Specific domain V reduction of beta-2-glycoprotein I induces protein flexibility and alters pathogenic antibody binding.

Beta-2-glycoprotein I (ß2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). ß2GPI exists mainly in closed or open conformations and comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide bond at domain V has been associated with different cysteine redox states. The role of this disulfide bond in conformational dynamics of this protein has not been investigated so far. Here, we report on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine; TCEP) of ß2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry analyses confirming majority targeting to the fifth domain of ß2GPI. Atomic force microscopy images suggested that reduced ß2GPI shows a slightly higher proportion of open conformation and is more flexible compared to the untreated protein as confirmed by modelling studies. We have determined a strong increase in the binding of pathogenic APS autoantibodies to reduced ß2GPI as demonstrated by ELISA. Our study is relevant for understanding the effect of ß2GPI reduction on the protein structure and its implications for antibody binding in APS patients.

New insights into the epidemiology of ANCA-associated vasculitides in Germany: results from a claims data study.

OBJECTIVE: ANCA-associated vasculitides (AAV) are rare, potentially life-threatening autoimmune diseases characterized by systemic inflammation and organ damage. AAV prevalence rates reported in Europe vary considerably and robust data sources are often lacking. This study aimed to examine the feasibility of claims data analysis as a complementary method to registry-based studies to assess the epidemiology of AAV. METHODS: In this retrospective observational study, anonymized longitudinal claims data from years 2013-2016 from German statutory health insurance companies (data source: InGef, Institute for Applied Health Research) have been analysed on an age- and gender-stratified cohort of ∼3 million persons representative of the German population. In this cohort, granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) patients were identified. RESULTS: The study cohort revealed a prevalence for GPA and MPA of 210 and 46 cases per million people, respectively. The annual incidence comprised 34 GPA cases and 13 MPA cases per million people per year. Hence, 17 500 AAV patients (GPA and MPA) are estimated to live in Germany, with an annual increase of 3200 patients. According to their demographic and disease-specific characteristics, AAV patients identified in this claims data approach are representative. CONCLUSION: This is the first study using claims data to assess the epidemiology of AAV. In Germany, AAV was diagnosed more frequently than it was estimated by previous self-reporting registry-based studies. The findings indicate that epidemiological data of AAV may have been underestimated but may also reflect improved diagnostic methods and disease recognition.

Thermodynamics, cooperativity and stability of the tetracycline repressor (TetR) upon tetracycline binding.

Allosteric regulation of the Tet repressor (TetR) homodimer relies on tetracycline binding that abolishes the affinity for the DNA operator. Previously, interpretation of circular dichroism data called for unfolding of the α-helical DNA-binding domains in absence of binding to DNA or tetracycline. Our small angle X-ray scattering of TetR(D) in solution contradicts this unfolding as a physiological process. Instead, in the core domain crystal structures analyses show increased immobilisation of helix α9 and two C-terminal turns of helix α8 upon tetracycline binding. Tetracycline complexes of TetR(D) and four single-site alanine variants were characterised by isothermal titration calorimetry, fluorescence titration, X-ray crystal structures, and melting curves. Five crystal structures confirm that Thr103 is a key residue for the allosteric events of induction, with the T103A variant lacking induction by any tetracycline. The T103A variant shows anti-cooperative inducer binding, and a melting curve of the tetracycline complex different to TetR(D) and other variants. For the N82A variant inducer binding is clearly anti-cooperative but triggers the induced conformation.

The role of beta-2-glycoprotein I in health and disease associating structure with function: More than just APS.

Beta-2-Glycoprotein I (ß2GPI) plays a number of essential roles throughout the body. ß2GPI, C-reactive protein and thrombomodulin are the only three proteins that possess the dual capability to up and down regulate the complement and coagulation systems depending upon external stimulus. Clinically, ß2GPI is the primary antigen in the autoimmune condition antiphospholipid syndrome (APS), which is typically characterised by pregnancy morbidity and vascular thrombosis. This protein is also capable of adopting at least two distinct structural forms, but it has been argued that several other intermediate forms may exist. Thus, ß2GPI is a unique protein with a key role in haemostasis, homeostasis and immunity. In this review, we examine the genetics, structure and function of ß2GPI in the body and how these factors may influence its contribution to disease pathogenesis. We also consider the clinical implications of ß2GPI in the diagnosis of APS and as a potentially novel therapeutic target.

The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant.

One of the most common mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene is the N34S variant which is strongly associated with chronic pancreatitis. Although it is assumed that N34S mutation constitutes a high-risk factor, the underlying pathologic mechanism is still unknown. In the present study, we investigated the impact of physiological stress factors on SPINK1 protein structure and trypsin inhibitor function using biophysical methods. Our circular dichroism spectroscopy data revealed differences in the secondary structure of SPINK1 and N34S mutant suggesting protein structural changes induced by the mutation as an impairment that could be disease-relevant. We further confirmed that both SPINK1 (KD of 0.15 ±â€¯0.06 nM) and its N34S variant (KD of 0.08 ±â€¯0.02 nM) have similar binding affinity and inhibitory effect towards trypsin as shown by surface plasmon resonance and trypsin inhibition assay studies, respectively. We found that stress conditions such as altered ion concentrations (i.e. potassium, calcium), temperature shifts, as well as environmental pH lead to insignificant differences in trypsin inhibition between SPINK1 and N34S mutant. However, we have shown that the environmental pH induces structural changes in both SPINK1 constructs in a different manner. Our findings suggest protein structural changes in the N34S variant as an impairment of SPINK1 and environmental pH shift as a trigger that could play a role in disease progression of pancreatitis.

Drug-induced activation of integrin alpha IIb beta 3 leads to minor localized structural changes.

Integrins are transmembrane proteins involved in hemostasis, wound healing, immunity and cancer. In response to intracellular signals and ligand binding, integrins adopt different conformations: the bent (resting) form; the intermediate extended form; and the ligand-occupied active form. An integrin undergoing such conformational dynamics is the heterodimeric platelet receptor αIIbß3. Although the dramatic rearrangement of the overall structure of αIIbß3 during the activation process is potentially related to changes in the protein secondary structure, this has not been investigated so far in a membrane environment. Here we examine the Mn2+- and drug-induced activation of αIIbß3 and the impact on the structure of this protein reconstituted into liposomes. By quartz crystal microbalance with dissipation monitoring and activation assays we show that Mn2+ induces binding of the conformation-specific antibody PAC-1, which only recognizes the extended, active integrin. Circular dichroism spectroscopy reveals, however, that Mn2+-treatment does not induce major secondary structural changes of αIIbß3. Similarly, we found that treatment with clinically relevant drugs (e.g. quinine) led to the activation of αIIbß3 without significant changes in protein secondary structure. Molecular dynamics simulation studies revealed minor local changes in the beta-sheet probability of several extracellular domains of the integrin. Our experimental setup represents a new approach to study transmembrane proteins, especially integrins, in a membrane environment and opens a new way for testing drug binding to integrins under clinically relevant conditions.

Lysine residues control the conformational dynamics of beta 2-glycoprotein I.

One of the major problems in the study of the dynamics of proteins is the visualization of changing conformations that are important for processes ranging from enzyme catalysis to signaling. A protein exhibiting conformational dynamics is the soluble blood protein beta 2-glycoprotein I (beta2GPI), which exists in two conformations: the closed (circular) form and the open (linear) form. It is hypothesized that an increased proportion of the open conformation leads to the autoimmune disease antiphospholipid syndrome (APS). A characteristic feature of beta2GPI is the high content of lysine residues. However, the potential role of lysine in the conformational dynamics of beta2GPI has been poorly investigated. Here, we report on a strategy to permanently open up the closed protein conformation by chemical acetylation of lysine residues using acetic acid N-hydroxysuccinimide ester (NHS-Ac). Specific and complete acetylation was demonstrated by the quantification of primary amino groups with fluoraldehyde o-phthalaldehyde (OPA) reagent, as well as western blot analysis with an anti-acetylated lysine antibody. Our results demonstrate that acetylated beta2GPI preserves its secondary and tertiary structures, as shown by circular dichroism spectroscopy. We found that after lysine acetylation, the majority of proteins are in the open conformation as revealed by atomic force microscopy high-resolution images. Using this strategy, we proved that the electrostatic interaction of lysine residues plays a major role in stabilizing the beta2GPI closed conformation, as confirmed by lysine charge distribution calculations. We foresee that our approach will be applied to other lysine-rich proteins (e.g. histones) undergoing conformational transitions. For instance, conformational dynamics can be triggered by environmental conditions (e.g. pH, ion concentration, post-translational modifications, and binding of ligands). Therefore, our study may be relevant for investigating the equilibrium of protein conformations causing diseases.

Selective Targeting of G-Quadruplex Structures by a Benzothiazole-Based Binding Motif.

A benzothiazole derivative was identified as potent ligand for DNA G-quadruplex structures. Fluorescence titrations revealed selective binding to quadruplexes of different topologies including parallel, antiparallel, and (3+1) hybrid structures. The parallel c-MYC sequence was found to constitute the preferred target with dissociation constants in the micromolar range. Binding of the benzothiazole-based ligand to c-MYC was structurally and thermodynamically characterized in detail by employing a comprehensive set of spectroscopic and calorimetric techniques. Job plot analyses and mass spectral data indicate noncooperative ligand binding to form complexes with 1:1 and 2:1 stoichiometries. Whereas stacking interactions are suggested by optical methods, NMR chemical shift perturbations also indicate significant rearrangements of both 5'- and 3'-flanking sequences upon ligand binding. Additional isothermal calorimetry studies yield a thermodynamic profile of the ligand-quadruplex association and reveal enthalpic contributions to be the major driving force for binding. Structural and thermodynamic information obtained in the present work provides the basis for the rational development of benzothiazole derivatives as promising quadruplex binding agents.

You are what you talk: quorum sensing induces individual morphologies and cell division modes in Dinoroseobacter shibae.

Dinoroseobacter shibae, a member of the Roseobacter clade abundant in marine environments, is characterized by a pronounced pleomorphism. Cell shapes range from variable-sized ovoid rods to long filaments with a high copy number of chromosomes. Time-lapse microscopy shows cells dividing either by binary fission or by budding from the cell poles. Here we demonstrate that this morphological heterogeneity is induced by quorum sensing (QS). D. shibae utilizes three acylated homoserine lactone (AHL) synthases (luxI1-3) to produce AHLs with unsaturated C18 side chains. A ΔluxI1-knockout strain completely lacking AHL biosynthesis was uniform in morphology and divided by binary fission only. Transcriptome analysis revealed that expression of genes responsible for control of cell division was reduced in this strain, providing the link between QS and the observed phenotype. In addition, flagellar biosynthesis and type IV secretion system (T4SS) were downregulated. The wild-type phenotype and gene expression could be restored through addition of synthetic C18-AHLs. Their effectiveness was dependent on the number of double bonds in the acyl side chain and the regulated trait. The wild-type expression level of T4SS genes was fully restored even by an AHL with a saturated C18 side chain that has not been detected in D. shibae. QS induces phenotypic individualization of D. shibae cells rather than coordinating the population. This strategy might be beneficial in unpredictably changing environments, for example, during algal blooms when resource competition and grazing exert fluctuating selective pressures. A specific response towards non-native AHLs might provide D. shibae with the capacity for complex interspecies communication.

Complete sequence of the suicide vector pJP5603.

We have sequenced the complete R6K-based and mobilizable suicide vector pJP5603. For the replication of the vector a trans supply of the pir-encoded π protein of plasmid R6K is essential. The 3.126 kb plasmid encodes a kanamycin resistance cassette for selection and contains a lacZ-α-system that allows a blue-white selection of cloned fragments.

Constitutive expression of the proteorhodopsin gene by a flavobacterium strain representative of the proteorhodopsin-producing microbial community in the North Sea.

Proteorhodopsin (PR), a photoactive proton pump containing retinal, is present in approximately half of all bacteria in the ocean, but its physiological role is still unclear, since very few strains carrying the PR gene have been cultured. The aim of this work was to characterize PR diversity in a North Sea water sample, cultivate a strain representative of North Sea PR clusters, and study the effects of light and carbon concentration on the expression of the PR gene. A total of 117 PR sequences, of which 101 were unique, were obtained from a clone library of PCR-amplified PR gene fragments. Of the North Sea PRs, 97% were green light absorbing, as inferred from the amino acid at position 105; 67% of the PR protein fragments showed closest similarity to PRs from Alphaproteobacteria, 4% showed closest similarity to PRs from Gammaproteobacteria, and 29% showed closest similarity to PRs from "Bacteroidetes"/Flavobacteria. The dominant PR cluster (comprising 18% of all PRs) showed a high degree of similarity to the PR from the cultivated Roseobacter strain HTCC2255. The relative abundances of the North Sea PR clusters were confirmed by quantitative PCR. They were detected in metagenomic fragments from coastal oceans worldwide with various degrees of abundance. Several hundred bacterial strains from the North Sea water sample were cultivated on oligocarbophilic media. By screening with degenerate primers, two strains carrying the PR gene were identified. Their 16S rRNA gene sequences were identical and affiliated with a Bacteroidetes subcluster from the North Sea. The PR sequence of isolate PRO95 was completed by chromosomal walking. It was 76% identical to that of Dokdonia donghaensis MED134 and was functional, as indicated by the signature amino acids. PRO95 expressed its PR gene in liquid media containing between 9.7 and 121 mM carbon, both in the light and in the dark. Growth was not enhanced by light. Thus, the detection of the physiological role of PR may require more sensitive methods.

The complete genome sequence of the algal symbiont Dinoroseobacter shibae: a hitchhiker's guide to life in the sea.

Dinoroseobacter shibae DFL12(T), a member of the globally important marine Roseobacter clade, comprises symbionts of cosmopolitan marine microalgae, including toxic dinoflagellates. Its annotated 4 417 868 bp genome sequence revealed a possible advantage of this symbiosis for the algal host. D. shibae DFL12(T) is able to synthesize the vitamins B(1) and B(12) for which its host is auxotrophic. Two pathways for the de novo synthesis of vitamin B(12) are present, one requiring oxygen and the other an oxygen-independent pathway. The de novo synthesis of vitamin B(12) was confirmed to be functional, and D. shibae DFL12(T) was shown to provide the growth-limiting vitamins B(1) and B(12) to its dinoflagellate host. The Roseobacter clade has been considered to comprise obligate aerobic bacteria. However, D. shibae DFL12(T) is able to grow anaerobically using the alternative electron acceptors nitrate and dimethylsulfoxide; it has the arginine deiminase survival fermentation pathway and a complex oxygen-dependent Fnr (fumarate and nitrate reduction) regulon. Many of these traits are shared with other members of the Roseobacter clade. D. shibae DFL12(T) has five plasmids, showing examples for vertical recruitment of chromosomal genes (thiC) and horizontal gene transfer (cox genes, gene cluster of 47 kb) possibly by conjugation (vir gene cluster). The long-range (80%) synteny between two sister plasmids provides insights into the emergence of novel plasmids. D. shibae DFL12(T) shows the most complex viral defense system of all Rhodobacterales sequenced to date.

Genetic tools for the investigation of Roseobacter clade bacteria.

BACKGROUND: The Roseobacter clade represents one of the most abundant, metabolically versatile and ecologically important bacterial groups found in marine habitats. A detailed molecular investigation of the regulatory and metabolic networks of these organisms is currently limited for many strains by missing suitable genetic tools. RESULTS: Conjugation and electroporation methods for the efficient and stable genetic transformation of selected Roseobacter clade bacteria including Dinoroseobacter shibae, Oceanibulbus indolifex, Phaeobacter gallaeciensis, Phaeobacter inhibens, Roseobacter denitrificans and Roseobacter litoralis were tested. For this purpose an antibiotic resistance screening was performed and suitable genetic markers were selected. Based on these transformation protocols stably maintained plasmids were identified. A plasmid encoded oxygen-independent fluorescent system was established using the flavin mononucleotide-based fluorescent protein FbFP. Finally, a chromosomal gene knockout strategy was successfully employed for the inactivation of the anaerobic metabolism regulatory gene dnr from D. shibae DFL12T. CONCLUSION: A genetic toolbox for members of the Roseobacter clade was established. This provides a solid methodical basis for the detailed elucidation of gene regulatory and metabolic networks underlying the ecological success of this group of marine bacteria.