The predominant roles of the sequence periodicity in the self‐assembly of collagen‐mimetic mini‐fibrils

Collagen fibrils represent a unique case of protein folding and self‐association. We have recently successfully developed triple‐helical peptides that can further self‐assemble into collagen‐mimetic mini‐fibrils. The 35 nm axially repeating structure of the mini‐fibrils, which is designated the d‐period, is highly reminiscent of the well‐known 67 nm D‐period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo‐identical repeating sequence units in the primary structure of the designed peptides that give rise to the d‐period of the quaternary structure of the mini‐fibrils. In this work, we characterize the self‐assembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple‐helix domain of Col877 consists of three pseudo‐identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 self‐associate laterally under fibril forming conditions to form mini‐fibrils having the predicted d‐period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self‐assembly of collagen mini‐fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen‐mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications.     

Sequence dependence of kinetics and morphology of collagen model peptide self-assembly into higher order structures

The process of self-assembly of the triple-helical peptide (Pro-Hyp-Gly)(10) into higher order structure resembles the nucleation-growth mechanism of collagen fibril formation in many features, but the irregular morphology of the self-assembled peptide contrasts with the ordered fibers and networks formed by collagen in vivo. The amino acid sequence in the central region of the (Pro-Hyp-Gly)(10) peptide was varied and found to affect the kinetics of self-assembly and nature of the higher order structure formed. Single amino acid changes in the central triplet produced irregular higher order structures similar to (Pro-Hyp-Gly)(10), but the rate of self-association was markedly delayed by a single change in one Pro to Ala or Leu. The introduction of a Hyp-rich hydrophobic sequence from type IV collagen resulted in a more regular suprastructure of extended fibers that sometimes showed supercoiling and branching features similar to those seen for type IV collagen in the basement membrane network. Several peptides, where central Pro-Hyp sequences were replaced by charged residues or a nine-residue hydrophobic region from type III collagen, lost the ability to self-associate under standard conditions. The inability to self-assemble likely results from loss of imino acids, and lack of an appropriate distribution of hydrophobic/electrostatic residues. The effect of replacement of a single Gly residue was also examined, as a model for collagen diseases such as osteogenesis imperfecta and Alport syndrome. Unexpectedly, the Gly to Ala replacement interfered with self-assembly of (Pro-Hyp-Gly)(10), while the peptide with a Gly to Ser substitution self-associated to form a fibrillar structure.     

To achieve self‐assembled collagen mimetic fibrils using designed peptides

It has proven challenging to obtain collagen‐mimetic fibrils by protein design. We recently reported the self‐assembly of a mini‐fibril showing a 35 nm, D‐period like, axially repeating structure using the designed triple helix Col108. Peptide Col108 was made by bacterial expression using a synthetic gene; its triple helix domain consists of three pseudo‐identical units of amino acid sequence arranged in tandem. It was postulated that the 35 nm d‐period of Col108 mini‐fibrils originates from the periodicity of the Col108 primary structure. A mutual staggering of one sequence unit of the associating Col108 triple helices can maximize the inter‐helical interactions and produce the observed 35 nm d‐period. Based on this unit‐staggered model, a triple helix consisting of only two sequence units is expected to have the potential to form the same d‐periodic mini‐fibrils. Indeed, when such a peptide, peptide 2U108, was made it was found to self‐assemble into mini‐fibrils having the same d‐period of 35 nm. In contrast, no d‐periodic mini‐fibrils were observed for peptide 1U108, which does not have long‐range repeating sequences in its primary structure. The findings of the periodic mini‐fibrils of Col108 and 2U108 suggest a way forward to create collagen‐mimetic fibrils for biomedical and industrial applications.     

Morphology of self‐assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac‐VQIVYK‐amide (AcPHF6) and Ac‐QIVYK‐amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self‐association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly β‐conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for β‐structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and β‐structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Streptococcus pyogenes cytolysin‐mediated translocation does not require pore formation by streptolysin O

Bacterial toxin injection into the host cell is required for the virulence of numerous pathogenic bacteria. Cytolysin‐mediated translocation (CMT) of Streptococcus pyogenes uses streptolysin O (SLO) to translocate the S. pyogenes nicotinamide adenine dinucleotide‐glycohydrolase (SPN) into the host cell cytosol, resulting in the death of the host cell. Although SLO is a pore‐forming protein, previous studies have shown that pore formation alone is not sufficient for CMT to occur. Thus, the role and requirement of the SLO pore remains unclear. In this study, we constructed various S. pyogenes strains expressing altered forms of SLO to assess the importance of pore formation. We observed that SLO mutants that are unable to form pores retain the ability to translocate SPN. In addition, SPN translocation occurs after inhibition of actin polymerization, suggesting that CMT occurs independently of clathrin‐mediated endocytosis. Moreover, despite the ability of mutants to translocate SPN, their cytotoxic effect requires SLO pore formation.     

Effect of anti-TNF-α treatment in an antibiotic treated murine model of shock due to Streptococcus pyogenes

Abstract Experimentally, Gram-negative septic shock can be prevented by the prophylactic use of an anti-TNF-α monoclonal antibody. The clinical similarity between Gram-negative and Gram-positive septic shock suggested that anti-TNF-α therapy might have a wide application. Increased levels of TNF-α were seen in a murine model of septic shock due to Streptococcus pyogenes but administration of an anti-TNF-α monoclonal antibody had no beneficial effect on the outcome.     

Self‐association of TPR domains: Lessons learned from a designed,consensus‐based TPR oligomer

The tetratricopeptide repeat (TPR) motif is a protein–protein interaction module that acts as an organizing centre for complexes regulating a multitude of biological processes. Despite accumulating evidence for the formation of TPR oligomers as an additional level of regulation there is a lack of structural and solution data explaining TPR self‐association. In the present work we characterize the trimeric TPR‐containing protein YbgF, which is linked to the Tol system in Gram‐negative bacteria. By subtracting previously identified TPR consensus residues required for stability of the fold from residues conserved across YbgF homologs, we identified residues involved in oligomerization of the C‐terminal YbgF TPR domain. Crafting these residues, which are located in loop regions between TPR motifs, onto the monomeric consensus TPR protein CTPR3 induced the formation of oligomers. The crystal structure of this engineered oligomer shows an asymmetric trimer where stacking interactions between the introduced tyrosines and displacement of the C‐terminal hydrophilic capping helix, present in most TPR domains, are key to oligomerization. Asymmetric trimerization of the YbgF TPR domain and CTPR3Y3 leads to the formation of higher order oligomers both in the crystal and in solution. However, such open‐ended self‐association does not occur in full‐length YbgF suggesting that the protein's N‐terminal coiled‐coil domain restricts further oligomerization. This interpretation is borne out in experiments where the coiled‐coil domain of YbgF was engineered onto the N‐terminus of CTPR3Y3 and shown to block self‐association beyond trimerization. Our study lays the foundations for understanding the structural basis for TPR domain self‐association and how such self‐association can be regulated in TPR domain‐containing proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Lipopolysaccharide accelerates collagen‐induced arthritis in association with rapid and continuous production of inflammatory mediators and anti‐type II collagen antibody

Collagen‐induced arthritis (CIA) is an animal model for rheumatoid arthritis (RA). Lipopolysaccharide (LPS) is known to accelerate CIA; however, the pathogenetic mechanisms are not yet fully understood. In this study, type II collagen (CII)‐immunized mice were found to have marked increases in degree of expression of mRNA of inflammatory mediators such as tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐1β, and macrophage inflammatory protein‐2 (MIP‐2) in their arthritic paws and of serum anti‐CII antibody concentration before the onset of arthritis induced by LPS injection. The gene expression was rapid and continuous after direct activation of nuclear factor κB. The amounts of mRNA of TNF‐α, IL‐1β, and MIP‐2, as well as of matrix metalloproteinases and the receptor activator of nuclear factor κB ligand, increased with the development of arthritis, correlated positively with clinical severity and corresponded with histopathological changes. Moreover, anti‐TNF‐α neutralizing antibody inhibited the development of LPS‐accelerated CIA and a single injection of recombinant mouse TNF‐α induced increases in anti‐CII antibody concentrations, suggesting TNF‐α may contribute to the development of arthritis by both initiation of inflammation and production of autoantibodies. These data suggest that exacerbation of RA by LPS is associated with rapid and continuous production of inflammatory mediators and autoantibodies.     

Ethanol induced the formation of β‐sheet and amyloid‐like fibrils by surfactant‐like peptide A6K

Self‐assembly of natural or designed peptides into fibrillar structures based on β‐sheet conformation is a ubiquitous and important phenomenon. Recently, organic solvents have been reported to play inductive roles in the process of conformational change and fibrillization of some proteins and peptides. In this study, we report the change of secondary structure and self‐assembling behavior of the surfactant‐like peptide A6K at different ethanol concentrations in water. Circular dichroism indicated that ethanol could induce a gradual conformational change of A6K from unordered secondary structure to β‐sheet depending upon the ethanol concentration. Dynamic light scattering and atomic force microscopy revealed that with an increase of ethanol concentration the nanostructure formed by A6K was transformed from nanosphere/string‐of‐beads to long and smooth fibrils. Furthermore, Congo red staining/binding and thioflavin‐T binding experiments showed that with increased ethanol concentration, the fibrils formed by A6K exhibited stronger amyloid fibril features. These results reveal the ability of ethanol to promote β‐sheet conformation and fibrillization of the surfactant‐like peptide, a fact that may be useful for both designing self‐assembling peptide nanomaterials and clarifying the molecular mechanism behind the formation of amyloid fibrils. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Hexafluoroisopropanol induces self‐assembly of β‐amyloid peptides into highly ordered nanostructures

Deposition of insoluble fibrillar aggregates of β‐amyloid (Aβ) peptides in the brain is a hallmark of Alzheimer's disease. Apart from forming fibrils, these peptides also exist as soluble aggregates. Fibrillar and a variety of nonfibrillar aggregates of Aβ have also been obtained in vitro. Hexafluoroisopropanol (HFIP) has been widely used to dissolve Aβ and other amyloidogenic peptides. In this study, we show that the dissolution of Aβ40, 42, and 43 in HFIP followed by drying results in highly ordered aggregates. Although α‐helical conformation is observed, it is not stable for prolonged periods. Drying after prolonged incubation of Aβ40, 42, and 43 peptides in HFIP leads to structural transition from α‐helical to β‐conformation. The peptides form short fibrous aggregates that further assemble giving rise to highly ordered ring‐like structures. Aβ16–22, a highly amyloidogenic peptide stretch from Aβ, also formed very similar rings when dissolved in HFIP and dried. HFIP could not induce α‐helical conformation in Aβ16–22, and rings were obtained from freshly dissolved peptide. The rings formed by Aβ40, 42, 43, and Aβ16–22 are composed of the peptides in β‐conformation and cause enhancement in thioflavin T fluorescence, suggesting that the molecular architecture of these structures is amyloid‐like. Our results clearly indicate that dissolution of Aβ40, 42 and 43 and the amyloidogenic fragment Aβ16–22 in HFIP results in the formation of annular amyloid‐like structures. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Prediction of surface exposed proteins in Streptococcus pyogenes,with a potential application to other Gram‐positive bacteria

The in silico prediction of bacterial surface exposed proteins is of growing interest for the rational development of vaccines and in the study of bacteria–host relationships, whether pathogenic or host beneficial. This interest is driven by the increase in the use of DNA sequencing as a major tool in the early characterization of pathogenic bacteria and, more recently, even of complex ecosystems at the host–environment interface in metagenomics approaches. Current protein localization protocols are not suited to this prediction task as they ignore the potential surface exposition of many membrane‐associated proteins. Therefore, we developed a new flow scheme, SurfG+, for the processing of protein sequence data with the particular aim of identification of potentially surface exposed (PSE) proteins from Gram‐positive bacteria, which was validated for Streptococcus pyogenes. The results of an exploratory case study on closely related lactobacilli of the acidophilus group suggest that the yogurt bacterium Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) dedicates a relatively important fraction of its coding capacity to secreted proteins, while the probiotic gastrointestinal (GI) tract bacteria L. johnsonii and L. gasseri appear to encode a larger variety of PSE proteins, that may play a role in the interaction with the host.     

Bacterial collagen‐binding domain targets undertwisted regions of collagen

Clostridium histolyticum collagenase causes extensive degradation of collagen in connective tissue that results in gas gangrene. The C‐terminal collagen‐binding domain (CBD) of these enzymes is the minimal segment required to bind to a collagen fibril. CBD binds unidirectionally to the undertwisted C‐terminus of triple helical collagen. Here, we examine whether CBD could also target undertwisted regions even in the middle of the triple helix. Collageneous peptides with an additional undertwisted region were synthesized by introducing a Gly → Ala substitution [(POG)_xPOA(POG)_y]₃, where x + y = 9 and x > 3). ¹H–¹⁵N heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) titration studies with ¹⁵N‐labeled CBD demonstrated that the minicollagen binds to a 10 Å wide 25 Å long cleft. Six collagenous peptides each labeled with a nitroxide radical were then titrated with ¹⁵N‐labeled CBD. CBD binds to either the Gly → Ala substitution site or to the C‐terminus of each minicollagen. Small‐angle X‐ray scattering measurements revealed that CBD prefers to bind the Gly → Ala site to the C‐terminus. The HSQC NMR spectra of ¹⁵N‐labeled minicollagen and minicollagen with undertwisted regions were unaffected by the titration of unlabeled CBD. The results imply that CBD binds to the undertwisted region of the minicollagen but does not actively unwind the triple helix.     

Application of an enzyme‐labeled antigen method for visualizing plasma cells producing antibodies against Strep A,a carbohydrate antigen of Streptococcus pyogenes,in recurrent tonsillitis

Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme‐labeled antigen method is a novel histochemical technique that visualizes specific antibody‐producing cells in tissue sections by employing a biotin‐labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde‐fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme‐labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR‐detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti‐Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A‐reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.     

Self‐assembling amyloid‐like peptides as exogenous second harmonic probes for bioimaging applications

Amyloid‐like peptides are an ideal model for the mechanistic study of amyloidosis, which may lead to many human diseases, such as Alzheimer disease. This study reports a strong second harmonic generation (SHG) effect of amyloid‐like peptides, having a signal equivalent to or even higher than those of endogenous collagen fibers. Several amyloid‐like peptides (both synthetic and natural) were examined under SHG microscopy and shown they are SHG‐active. These peptides can also be observed inside cells (in vitro). This interesting property can make these amyloid‐like peptides second harmonic probes for bioimaging applications. Furthermore, SHG microscopy can provide a simple and label‐free approach to detect amyloidosis. Lattice corneal dystrophy was chosen as a model disease of amyloidosis. Morphological difference between normal and diseased human corneal biopsy samples can be easily recognized, proving that SHG can be a useful tool for disease diagnosis.     

Noncollagenous region of the streptococcal collagen‐like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

Proper folding of the (Gly‐Xaa‐Yaa)_n sequence of animal collagens requires adjacent N‐ or C‐terminal noncollagenous trimerization domains which often contain coiled‐coil or beta sheet structure. Collagen‐like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen‐like protein from Streptococcus pyogenes has an N‐terminal globular domain, designated V_sp, adjacent to its triple‐helix domain. The V_sp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V_sp domain alone is shown to form trimers with a significant α‐helix content and to have a thermal stability of T_m = 45°C. Examination of a new construct shows that the V_sp domain facilitates efficient in vitro refolding only when it is located N‐terminal to the triple‐helix domain but not when C‐terminal to the triple‐helix domain. Fusion of the V_sp domain N‐terminal to a heterologous (Gly‐Xaa‐Yaa)_n sequence from Clostridium perfringens led to correct folding and refolding of this triple‐helix, which was unable to fold into a triple‐helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly‐Xaa‐Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple‐helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.     

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3^SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.     

Characterization of a novel collagen‐like protein TrpA in the cyanobacterium Trichodesmium erythraeum IMS101

The collagen protein family is diverse and its membership is continually expanding as new collagen‐like molecules are identified. Identification of collagen in unicellular eukaryotes and prokaryotes has opened discussion on the function of these collagens and their role in the emergence of multicellularity. The previous identification of a collagen gene in Trichodesmium erythraeum raises the question of function of this structural protein in a prokaryote. In this study, we show that this gene is expressed during all phases of growth, indicating that it may be required for all phases of growth. Using immunofluorescence techniques, we demonstrate that the collagen‐like protein is localized in a specific manner between adjacent cells along the trichome of T. erythraeum. Trichomes treated with the enzyme collagenase exhibited fragmentation, supporting our immunofluorescence localization data that this collagen‐like protein is found between adjacent cells. Our data strongly suggest that the collagen‐like protein found in T. erythraeum functions to maintain the structural integrity of the trichome through the adhesion of adjacent cells.