In autoimmune bullous dermatoses (AIBD), autoantibodies induce blisters on skin or mucous membranes, or both. Mechanisms of continued autoantibody production and blistering have been well characterized using AIBD animal models. Mechanisms leading to the initial autoantibody production, however, have not been investigated in detail. Epidermolysis bullosa acquisita (EBA) is an AIBD associated with autoantibodies to type VII collagen (COL7). The majority of EBA patients’ sera recognize the noncollagenous domain 1, including the von Willebrand factor A–like domain 2 (vWFA2). In experimental EBA induced by immunization with GST-COL7, disease manifestation depended on the genetic background, a Th1 polarization, and the GST-tag. In this model, nude mice neither produced autoantibodies nor blisters. It has remained uncertain which APC and T cell subsets are required for EBA induction. We established a novel EBA model by immunization with vWFA2 fused to intein (lacking the GST-tag). All tested mouse strains developed autoantibodies, but blisters were exclusively observed in mice carrying H2s. In immunized mice, CD4 T cells specific for vWFA2 were detected, and their induction required presence of B cells, dendritic cells, and macrophages. Anti-vWFA2 autoantibodies located at the lamina densa bound to the dermal side of salt-split skin and induced blisters when transferred into healthy mice. Absence of CD8 T cells at time of immunization had no effect, whereas depletion of CD4 T cells during the same time period delayed autoantibody production and blisters. Collectively, we demonstrate the pathogenic relevance of Abs targeting the vWFA2 domain of COL7 and show the requirement of APC-induced CD4 T cells to induce experimental EBA.

Animal models have enhanced our understanding of the pathogenesis of autoimmune diseases. For these models, genetically identical, inbred mice have commonly been used. Different inbred mouse strains, however, show a high variability in disease manifestation. Identifying the factors that influence this disease variability could provide unrecognized insights into pathogenesis. We established a novel Ab transfer-induced model of epidermolysis bullosa acquisita (EBA), an autoimmune disease characterized by (muco)-cutaneous blistering caused by anti-type VII collagen (COL7) autoantibodies. Blistering after anti-COL7 IgG (directed against the von Willebrand factor A–like domain 2) transfer showed clear variability among inbred mouse strains, that is, severe cutaneous blistering and inflammation in C57BL/6J and absence of skin lesions in MRL/MpJ mice. The transfer of anti-COL7 IgG into irradiated, EBA-resistant MRL/MpJ mice, rescued by transplantation with bone marrow from EBA-susceptible B6.AK-H2k mice, induced blistering. To the contrary, irradiated EBA-susceptible B6.AK-H2k mice that were rescued using MRL/MpJ bone marrow were devoid of blistering. In vitro, immune complex activation of neutrophils from C57BL/6J or MRL/MpJ mice showed an impaired reactive oxygen species release from the latter, whereas no differences were observed after PMA activation. This finding was paralleled by divergent expression profiles of immune complex–activated neutrophils from either C57BL/6J or MRL/MpJ mice. Collectively, we demonstrate that radiosensitive cells determine the varying extent of skin inflammation and blistering in the end-stage effector phase of EBA.

Neutrophil activation is an important mechanism of host defense against pathogens. Chronic inflammation and autoimmunity are often associated with abnormalities in phenotype and functions of neutrophils. Since effector functions of immune cells during inflammation are tightly linked to their metabolic state, changes in neutrophil metabolome upon activation have been investigated in this study. Human neutrophils from healthy blood donors (n = 6) were treated either with tumor necrosis factor α (TNF-α) or lipopolysaccharide (LPS), whereas untreated neutrophils were used as control. Since apoptotic cells are abundant at sites of inflammation, the metabolome of aged, mainly apoptotic neutrophils was analyzed too. NMR spectroscopy of water-soluble metabolites revealed a clear distinction between aged neutrophils and neutrophils in control and activated samples. Higher levels of NAD (4- to 9-fold) and lower levels of ATP (0.3-fold), glutathione (0.8-fold), hypotaurine (0.8-fold), and phosphocholine (0.6-fold) were detected in aged neutrophils than in the other samples. Differences in metabolic profiles between LPS and TNF-α-stimulated cells as well as between stimulated and control neutrophils were statistically not significant. Replication with additional six blood donors confirmed increased NAD levels in aged cells compared to activated and control neutrophils.

The histone chaperone Asf1 and the checkpoint kinase Rad53 are found in a complex in budding yeast cells in the absence of genotoxic stress. Our data suggest that this complex involves at least three interaction sites. One site involves the H3-binding surface of Asf11 with an as yet undefined surface of Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-T 270 phosphorylated by casein kinase II. The third site involves the C-terminal 21 amino acids of Rad53 bound to the conserved Asf1 N-terminal domain. The structure of this site showed that the Rad53 C-terminus binds Asf1 in a remarkably similar manner to peptides derived from the histone cochaperones HirA and CAF-I. We call this binding motif, ðR∕KÞRðI∕A∕VÞ × ðL∕PÞ, the AIP box for Asf1-Interacting Protein box. Furthermore, C-terminal Rad53-F 820 binds the same pocket of Asf1 as does histone H4-F 100 . Thus Rad53 competes with histones H3-H4 and cochaperones HirA/CAF-I for binding to Asf1. Rad53 is phosphorylated and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when cells were treated with hydroxyurea but not methyl-methane-sulfonate, suggesting a regulation of the complex as a function of the stress. We identified a rad53 mutation that destabilized the Asf1-Rad53 complex and increased the viability of rad9 and rad24 mutants in conditions of genotoxic stress, suggesting that complex stability impacts the DNA damage response.checkpoint | chromatin | DNA damage | NMR | X-ray structure

We report here an advanced approach for the characterization of the folding pattern of a de novo designed antiparallel coiled coil peptide by high-resolution methods. Incorporation of two fluorescence labels at the C- and N-terminus of the peptide chain as well as modification of two hydrophobic core positions by Phe/[15N,13C]Leu enable the study of the folding characteristics and of distinct amino acid side chain interactions by fluorescence resonance energy transfer (FRET) and NMR spectroscopy. Results of both experiments reveal the antiparallel alignment of the helices and thus prove the design concept. This finding is also supported by molecular dynamics simulations. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in combination with NMR experiments was used for verification of the oligomerization equilibria of the coiled coil peptide.

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Edited by Gianni CesareniKeywords: vWFA Domain interaction Skin structure NMR a b s t r a c t Type VII collagen as component of anchoring fibrils plays an important role in skin architecture, however, no detailed structural information is available. Here, we describe the recombinant expression, isotope labeling, and 1 H, 15 N, 13 C chemical shift assignment of a subdomain of the murine type VII collagen -the von-Willebrand-factor-A-like domain 2 (mvWFA2). vWFA2 interacts with type I collagen and plays a central role in certain skin blistering diseases. Based on these assignments a secondary structure prediction was performed showing a properly folded protein. An interaction of mvWFA2 with its neighboring domain mFNIII-9 was characterized with NMR spectroscopy and SPR.Structured summary of protein interactions: mFNIII-9 and mvWFA2 bind by nuclear magnetic resonance (View interaction) mvWFA2 binds to mFNIII-9 by surface plasmon resonance (View interaction)