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Professional secretory cells can produce large amounts of high-quality complex molecules, including IgM antibodies. Owing to their multivalency, polymeric IgM antibodies provide an efficient first-line of defense against pathogens. To decipher the mechanisms of IgM assembly, we investigated its biosynthesis in living cells and faithfully reconstituted the underlying processes in vitro. We find that a conserved peptide extension at the C-terminal end of the IgM heavy (Ig-μ) chains, termed the tailpiece, is necessary and sufficient to establish the correct geometry. Alanine scanning revealed that hydrophobic amino acids in the first half of the tailpiece contain essential information for generating the correct topology. Assembly is triggered by the formation of a disulfide bond linking two tailpieces. This induces conformational changes in the tailpiece and the adjacent domain, which drive further polymerization. Thus, the biogenesis of large and topologically challenging IgM complexes is dictated by a local conformational switch in a peptide extension., Competing Interests: The authors declare no conflict of interest.

Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC.

Single point mutations in the Alzheimer's disease associated Aβ42 peptide are found to alter significantly its neurotoxic properties in vivo and have been associated with early onset forms of this devastating condition. We show that such mutations can induce structural changes in Aβ42 fibrils and are associated with a dramatic switch in the fibril-dependent mechanism by which Aβ42 aggregates. These observations reveal how subtle perturbations to the physicochemical properties of the Aβ peptide, and the structural properties of fibrils that it forms, can have profound effects on the mechanism of its aggregation and pathogenicity.

In recent years an increasing number of neurodegenerative diseases has been linked to the misfolding of a specific protein and its subsequent accumulation into aggregated species, often toxic to the cell. Of all the factors that affect the behavior of these proteins, disulfide bonds are likely to be important, being very conserved in protein sequences and being the enzymes devoted to their formation among the most conserved machineries in mammals. Their crucial role in the folding and in the function of a big fraction of the human proteome is well established. The role of disulfide bonding in preventing and managing protein misfolding and aggregation is currently under investigation. New insights into their involvement in neurodegenerative diseases, their effect on the process of protein misfolding and aggregation, and into the role of the cellular machineries devoted to disulfide bond formation in neurodegenerative diseases are emerging. These studies mark a step forward in the comprehension of the biological base of neurodegenerative disorders and highlight the numerous questions that still remain open.

An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils reveals that this structural form is likely to be the most stable one that protein molecules can adopt even under physiological conditions. This result challenges the conventional assumption that functional forms of proteins correspond to the global minima in their free energy surfaces and suggests that living systems are conformationally as well as chemically metastable.

The propensity of protein molecules to self-assemble into highly ordered, fibrillar aggregates lies at the heart of understanding many disorders ranging from Alzheimer's disease to systemic lysozyme amyloidosis. In this paper we use highly accurate kinetic measurements of amyloid fibril growth in combination with spectroscopic tools to quantify the effect of modifications in solution conditions and in the amino acid sequence of human lysozyme on its propensity to form amyloid fibrils under acidic conditions. We elucidate and quantify the correlation between the rate of amyloid growth and the population of nonnative states, and we show that changes in amyloidogenicity are almost entirely due to alterations in the stability of the native state, while other regions of the global free-energy surface remain largely unmodified. These results provide insight into the complex dynamics of a macromolecule on a multidimensional energy landscape and point the way for a better understanding of amyloid diseases.

The partial unfolding of human lysozyme underlies its conversion from the soluble state into amyloid fibrils observed in a fatal hereditary form of systemic amyloidosis. To understand the molecular origins of the disease, it is critical to characterize the structural and physicochemical properties of the amyloidogenic states of the protein. Here we provide a high-resolution view of the unfolding process at low pH for three different lysozyme variants, the wild-type protein and the mutants I56T and I59T, which show variable stabilities and propensities to aggregate in vitro. Using a range of biophysical techniques that includes differential scanning calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate that thermal unfolding under amyloidogenic solution conditions involves a cooperative loss of native tertiary structure, followed by progressive unfolding of a compact, molten globule-like denatured state ensemble as the temperature is increased. The width of the temperature window over which the denatured ensemble progressively unfolds correlates with the relative amyloidogenicity and stability of these variants, and the region of lysozyme that unfolds first maps to that which forms the core of the amyloid fibrils formed under similar conditions. Together, these results present a coherent picture at atomic resolution of the initial events underlying amyloid formation by a globular protein.

Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-β core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The aggregation process of wild-type human lysozyme at pH3.0 and 60 degrees C has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transform infrared spectroscopy, and controlled proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8-17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solvent-exposed hydrophobic patches able to bind the fluorescent probe 1-anilino-naphthalene-8-sulfonate. Fourier transform infrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the beta-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes.

The conversion of specific proteins or protein fragments into insoluble, ordered fibrillar aggregates is a fundamental process in protein chemistry, biology, medicine and biotechnology. As this structural conversion seems to be a property shared by many proteins, understanding the mechanism of this process will be of extreme importance. Here we present a structural characterisation of a conformational state populated at low pH by the N-terminal domain of Escherichia coli HypF. Combining different biophysical and biochemical techniques, including near- and far-UV circular dichroism, intrinsic and 8-anilinonaphthalene-1-sulfonate-derived fluorescence, dynamic light scattering and limited proteolysis, we will show that this state is largely unfolded but contains significant secondary structure and hydrophobic clusters. It also appears to be more compact than a random coil-like state but less organised than a molten globule state. Increase of the total ionic strength of the solution induces aggregation of such a pre-molten globule state into amyloid-like protofibrils, as revealed by thioflavin T fluorescence and atomic force microscopy. These results show that a pre-molten globule state can be, among other possible conformational states, one of the precursor states of amyloid formation. In addition, the possibility of triggering aggregation by modulating the ionic strength of the solution provides one a unique opportunity to study both the initial precursor state and the aggregation process.

Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be induced to form amyloid fibrils when incubated under appropriate conditions. In this study, fibrils of wild-type human lysozyme formed at low pH have been analyzed by a combination of limited proteolysis and Fourier-transform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates. After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a significant minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive beta-sheet structure and a substantial element of non beta-sheet or random structure that is reduced significantly in the fibrils after digestion. The sequence 32-108 includes the beta-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme. The present proteolytic data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain. The majority of amyloid fibrils formed from lysozyme under the conditions used here contain a core structure involving some 50% of the polypeptide chain that is flanked by proteolytically accessible N and C-terminal regions.

Alpha-lactalbumin (LA) in its molten globule (MG) state at low pH forms amyloid fibrils. Here, we have studied the aggregation propensities of LA derivatives characterized by a single peptide bond fission (1-40/41-123, named Th1-LA) or a deletion of a chain segment of 12 amino acid residues located at the level of the beta-subdomain of the native protein (1-40/53-123, named desbeta-LA). We have also compared the early stages of the aggregation process of these LA derivatives with those of intact LA. Th1-LA and desbeta-LA aggregate at pH 2.0 much faster than the intact protein and form long and well-ordered fibrils. Furthermore, in contrast to intact LA, the LA derivatives form regular fibrils also at neutral pH, even if at much reduced rate. In acidic solution, Th1-LA and desbeta-LA adopt a MG state which appears to be similar to that of intact LA, as given by spectroscopic criteria. At neutral pH, both Th1-LA and desbeta-LA are able to bind the hydrophobic dye 1-anilinonaphtalene-8-sulfonate, thus indicating the presence of exposed hydrophobic patches. It is concluded that nicked Th1-LA and gapped desbeta-LA are more relaxed and expanded than intact LA and, consequently, that they are more suitable protein species to allow the large conformational transitions required for the polypeptide chain to form the amyloid cross-beta structure. As a matter of fact, the MG of LA attains an even more flexible conformational state during the early phases of the aggregation process at acidic pH, as deduced from the enhancement of its susceptibility to proteolysis by pepsin. Our data indicate that deletion of the beta-subdomain in LA does not alter the ability of the protein to assemble into well-ordered fibrils, implying that this chain region is not essential for the amyloid formation. It is proposed that a proteolytic hydrolysis of a protein molecule at the cellular level can trigger an easier formation of amyloid precipitates and therefore that limited proteolysis of proteins can be a causative mechanism of protein aggregation and fibrillogenesis. Indeed, a vast majority of protein deposits in amyloid diseases are given by protein fragments derived from larger protein precursors.