Silencing of SbPPCK1-3 Negatively Affects Development, Stress Responses and Productivity in Sorghum.

Phosphoenolpyruvate carboxylase (PEPC) plays central roles in photosynthesis, respiration, amino acid synthesis, and seed development. PEPC is regulated by different post-translational modifications. Between them, the phosphorylation by PEPC-kinase (PEPCk) is widely documented. In this work, we simultaneously silenced the three sorghum genes encoding PEPCk (SbPPCK1-3) by RNAi interference, obtaining 12 independent transgenic lines (Ppck1-12 lines), showing different degrees of SbPPCK1-3 silencing. Among them, two T2 homozygous lines (Ppck-2 and Ppck-4) were selected for further evaluation. Expression of SbPPCK1 was reduced by 65% and 83% in Ppck-2 and Ppck-4 illuminated leaves, respectively. Expression of SbPPCK2 was higher in roots and decreased by 50% in Ppck-2 and Ppck-4 in this tissue. Expression of SbPPCK3 was low and highly variable. Despite the incomplete gene silencing, it decreased the degree of phosphorylation of PEPC in illuminated leaves, P-deficient plants, and NaCl-treated plants. Both leaves and seeds of Ppck lines had altered metabolic profiles and a general decrease in amino acid content. In addition, Ppck lines showed delayed flowering, and 20% of Ppck-4 plants did not produce flowers at all. The total amount of seeds was lowered by 50% and 36% in Ppck-2 and Ppck-4 lines, respectively. The quality of seeds was lower in Ppck lines: lower amino acid content, including Lys, and higher phytate content. These data confirm the relevance of the phosphorylation of PEPC in sorghum development, stress responses, yield, and quality of seeds.

Knock-down of phosphoenolpyruvate carboxylase 3 negatively impacts growth, productivity, and responses to salt stress in sorghum (Sorghum bicolor L.).

Phosphoenolpyruvate carboxylase (PEPC) is a carboxylating enzyme with important roles in plant metabolism. Most studies in C4 plants have focused on photosynthetic PEPC, but less is known about non-photosynthetic PEPC isozymes, especially with respect to their physiological functions. In this work, we analyzed the precise roles of the sorghum (Sorghum bicolor) PPC3 isozyme by the use of knock-down lines with the SbPPC3 gene silenced (Ppc3 lines). Ppc3 plants showed reduced stomatal conductance and plant size, a delay in flowering time, and reduced seed production. In addition, silenced plants accumulated stress indicators such as Asn, citrate, malate, and sucrose in roots and showed higher citrate synthase activity, even in control conditions. Salinity further affected stomatal conductance and yield and had a deeper impact on central metabolism in silenced plants compared to wild type, more notably in roots, with Ppc3 plants showing higher nitrate reductase and NADH-glutamate synthase activity in roots and the accumulation of molecules with a higher N/C ratio. Taken together, our results show that although SbPPC3 is predominantly a root protein, its absence causes deep changes in plant physiology and metabolism in roots and leaves, negatively affecting maximal stomatal opening, growth, productivity, and stress responses in sorghum plants. The consequences of SbPPC3 silencing suggest that this protein, and maybe orthologs in other plants, could be an important target to improve plant growth, productivity, and resistance to salt stress and other stresses where non-photosynthetic PEPCs may be implicated.

Genetic and Pharmacological Inhibition of Autophagy increases the Monoubiquitination of Non-Photosynthetic Phosphoenolpyruvate Carboxylase.

Phosphoenolpyruvate carboxylase (PEPC) is an enzyme with key roles in carbon and nitrogen metabolisms. The mechanisms that control enzyme stability and turnover are not well known. This paper investigates the degradation of PEPC via selective autophagy, including the role of the monoubiquitination of the enzyme in this process. In Arabidopsis, the genetic inhibition of autophagy increases the amount of monoubiquitinated PEPC in the atg2, atg5, and atg18a lines. The same is observed in nbr1, which is deficient in a protein that recruits monoubiquitinated substrates for selective autophagy. In cultured tobacco cells, the chemical inhibition of the degradation of autophagic substrates increases the quantity of PEPC proteins. When the formation of the autophagosome is blocked with 3-methyladenine (3-MA), monoubiquitinated PEPC accumulates as a result. Finally, pull-down experiments with a truncated version of NBR1 demonstrate the recovery of intact and/or fragmented PEPC in Arabidopsis leaves and roots, as well as cultured tobacco cells. Taken together, the results show that a fraction of PEPC is cleaved via selective autophagy and that the monoubiquitination of the enzyme has a role in its recruitment towards this pathway. Although autophagy seems to be a minor pathway, the results presented here increase the knowledge about the role of monoubiquitination and the regulation of PEPC degradation.

Unraveling the role of the CbrA histidine kinase in the signal transduction of the CbrAB two-component system in Pseudomonas putida.

The histidine kinase CbrA of the CbrAB two-component system of Pseudomonas putida is a key element to recognise the activating signal and mediate auto- and trans-phosphorylation of the response element CbrB. CbrA is encoded by the gene cbrA which is located downstream of a putative open reading frame we have named cbrX. We describe the role of the CbrX product in the expression of CbrA and show there is translational coupling of the genes. We also explore the role of the transmembrane (TM) and PAS domains of CbrA in the signal recognition. A ΔcbrXA mutant lacking its TM domains is uncoupled in its growth in histidine and citrate as carbon sources, but its overexpression restores the ability to grow in such carbon sources. In these conditions ΔTM-CbrA is able to respond to carbon availability, thus suggesting an intracellular nature for the signal sensed.

Anionic Phospholipids Induce Conformational Changes in Phosphoenolpyruvate Carboxylase to Increase Sensitivity to Cathepsin Proteases.

Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that serves a variety of functions in plants, acting as the primary form of CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). In a previous work we have shown that C4-PEPC bind anionic phospholipids, resulting in PEPC inactivation. Also, we showed that PEPC can associate with membranes and to be partially proteolyzed. However, the mechanism controlling this remains unknown. Using semi purified-PEPC from sorghum leaf and a panel of PEPC-specific antibodies, we analyzed the conformational changes in PEPC induced by anionic phospholipids to cause the inactivation of the enzyme. Conformational changes observed involved the exposure of the C-terminus of PEPC from the native, active enzyme conformation. Investigation of the protease activity associated with PEPC demonstrated that cysteine proteases co-purify with the enzyme, with protease-specific substrates revealing cathepsin B and L as the major protease species present. The anionic phospholipid-induced C-terminal exposed conformation of PEPC appeared highly sensitive to the identified cathepsin protease activity and showed initial proteolysis of the enzyme beginning at the N-terminus. Taken together, these data provide the first evidence that anionic phospholipids promote not only the inactivation of the PEPC enzyme, but also its proteolysis.

HuR biological function involves RRM3-mediated dimerization and RNA binding by all three RRMs.

HuR/ELAVL1 is an RNA-binding protein involved in differentiation and stress response that acts primarily by stabilizing messenger RNA (mRNA) targets. HuR comprises three RNA recognition motifs (RRMs) where the structure and RNA binding of RRM3 and of full-length HuR remain poorly understood. Here, we report crystal structures of RRM3 free and bound to cognate RNAs. Our structural, NMR and biochemical data show that RRM3 mediates canonical RNA interactions and reveal molecular details of a dimerization interface localized on the α-helical face of RRM3. NMR and SAXS analyses indicate that the three RRMs in full-length HuR are flexibly connected in the absence of RNA, while they adopt a more compact arrangement when bound to RNA. Based on these data and crystal structures of tandem RRM1,2-RNA and our RRM3-RNA complexes, we present a structural model of RNA recognition involving all three RRM domains of full-length HuR. Mutational analysis demonstrates that RRM3 dimerization and RNA binding is required for functional activity of full-length HuR in vitro and to regulate target mRNAs levels in human cells, thus providing a fine-tuning for HuR activity in vivo.

The CbrB Regulon: Promoter dissection reveals novel insights into the CbrAB expression network in Pseudomonas putida.

CbrAB is a high ranked global regulatory system exclusive of the Pseudomonads that responds to carbon limiting conditions. It has become necessary to define the particular regulon of CbrB and discriminate it from the downstream cascades through other regulatory components. We have performed in vivo binding analysis of CbrB in P. putida and determined that it directly controls the expression of at least 61 genes; 20% involved in regulatory functions, including the previously identified CrcZ and CrcY small regulatory RNAs. The remaining are porines or transporters (20%), metabolic enzymes (16%), activities related to protein translation (5%) and orfs of uncharacterised function (38%). Amongst the later, we have selected the operon PP2810-13 to make an exhaustive analysis of the CbrB binding sequences, together with those of crcZ and crcY. We describe the implication of three independent non-palindromic subsites with a variable spacing in three different targets; CrcZ, CrcY and operon PP2810-13 in the CbrAB activation. CbrB is a quite peculiar σN-dependent activator since it is barely dependent on phosphorylation for transcriptional activation. With the depiction of the precise contacts of CbrB with the DNA, the analysis of the multimerisation status and its dependence on other factors such as RpoN o IHF, we propose a model of transcriptional activation.

Oxidative stress is tightly regulated by cytochrome c phosphorylation and respirasome factors in mitochondria.

Respiratory cytochrome c has been found to be phosphorylated at tyrosine 97 in the postischemic brain upon neuroprotective insulin treatment, but how such posttranslational modification affects mitochondrial metabolism is unclear. Here, we report the structural features and functional behavior of a phosphomimetic cytochrome c mutant, which was generated by site-specific incorporation at position 97 of p-carboxymethyl-l-phenylalanine using the evolved tRNA synthetase method. We found that the point mutation does not alter the overall folding and heme environment of cytochrome c, but significantly affects the entire oxidative phosphorylation process. In fact, the electron donation rate of the mutant heme protein to cytochrome c oxidase, or complex IV, within respiratory supercomplexes was higher than that of the wild-type species, in agreement with the observed decrease in reactive oxygen species production. Direct contact of cytochrome c with the respiratory supercomplex factor HIGD1A (hypoxia-inducible domain family member 1A) is reported here, with the mutant heme protein exhibiting a lower affinity than the wild-type species. Interestingly, phosphomimetic cytochrome c also exhibited a lower caspase-3 activation activity. Altogether, these findings yield a better understanding of the molecular basis for mitochondrial metabolism in acute diseases, such as brain ischemia, and thus could allow the use of phosphomimetic cytochrome c as a neuroprotector with therapeutic applications.

A putative RNA binding protein from Plasmodium vivax apicoplast.

Malaria is caused by Apicomplexa protozoans from the Plasmodium genus entering the bloodstream of humans and animals through the bite of the female mosquitoes. The annotation of the Plasmodium vivax genome revealed a putative RNA binding protein (apiRBP) that was predicted to be trafficked into the apicoplast, a plastid organelle unique to Apicomplexa protozoans. Although a 3D structural model of the apiRBP corresponds to a noncanonical RNA recognition motif with an additional C-terminal α-helix (α3), preliminary protein production trials were nevertheless unsuccessful. Theoretical solvation analysis of the apiRBP model highlighted an exposed hydrophobic region clustering α3. Hence, we used a C-terminal GFP-fused chimera to stabilize the highly insoluble apiRBP and determined its ability to bind U-rich stretches of RNA. The affinity of apiRBP toward such RNAs is highly dependent on ionic strength, suggesting that the apiRBP-RNA complex is driven by electrostatic interactions. Altogether, apiRBP represents an attractive tool for apicoplast transcriptional studies and for antimalarial drug design.

The E. coli S30 lysate proteome: A prototype for cell-free protein production.

Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a "black box". Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures. This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis.

Microalgae-derived oxylipins decrease inflammatory mediators by regulating the subcellular location of NFκB and PPAR-γ.

Oxylipins (OXLs) are bioactive molecules generated by the oxidation of fatty acids that promote the resolution of acute inflammation and prevent chronic inflammatory processes through molecular mechanisms that are not well known. We have previously reported the anti-inflammatory activity of microalgae-derived OXLs and OXL-containing biomass in two inflammatory bowel disease (IBD) models: 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced acute colitis and TNBS-induced recurrent colitis. In this study, we examined the in vitro anti-inflammatory mechanism of action of the most abundant OXLs isolated from Chlamydomonas debaryana (13S-HOTE and 13S-HODE) and Nannochloropsis gaditana (15S-HEPE). These OXLs decreased IL-1ß and IL-6 pro-inflammatory cytokines production as well as iNOS and COX-2 expression levels in THP-1 macrophages. In addition, OXLs decreased IL-8 production in HT-29 colon cells, the major chemokine produced by these cells. The interaction of OXLs with NFκB and PPAR-γ signaling pathways was studied by confocal microscopy. In THP-1 macrophages and HT-29 colon cells, stimulated by LPS and TNFα respectively, a pre-treatment with 13S-HOTE, 13S-HODE and 15S-HEPE (100µM) resulted in a lower nuclear presence of NFκB in both cell lines. The study of the subcellular localization of PPAR-γ showed that the treatment of THP-1 and HT-29 cells with these OXLs caused the migration of PPAR-γ into the nucleus. Colocalization analysis of both transcription factors in LPS-stimulated THP-1 macrophages showed that the pre-treatment with 13S-HOTE, 13S-HODE or 15S-HEPE lowered nuclear colocalization similar to control value, and increased cytosolic localization above control level. These results indicate that these OXLs could act as agonist of PPAR-γ and consequently inhibit NFκB signaling pathway activation, thus lowering the production of inflammatory markers, highlighting the therapeutic potential of these OXLs in inflammatory diseases such as IBD.

RNA Binding Protein Regulation and Cross-Talk in the Control of AU-rich mRNA Fate.

mRNA metabolism is tightly orchestrated by highly-regulated RNA Binding Proteins (RBPs) that determine mRNA fate, thereby influencing multiple cellular functions across biological contexts. Here, we review the interplay between six well-known RBPs (TTP, AUF-1, KSRP, HuR, TIA-1, and TIAR) that recognize AU-rich elements (AREs) at the 3' untranslated regions of mRNAs, namely ARE-RBPs. Examples of the links between their cross-regulations and modulation of their targets are analyzed during mRNA processing, turnover, localization, and translational control. Furthermore, ARE recognition can be self-regulated by several factors that lead to the prevalence of one RBP over another. Consequently, we examine the factors that modulate the dynamics of those protein-RNA transient interactions to better understand the final consequences of the regulation mediated by ARE-RBPs. For instance, factors controlling the RBP isoforms, their conformational state or their post-translational modifications (PTMs) can strongly determine the fate of the protein-RNA complexes. Moreover, mRNA specific sequence and secondary structure or subtle environmental changes are also key determinants to take into account. To sum up, the whole understanding of such a fine tuned regulation is a challenge for future research and requires the integration of all the available structural and functional data by in vivo, in vitro and in silico approaches.

Salinity promotes opposite patterns of carbonylation and nitrosylation of C4 phosphoenolpyruvate carboxylase in sorghum leaves.

MAIN CONCLUSION: Carbonylation inactivates sorghum C 4 PEPCase while nitrosylation has little impact on its activity but holds back carbonylation. This interplay could be important to preserve photosynthetic C4 PEPCase activity in salinity. Previous work had shown that nitric acid (NO) increased phosphoenolpyruvate carboxylase kinase (PEPCase-k) activity, promoting the phosphorylation of phosphoenolpyruvate carboxylase (PEPCase) in sorghum leaves (Monreal et al. in Planta 238:859-869, 2013b). The present work investigates the effect of NO on C4 PEPCase in sorghum leaves and its interplay with carbonylation, an oxidative modification frequently observed under salt stress. The PEPCase of sorghum leaves could be carbonylated in vitro and in vivo, and this post-translational modification (PTM) was accompanied by a loss of its activity. Similarly, PEPCase could be S-nitrosylated in vitro and in vivo, and this PTM had little impact on its activity. The S-nitrosylated PEPCase showed increased resistance towards subsequent carbonylation, both in vitro and in vivo. Under salt shock, carbonylation of PEPCase increased in parallel with decreased S-nitrosylation of the enzyme. Subsequent increase of S-nitrosylation was accompanied by decreased carbonylation. Taken together, the results suggest that S-nitrosylation could contribute to maintain C4 PEPCase activity in stressed sorghum plants. Thus, salt-induced NO synthesis would be protecting photosynthetic PEPCase activity from oxidative inactivation while promoting its phosphorylation, which will guarantee its optimal functioning in suboptimal conditions.

Enzymatic activity, gene expression and posttranslational modifications of photosynthetic and non-photosynthetic phosphoenolpyruvate carboxylase in ammonium-stressed sorghum plants.

Sorghum plants grown with 5mM (NH4)2SO4 showed symptoms of stress, such as reduced growth and photosynthesis, leaf chlorosis, and reddish roots. Phosphoenolpyruvate carboxylase (PEPC) activity, by supplying carbon skeletons for ammonium assimilation, plays a pivotal role in tolerance to ammonium stress. This work investigated the effect of ammonium nutrition on PPC and PPCK gene expression, on PEPC activity, and on post-translational modifications (PTMs) of PEPC in leaves and roots of sorghum plants. Ammonium increased PEPC kinase (PEPCk) activity and the phosphorylation state of PEPC in leaves, both in light and in the dark, due to increased PPCK1 expression in leaves. This result resembled the effect of salinity on sorghum leaf PEPC and PEPCk, which is thought to allow a better functioning of PEPC in conditions that limit the income of reduced C. In roots, ammonium increased PEPC activity and the amount of monoubiquitinated PEPC. The first effect was related to increased PPC3 expression in roots. These results highlight the relevance of this specific isoenzyme (PPC3) in sorghum responses to ammonium stress. Although the role of monoubiquitination is not fully understood, it also increased in germinating seeds along with massive mobilization of reserves, a process in which the anaplerotic function of PEPC is of major importance.

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48.

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation-in particular, at tyrosine 48-is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-l-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects.

TIA-1 RRM23 binding and recognition of target oligonucleotides.

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Šresolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression.

Anti-inflammatory effects of an oxylipin-containing lyophilised biomass from a microalga in a murine recurrent colitis model.

Diet and nutritional factors have emerged as possible interventions for inflammatory bowel diseases (IBD), which are characterised by chronic uncontrolled inflammation of the intestinal mucosa. Microalgal species are a promising source of n-3 PUFA and derived oxylipins, which are lipid mediators with a key role in the resolution of inflammation. The aim of the present study was to investigate the effects of an oxylipin-containing lyophilised biomass from Chlamydomonas debaryana on a recurrent 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis mice model. Moderate chronic inflammation of the colon was induced in BALB/c mice by weekly intracolonic instillations of low dose of TNBS. Administration of the lyophilised microalgal biomass started 2 weeks before colitis induction and was continued throughout colitis development. Mice were killed 48 h after the last TNBS challenge. Oral administration of the microalgal biomass reduced TNBS-induced intestinal inflammation, evidenced by an inhibition of body weight loss, an improvement in colon morphology and a decrease in pro-inflammatory cytokines TNF-α, IL-1ß, IL-6 and IL-17. This product also down-regulated colonic expressions of inducible nitric oxide, cyclo-oxygenase 2 and NF-κB, as well as increased PPAR-γ. In addition, lyophilised microalgal biomass up-regulated the expressions of the antioxidant transcription factor nuclear factor E2-related factor 2 and the target gene heme oxygenase 1. This study describes for the first time the prophylactic effects of an oxylipin-containing lyophilised microalgae biomass from C. debaryana in the acute phase of a recurrent TNBS-induced colitis model in mice. These findings suggest the potential use of this microalga, or derived oxylipins, as a nutraceutical in the treatment of IBD.

Phosphoenolpyruvate carboxylase (PEPC) and PEPC-kinase (PEPC-k) isoenzymes in Arabidopsis thaliana: role in control and abiotic stress conditions.

MAIN CONCLUSION: Arabidopsis ppc3 mutant has a growth-arrest phenotype and is affected in phosphate- and salt-stress responses, showing that this protein is crucial under control or stress conditions. Phosphoenolpyruvate carboxylase (PEPC) and its dedicated kinase (PEPC-k) are ubiquitous plant proteins implicated in many physiological processes. This work investigates specific roles for the three plant-type PEPC (PTPC) and the two PEPC-k isoenzymes in Arabidopsis thaliana. The lack of any of the PEPC isoenzymes reduced growth parameters under optimal growth conditions. PEPC activity was decreased in shoots and roots of ppc2 and ppc3 mutants, respectively. Phosphate starvation increased the expression of all PTPC and PPCK genes in shoots, but only PPC3 and PPCK2 in roots. The absence of any of these two proteins was not compensated by other isoforms in roots. The effect of salt stress on PTPC and PPCK expression was modest in shoots, but PPC3 was markedly increased in roots. Interestingly, both stresses decreased root growth in each of the mutants except for ppc3. This mutant had a stressed phenotype in control conditions (reduced root growth and high level of stress molecular markers), but was unaffected in their response to high salinity. Salt stress increased PEPC activity, its phosphorylation state, and L-malate content in roots, all these responses were abolished in the ppc3 mutant. Our results highlight the importance of the PPC3 isoenzyme for the normal development of plants and for root responses to stress.

Expression patterns of sirtuin 1-AMPK-autophagy pathway in chronic colitis and inflammation-associated colon neoplasia in IL-10-deficient mice.

BACKGROUND: Interleukin-10-deficient (IL-10 (-/-)) mice spontaneously develop chronic colitis and adenocarcinoma through the dysplasia sequence. Autophagy malfunction is associated to inflammatory bowel disease (IBD) and colorectal cancer (CRC) pathogenesis. Autophagy is regulated by silent information regulator-1 (SIRT1), a NAD+-dependent histone deacetylase. Our aim was to investigate the expression changes of SIRT1-AMPK-autophagy pathway in the progression from chronic colitis to CRC. METHODS: We studied C57BL/6-IL-10-deficient mice between 6 and 18weeks of age. Macroscopic and histological analysis, and characterization of inflammatory and tumor biomarkers were performed. RESULTS: IL-10-deficient mice developed colitis from the age of 6weeks onward. The severity of inflammation and dysplasia, and the proliferative activity increased gradually with age. IL-10 (-/-) mice were characterized by improved levels of TNF-α and decreased expression of SIRT1. Moreover, our findings show an increase in p-AMPK expression and an activation of the autophagy in IL-10 (-/-) mice from all stages, evidenced by the accumulation of LC3-II protein, the increase in Beclin 1 expression and the reduction in Bcl-2 levels. CONCLUSIONS: SIRT1-AMPK-autophagy pathway may be involved in the maintenance of chronic inflammation and dysplasia development in the IL-10-deficient mice model. Modulation of this pathway could be a novel strategy for IBD and CRC treatment.

Goniothalamin prevents the development of chemically induced and spontaneous colitis in rodents and induces apoptosis in the HT-29 human colon tumor cell line.

Colon cancer is the third most incident type of cancer worldwide. One of the most important risk factors for colon cancer development are inflammatory bowel diseases (IBD), thus therapies focusing on IBD treatment have great potential to be used in cancer prevention. Nature has been a source of new therapeutic and preventive agents and the racemic form of the styryl-lactone goniothalamin (GTN) has been shown to be a promising antiproliferative agent, with gastroprotective, antinociceptive and anti-inflammatory effects. As inflammation is a well-known tumor promoter, the major goal of this study was to evaluate the therapeutic and preventive potentials of GTN on chemically induced and spontaneous colitis, as well as the cytotoxic effects of GTN on a human colon tumor cell line (HT-29). GTN treatments inhibited TNBS-induced acute and chronic colitis development in Wistar rats, reducing myeloperoxidase levels and inflammatory cells infiltration in the mucosa. In spontaneous-colitis using IL-10 deficient mice (C57BL/6 background), GTN prevented colitis development through downregulation of TNF-α, upregulation of SIRT-1 and inhibition of proliferation (PCNA index), without signs of toxicity after three months of treatment. In HT-29 cells, treatment with 10µM of GTN induced apoptosis by increasing BAX/BCL2, p-JNK1/JNK1, p-P38/P38 ratios as well as through ROS generation. Caspase 8, 9 and 3 activation also occurred, suggesting caspase-dependent apoptotic pathway, culminating in PARP-1 cleavage. Together with previous data, these results show the importance of GTN as a pro-apoptotic, preventive and therapeutic agent for IBD and highlight its potential as a chemopreventive agent for colon cancer.