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Soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against enzymes may behave as potent inhibitors. The immunization of alpacas with the CMY-2 β-lactamase led to the isolation of three VHHs that specifically recognized and inhibited CMY-2. The structure of the complex VHH cAbCMY-2(254)/CMY-2 was determined by X-ray crystallography. We showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes in the catalytic site. The β-lactamase inhibition was found to follow a mixed profile with a predominant non-competitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binder. Our study identified a binding site that can be targeted by a new class of β-lactamase’s inhibitors designed with the help of a peptidomimetic approach. Furthermore, the use of mono or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enable the development of the first generation of ELISA test for the detection of CMY-2 produced by resistant bacteria.IMPORTANCEThe still increasing antimicrobial resistance in human clinic or veterinary medicine is a major threat for modern chemotherapy. Beside the major caution in the use of current antibiotics, it is important to develop new classes of antibiotics. This work was focused on β-lactamases that are the enzymes involved in the hydrolysis of the major class of antibiotics, the β-lactam compounds. We selected camelid antibodies that inhibit CMY-2, a class C β-lactamase produced by bacteria isolated from the veterinary and human settings. We characterized the conformational epitope present in CMY-2 in order to create a new family of inhibitors based on the paratope of the antibody. Finally, we designed a primary version of a detection system based on an ELISA using VHH and polyclonal antibodies.

I have been invited to summarize my career with an emphasis on the time I spent in the laboratory of Prof Christopher M. Dobson, who sadly passed away on September 8th 2019, and to describe his role as a mentor. I accepted this slightly unusual request as it constitutes a unique way for me to express my deep gratitude and admiration for Chris.

Conjugation to polyethylene glycol (PEG) is a widely used approach to improve the therapeutic value of proteins essentially by prolonging their body residence time. PEGylation may however induce changes in the structure and/or the stability of proteins and thus on their function(s). The effects of PEGylation on the thermodynamic stability can either be positive (stabilization), negative (destabilization), or neutral (no effect). Moreover, various factors such as the PEG length and PEGylation site can influence the consequences of PEGylation on the structure and stability of proteins. In this study, the effects of PEGylation on the structure, stability, and polymerization of alpha1-antitrypsin (AAT) were investigated, using PEGs with different lengths, different structures (linear or 2-armed) and different linking chemistries (via amine or thiol) at two distinct positions of the sequence. The results show that whatever the size, position, and structure of PEG chains, PEGylation (a) does not induce significant changes in AAT structure (either at the secondary or tertiary level); (b) does not alter the stability of the native protein upon both chemical- and heat-induced denaturation; and (c) does not prevent AAT to fully refold and recover its activity following chemical denaturation. However, the propensity of AAT to aggregate upon heat treatment was significantly decreased by PEGylation, although PEGylation did not prevent the irreversible inactivation of the enzyme. Moreover, conjugation to PEG, especially 2-armed 40 kDa PEG, greatly improved the proteolytic resistance of AAT. PEGylation of AAT could be a promising strategy to prolong its half-life after infusion in AAT-deficient patients and thereby decrease the frequency of infusions.

Background Antibody-based therapies targeting CD38 are currently used as single agents as well as in combination regimens for multiple myeloma, a malignant plasma cell disorder. In this study, we aimed to develop anti-CD38 single-domain antibodies (sdAbs) that can be used to trace CD38+ tumour cells and subsequently used for targeted radionuclide therapy. SdAbs are derived from Camelidae heavy-chain antibodies and have emerged as promising theranostic agents due to their favourable pharmacological properties. Methods Four different anti-CD38 sdAbs were produced, and their binding affinities and potential competition with the monoclonal antibody daratumumab were tested using biolayer interferometry. Their binding kinetics and potential cell internalisation were further studied after radiolabelling with the diagnostic radioisotope Indium-111. The resulting radiotracers were evaluated in vivo for their tumour-targeting potential and biodistribution through single-photon emission computed tomography (SPECT/CT) imaging and serial dissections. Finally, therapeutic efficacy of a lead anti-CD38 sdAb, radiolabelled with the therapeutic radioisotope Lutetium-177, was evaluated in a CD38+ MM xenograft model. Results We retained anti-CD38 sdAb #2F8 as lead based on its excellent affinity and superior stability, the absence of competition with daratumumab and the lack of receptor-mediated internalisation. When intravenously administered to tumour-xenografted mice, radiolabelled sdAb #2F8 revealed specific and sustained tumour retention with low accumulation in other tissues, except kidneys, resulting in high tumour-to-normal tissue ratios. In a therapeutic setting, myeloma-bearing mice received three consecutive intravenous administrations of a high (18.5 MBq) or a low radioactive dose (9.3 MBq) of 177Lu-DTPA-2F8 or an equal volume of vehicle solution. A dose-dependent tumour regression was observed, which translated into a prolonged median survival from 43 days for vehicle-treated mice, to 62 days (p = 0.027) in mice receiving the low and 65 days in mice receiving the high (p = 0.0007) radioactive dose regimen, respectively. Conclusions These results highlight the theranostic potential of radiolabelled anti-CD38 sdAbs for the monitoring and treatment of multiple myeloma.

Herein, we compared the ability of linear and cyclic peptides generated in silico to target different protein sites: internal pockets and solvent-exposed sites. We selected human lysozyme (HuL) as a model target protein combined with the computational evolution of linear and cyclic peptides. The sequence evolution of these peptides was based on the PARCE algorithm. The generated peptides were screened based on their aqueous solubility and HuL binding affinity. The latter was evaluated by means of scoring functions and atomistic molecular dynamics (MD) trajectories in water, which allowed prediction of the structural features of the protein-peptide complexes. The computational results demonstrated that cyclic peptides constitute the optimal choice for solvent exposed sites, while both linear and cyclic peptides are capable of targeting the HuL pocket effectively. The most promising binders found in silico were investigated experimentally by surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS) techniques. All tested peptides displayed dissociation constants in the micromolar range, as assessed by SPR; however, both NMR and ESI-MS suggested multiple binding modes, at least for the pocket binding peptides. A detailed NMR analysis confirmed that both linear and cyclic pocket peptides correctly target the binding site they were designed for.

The accumulation in vital organs of amyloid fibrils made of mutational variants of lysozyme (HuL) is associated with a human systemic amyloid disease. The detailed comparison of the in vitro properties of the I56T and D67H amyloidogenic variants to those of the T70N non-amyloidogenic variant and the wild-type (WT) protein suggested that the deposition of large amounts of aggregated disease-related lysozyme variants is initiated by the formation of transient intermediate species. The ability to populate such intermediates is essentially due to the destabilisation of the protein and the loss of the global structural cooperativity under physiologically relevant conditions. Here, we report the characterisation of a third naturally occurring amyloidogenic lysozyme variant, W64R, in comparison with the I56T and WT proteins. The X-ray crystal structure of the W64R variant at 1.15 Å resolution is very similar to that of the WT protein; a few interactions within the β-domain and at the interface between the α- and β-domains differ, however, from those in the WT protein. Consequently, the W64R mutation destabilizes the protein to an extent that is similar to that observed for the I56T and D67H mutations. The ΔG°

Parkinson's disease (PD) is a frequent degenerative disorder that is diagnosed based on clinical symptoms. When the first symptoms appear, more than 70% of the dopaminergic cells are already lost. Therefore, it is of utmost importance to have reliable biomarkers to diagnose much earlier PD. In this context, alpha-synuclein (aSyn) is a protein of high interest because of its tendency to form oligomers and amyloid fibrils. The oligomeric forms seem to play a critical pathological role in PD. To date, most of studies aiming at detecting and quantifying aSyn oligomers were performed by immunoassays, mainly by ELISA using specific antibodies. In this study a capillary gel electrophoresis (CGE) coupled with fluorescence detection method was developed to detect and quantify the oligomeric forms of aSyn formed in vitro. All the results obtained were supported by SDS-PAGE analysis, a widely used and well-known technique but exhibiting a main drawback since it is not an automated technique. The repeatability and the intermediate precision of the method were evaluated, as well as the stability of the labeled and non-labeled aSyn samples. After careful screening and optimization of various labeling reagents, 4-fluoro-7-nitrobenzofurazan (NBD-F) was selected and used to establish a calibration curve with monomeric fluorescently-labeled aSyn. Finally, the method was used to study the effect of doxycycline on the oligomerization process. Altogether, our results show that CGE is a very promising automated technique to analyze aSyn monomers, as well as small oligomers.

International audience; Heterogeneity and polymorphism are generic features of amyloid fibers with some important effects on the related disease development. We report here the characterization, by charge detection mass spectrometry, of amyloid fibers made of three polypeptides involved in neurodegenerative diseases: Aβ1–42 peptide, tau and α-synuclein. Beside the mass of individual fibers, this technique enables to characterize the heterogeneity and the polymorphism of the population. In the case of Aβ1–42 peptide and tau protein, several coexisting species could be distinguished and characterized. In the case of α-synuclein, we show how the polymorphism affects the mass and charge distributions.

Background Theranostic agents include molecules with a combined diagnostic and therapeutic capability and nanobodies (Nbs) are increasingly used in this field. Nbs are single-domain antigen-binding fragments that are derived from Camelidae heavy-chain antibodies and have several advantages such as their small size leading to better tissue penetration, their favourable pharmacological properties and their ability to recognise small, buried epitopes. Methods The current project aims to use nanobodies directed against CD38 as diagnostic and therapeutic tools in the management of multiple myeloma (MM) disease. The differentiation marker CD38 is highly expressed on myeloma cells isolated from patients with a newly diagnosed or a relapsing disease. Our Nb, retained for its affinity, stability, its favorable biodistribution and its capacity to recognize myeloma cells in a xenograft model of MM, was coupled to Indium-111 and Lutetium-177 and intravenously injected in tumor bearing mice to assess its diagnostic and therapeutic value. Tumor development was assessed by bioluminescence. Results The binding capacities of 111In-DTPA-Nb2F8 was verified by saturation binding experiments (using serial dilutions of the conjugated Nb) on CD38+ RPMI-8226 cells and internalisation experiments were performed in order to assess their therapeutic potential. A minor internalisation (about 20% of the initial bound activity) was observed in the first hours and remained stable during 24H. Micro-SPECT/CT images of mice bearing CD38+ RPMI-8226 tumors and injected with 111In-DPTA-Nb2F8 showed specific tumor targeting 1 hour and at least until 48 hours after injection with a low background signal already 1 hour post-injection (p.i.), except kidneys. The in and ex vivo biodistribution data revealed uptake values in tumor of 3.1% IA/g at 1h post injection, while the uptake of a labelled control Nb was low 0.54% IA/g confirming the specific targeting of our anti-CD38 Nb. Similar biodistributions were found with 177Lu-DTPA-Nb2F8. In order to assess the therapeutic potential of this nanobody, mice with subcutaneously injected RPMI-8226 cells were randomly categorised into 3 groups (n=10). Mice in each group received 3 consecutive intravenous administrations of a high (17.5MBq) or a low radioactive dose (8.6MBq) 177Lu-DTPA-2F8 or an equal volume of vehicle solution. A dose-dependent regression was observed for both therapeutic regimens, which translated in a prolonged median survival from 43 days for vehicle-treated mice, compared to 62 days (p=0.027) for the low and 65 days for the high (p=0.0007) radioactive dose regimen respectively. Conclusions In conclusion, this is the first report on CD38-binding Nbs used for the identification and treatment of MM cells by conjugating them to diagnostic and therapeutic isotopes.

Recent advances in transcriptome sequencing and analysis have revealed the complexity of the human genome. The majority (≈ 98%) of cellular transcripts is not translated into proteins and represents a vast, unchartered world of functional non-coding RNAs. Most of them adopt a well-defined three-dimensional structure to achieve their biological functions. However, only very few RNA structures are currently available which reflects the challenges associated with RNA crystallization. Nevertheless, these structures would represent a critical step in understanding functions of non-coding RNAs and their molecular mechanisms in the cell. The overall goal of this study is to develop an innovative and versatile tool to facilitate the functional study and crystallization of structured RNAs (stRNAs). In this work, we have engineered an antibody fragment from camelid heavy-chain antibody (nanobody) able to specifically bind with low nanomolar affinity to stRNA, while no binding could be detected for single-stranded DNA/RNA, double-stranded DNA/RNA or a negatively charged protein. However, this nanobody recognizes different and non-related stRNAs, this observation suggests that it binds to an epitope shared by these stRNAs. Finally, our data also show that the binding of the nanobody does not alter the secondary structure content of the stRNA as well as its unfolding/refolding processes during heat treatment. This work constitutes a successful proof of concept demonstrating that nanobodies can be engineered to recognize RNA-related epitopes.

The deposition of misfolded peptides and proteins in the form of amyloid fibrils is the hallmark of nearly fifty medical disorders, including Alzheimer's disease, Parkinson's disease, prion diseases and type II diabetes. These disorders, referred to as amyloidoses, generally become apparent late in life. Their psycho-sociological and economic incidence in western societies will be therefore considerable in the coming decades due to the ageing of the population. Neither preventing nor curative treatments are available yet. These disorders constitute therefore a medical challenge of great importance. Thus, an extensive research is being carried out to understand, at the molecular level, (i) how amyloidogenic proteins misfold and convert from their soluble form into amyloid fibrils, and (ii) how these aggregates or some of their oligomeric precursor species are toxic. The formation of amyloid fibrils proceeds through a complex nucleation/polymerisation mechanism with the formation of various species, including small oligomers. In this review, we focus on how VHHs or nanobodies, the antigen-binding domains of camelid heavy-chain antibodies, are being increasingly used to characterise each of the species formed on the pathway of fibril formation in terms of structure, stability, kinetics of formation and toxicity. We first introduce the characteristic features of nanobodies compared to those of conventional antibody fragments. Thereafter, we discuss how nanobodies, due to their unique properties, are used as probes to dissect the molecular mechanisms of misfolding and aggregation of six proteins associated with diseases, i.e. human lysozyme, β2-microglobulin, α-synuclein, prion, polyadenylate binding protein nuclear 1 and amyloid β-peptide. A brief general presentation of each disease and the associated peptide/protein is also provided. In addition, we discuss how nanobodies could be used as early diagnostic tools and as novel strategies to treat diseases associated with protein misfolding and aggregation.

Background A series of human diseases are caused by the misfolding and aggregation of specific proteins or peptides into amyloid fibrils; nine of these diseases, referred to as polyglutamine diseases, are associated with proteins carrying an expanded polyglutamine (polyQ) region. While the presence of this latter is thought to be the determinant factor for the development of polyQ diseases, the non-polyQ regions of the host proteins are thought to play a significant modulating role. Method In order to better understand the role of non-polyQ regions, the toxic effects of model proteins bearing different polyQ regions (containing up to 79 residues) embedded at two distinct locations within the β-lactamase (BlaP) host enzyme were evaluated in Caenorhabditis elegans. This small organism can be advantageous for the validation of in vitro findings, as it provides a multicellular context yet avoids the typical complexity of common studies relying on vertebrate models. Several phenotypic assays were performed in order to screen for potential toxic effects of the different BlaP-polyQ proteins. Results Despite the significant in vitro aggregation of BlaP-polyQ proteins with long polyQ regions, none of the BlaP-polyQ chimeras aggregated in the generated transgenic in vivo models. Conclusion The absence of a toxic effect of the expression of BlaP-polyQ chimeras may find its cause in biochemical mechanisms present in vivo to cope with protein aggregation (e.g. presence of chaperones) or in C. elegans’ limitations such as its short lifespan. It is plausible that the aggregation propensities of the different BlaP chimeras containing embedded polyQ sequences are too low in this in vivo environment to permit their aggregation. These experiments emphasize the need for several comparative and in vivo verification studies of biologically relevant in vitro findings, which reveal both the strengths and limitations of widely used model systems. Electronic supplementary material The online version of this article (doi:10.1186/s12952-017-0080-5) contains supplementary material, which is available to authorized users.

Aim: Gadolinium-based nanoparticles were functionalized with either the Pittsburgh compound B or a nanobody (B10AP) in order to create multimodal tools for an early diagnosis of amyloidoses. Materials & methods: The ability of the functionalized nanoparticles to target amyloid fibrils made of β-amyloid peptide, amylin or Val30Met-mutated transthyretin formed in vitro or from pathological tissues was investigated by a range of spectroscopic and biophysics techniques including fluorescence microscopy. Results: Nanoparticles functionalized by both probes efficiently interacted with the three types of amyloid fibrils, with KD values in 10 micromolar and 10 nanomolar range for, respectively, Pittsburgh compound B and B10AP nanoparticles. Moreover, they allowed the detection of amyloid deposits on pathological tissues. Conclusion: Such functionalized nanoparticles could represent promising flexible and multimodal imaging tools for the early diagnostic of amyloid diseases, in other words, Alzheimer's disease, Type 2 diabetes mellitus and the familial amyloidotic polyneuropathy.

Class A β-lactamases have been widely used as versatile scaffolds to create hybrid (or chimeric) proteins for a series of applications ranging from basic research to medicine. We have, in particular, used the β-lactamase BlaP from Bacillus licheniformis 749/C (BlaP) as a protein scaffold to create model polyglutamine (polyQ) proteins in order to better understand the mechanism(s) by which an expanded polyQ sequence triggers the formation of amyloid fibrils. The model chimeras were designed by inserting a polyQ sequence of various lengths at two different locations within BlaP (i.e. position 197 or position 216) allowing a detailed comparison of the effects of subtle differences in the environment of the polyQ sequence on its ability to trigger protein aggregation. In order to investigate the effects of the polyQ insertion at both positions on the structure, stability and dynamics of BlaP, a series of NMR experiments including H/D exchange are foreseen. Accordingly, as necessitated by these studies, here we report the NMR assignment of the wild-type BlaP (BlaP-WT) and of the two reference proteins, BlaP197Q0 and BlaP216Q0, wherein a Pro-Gly dipeptide has been introduced at position 197 and 216, respectively; this dipeptide originates from the addition of the Sma1 restriction site at the genetic level to allow further polyQ sequence insertion.

Inflammation occurs in many amyloidoses, but its underlying mechanisms remain enigmatic. Here we show that amyloid fibrils of human lysozyme, which are associated with severe systemic amyloidoses, induce the secretion of pro-inflammatory cytokines through activation of the NLRP3 (NLR, pyrin domain containing 3) inflammasome and the Toll-like receptor 2, two innate immune receptors that may be involved in immune responses associated to amyloidoses. More importantly, our data clearly suggest that the induction of inflammatory responses by amyloid fibrils is linked to their intrinsic structure, because the monomeric form and a non-fibrillar type of lysozyme aggregates are both unable to trigger cytokine secretion. These lysozyme species lack the so-called cross-β structure, a characteristic structural motif common to all amyloid fibrils irrespective of their origin. Since fibrils of other bacterial and endogenous proteins have been shown to trigger immunological responses, our observations suggest that the cross-β structural signature might be recognized as a generic danger signal by the immune system.

We have studied the effects of the extracellular molecular chaperone, clusterin, on the in vitro aggregation of mutational variants of human lysozyme, including one associated with familial amyloid disease. The aggregation of the amyloidogenic variant I56T is inhibited significantly at clusterin to lysozyme ratios as low as 1:80 (i.e. one clusterin molecule per 80 lysozyme molecules). Experiments indicate that under the conditions where inhibition of aggregation occurs, clusterin does not bind detectably to the native or fibrillar states of lysozyme, or to the monomeric transient intermediate known to be a key species in the aggregation reaction. Rather, it seems to interact with oligomeric species that are present at low concentrations during the lag (nucleation) phase of the aggregation reaction. This behavior suggests that clusterin, and perhaps other extracellular chaperones, could have a key role in curtailing the potentially pathogenic effects of the misfolding and aggregation of proteins that, like lysozyme, are secreted into the extracellular environment.

Background Amyloidoses are characterized by the extracellular deposition of insoluble fibrillar proteinaceous aggregates highly organized into cross-β structure and referred to as amyloid fibrils. Nowadays, the diagnosis of these diseases remains tedious and involves multiple examinations while an early and accurate protein typing is crucial for the patients’ treatment. Routinely used neuroimaging techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) using Pittsburgh compound B, [11C]PIB, provide structural information and allow to assess the amyloid burden, respectively, but cannot discriminate between different amyloid deposits. Therefore, the availability of efficient multimodal imaging nanoparticles targeting specific amyloid fibrils would provide a minimally-invasive imaging tool useful for amyloidoses typing and early diagnosis. In the present study, we have functionalized gadolinium-based MRI nanoparticles (AGuIX) with peptides highly specific for Aβ amyloid fibrils, LPFFD and KLVFF. The capacity of such nanoparticles grafted with peptide to discriminate among different amyloid proteins, was tested with Aβ(1–42) fibrils and with mutated-(V30M) transthyretin (TTR) fibrils. Results The results of surface plasmon resonance studies showed that both functionalized nanoparticles interact with Aβ(1–42) fibrils with equilibrium dissociation constant (Kd) values of 403 and 350 µM respectively, whilst they did not interact with V30M-TTR fibrils. Similar experiments, performed with PIB, displayed an interaction both with Aβ(1–42) fibrils and V30M-TTR fibrils, with Kd values of 6 and 10 µM respectively, confirming this agent as a general amyloid fibril marker. Thereafter, the ability of functionalized nanoparticle to target and bind selectively Aβ aggregates was further investigated by immunohistochemistry on AD like-neuropathology brain tissue. Pictures clearly indicated that KLVFF-grafted or LPFFD-grafted to AGuIX nanoparticle recognized and bound the Aβ amyloid plaque localized in the mouse hippocampus. Conclusion These results constitute a first step for considering these functionalized nanoparticles as a valuable multimodal imaging tool to selectively discriminate and diagnose amyloidoses. Electronic supplementary material The online version of this article (doi:10.1186/s12951-016-0212-y) contains supplementary material, which is available to authorized users.

Metallo-β-lactamases catalyse the hydrolysis of most β-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus β-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (∆G°) of 32 ± 2 kJ·mol^−1 . For holoBcII, a first noncooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site and the protein displays a well-organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. 2D NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ∆G° value of 65 ± 1.4 kJ·mol^−1 . These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well-defined conformation for both active site loops in order to maintain enzymatic activity.

The conversion of human lysozyme into amyloid fibrils is associated with a rare but fatal hereditary form of nonneuropathic systemic amyloidosis. The accumulation of large amounts of aggregated protein is thought to be initiated by the formation of transient intermediate species of disease-related lysozyme variants, essentially due to the loss of global cooperativity under physiologically relevant conditions. Interestingly, all five naturally occurring, amyloidogenic, single-point mutations are located in the β-domain of lysozyme, the region that is predominantly unfolded during the formation of the transient intermediate species. Given the lack of known naturally occurring, amyloidogenic, single-point mutations in the α-domain, we chose three specific mutations to address the effects that location may have on native-state dynamics, as studied by hydrogen-deuterium (HD) exchange experiments analyzed by NMR spectroscopy, and mass spectrometry. We compared the effect of a destabilizing α-domain mutation (I23A) with that of the well-characterized I59T β-domain variant. We also investigated the effect of a mutation that has minor effects on native-state stability at the domain interface (I56V) and compared it with that of a variant with similar stability within the C-helix (I89V). We show that when variants have similar reduced native-state stabilities, the location of the mutation (I23A versus I59T) is crucial to the native-state dynamics, with the α-domain mutation having a significantly lower ability to populate transient intermediate species under physiologically relevant conditions. Interestingly, the mutation at the interface (I56V) has a greater effect in facilitating the formation of transient intermediate species at elevated temperatures compared with the variants containing α-domain mutations, even though this mutation results in only minor changes to the native-state stability of lysozyme. These findings reveal that the location of specific mutations is an important factor in determining the native-state dynamical properties of human lysozyme in the context of its propensity to populate the aggregation-prone transient intermediate species associated with pathogenic amyloid formation.

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.-Kumita, J. R., Helmfors, L., Williams, J., Luheshi, L. M., Menzer, L., Dumoulin, M., Lomas, D. A., Crowther, D. C., Dobson, C. M., Brorsson, A.-C. Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster. funding agencies|Swedish Research Council||Biotechnology and Biological Sciences Research Council| BB/E019927/1 BBH0038431 |Wellcome Trust||Leverhulme Trust||European Commission| LSHM-CT-2006-037525/EURAMY |Medical Research Council| G0700990 |VINNOVA

Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from diseases (such as Alzheimer's and Parkinson's diseases) to the production (e.g. inclusion bodies), stability, storage and delivery of protein drugs. β-Cyclodextrin (β-CD) is a circular heptasaccharide characterized by a hydrophilic exterior and a hydrophobic interior ring structure. In this research, we studied the effects of a chemically modified β-CD (BCD07056), on the aggregating and refolding properties of BlaPChBD, a hybrid protein obtained by inserting the chitin binding domain of the human macrophage chitotriosidase into the class A β-lactamase BlaP from Bacillus licheniformis 749/I during its thermal denaturation. The results show that BCD07056 strongly increases the refolding yield of BlaPChBD after thermal denaturation and constitutes an excellent additive to stabilize the protein over time at room temperature. Our data suggest that BCD07056 acts early in the denaturation process by preventing the formation of an intermediate which leads to an aggregated state. Finally, the role of β-CD derivatives on the stability of proteins is discussed.

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.

The aggregation of the intrinsically disordered protein alpha-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's disease. The molecular mechanism of alpha-synuclein aggregation and toxicity is not yet understood in any detail, not least because of the paucity of structural probes through which to study the behavior of such a disordered system. Here, we describe an investigation involving a single-domain camelid antibody, NbSyn2, selected by phage display techniques to bind to alpha-synuclein, including the exploration of its effects on the in vitro aggregation of the protein under a variety of conditions. We show using isothermal calorimetric methods that NbSyn2 binds specifically to monomeric alpha-synuclein with nanomolar affinity and by means of NMR spectroscopy that it interacts with the four C-terminal residues of the protein. This latter finding is confirmed by the determination of a crystal structure of NbSyn2 boundto a peptide encompassing the nine C-terminal residues of alpha-synuclein. The NbSyn2:alpha-synuclein interaction is mediated mainly by side-chain interactions while water molecules cross-link the main-chain atoms of alpha-synuclein to atoms of NbSyn2, a feature we believe could be important in intrinsically disordered protein interactions more generally. The aggregation behavior of alpha-synuclein at physiological pH, including the morphology of the resulting fibrillar structures, is remarkably unaffected by the presence of NbSyn2 and indeed we show that NbSyn2 binds strongly to the aggregated as well as to the soluble forms of alpha-synuclein. These results give strong support to the conjecture that the C-terminal region of the protein is not directly involved in the mechanism of aggregation and suggest that binding of NbSyn2 could be a useful probe for the identification of alpha-synuclein aggregation in vitro and possibly in vivo.

Using genetic engineering technologies, the chitin-binding domain (ChBD) of the human macrophage chitotriosidase has been inserted into the host protein BlaP, a class A beta-lactamase produced by Bacillus licheniformis. The product of this construction behaved as a soluble chimeric protein that conserves both the capacity to bind chitin and to hydrolyze beta-lactam moiety. Here we describe the biochemical and biophysical properties of this protein (BlaPChBD). This work contributes to a better understanding of the reciprocal structural and functional effects of the insertion on the host protein scaffold and the heterologous structured protein fragments. The use of BlaP as a protein carrier represents an efficient approach to the functional study of heterologous protein fragments.

Studies of lysozyme have played a major role over several decades in defining the general principles underlying protein structure, folding, and stability. Following the discovery some 10 years ago that two mutational variants of lysozyme are associated with systemic amyloidosis, these studies have been extended to investigate the mechanism of amyloid fibril formation. This Account describes our present knowledge of lysozyme folding and misfolding, and how the latter can give rise to amyloid disease. It also discusses the significance of these studies for our general understanding of normal and aberrant protein folding in the context of human health and disease.

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5A. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70-74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the beta-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the alpha-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the beta-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo.

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data that permit a detailed comparison to be made of the behaviour of two of these amyloidogenic variants, I56T and D67H, under identical conditions. Hydrogen/deuterium exchange experiments monitored by NMR and mass spectrometry reveal that, despite their different locations and the different effects of the two mutations on the structure of the native state of lysozyme, both mutations cause a cooperative destabilisation of a remarkably similar segment of the structure, comprising in both cases the beta-domain and the adjacent C-helix. As a result, both variant proteins populate transiently a closely similar, partially unstructured intermediate in which the beta-domain and the adjacent C-helix are substantially and simultaneously unfolded, whereas the three remaining alpha-helices that form the core of the alpha-domain still have their native-like structure. We show, in addition, that the binding of a camel antibody fragment, cAb-HuL6, which was raised against wild-type lysozyme, restores to both variant proteins the stability and cooperativity characteristic of the wild-type protein; as a consequence, it inhibits the formation of amyloid fibrils by both variants. These results indicate that the reduction in global cooperativity, and the associated ability to populate transiently a specific, partly unfolded intermediate state under physiologically relevant conditions, is a common feature underlying the behaviour of these two pathogenic mutations. The formation of intermolecular interactions between lysozyme molecules that are in this partially unfolded state is therefore likely to be the fundamental trigger of the aggregation process that ultimately leads to the formation and deposition in tissue of amyloid fibrils.

Summary Background The ability of an intact protein to reach the circulatory system may be a prerequisite toallergenicity and many allergens, particularly those from plant foods, have been found to beconsistently more resistant to digestion by pepsin than other proteins.Objective This study assessed the pepsinolytic stability of native 2S albumins from Brazil nut andsunflower seed and their recombinant versions produced in Pichia pastoris. The physicochemicalstability of native and recombinant Brazil nut 2S albumins and recombinant sunflower seed 2Salbumin was also assessed. The immunoreactivity of native Brazil nut 2S albumin and recombinant2S albumins was compared using serum from patients allergic to Brazil nuts and animals immunizedwith native 2S albumins.Methods Digestibility was measured in simulated gastric fluid followed by SDS-PAGE. Circulardichroism spectra were used to analyse unfolding, as proteins were denatured by temperature, pHand guanidinium chloride. Immunoreactivity was assessed by immunoblot, RAST and ELISA.Results Brazil nut 2S albumin was significantly more resistant to proteolytic digestion than otherBrazil nut proteins. It was also resistant to thermally and chemically induced denaturation. Equallyhigh resistance to proteolytic digestion was observed with sunflower seed 2S albumin. Therecombinant albumins mirrored their native counterparts in stability and immunoreactivity.Conclusion The important food allergen Brazil nut 2S albumin is as stable to digestion as issunflower seed 2S albumin, whose allergenicity has yet to be determined. The 2S albumins and theirrecombinant counterparts could not be easily denatured by physicochemical treatments. The resultssuggest that 2S albumin is the only Brazil nut protein to reach the gut immune system intact. Theproduction of properly folded recombinant proteins will facilitate mechanistic studies as well asdiagnostic testing and antigen-based therapies.Keywords allergy, Brazil nut, recombinant allergens, 2S albumins, stability, structure, sunflowerSubmitted 28 November 2002; revised 19 February 2003; accepted 13 May 2003

Polyglutamine (polyQ) diseases, including Huntington's disease, are neurodegenerative disorders associated with the abnormal expansion of a polyQ tract within nine proteins. The polyQ expansion is thought to be a major determinant in the development of neurotoxicity, triggering protein aggregation into amyloid fibrils, although non-polyQ regions play a modulating role. In this work, we investigate the relative importance of the polyQ length, its location within a host protein, and the conformational state of the latter in the amyloid fibril elongation. Model polyQ proteins made of the β-lactamase BlaP containing up to 79Q inserted at two different positions, and quartz crystal microbalance and atomic force microscopy were used for this purpose. We demonstrate that, independently of the polyQ tract location and the conformational state of the host protein, the relative elongation rate of fibrils increases linearly with the polyQ length. The slope of the linear fit is similar for both sets of chimeras (i.e., the elongation rate increases by ~ 1.9% for each additional glutamine), and is also similar to that previously observed for polyQ peptides. The elongation rate is, however, strongly influenced by the location of the polyQ tract within BlaP and the conformational state of BlaP. Moreover, comparison of our results with those previously reported for aggregation in solution indicates that these two parameters also modulate the ability of BlaP-polyQ chimeras to form the aggregation nucleus. Altogether our results suggest that non-polyQ regions are valuable targets in order to interfere with the process of amyloid fibril formation associated with polyQ diseases.

High-pressure-induced gelation of ovalbumin, egg yolk and soy protein was studied by pressurization up to 500 MPa for 30 min at -20 to 50°C; hardness, cohesiveness and gumminess of the gels were compared to those of gels obtained by heating at 100°C for 10 min. In the range -5 to 50°C, ovalbumin gelation showed a minimum at 25°C at all pressures applied. Operating at low temperatures allowed a reduction in pressure required to provide rather hard gels. With egg yolk and soy protein, gelation effectiveness progressively and continuously decreased with decreasing temperatures; the decrease was sharper with egg yolk.

High pressure is used to texturize food with two different strategies. The first strategy consists of using high pressure of several hundred MPa as a gelling agent per se to replace any other gelling or coagulating agents. In the second strategy, a moderate pressure of 100-300 MPa is used as a pretreatment to modify the food components properties in order to improve the efficiency of the traditional texturing treatment such as heat texturization, acid or rennet coagulation of milk, and so on. In both cases, pressure effects depend on many parameters such as the nature and the concentration of the macromolecules, the temperature of pressurization, the holding time and pressure, the pH, and the presence of additives. The behavior of real foods upon pressurization is even more complex compared to that of macromolecular model solutions due to their complex composition and, especially, to the presence of enzymes which are sometimes hardly affected by pressurization. Findings exposed in this article demonstrate that high-pressure technology is a unique tool to texturize food and provide products with unique properties.

We report the effects of the interaction of two camelid antibody fragments, generally called nanobodies, namely cAb-HuL5 and a stabilized and more aggregation-resistant variant cAb-HuL5G obtained by protein engineering, on the properties of two amyloidogenic variants of human lysozyme, I56T and D67H, whose deposition in vital organs including the liver, kidney, and spleen is associated with a familial non-neuropathic systemic amyloidosis. Both NMR spectroscopy and X-ray crystallographic studies reveal that cAb-HuL5 binds to the ?-domain, one of the two lobes of the native lysozyme structure. The binding of cAb-HuL5/cAb-HuL5G strongly inhibits fibril formation by the amyloidogenic variants; it does not, however, suppress the locally transient cooperative unfolding transitions, characteristic of these variants, in which the ?-domain and the C-helix unfold and which represents key early intermediate species in the formation of amyloid fibrils. Therefore, unlike two other nanobodies previously described, cAb-HuL5/cAb-HuL5G does not inhibit fibril formation via the restoration of the global cooperativity of the native structure of the lysozyme variants to that characteristic of the wild-type protein. Instead, it inhibits a subsequent step in the assembly of the fibrils, involving the unfolding and structural reorganization of the ?-domain. These results show that nanobodies can protect against the formation of pathogenic aggregates at different stages in the structural transition of a protein from the soluble native state into amyloid fibrils, illustrating their value as structural probes to study the molecular mechanisms of amyloid fibril formation. Combined with their amenability to protein engineering techniques to improve their stability and solubility, these findings support the suggestion that nanobodies can potentially be developed as therapeutics to combat protein misfolding diseases.

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications from basic research to medical applications. This review focuses on the use of class Aβ-lactamases as versatile scaffolds to design hybrid enzymes (referred to asβ-lactamase hybrid proteins, BHPs) in which an exogenous peptide, protein or fragment thereof is inserted at various permissive positions. We discuss how BHPs can be specifically designed to create bifunctional proteins, to produce and to characterize proteins that are otherwise difficult to express, to determine the epitope of specific antibodies, to generate antibodies against nonimmunogenic epitopes, and to better understand the structure/function relationship of proteins.

Two well known 2 S albumins, Ber e 1 from brazil nut and sunflower 2 S albumin 8 (SFA-8), have been expressed in a eukaryotic system and purified. Analysis of recombinant versions of Ber e 1 and SFA-8 revealed them to be significantly more resistant to digestion by pepsin than BSA, and to be stable for up to 30 min in simulated gastric fluid. Unfolding monitored by CD indicated that both proteins were also very resistant to denaturation induced by heat and low pH. These results suggest that, although the ability of 2 S albumins to reach the circulatory system may be a prerequisite for the allergenicity of this group of proteins, stability is just one of a number of characteristics that provoke a selective immune response.

We report here the detailed characterisation of a non-naturally occurring variant of human lysozyme, I59T, which possesses a destabilising point mutation at the interface of the alpha- and beta-domains. Although more stable in its native structure than the naturally occurring variants that give rise to a familial form of systemic amyloidosis, I59T possesses many attributes that are similar to these disease-associated species. In particular, under physiologically relevant conditions, I59T populates transiently an intermediate in which a region of the structure unfolds cooperatively; this loss of global cooperativity has been suggested to be a critical feature underlying the amyloidogenic nature of the disease-associated lysozyme variants. In the present study, we have utilised this variant to provide direct evidence for the generic nature of the conformational transition that precedes the ready formation of the fibrils responsible for lysozyme-associated amyloid disease. This non-natural variant can be expressed at higher levels than the natural amyloidogenic variants, enabling, for example, singly isotopically labelled protein to be generated much more easily for detailed structural studies by multidimensional NMR spectroscopy. Moreover, we demonstrate that the I59T variant can readily form fibrils in vitro, similar in nature to those of the amyloidogenic I56T variant, under significantly milder conditions than are needed for the wild-type 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.

Background Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes. Methods To have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1. Results We showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen. General significance Finally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis.

Nanobodies are single chain antibodies that are uniquely produced in Camelidae, e.g. camels and llamas. They have the desirable features of small sizes (Mw14 kDa) and high affinities against antigens (Kd approximately nM), making them ideal as structural probes for biomedically relevant motifs both in vitro and in vivo. We have previously shown that nanobody binding to amyloidogenic human lysozyme variants can effectively inhibit their aggregation, the process that is at the origin of systemic amyloid disease. Here we report the NMR assignments of a new nanobody, termed NbSyn2, which recognises the C-terminus of the intrinsically disordered protein, human alpha-synuclein (aS), whose aberrant self-association is implicated in Parkinson's disease.

The aggregation process of wild-type human lysozyme at pH 3.0 and 60 °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 β-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.

Thioredoxins are small, ubiquitous redox enzymes that reduce protein disulfide bonds by using a pair of cysteine residues present in a strictly conserved WCGPC catalytic motif. The Escherichia coli cytoplasm contains two thioredoxins, Trx1 and Trx2. Trx2 is special because it is induced under oxidative stress conditions and it has an additional N-terminal zinc-binding domain. We have determined the redox potential of Trx2, the pK(a) of the active site nucleophilic cysteine, as well as the stability of the oxidized and reduced form of the protein. Trx2 is more oxidizing than Trx1 (-221 mV versus -284 mV, respectively), which is in good agreement with the decreased value of the pK(a) of the nucleophilic cysteine (5.1 versus 7.1, respectively). The difference in stability between the oxidized and reduced forms of an oxidoreductase is the driving force to reduce substrate proteins. This difference is smaller for Trx2 (Delta Delta G degrees(H2O) = 9 kJ/mol and Delta T-m = 7.4 degrees C) than for Trx1 (Delta Delta G degrees(H2O) = 15 kJ/mol and Delta T-m = 13 degrees C). Altogether, our data indicate that Trx2 is a significantly less reducing enzyme than Trx1, which suggests that Trx2 has a distinctive function. We disrupted the zinc center by mutating the four Zn2+-binding cysteines to serine. This mutant has a more reducing redox potential (-254 mV) and the pK(a) of its nucleophilic cysteine shifts from 5.1 to 7.1. The removal of Zn2+ also decreases the overall stability of the reduced and oxidized forms by 3.2 kJ/mol and 5.8 kJ/mol, respectively. In conclusion, our data show that the Zn2+-center of Trx2 fine-tunes the properties of this unique thioredoxin.

A single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic variants of human lysozyme, which are associated with a form of systemic amyloidosis, have been investigated by a wide range of biophysical techniques. Pulse-labeling hydrogen-deuterium exchange experiments monitored by mass spectrometry reveal that binding of the antibody fragment strongly inhibits the locally cooperative unfolding of the I56T and D67H variants and restores their global cooperativity to that characteristic of the wild-type protein. The antibody fragment was, however, not stable enough under the conditions used to explore its ability to perturb the aggregation behavior of the lysozyme amyloidogenic variants. We therefore engineered a more stable version of cAb-HuL22 by adding a disulfide bridge between the two beta-sheets in the hydrophobic core of the protein. The binding of this engineered antibody fragment to the amyloidogenic variants of lysozyme inhibited their aggregation into fibrils. These findings support the premise that the reduction in global cooperativity caused by the pathogenic mutations in the lysozyme gene is the determining feature underlying their amyloidogenicity. These observations indicate further that molecular targeting of enzyme active sites, and of protein binding sites in general, is an effective strategy for inhibiting or preventing the aberrant self-assembly process that is often a consequence of protein mutation and the origin of pathogenicity. Moreover, this work further demonstrates the unique properties of camelid single-domain antibody fragments as structural probes for studying the mechanism of aggregation and as potential inhibitors of fibril formation.

We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods. Both variants have a substantially decreased thermostability compared with wild-type human lysozyme, a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. The secreted yields of the F57I and W64R variants from P. pastoris are 200- and 30-fold lower, respectively, than that of wild-type human lysozyme. More comprehensive analysis of the secretion levels of 10 lysozyme variants shows that the low yields of these secreted proteins, under controlled conditions, can be directly correlated with a reduction in the thermostability of their native states. Analysis of mRNA levels in this selection of variants suggests that the lower levels of secretion are due to post-transcriptional processes, and that the reduction in secreted protein is a result of degradation of partially folded or misfolded protein via the yeast quality control system. Importantly, our results show that the human disease-associated mutations do not have levels of expression that are out of line with destabilizing mutations at other sites. These findings indicate that a complex interplay between reduced native-state stability, lower secretion levels, and protein aggregation propensity influences the types of mutation that give rise to familial forms of amyloid disease.