleation and retards the growth rate of Aβ42 species required for fibril formation. We speculate that inhibition of the growth rate of potent Aβ42 species by MBP suppresses Aβ42-mediated toxicity in SH-SY5Y cells.     

Enrichment of fibrillar cytoplasmic actomyosin in protoplasmic strands of Physarum polycephalum for the production of cell-free models

Summary The treatment of isolated protoplasmic strands of Physarum polycephalum with 2.5% ethanol in a physioogical salt solution under isometric conditions induces the formation of a large amount of mostly longitudinally organized actomyosin fibrils in the endoplasmic channel, a region normally free of actomyosin fibrils. The quantity of fibrillogenesis as well as the concomitant force output during the induced contractures are dependent on the Ca^{+ +}- content and the temperature of the test solution. The method was developed to optimize the structure of the plasmodial strands before their subsequent transformation into cell-free models by permeabilization and extraction of the strands.Cryosections of plasmodial strands containing cytoplasmic actomyosin fibrils stained with fluorescently labeled phallotoxins offer a further assay for the study of their contraction physiology under cell-free conditions.     

Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

Dialysis related amyloidosis (DRA) is a serious complication to long-term hemodialysis treatment which causes clinical symptoms such as carpal tunnel syndrome and destructive arthropathies. The disease is characterized by the assembly and deposition of β₂-microglobulin (β₂m) predominantly in the musculoskeletal system, but the initiating events leading to β₂m amyloidogenesis and the molecular mechanisms underlying amyloid fibril formation are still unclear. Glycosaminoglycans (GAGs) and metal ions have been shown to be related to the onset of protein aggregation and to promote de novo fiber formation. In this study, we show that fibrillogenesis of a cleavage variant of β₂m, ΔK58-β₂m, which can be found in the circulation of hemodialysis patients and is able to fibrillate at near-physiological pH in vitro, is affected by the presence of copper ions and heparan sulfate. It is found that the fibrils generated when heparan sulfate is present have increased length and diameter, and possess enhanced stability and seeding properties. However, when copper ions are present the fibrils are short, thin and less stable, and form at a slower rate. We suggest that heparan sulfate stabilizes the cleaved monomers in the early aggregates, hereby promoting the assembly of these into fibrils, whereas the copper ions appear to have a destabilizing effect on the monomers. This keeps them in a structure forming amorphous aggregates for a longer period of time, leading to the formation of spherical bodies followed by the assembly of fibrils. Hence, the in vivo formation of amyloid fibrils in DRA could be initiated by the generation of ΔK58-β₂m which spontaneously aggregate and form fibrils. The fibrillogenesis is enhanced by the involvement of GAGs and/or metal ions, and results in amyloid-like fibrils able to promote the de novo formation of β₂m amyloid by a scaffold mechanism.     

Actinidain-hydrolyzed Type I Collagen Reveals a Crucial Amino Acid Sequence in Fibril Formation

We investigated the ability of type I collagen telopeptides to bind neighboring collagen molecules, which is thought to be the initial event in fibrillogenesis. Limited hydrolysis by actinidain protease produced monomeric collagen, which consisted almost entirely of α1 and α2 chains. As seen with ultrahigh resolution scanning electron microscopy, actinidain-hydrolyzed collagen exhibited unique self-assembly, as if at an intermediate stage, and formed a novel suprastructure characterized by poor fibrillogenesis. Then, the N- and C-terminal sequences of chicken type I collagen hydrolyzed by actinidain or pepsin were determined by Edman degradation and de novo sequence analysis with matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry, respectively. In the C-telopeptide region of the α1 chain, pepsin cleaved between Asp¹⁰³⁵ and Phe¹⁰³⁶, and actinidain between Gly¹⁰³² and Gly¹⁰³³. Thus, the actinidain-hydrolyzed α1 chain is shorter at the C terminus by three residues, Gly¹⁰³³, Phe¹⁰³⁴, and Asp¹⁰³⁵. In the α2 chain, both proteases cleaved between Glu¹⁰³⁰ and Val¹⁰³¹. We demonstrated that a synthetic nonapeptide mimicking the α1 C-terminal sequence including GFD weakly inhibited the self-assembly of pepsin-hydrolyzed collagen, whereas it remarkably accelerated that of actinidain-hydrolyzed collagen. We conclude that the specific GFD sequence of the C-telopeptide of the α1 chain plays a crucial role in stipulating collagen suprastructure and in subsequent fibril formation.     

Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance

Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing lysyl hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846-8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization.     

Potentiation of β-amyloid polymerisation by low-density lipoprotein enhances the peptide's vasoactivity

Alzheimer's disease (AD) is characterised by the accumulation of insoluble β-amyloid (Aβ) fibrils in the brain. Factors that promote Aβ fibrillogenesis may influence the pathogenesis of AD and represent targets for therapeutic intervention. Some Aβ deposited in AD may originate in the circulation and plasma factors could promote Aβ deposition, particularly in the cerebrovasculature. We investigated the effects of plasma low-density lipoprotein (LDL), in both its native and oxidised forms, on Aβ_1–40 fibrillogenesis and vasoactivity. LDL enhanced Aβ fibrillogenesis in a process dependent on LDL concentration and the oxidative state of the lipoprotein, as indicated by measurements of thiobarbituric acid reactive substances (TBARS) and conjugated dienes. LDL's actions were inhibited by the iAβ5 peptide, suggesting that LDL-induced Aβ polymerisation involved β-pleated sheet formation. Potentiated Aβ polymerisation was reflected by enhanced Aβ-mediated vascular responses. Human endothelial cells exposed to fibrillar Aβ generated with LDL, especially oxidised LDL, exhibited decreased 20S proteasome activity. Rat aortic ring constriction induced by noradrenaline was enhanced by Aβ fibrils generated with LDL, with oxidised LDL producing the more marked effects. Should plasma lipoproteins prove to play a role in cerebral Aβ deposition their modification with statins or antioxidants may offer therapeutic benefit.