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It has been observed that monoclonal immunoglobulin proteins of the lambda VI subgroup have a high propensity to form amyloid deposits. To ascertain whether lambda VI proteins have unique structure determinants that would account for self association and resultant fibril formation, we have determined the complete amino acid sequence of the AL amyloid protein WLT. This protein, isolated from the spleen of a patient with AL amyloid, has 134 amino acid residues and contains the entire variable region, the joining segment, and the first tryptic peptide of the constant region. Comparison of the structure of this protein with the 3 completely sequenced lambda VI proteins reveals that they are highly homologous and contain a 2-residue insertion at positions 68 and 69. Phylogenetic comparisons of the variable domain of all lambda VI proteins reveal that the 3 amyloid proteins WLT, SUT, and AR are all more closely related to each other than to the myeloma protein NIG48. Separating the variable domains into framework (FR) and complementarity-determining regions (CD) and recalculating the phylogenetic comparisons, we identify major substitutions in the FR regions of NIG48 in relation to the amyloid proteins. This supports the hypothesis that the formation of AL amyloid is a result of the secondary structure of the FR regions of the precursor molecules.

Collagen and elastin are the essential structural proteins of the arterial wall; the first one is being responsible for tensile strength, the other one for elasticity.

Reconstituted cartilage collagen fibrils with an oblique banding pattern or with two types of symmetrical patterns, and reconstituted rattail tendon fibrils with a third type of symmetrical pattern were examined by electron microscopy and found to consist of narrow subfibrils having native-type cross-striations. Analysis of the four types of patterns by a graphic method of specific band matching revealed the orientation and axial relation of individual subfibrils and their component molecules. In fibrils with an oblique pattern, subfibrils have the same orientation and a regular 100A axial displacement. Observations on staining characteristics, folded fibrils, and transverse sections of embedded fibrils suggest that the obliquely banded fibrils are ribbonlike or layered structures. In the three types of fibrils with a symmetrical pattern, adjacent subfibrils are oppositely oriented and aligned within a 119-A segment of the 670-A major period. Considered together, the observations suggest that interaction sites on the surface of subfibrils (and perhaps on the surface of native collagen fibrils) occur in various patterns that are manifested accouding to the nature of the environment during fibril formation, and that such patterns can be mapped on the surface of subfibrils by noting the arrangement of subfibrils in polymorphic forms.

Publisher Summary This chapter discusses a study to analyze the effect of high-density lipoproteins (HDL) and its fractions on in vitro collagen fibril formation. The effect of low density lipoproteins (LDL) and HDL on collagen fibril formation was studied by the heat gelation method. Collagen type I and III were extracted from rat skin and fractionated. Collagen type II was extracted from nasal cartilage. LDL and HDL fractions were prepared according to Kostner and Holasek (1977) from pooled serum. LDL and very low-density lipoproteins (VLDL) were precipitated by phosphotungstic acid (PTA)-magnesiumchloride. HDL was obtained from the supernatant after centrifugation at the specific gravity of 1.220, for 48 h in ultracentrifuge Janetzky, type VAC 402 at 200,000× g . The supernatant was called unfractionated HDL. After washing, it was fractionated at the specific gravity of 1.110 for 24 h at 150,000×g. It was found that LDL did not influence, not even slightly, the rate of fibril formation. On the contrary, HDL used at the same concentration delayed in vitro fibril formation.

Rat tail tendon collagen-initiated platelet aggregation exhibits a collagen age-dependent lag time. This lag time is an inverse function of the previously determined rate of fibril formation of collagen, and corresponds to the elapsed time necessary to form a collagen fibril of requisite size under the platelet aggregation conditions chosen. Such fibers exhibit native spacing and appear to be 45 to 90 A in diameter. Fibers preformed to that size (less than 2 min for 21- to 1,100-day-old collagen), no matter what the age of the collagen, give rise to identical platelet aggregations. Fibers formed after more prolonged incubation, greater than or equal to 20 min, have impaired platelet aggregating ability.

In our earlier analysis of intermolecular interactions between collagen molecules, a major concern with the program employed is that it compared numbers of interactions between residues located on edges of defined, identical width and thus would not necessarily compare the same number of residues in each edge. This would be particularly true of some values of θ where well-defined vertical ranking of residues occurs. We have examined ranking of residues in relation to intermolecular edge association between bovine skin [α1(I)]3 model collagen molecules by utilizing two different methods of counting intermolecular interactions between residues. The interaction peaks at θ = 27.69° and 36.00° are absent or relatively less intense in the plots obtained by utilizing radial distances between interacting residues instead of vertical bands of defined width. These studies suggest caution in accepting recently reported analyses of superhelix coiling of the collagen molecule which point to values of 27.69° or 36.00° for the twist of the superhelix. Although intramolecular interactions clearly point to interaction of collagen molecules at D intervals, they are insufficiently restricted in distribution to provide a reliable estimate of the superhelix angle by procedures so far employed.

Native or heat-denatured collagens were incubated under controlled conditions of temperature and pH with variable molar ratios of KCNO or 2,4,6-trinitrobenzene sulphonic acid. The results obtained suggest that a small number of free amino groups are available for reaction on the native protein, while all the free amino groups react on the denatured protein. The highly reactive free amino groups in the native protein are hydroxylysine residues and have an abnormally low pK of 8.5 which is conformation dependent; this pK becomes normalized upon denaturation of the protein. The reactive hydroxylysines appear to be located in basic regions that could be the nucleation sites needed for fiber formation in the heat-gelation assay; the modified protein does not form stable fibrils upon heating at 37 degrees C and the few fibers formed are not stabilized after reduction with NaBH4. Our results also suggest that the triple helix in collagen is heterogeneous with respect to the reactivity of free amino groups and that several discrete transition temperatures are observed with two main breaks at 30 degrees C and at 37 degrees C, respectively.

Glucose inhibits collagen fibril formation in vitro. A linear dose response was observed, with half-maximum inhibition of fibril formation occurring at 50 mM glucose. Nonfibrillar collagen cannot be cross-linked by lysyl oxidase, an enzyme that catalyzes the initial cross-linking reaction. The degree of decreased fibril formation correlated with the loss of ability of the collagen to serve as a substrate for lysyl oxidase. Collagen that is not cross-linked is unstable and more susceptible to collagenolytic attack. Interference with collagen cross-linking and more rapid degradation may explain the decreased amounts of interstitial collagen and the poor healing of wounds associated with diabetes mellitus.

Vertebrate collagens constitute a family of at least twelve genetic types that shows remarkable diversity in molecular structure and supramolecular assembly (Mayne & Burgeson, 1987). Types I, II and III collagens assemble in vivo to form fibrils of uniform diameter, near circular cross-section and with a characteristic axial periodicity of 65 to 67 nm (D). Fibrils in vivo are long (several µm) and diameters range from 8 nm to 500 nm, depending on collagen type, species, age and tissue of origin (Parry & Craig, 1984). The mechanisms that control fibril shape and diameter in vivo are poorly understood.

Type XI collagen (1 alpha,2 alpha,3 alpha) from bovine articular cartilage form fibrils at 4 degrees C in 0.15 M NaCl at pH 7.4, but fibrillogenesis is inhibited by the addition of 1 M glucose or by raising the NaCl concentration to 1 M. Removal of the glucose or NaCl by dialysis allows fibril formation. When proteoglycans, heparin, or chondroitin sulfate were added to type XI collagen in 1 M NaCl both fibrillogenesis and polyanion-collagen interaction were inhibited by the high NaCl concentration. When the mixture was dialysed against 0.15 M NaCl, a new aggregate type was seen, scattered among shortened and branched fibers. The new aggregates were either X-, Y-, or wheel-shaped structures with electron dense cores. They were apparently formed by collagen molecules intersecting approximately 200 nm from one end. In contrast, when the polyanion was mixed with the collagen in 1 M glucose, which inhibits fibrillogenesis but not polyanion-collagen interaction, a different type of aggregate appeared following dialysis. These aggregates were discrete 280 X 40 nm structures with an asymmetric banding pattern. They are similar to SLS aggregates, and probably are composed of collagen molecules lined up in register. The results are different from those seen with the interstitial collagens and emphasize the unique character of the interaction of polyanions, including proteoglycan, with type XI collagen.

Electron microscopic autoradiographic observations on collagen fibrils grown in vitro allow growth rates in the N- and C-terminal directions to be measured on individual fibrils. Such observations, made on normal and iodinated collagen, show that normal fibrils grow at both ends (although rather more rapidly at the N-terminal end), whereas fully-iodinated collagen fibrils grow only at the N-terminal end. Measurements of growth rates at different temperatures provide estimates of the activation enthalpy (ΔH≠) and entropy (ΔS≠) of precipitation for the two types of collagen. Solubility measurements have also yielded values for the thermodynamic enthalpy (ΔH) and entropy (ΔS) of precipitation. Results show that the activated (rate-limiting) state is characterized by a large positive ΔH≠ and ΔS≠ similar in magnitude to the ΔH and ΔS of transition from solution to fibril. It is also concluded that the different rates of precipitation of normal and iodinated collagen cannot be explained in terms of fibril formation requiring ionization of the tyrosine residues.

Electron microscopy techniques were used to study the attachment matrix of Pseudomonas fragi used to demonstrate the presence of attachment fibrils of P. fragi adhered to stainless steel. Transmission electron microscopy (TEM) was used to examine thin sectioned cells strained with ruthenium red or alcian blue. Extracellular substances surrounding cells stained with ruthenium red or alcian blue had a mat- or spike-like morphology. This indicated that these substances were acidic mucopolysaccharides that may be involved in the attachment of P. fragi to food contact surfaces.

Summary The effect of glycosaminoglycans (GAG) on the in vitro collagen fibril formation of type I and type III collagen extracted from rat skin was studied by measuring the optical absorbancy change of collagen-GAG solution during the fibrillary precipitation and following the electronmicroscopic structure of collagen fibrils. The main difference between type I and type III collagen was found in the kinetic of fibrillary precipitation mainly during the logarithmic growth (“log”) period of fibril formation. Chondroitin sulphate-A (CSA) shortened this phase and affected especially type III collagen which has a longer “log” period. No difference was found in the electronmicroscopic structure of collagen fibrils. The supposed biological significance of the results is briefly discussed.

Electron micrographs of the fibrils of F-insulin, produced by heating soluble insulin at low p H , show that these consist of fine fibrils of minimum width 50–80 A and indefinite length. No periodic structure was found in the fibrils, but the tendency to split transversely is in agreement with the view that they are formed by linear aggregation of particles. The particle, which participates in the actual linear aggregation leading to fibril formation, seems to be larger than the 12,000 M.W. insulin molecule present in acid solution, and this suggests that a preliminary association of several molecules occurs before the actual linear polymerisation.

1. Salt-soluble rat skin collagen was precipitated from solution at neutral pH and 37°. On cooling, a portion of the collagen returned into solution. The fractions were separated, the supernatant was concentrated and the precipitate was redissolved in dilute acetic acid. 2. Solutions of supernatant and precipitate were subjected to the same fractionation procedure, giving four fractions. 3. Each fraction was examined by starch-gel electrophoresis and a relationship between subunit composition and the fractionation procedure was noted. The collagen that redissolved on cooling contained less of the more highly cross-linked components than did either the fraction remaining in the precipitate or the starting material.

Three distinct phases of growth of a line of mouse-lung fibroblast-like cells adapted to a serum-free medium are described. These are, attachment and growth, “elution” and growth, and fibril formation. Attachment and elution were shown to be independent of the glass surface per se and the pH (6.8–7.2) of the original medium. The conditions and reasons for fibril formation are unknown at this time but are under investigation.

Incubation conditions are an important factor to consider when studying protein aggregation in vitro. Here, we employed biophysical methods and atomic force microscopy to show that agitation dramatically alters the morphology of medin, an amyloid protein deposited in the aorta. Agitation reduces the lag time for fibrillation by ~18-fold, suggesting that the rate of fibril formation plays a key role in directing the protein packing arrangement within fibrils. Utilising preformed sonicated fibrils as seeds, we probed the role of seeding on medin fibrillation and revealed three distinct fibril morphologies, with biophysical modelling explaining the salient features of experimental observations. We showed that nucleation pathways to distinct fibril morphologies may be switched on and off depending on the properties of the seeding fibrils and growth conditions. These findings may impact on the development of amyloid-based biomaterials and enhance understanding of seeding as a pathological mechanism.

Aggregation and fibril formation of the amyloid-β (Aβ) peptides play a pivotal role in the pathogenesis of Alzheimer's disease (AD). The missing links on the pathway to Aβ oligomerization, fibril formation, and neurotoxicity in AD remain the identity of the toxic Aβ species and mechanism(s) of their toxicity. Such information is crucial for the development of mechanism-based therapeutics to treat AD and tools to diagnose and/or monitor the disease progression. Herein, we describe a simple approach that combines standard biophysical methods with cell biology assays to correlate the aggregation state of Aβ peptides with their cytotoxicity in vitro. The individual assays are well-established, commonly used, rely on easily accessible materials and can be performed within 24 h.

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of alpha-synuclein (alpha-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type alpha-syn and alpha-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of alpha-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric alpha-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric alpha-syn composed of either wild-type or Gln --> Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of alpha-syn. These studies demonstrate for the first time that Gln(79) and Gln(109) serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of alpha-syn and tTG-induced inhibition of alpha-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln(79) and Gln(109). This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on alpha-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of alpha-syn.

(1995). Etiology of Osteogenesis Imperfecta: An Overview of Biochemical and Molecular Genetic Analyses. Connective Tissue Research: Vol. 31, No. 4, pp. 257-259.

Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions.

Protein aggregation is associated with many debilitating diseases including Alzheimer’s, Parkinson’s, and light-chain amyloidosis (AL). Additionally, such aggregation is a major problem in an industrial setting where antibody therapeutics often require high local concentrations of protein domains to be stable for substantial periods of time. However, despite a plethora of research in this field, dating back over 50 years, there is still no consensus on the mechanistic basis for protein aggregation. Here we use experimental data to derive a mechanistic model that well describes the aggregation of Titin I27, an immunoglobulin-like domain. Importantly, we find that models that are suitable for nucleated fibril formation do not fit our aggregation data. Instead, we show that aggregation proceeds via the addition of activated dimers, and that the rate of aggregation is dependent on the surface area of the aggregate. Moreover, we suggest that the “lag time” seen in these studies is not the time needed for a nuc...

alpha-Synuclein aggregation and accumulation in Lewy bodies are implicated in progressive loss of dopaminergic neurons in Parkinson disease and related disorders. In neurons, the Hsp70s and their Hsp40-like J-domain co-chaperones are the only known components of chaperone network that can use ATP to convert cytotoxic protein aggregates into harmless natively refolded polypeptides. Here we developed a protocol for preparing a homogeneous population of highly stable beta-sheet enriched toroid-shaped alpha-Syn oligomers with a diameter typical of toxic pore-forming oligomers. These oligomers were partially resistant to in vitro unfolding by the bacterial Hsp70 chaperone system (DnaK, DnaJ, GrpE). Moreover, both bacterial and human Hsp70/Hsp40 unfolding/refolding activities of model chaperone substrates were strongly inhibited by the oligomers but, remarkably, not by unstructured alpha-Syn monomers even in large excess. The oligomers acted as a specific competitive inhibitor of the J-domain co-chaperones, indicating that J-domain co-chaperones may preferably bind to exposed bulky misfolded structures in misfolded proteins and, thus, complement Hsp70s that bind to extended segments. Together, our findings suggest that inhibition of the Hsp70/Hsp40 chaperone system by alpha-Syn oligomers may contribute to the disruption of protein homeostasis in dopaminergic neurons, leading to apoptosis and tissue loss in Parkinson disease and related neurodegenerative diseases.

In the early stages of amyloid formation, heterogeneous populations of oligomeric species are generated, the affinity, specificity, and nature of which may promote, inhibit, or define the course of assembly. Despite the importance of the intermolecular interactions that initiate amyloid assembly, our understanding of these events remains poor. Here, using amyloidogenic and nonamyloidogenic variants of β2-microglobulin, we identify the interactions that inhibit or promote fibril formation in atomic detail. The results reveal that different outcomes of assembly result from biomolecular interactions involving similar surfaces. Specifically, inhibition occurs via rigid body docking of monomers in a head-to-head orientation to form kinetically trapped dimers. By contrast, the promotion of fibrillation involves relatively weak protein association in a similar orientation, which results in conformational changes in the initially nonfibrillogenic partner. The results highlight the complexity of interactions early in amyloid assembly and reveal atomic-level information about species barriers in amyloid formation.

Prion diseases are fatal neurodegenerative disorders that affect humans and animals and for which no pharmacological treatment is available. Compounds consisting of two identical moieties joined via an appropriate spacer (i.e. bivalent compounds) have turned out to be effective tools to prevent prion fibril formation and exhibit an improved biological profile with regard to the corresponding monovalent derivatives. In this review we discuss the importance of the bivalent strategy as a viable approach to design new chemical entities to combat prion diseases.

This review will focus on the process of amyloid-type protein aggregation. Amyloid fibrils are an important hallmark of protein misfolding diseases and therefore have been investigated for decades. Only recently, however, atomic or near-atomic resolution structures have been elucidated from various in vitro and ex vivo obtained fibrils. In parallel, the process of fibril formation has been studied in vitro under highly artificial but comparatively reproducible conditions. The review starts with a summary of what is known and speculated from artificial in vitro amyloid-type protein aggregation experiments. A partially hypothetic fibril selection model will be described that may be suitable to explain why amyloid fibrils look the way they do, in particular, why at least all so far reported high resolution cryo-electron microscopy obtained fibril structures are in register, parallel, cross-β-sheet fibrils that mostly consist of two protofilaments twisted around each other. An intrinsic feature of the model is the prion-like nature of all amyloid assemblies. Transferring the model from the in vitro point of view to the in vivo situation is not straightforward, highly hypothetic, and leaves many open questions that need to be addressed in the future.

1. Thermal inactivation of insulin (in particular, fibril formation) does not change the reactivity of the guanidine group of insulin in the Sakaguchi reaction. 2. Sparingly soluble protein samples, in particular, thermally inactivated insulin, can be analyzed for their content of guanidine groups according to the Sakaguchi reaction, by dissolving the sample in the form of the copper biuret complex.

A number of intrinsically disordered proteins, including alpha-synuclein and beta-amyloid, are known to form amyloids in neurodegenerative diseases such as Parkison's and Alzheimer's diseases, respectively, indicating generic behaviour of this class of proteins. The accompanying large conformational transition, from disordered to a beta-sheet, makes it difficult and extremely time-consuming to study the aggregation process by standard simulation methods. We have developed a Lambda Dynamics technique in which the coarse-grained particles, representing sequences of consecutive amino acids, respond to their environment by changing shape and interaction properties [1]. The evolving states of the particles are determined by internal and external interactions. The translational and rotational motion of the anisotropic particles are simulated with a newly developed concise Brownian Dynamics algorithm [2,3]. We present results on the aggregation of solvated spherical, disordered proteins into fibrils of elongated, beta-sheet forming proteins.[1] I.M. Ilie, W.K. den Otter and W.J. Briels, in preparation[2] I.M. Ilie, W.J. Briels and W.K. den Otter, in preparation[3] I.M. Ilie, W.K. den Otter and W.J. Briels, J. Chem. Phys. 141, 065101 (2014)

α-synuclein (α-syn) is the central protein in the etiology of Parkinson's diseases. The development of this disorder has indeed been associated with the formation of insoluble α-syn amyloid fibrils. When isolated in solution, α-syn behaves an intrinsically disordered protein, however, in vivo monomeric α-syn exists in equilibrium between free and membrane-bound states, the latter featuring an increased level of alpha-helix structure. α-syn binding to lipid membranes is a key process with functional relevance in synaptic regulation. This interaction also impacts α-syn fibril formation, i.e. from the inhibition to acceleration. Understanding the molecular basis of α-syn/membrane interaction is therefore a top current challenge. We here adopt advanced techniques of nuclear magnetic resonance to dissect the underlying principles governing this binding and its regulation under physiological conditions.

Huntington's disease is associated with an expanded polyglutamine (polyQ) tract in huntingtin. The relevant aggregation prone toxic species encompasses an N-terminal 17-residue amphipathic stretch (N17), the polyQ tract, and a C-terminal 38-residue proline-rich stretch (C38). Experiments have shown that C38 decreases the overall driving force to form aggregates, whereas N17 decreases the overall solubility and accelerates fibril formation by destabilizing non-fibrillar species. We present results from multiscale simulations to explain how N17 and C38 accomplish their distinct modulatory behaviors. Our inverse Boltzmann procedure yields a systematic coarse-graining method whereby we bootstrap against atomistic simulations of monomers and reversible associations for pairs of molecules. We demonstrate that the species distributions and kinetics of forming early-stage aggregates are governed by the relative favorability of intermolecular interactions between flanking sequence regions and the polyQ tract. Specifically, the two flanking sequences reduce the favorable polyQ interaction surface and hence reduce the frequency of intermolecular interactions between and entanglements of the polyQ tract, thus slowing aggregation kinetics regardless of the molecular architecture at the monomer level. The latter affects the morphologies and number of aggregates that form during early aggregation events. Overall, we show that N17 and C38 diminish the formation of large, polydisperse early-stage aggregates that are intrinsic to polyQ. Our results also show how polyQ expansions can override the effects of flanking sequences. Taken together with cellular data, we propose that flanking sequences help diminish the formation of toxic species and this functionality is overwhelmed by mutational expansion of polyQ tracts. We use the conceptual framework of Janus colloids to propose a connection between our results and other aggregation prone systems because they can be partitioned into cores that drive aggregation and contexts that control the aggregation process.