Class A β -Lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications frombasic research to medical applications. This review focuses on the use of class A -lactamases as versatile scaffolds to design hybrid enzymes (referred to as -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.Peer reviewe

Predictive factors for the presence and long-term persistence of SARS-CoV-2 antibodies in healthcare and university workers

peer reviewedWhile patient groups at risk for severe COVID-19 infections are now well identified, the risk factors associated with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) transmission and immunization are still poorly understood. In a cohort of staff members of a Belgian tertiary academic hospital tested for SARS-CoV-2 antibodies during the early phase of the pandemic and followed-up after 6 weeks, 3 months and 10 months, we collected personal, occupational and medical data, as well as symptoms based on which we constructed a COVID-19 score. Seroprevalence was higher among participants in contact with patients or with COVID-19 confirmed subjects or, to a lesser extent, among those handling respiratory specimens, as well as among participants reporting an immunodeficiency or a previous or active hematological malignancy, and correlated with several symptoms. In multivariate analysis, variables associated with seropositivity were: contact with COVID-19 patients, immunodeficiency, previous or active hematological malignancy, anosmia, cough, nasal symptoms, myalgia, and fever. At 10 months, participants in contact with patients and those with higher initial COVID-19 scores were more likely to have sustained antibodies, whereas those with solid tumors or taking chronic medications were at higher risk to become seronegative

Long-term longitudinal evaluation of the prevalence of SARS-CoV-2 antibodies in healthcare and university workers.

peer reviewedAsymptomatic and pauci-symptomatic cases contribute to underestimating the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Moreover, we have few studies available on the longitudinal follow-up of SARS-CoV-2 antibodies after natural infection. We tested staff members of a Belgian tertiary academic hospital for SARS-CoV-2 IgG, IgM, and IgA antibodies. We analyzed the evolution of IgM and IgG after 6 weeks, and the persistence of IgG after 3 and 10 months. At the first evaluation, 409/3776 (10.8%) participants had a positive SARS-CoV-2 serology. Among initially seropositive participants who completed phases 2 and 3, IgM were still detected after 6 weeks in 53.1% and IgG persisted at 12 weeks in 82.0% (97.5% of those with more than borderline titers). IgG levels were higher and increased over time in symptomatic but were lower and stable in asymptomatic participants. After 10 months, 88.5% of participants had sustained IgG levels (97.0% of those with more than borderline titers)

Evaluation of Screening Program and Phylogenetic Analysis of SARS-CoV-2 Infections among Hospital Healthcare Workers in Liège, Belgium

Healthcare workers (HCWs) are known to be at higher risk of developing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections although whether these risks are equal across all occupational roles is uncertain. Identifying these risk factors and understand SARS-CoV-2 transmission pathways in healthcare settings are of high importance to achieve optimal protection measures. We aimed to investigate the implementation of a voluntary screening program for SARS-CoV-2 infections among hospital HCWs and to elucidate potential transmission pathways though phylogenetic analysis before the vaccination era. HCWs of the University Hospital of Liège, Belgium, were invited to participate in voluntary reverse transcriptase-polymerase chain reaction (RT-PCR) assays performed every week from April to December 2020. Phylogenetic analysis of SARS-CoV-2 genomes were performed for a subgroup of 45 HCWs. 5095 samples were collected from 703 HCWs. 212 test results were positive, 15 were indeterminate, and 4868 returned negative. 156 HCWs (22.2%) tested positive at least once during the study period. All SARS-CoV-2 test results returned negative for 547 HCWs (77.8%). Nurses (p < 0.05), paramedics (p < 0.05), and laboratory staff handling respiratory samples (p < 0.01) were at higher risk for being infected compared to the control non-patient facing group. Our phylogenetic analysis revealed that most positive samples corresponded to independent introduction events into the hospital. Our findings add to the growing evidence of differential risks of being infected among HCWs and support the need to implement appropriate protection measures based on each individual’s risk profile to guarantee the protection of both HCWs and patients. Furthermore, our phylogenetic investigations highlight that most positive samples correspond to distinct introduction events into the hospital

Model polyQ proteins based on the beta-lactamase BlaP: How non-polyQ regions influence the polyQ length-dependent aggregation process

Neurodegenerative amyloid diseases are fatal disorders representing an important human health and economic burden. Amongst them, nine disorders are classified as polyglutamine (polyQ) diseases, for which no treatment is yet available. They are all characterized by the pathological expansion of a poly(CAG) sequence, above a specific threshold, within the coding sequence of nine unrelated genes, translated in nine polyQ proteins. The polyQ expansion, the only common point, is the critical determinant for polyQ disease development by triggering protein aggregation into amyloid fibrils. More recent studies however indicate that the protein context modulates the polyQ-induced aggregation process and the disease phenotype. The objectives of my thesis are to deeper investigate the molecular determinants of the complex interplay between the propensity of the polyQ tract to trigger protein aggregation and the modulating role of non-polyQ regions in order to allow the identification of strategies to interfere with the pathological aggregation process. For that purpose, model polyQ proteins, referred to as BlaP-polyQ chimeras, based on the β-lactamase BlaP and polyQ sequences (23 – 79Q) inserted at two positions (197, in between or not unstructured peptides, or 216), are used and their aggregation properties are characterized under several conditions. We first observe that the polyQ length is determinant for BlaP-polyQ chimera aggregation. Indeed, there is a Q-threshold for the aggregation into amyloid fibrils and for fibril elongation. Above this threshold, the aggregation in solution and the elongation rate increase with the length of the polyQ tract, with an exponential rise-to-maximum and a linear regression, respectively, independently of the conformation of the BlaP moiety, and of the position of the polyQ tract within BlaP. Longer polyQ tracts are likely to have a larger conformational flexibility allowing them to more easily adopt an amyloid-aggregation prone conformation. However, the Q-threshold for fibril elongation is much lower than for fibril formation in solution: the polyQ tract requires a larger conformational flexibility to nucleate the formation of fibrils than to elongate them. Secondly, the Q-threshold for fibril formation is lower once the BlaP moiety is unfolded, and BlaP-polyQ chimeras aggregate faster into amyloid fibrils under conditions favoring the unfolding of the BlaP moiety. The native structure of BlaP is likely to impose conformational constraints to moderate and long polyQ tracts that block and decrease, respectively, their propensity to form fibrils. The effects of constraints decrease with the polyQ length. Thirdly, BlaP chimeras with the polyQ tract in position 216 have an increased propensity to trigger the nucleation and the elongation of amyloid fibrils compared to chimeras with the polyQ in position 197. Advanced studies confirm that the propensity of the polyQ tract to aggregate into amyloid fibrils is linked to the conformational flexibility of the polyQ tract, which depends on (i) the polyQ length, (ii) the location of the polyQ tract within BlaP, i.e., a terminal location or embedded within a protein domain, and (iii) the structural properties of the polyQ flanking regions. Finally, we observe that the whole region flanking the polyQ tract in position 197 at its N-terminus has an anti-aggregating property that fully counterbalances the pro-aggregating property of that flanking at the C-terminus. The former imposes strong conformational constraints to the polyQ tract that reduce its conformational flexibility and hence its aggregation propensity. Moreover, these regions are likely to differently affect the solubility of the polyQ protein and hence the driving force for insoluble aggregation. Altogether, our data also suggest that BlaP chimeras aggregate via the commonly described nucleation-dependent polymerization mechanism during which the aggregation is first triggered by polyQ-polyQ interactions, and then a subsequent slight reorganization of the BlaP moiety is required for conversion of aggregates into amyloid fibrils. Based on these results, we should allow the development of therapeutic strategies, targeting specifically the molecular features of the complex interplay between the polyQ and non-polyQ regions during the nucleation and/or elongation of the pathological aggregation of polyQ proteins

Role of non-polyQ regions on the different steps of amyloid fibril formation by polyQ proteins

Ten neurodegenerative diseases, referred to as polyglutamine (polyQ) diseases, are associated with the aggregation into amyloid fibrils of ten different proteins containing a polyQ expansion higher than a pathological threshold comprised between 35 to 45Q (1, 2). A large body of evidence indicate that the polyQ expansion is the critical determinant for the aggregation of these polyQ proteins. The aggregation process of polyQ proteins is, however, still not well understood. To better understand this mechanism at a molecular level, we have characterized model polyQ proteins made of the β-lactamase BlaP from Bacillus licheniformis 749/C and a polyQ tract of 0 to 79Q inserted either at position 197 or position 216 of BlaP. Those chimeras recapitulate the same aggregation behaviours than that of disease-associated polyQ proteins: there is a glutamine threshold for the aggregation into amyloid fibrils and the anticipation phenomenon. Most importantly, the threshold critically depends on the structural integrity of BlaP (3) which would impose some conformational and/or sterical constraints to the polyQ tract. Moreover the position of the polyQ insertion into BlaP modifies the aggregation propensity of BlaP chimeras. The present work aims to further investigate (i) how the protein context affects the different phases of the aggregation phenomenon (i.e. the nucleation and elongation phases) and (ii) the role of the oligomers formed during the early time of the aggregation process. The techniques used are mainly (1) quartz crystal microbalance (QCM) and atomic force microscopy (AFM) to study the elongation step of amyloid fibril formation and (2) dynamic light scattering (DLS) to study the evolution of the different populations formed during the aggregation time course. The results of these experiments indicate that the native conformation of BlaP197(Gln)55 interferes mainly with the nucleation but not with the elongation step of amyloid fibril formation. Moreover, these results demonstrate that the sequences flanking the polyQ tract significantly influence its propensity to elongate amyloid fibrils

Influence of protein context on the propensity of polyglutamine tracts to induce protein aggregation into amyloid fibrils

Nine neurodegenerative diseases, referred to as polyglutamine diseases, are associated with nine proteins containing an expanded polyglutamine (polyQ) tract. PolyQ tracts are encoded by a repetition of the CAG codon in the corresponding genes, and are present in proteins of healthy people. They are however pathogenic when their length, due to mutations, becomes higher than a threshold generally comprised between 35 and 45Q. Such pathogenic tracts trigger the aggregation of the proteins into amyloid-like aggregates that could play an important role in the disease. It is therefore necessary to investigate at a molecular level the aggregation process of polyQ proteins. Since proteins associated with polyQ diseases are generally big and relatively insoluble, they are difficult to produce and manipulate. We have therefore decided to study the aggregating properties of polyQ proteins by designing and characterizing model proteins made of a well-characterized host protein, the β-lactamase BlaP, and polyQ tracts of different lengths (23 to 79Q) inserted at position 197 of BlaP. The aggregating properties of these BlaP chimeras recapitulate those of proteins associated with polyQ diseases. We indeed observed that there is a minimal number of glutamines (threshold) required for chimeras aggregation into amyloid-like fibrils and that the kinetics of aggregation are faster with longer glutamine repeats. Most importantly, the value of the threshold for amyloid-like fibril formation seems to critically depend on the structural integrity of BlaP and thus on the constraints applied to the polyQ tract. In the present work, we investigate more deeply the role of the protein context and the role of oligomers in the process of aggregation of BlaP chimeras. This study is mainly based on the use of two techniques: the quartz crystal microbalance with dissipation (QCM-D) in combination with atomic force microscopy (AFM), and the dynamic light scattering (DLS). Preliminary QCM-D results indicate that in native condition there is a minimal number of glutamines required for the elongation of BlaP197(Gln)79 fibrils by BlaP chimeras. This threshold is different to that observed for the aggregation monitored in solution in absence of seed. Most interestingly it corresponds to the threshold observed in denaturing conditions. These observations suggest that the conformation of BlaP is the limiting step for amyloid fibril formation by interfering with the nucleation step. Preliminary DLS experiments indicate that the oligomers formed by BlaP197(Gln)79 in native condition are on the pathway of amyloid fibril formation and could act as aggregation nuclei.Mécanisme d'agrégation de protéines chimères composées de la béta-lactamase BlaP et de séquences polyglutamine: étude des contraintes stériques et/ou conformationelles imposées par BlaP sur les propriétés des stretchs polyglutamin

Role of non-polyQ regions on the aggregation process by polyQ proteins into amyloid fibrils

Nine neurodegenerative disorders, referred to as polyglutamine diseases and including Huntington’s disease, are associated with the abnormal expansion of a polyglutamine tract inside nine unrelated proteins. This polyQ expansion is thought to be the major determinant in the development of neurotoxicity, triggering protein aggregation into amyloid fibrils. A large body of evidence however indicates that non-polyQ regions modulate the aggregation process triggered by polyQ expansions. The interplay between the polyQ tract and non-polyQ regions is complex and still not fully understood. In order to better understand it, we previously designed and characterized model polyQ proteins made of the beta-lactamase BlaP and a 23, 30, 55 or 79Q tract inserted in position 197 or 216. These chimeras recapitulate the aggregation properties of polyQ disease-associated proteins: there is a Q threshold for the formation of amyloid fibrils, and above the threshold, the longer the polyQ, the faster the aggregation. Moreover, the structure of BlaP (native or unfolded) and the position of insertion of the polyQ tract (197 versus 216) influence their aggregation properties. In this work, (i) we will discuss the role of the conformation of the host protein, BlaP, and of the location of the polyQ within BlaP on the different phases of amyloid fibril formation, the nucleation and elongation steps, using mainly quartz crystal microbalance (QCM), atomic force microscopy (AFM) and dynamic light scattering (DLS); and (ii) we will investigate the precise aggregation threshold and the modulating role of the N- and C-terminal polyQ flanking sequences in position 197 of BlaP by creating and characterizing new chimeras containing intermediate length polyQ tracts in position 197, or polyQ tracts inserted between two cleavage sites in position 197, respectively. Our results highlight a linear dependence of the polyQ length on the elongation rate whatever the insertion site and the conformation of BlaP. These two parameters however drastically influence the ability of a polyQ tract to trigger the nucleation and the elongation steps of amyloid fibril formation. Finally, we observed that the propensity to form amyloid fibrils and its rate seems to be largely dependent on the polyQ length and on the polyQ flanking sequences. Altogether our results contribute to identify the important species and elements (polyQ or non-polyQ regions, monomers, oligomers or fibrils) during the aggregation process into amyloid fibrils to interfere with the latter associated with neurotoxicity

Production et caractérisation des propriétés physico-chimiques du peptide AB42 (associé avec la maladie d'Alzheimer) replacé dans un environnement protéique

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Influence of the protein context in the aggregation of polyQ proteins into amyloid fibrils

Ten neurodegenerative diseases, referred to as polyglutamine (polyQ) diseases, are associated with the aggregation into amyloid fibrils of ten different proteins containing a polyQ expansion higher than a pathological threshold comprised between 35 to 45Q (1, 2). A large body of evidence indicate that the polyQ expansion is the critical determinant for the aggregation of these polyQ proteins. The aggregation process of polyQ proteins is, however, still not well understood. To better understand this mechanism at a molecular level, we have characterized model polyQ proteins made of the β-lactamase BlaP from Bacillus licheniformis 749/C and a polyQ tract of 0 to 79Q inserted either at position 197 or position 216 of BlaP. Those chimeras recapitulate the same aggregation behaviours than that of disease-associated polyQ proteins: there is a glutamine threshold for the aggregation into amyloid fibrils and the anticipation phenomenon. Most importantly, the threshold critically depends on the structural integrity of BlaP (3) which would impose some conformational and/or sterical constraints to the polyQ tract. Moreover the position of the polyQ insertion into BlaP modifies the aggregation propensity of BlaP chimeras. The present work aims to further investigate (i) how the protein context affects the different phases of the aggregation phenomenon (i.e. the nucleation and elongation phases) and (ii) the role of the oligomers formed during the early time of the aggregation process. The techniques used are mainly (1) quartz crystal microbalance (QCM) and atomic force microscopy (AFM) to study the elongation step of amyloid fibril formation and (2) dynamic light scattering (DLS) to study the evolution of the different populations formed during the aggregation time course. The results of these experiments indicate that the native conformation of BlaP197(Gln)55 interferes mainly with the nucleation but not with the elongation step of amyloid fibril formation. Moreover, these results demonstrate that the sequences flanking the polyQ tract significantly influence its propensity to elongate amyloid fibrils. Finally, they clearly indicate that the oligomers of BlaP197(Gln)79 observed at the early stage of the aggregation process are on the pathway of amyloid fibril formation, and likely constitute the aggregation nucleus