Structural basis of IL-23 antagonism by an Alphabody protein scaffold

Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein-protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies

Cell-penetrating Alphabody protein scaffolds for intracellular drug targeting

The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold’s termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics

Algorithm for backrub motions in protein design

Motivation: The Backrub is a small but kinematically efficient side-chain-coupled local backbone motion frequently observed in atomic-resolution crystal structures of proteins. A backrub shifts the Cα–Cβ orientation of a given side-chain by rigid-body dipeptide rotation plus smaller individual rotations of the two peptides, with virtually no change in the rest of the protein. Backrubs can therefore provide a biophysically realistic model of local backbone flexibility for structure-based protein design. Previously, however, backrub motions were applied via manual interactive model-building, so their incorporation into a protein design algorithm (a simultaneous search over mutation and backbone/side-chain conformation space) was infeasible

Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells

Background: The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection. Methods and Findings: Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM+ memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge. Conclusions: The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM+ memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens

Per- and polyfluoroalkyl substances (PFAS) in private gardens : factors affecting accumulation in homegrown food and characterization of human exposure risk

Abstract: In the past decade, homegrown food consumption has surged in rural, urban, and industrial areas. However, organic pollutants in private gardens, including per- and polyfluoroalkylated substances (PFAS), pose health risks by entering the food chain through bioaccumulation. Very little is known about the driving factors of PFAS accumulation in homegrown food. Therefore, this thesis project aimed to assess PFAS accumulation in various homegrown food categories and related human exposure risks, exploring factors affecting PFAS bioavailability. The results showed that multiple PFAS are omnipresent in homegrown food and can accumulate to concentrations that frequently exceed available health guidelines, even under modest consumption scenarios, especially with regard to egg intake. Within the crop category, higher accumulation was noticed in annual crops in comparison to perennial crops, potentially linked with differences in terms of life-history strategies between these two plant taxa. Large spatial and temporal differences in soil PFAS profile and concentrations were found within private gardens, suggesting that site-specific characteristics and functional usage play a major role in shaping local PFAS contamination. Predictive models could be constructed for some major PFAS in eggs, which show promising potential for applicability in risk assessment by policy makers. Moreover, mitigation and remediation measures could be formulated that should be readily usable for private gardeners to ultimately lower PFAS exposure via homegrown food. PFAS pollution in gardens within \ub1 4 km from the fluorochemical plant in Antwerp could be strongly linked with both historical and recent fluorochemical emissions. On the other hand, diffusive mechanisms (e.g. atmospheric transport) and site-specific soil management may be mainly affecting levels at gardens further away from point sources. The accumulation in chicken eggs was generally higher closer to the major fluorochemical plant, although soil characteristics (e.g. organic matter, clay content and pH) could strongly affect this pattern. Conversely, the PFAS accumulation in the crops was not affected by the distance from the plant site and soil characteristics played only a minor role in governing crop accumulation. Long-term declining concentrations in soil and eggs could be observed for some PFAS, although this trend stagnated over recent years. Short-term increases of short-chain and long-chain PFAS concentrations could be observed, mainly in the soil from the chicken enclosure. These findings underpin that homegrown food cannot be neglected as a relevant human exposure source to PFAS and show the urgent necessity for further regulation steps and monitoring efforts

Explanation of Benveniste

SCOPUS: le.jinfo:eu-repo/semantics/publishe

Reduction methods for logical control networks

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

The design of idealized α/β-barrels: Analysis of β-sheet closure requirements

SCOPUS: ar.jFLWNAinfo:eu-repo/semantics/publishe