Schlafen 11 Is a Novel Target for Mucosal Regeneration in Ulcerative Colitis.

BACKGROUND AND AIMS: Ulcerative colitis [UC] is a chronic inflammatory disease of the colon with an intractable course. Although the goal of UC therapy is to achieve mucosal healing, the pathogenesis of mucosal injury caused by chronic inflammation remains unknown. We therefore aim to elucidate molecular mechanisms of mucosal injury by establishing in vitro and in vivo humanised UC-mimicking models. METHODS: An in vitro model using human colon organoids was established by 60 weeks of inflammatory stimulation. The key gene for mucosal injury caused by long-term inflammation was identified by microarray analysis. An in vivo model was established by xenotransplantation of organoids into mouse colonic mucosa. RESULTS: An in vitro model demonstrated that long-term inflammation induced irrecoverable changes in organoids: inflammatory response and apoptosis with oxidative stress and suppression of cell viability. This model also mimicked organoids derived from patients with UC at the gene expression and phenotype levels. Microarray analysis revealed Schlafen11 [SLFN11] was irreversibly induced by long-term inflammation. Consistently, SLFN11 was highly expressed in UC mucosa but absent in normal mucosa. The knockdown of SLFN11 [SLFN11-KD] suppressed apoptosis of intestinal epithelial cells [IECs] induced by inflammation. Moreover, SLFN11-KD improved the take rates of xenotransplantation and induced the regenerative changes of crypts observed in patients with UC in remission. CONCLUSIONS: In vitro and in vivo UC-mimicking models were uniquely established using human colonic organoids. They revealed that SLFN11 is significant for mucosal injury in UC, and demonstrated its potential as a novel target for mucosal regeneration.

TP53 Mutation by CRISPR System Enhances the Malignant Potential of Colon Cancer.

Tumor protein p53 (TP53) mutation is a well-known occurrence at the late phase of carcinogenesis during the adenoma-carcinoma sequence of a sporadic colon cancer. Although numerous reports about clinical information of the patients with colon cancer have suggested that TP53 mutation might be related to various types of malignant potential, the direct effects of this mutation on the malignant potential of colon cancer remain unknown. Notably, no previous report has described a relationship between TP53 mutation and cancer stemness. We therefore aimed to assess the function of a TP53 mutant induced by the CRISPR-Cas9 system in colon cancer cells. In this study, two TP53 mutations, corresponding to exon 3 (TP53E3) and 10 (TP53E10), were generated in LS174T cells derived from a wild-type TP53 human colon cancer via a lentiviral CRISPR-Cas9 system. The loss of function of TP53 resulting from both mutations manifested as resistance to Nutlin3a-induced apoptosis and the downregulation of target genes of TP53. TP53 mutants exhibited an enhanced malignant potential, characterized by accelerated cell growth, invasiveness, chemoresistance, and cancer stemness. Interestingly, TP53E10 but not TP53E3 cells exhibited aberrant transcriptional activity of regenerating family member 1-α (REG1A) and expression of REG1A, resulting in the acquisition of enhanced malignant potential. In conclusion, we demonstrated for the first time that TP53 genomic mutation into human colon cancer cells affects the malignant potential. IMPLICATIONS: These findings suggest that both a loss of function and an aberrant gain of function of TP53 might promote high malignant potentials at the late phase of carcinogenesis in colon cancer.

Establishment of a system to evaluate the therapeutic effect and the dynamics of an investigational drug on ulcerative colitis using human colonic organoids.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with an intractable, recurrent course. The goal of UC therapy is to target mucosal healing because immune-suppressive therapy for UC frequently results in relapse. However, few drugs directly target mucosal healing. We, therefore, aim to evaluate the therapeutic effect of an investigational drug on intestinal epithelial cells in an in vitro UC model using human colonic organoids. METHODS: Colonic organoids were isolated from human colon and cultured. A mixture of cytokines and bacterial components were used to mimic UC in humans. The effect of the investigational drug on colonic organoid was evaluated by microarray analysis and 3D immunofluorescence. The enrichment of stem cells was assessed with a colony formation assay. RESULTS: Inflammatory stimulation resulted in a significant induction of inflammatory-related genes in colonic organoids whereas cell differentiation was suppressed. Treatment with the investigational drug KAG-308 showed reciprocal dynamics of gene expression to inflammatory stimulation, which resulted in not only the suppression of immune response but also the promotion of cellular differentiation towards secretory lineages. Moreover, SPDEF and Reg4 were identified as novel targets for the enrichment of intestinal epithelial stem cells and mucosal healing. CONCLUSIONS: The establishment of in vitro UC model using human colonic organoid could reveal the effects and targets of investigational drugs in intestinal epithelial cells under inflammation conditions. Further maturation of this system might be more efficient to predict the effect on UC, as compared with the use of animal model, for the development of new drugs.

Synergistic Effect of Distal Polar Interactions in Myoglobin and Their Structural Consequences.

In the L29F variant of myoglobin (Mb), the coordination of oxygen (O2) to the heme Fe atom is stabilized by favorable electrostatic interactions between the polar Fe-O2 moiety and the multipole of the phenyl ring of the Phe29 side chain (Phe29 interaction), in addition to the well-known hydrogen bond (H-bond) between the Fe-bound O2 and the 64th residue (distal H-bond; Carver, T. E.; Brantley, R. E., Jr.; Singleton, E. W.; Arduini, R. M.; Quillin, M. L.; Phillips, G. N., Jr.; Olson, J. S. J. Biol. Chem. 1992, 267, 14443-14450). The O2 and carbon monoxide (CO) binding properties and autoxidation of the L29F/H64L and L29F/H64Q variants reconstituted with a series of chemically modified heme cofactors were analyzed and then compared with those of native Mb, and the L29F, H64Q, and H64L variants similarly reconstituted with the chemically modified heme cofactors in order to elucidate the relationship between the Phe29 interaction and the distal H-bond that critically contributes to stabilization of Fe-bound O2. We found that the Phe29 interaction and distal H-bond act cooperatively to stabilize the Fe-bound O2 in such a manner that the Phe29 interaction strengthens with increasing strength of the distal H-bond. Comparison of the functional properties between the L29F and H64L variants indicated that the synergistic effect of the two interactions decreases the O2 dissociation and autoxidation rate constants of the protein by factors of ∼1/2000 and ∼1/400, respectively. Although the CO binding properties of the proteins were not greatly affected by the distal polar interactions, their synergistic effects were clearly and sharply manifested in the vibrational frequencies of the Fe-bound C-O stretching of the proteins.

Characterization of Heme Orientational Disorder in a Myoglobin Reconstituted with a Trifluoromethyl-Group-Substituted Heme Cofactor.

The orientation of a CF3-substituted heme in sperm whale myoglobin and L29F, H64L, L29F/H64Q, and H64Q variant proteins has been investigated using 19F NMR spectroscopy to elucidate structural factors responsible for the thermodynamic stability of the heme orientational disorder, i.e., the presence of two heme orientations differing by a 180° rotation about the 5-15 meso axis, with respect to the protein moiety. Crystal structure of the met-aquo form of the wild-type myoglobin reconstituted with 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-trifluoromethylporphyrinatoiron(III), determined at resolution of 1.25 Å, revealed the presence of the heme orientational disorder. Alterations of the salt bridge between the heme 13-propionate and Arg45(CD3) side chains due to the mutations resulted in equilibrium constants of the heme orientational disorder ranging between 0.42 and 1.4. Thus, the heme orientational disorder is affected by the salt bridge associated with the heme 13-propionate side chain, confirming the importance of the salt bridge in the heme binding to the protein.

Long-term Inflammation Transforms Intestinal Epithelial Cells of Colonic Organoids.

BACKGROUND AND AIMS: Patients with ulcerative colitis [UC] are at an increased risk of developing colitis-associated cancer [CAC], suggesting that continuous inflammation in the colon promotes the transformation of colonic epithelial cells. However, the mechanisms underlying cell transformation in UC remain unknown. We therefore aimed to investigate the effect of long-term inflammation on intestinal epithelial cells [IECs] using organoid culture. METHODS: IECs were isolated from mouse colon, and were cultured according to a method for a three-dimensional [3D] organoid culture. To mimic chronic inflammation, a mixture of cytokines and bacterial components were added to the medium for over a year. Cell signal intensity was assessed by 3D immunofluorescence. Cell transformation was assessed by microarray with gene set enrichment analysis. RESULTS: Stimulation with cytokines resulted in a significant induction of target genes for the nuclear factor [NF]-κB pathway in colonic organoids. Following 60 weeks of continuous stimulation, cell differentiation was suppressed. Continuous stimulation also resulted in significant amplification of NF-κB signalling. Amplified NF-κB signalling by long-term stimulation remained in colonic organoids even 11 weeks after the removal of all cytokines. Some genes were specifically upregulated only in colonic organoids after the removal all cytokines following long-term stimulation. CONCLUSIONS: Colonic organoids stimulated with cytokines for a prolonged period were established as in vitro model to assess long-term epithelial responses to inflammatory cytokines. Chronic inflammation led to sustained NF-κB signalling activation in colonic organoids, resulting in cell transformation that might be related to the carcinogenesis of CAC in UC.

Characterization of Ground State Electron Configurations of High-Spin Quintet Ferrous Heme Iron in Deoxy Myoglobin Reconstituted with Trifluoromethyl Group-Substituted Heme Cofactors.

We introduced trifluoromethyl (CF3) group(s) as heme side chain(s) of sperm whale myoglobin (Mb) in order to characterize the electronic nature of heme Fe(II) in deoxy Mb using 19F NMR spectroscopy. On the basis of the anti-Curie behavior of CF3 signals, we found that the deoxy Mb is in thermal equilibrium between the 5B2, (dxy)2(dxz)(dyz)(dz2)(dx2-y2), and 5E, (dxy)(dxz)2(dyz)(dz2)(dx2-y2), states of the heme Fe(II), i.e., 5B2 ⇆ 5E. Analysis of the curvature in Curie plots has yielded for the first time ΔH and ΔS values of ∼-20 kJ mol-1 and ∼-60 J K-1 mol-1, respectively, for the thermal equilibrium. Thus, the 5E state is slightly dominant over the 5B2 one at 25 °C. These findings provide not only valuable information about the ground state electronic structure of the high-spin heme Fe(II) in deoxy native Mb but also an important clue for elucidating the mechanism responsible for acceleration of the spin-forbidden oxygenation of the protein.

Effects of Heme Electronic Structure and Distal Polar Interaction on Functional and Vibrational Properties of Myoglobin.

We analyzed the oxygen (O2) and carbon monoxide (CO) binding properties, autoxidation reaction rate, and FeO2 and FeCO vibrational frequencies of the H64Q mutant of sperm whale myoglobin (Mb) reconstituted with chemically modified heme cofactors possessing a variety of heme Fe electron densities (ρ(Fe)), and the results were compared with those for the previously studied native [Shibata, T. et al. J. Am. Chem. Soc. 2010, 132, 6091-6098], and H64L [Nishimura, R. et al. Inorg. Chem. 2014, 53, 1091-1099], and L29F [Nishimura, R. et al. Inorg. Chem. 2014, 53, 9156-9165] mutants in order to elucidate the effect of changes in the heme electronic structure and distal polar interaction contributing to stabilization of the Fe-bound ligand on the functional and vibrational properties of the protein. The study revealed that, as in the cases of the previously studied native protein [Shibata, T. et al. Inorg. Chem. 2012, 51, 11955-11960], the O2 affinity and autoxidation reaction rate of the H64Q mutant decreased with a decrease in ρ(Fe), as expected from the effect of a change in ρ(Fe) on the resonance between the Fe(2+)-O2 bond and Fe(3+)-O2(-)-like species in the O2 form, while the CO affinity of the protein is independent of a change in ρ(Fe). We also found that the well-known inverse correlation between the frequencies of Fe-bound CO (ν(CO)) and Fe-C (ν(FeC)) stretching [Li, X.-Y.; Spiro, T. G. J. Am. Chem. Soc. 1988, 110, 6024-6033] is affected differently by changes in ρ(Fe) and the distal polar interaction, indicating that the effects of the two electronic perturbations due to the chemical modification of a heme cofactor and the replacement of nearby amino acid residues on the resonance between the two alternative canonical forms of the FeCO fragment in the protein are slightly different from each other. These findings provide a new insight for deeper understanding of the functional regulation of the protein.

Electronic control of ligand-binding preference of a myoglobin mutant.

The L29F mutant of sperm whale myoglobin (Mb), where the leucine 29 residue was replaced by phenylalanine (Phe), was shown to exhibit remarkably high affinity to oxygen (O2), possibly due to stabilization of the heme Fe atom-bound O2 in the mutant protein through a proposed unique electrostatic interaction with the introduced Phe29, in addition to well-known hydrogen bonding with His64 [Carver, T. E.; Brantley, R. E.; Singleton, E. W.; Arduini, R. M.; Quillin, M. L.; Phillips, G. N., Jr.; Olson, J. S. J. Biol. Chem., 1992, 267, 14443-14450]. We analyzed the O2 and carbon monoxide (CO) binding properties of the L29F mutant protein reconstituted with chemically modified heme cofactors possessing a heme Fe atom with various electron densities, to determine the effect of a change in the electron density of the heme Fe atom (ρ(Fe)) on the O2 versus CO discrimination. The study demonstrated that the preferential binding of O2 over CO by the protein was achieved through increasing ρ(Fe), and the ordinary ligand-binding preference, that is, the preferential binding of CO over O2, by the protein was achieved through decreasing ρ(Fe). Thus, the O2 and CO binding preferences of the L29F mutant protein could be controlled through electronic modulation of intrinsic heme Fe reactivity through a change in ρ(Fe). The present study highlighted the significance of the tuning of the intrinsic heme Fe reactivity through the heme electronic structure in functional regulation of Mb.

Electronic control of discrimination between O2 and CO in myoglobin lacking the distal histidine residue.

We analyzed the oxygen (O2) and carbon monoxide (CO) binding properties of the H64L mutant of myoglobin reconstituted with chemically modified heme cofactors possessing a heme Fe atom with a variety of electron densities, in order to elucidate the effect of the removal of the distal His64 on the control of both the O2 affinity and discrimination between O2 and CO of the protein by the intrinsic heme Fe reactivity through the electron density of the heme Fe atom (ρFe). The study revealed that, as in the case of the native protein, the O2 affinity of the H64L mutant protein is regulated by the ρFe value in such a manner that the O2 affinity of the protein decreases, due to an increase in the O2 dissociation rate constant, with a decrease in the ρFe value, and that the O2 affinities of the mutant and native proteins are affected comparably by a given change in the ρFe value. On the other hand, the CO affinity of the H64L mutant protein was found to increase, due to a decrease in the CO dissociation rate constant, with a decrease in the ρFe value, whereas that of the native protein was essentially independent of a change in the ρFe value. As a result, the regulation of the O2/CO discrimination in the protein through the ρFe value is affected by the distal His64. Thus, the study revealed that the electronic tuning of the intrinsic heme Fe reactivity through the ρFe value plays a vital role in the regulation of the protein function, as the heme environment furnished by the distal His64 does.

Relationship between the electron density of the heme Fe atom and the vibrational frequencies of the Fe-bound carbon monoxide in myoglobin.

We analyzed the vibrational frequencies of the Fe-bound carbon monoxide (CO) of myoglobin reconstituted with a series of chemically modified heme cofactors possessing a heme Fe atom with a variety of electron densities. The study revealed that the stretching frequency of Fe-bound CO (ν(CO)) increases with decreasing electron density of the heme Fe atom (ρ(Fe)). This finding demonstrated that the ν(CO) value can be used as a sensitive measure of the ρ(Fe) value and that the π back-donation of the heme Fe atom to CO is affected by the heme π-system perturbation induced through peripheral side chain modifications.

Relationship between oxygen affinity and autoxidation of myoglobin.

Studies using myoglobins reconstituted with a variety of chemically modified heme cofactors revealed that the oxygen affinity and autoxidation reaction rate of the proteins are highly correlated to each other, both decreasing with decreasing the electron density of the heme iron atom. An Fe(3+)-O(2)(-)-like species has been expected for the Fe(2+)-O(2) bond in the protein, and the electron density of the heme iron atom influences the resonance process between the two forms. A shift of the resonance toward the Fe(2+)-O(2) form results in lowering of the O(2) affinity due to an increase in the O(2) dissociation rate. On the other hand, a shift of the resonance toward the Fe(3+)-O(2)(-)-like species results in acceleration of the autoxidation through increasing H(+) affinity of the bound ligand.

Structural characterization of a carbon monoxide adduct of a heme-DNA complex.

The structure of a carbon monoxide (CO) adduct of a complex between heme and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized using ¹H and ¹³C NMR spectroscopy and density function theory calculations. The study revealed that the heme binds to the 3'-terminal G-quartet of the DNA though a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The π-π stacking interaction between the pseudo-C2-symmetric heme and the C4-symmetric G-quartet in the complex resulted in the formation of two isomers possessing heme orientations differing by 180° rotation about the pseudo-C2 axis with respect to the DNA. These two slowly interconverting heme orientational isomers were formed in a ratio of approximately 1:1, reflecting that their thermodynamic stabilities are identical. Exogenous CO is coordinated to heme Fe on the side of the heme opposite the G-quartet in the complex, and the nature of the Fe-CO bond in the complex is similar to that of the Fe-CO bonds in hemoproteins. These findings provide novel insights for the design of novel DNA enzymes possessing metalloporphyrins as prosthetic groups.