Melatonin receptor structure and signaling.

Melatonin (5-methoxy-N-acetyltryptamine) binds with high affinity and specificity to membrane receptors. Several receptor subtypes exist in different species, of which the mammalian MT1 and MT2 receptors are the best-characterized. They are members of the G protein-coupled receptor superfamily, preferentially coupling to Gi/o proteins but also to other G proteins in a cell-context-depending manner. In this review, experts on melatonin receptors will summarize the current state of the field. We briefly report on the discovery and classification of melatonin receptors, then focus on the molecular structure of human MT1 and MT2 receptors and highlight the importance of molecular simulations to identify new ligands and to understand the structural dynamics of these receptors. We then describe the state-of-the-art of the intracellular signaling pathways activated by melatonin receptors and their complexes. Brief statements on the molecular toolbox available for melatonin receptor studies and future perspectives will round-up this review.

RING induces cell cycle arrest and apoptosis in human breast cancer cells by regulating the HSF1/MT2A axis.

It was reported that lowly expressed RING1 indicates poor prognosis in breast cancer (BC) patients, while the mechanism by which RING1 is involved in BC progression is not fully understood. Here, we found that RING1 was lowly expressed in BC tissues and cells than in normal mammary tissues and epithelial cells. Overexpression of RING1 suppressed the cell proliferative and colony formation abilities, and facilitated cell cycle arrest and cell apoptosis in BC cells (T47D and MCF-7 cells). Mechanistically, as an ubiquitin ligase, RING1 bound to HSF1 and induced its proteasome-dependent degradation. HSF1 could bind to the promoter region of MT2A to promote the transcriptional level of MT2A. While RING1 overexpression hindered the transcriptional activation of MT2A induced by HSF1. Moreover, ectopic expression of MT2A reversed the inhibitory effect of RING1 on cell proliferation and clonogenesis, and antagonized the promotion effect of RING1 on cell cycle arrest and apoptosis in BC cells. Additionally, T47D cells infected with or without lentivirus-mediated RING1 overexpression vector (LV-RING1) were injected subcutaneously into the right back of nude mice to evaluate tumorigenicity. And overexpression of RING1 impeded the growth of BC xenografts in mice. In conclusion, RING1 suppressed the transcriptional activation of MT2A induced by HSF1 by facilitating the ubiquitination degradation of HSF1, resulting in cell cycle arrest and apoptosis in BC cells.

Single-cell transcriptome analysis revealing the intratumoral heterogeneity of ccRCC and validation of MT2A in pathogenesis.

Clear-cell renal cell carcinoma (ccRCC) appears as the most common type of kidney cancer, the carcinogenesis of which has not been fully elucidated. Tumor heterogeneity plays a crucial role in cancer progression, which could be largely deciphered by the implement of scRNA-seq. The bulk and single-cell RNA expression profile is obtained from TCGA and study conducted by Young et al. We utilized UMAP, TSNE, and clustering algorithm Louvain for dimensionality reduction and FindAllMarkers function for determining the DEGs. Monocle2 was utilized to perform pseudo-time series analysis. SCENIC was implemented for transcription factor analysis of each cell subgroup. A series of WB, CFA, CCK-8, and EDU analysis was utilized for the validation of the role of MT2A in ccRCC carcinogenesis. We observed higher infiltration of T/NK and B cells in tumorous tissues, indicating the role of immune cells in ccRCC carcinogenesis. Transcription factor analysis revealed the activation of EOMES and ETS1 in CD8 + T cells, while CAFs were divided into myo-CAFs and i-CAFs, with i-CAFs showing distinct enrichment of ATF3, JUND, JUNB, EGR1, and XBP1. Through cell trajectory analysis, we discerned three distinct stages of cellular evolution, where State2 symbolizes normal renal tubular cells that underwent transitions into State1 and State3 as the CNV score ascended. Functional enrichment examination revealed an amplification of interferon gamma and inflammatory response pathways within tumor cells. The consensus clustering algorithm yielded two molecular subtypes, with cluster 2 being associated with advanced tumor stages and an abundance of infiltrated immune cells. We identified 17 prognostic genes through Cox and LASSO regression models and used them to construct a prognostic model, the efficacy of which was verified in multiple cohorts. Furthermore, we investigated the role of MT2A, one of our hub genes, in ccRCC carcinogenesis, and found it to regulate proliferation and migration of malignant cells. We depicted a detailed single-cell landscape of ccRCC, with special focus on CAFs, endothelial cells, and renal tubular cells. A prognostic model of high stability and accuracy was constructed based on the DEGs. MT2A was found to be actively implicated in ccRCC carcinogenesis, regulating proliferation and migration of the malignant cells.

lncRNA GHET1 regulates extravillous trophoblastic phenotype via EZH2/LSD1-mediated MT2A epigenetic suppression in pre-eclampsia.

Pre-eclampsia (PE) is usually defined as new-onset hypertension with albuminuria or other organ damage. Herein, the role and mechanism of long noncoding RNA (lncRNA) gastric carcinoma high expressed transcript 1 (GHET1) during PE are investigated. Expression of GHET1 in PE pregnancies was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). Proliferation and cell cycle of extravillous trophoblasts were assessed by Cell Counting Kit-8 (CCK-8), colony formation, 5-Ethynyl-2'-deoxyuridine (EdU) assays, and flow cytometry, respectively. Migration, invasion, and network formation of trophoblasts were measured by wound healing, transwell system, and tube formation assays. RNA immunoprecipitation (RIP), RNA pull-down, and chromatin immunoprecipitation (ChIP) assays were used to confirm the molecular interaction. GHET1 was markedly decreased in the placenta of PE patients. GHET1 promoted the proliferation and cell cycle of extravillous trophoblasts, as well as migration, invasion, and network formation in vitro. Metallothionein 2A (MT2A) functioned as a downstream effector of GHET1, which was negatively correlated with GHET1 in PE. GHET1 directly bound with zeste 2 polycomb repressive complex 2/lysine-specific demethylase 1 (EZH2/LSD1). Knockdown of GHET1 reduced the occupancies of H3K27me3 and H3K4me2 in the MT2A promoter region by recruiting EZH2 and LSD1. MT2A knockdown reversed GHET1 inhibition mediated biological functions. GHET1 regulates extravillous trophoblastic phenotype via EZH2/LSD1-mediated MT2A epigenetic suppression in PE.

Evaluating the microcystin-LR-degrading potential of bacteria growing in extreme and polluted environments.

Inhabitants of extreme and polluted environments are attractive as candidates for environmental bioremediation. Bacteria growing in oil refinery effluents, tannery dumpsite soils, car wash effluents, salt pans and hot springs were screened for microcystin-LR biodegradation potentials. Using a colorimetric BIOLOG MT2 assay; Arthrobacter sp. B105, Arthrobacter junii, Plantibacter sp. PDD-56b-14, Acinetobacter sp. DUT-2, Salinivibrio sp. YH4, Bacillus sp., Bacillus thuringiensis and Lysinibacillus boronitolerans could grow in the presence of microcystin-LR at 1, 10 and 100 µg L-1. Most bacteria grew optimally at 10 µg L-1 microcystin-LR under alkaline pH (8 and 9). The ability of these bacteria to use MC-LR as a growth substrate depicts their ability to metabolize the toxin, which is equivalent to its degradation. Through PCR screening, these bacteria were shown to lack the mlr genes implying possible use of a unique microcystin-LR degradation pathway. The study highlights the wide environmental and taxonomic distribution of microcystin-LR degraders.

Differentiation of T cells producing interleukin-17 (Th17) against the background of exogenous melatonin during pregnancy.

The population of T lymphocytes producing IL-17 (Th17) plays a dual role during pregnancy and its activity is tightly controlled during this period. One of the factors involved in this process may be the pineal hormone melatonin, which can effectively regulate this T cell population. Here we have shown that exogenous melatonin in pharmacological concentrations is able to enhance the differentiation of Th17 cells of pregnant women in vitro. The stimulatory effects of melatonin were limited to in the first trimester of pregnancy and were apparently mediated by both membrane and nuclear melatonin receptors. Since exogenous melatonin is currently considered as a promising drug in solving various problems associated with reproduction, it is necessary to take into account its immunoregulatory effects.

The Uterine Melatonergic Systems of AANAT and Melatonin Membrane Receptor 2 (MT2) Are Essential for Endometrial Receptivity and Early Implantation in Mice.

In the current study, using Aanat and Mt2 KO mice, we observed that the preservation of the melatonergic system is essential for successful early pregnancy in mice. We identified that aralkylamine N-acetyltransferase (AANAT), melatonin receptor 1A (MT1), and melatonin receptor 1B (MT2) were all expressed in the uterus. Due to the relatively weak expression of MT1 compared to AANAT and MT2, this study focused on AANAT and MT2. Aanat and Mt2 KO significantly reduced the early implantation sites and the abnormal morphology of the endometrium of the uterus. Mechanistical analysis indicated that the melatonergic system is the key player in the induction of the normal nidatory estrogen (E2) response for endometrial receptivity and functions by activating the STAT signaling pathway. Its deficiency impaired the interactions between the endometrium, the placenta, and the embryo. The reduction in melatonin production caused by Aanat KO and the impairment of signal transduction caused by Mt2 KO reduced the uterine MMP-2 and MMP-9 activity, resulting in a hyperproliferative endometrial epithelium. In addition, melatonergic system deficiency also increased the local immunoinflammatory reaction with elevated local proinflammatory cytokines leading to early abortion in the Mt2 KO mice compared to the WT mice. We believe that the novel data obtained from the mice might apply to other animals including humans. Further investigation into the interaction between the melatonergic system and reproductive effects in different species would be worthwhile.

The Antifungal Effect of Pyroligneous Acid on the Phytopathogenic Fungus Botrytis cinerea.

In recent years, climate change has intensified harsh periods of rain alternating with periods of drought, leading to an increase in the presence of phytopathogenic fungi. In this study, we want to analyse the antifungal properties of pyroligneous acid against the fungal phytopathogen Botrytis cinerea. Through the inhibition test, we observed that the application of different dilutions of pyroligneous acid rarefied the growth of the fungal mycelium. Furthermore, we have seen through the metabolic profile that B. cinerea is not able to use pyroligneous acid as a resource or even grow in close contact with this resource. Moreover, we observed that the pre-incubation of the fungus in pyroligneous acid leads to a reduction in biomass production. These results give us hope for the possible use of this natural substance as a possible substance to protect plantations from pathogen attacks.

Metallothionein expression in the central nervous system in response to chronic heavy metal exposure: possible neuroprotective mechanism.

It has been reported that volcanoes release several tonnes of mercury per year among other heavy metals through eruptions, fumaroles, or diffuse soil degassing. Since a high percentage of the world's population lives in the vicinity of an active volcano, the aim of this study is to evaluate the accumulation of these metals in the central nervous system and the presence of cellular mechanisms of heavy metal detoxification such as metallothioneins. To carry out this study, wild mice (Mus musculus) chronically exposed to an active volcanic environment were captured in Furnas village (Azores, Portugal) and compared with those trapped in a reference area (Rabo de Peixe, Azores, Portugal). On the one hand, the heavy metal load has been evaluated by analyzing brain and cerebellum using ICP-MS and a mercury analyzer and on the other hand, the presence of metallothionein 2A has been studied by immunofluorescence assays. Our results show a higher load of metals such as mercury, cadmium and lead in the central nervous system of exposed mice compared to non-exposed individuals and, in addition, a higher immunoreactivity for metallothionein 2A in different areas of the cerebrum and cerebellum indicating a possible neuroprotection process.

Metallothionein 2A (MT2A) controls cell proliferation and liver metastasis by controlling the MST1/LATS2/YAP1 signaling pathway in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the three major cancers in the world and is the cancer with the most liver metastasis. The present study aimed to investigate the role of metallothionein 2A (MT2A) in the modulation of CRC cell proliferation and liver metastasis, as well as its molecular mechanisms. METHODS: The expression profile of metallothionein 2A (MT2A) in colorectal cancer retrieved from TCGA, GEO and Oncomine database. The biological effect of MT2A overexpression was investigated mainly involving cell proliferation and migration in CRC cells as well as growth and metastasis in CRC animal models. To explore the specific mechanism of MT2A metastasis in CRC, transcriptome sequencing was used to compare the overall expression difference between the control group and the MT2A overexpression group. RESULTS: Metallothionein 2A (MT2A) was downregulated in the tumor tissues of patients with CRC compared to adjacent normal tissues and was related to the tumor M stage of patients. MT2A overexpression inhibited CRC cell proliferation and migration in cells, as well as growth and metastasis in CRC animal models. While knockdown of MT2A had the opposite effect in cells. Western blotting confirmed that MT2A overexpression promoted the phosphorylation of MST1, LAST2 and YAP1, thereby inhibiting the Hippo signaling pathway. Additionally, specific inhibitors of MST1/2 inhibited MT2A overexpression-mediated phosphorylation and relieved the inhibition of the Hippo signaling pathway, thus promoting cell proliferation. Immunohistochemistry in subcutaneous grafts and liver metastases further confirmed this result. CONCLUSIONS: Our results suggested that MT2A is involved in CRC growth and liver metastasis. Therefore, MT2A and MST1 may be potential therapeutic targets for patients with CRC, especially those with liver metastases.

High MT2A Expression Predicts Worse Prognosis in Patients with Urothelial Carcinoma.

Introduction: Dysregulation of metal ion homeostasis is associated with urothelial carcinogenesis. From a published urinary bladder urothelial carcinoma (UBUC) transcriptome, we identified metallothionein 2A (MT2A) as the most significantly upregulated gene implicated in cancer progression among metal ion binding-related genes. Therefore, we analyzed the association between MT2A expression and clinical significance in our well-characterized cohort of patients with upper tract urothelial carcinoma (UTUC) and UBUC. Methods: We retrospectively reviewed the clinicopathological characteristics of 295 and 340 patients with UBUC and UTUC, respectively. MT2A expression was assessed using real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. We further correlated MT2A expression with clinicopathological factors, disease-specific survival (DSS) and metastasis-free survival (MFS) using the Pearson's χ2 test, Kaplan-Meier analysis, and multivariate Cox proportional hazards model. Results: High MT2A expression was significantly associated with aggressive pathological features including high tumor stage, lymph node metastasis, high tumor grade, vascular invasion, and perineural invasion. In the Kaplan-Meier analysis, high MT2A expression was significantly correlated with poor DSS (p < 0.0001) and MFS (p < 0.0001); in the multivariate analysis, it was an independent predictor of CSS (p < 0.001) and MFS (p = 0.001). Gene coexpression analysis demonstrated that MT2A overexpression promotes UC progression through complement activation. Conclusion: High MT2A expression correlated with aggressive UC features and was an independent predictor of cancer metastasis and patient survival, suggesting its role in risk stratification and decision-making in patients with UTUC and UBUC.

Coadministration of Melatonin and Insulin Improves Diabetes-Induced Impairment of Rat Kidney Function.

INTRODUCTION: The present study was designed to evaluate the therapeutic efficacy of melatonin and insulin coadministration in diabetes-induced renal injury in rats. RESEARCH DESIGN AND METHODS: Diabetes was achieved by giving streptozotocin (15 mg/kg) for 6 consecutive days. The diabetic condition was confirmed by assessing the blood glucose level; animals having blood glucose levels above 250 mg were considered as diabetic. Following the confirmation, animals were randomly divided into different experimental groups, viz group I served as the control (CON), group II diabetic (D), group III D+melatonin (MEL), group IV D+insulin (INS), group V D+MEL+INS, group VI D+glibenclamide (GB), group VII CON+MEL, group VIII CON+INS, and group IX CON+GB. Following the completion of the experimental period, animals were sacrificed, blood was collected via a retro-orbital puncture, and kidneys were harvested. Diabetic rats exhibited a significant increment in blood glucose and biochemical indexes of renal injury (tubular disruption, swollen glomeruli with loss of glomerular spaces, and distortion of the endothelial lining) including augmented levels of serum creatinine, urea, uric acid, Na+, and K+, and inhibition/suppression of the activity of glutathione (GSH) peroxidase, GSH reductase, glucose-6-phosphate dehydrogenase, and GSH-S-transferase in the renal cortex. RESULTS: By examining thiobarbiturate reactive substances, reduced GSH, superoxide dismutase activity, and catalase activity in the renal cortex of control and diabetic rats, it was documented that treatment with melatonin or insulin alone or in combination showed a significant ad integrum recovery of GSH-dependent antioxidative enzymatic activities. Melatonin and insulin coadministration caused greater reductions in circulating tumor necrosis factor-α, tumor growth factor-ß1, interleukin (IL)-1ß, and IL-6 levels in diabetic rats, whereas IL-10 levels increased, as compared to each treatment alone. Diabetic rats showed a significant increase in the expression of both MT1 and MT2 melatonin receptor genes. Melatonin or insulin treatment alone or in combination resulted in significant restoration of the relative expression of both melatonin receptors in the renal cortex. CONCLUSION: The coadministration of exogenous melatonin and insulin abolished many of the deleterious effects of type 1 diabetes on rat renal function.

Supraspinal melatonin MT2 receptor agonism alleviates pain via a neural circuit that recruits mu opioid receptors.

Melatonin, through its G protein-coupled receptor (GPCR) (MTNR1B gene) MT2 , is implicated in analgesia, but the relationship between MT2 receptors and the opioid system remains elusive. In a model of rodent neuropathic pain (spared nerve injured [SNI]), the selective melatonin MT2 agonist UCM924 reversed the allodynia (a pain response to a non-noxious stimulus), and this effect was nullified by the pharmacological blockade or genetic inactivation of the mu opioid receptor (MOR), but not the delta opioid receptor (DOR). Indeed, SNI MOR, but not DOR knockout mice, did not respond to the antiallodynic effects of the UCM924. Similarly, the nonselective opioid antagonist naloxone and the selective MOR antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) blocked the effects of UCM924 in SNI rats, but not the DOR antagonist naltrindole (NTI). Electrophysiological recordings in the rostral-ventromedial medulla (RVM) revealed that the typical reduction of the firing activity of pronociceptive ON-cells, and the enhancement of the firing of the antinociceptive OFF-cells, induced by the microinjection of the MT2 agonist UCM924 into the ventrolateral periaqueductal gray (vlPAG) were blocked by MOR, but not DOR, antagonism. Immunohistochemistry studies showed that MT2 receptors are expressed in both excitatory (CaMKIIα+ ) and inhibitory (GAD65+ ) neuronal cell bodies in the vlPAG (~2.16% total), but not RVM. Only 0.20% of vlPAG neurons coexpressed MOR and MT2 receptors. Finally, UCM924 treatment induced an increase in the enkephalin precursor gene (PENK) in the PAG of SNI mice. Collectively, the melatonin MT2 receptor agonism requires MORs to exert its antiallodynic effects, mostly through an interneuronal circuit involving MOR and MT2 receptors.

An Overlooked Hepcidin-Cadmium Connection.

Hepcidin (DTHFPICIFCCGCCHRSKCGMCCKT), an iron-regulatory hormone, is a 25-amino-acid peptide with four intramolecular disulfide bonds circulating in blood. Its hormonal activity is indirect and consists of marking ferroportin-1 (an iron exporter) for degradation. Hepcidin biosynthesis involves the N-terminally extended precursors prepro-hepcidin and pro-hepcidin, processed by peptidases to the final 25-peptide form. A sequence-specific formation of disulfide bonds and export of the oxidized peptide to the bloodstream follows. In this study we considered the fact that prior to export, reduced hepcidin may function as an octathiol ligand bearing some resemblance to the N-terminal part of the α-domain of metallothioneins. Consequently, we studied its ability to bind Zn(II) and Cd(II) ions using the original peptide and a model for prohepcidin extended N-terminally with a stretch of five arginine residues (5R-hepcidin). We found that both form equivalent mononuclear complexes with two Zn(II) or Cd(II) ions saturating all eight Cys residues. The average affinity at pH 7.4, determined from pH-metric spectroscopic titrations, is 1010.1 M-1 for Zn(II) ions; Cd(II) ions bind with affinities of 1015.2 M-1 and 1014.1 M-1. Using mass spectrometry and 5R-hepcidin we demonstrated that hepcidin can compete for Cd(II) ions with metallothionein-2, a cellular cadmium target. This study enabled us to conclude that hepcidin binds Zn(II) and Cd(II) sufficiently strongly to participate in zinc physiology and cadmium toxicity under intracellular conditions.

Light-Induced Functional Pinealectomy: Expression of MT2 Receptors in Liver Cells of C57BL/6 Mice after Melatonin Treatment.

We performed an immunohistochemical study of MT2 melatonin receptor expression in the liver of C57BL/6 mice with modeled light-induced functional pinealectomy and after melatonin administration by the indirect avidin-biotin peroxidase ABC method. The animals were kept for 14 days under constant lighting. Intragastric administration of melatonin in physiological doses (1 mg/kg body weight for 14 days) to mice with light-induced functional pinealectomy resulted in a 2-fold increase in the relative expression area of MT2 receptors in liver cells in comparison with that in animals kept under standard lighting conditions, 24-h lighting for 14 days, or 24-h lighting receiving placebo (intragastric administration of 200 ml distilled water). Melatonin treatment had practically no effect on MT2 staining intensity. Our results attest to the important role of MT2 receptors in melatonin synthesis disorders and can serve as the basis for the development of therapeutic strategies aimed at melatonin receptors.

Melatonergic systems of AANAT, melatonin, and its receptor MT2 in the corpus luteum are essential for reproductive success in mammals†.

Corpus luteum (CL) plays a critical role in mammalian reproductive physiology. Its dysfunction will lead to infertility or habitual abortion. In the current study, by use of melatonin specific membrane receptor 2 (MT2) knocking out (KO) mice model combined with RNA-Seq, immunohistochemistry, and immunofluorescence analyses, the genes of melatonin synthetic enzyme arylalkylamine N-acetyltransferase (AANAT) and MT2 were identified to strongly express in the CL of sows and mice. KO MT2 significantly impaired the reproductive performance in mice indicated by the reduced litter sizes. Melatonin treatment elevated the progesterone production in sows suggesting the improved CL function. Mechanistic analysis showed that melatonin upregulated a set of progesterone synthesis-related genes including cytochrome P450 family 11 subfamily A member 1 (Cyp11a1), aldo-keto reductase family 1, member C18 (Akr1c18), isopentenyl-diphosphate delta isomerase 1 (Idi1), and luteinizing hormone/choriogonadotropin receptor (Lhcgr). The upregulation of these genes directly related to the increased progesterone production. The regulatory effects of melatonin on these gene expressions were mediated by MT2 and MT2KO diminished the effects of melatonin in this respect. Thus, the presence of melatonergic system of AANAT, melatonin, and its receptor MT2 in CL is essential for reproductive success in mammals.

Major role of MT2 receptors in the beneficial effect of melatonin on long-term recognition memory in C57BL/6J male mice.

Melatonin, a major signal of the circadian system, is also involved in brain functions such as learning and memory. Chronic melatonin treatment is known to improve memory performances, but the respective contribution of its central receptors, MT1 and MT2, is still unclear. Here, we used new single receptor deficient MT1-/- and MT2-/- mice to investigate the contribution of each receptor in the positive effect of chronic melatonin treatment on long-term recognition memory. The lack of MT2 receptor precluded memory-enhancing effect of melatonin in the object recognition task and to a lesser extent in the object location task, whereas the lack of MT1 receptor mitigated its effect in the object location task only. Our findings support a key role of MT2 in mediating melatonin's beneficial action on long-term object recognition memory, whereas MT1 may contribute to the effect on object location memory.

Melatonin promotes regeneration of injured motor axons via MT1 receptors.

Melatonin is an ancient multi-tasking molecule produced by the pineal gland and by several extrapineal tissues. A variety of activities has been ascribed to this hormone in different physiological and pathological contexts, but little is known about its role in peripheral neuroregeneration. Here, we have exploited two different types of injury to test the capability of melatonin to stimulate regeneration of motor axons: (a) the acute and reversible presynaptic degeneration induced by the spider neurotoxin α-Latrotoxin and (b) the compression/transection of the sciatic nerve. We found that in both cases melatonin administration accelerates the process of nerve repair. This pro-regenerative action is MT1 -mediated, and at least in part due to a sustained activation of the ERK1/2 pathway. These findings reveal a receptor-mediated, pro-regenerative action of melatonin in vivo that holds important clinical implications, as it posits melatonin as a safe candidate molecule for the treatment of a number of peripheral neurodegenerative conditions.

Effect of MT2A on apoptosis and proliferation in HL60 cells.

Although accumulating evidence has revealed that metallothioneins (MTs) and its family member MT2A are strongly linked to the risk of various solid tumors, researches on the occurrence and development of acute myeloid leukemia (AML) have rarely been investigated. Here, we constructed a lentiviral vector with MT2A over-expression and the interfering plasmids with MT2A expression inhibition to study the influence of MT2A on the bioactivities of HL60 cells. After cells were infected with a lentiviral vector containing the MT2A gene, both transcription and translation levels of MT2A were significantly increased in the over-expressed group in comparison with control groups. In vitro experiments, all results demonstrated that cell reproductive capacity was inhibited, but cell apoptosis rate was significantly increased. Together, the expression of apoptosis-related protein Bcl2 was remarkably reduced, while a high expression level of Bax protein was detected. Further experiments revealed that up-regulation of MT2A induced cell apoptosis and promoted G2/M phase arrest. The mechanism may be associated with down-regulated p-IκB-α and cyclinD1 expression and up-regulated IκB-α expression in the nuclear factor-kappaB (NF-κB) pathway. On the contrary, MT2A expression was down-regulated by interfering plasmids. We found that cell proliferative potential was notably increased in the interfering group compared with the negative and untreated group. What's more, MT2A may be closely related to AML cell proliferation and function via the NF-κB signal pathway.

Structural and biochemical analyses of the LdtMt2-panipenem adduct provide new insights into the effect of the 1-ß-methyl group on carbapenems.

Tuberculosis has attracted increased attention worldwide due to its high morality and its resistance to treatment with traditional antibacterial drugs. The l,d-transpeptidase LdtMt2 confers resistance to traditional ß-lactams and is considered a target for anti-Tuberculosis treatment. Carbapenems are proposed to inhibit Mycobacterium tuberculosis by repressing the activity of LdtMt2. The interaction mechanisms between LdtMt2 and carbapenems have been revealed by LdtMt2-carbapenem adduct structures along with various biochemical assays. Interestingly, the lack of the 1-ß-methyl group in imipenem may be related to its high binding ability to LdtMt2. However, there is limited evidence on the interaction mode of LdtMt2 and panipenem, another carbapenem lacking the 1-ß-methyl group. Herein, we identified the biochemical features of panipenem binding to LdtMt2. We further suggest that the presence of the 1-ß-methyl group in carbapenems is indeed related to the ligand affinity of LdtMt2 and that the presence of the Y308 and Y318 residues in LdtMt2 stabilized the conformation of the LdtMt2-carbepenem adduct. Our research provides a structural basis for the development of novel carbapenems against L,D-transpeptidases.