Supramolecular structures of NiII and CuII with the sterically demanding Schiff base dyes driven by cooperative action of preagostic and other non-covalent interactions.

This work reports on synthesis and extensive experimental and theoretical investigations on photophysical, structural and thermal properties of the NiII and CuII discrete mononuclear homoleptic complexes [Ni(L I,II)2] and [Cu(L I,II)2] fabricated from the Schiff base dyes o-HOC6H4-CH=N-cyclo-C6H11 (HL I) and o-HOC10H6-CH=N-cyclo-C6H11 (HL II), containing the sterically crowding cyclo-hexyl units. The six-membered metallocycles adopt a clearly defined envelope conformation in [Ni(L II)2], while they are much more planar in the structures of [Ni(L I)2] and [Cu(L I,II)2]. It has been demonstrated by in-depth bonding analyses based on the ETS-NOCV and Interacting Quantum Atoms energy-decomposition schemes that application of the bulky substituents, containing several C-H groups, has led to the formation of a set of classical and unintuitive intra- and inter-molecular interactions. All together they are responsible for the high stability of [Ni(L I,II)2] and [Cu(L I,II)2]. More specifically, London dispersion dominated intramolecular C-H⋯O, C-H⋯N and C-H⋯H-C hydrogen bonds are recognized and, importantly, the attractive, chiefly the Coulomb driven, preagostic (not repulsive anagostic) C-H⋯Ni/Cu interactions have been discovered despite their relatively long distances (∼2.8-3.1 Å). All the complexes are further stabilized by the extremely efficient intermolecular C-H⋯π(benzene) and C-H⋯π(chelate) interactions, where both the charge-delocalization and London dispersion constituents appear to be crucial for the crystal packing of the obtained complexes. All the complexes were found to be photoluminescent in CH2Cl2, with [Cu(L II)2] exhibiting the most pronounced emission - the time-dependent density-functional-theory computations revealed that it is mostly caused by metal-to-ligand charge-transfer transitions.

Resonance Assisted Hydrogen Bonding Phenomenon Unveiled through Both Experiments and Theory: A New Family of Ethyl N-Salicylideneglycinate Dyes.

Extensive experimental and theoretical investigations are reported on the nature of resonance-assisted hydrogen bonding phenomenon (RAHB) and its influence on photophysical properties of the newly designed dyes differing in donor-acceptor properties, namely ethyl N-salicylideneglycinate (1), ethyl N-(5-methoxysalicylidene)glycinate (2), ethyl N-(5-bromosalicylidene)glycinate (3) and ethyl N-(5-nitrosalicylidene)glycinate (4). All compounds are thermochromic in the solid state and they contain a typical intramolecular O-H⋅⋅⋅N hydrogen bond formed between the hydroxyl hydrogen atom and the imine nitrogen atom, yielding the enol form in the solid state. It is unveiled, that the magnitude of RAHB effect fine tunes the strength of the O-H⋅⋅⋅N bonding and accordingly the relative populations of the enol, cis-keto and trans-keto forms leading to variation of the photophysical properties of 1-4. It is determined, that the electron-withdrawing NO2 in 4 amplifies the most RAHB effect causing the breaking of the O-H⋅⋅⋅N hydrogen bond and accordingly formation of the dominant cis-keto isomer in both the solid state and EtOH. To this end, the UV/Vis spectra of 1-3 in EtOH revealed the exclusive presence of the enol form, while the prevalent contribution of the cis-keto form was found for 4. Furthermore, only compound 4 is emissive in the solid state in ambient condition due to dual emission arising from the cis-keto* and trans-keto* forms, while 2 was found to be highly emissive in EtOH. It is revealed qualitatively and quantitatively, based on the ETS-NOCV charge and energy decomposition scheme and the EDDB population-based method, that RAHB is strongly a non-local phenomenon based on electrons pumping or sucking through both the π- and σ-channels, which accordingly exerts chemical bonding changes at both the phenyl ring and predominantly a distant O-H⋅⋅⋅N area.

Elevated interleukin-8 in bile of patients with primary sclerosing cholangitis.

BACKGROUND & AIMS: To better understand the pathogenesis of primary sclerosing cholangitis, anti- and pro-inflammatory factors were studied in bile. METHODS: Ductal bile of PSC patients (n = 36) and controls (n = 20) was collected by endoscopic retrograde cholangiography. Gallbladder bile was collected at liver transplantation. Bile samples were analysed for cytokines, FGF19 and biliary lipids. Hepatobiliary tissues of PSC and non-PSC patients (n = 8-11 per patient group) were collected at transplantation and were analysed for IL8 and FGF19 mRNA expression and IL8 localization. The effect of IL8 on proliferation of primary human cholangiocytes and expression of pro-fibrotic genes was studied. RESULTS: In PSC patients, median IL8 in ductal bile was 6.6 ng/ml vs. 0.24 ng/ml in controls. Median IL8 in gallbladder bile was 7.6 ng/ml in PSC vs. 2.2 and 0.3 ng/ml in two control groups. IL8 mRNA in PSC gallbladder was increased and bile ducts stained positive for IL8. In vitro, IL8 induced proliferation of primary human cholangiocytes and increased the expression of pro-fibrotic genes. CONCLUSION: Elevation of IL8 in bile of PSC patients, collected at different stages of disease, indicates an ongoing inflammatory stimulus that drives IL8 production. This challenges the idea that advanced PSC is a burned-out disease, and calls for reconsideration of anti-inflammatory therapy in PSC.

Genome-wide association analysis in primary sclerosing cholangitis and ulcerative colitis identifies risk loci at GPR35 and TCF4.

UNLABELLED: Approximately 60%-80% of patients with primary sclerosing cholangitis (PSC) have concurrent ulcerative colitis (UC). Previous genome-wide association studies (GWAS) in PSC have detected a number of susceptibility loci that also show associations in UC and other immune-mediated diseases. We aimed to systematically compare genetic associations in PSC with genotype data in UC patients with the aim of detecting new susceptibility loci for PSC. We performed combined analyses of GWAS for PSC and UC comprising 392 PSC cases, 987 UC cases, and 2,977 controls and followed up top association signals in an additional 1,012 PSC cases, 4,444 UC cases, and 11,659 controls. We discovered novel genome-wide significant associations with PSC at 2q37 [rs3749171 at G-protein-coupled receptor 35 (GPR35); P = 3.0 × 10(-9) in the overall study population, combined odds ratio [OR] and 95% confidence interval [CI] of 1.39 (1.24-1.55)] and at 18q21 [rs1452787 at transcription factor 4 (TCF4); P = 2.61 × 10(-8) , OR (95% CI) = 0.75 (0.68-0.83)]. In addition, several suggestive PSC associations were detected. The GPR35 rs3749171 is a missense single nucleotide polymorphism resulting in a shift from threonine to methionine. Structural modeling showed that rs3749171 is located in the third transmembrane helix of GPR35 and could possibly alter efficiency of signaling through the GPR35 receptor. CONCLUSION: By refining the analysis of a PSC GWAS by parallel assessments in a UC GWAS, we were able to detect two novel risk loci at genome-wide significance levels. GPR35 shows associations in both UC and PSC, whereas TCF4 represents a PSC risk locus not associated with UC. Both loci may represent previously unexplored aspects of PSC pathogenesis.

Septin 8 is an interaction partner and in vitro substrate of MK5.

AIM: To identify novel substrates for the mitogen-activated protein kinase-activated protein kinase 5 (MK5). METHODS: Yeast two-hybrid screening with MK5 as bait was used to identify novel possible interaction partners. The binding of putative partner was further examined by glutathione S-transferase (GST) pull-down, co-immunoprecipitation and fluorescence resonance energy transfer (FRET) analysis. In vitro kinase and peptide array assays were used to map MK5 phosphoacceptor sites on the new partner. Confocal microscopy was performed to study the subcellular localization of MK5 and its partners. RESULTS: Septin 8 was identified as a novel interaction partner for MK5 by yeast two-hybrid screening. This interaction was confirmed by GST pull-down, co-immunoprecipitation and FRET analysis. Septin 5, which can form a complex with septin 8, did not interact with MK5. Serine residues 242 and 271 on septin 8 were identified as in vitro MK5 phosphorylation sites. MK5 and septin 8 co-localized in the perinuclear area and in cell protrusions. Moreover, both proteins co-localized with vesicle marker synaptophysin. CONCLUSION: Septin 8 is a bona fide interaction partner and in vitro substrate for MK5. This interaction may be implicated in vesicle trafficking.

Genetics in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic disorder with a progressive course. PSC is strongly associated with inflammatory bowel disease and is often complicated by cholangiocarcinoma development. Etiology and pathogenesis remain obscure, but the diverse clinical manifestation of the disease might, to some extent, indicate different genetic susceptibility in subgroups of patients. In recent years, genome-wide association studies performed in PSC have identified a number of genetic susceptibility loci. In this mini-review, we suggest that the genetic associations established can be grouped according to four pathogenic aspects relating to inflammation, cholangiocyte function, fibrosis and carcinogenesis. Subclassification of PSC patients according to their genetic predisposition could be a valuable tool in future functional and clinical studies.

Extended analysis of a genome-wide association study in primary sclerosing cholangitis detects multiple novel risk loci.

BACKGROUND & AIMS: A limited number of genetic risk factors have been reported in primary sclerosing cholangitis (PSC). To discover further genetic susceptibility factors for PSC, we followed up on a second tier of single nucleotide polymorphisms (SNPs) from a genome-wide association study (GWAS). METHODS: We analyzed 45 SNPs in 1221 PSC cases and 3508 controls. The association results from the replication analysis and the original GWAS (715 PSC cases and 2962 controls) were combined in a meta-analysis comprising 1936 PSC cases and 6470 controls. We performed an analysis of bile microbial community composition in 39 PSC patients by 16S rRNA sequencing. RESULTS: Seventeen SNPs representing 12 distinct genetic loci achieved nominal significance (p(replication) <0.05) in the replication. The most robust novel association was detected at chromosome 1p36 (rs3748816; p(combined)=2.1 × 10(-8)) where the MMEL1 and TNFRSF14 genes represent potential disease genes. Eight additional novel loci showed suggestive evidence of association (p(repl) <0.05). FUT2 at chromosome 19q13 (rs602662; p(comb)=1.9 × 10(-6), rs281377; p(comb)=2.1 × 10(-6) and rs601338; p(comb)=2.7 × 10(-6)) is notable due to its implication in altered susceptibility to infectious agents. We found that FUT2 secretor status and genotype defined by rs601338 significantly influence biliary microbial community composition in PSC patients. CONCLUSIONS: We identify multiple new PSC risk loci by extended analysis of a PSC GWAS. FUT2 genotype needs to be taken into account when assessing the influence of microbiota on biliary pathology in PSC.

Distinct roles of MK2 and MK5 in cAMP/PKA- and stress/p38MAPK-induced heat shock protein 27 phosphorylation.

BACKGROUND: Classical mammalian mitogen-activated protein kinase (MAPK) pathways consist of a cascade of three successive phosphorylation events resulting in the phosphorylation of a variety of substrates, including another class of protein kinases referred to as MAPK-activating protein kinases (MAPKAPKs). The MAPKAPKs MK2, MK3 and MK5 are closely related, but MK2 and MK3 are the major downstream targets of the p38MAPK pathway, while MK5 can be activated by the atypical MAPK ERK3 and ERK4, protein kinase A (PKA), and maybe p38MAPK. MK2, MK3, and MK5 can phosphorylate the common substrate small heat shock protein 27 (HSP27), a modification that regulates the role of HSP27 in actin polymerization. Both stress and cAMP elevating stimuli can cause F-actin remodeling, but whereas the in vivo role of p38MAPK-MK2 in stress-triggered HSP27 phosphorylation and actin reorganization is well established, it is not known whether MK2 is involved in cAMP/PKA-induced F-actin rearrangements. On the other hand, MK5 can phosphorylate HSP27 and cause cytoskeletal changes in a cAMP/PKA-dependent manner, but its role as HSP27 kinase in stress-induced F-actin remodeling is disputed. Therefore, we wanted to investigate the implication of MK2 and MK5 in stress- and PKA-induced HSP27 phosphorylation. RESULTS: Using HEK293 cells, we show that MK2, MK3, and MK5 are expressed in these cells, but MK3 protein levels are very moderate. Stress- and cAMP-elevating stimuli, as well as ectopic expression of active MKK6 plus p38MAPK or the catalytic subunit of PKA trigger HSP27 phosphorylation, and specific inhibitors of p38MAPK and PKA prevent this phosphorylation. Depletion of MK2, but not MK3 and MK5 diminished stress-induced HSP27 phosphorylation, while only knockdown of MK5 reduced PKA-induced phosphoHSP27 levels. Stimulation of the p38MAPK, but not the PKA pathway, caused activation of MK2. CONCLUSION: Our results suggest that in HEK293 cells MK2 is the HSP27 kinase engaged in stress-induced, but not cAMP-induced phosphorylation of HSP27, while MK5 seems to be the sole MK to mediate HSP27 phosphorylation in response to stimulation of the PKA pathway. Thus, despite the same substrate specificity towards HSP27, MK2 and MK5 are implicated in different signaling pathways causing actin reorganization.

Cross-talk between protein kinase A and the MAPK-activated protein kinases RSK1 and MK5.

Typical mammalian mitogen-activated protein kinase (MAPK) pathways consist of a cascade of three consecutive phosphorylation events exerted by a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK), and finally a MAPK. MAPKs not only target non-protein kinase substrates, they can also phosphorylate other protein kinases designated as MAPK-activated protein kinases (MAPKAPK). The MAPKAPK family includes the ribosomal-S6-kinases (RSK1-4), the MAPK-interacting kinases (MNK1 and 2), the mitogen-and stress-activated kinases (MSK1 and 2), and the MAPKAPK (MK2, 3, and 5) subfamilies. Although several reports indicate extensive cross-talk between the MAPK and protein kinase A (PKA) pathways, evidence of a direct interaction at the level of the MAPKAPK only appeared recently. The MAPKAPKs RSK1 and MK5 can bind to PKA, but the features of these interactions are distinct. This review discusses the different characteristics of regulating the activity and subcellular localization of MK5 and RSK1 by PKA and the functional implications of these interactions.

Serine residue 115 of MAPK-activated protein kinase MK5 is crucial for its PKA-regulated nuclear export and biological function.

The mitogen-activated protein kinase-activated protein kinase-5 (MK5) resides predominantly in the nucleus of resting cells, but p38(MAPK), extracellular signal-regulated kinases-3 and -4 (ERK3 and ERK4), and protein kinase A (PKA) induce nucleocytoplasmic redistribution of MK5. The mechanism by which PKA causes nuclear export remains unsolved. In the study reported here we demonstrated that Ser-115 is an in vitro PKA phosphoacceptor site, and that PKA, but not p38(MAPK), ERK3 or ERK4, is unable to redistribute MK5 S115A to the cytoplasm. However, the phospho-mimicking MK5 S115D mutant resides in the cytoplasm in untreated cells. While p38(MAPK), ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells. Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5. Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5.

Comment on "Increased MKK4 abundance with replicative senescence is linked to the joint reduction of multiple microRNAs".

Marasa et al. (Research Article, 27 October 2009, DOI: 10.1126/scisignal.2000442) reported that the human kinase p38-regulated/activated protein kinase (PRAK) was phosphorylated on residue Ser(93) in senescent cells. We have been unable to detect phosphorylation at this site with the antibody that they used, and the commercial supplier of this antibody has discontinued its availability, which casts doubt on whether this residue of PRAK is phosphorylated.

Mitogen-activated protein kinase p38 and MK2, MK3 and MK5: ménage à trois or ménage à quatre?

The mitogen-activated protein kinase (MAPK) signalling pathways play pivotal roles in cellular processes such as proliferation, apoptosis, gene regulation, differentiation, and cell motility. The typical mammalian MAPK pathways ERK1/2, JNK, p38(MAPK), and ERK5 operate through a concatenation of three successive phosphorylation events mediated by a MAPK kinase kinase, a MAPK kinase, and a MAPK. MAPKs phosphorylate substrates with distinct functions, including other protein kinases referred to as MAPK-activated protein kinases. One family of related MAPK-activated protein kinases includes MK2, MK3, and MK5. While it is generally accepted that MK2 and MK3 are bona fide substrates for p38(MAPK), the genuineness of MK5 as a p38(MAPK) substrate is disputed. This review summarizes the findings pro and contra an authentic p38(MAPK)-MK5 relationship, discusses possible explanations for these discrepancies, and proposes experiments that may help to unequivocally clarify whether MK5 is indeed a substrate for p38(MAPK).

The transcriptional regulation and cell-specific expression of the MAPK-activated protein kinase MK5.

The mitogen-activated protein kinase (MAPK) cascades regulate important cellular processes, including growth, differentiation, apoptosis, embryogenesis, motility and gene expression. Although MAPKs mostly appear to be constitutively expressed, the transcript levels of some MAPK-encoding genes increase upon treatment with specific stimuli. This applies to the MAPK-activated protein kinases MK2 and MK3. By contrast, the transcriptional regulation of the related MK5 has not yet been studied. The MK5 promoters of mouse, rat and human contain a plethora of putative transcription factor sites, and the spatio-temporal expression of MK5 suggests inducible transcription of the gene. We examined the transcription pattern of MK5 in different tissues, and studied the kinetics of MK5 expression at the transcriptional and/or translation level in PC12 cells exposed to arsenite, forskolin, KCl, lipopolysaccharide, spermine NONOate, retinoic acid, serum, phorbol ester, temperature shock, and vanadate. Cells exposed to forskolin display a transient increase in MK5 mRNA, despite their unaltered MK5 protein levels. The MK5 promoters of human, mouse and rat contain a cAMP-responsive element that binds the cAMP-responsive element-binding protein (CREB) in vitro. Luciferase reporter constructs containing an 850-base pair human MK5 promoter fragment encompassing the CRE showed a basal activity that was 10-fold higher than the corresponding construct in which the CRE motif was deleted. siRNA-mediated depletion of CREB had no effect on the endogenous MK5 protein levels. Several binding motifs for heat shock factor are dispersed in the mouse and rat promoter, and temperature shock transiently enhanced the MK5 transcript levels. None of the other tested stimuli had an effect on the MK5 mRNA or protein levels. Our results indicate an inducible regulation of MK5 transcription in response to specific stimuli. However, the MK5 protein levels remained unaffected by all the stimuli tested. There is still no explanation for the discrepancy between the increased mRNA and unchanged MK5 protein levels.

Relations between the mitogen-activated protein kinase and the cAMP-dependent protein kinase pathways: comradeship and hostility.

Inter- and intracellular communications and responses to environmental changes are pivotal for the orchestrated and harmonious operation of multi-cellular organisms. These well-tuned functions in living organisms are mediated by the action of signal transduction pathways, which are responsible for receiving a signal, transmitting and amplifying it, and eliciting the appropriate cellular responses. Mammalian cells posses numerous signal transduction pathways that, rather than acting in solitude, interconnect with each other, a phenomenon referred to as cross-talk. This allows cells to regulate the distribution, duration, intensity and specificity of the response. The cAMP/cAMP-dependent protein kinase (PKA) pathway and the mitogen-activated protein kinase (MAPK) cascades modulate common processes in the cell and multiple levels of cross-talk between these signalling pathways have been described. The first- and best-characterized interconnections are the PKA-dependent inhibition of the MAPKs ERK1/2 mediated by RAF-1, and PKA-induced activation of ERK1/2 interceded through B-RAF. Recently, novel interactions between components of these pathways and new mechanisms for cross-talk have been elucidated. This review discusses both known and novel interactions between compounds of the cAMP/PKA and MAPKs signalling pathways in mammalian cells.

Modulation of F-actin rearrangement by the cyclic AMP/cAMP-dependent protein kinase (PKA) pathway is mediated by MAPK-activated protein kinase 5 and requires PKA-induced nuclear export of MK5.

The MAPK-activated protein kinases belong to the Ca2+/calmodulin-dependent protein kinases. Within this group, MK2, MK3, and MK5 constitute three structurally related enzymes with distinct functions. Few genuine substrates for MK5 have been identified, and the only known biological role is in ras-induced senescence and in tumor suppression. Here we demonstrate that activation of cAMP-dependent protein kinase (PKA) or ectopic expression of the catalytic subunit Calpha in PC12 cells results in transient nuclear export of MK5, which requires the kinase activity of both Calpha and MK5 and the ability of Calpha to enter the nucleus. Calpha and MK5, but not MK2, interact in vivo, and Calpha increases the kinase activity of MK5. Moreover, Calpha augments MK5 phosphorylation, but not MK2, whereas MK5 does not seem to phosphorylate Calpha. Activation of PKA can induce actin filament accumulation at the plasma membrane and formation of actin-based filopodia. We demonstrate that small interfering RNA-triggered depletion of MK5 interferes with PKA-induced F-actin rearrangement. Moreover, cytoplasmic expression of an activated MK5 variant is sufficient to mimic PKA-provoked F-actin remodeling. Our results describe a novel interaction between the PKA pathway and MAPK signaling cascades and suggest that MK5, but not MK2, is implicated in PKA-induced microfilament rearrangement.