Vascular adhesion protein-1-targeted PET imaging in autoimmune myocarditis.

BACKGROUND: Vascular adhesion protein-1 (VAP-1) is an adhesion molecule and primary amine oxidase, and Gallium-68-labeled 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetra-acetic acid conjugated sialic acid-binding immunoglobulin-like lectin 9 motif containing peptide ([68Ga]Ga-DOTA-Siglec-9) is a positron emission tomography (PET) tracer targeting VAP-1. We evaluated the feasibility of PET imaging with [68Ga]Ga-DOTA-Siglec-9 for the detection of myocardial lesions in rats with autoimmune myocarditis. METHODS: Rats (n = 9) were immunized twice with porcine cardiac myosin in complete Freund's adjuvant. Control rats (n = 6) were injected with Freund's adjuvant alone. On day 21, in vivo PET/computed tomography (CT) imaging with [68Ga]Ga-DOTA-Siglec-9 was performed, followed by ex vivo autoradiography, histology, and immunohistochemistry of tissue sections. In addition, myocardial samples from three patients with cardiac sarcoidosis were studied. RESULTS: [68Ga]Ga-DOTA-Siglec-9 PET/CT images of immunized rats showed higher uptake in myocardial lesions than in myocardium outside lesions (SUVmean, 0.5 ± 0.1 vs 0.3 ± 0.1; P = .003) or control rats (SUVmean, 0.2 ± 0.03; P < .0001), which was confirmed by ex vivo autoradiography of tissue sections. Immunohistochemistry showed VAP-1-positive staining in lesions of rats with myocarditis and in patients with cardiac sarcoidosis. CONCLUSION: VAP-1-targeted [68Ga]Ga-DOTA-Siglec-9 PET is a potential novel technique for the detection of myocardial lesions.

PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion.

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.

Revealing secrets of the enigmatic omega subunit of bacterial RNA polymerase.

The conserved omega (ω) subunit of RNA polymerase (RNAP) is the only nonessential subunit of bacterial RNAP core. The small ω subunit (7 kDa-11.5 kDa) contains three conserved α helices, and helices α2 and α3 contain five fully conserved amino acids of ω. Four conserved amino acids stabilize the correct folding of the ω subunit and one is located in the vicinity of the ß' subunit of RNAP. Otherwise ω shows high variation between bacterial taxa, and although the main interaction partner of ω is always ß', many interactions are taxon-specific. ω-less strains show pleiotropic phenotypes, and based on in vivo and in vitro results, a few roles for the ω subunits have been described. Interactions of the ω subunit with the ß' subunit are important for the RNAP core assembly and integrity. In addition, the ω subunit plays a role in promoter selection, as ω-less RNAP cores recruit fewer primary σ factors and more alternative σ factors than intact RNAP cores in many species. Furthermore, the promoter selection of an ω-less RNAP holoenzyme bearing the primary σ factor seems to differ from that of an intact RNAP holoenzyme.

Conserved lysine residues in decorin binding proteins of Borrelia garinii are critical in adhesion to human brain microvascular endothelial cells.

Lyme borreliosis is a tick-borne disease caused by Borrelia burgdorferi sensu lato spirochetes (Lyme borreliae). When the disease affects the central nervous system, it is referred to as neuroborreliosis. In Europe, neuroborreliosis is most often caused by Borrelia garinii. Although it is known that in the host Lyme borreliae spread from the tick bite site to distant tissues via the blood vasculature, the adherence of Lyme borreliae to human brain microvascular endothelial cells has not been studied before. Decorin binding proteins are adhesins expressed on Lyme borreliae. They mediate the adhesion of Lyme borreliae to decorin and biglycan, and the lysine residues located in the binding site of decorin binding proteins are important to the binding activity. In this study, we show that lysine residues located in the canonical binding site can also be found in decorin binding proteins of Borrelia garinii, and that these lysines contribute to biglycan and decorin binding. Most importantly, we show that the lysine residues are crucial for the binding of Lyme borreliae to decorin and biglycan expressing human brain microvascular endothelial cells, which in turn suggests that they are involved in the pathogenesis of neuroborreliosis.

Nafamostat is a Potent Human Diamine Oxidase Inhibitor Possibly Augmenting Hypersensitivity Reactions during Nafamostat Administration.

Nafamostat is an approved short-acting serine protease inhibitor. However, its administration is also associated with anaphylactic reactions. One mechanism to augment hypersensitivity reactions could be inhibition of diamine oxidase (DAO). The chemical structure of nafamostat is related to the potent DAO inhibitors pentamidine and diminazene. Therefore, we tested whether nafamostat is a human DAO inhibitor. Using different activity assays, nafamostat reversibly inhibited recombinant human DAO with an IC50 of 300-400 nM using 200 µM substrate concentrations. The Ki of nafamostat for the inhibition of putrescine and histamine deamination is 27 nM and 138 nM, respectively For both substrates, nafamostat is a mixed mode inhibitor with P values of <0.01 compared with other inhibition types. Using 80-90% EDTA plasma, the IC50 of nafamostat inhibition was approximately 360 nM using 20 µM cadaverine. In 90% EDTA plasma, the IC50 concentrations were 2-3 µM using 0.9 µM and 0.18 µM histamine as substrate. In silico modeling showed a high overlap compared with published diminazene crystallography data, with a preferred orientation of the guanidine group toward topaquinone. In conclusion, nafamostat is a potent human DAO inhibitor and might increase severity of anaphylactic reaction by interfering with DAO-mediated extracellular histamine degradation. SIGNIFICANCE STATEMENT: Treatment with the short-acting anticoagulant nafamostat during hemodialysis, leukocytapheresis, extracorporeal membrane oxygenator procedures, and disseminated intravascular coagulation is associated with severe anaphylaxis in humans. Histamine is a central mediator in anaphylaxis. Potent inhibition of the only extracellularly histamine-degrading enzyme diamine oxidase could augment anaphylaxis reactions during nafamostat treatment.

Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent.

Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possesses an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition and identified VgrG4 as a T6SS effector. The DUF2345 domain of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition.

Human diamine oxidase cellular binding and internalization in vitro and rapid clearance in vivo are not mediated by N-glycans but by heparan sulfate proteoglycan interactions.

Human diamine oxidase (hDAO) rapidly inactivates histamine by deamination. No pharmacokinetic data are available to better understand its potential as a new therapeutic modality for diseases with excess local and systemic histamine, like anaphylaxis, urticaria or mastocytosis. After intravenous administration of recombinant hDAO to rats and mice, more than 90% of the dose disappeared from the plasma pool within 10 min. Human DAO did not only bind to various endothelial and epithelial cell lines in vitro, but was also unexpectedly internalized and visible in granule-like structures. The uptake of rhDAO into cells was dependent on neither the asialoglycoprotein-receptor (ASGP-R) nor the mannose receptor (MR) recognizing terminal galactose or mannose residues, respectively. Competition experiments with ASGP-R and MR ligands did not block internalization in vitro or rapid clearance in vivo. The lack of involvement of N-glycans was confirmed by testing various glycosylation mutants. High but not low molecular weight heparin strongly reduced the internalization of rhDAO in HepG2 cells and HUVECs. Human DAO was readily internalized by CHO-K1 cells, but not by the glycosaminoglycan- and heparan sulfate-deficient CHO cell lines pgsA-745 and pgsD-677, respectively. A docked heparin hexasaccharide interacted well with the predicted heparin binding site 568RFKRKLPK575. These results strongly imply that rhDAO clearance in vivo and cellular uptake in vitro is independent of N-glycan interactions with the classical clearance receptors ASGP-R and MR, but is mediated by binding to heparan sulfate proteoglycans followed by internalization via an unknown receptor.

Genetic and functional implications of an exonic TRIM55 variant in heart failure.

BACKGROUND: To tackle the missing heritability of sporadic heart failure, we screened for novel heart failure-associated genetic variants in the Finnish population and functionally characterized a novel variant in vitro and in vivo. METHODS AND RESULTS: Heart failure-associated variants were screened in genotyping array data of the FINRISK study, consisting of 994 cases and 20,118 controls. Based on logistic regression analysis, a potentially damaging variant in TRIM55 (rs138811034), encoding an E140K variant, was selected for validations. In HL-1 cardiomyocytes, we used CRISPR/Cas9 technology to introduce the variant in the endogenous locus, and additionally TRIM55 wildtype or E140K was overexpressed from plasmid. Functional responses were profiled using whole-genome RNA sequencing, RT-PCR and Western analyses, cell viability and cell cycle assays and cell surface area measurements. In zebrafish embryos, cardiac contractility was measured using videomicroscopy after CRISPR-mediated knockout of trim55a or plasmid overexpression of TRIM55 WT or E140K. Genes related to muscle contraction and cardiac stress were highly regulated in Trim55 E140K/- cardiomyocytes. When compared to the WT/WT cells, the variant cells demonstrated reduced viability, significant hypertrophic response to isoproterenol, p21 protein overexpression and impaired cell cycle progression. In zebrafish embryos, the deletion of trim55a or overexpression of TRIM55 E140K reduced cardiac contractility as compared to embryos with wildtype genotype or overexpression of WT TRIM55, respectively. CONCLUSIONS: A previously uncharacterized TRIM55 E140K variant demonstrated a number of functional implications for cardiomyocyte functions in vitro and in vivo. These findings suggest a novel role for TRIM55 polymorphism in predisposing to heart failure.

Structural and Biomolecular Analyses of Borrelia burgdorferi BmpD Reveal a Substrate-Binding Protein of an ABC-Type Nucleoside Transporter Family.

Borrelia burgdorferisensu lato, the causative agent of tick-borne Lyme borreliosis (LB), has a limited metabolic capacity and needs to acquire nutrients, such as amino acids, fatty acids, and nucleic acids, from the host environment. Using X-ray crystallography, liquid chromatography-mass spectrometry, microscale thermophoresis, and cellular localization studies, we show that basic membrane protein D (BmpD) is a periplasmic substrate-binding protein of an ABC transporter system binding to purine nucleosides. Nucleosides are essential for bacterial survival in the host organism, and these studies suggest a key role for BmpD in the purine salvage pathway of B. burgdorferi sensu lato Because B. burgdorferisensu lato lacks the enzymes required for de novo purine synthesis, BmpD may play a vital role in ensuring access to the purines needed to sustain an infection in the host. Furthermore, we show that, although human LB patients develop anti-BmpD antibodies, immunization of mice with BmpD does not confer protection against B. burgdorferi sensu lato infection.

Crystal structure of dimeric Synechococcus spermidine synthase with bound polyamine substrate and product.

Spermidine is a ubiquitous polyamine synthesized by spermidine synthase (SPDS) from the substrates, putrescine and decarboxylated S-adenosylmethionine (dcAdoMet). SPDS is generally active as homodimer, but higher oligomerization states have been reported in SPDS from thermophiles, which are less specific to putrescine as the aminoacceptor substrate. Several crystal structures of SPDS have been solved with and without bound substrates and/or products as well as inhibitors. Here, we determined the crystal structure of SPDS from the cyanobacterium Synechococcus (SySPDS) that is a homodimer, which we also observed in solution. Unlike crystal structures reported for bacterial and eukaryotic SPDS with bound ligands, SySPDS structure has not only bound putrescine substrate taken from the expression host, but also spermidine product most probably as a result of an enzymatic reaction. Hence, to the best of our knowledge, this is the first structure reported with both amino ligands in the same structure. Interestingly, the gate-keeping loop is disordered in the putrescine-bound monomer while it is stabilized in the spermidine-bound monomer of the SySPDS dimer. This confirms the gate-keeping loop as the key structural element that prepares the active site upon binding of dcAdoMet for the catalytic reaction of the amine donor and putrescine.

Human Copper-Containing Amine Oxidases in Drug Design and Development.

Two members of the copper-containing amine oxidase family are physiologically important proteins: (1) Diamine oxidase (hDAO; AOC1) with a preference for diamines is involved in degradation of histamine and (2) Vascular adhesion protein-1 (hVAP-1; AOC3) with a preference for monoamines is a multifunctional cell-surface receptor and an enzyme. hVAP-1-targeted inhibitors are designed to treat inflammatory diseases and cancer, whereas the off-target binding of the designed inhibitors to hDAO might result in adverse drug reactions. The X-ray structures for both human enzymes are solved and provide the basis for computer-aided inhibitor design, which has been reported by several research groups. Although the putative off-target effect of hDAO is less studied, computational methods could be easily utilized to avoid the binding of VAP-1-targeted inhibitors to hDAO. The choice of the model organism for preclinical testing of hVAP-1 inhibitors is not either trivial due to species-specific binding properties of designed inhibitors and different repertoire of copper-containing amine oxidase family members in mammalian species. Thus, the facts that should be considered in hVAP-1-targeted inhibitor design are discussed in light of the applied structural bioinformatics and structural biology approaches.

Candida antarctica Lipase A-Based Enantiorecognition of a Highly Strained 4-Dibenzocyclooctynol (DIBO) Used for PET Imaging.

The enantiomers of aromatic 4-dibenzocyclooctynol (DIBO), used for radiolabeling and subsequent conjugation of biomolecules to form radioligands for positron emission tomography (PET), were separated by kinetic resolution using lipase A from Candida antarctica (CAL-A). In optimized conditions, (R)-DIBO [(R)-1, ee 95%] and its acetylated (S)-ester [(S)-2, ee 96%] were isolated. In silico docking results explained the ability of CAL-A to differentiate the enantiomers of DIBO and to accommodate various acyl donors. Anhydrous MgCl2 was used for binding water from the reaction medium and, thus, for obtaining higher conversion by preventing hydrolysis of the product (S)-2 into the starting material. Since the presence of hydrated MgCl26H2O also allowed high conversion or effect on enantioselectivity, Mg2+ ion was suspected to interact with the enzyme. Binding site predictions indicated at least two sites of interest; one in the lid domain at the bottom of the acyl binding pocket and another at the interface of the hydrolase and flap domains, just above the active site.

Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms.

Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure. Hydroxylation of proline residues is critical for helix stability, and diminished prolyl hydroxylase activity causes wide-spread defects in connective tissues. Still, the role of proline hydroxylation in the binding of collagen receptors such as integrins is unclear. Here, we isolated skin collagen from genetically modified mice having reduced prolyl 4-hydroxylase activity. At room temperature, the reduced proline hydroxylation did not affect interactions with the recombinant integrin α2I domain, but at 37 °C, collagen hydroxylation correlated with the avidity of α2I domain binding. Of note, LC-MS/MS analysis of isolated skin collagens revealed no major changes in the hydroxyproline content of the main integrin-binding sites. Thus, the disrupted α2I domain binding at physiological temperatures was most likely due to structural destabilization of the collagenous helix. Integrin α2I binding to the triple-helical GFPGER motif was slightly weaker than to GFOGER (O = hydroxyproline). This phenomenon was more prominent when α1 integrin was tested. Integrin α1ß1 expressed on CHO cells and recombinant α1I domain showed remarkably slower binding velocity and weaker avidity to GFPGER when compared with GFOGER. Structural modeling revealed the critical interaction between Arg-218 in α1I and the hydroxyproline residue in the integrin-binding motif. The role of Arg-218 was further validated by testing a variant R218D α1I domain in solid-phase binding assays. Thus, our results show that the lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via structural destabilization of the triple helix.

Oligomannosidic glycans at Asn-110 are essential for secretion of human diamine oxidase.

N-Glycosylation plays a fundamental role in many biological processes. Human diamine oxidase (hDAO), required for histamine catabolism, has multiple N-glycosylation sites, but their roles, for example in DAO secretion, are unclear. We recently reported that the N-glycosylation sites Asn-168, Asn-538, and Asn-745 in recombinant hDAO (rhDAO) carry complex-type glycans, whereas Asn-110 carries only mammalian-atypical oligomannosidic glycans. Here, we show that Asn-110 in native hDAO from amniotic fluid and Caco-2 cells, DAO from porcine kidneys, and rhDAO produced in two different HEK293 cell lines is also consistently occupied by oligomannosidic glycans. Glycans at Asn-168 were predominantly sialylated with bi- to tetra-antennary branches, and Asn-538 and Asn-745 had similar complex-type glycans with some tissue- and cell line-specific variations. The related copper-containing amine oxidase human vascular adhesion protein-1 also exclusively displayed high-mannose glycosylation at Asn-137. X-ray structures revealed that the residues adjacent to Asn-110 and Asn-137 form a highly conserved hydrophobic cleft interacting with the core trisaccharide. Asn-110 replacement with Gln completely abrogated rhDAO secretion and caused retention in the endoplasmic reticulum. Mutations of Asn-168, Asn-538, and Asn-745 reduced rhDAO secretion by 13, 71, and 32%, respectively. Asn-538/745 double and Asn-168/538/745 triple substitutions reduced rhDAO secretion by 85 and 94%. Because of their locations in the DAO structure, Asn-538 and Asn-745 glycosylations might be important for efficient DAO dimer formation. These functional results are reflected in the high evolutionary conservation of all four glycosylation sites. Human DAO is abundant only in the gastrointestinal tract, kidney, and placenta, and glycosylation seems essential for reaching high enzyme expression levels in these tissues.

Evolution and functional classification of mammalian copper amine oxidases.

Mammalian copper-containing amine oxidases (CAOs), encoded by four genes (AOC1-4) and catalyzing the oxidation of primary amines to aldehydes, regulate many biological processes and are linked to various diseases including inflammatory conditions and histamine intolerance. Despite the known differences in their substrate preferences, CAOs are currently classified based on their preference for either primary monoamines (EC 1.4.3.21) or diamines (EC 1.4.3.22). Here, we present the first extensive phylogenetic study of CAOs that, combined with structural analyses of the CAO active sites, provides in-depth knowledge of their relationships and guidelines for classification of mammalian CAOs into AOC1-4 sub-families. The phylogenetic results show that CAOs can be classified based on two residues, X1 and X2, from the active site motif: T/S-X1-X2-N-Y-D. Residue X2 discriminates among the AOC1 (Tyr), AOC2 (Gly), and AOC3/AOC4 (Leu) proteins, while residue X1 further classifies the AOC3 (Leu) and AOC4 (Met) proteins that so far have been poorly identified and annotated. Residues X1 and X2 conserved within each sub-family and located in the catalytic site seem to be the key determinants for the unique substrate preference of each CAO sub-family. Furthermore, one residue located at 10 Šdistance from the catalytic site is different between the sub-families but highly conserved within each sub-family (Asp in AOC1, His in AOC2, Thr in AOC3 and Asn in AOC4) and likely contributes to substrate selectivity. Altogether, our results will benefit the design of new sub-family specific inhibitors and the design of in vitro tests to detect individual CAO levels for diagnostic purposes.

Predicting the ligand-binding properties of Borrelia burgdorferi s.s. Bmp proteins in light of the conserved features of related Borrelia proteins.

Bacteria of the genus Borrelia cause vector-borne infections like the most important hard tick-borne disease in the northern hemisphere, Lyme borreliosis (LB), and soft tick or louse transmitted relapsing fevers (RF), prevalent in temperate and tropical areas. Borrelia burgdorferi sensu lato (s.l.) includes several genospecies and causes LB in humans. In infected patients, Borrelia burgdorferi sensu stricto (s.s.) expresses the BmpA, BmpB, BmpC and BmpD proteins. The role of these proteins in the pathogenesis of LB remains incompletely characterized, but they are, however, closely related to Treponema pallidum PnrA (Purine nucleoside receptor A), a substrate-binding lipoprotein of the ATP-binding cassette (ABC) transporter family preferentially binding purine nucleosides. Based on 3D homology modeling, the Bmp proteins share the typical fold of the substrate-binding protein family and the ligand-binding properties of BmpA, BmpB and BmpD are highly similar, whereas those of BmpC differ markedly. Nevertheless, these residues are highly conserved within the genus Borrelia and the inferred phylogenetic tree also reveals that the RF Borrelia lack BmpB proteins but has an additional Bmp protein (BmpA2) missing in LB-causing Borrelia burgdorferi s.l. Our results indicate that the Bmp proteins could bind nucleosides, although BmpC might have a different ligand-binding specificity and, therefore, a distinct function. Furthermore, the work provides a means for classifying the Bmp proteins and supports further elucidation of the roles of these proteins.

Plant lipid transfer proteins: are we finally closing in on the roles of these enigmatic proteins?

The nonspecific lipid transfer proteins (LTPs) are small compact proteins folded around a tunnel-like hydrophobic cavity, making them suitable for lipid binding and transport. LTPs are encoded by large gene families in all land plants, but they have not been identified in algae or any other organisms. Thus, LTPs are considered key proteins for plant survival on and colonization of land. LTPs are abundantly expressed in most plant tissues, both above and below ground. They are usually localized to extracellular spaces outside the plasma membrane. Although the in vivo functions of LTPs remain unclear, accumulating evidence suggests a role for LTPs in the transfer and deposition of monomers required for assembly of the waterproof lipid barriers, such as cutin and cuticular wax, suberin, and sporopollenin, formed on many plant surfaces. Some LTPs may be involved in other processes, such as signaling during pathogen attacks. Here, we present the current status of LTP research with a focus on the role of these proteins in lipid barrier deposition and cell expansion. We suggest that LTPs facilitate extracellular transfer of barrier materials and adhesion between barriers and extracellular materials. A growing body of research may uncover the true role of LTPs in plants.

Arabidopsis FNRL protein is an NADPH-dependent chloroplast oxidoreductase resembling bacterial ferredoxin-NADP+ reductases.

Plastidic ferredoxin-NADP+ oxidoreductases (FNRs; EC:1.18.1.2) together with bacterial type FNRs (FPRs) form the plant-type FNR family. Members of this group contain a two-domain scaffold that forms the basis of an extended superfamily of flavin adenine dinucleotide (FAD) dependent oxidoreductases. In this study, we show that the Arabidopsis thaliana At1g15140 [Ferredoxin-NADP+ oxidoreductase-like (FNRL)] is an FAD-containing NADPH dependent oxidoreductase present in the chloroplast stroma. Determination of the kinetic parameters using the DCPIP NADPH-dependent diaphorase assay revealed that the reaction catalysed by a recombinant FNRL protein followed a saturation Michaelis-Menten profile on the NADPH concentration with kcat = 3.2 ± 0.2 s-1 , KmNADPH = 1.6 ± 0.3 µM and kcat /KmNADPH = 2.0 ± 0.4 µM-1 s-1 . Biochemical assays suggested that FNRL is not likely to interact with Arabidopsis ferredoxin 1, which is supported by the sequence analysis implying that the known Fd-binding residues in plastidic FNRs differ from those of FNRL. In addition, based on structural modelling FNRL has an FAD-binding N-terminal domain built from a six-stranded ß-sheet and one α-helix, and a C-terminal NADP+ -binding α/ß domain with a five-stranded ß-sheet with a pair of α-helices on each side. The FAD-binding site is highly hydrophobic and predicted to bind FAD in a bent conformation typically seen in bacterial FPRs.

Deciphering tissue-induced Klebsiella pneumoniae lipid A structure.

The outcome of an infection depends on host recognition of the pathogen, hence leading to the activation of signaling pathways controlling defense responses. A long-held belief is that the modification of the lipid A moiety of the lipopolysaccharide could help Gram-negative pathogens to evade innate immunity. However, direct evidence that this happens in vivo is lacking. Here we report the lipid A expressed in the tissues of infected mice by the human pathogen Klebsiella pneumoniae. Our findings demonstrate that Klebsiella remodels its lipid A in a tissue-dependent manner. Lipid A species found in the lungs are consistent with a 2-hydroxyacyl-modified lipid A dependent on the PhoPQ-regulated oxygenase LpxO. The in vivo lipid A pattern is lost in minimally passaged bacteria isolated from the tissues. LpxO-dependent modification reduces the activation of inflammatory responses and mediates resistance to antimicrobial peptides. An lpxO mutant is attenuated in vivo thereby highlighting the importance of this lipid A modification in Klebsiella infection biology. Colistin, one of the last options to treat multidrug-resistant Klebsiella infections, triggers the in vivo lipid A pattern. Moreover, colistin-resistant isolates already express the in vivo lipid A pattern. In these isolates, LpxO-dependent lipid A modification mediates resistance to colistin. Deciphering the lipid A expressed in vivo opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern.

Slr0006-like proteins: A TsaC/TsaC2/YciO subfamily exclusive to cyanobacteria.

The universally conserved TsaC/TsaC2/YciO family of proteins is essential for the N6-threonylcarbamoyladenosine modification present in almost all ANN-decoding tRNAs. Previously, the family has been grouped into the TsaC/TsaC2 and YciO subfamilies. We used sequence analysis, phylogenetic methods and homology modeling to show that a third subfamily, the Slr0006-like subfamily, exists exclusively in some cyanobacteria. The Slr0006-like proteins are solely found together with both TsaC and YciO homologs, indicating a distinct function for the Slr0006-like subfamily. Accordingly, the homology models show that the amino acids in their putative binding clefts differ significantly. Hence, we introduce a new cyanobacterial subfamily of proteins with the TsaC-domain fold, along with the generated classification rules to assign new members to the correct cyanobacterial subfamily.