Enhanced S-adenosyl-l-methionine production in Saccharomyces cerevisiae by spaceflight culture, overexpressing methionine adenosyltransferase and optimizing cultivation

Aims: S‐adenosyl‐l ‐methionine (SAM) is an important biochemical molecule with great potential in the pharmacological and chemotherapeutic fields. In this study, our aims were to enhance SAM production in Saccharomyces cerevisiae. Methods and Results: Through spaceflight culture, a SAM‐accumulating strain, S. cerevisiae H5M147, was isolated and found to produce 86·89% more SAM than its ground control strain H5. Amplified fragment length polymorphism (AFLP) analysis demonstrated that there were genetic variations between strain H5M147 and its ground control. Through recombinant DNA technology, the heterologous gene encoding methionine adenosyltransferase was integrated into the genome of strain H5M147. The recombinant strain H5MR83 was selected because its SAM production was increased by 42·98% when compared to strain H5M147. Furthermore, cultivation conditions were optimized using the one‐factor‐at‐a‐time and Taguchi methods. Under optimal conditions, strain H5MR83 yielded 7·76 g l^?1 of SAM in shake flask, an increase of 536·07% when compared to the strain H5. Furthermore, 9·64 g l^?1 of SAM was produced in fermenter cultivation. Conclusions: A new SAM‐accumulating strain, S. cerevisiae H5MR83, was obtained through spaceflight culture and genetic modification. Under optimal conditions, SAM production was increased to a relative high level in our study. Significance and Impact of the Study: Through comprehensive application of multiple methods including spaceflight culture, genetic modification and optimizing cultivation, the yield of SAM could be increased by 6·4 times compared to that in the control strain H5. The obtained S. cerevisiae H5MR83 produced 7·76 g l^?1 of SAM in the flask cultures, a significant improvement on previously reported results. The SAM production period with S. cerevisiae H5MR83 was 84 h, which is shorter than previously reported results. Saccharomyces cerevisiae H5MR83 has considerable potential for use in industrial applications.     

cAMP inhibits mammalian target of rapamycin complex-1 and -2 (mTORC1 and 2) by promoting complex dissociation and inhibiting mTOR kinase activity

cAMP and mTOR signalling pathways control a number of critical cellular processes including metabolism, protein synthesis, proliferation and cell survival and therefore understanding the signalling events which integrate these two signalling pathways is of particular interest. In this study, we show that the pharmacological elevation of [cAMP]_i in mouse embryonic fibroblasts (MEFs) and human embryonic kidney 293 (HEK293) cells inhibits mTORC1 activation via a PKA-dependent mechanism. Although the inhibitory effect of cAMP on mTOR could be mediated by impinging on signalling cascades (i.e. PKB, MAPK and AMPK) that inhibit TSC1/2, an upstream negative regulator of mTORC1, we show that cAMP inhibits mTORC1 in TSC2 knockout (TSC2^−/−) MEFs. We also show that cAMP inhibits insulin and amino acid-stimulated mTORC1 activation independently of Rheb, Rag GTPases, TSC2, PKB, MAPK and AMPK, indicating that cAMP may act independently of known regulatory inputs into mTOR. Moreover, we show that the prolonged elevation in [cAMP]_i can also inhibit mTORC2. We provide evidence that this cAMP-dependent inhibition of mTORC1/2 is caused by the dissociation of mTORC1 and 2 and a reduction in mTOR catalytic activity, as determined by its auto-phosphorylation on Ser2481. Taken together, these results provide an important insight into how cAMP signals to mTOR and down-regulates its activity, which may lead to the identification of novel drug targets to inhibit mTOR that could be used for the treatment and prevention of human diseases such as cancer.     

mTORC1 stimulates cell growth through SAM synthesis and m6A mRNA-dependent control of protein synthesis

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Deficiency in mTORC1‐controlled C/EBPβ‐mRNA translation improves metabolic health in mice

The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of physiological adaptations in response to changes in nutrient supply. Major downstream targets of mTORC1 signalling are the mRNA translation regulators p70 ribosomal protein S6 kinase 1 (S6K1p70) and the 4E‐binding proteins (4E‐BPs). However, little is known about vertebrate mRNAs that are specifically controlled by mTORC1 signalling and are engaged in regulating mTORC1‐associated physiology. Here, we show that translation of the CCAAT/enhancer binding protein beta (C/EBPβ) mRNA into the C/EBPβ‐LIP isoform is suppressed in response to mTORC1 inhibition either through pharmacological treatment or through calorie restriction. Our data indicate that the function of 4E‐BPs is required for suppression of LIP. Intriguingly, mice lacking the cis‐regulatory upstream open reading frame (uORF) in the C/EBPβ‐mRNA, which is required for mTORC1‐stimulated translation into C/EBPβ‐LIP, display an improved metabolic phenotype with features also found under calorie restriction. Thus, our data suggest that translational adjustment of C/EBPβ‐isoform expression is one of the key processes that direct metabolic adaptation in response to changes in mTORC1 activity.     

Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism

Here we report that the specificity of peptide release in the ribosome on a nonstop mRNA by ArfA and RF2 is achieved by an induced-fit mechanism. Using RF2 that is methylated on the glutamine of its GGQ motif (RF2^m), we show that methylation substantially increases the rate of ArfA/RF2-catalyzed peptide release on a nonstop mRNA that does not occupy the ribosomal A site, but has only a modest effect on k_cat by the same proteins on longer nonstop mRNAs occupying the A site of the mRNA channel in the ribosome. Our data suggest that enhancement in the k_cat of peptide release by ArfA and RF2 under the cognate decoding condition is the result of favorable conformational changes in the nonstop complex. We demonstrate a shared mechanism between canonical and nonstop termination, supported by similarities in the kinetic mechanisms in antibiotic inhibition and methylation-correlated enhancement in the rate of peptide release. Despite these similarities, our data suggest that nonstop termination differs from canonical pathway in the downstream event of recycling.     

Targeting of mTORC1/2 by dihydroevocarpine induces cytotoxicity in acute myeloid leukemia

Interactions between the tumor cells and bone marrow (BM) microenvironment promote survival, growth, and chemoresistance of acute myeloid leukemia (AML). The mTOR pathway plays a key role in mediating the AML-BM microenvironment interactions. Here, we report the anti-AML activity of a natural monomer extracted from the Chinese medicinal herb Evodia rutaecarpa, dihydroevocarpine. Our results showed that dihydroevocarpine-induced cytotoxicity, apoptosis, and G0/G1 arrest in AML cells, and inhibited the tumor growth in an AML xenograft model. Importantly, our study revealed that the dihydroevocarpine treatment inhibited the mTOR pathway via suppressing the mTORC1/2 activity, and thus overcame the protective effect of the BM microenvironment on AML cells. Taken together, our findings suggest that dihydroevocarpine could be used as a potential anti-AML agent alone or a therapeutic adjunct in AML therapy, particularly in the presence of the BM microenvironment.     

Elp3‐mediated codon‐dependent translation promotes mTORC2 activation and regulates macrophage polarization

Macrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1‐activating signals in myeloid cells, where it limits the production of pro‐inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2‐activating signals upregulate Elp3 expression through a PI3K‐ and STAT6‐dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon‐dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt‐driven tumor initiation in the intestine by maintaining a pool of tumor‐associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization.     

Dramatic suppression of colorectal cancer cell growth by the dual mTORC1 and mTORC2 inhibitor AZD-2014

Colorectal cancer is a major contributor of cancer-related mortality. The mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in colorectal cancers, promoting cancer progression and chemo-resistance. In the current study, we investigated the anti-colorectal cancer effect of a novel mTOR complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1 (S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the mice survival was dramatically improved. At the same time, in xenografted tumors administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely inhibited, and autophagic markers were significantly increased. Thus, AZD-2014 inhibits colorectal cancer cell growth both in vivo and in vitro. Our results suggest that AZD-2014 may be further investigated for colorectal cancer therapy in clinical trials.     

Imidazo[1,5-a]pyrazines: orally efficacious inhibitors of mTORC1 and mTORC2

The discovery and optimization of a series of imidazo[1,5-a]pyrazine inhibitors of mTOR is described. HTS hits were optimized for potency, selectivity and metabolic stability to provide the orally bioavailable proof of concept compound 4c that demonstrated target inhibition in vivo and concomitant inhibition of tumor growth in an MDA-MB-231 xenograft model.     

Discovery and optimization of 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives as mTORC1/mTORC2 dual inhibitors

New potent mTORC1/mTORC2 dual inhibitors, 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives, were obtained by optimizing functional groups on our previously reported PI3Kα inhibitor. All the target compounds were synthesized and structural optimization on the structure of the lead compound based on cytotoxic activity. The results showed that some of the target compounds exhibited moderate to high cytotoxic activity against cell line U87MG and PC-3. The activities against mTOR kinase were investigated and the compound 12q showed excellent activity with an IC₅₀ value of 54 nM in the same level of the positive control BEZ235 with IC₅₀ value of 55 nM under the same test conditions. The western blot and cell cycle results demonstrate that compound 12q is a candidate as an mTORC1/mTORC2 dual-target inhibitor. The theoretical calculations were also performed to better understanding the binding modes of the compound 12q in the mTOR active site.     

Curcumin induces apoptosis in JAK2‐mutated cells by the inhibition of JAK2/STAT and mTORC1 pathways

Myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive and negative. The JAK2 V617F is the most common mutation in Philadelphia negative patients and results in a constitutive activation of the JAK/STAT pathway, conferring a proliferative advantage and apoptosis inhibition. Recent studies identified a functional crosstalk between the JAK/STAT and mTOR pathways. The identification of an effective therapy is often difficult, so the availability of new therapeutic approaches might be attractive. Previous studies showed that curcumin, the active principle of the Curcuma longa, can suppress JAK2/STAT pathways in different type of cancer and injuries. In this study, we investigated the anti‐proliferative and pro‐apoptotic effects of curcumin in JAK2 V617F‐mutated cells. HEL cell line and cells from patients JAK2 V617F mutated have been incubated with increasing concentrations of curcumin for different time. Apoptosis and proliferation were evaluated. Subsequently, JAK2/STAT and AKT/mTOR pathways were investigated at both RNA and protein levels. We found that curcumin induces apoptosis and inhibition of proliferation in HEL cells. Furthermore, we showed that curcumin inhibits JAK2/STAT and mTORC1 pathways in JAK2 V617F‐mutated cells. This inhibition suggests that curcumin could represent an alternative strategy to be explored for the treatment of patients with myeloproliferative neoplasms.     

A conformational change in the N terminus of SLC38A9 signals mTORC1 activation

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mTORC1 promotes cell growth via m6A-dependent mRNA degradation

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PIK3CA‐mutated melanoma cells rely on cooperative signaling through mTORC1/2 for sustained proliferation

Malignant conversion of BRAF‐ or NRAS‐mutated melanocytes into melanoma cells can be promoted by PI3′‐lipid signaling. However, the mechanism by which PI3′‐lipid signaling cooperates with mutationally activated BRAF or NRAS has not been adequately explored. Using human NRAS‐ or BRAF‐mutated melanoma cells that co‐express mutationally activated PIK3CA, we explored the contribution of PI3′‐lipid signaling to cell proliferation. Despite mutational activation of PIK3CA, melanoma cells were more sensitive to the biochemical and antiproliferative effects of broader spectrum PI3K inhibitors than to an α‐selective PI3K inhibitor. Combined pharmacological inhibition of MEK1/2 and PI3K signaling elicited more potent antiproliferative effects and greater inhibition of the cell division cycle compared to single‐agent inhibition of either pathway alone. Analysis of signaling downstream of MEK1/2 or PI3K revealed that these pathways cooperate to regulate cell proliferation through mTORC1‐mediated effects on ribosomal protein S6 and 4E‐BP1 phosphorylation in an AKT‐dependent manner. Although PI3K inhibition resulted in cytostatic effects on xenografted NRAS^Q61H/PIK3CA^H1047R melanoma, combined inhibition of MEK1/2 plus PI3K elicited significant melanoma regression. This study provides insights as to how mutationally activated PIK3CA acts in concert with MEK1/2 signaling to cooperatively regulate mTORC1/2 to sustain PIK3CA‐mutated melanoma proliferation.     

Potent antitumour of the mTORC1/2 dual inhibitor AZD2014 in docetaxel-sensitive and docetaxel-resistant castration-resistant prostate cancer cells

Recent studies indicate mammalian target of rapamycin (mTOR) may play an important role in PCa progression and drug resistance. Here, we investigated the effects of a novel mTORC1/C2 dual inhibitor, AZD2014, on naive and docetaxel (Doc)-pre-treated castration-resistant PCa (CRPC) cells and explored its therapeutic potential in CRPCs. In the current study, AZD2014 has a greater inhibitory effect against 4EBP1 and AKT phosphorylation than rapamycin in CRPC cells and prevented the feedback activation of AKT signalling. Importantly, AZD2014 suppressed CRPC cell growth in vitro by suppressing proliferation, apoptosis, cell cycle arrest at G1 phase and autophagy to a greater extent than rapamycin. Moreover, AZD2014 was more efficacious than rapamycin in inhibiting migration, invasion and EMT progression in Doc-sensitive and Doc-resistant CRPC cells. Overall, AZD2014 showed significant antitumour effects. Thereby, the current study highlights a reliable theoretical basis for the clinical application of AZD2014 in both Doc-sensitive and Doc-resistant CRPCs.