NADPH Complex Research Articles

Balanced regulation of ROS production and inflammasome activation in preventing early development of colorectal cancer.

Reactive oxygen species (ROS) production and inflammasome activation are the key components of the innate immune response to microbial infection and sterile insults. ROS are at the intersection of inflammation and immunity during cancer development. Balanced regulation of ROS production and inflammasome activation serves as the central hub of innate immunity, determining whether a cell will survive or undergo cell death. However, the mechanisms underlying this balanced regulation remain unclear. Mitochondria and NADPH oxidases are the two major sources of ROS production. Recently, NCF4, a component of the NADPH oxidase complex that primarily contributes to ROS generation in phagocytes, was reported to balance ROS production and inflammasome activation in macrophages. The phosphorylation and puncta distribution of NCF4 shifts from the membrane-bound NADPH complex to the perinuclear region, promoting ASC speck formation and inflammasome activation, which triggers downstream IL-18-IFN-γ signaling to prevent the progression of colorectal cancer (CRC). Here, we review ROS signaling and inflammasome activation studies in colitis-associated CRC and propose that NCF4 acts as a ROS sensor that balances ROS production and inflammasome activation. In addition, NCF4 is a susceptibility gene for Crohn's disease (CD) and CRC. We discuss the evidence demonstrating NCF4's crucial role in facilitating cell-cell contact between immune cells and intestinal cells, and mediating the paracrine effects of inflammatory cytokines and ROS. This coordination of the signaling network helps create a robust immune microenvironment that effectively prevents epithelial cell mutagenesis and tumorigenesis during the early stage of colitis-associated CRC.

NCF4 attenuates colorectal cancer progression by modulating inflammasome activation and immune surveillance

The spatiotemporal regulation of inflammasome activation remains unclear. To examine the mechanism underlying the assembly and regulation of the inflammasome response, here we perform an immunoprecipitation-mass spectrometry analysis of apoptosis-associated speck-like protein containing a CARD (ASC) and identify NCF4/1/2 as ASC-binding proteins. Reduced NCF4 expression is associated with colorectal cancer development and decreased five-year survival rate in patients with colorectal cancer. NCF4 cooperates with NCF1 and NCF2 to promote NLRP3 and AIM2 inflammasome activation. Mechanistically, NCF4 phosphorylation and puncta distribution switches from the NADPH complex to the perinuclear region, mediating ASC oligomerization, speck formation and inflammasome activation. NCF4 functions as a sensor of ROS levels, to establish a balance between ROS production and inflammasome activation. NCF4 deficiency causes severe colorectal cancer in mice, increases transit-amplifying and precancerous cells, reduces the frequency and activation of CD8+ T and NK cells, and impairs the inflammasome-IL-18-IFN-γ axis during the early phase of colorectal tumorigenesis. Our study implicates NCF4 in determining the spatial positioning of inflammasome assembly and contributing to inflammasome-mediated anti-tumor responses.

New insights into the substrate inhibition of human 17β-hydroxysteroid dehydrogenase type 1

Human type 1 17β-hydroxysteroid dehydrogenase (17β-HSD1),a member of the short-chain dehydrogenase/reductase family, catalyzes the last step in the bioactivation of the most potent estrogen estradiol with high specificity and is thus involved in estrogen-dependent diseases. As an oxidoreductase, 17β-HSD1 can utilize both triphosphate and diphosphate cofactors in reaction at the molecular level, but more specific with triphosphate cofactor. The NADPH is much higher than NADP+ in living cells leading to preliminary reduction action. The enzyme also showed substrate-induced inhibition unprecedented in other members of 17β-HSDs. Our previous study elucidated the structural mechanism of substrate inhibition is due to the reversely bound estrone (E1) in the substrate-binding pocket of the enzyme resulting in a dead-end complex. However, the effect of the cofactor preference on the substrate inhibition of the enzyme is not yet clear. In the present study, we solved the ternary crystal structures of 17β-HSD1 in complex with E1 and cofactor analog NAD+ . Combined with molecular dynamics simulation using the enzyme with NADH/NADPH and different oriented E1 (normally oriented, E1N; reversely oriented, E1R), such ternary structure provides a complete picture of enzyme-substrate-cofactor interactions. The results reveal that different cofactors and substrate binding mode affect the allosteric effect between the two subunits of the enzyme. And the results from MD simulations confirmed that His221 plays a key role in the formation of dead-end complex in NADPH complex, and the absence of stable interaction between His221 and E1R in the NADH complex should be the main reason for its lack of substrate inhibition.

GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis.

Neutrophils are the most prevalent immune cells in circulation, but the repertoire of canonical inflammasomes in neutrophils and their respective involvement in neutrophil IL-1β secretion and neutrophil cell death remain unclear. Here, we show that neutrophil-targeted expression of the disease-associated gain-of-function Nlrp3A350V mutant suffices for systemic autoinflammatory disease and tissue pathology in vivo. We confirm the activity of the canonical NLRP3 and NLRC4 inflammasomes in neutrophils, and further show that the NLRP1b, Pyrin and AIM2 inflammasomes also promote maturation and secretion of interleukin (IL)-1β in cultured bone marrow neutrophils. Notably, all tested canonical inflammasomes promote GSDMD cleavage in neutrophils, and canonical inflammasome-induced pyroptosis and secretion of mature IL-1β are blunted in GSDMD-knockout neutrophils. In contrast, GSDMD is dispensable for PMA-induced NETosis. We also show that Salmonella Typhimurium-induced pyroptosis is markedly increased in Nox2/Gp91Phox -deficient neutrophils that lack NADPH oxidase activity and are defective in PMA-induced NETosis. In conclusion, we establish the canonical inflammasome repertoire in neutrophils and identify differential roles for GSDMD and the NADPH complex in canonical inflammasome-induced neutrophil pyroptosis and mitogen-induced NETosis, respectively.

Evidence of off-target effects of bosutinib that promote retinoic acid-induced differentiation of non-APL AML cells

ABSTRACT In the present study, we determined the effects of the Src family kinase (SFK) inhibitor, Bosutinib, and the engineered loss of the Lyn SFK on all-trans retinoic acid-induced leukemic cell differentiation. Retinoic acid (RA) is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing differentiation of a non-APL AML cell model, the HL-60 human myeloblastic (FAB-M2) leukemia cell line, via activation of a novel signalsome containing an ensemble of signaling molecules that drive differentiation. Bosutinib is an inhibitor of SFKs used to treat myeloid leukemias where prominent high expression of SFKs, in particular Lyn, has been observed. Using either Bosutinib or loss of Lyn expression due to shRNA promoted RA-induced phenotypic differentiation, G0 arrest, and respiratory burst (functional differentiation) of HL-60 cells. Signaling events putatively seminal to RA-induced differentiation, the expression of Fgr, Cbl, Slp-76 and Vav, and the phosphorylation of c-Raf (pS259), Vav (p-tyr), and Slp76 (p-tyr) were not inhibited by Bosutinib or loss of Lyn. Nor was RA-induced upregulation of p-tyr phosphorylation of p47phox, a member of the NADPH complex that produces ROS, a putative phosphorylation dependent signaling regulator. Surprisingly, Bosutinib still works in the absence of Lyn to enhance RA-induced differentiation and neither compromised RA-induced expression, nor phosphorylation of signaling molecules that drive differentiation. These findings suggested there is a novel, off-target, Lyn-independent effect of Bosutinib that is of therapeutic significance to differentiation therapy.

Novel mutations in the CYBB gene causing X-linked chronic granulomatous disease: a case report of 2 patients

Introduction: Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency caused by mutations in the NADPH complex characterized by recurrent infections, inflammation and autoimmunity. While autosomal recessive forms exist, X-linked CGD makes up the majority of cases, which is caused by mutations in the CYBB gene. Patients are at high risk for infections with catalase positive bacteria and fungi. The prognosis has improved significantly with improvements in disease detection and management, including prophylactic antibiotic and antifungal therapy. Hematopoietic stem cell transplantation (HSCT) is a curative option for patients with a suitable donor. Aim: To report the clinical presentation, immune features and genetic mutations in 2 patients with novel mutations in the CYBB gene causing X-linked CGD who underwent HSCT. Results: Case 1: Patient 1 is a 14-year-old patient who initially presented with disseminated aspergillosis at the age of 3. He was noted to have an abnormal neutrophil oxidative burst index (NOBI) and genetic testing revealed a mutation in the CYBB gene (c.883_87dupGTGGT) consistent with CGD. He successfully underwent HSCT at age 4. At age 10 he developed a primary intracranial rhabdomyosarcoma in the posterior cranial fossa. Case 2: Patient 2 is a 4-year-old male who was worked up for CGD after developing a perianal abscess at 1 month of age followed by Moraxella bacteremia at 2 months of age. He had 2 abnormal NOBIs and genetic testing identified a novel mutation in the CYBB gene that was thought to explain his phenotype (c.941delA). He underwent an HSCT (10/10 HLA matched unrelated donor). Both patients have had normalization of their NOBI post-transplant and remain free of significant infections. Discussion: We report the clinical presentation, immune features and genetic mutations in 2 patients with novel mutations in the CYBB gene causing X-linked CGD. Identifying pathogenic mutations causing CGD is important for a better understanding of genotype–phenotype associations and disease course in this patient population. Statement of novelty: We describe 2 pediatric patients diagnosed with X-linked chronic granulomatous disease due to novel mutations in the CYBB gene.

Genome Sequencing of the Endangered Kingdonia uniflora (Circaeasteraceae, Ranunculales) Reveals Potential Mechanisms of Evolutionary Specialization.

SummaryKingdonia uniflora, an alpine herb, has an extremely narrow distribution and represents a model for studying evolutionary mechanisms of species that have adapted to undisturbed environments for evolutionarily long periods of time. We assembled a 1,004.7-Mb draft genome (encoding 43,301 genes) of K. uniflora and found significant overrepresentation in gene families associated with DNA repair, underrepresentation in gene families associated with stress response, and loss of most plastid ndh genes. During the evolutionary process, the overrepresentation of gene families involved in DNA repair could help asexual K. uniflora reduce the accumulation of deleterious mutations, while reducing genetic diversity, which is important in responding to environment fluctuations. The underrepresentation of gene families related to stress response and functional loss of ndh genes could be due to lack or loss of ability to respond to environmental changes caused by long-term adaptation to a relatively stable ecological environment.

Identification of SclB, a Zn(II)2Cys6 transcription factor involved in sclerotium formation in Aspergillus niger

Certain Aspergillus species such as Aspergillus flavus and A. parasiticus are well known for the formation of sclerotia. These developmental structures are thought to act as survival structures during adverse environmental conditions but are also a prerequisite for sexual reproduction. We previously described an A. niger mutant (scl-2) which formed sclerotium-like structures, suggesting a possible first stage of sexual development in this species. Several lines of evidence presented in this study support the previous conclusion that the sclerotium-like structures of scl-2 are indeed sclerotia. These included the observations that: (i) safranin staining of the sclerotia-like structures produced by the scl-2 mutant showed the typical cellular structure of a sclerotium; (ii) metabolite analysis revealed specific production of indoloterpenes, which have previously been connected to sclerotium formation; (iii) formation of the sclerotium-like structures is dependent on a functional NADPH complex, as shown for other fungi forming sclerotia. The mutation in scl-2 responsible for sclerotium formation was identified using parasexual crossing and bulk segregant analysis followed by high throughput sequencing and subsequent complementation analysis. The scl-2 strain contains a mutation that introduces a stop codon in the putative DNA binding domain of a previously uncharacterized Zn(II)2Cys6 type transcription factor (An08g07710). Targeted deletion of this transcription factor (sclB) confirmed its role as a repressor of sclerotial formation and in the promotion of asexual reproduction in A. niger. Finally, a genome-wide transcriptomic comparison of RNA extracted from sclerotia versus mycelia revealed major differences in gene expression. Induction of genes related to indoloterpene synthesis was confirmed and also let to the identification of a gene cluster essential for the production of aurasperones during sclerotium formation. Expression analysis of genes encoding other secondary metabolites, cell wall related genes, transcription factors, and genes related to reproductive processes identified many interesting candidate genes to further understand the regulation and biosynthesis of sclerotia in A. niger. The newly identified SclB transcription factor acts as a repressor of sclerotium formation and manipulation of sclB may represent a first prerequisite step towards engineering A. niger strains capable of sexual reproduction. This will provide exciting opportunities for further strain improvement in relation to protein or metabolite production in A. niger.

396PD - Characterization of a novel BBB-permeable mutant IDH1 inhibitor, DS-1001b

BackgroundMutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in glioma, acute myeloid leukemia (AML) and many other cancers. While wild-type IDHs convert isocitrate to α-ketoglutarate (α-KG), mutant IDHs convert α-KG to onco-metabolite 2-hydroxyglutarate (2-HG). Inhibitors of mutated IDH1 or IDH2 showed clinical benefits and were approved for AML patients, supporting IDH1/2 mutations are bona fide oncogenes. However, it remains unanswered whether inhibition of IDH1 mutant activity in glioma shows clinical benefits. MethodsSmall-molecule inhibitors for mutant IDH1 were synthesized and tested by in vitro IDH1 mutant enzymes and cell-based assays. DS-1001b is a tert-butylamine salt of DS-1001a. X-ray crystallography was applied to get the insight of inhibition mechanism. We used 14C-labelled DS-1001a to check the brain exposure of the compound. We used a glioblastoma patient-derived xenograft (PDX) model with heterozygous IDH1R132H to monitor the anti-tumor activity of DS-1001b. ResultsDS-1001b inhibited the enzymatic activities of IDH1R132H, IDH1R132C, and wild type IDH1 with IC50 values of 8, 11 and 180nM, respectively, while it did not inhibit IDH2R140Q, IDH2R172Q, or wild type IDH2 (IC50 values of>10000nM). Through the analysis of X-ray crystallography of the ternary complex of IDH1R132C, NADPH, and compound A, a DS-1001b derivative, compound A was found in the allosteric pocket located at the dimer surface and IDH1R132C was in "open" inactive form. DS-1001b treatment inhibited production of 2-HG from cells with IDH1R132H or IDH1R132C at 20 – 50nM. Brain exposure of the compound was tested in mice with radioactivity of [14C]DS-1001a, and the results suggested DS-1001a penetrates blood-brain barrier (BBB). Administration of DS-1001b showed great reduction of 2-HG in the tumor and clear anti-tumor effects against subcutaneous A1074 PDX model with heterozygous IDH1R132H. ConclusionsOur results indicate that DS-1001b, which is currently in a phase I clinical trial for treating glioma with IDH1 mutations (NCT03030066), is BBB-permeable and effective against the PDX model of IDH1 mutant glioma through inhibition of IDH1 mutant proteins. Clinical trial identificationNCT03030066. Legal entity responsible for the studyDaiichi Sankyo Co., Ltd. FundingDaiichi Sankyo Co., Ltd. DisclosureH. Matsunaga: Full / Part-time employment: Daiichi Sankyo Co., Ltd. Y. Machida: Research grant / Funding (institution): Daiichi Sankyo Co., Ltd. M. Nakagawa: Research grant / Funding (institution): Daiichi Sankyo Co., Ltd. M. Yamaguchi: Research grant / Funding (institution): Daiichi Sankyo Co., Ltd. Y. Ogawara: Research grant / Funding (institution): Daiichi Sankyo Co., Ltd. Y. Shima: Research grant / Funding (institution): Daiichi Sankyo Co., Ltd. K. Yamagata: Research grant / Funding (institution): Daiichi Sankyo Co., Ltd. T. Katsumoto: Research grant / Funding (institution): Daiichi Sankyo Co., Ltd. A. Hattori: Research grant / Funding (institution): Daiichi Sankyo Co., Ltd. M. Itoh: Full / Part-time employment: Daiichi Sankyo Co., Ltd. T. Seki: Full / Part-time employment: Daiichi Sankyo Co., Ltd. Y. Nishiya: Full / Part-time employment: Daiichi Sankyo Co., Ltd. K. Nakamura: Full / Part-time employment: Daiichi Sankyo Co., Ltd. K. Suzuki: Full / Part-time employment: Daiichi Sankyo Co., Ltd. T. Imaoka: Full / Part-time employment: Daiichi Sankyo Co., Ltd. M. Suzuki: Full / Part-time employment: Daiichi Sankyo RD Novare Co., Ltd. K. Ichimura: Research grant / Funding (institution): Daiichi Sankyo. I. Kitabayashi: Research grant / Funding (institution): Daiichi Sankyo Co., Ltd. All other authors have declared no conflicts of interest.

Endogenous Synthesis of Erythritol, a Novel Biomarker of Weight Gain (P15-016-19)

ObjectivesSerum erythritol is associated with central adiposity gain in young adults. Erythritol, a 4-carbon polyol, is synthesized endogenously from erythrose through the pentose phosphate pathway. We have identified two enzymes which catalyze this reaction: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Interestingly, ADH1 isoforms ADH1B1 and ADH1C2 catalyze NADPH-dependent synthesis of erythritol in vitro, but ADH1B1 does not. In A549 cells, siRNA knockdown of SORD levels to less than 15% of control levels reduced erythritol by 50%. A549 cells also have low levels of ADH1 expression. This indicates that other enzymes may be capable of endogenous erythritol production. Based on its high degree of homology to ADH1, we hypothesize that ADH4 also catalyzes erythritol synthesis. The objective of this study was to further elucidate the mechanism of erythritol synthesis by: determining key differences between the active site of ADH1B2 compared to ADH1B1 and SORD, and screening a new candidate enzyme, ADH4. MethodsThis study used molecular modeling to evaluate the enzyme, erythrose, NADPH complex. Site-directed mutagenesis was performed to replace Arg208 (R208) of recombinant human SORD with histidine. Recombinant SORD R208H mutant and ADH4 were then expressed, purified, and their erythrose reduction activity was kinetically characterized. ResultsMolecular modeling of the active sites of ADH1B1 and ADH1B2 revealed that the Arg48 residue of ADH1B1 makes three contacts with NADPH, whereas His48 of ADH1B2 only makes one contact. Mutation of the SORD active-site Arg208 to histidine reduced the kcat by more than 80% compared to wild-type (95 ± 21 min−1 wild-type, 16 ± 2 min−1 R208H, P < 0.05). ADH4 erythrose reduction activity reduced by more than 95% compared to ADH1 variant proteins in vitro (kcat 2 ± 1 min−1). ConclusionsMutation of arginine 208 to histidine in the active site of SORD significantly reduced the kcat, indicating that the presence of an arginine residue is essential for binding NADPH, the cofactor required in vivo for erythritol synthesis. The low erythrose reduction activity of ADH4 suggests that ADH4 does not contribute to erythritol synthesis in humans. Funding SourcesNone.

The metabolites NADP+ and NADPH are the targets of the circadian protein Nocturnin (Curled)

Nocturnin (NOCT) is a rhythmically expressed protein that regulates metabolism under the control of circadian clock. It has been proposed that NOCT deadenylates and regulates metabolic enzyme mRNAs. However, in contrast to other deadenylases, purified NOCT lacks the deadenylase activity. To identify the substrate of NOCT, we conducted a mass spectrometry screen and report that NOCT specifically and directly converts the dinucleotide NADP+ into NAD+ and NADPH into NADH. Further, we demonstrate that the Drosophila NOCT ortholog, Curled, has the same enzymatic activity. We obtained the 2.7 Å crystal structure of the human NOCT•NADPH complex, which revealed that NOCT recognizes the chemically unique ribose-phosphate backbone of the metabolite, placing the 2′-terminal phosphate productively for removal. We provide evidence for NOCT targeting to mitochondria and propose that NADP(H) regulation, which takes place at least in part in mitochondria, establishes the molecular link between circadian clock and metabolism.

Crystal structure of archaeal HMG-CoA reductase: insights into structural changes of the C-terminal helix of the class-I enzyme.

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyses the last step in mevalonate biosynthesis. HMGR is the target of statin inhibitors that regulate cholesterol concentration in human blood. Here, we report the properties and structures of HMGR from an archaeon Methanothermococcus thermolithotrophicus (mHMGR). The structures of the apoenzyme and the NADPH complex are highly similar to those of human HMGR. A notable exception is C-terminal helix (Lα10-11) that is straight in both mHMGR structures. This helix is kinked and closes the active site in the human enzyme ternary complex, pointing to a substrate-induced structural rearrangement of C-terminal in class-I HMGRs during the catalytic cycle.

Nonequivalence of Second Sphere "Noncatalytic" Residues in Pentaerythritol Tetranitrate Reductase in Relation to Local Dynamics Linked to H-Transfer in Reactions with NADH and NADPH Coenzymes.

Many enzymes that catalyze hydride transfer reactions work via a mechanism dominated by quantum mechanical tunneling. The involvement of fast vibrational modes of the reactive complex is often inferred in these reactions, as in the case of the NAD(P)H-dependent pentaerythritol tetranitrate reductase (PETNR). Herein, we interrogated the H-transfer mechanism in PETNR by designing conservative (L25I and I107L) and side chain shortening (L25A and I107A) PETNR variants and using a combination of experimental approaches (stopped-flow rapid kinetics, X-ray crystallography, isotope/temperature dependence studies of H-transfer and NMR spectroscopy). X-ray data show subtle changes in the local environment of the targeted side chains but no major structural perturbation caused by mutagenesis of these two second sphere active site residues. However, temperature dependence studies of H-transfer revealed a coenzyme-specific and complex thermodynamic equilibrium between different reactive configurations in PETNR–coenzyme complexes. We find that mutagenesis of these second sphere “noncatalytic” residues affects differently the reactivity of PETNR with NADPH and NADH coenzymes. We attribute this to subtle, dynamic structural changes in the PETNR active site, the effects of which impact differently in the nonequivalent reactive geometries of PETNR−NADH and PETNR−NADPH complexes. This inference is confirmed through changes observed in the NMR chemical shift data for PETNR complexes with unreactive 1,4,5,6-tetrahydro-NAD(P) analogues. We show that H-transfer rates can (to some extent) be buffered through entropy–enthalpy compensation, but that use of integrated experimental tools reveals hidden complexities that implicate a role for dynamics in this relatively simple H-transfer reaction. Similar approaches are likely to be informative in other enzymes to understand the relative importance of (distal) hydrophobic side chains and dynamics in controlling the rates of enzymatic H-transfer.

Backbone 1H, 13C and 15N chemical shift assignments for the binary L28F ecDHFR:NADPH complex

Backbone 1H, 13C and 15N chemical shift assignments for the binary L28F ecDHFR:NADPH complex

The activity of superoxide dismutases (SODs) at the early stages of wheat deetiolation.

Unbound tetrapyrroles, i.e. protochlorophyllide (Pchlide), chlorophyllide and chlorophylls, bring the risk of reactive oxygen species (ROS) being generated in the initial stages of angiosperm deetiolation due to inefficient usage of the excitation energy for photosynthetic photochemistry. We analyzed the activity of superoxide dismutases (SODs) in etiolated wheat (Triticum aestivum) leaves and at the beginning of their deetiolation. Mn-SOD and three isoforms of Cu/Zn-SODs were identified both in etiolated and greening leaves of T. aestivum. Two Cu/Zn-SODs, denoted as II and III, were found in plastids. The activity of plastidic Cu/Zn-SOD isoforms as well as that of Mn-SOD correlated with cell aging along a monocot leaf, being the highest at leaf tips. Moreover, a high Pchlide content at leaf tips was observed. No correlation between SOD activity and the accumulation of photoactive Pchlide, i.e. Pchlide bound into ternary Pchlide:Pchlide oxidoreductase:NADPH complexes was found. Cu/Zn-SOD I showed the highest activity at the leaf base. A flash of light induced photoreduction of the photoactive Pchlide to chlorophyllide as well as an increase in all the SODs activity which occurred in a minute time-scale. In the case of seedlings that were deetiolated under continuous light of moderate intensity (100 μmol photons m-2 s-1), only some fluctuations in plastidic Cu/Zn-SODs and Mn-SOD within the first four hours of greening were noticed. The activity of SODs is discussed with respect to the assembly of tetrapyrroles within pigment-protein complexes, monitored by fluorescence spectroscopy at 77 K.

P40 phox -Deficient Mice Exhibit Impaired Bacterial Clearance and Enhanced Pro-inflammatory Responses during Salmonella enterica serovar Typhimurium Infection.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of acute gastroenteritis in humans. During infection, reactive oxygen species (ROS), generated from NADPH oxidase (a multisubunit enzyme complex), are required for pathogen killing upon phagocytosis and for regulating pro-inflammatory signaling in phagocytic cells. Mutations in subunits forming the NADPH complex may lead to enhanced susceptibility to infection and inflammatory disease. Compared to other NADPH oxidase subunits, the function of p40phox is relatively understudied, particularly in the context of intestinal bacterial infection. In this study, we utilized genetically engineered mice to determine the role of p40phox in the response to S. Typhimurium infection. We show that mice lacking p40phox are more susceptible to oral infection with S. Typhimurium, as demonstrated by significantly enhanced bacterial dissemination to spleen and liver, and development of exacerbated bacterial colitis. Moreover, we demonstrate that the increased infection and disease severity are correlated with markedly increased F4/80+ macrophage and Ly6G+ neutrophil infiltration in the infected tissues, coincident with significantly elevated pro-inflammatory cytokines (IL-1β and TNF-α) and chemoattractant molecules in the infected tissues. Functional analysis of macrophages and neutrophils further shows that p40phox deficiency impairs bacteria- or PMA-induced intracellular ROS production as well as intracellular killing of Salmonella. These observations indicate that the p40phox subunit of NADPH oxidase plays an essential role in suppressing intracellular multiplication of Salmonella in macrophages and in the regulation of both systemic and mucosal inflammatory responses to bacterial infection.

MGDG, PG and SQDG regulate the activity of light-dependent protochlorophyllide oxidoreductase.

Light-dependent protochlorophyllide oxidoreductase (POR) is a plant enzyme involved in the chlorophyll biosynthesis pathway. POR reduces one of the double bonds of the protochlorophyllide (Pchlide) using NADPH and light. In the present study, we found out that phosphatidylglycerol and sulfoquinovosyl diacylglycerol are allosteric regulators of the nucleotide binding, which increase the affinity towards NADPH a 100-fold. Moreover, we showed for the first time that NADH can, like NADPH, form active complexes with Pchlide and POR, however, at much higher concentrations. Additionally, monogalactosyldiacylglycerol (MGDG) was shown to be the main factor responsible for the red shift of the fluorescence emission maximum of Pchlide:POR:NADPH complexes. Importantly, the emission maximum at 654 nm was obtained only for the reaction mixtures supplemented with MGDG and at least one of the negatively charged plant lipids. Moreover, the site-directed mutagenesis allowed us to identify amino acid residues that may be responsible for lipid binding and Pchlide coordination. Our experiments allowed us to identify six different Pchlide:POR complexes that differ in the fluorescence emission maxima of the pigment. The results presented here reveal the contribution of thylakoid lipids in the regulation of the chlorophyll biosynthesis pathway; however, the molecular mechanisms of this process are to be clarified.

Cofactor-Mediated Conformational Dynamics Promote Product Release From Escherichia coli Dihydrofolate Reductase via an Allosteric Pathway.

The enzyme dihydrofolate reductase (DHFR, E) from Escherichia coli is a paradigm for the role of protein dynamics in enzyme catalysis. Previous studies have shown that the enzyme progresses through the kinetic cycle by modulating the dynamic conformational landscape in the presence of substrate dihydrofolate (DHF), product tetrahydrofolate (THF), and cofactor (NADPH or NADP+). This study focuses on the quantitative description of the relationship between protein fluctuations and product release, the rate-limiting step of DHFR catalysis. NMR relaxation dispersion measurements of millisecond time scale motions for the E:THF:NADP+ and E:THF:NADPH complexes of wild-type and the Leu28Phe (L28F) point mutant reveal conformational exchange between an occluded ground state and a low population of a closed state. The backbone structures of the occluded ground states of the wild-type and mutant proteins are very similar, but the rates of exchange with the closed excited states are very different. Integrated analysis of relaxation dispersion data and THF dissociation rates measured by stopped-flow spectroscopy shows that product release can occur by two pathways. The intrinsic pathway consists of spontaneous product dissociation and occurs for all THF-bound complexes of DHFR. The allosteric pathway features cofactor-assisted product release from the closed excited state and is utilized only in the E:THF:NADPH complexes. The L28F mutation alters the partitioning between the pathways and results in increased flux through the intrinsic pathway relative to the wild-type enzyme. This repartitioning could represent a general mechanism to explain changes in product release rates in other E. coli DHFR mutants.

Effects of Molecular Crowding on the Binding Affinity of Dihydrofolate for Dihydrofolate Reductase

The reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) by the enzyme dihydrofolate reductase (DHFR), using NADPH as a cofactor, is an essential part of the folate cycle. The inhibition of DHFR leads to interruption of DNA synthesis and consequently cell death, making this enzyme a crucial target in the treatment of cancer and other diseases. Previous studies examined the effects of small molecule osmolytes on the substrate interactions with two non-homologous DHFRs, E. coli chromosomal DHFR (EcDHFR) and R67 DHFR, with vastly different active site structures. The results indicated that DHF weakly interacts with the osmolytes in solution, shifting the binding equilibrium from DHF bound to DHFR to unbound DHF. It is hypothesized that similar weak, nonspecific interactions may also occur between cellular proteins and DHF. Weak interactions between cellular proteins and DHF would have consequences in vivo, where the concentration of the cellular milieu is approximately 300 g/L. Under the crowded conditions in the cell, there is a higher propensity for intermolecular interaction.Crowding effects of macromolecules in concentrations similar to those in vivo were examined. Isothermal titration calorimetry (ITC) and enzyme kinetic assays were used to detect effects of molecular crowders by monitoring activity of the (DHFR)-NADPH or DHF complex and the ternary DHFR-DHF-NADPH complex in the presence of these crowders. To recreate the conditions of molecular crowding in vivo, the binding of the enzyme-ligand complexes in the presence of molecular weight crowding agents (lysozyme or casein) was examined. Analysis of the Kd's and Km's indicated a correlation between increased molecular crowding in the solution and weakened binding of the DHFR-substrate complexes. These findings indicate an importance of molecular crowding on EcDHFR activity in vivo.

Structure–activity relationship of flavonoids as potent inhibitors of carbonyl reductase 1 (CBR1)

Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase superfamily, reduces a variety of carbonyl compounds including therapeutic drugs. CBR1 is involved in the reduction of the anthracycline anticancer drugs to their less anticancer C-13 hydroxy metabolites, which are cardiotoxic. CBR1 inhibitors are thought to be promising agents for adjuvant therapy with twofold beneficial effect in prolonging the anticancer efficacy of the anthracyclines while decreasing cardiotoxicity, a side effect of the drugs. In this study, we evaluated 27 flavonoids for their inhibitory activities of CBR1 in order to explore the structure–activity relationship (SAR). Among them, luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one) showed the most potent inhibition (IC5095nM), which is also more potent compared to all known classes of CBR1 inhibitors. The inhibition of luteolin was noncompetitive with respect to the substrate in the NADPH-dependent reduction direction, but CBR1 exhibited moderate NADP+-dependent dehydrogenase activity for some alicyclic alcohols, in which the luteolin inhibition was competitive with respect to the alcohol substrate (Ki59nM). The SAR of the flavonoids indicated that the 7-hydroxy group of luteolin was responsible for the potent inhibition of CBR1. The molecular docking of luteolin in CBR1–NADPH complex showed that theflavonoid binds to the substrate-binding cleft, in which its 7-hydroxy group formed a H-bond with main-chain oxygen of Met234, in addition to H-bond interactions (of its 5-hydroxy and 4-carbonyl groups with catalytically important residues Tyr193 and/or Ser139) and a π-stacking interaction (between its phenyl ring and Trp229).