Using in-tube extraction and slice selective NMR experiments allow imaging via statistical analysis of metabolic profiles.

Imaging the distribution of metabolites is very powerful in diagnostics but it is also employed in fundamental research. Although NMR spectroscopy is well established for determining metabolic profiles of biological samples, its application is limited to magnetic resonance imaging that can produce images of larger structures, but the number of detectable metabolites is very low. Mass spectrometry imaging on the other hand is well established with pixel sizes in the µm range. This limits the analysis of larger structures like tissue sections and detection of metabolites depends on their ionization properties. High resolution NMR metabolomics could complement these methods. However, this is prevented due to time consuming extraction procedures. To overcome these limitations, the following protocol was established and applied to two different ham slices: sampling is directly done into the NMR tube and after extraction of polar and non-polar metabolites in the NMR tube, slice selective NMR spectra are acquired. Multivariate analysis (PCA) of the NMR-spectra and subsequent visualization of the differences correlate well with structures visible in the ham slices. The proposed protocol can be used for metabolic imaging and could complement other imaging methods.

Structural and biophysical characterization of the selenoprotein SelW1 from Chlamydomonas reinhardtii.

Selenoprotein W is widespread among pro- and eukaryotic organisms. It possesses antioxidant activity and plays pivotal roles in mammalian embryonic development and cellular functions. A very simple, prototypical selenoprotein W is SelW1 from Chlamydomonas. The U14C mutant of SelW1 was isolated and biophysically characterized. It contains an intramolecular disulfide bond and is thermally stable up to 70 °C. NMR resonance assignment of reduced and oxidized SelW1 showed that SelW1 adopts a thioredoxin fold. Interestingly, both forms show two additional sets of resonance for amino acid residues near the termini and have basically identical dynamic behavior. Since SelW1 from Chlamydomonas resembles the ancestor of mammalian selenoproteins in certain aspects, this study lays the basis for future characterization of SelW1 function and possible interaction partners.

Simple and Rapid (Extraction) Protocol for NMR Metabolomics-Direct Measurement of Hydrophilic and Hydrophobic Metabolites Using Slice Selection.

Investigating the metabolic profiles of solid sample materials with solution nuclear magnetic resonance (NMR) spectroscopy requires the extraction of these metabolites. This is commonly done by using two immiscible solvents such as water and chloroform for extraction. Subsequent solvent removal makes these extraction procedures very time-consuming. To shorten the preparation time of the NMR sample, the following protocol is proposed: the metabolites from a solid or liquid sample are extracted directly in the NMR tube, the NMR tube is centrifuged, and the metabolite profiles in the aqueous and organic phases are determined by using slice-selective proton NMR experiments. This protocol was tested with 11 black teas and 11 green teas, which can be easily distinguished by their metabolic profiles in the aqueous phase. As a test case for liquid samples, 29 milk samples were investigated. The geographical origin of the diaries where the milk was processed could not be determined unequivocally from the metabolic profiles of the hydrophilic metabolites; however, this was easily seen in the lipid profiles. As shown for the different test samples, the extraction protocol in combination with slice-selection NMR experiments is suitable for metabolic investigations. Because samples are rapidly processed, this approach can be used to explore different extraction strategies for metabolite isolation.

Structural and biophysical characterization of the type VII collagen vWFA2 subdomain leads to identification of two binding sites.

Type VII collagen is an extracellular matrix protein, which is important for skin stability; however, detailed information at the molecular level is scarce. The second vWFA (von Willebrand factor type A) domain of type VII collagen mediates important interactions, and immunization of mice induces skin blistering in certain strains. To understand vWFA2 function and the pathophysiological mechanisms leading to skin blistering, we structurally characterized this domain by X-ray crystallography and NMR spectroscopy. Cell adhesion assays identified two new interactions: one with ß1 integrin via its RGD motif and one with laminin-332. The latter interaction was confirmed by surface plasmon resonance with a KD of about 1 mm. These data show that vWFA2 has additional functions in the extracellular matrix besides interacting with type I collagen.

Maternally Inherited Differences within Mitochondrial Complex I Control Murine Healthspan.

Mitochondrial complex I-the largest enzyme complex of the mitochondrial oxidative phosphorylation machinery-has been proposed to contribute to a variety of age-related pathological alterations as well as longevity. The enzyme complex-consisting proteins are encoded by both nuclear (nDNA) and mitochondrial DNA (mtDNA). While some association studies of mtDNA encoded complex I genes and lifespan in humans have been reported, experimental evidence and the functional consequence of such variants is limited to studies using invertebrate models. Here, we present experimental evidence that a homoplasmic mutation in the mitochondrially encoded complex I gene mt-Nd2 modulates lifespan by altering cellular tryptophan levels and, consequently, ageing-related pathways in mice. A conplastic mouse strain carrying a mutation at m.4738C > A in mt-Nd2 lived slightly, but significantly, shorter than the controls did. The same mutation led to a higher susceptibility to glucose intolerance induced by high-fat diet feeding. These phenotypes were not observed in mice carrying a mutation in another mtDNA encoded complex I gene, mt-Nd5, suggesting the functional relevance of particular mutations in complex I to ageing and age-related diseases.

1H-NMR spectroscopy shows cellular uptake of HEPES buffer by human cell lines-an effect to be considered in cell culture experiments.

HEPES is commonly used in cell culture media as a buffering substance. Compared to the bicarbonate/CO2 buffer system, it does not require a CO2 atmosphere, thereby ensuring stable pH values during handling of cell culture media outside of an incubator. Due to its intrinsic charge, HEPES is considered not to be taken up by cells, which was a prerequisite during buffer development for cell culture by Good and colleagues. However, during the last years, evidence has emerged that HEPES seems to be taken up into cells and that it has major effects on cellular functions. Investigating three different cell lines (MCF-7, U2OS, HeLa) showed that all of them accommodated HEPES-containing medium, i.e., they survive and proliferate in the presence of HEPES. Determination of intracellular metabolites revealed the presence of HEPES for all cell lines. Further analysis of MCF-7 cells showed that even 48 h after medium exchange from HEPES-containing medium to HEPES-free medium, intracellular HEPES could still be detected. Thus, contrary to the common view, HEPES is taken up by cells which should be taken into consideration for studies of specific cellular functions. Graphical abstract ᅟ.

Changes in Neutrophil Metabolism upon Activation and Aging.

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.

Skin melanocytes and fibroblasts show different changes in choline metabolism during cellular senescence.

Unmodified cells undergo only a limited number of cell divisions until they enter a state termed cellular senescence. Other triggers like cytotoxic compounds can also induce cell senescence. Since cell senescence represents a major mechanism of tumor suppression this cellular state has attracted increasing attention. Different markers like senescence-associated ß-galactosidase (SAßGal), senescence-associated heterochromatic foci (SAHF) or certain metabolic changes have been identified to be characteristic for senescent cells; however, data is often limited to fibroblasts - the cardinal model system for cellular senescence. In order to investigate whether metabolic changes during senescence are cell type independent, skin fibroblasts and skin melanocytes have been examined. Expression of the senescence marker p16 could be detected in skin fibroblasts but not in melanocytes of this specific donor, rendering the senescent phenotype not fully ascertained for the melanocytes. Metabolic profiles of senescent cells and controls have been determined using NMR spectroscopy. Changes in metabolism are different for fibroblasts and melanocytes. Senescent melanocytes showed lower levels of phosphocholine whereas for fibroblasts in accordance with literature, levels of glycerophosphocholine were increased during senescence. Although no general metabolic marker for cellular senescence exists, the same metabolic pathway seems to be affected for both cell types.

A type VII collagen subdomain mutant is thermolabile and shows enhanced proteolytic degradability - Implications for the pathogenesis of recessive dystrophic epidermolysis bullosa?

Type VII collagen is the major constituent of anchoring fibrils. It has a central collagenous domain that is surrounded by a small C-terminal non-collagenous domain (NC2) and a large N-terminal non-collagenous (NC1) domain. Mutations in type VII collagen can lead to hereditary skin blistering disease dystrophic epidermolysis bullosa (DEB). Most of the pathogenic missense mutations are within the collagenous domain. NC1 domain mediates interactions with other extracellular matrix molecules and only very few missense mutations within NC1 causing DEB have been reported. Interestingly, fibronectin III like (FNIII) domain 8 in the human protein can harbour different mutations at position 886 with one (R886P) leading to recessive DEB, whereas the others do not. We characterized subdomains of murine NC1, the FNIII domains 7-8, and the individual domains FNIII7 and FNIII8 by NMR- and CD-spectroscopy. We analysed the influence on stability for a mutation causing DEB and a non-pathogenic mutation. Whereas the silent mutation behaves as the wild type, the pathogenic mutation leads to a dramatic decrease in thermal stability of the FNIII8 domain. The melting temperature lowered from 77°C to 40°C compared to the wild type protein. This renders the domain susceptible to protease cleavage which could be shown by degradation tests with cathepsin G, cathepsin K, and MMP9. Our data show partial unfolding of type VII collagen due to the mutation causes an increased degradation. This could lead to skin blistering and opens new concomitant treatment options in some types of type VII collagen related skin blistering diseases.

Metabolic Changes Investigated by Proton NMR Spectroscopy in Cells Undergoing Oncogene-Induced Senescence.

Investigating metabolic changes during different organismal or cellular states is of increasing interest. The combination of a data-rich analytical method like mass spectrometry or NMR spectroscopy with a statistical analysis identifies metabolites that are affected by a certain stimulus. Thus, important information on the underlying molecular pathways can be obtained. Here, we describe how to investigate metabolic changes in a model of oncogene-induced senescence. The water-soluble metabolites are isolated by a chloroform-methanol treatment and subsequently analyzed by NMR spectroscopy.

Chemical shift assignments of the fibronectin III like domains 7-8 of type VII collagen.

Type VII collagen (Col7) is important for skin stability. This is underlined by the severe skin blistering phenotype in the Col7 related diseases dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita (EBA). Col7 has a large N-terminal non-collagenous domain (NC1) that is followed by the triple helical collagenous domain. The NC1 domain has subdomains with homology to adhesion molecules and mediates important interactions within the extracellular matrix. An 185 amino acid long part of the NC1-subdomain termed fibronectin III like domains 7 and 8 (FNIII7-8) was investigated. Antibodies against this region are pathogenic in a mouse model of EBA and one reported missense mutations of Col7 lies within these domains. The nearly complete NMR resonance assignment of recombinant FNIII7-8 of Col7 is reported.

Radiosensitive Hematopoietic Cells Determine the Extent of Skin Inflammation in Experimental Epidermolysis Bullosa Acquisita.

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.

A recombinant fusion protein derived from dog hookworm inhibits autoantibody-induced dermal-epidermal separation ex vivo.

The proteins secreted by parasitic nematodes are evolutionarily optimized molecules with unique capabilities of suppressing the immune response of the host organism. Neutrophil inhibitory factor (NIF), which is secreted by the dog hookworm Ancylostoma caninum, binds to the ß2 integrin CD11b/CD18, which is expressed on human neutrophils, eosinophils, monocytes and macrophages and inhibits neutrophil-dependent lung injury and neutrophil invasion of ischaemic brain tissue. Neutrophils are key players in the pathogenesis of subepidermal autoimmune blistering diseases (sAIBDs), and their pathogenic activities are crucially dependent on ß2 integrin functionality. Based on the template of single-stranded, dimerizing antibody derivatives, which are already used in cancer treatment, we designed a novel biologic, NIF-IGHE-CH4, comprising NIF and the dimerizing but otherwise inert constant heavy subdomain 4 (CH4) of human IgE (IGHE). This molecule was evaluated in a variety of in vitro assays, demonstrating its ability to inhibit pathogenically relevant neutrophil functions such as migration, adhesion and spreading, and release of reactive oxygen species. Finally, we confirmed that NIF-IGHE-CH4 inhibits blister formation in an ex vivo assay of sAIBD. These results suggest that NIF-IGHE-CH4 is a novel potential anti-inflammatory drug for the treatment of neutrophil-mediated diseases such as sAIBDs. This study promotes the drugs from bugs concept and encourages further research and development focused on turning parasite proteins into useful anti-inflammatory biologics.

The hinge region of type VII collagen is intrinsically disordered.

Type VII collagen (Col7) is important for skin integrity. As a major component of the anchoring fibrils, Col7 is essential for linking different skin layers together. The central collagenous domain of Col7 contains several interruptions of the collagen triple helix. The longest interruption is 39 amino acids long and referred to as the hinge region. The hinge region is highly conserved between species. This region was predicted to adopt a coiled coil structure and to serve as the trimerization domain of Col7. To gain insight into the potential function of the hinge region we investigated a heterologous expressed peptide by CD and NMR spectroscopy. CD spectroscopy implies that the hinge region is intrinsically disordered. Resonance assignment was performed and allowed secondary structure analysis based on the chemical shift values. Seven amino acids in the N-terminal moiety show residual α-helical conformation. Subsequent investigation of temperature dependency of amide chemical shifts indicated participation in hydrogen bonding of amino acid residues in the C-terminal moiety of the hinge region. Therefore, the hinge region does not form a coiled coil structure under the employed experimental conditions. The intrinsic disorder of the hinge region might be desired for flexibility to serve as a "hinge" or the hinge region is an important interaction site as typically observed for intrinsically disordered proteins.

The cysteine-rich region of type VII collagen is a cystine knot with a new topology.

Collagens are a group of extracellular matrix proteins with essential functions for skin integrity. Anchoring fibrils are made of type VII collagen (Col7) and link different skin layers together: the basal lamina and the underlying connective tissue. Col7 has a central collagenous domain and two noncollagenous domains located at the N and C terminus (NC1 and NC2), respectively. A cysteine-rich region of hitherto unknown function is located at the transition of the NC1 domain to the collagenous domain. A synthetic model peptide of this region was investigated by CD and NMR spectroscopy. The peptide folds into a collagen triple helix, and the cysteine residues form disulfide bridges between the different strands. The eight cystine knot topologies that are characterized by exclusively intermolecular disulfide bridges have been analyzed by molecular modeling. Two cystine knots are energetically preferred; however, all eight disulfide bridge arrangements are essentially possible. This novel cystine knot is present in type IX collagen, too. The conserved motif of the cystine knot is CX3CP. The cystine knot is N-terminal to the collagen triple helix in both collagens and therefore probably impedes unfolding of the collagen triple helix from the N terminus.

B cells, dendritic cells, and macrophages are required to induce an autoreactive CD4 helper T cell response in experimental epidermolysis bullosa acquisita.

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.

Metabolite analysis distinguishes between mice with epidermolysis bullosa acquisita and healthy mice.

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is a rare skin blistering disease with a prevalence of 0.2/ million people. EBA is characterized by autoantibodies against type VII collagen. Type VII collagen builds anchoring fibrils that are essential for the dermal-epidermal junction. The pathogenic relevance of antibodies against type VII collagen subdomains has been demonstrated both in vitro and in vivo. Despite the multitude of clinical and immunological data, no information on metabolic changes exists. METHODS: We used an animal model of EBA to obtain insights into metabolomic changes during EBA. Sera from mice with immunization-induced EBA and control mice were obtained and metabolites were isolated by filtration. Proton nuclear magnetic resonance (NMR) spectra were recorded and analyzed by principal component analysis (PCA), partial least squares discrimination analysis (PLS-DA) and random forest. RESULTS: The metabolic pattern of immunized mice and control mice could be clearly distinguished with PCA and PLS-DA. Metabolites that contribute to the discrimination could be identified via random forest. The observed changes in the metabolic pattern of EBA sera, i.e. increased levels of amino acid, point toward an increased energy demand in EBA. CONCLUSIONS: Knowledge about metabolic changes due to EBA could help in future to assess the disease status during treatment. Confirming the metabolic changes in patients needs probably large cohorts.

Metabolic changes during cellular senescence investigated by proton NMR-spectroscopy.

Cellular senescence is of growing interest due to its role in tumour suppression and its contribution to organismic ageing. This cellular state can be reached by replicative loss of telomeres or certain stresses in cell culture and is characterized by the termination of cell division; however, the cells remain metabolically active. To identify metabolites that are characteristic for senescent cells, extracts of human embryonic lung fibroblast (WI-38 cell line) have been investigated with NMR spectroscopy. Three different types of senescence have been characterized: replicative senescence, DNA damage-induced senescence (etoposide treatment) and oncogene-induced senescence (hyperactive RAF kinase). The metabolite pattern allows (I) discrimination of senescent and control cells and (II) discrimination of the three senescence types. Senescent cells show an increased ratio of glycerophosphocholine to phosphocholine independent from the type of senescence. The increase in glycerophosphocholine implicates a key role of phospholipid metabolism in cellular senescence. The observed changes in the choline metabolism are diametrically opposite to the well-known changes in choline metabolism of tumour cells. As tumours responding to chemotherapeutic agents show a "glycerophosphocholine-to-phosphocholine switch" i.e. an increase in glycerophosphocholine, our metabolic data suggests that these malignant cells enter a senescent state emphasizing the role of senescence in tumour suppression.

The vWFA2 domain of type VII collagen is responsible for collagen binding.

Type VII collagen (Col7) is the major component of anchoring fibrils and very important for skin integrity. This is emphasized by the Col7 related skin blistering diseases dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita. Structural data that provides insights into the interaction network of Col7 and thus providing a basis for a better understanding of the pathogenesis of the diseases is missing. We proved that the von-Willebrand-factor A like domain 2 (vWFA2) of Col7 is responsible for type I collagen binding. The interaction has a K(D) value of 90 µM as determined by SPR and is enthalpy driven as derived from the van't Hoff equation. Furthermore, a hitherto unknown interaction of this domain with type IV collagen was identified. The interaction of vWFA2 with type I collagen is sensitive to the presence of magnesium ions, however, vWFA2 does not contain a magnesium binding site thus magnesium must bind to type I collagen. A lysine residue has been identified to be crucial for type I collagen binding. This allowed localization of the binding site. Mutational analysis suggests different interaction mechanisms in different species and that these interactions might be of covalent nature.

Surprising complexity of the Asf1 histone chaperone-Rad53 kinase interaction.

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, (R/K)R(I/A/V) (L/P), 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.