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SUMMARY PARAGRAPHDisease-causing mutations in genes encoding transcription factors (TFs) are a recurrent finding in hematopoietic malignancies and might involve key regulators of lineage adherence and cellular differentiation1–3. Such mutations can affect TF-interactions with their cognate DNA-binding motifs4, 5. Whether and how TF-mutations impact upon the nature of binding to TF composite elements (CE) and influence their interaction with other TFs is unclear. Here, we report a new mechanism of TF alteration in human lymphomas with perturbed B cell identity. It is caused by a recurrent somatic missense mutation c.295T>C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cell-differentiation and -activation6, 7. IRF4-C99R fundamentally alters IRF4 DNA-binding, with loss-of-binding to canonical IRF motifs and neomorphic gain-of-binding to canonical and non-canonical IRF composite elements (CEs). Furthermore, IRF4-C99R thoroughly modifies IRF4 function, by blocking IRF4-dependent plasma cell induction, and up-regulating disease-specific genes in a non-canonical Activator Protein-1 (AP-1)-IRF-CE (AICE)-dependent manner. Our data explain how a single arginine mutation creates a complex switch of TF specificity and gene regulation. These data open the possibility of designing specific inhibitors to block the neomorphic, disease-causing DNA-binding activities of a mutant transcription factor.

The IκB kinase (IKK)‐NF‐κB pathway is activated as part of the DNA damage response and controls both inflammation and resistance to apoptosis. How these distinct functions are achieved remained unknown. We demonstrate here that DNA double‐strand breaks elicit two subsequent phases of NF‐κB activation in vivo and in vitro, which are mechanistically and functionally distinct. RNA‐sequencing reveals that the first‐phase controls anti‐apoptotic gene expression, while the second drives expression of senescence‐associated secretory phenotype (SASP) genes. The rapidly activated first phase is driven by the ATM‐PARP1‐TRAF6‐IKK cascade, which triggers proteasomal destruction of inhibitory IκBα, and is terminated through IκBα re‐expression from the NFKBIA gene. The second phase, which is activated days later in senescent cells, is on the other hand independent of IKK and the proteasome. An altered phosphorylation status of NF‐κB family member p65/RelA, in part mediated by GSK3β, results in transcriptional silencing of NFKBIA and IKK‐independent, constitutive activation of NF‐κB in senescence. Collectively, our study reveals a novel physiological mechanism of NF‐κB activation with important implications for genotoxic cancer treatment., DNA damage induces two kinetically and functionally distinct phases of NF‐κB activation, the first being anti‐apoptotic and the non‐canonical second wave inducing the senescence‐associated secretory phenotype (SASP).

The IκB kinase (IKK)-NF-κB signaling pathway plays a multifaceted role in inflammatory bowel disease (IBD): on the one hand, it protects from apoptosis; on the other, it activates transcription of numerous inflammatory cytokines and chemokines. Although several murine models of IBD rely on disruption of IKK-NF-κB signaling, these involve either knockouts of a single family member of NF-κB or of upstream kinases that are known to have additional, NF-κB-independent, functions. This has made the distinct contribution of NF-κB to homeostasis in intestinal epithelium cells difficult to assess. To examine the role of constitutive NF-κB activation in intestinal epithelial cells, we generated a mouse model with a tissue-specific knockout of the direct inhibitor of NF-κB, Nfkbia/IκBα. We demonstrate that constitutive activation of NF-κB in intestinal epithelial cells induces several hallmarks of IBD including increased apoptosis, mucosal inflammation in both the small intestine and the colon, crypt hyperplasia, and depletion of Paneth cells, concomitant with aberrant Wnt signaling. To determine which NF-κB-driven phenotypes are cell-intrinsic, and which are extrinsic and thus require the immune compartment, we established a long-term organoid culture. Constitutive NF-κB promoted stem-cell proliferation, mis-localization of Paneth cells, and sensitization of intestinal epithelial cells to apoptosis in a cell-intrinsic manner. Increased number of stem cells was accompanied by a net increase in Wnt activity in organoids. Because aberrant Wnt signaling is associated with increased risk of cancer in IBD patients and because NFKBIA has recently emerged as a risk locus for IBD, our findings have critical implications for the clinic. In a context of constitutive NF-κB, our findings imply that general anti-inflammatory or immunosuppressive therapies should be supplemented with direct targeting of NF-κB within the epithelial compartment in order to attenuate apoptosis, inflammation, and hyperproliferation. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Persistent NF-κB activation is a hallmark of the malignant Hodgkin/Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL). Genomic lesions, Epstein-Barr virus infection, soluble factors, and tumor–microenvironment interactions contribute to this activation. Here, in an unbiased approach to identify the cHL cell-secreted key factors for NF-κB activation, we have dissected the secretome of cultured cHL cells by chromatography and subsequent mass spectrometry. We identified lymphotoxin-α (LTA) as the causative factor for autocrine and paracrine activation of canonical and noncanonical NF-κB in cHL cell lines. In addition to inducing NF-κB, LTA promotes JAK2/STAT6 signaling. LTA and its receptor TNFRSF14 are transcriptionally activated by noncanonical NF-κB, creating a continuous feedback loop. Furthermore, LTA shapes the expression of cytokines, receptors, immune checkpoint ligands and adhesion molecules, including CSF2, CD40, PD-L1/PD-L2, and VCAM1. Comparison with single-cell gene-activity profiles of human hematopoietic cells showed that LTA induces genes restricted to the lymphoid lineage, as well as those largely restricted to the myeloid lineage. Thus, LTA sustains autocrine NF-κB activation, impacts activation of several signaling pathways, and drives expression of genes essential for microenvironmental interactions and lineage ambiguity. These data provide a robust rationale for targeting LTA as a treatment strategy for cHL patients.

Abstract —Cytochrome P450 (P450)-dependent arachidonic acid metabolites may act as mediators in the regulation of vascular tone and renal function. We studied arachidonic acid hydroxylase activities in renal microsomes from normotensive NMRI mice, desoxycorticosterone acetate (DOCA)-salt hypertensive mice, and DOCA-salt mice treated with either lovastatin or bezafibrate, both of which improve hemodynamics in this model. Control renal microsomes had arachidonic acid hydroxylase activities of 175±12 pmol · min −1 · mg −1 . The metabolites formed were 20- and 19-hydroxyarachidonic acid, representing ≈80% and ≈20% of the total hydroxylation. Treatment with DOCA-salt resulted in significantly decreased hydroxylase activities (to 84±4 pmol · min −1 · mg −1 ) of the total microsomal P450 content and a decrease in immunodetectable Cyp4a proteins. Lovastatin had no effect on these variables, whereas bezafibrate increased arachidonic acid hydroxylase activities to 163±12 pmol · min −1 · mg −1 . In situ hybridization with probes for Cyp4a-10, 12, and 14 revealed that Cyp4a-14 was the P450 isoform most strongly induced by bezafibrate. The expression was concentrated in the cortical medullary junction and was localized predominantly in the proximal tubules. In conclusion, these results suggest that the capacity to produce 20-hydroxyarachidonic acid is impaired in the kidneys of DOCA-salt hypertensive mice. Furthermore, bezafibrate may ameliorate hemodynamics in this model by restoring P450-dependent arachidonic acid hydroxylase activities. Lovastatin, on the other hand, exerts its effects via P450-independent mechanisms.

Most of the eukaryotic cytochrome P450 forms are integral membrane proteins of the endoplasmic reticulum (ER). They are targeted to the ER and inserted into the membrane by uncleavable signal-ancho...

The electron exchange reaction of cytochrome c with NADPH-cytochrome P450 reductase was investigated electrochemically. For this purpose the electrochemical behavior of cytochrome c in solution, adsorbed or covalently immobilized on a modified gold electrode surface was studied. In the case when cytochrome c was in solution or only electrostatically adsorbed on the electrode surface, fast electron transfer was observed with NADPH-cytochrome P450 reductase. When cytochrome c was covalently bound to the electrode surface, in spite of quasi-reversible electron exchange with the electrode, no electron transfer was observed with NADPH-cytochrome P450 reductase. These results suggest that electrostatically adsorbed cytochme c on the modified electrode surface has some mobility that allows re-orientation as required to interact both with the electrode and with the NADPH-cytochrome P450 reductase. In contrast, covalent binding of cytochrome c leads to permanent orientation towards the electrode surface and thus blocks the cytochrome c electron accepting site from the reductase.

Cytochrome P450 52A3 (P450Cm1) is one of the membrane proteins known to trigger by its high-level expression a marked proliferation of the endoplasmic reticulum, (ER). To gain insight into the relationship between the expression of a membrane protein and the induction of ER proliferation we have characterized the membrane topology of P450Cm1 and identified the structural determinants required for ER targeting, formation of correct membrane orientation, and ER retention. We show that all these features are interrelated and determined by sequence elements within the NH2-terminal region of P450Cm1. Using several approaches--a protease protection assay followed by probing with peptide-specific antibodies, immunolabeling of the intact membrane-bound P450 protein, and expression of fusion proteins in Saccharomyces cerevisiae--membrane topology was defined as follows: residues 2-16 are located in the ER lumen, only the first hydrophobic segment (residues 17-34) spans the membrane, a second hydrophobic segment (48-66) is exposed at the cytoplasmic side, and the remaining part (67-523) forms a large cytosolic domain. Fused to a cytosolic reporter protein, the first 44-amino-acid sequence of P450Cm1 was sufficient to mediate ER targeting, wild-type membrane orientation, and retention in the ER. Similar to wild-type P450Cm1, various fusion proteins were able to induce distinctly organized structures of proliferated ER provided that they were either permanently retained in the ER or accumulated in this compartment due to a delay in further transportation. Thus, we conclude that membrane insertion of the first hydrophobic segment is sufficient to deliver a signal for increased membrane formation.

Two enzymes, P450 52A3 (P450Cm1) and 52A4 (P450Cm2), the genes of which belong to the CYP52 multigene family occuring in the alkane-assimilating yeast Candida maltosa, have been characterized biochemically and compared in terms of their substrate specificities. For this purpose, both the P450 proteins and the corresponding C. maltosa NADPH-cytochrome P450 reductase were separately produced by expressing their cDNAs in Saccharomyces cerevisiae, purified, and reconstituted to active monooxygenase systems. Starting from microsomal fractions with a specific content of 0.75 nmol P450Cm1, 0.34 nmol P450Cm2, and 10.5 units reductase per milligram of protein, respectively, each individual recombinant protein was purified to homogeneity. P450 substrate difference spectra indicated strong type I spectral changes and high-affinity binding of n -hexadecane ( K s = 26 μ M ) and n -octadecane ( K s = 27 μ M ) to P450Cm1, whereas preferential binding to P450Cm2 was observed using lauric acid ( K s = 127 μ M ) and myristic acid ( K s = 134 μ M ) as substrates. These substrate selectivities were further substantiated by kinetic parameters, determined for n -alkane and fatty acid hydroxylation in a reconstituted system, which was composed of the purified components and phospholipid, as well as in microsomes obtained after coexpressing each of the P450 proteins with the reductase. The highest catalytic activities within the reconstituted system were measured for n -hexadecane hydroxylation to 1-hexadecanol by P450Cm1 ( V max = 27 μ M × min −1 , K m = 54 μ M ) and oxidation of lauric acid to 16-hydroxylauric acid by P450Cm2 ( V max = 30 μ M × min −1 , K m = 61 μ M ). We conclude that P450Cm1 and P450Cm2 exhibit overlapping but distinct substrate specificities due to different chain-length dependencies and preferences for either n -alkanes or fatty acids.

A full-length cDNA for NADPH-cytochrome P450 reductase from Candida maltosa was cloned and sequenced. The derived amino acid sequence showed a high similarity to the reductases from other eukaryotes. Expression in Saccharomyces cerevisiae under control of the GAL10 promoter resulted in an approximately 70-fold increase in NADPH-cytochrome c reductase activity in the microsomal fraction. The functional integrity of the heterologously expressed reductase as an electron transfer component for alkane hydroxylating cytochrome P450 from C. maltosa was shown in a reconstituted system containing both enzymes in a highly purified state. The signal-anchor sequence of the reductase was identified within the N-terminal region of the protein by means of constructing and expressing fusion proteins with the cytosolic form of yeast invertase. The first 33 amino acids turned out to be sufficient for stable membrane insertion, wild-type membrane orientation and retention in the endoplasmic reticulum. As shown by immunoelectron microscopy, the heterologously expressed reductase was integrated into the endoplasmic reticulum of the host organism. It triggered a strong proliferation of the membrane system. This membrane-inducing property of the reductase was transferable to the cytosolic reporter protein with the same N-terminal sequences that confer membrane insertion. The nucleotide sequence of the cDNA of NADPH-cytochrome P450 reductase from C. maltosa is available from the EMBL data library under Accession Number X76226.

P450-dependent arachidonic acid (AA) metabolites regulate arterial tone by modulating calcium-activated (BK) potassium channels in vascular smooth muscle cells (VSMC). Because eicosapentaenoic acid (EPA) has been reported to improve vascular function, we tested the hypothesis that P450-dependent epoxygenation of EPA produces alternative vasoactive compounds. We synthesized the 5 regioisomeric epoxyeicosattrienoic acids (EETeTr) and examined them for effects on K + currents in rat cerebral artery VSMCs with the patch-clamp technique. 11(R),12(S)-epoxyeicosatrienoic acid (50 nmol/L) was used for comparison and stimulated K + currents 6-fold at +60 mV. However, 17(R),18(S)-EETeTr elicited a more than 14-fold increase. 17(S),18(R)-EET and the remaining four regioisomers were inactive. The effect of 17(R),18(S)-EETeTr was blocked by tetraethylammonium but not by 4-aminopyridine. VSMCs expressed P450s 4A1 and 4A3. Recombinant P450 4A1 hydroxylated EPA at C-19 and C-20 and epoxygenated the 17,18-double bond, yielding the R, S- and S, R-enantiomers in a ratio of 64:36. We conclude that 17(R),18(S)-EETeTr represents a novel, potent activator of BK potassium channels. Furthermore, this metabolite can be directly produced in VSMCs. We suggest that 17(R),18(S)-EETeTr may function as an important hyperpolarizing factor, particularly with EPA-rich diets.

Overproduction of an endoplasmic reticulum (ER)-resident membrane protein (cytochrome P450 52A3) and of a secretory protein (invertase) was used to study the regulation of the luminal ER protein Kar2p under conditions that lead to ER proliferation and secretory overload, respectively. In both cases we found (i) a significant increase of Kar2 protein and mRNA levels, (ii) a transcriptional regulation based on the the function of the 22 bp unfolded-protein-response element of the KAR2 promoter and (iii) an essential role of the transmembrane kinase Ire1p for upregulation of KAR2 gene expression. These results show that the same mechanism operates when KAR2 induction is triggered by overproduction of cytochrome P450 or invertase and that this mechanism shares the known features of the unfolded-protein-response pathway. Disruption of the IRE1 gene resulted in a marked decrease of the invertase protein levels produced. In contrast, a functional IRE1 gene was not required to reach high-level production of the integral membrane protein cytochrome P450 52A3, Moreover, IRE1 gene disruption did not prevent P450-induced ER proliferation. We suggest that Ire1p-mediated KAR2 induction is, in the case of cytochrome P450 52A3 overproduction, a process which follows on ER proliferation, thereby monitoring the increase of ER size and adjusting the level of Kar2p accordingly.

After spheroplast lysis and differential centrifugation the alkane monooxygenase system consisting of cytochrome P-450 and the NADPH-cytochrome P-450 reductase of alkane-grown Candida maltosa cells is enriched in the microsomal fraction. This membrane fraction is nearly free of intact mitochondria (cytochrome oxidase) and peroxisomes (catalase), but contains considerable amounts of plasma membrane fractures (azide insensitive, vanadate-sensitive Mg2+-ATPase) as demonstrated by biochemical an electron microscopic examinations. By means of sucrose density gradient centrifugation it was possible to separate the cytochrome P-450 containing membranes ( = 1,11 g/cm3) from the plasma membranes ( = 1,18 g/cm3). Therefore the cytochrome P-450 alkane monooxygenase system is most likely localized in the endoplasmic reticulum of the yeast cells. For the following enzymatic steps of terminal alkane oxidation to the corresponding fatty acid a quite different subcellular distribution was observed. The fatty alcohol oxidase and aldehyde dehydrogenase activities are mainly localized in the mitochondrial peroxisomal membrane fraction. During the oxidation of n-alkanes by yeast cells the fatty alcohol should be regarded as an intracellular transport from between the cytochrome P-450 containing endoplasmic reticulum and the sites of its further oxidation in peroxisomes and mitochondria.

A radioimwunoassay for the quantitation of cytochrome P-450 (cyt P-450) from the yeast Candida maltosa in the microsomab fraction was established using a purified secondary antibody adsorbed to cellulose nitrate discs, a primary rabbit anti (cyt P-450) serum and [125I] labeled cyt P-450. The present assay is highly sensitive, specific and simple, It allows the detection of as little as 5 f moles cyt P-450 in a volume of 40 μ1. Equal immunological reactivities were found for the highly purified, solubilized microsomal, denatured and the apo-form of the yeast cyt P-450.

A cDNA library was established starting from poly(A) RNA of n-alkane-grown Candida maltosa cells and cDNA clones were isolated containing the entire coding sequence for the alkane hydroxylating cytochrome P-450. The deduced protein consists of 521 amino acids, contains two putative transmembrane segments in the N-terminal region and has a characteristic heme-binding sequence in the C-terminal part. Sequence alignments with members of 11 reported cytochrome P-450 families revealed a strong homology to an alkane-inducible cytochrome P-450 from Candida tropicalis.

Transition of n-hexadecane utilizing cultures of Candida maltosa to oxygen-limited growth caused an up to 6-fold increase of the cellular cytochrome P-450 content. Enhanced cytochrome P-450 formation required protein de novo synthesis and was not due to a change of the apo/holo-enzyme ratio as demonstrated by cycloheximide inhibition and immunological quantitation. The effect of low oxygen concentration (pO2=3–5%) was simulated by selective inhibition of alkane hydroxylation with carbon monoxide (at a pO2 of 70–75%). Enhanced cytochrome P-450 formation occurred even when a constant growth rate was maintained through utilization of a second non-repressive growth substrate. However, the presence of n-alkanes was an essential precondition. It was concluded, that the cytochrome P-450 formation was mainly regulated by the intracellular inducer concentration which depends on the relative rates of alkane transport into the cell and the actual alkane hydroxylating activity of the enzyme system.

Inhibition of pressure natriuresis in mice lacking the AT2 receptorBackgroundAngiotensin II type 2 (AT2) receptor knockout mice have higher blood pressures than wild-type mice; however, the hypertension is imperfectly defined. We tested the hypothesis that renal mechanisms could be contributory.MethodsWe conducted pressure-natriuresis-diuresis experiments, measured renal cortical and medullary blood flow by laser Doppler methods, and explored cytochrome P450-dependent arachidonic acid metabolism by means of reverse transcription-polymerase chain reaction.ResultsBlood pressure was 15 mm Hg higher in AT2 receptor knockout mice than in controls, and pressure diuresis and natriuresis curves were shifted rightward. At similar renal perfusion pressures (113 to 118 mm Hg), wild-type mice excreted threefold more sodium and water than AT2 receptor knockout mice. Fractional sodium and water excretion curves were shifted rightward in parallel. Renal blood flow ranged between 6.72 and 7.88 mL/min/g kidney wet weight (kwt) in wild-type and between 5.84 and 6.15 mL/min/g kwt in AT2 receptor knockout mice. Renal vascular resistance was increased in AT2A receptor knockout mice. Cortical blood flow readings leveled at 2.5 V in wild-type and 1.5 V in AT2 receptor knockout mice. Medullary blood flow readings ranged between 0.8 and 1.0 V and increased 116% in wild-type mice as renal perfusion pressure was increased. This increase did not occur in AT2 receptor knockout mice. The glomerular filtration rate (GFR) was similar in both groups at approximately 1 mL/min/g kwt. Renal microsomes from AT2 receptor knockout mice had less activity in hydroxylating arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-meter) than controls, whereas renal AT1 receptor gene expression was increased in AT2 receptor knockout mice.ConclusionsHemodynamic and tubular factors modify renal sodium handling in AT2 receptor knockout mice and may cause hypertension. AT2 receptor disruption induces alterations of other regulatory systems, including altered arachidonic acid metabolism, that may contribute to the intrarenal differences observed between AT2 receptor knockout and wild-type mice.

Nitric oxide synthase expression in AT 2 receptor–deficient mice after DOCA-salt. Background Angiotensin II type 2 receptor–deficient mice (AT 2 -/y) provide an opportunity to study the relationship between the angiotensin II type 1 receptor (AT 1 ) and nitric oxide synthase (NOS) isoforms without concomitant AT 2 receptor–related effects. To test this relationship, the expression of renal NOS isoforms (neural, inducible, and endothelial) in AT 2 -/y and AT 2 +/y mice was examined. The mice were challenged with deoxycorticosterone acetate (DOCA)-salt to stimulate NO generation. Methods Gene expression analyses by real-time polymerase chain reaction (PCR) (TaqMan) were performed in kidneys to characterize neuronal nitric oxide synthase (nNOS), epithelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and the AT 1 receptor. Pressure-natriuresis experiments were done to determine the physiologic background. Results AT 2 -/y mice showed nNOS and iNOS up-regulation. DOCA-salt increased iNOS expression more in AT 2 -/y mice than in AT 2 +/y mice. Immunohistochemistry localized the iNOS expression with DOCA-salt mainly in the glomeruli. eNOS was not different between the groups, and was not affected by DOCA-salt. DOCA-salt increased mean arterial pressure more in AT 2 -/y mice than in AT 2 +/y mice. Concomitantly, the pressure-natriuresis relationship was shifted to the right in AT 2 -/y and AT 2 +/y mice after DOCA-salt. DOCA-salt decreased renal blood flow (RBF) and glomerular filtration rate (GFR) in both groups. iNOS blockade did not lower blood pressure. Conclusion We conclude that AT 2 receptor deletion and concomitant up-regulation of the AT 1 receptor is associated with up-regulation of nNOS and iNOS. Under DOCA-salt, renal iNOS expression was further increased. Because iNOS inhibition did not change blood pressure, iNOS may not be involved in the hemodynamics, but may contribute to organ damage.

In the yeast Candida maltosa the level of cytochrome P-450 was 100–300-fold higher in alkane-grown cells than in glucose-grown ones (detected by a radioimmunoassay). It was shown immunochemically (1) that this was not the result of an assembly of preexisting apoenzyme with the prosthetic heme group. By cell-free translation of total poly(A)RNA in a wheat germ system and subsequent immunoprecipitation it was shown that the amount of mRNA coding for cytochrome P-450 paralleled its concentration in the cell.

The immunological relations of the cytochrome P-450 from the n-alkane utilizing yeast Candida maltosa to cytochrome P-450 forms of other organisms - yeasts, bacteria and mammalia - were investigated using a solid-phase double-antibody radioimmunoassay. Only the microsomal fraction of other n-alkane utilizing yeasts shows a distinct cross-reaction with an antiserum against cytochrome P-450 from Candida maltosa. Neither the tested bacterial nor the mammalian cytochromes P-450 cross-react with the antiserum.

In the yeast Candida maltosa both the substrate (n-alkane) and oxygen mediated induction of the alkane hydroxylating P-450 were shown to be regulated transcriptionally. That was concluded from good correlations of P-450 protein content (spectral determination of intact holo-enzyme and radioimmunological quantification of apo-enzyme) and relative amount of P-450 mRNA (in vitro translation with subsequent immunoprecipitation).