Reduction of disulphide bonds unmasks potent antimicrobial activity of human ß-defensin 1.

Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of -200 mV to -300 mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces. Human ß-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.

An unfolded CH1 domain controls the assembly and secretion of IgG antibodies.

A prerequisite for antibody secretion and function is their assembly into a defined quaternary structure, composed of two heavy and two light chains for IgG. Unassembled heavy chains are actively retained in the endoplasmic reticulum (ER). Here, we show that the C(H)1 domain of the heavy chain is intrinsically disordered in vitro, which sets it apart from other antibody domains. It folds only upon interaction with the light-chain C(L) domain. Structure formation proceeds via a trapped intermediate and can be accelerated by the ER-specific peptidyl-prolyl isomerase cyclophilin B. The molecular chaperone BiP recognizes incompletely folded states of the C(H)1 domain and competes for binding to the C(L) domain. In vivo experiments demonstrate that requirements identified for folding the C(H)1 domain in vitro, including association with a folded C(L) domain and isomerization of a conserved proline residue, are essential for antibody assembly and secretion in the cell.

The structure of a folding intermediate provides insight into differences in immunoglobulin amyloidogenicity.

Folding intermediates play a key role in defining protein folding and assembly pathways as well as those of misfolding and aggregation. Yet, due to their transient nature, they are poorly accessible to high-resolution techniques. Here, we made use of the intrinsically slow folding reaction of an antibody domain to characterize its major folding intermediate in detail. Furthermore, by a single point mutation we were able to trap the intermediate in equilibrium and characterize it at atomic resolution. The intermediate exhibits the basic beta-barrel topology, yet some strands are distorted. Surprisingly, two short strand-connecting helices conserved in constant antibody domains assume their completely native structure already in the intermediate, thus providing a scaffold for adjacent strands. By transplanting these helical elements into beta(2)-microglobulin, a highly homologous member of the same superfamily, we drastically reduced its amyloidogenicity. Thus, minor structural differences in an intermediate can shape the folding landscape decisively to favor either folding or misfolding.