Apigenin: Molecular Mechanisms and Therapeutic Potential against Cancer Spreading.

Due to its propensity to metastasize, cancer remains one of the leading causes of death worldwide. Thanks in part to their intrinsic low cytotoxicity, the effects of the flavonoid family in the prevention and treatment of various human cancers, both in vitro and in vivo, have received increasing attention in recent years. It is well documented that Apigenin (4',5,7-trihydroxyflavone), among other flavonoids, is able to modulate key signaling molecules involved in the initiation of cancer cell proliferation, invasion, and metastasis, including JAK/STAT, PI3K/Akt/mTOR, MAPK/ERK, NF-κB, and Wnt/ß-catenin pathways, as well as the oncogenic non-coding RNA network. Based on these premises, the aim of this review is to emphasize some of the key events through which Apigenin suppresses cancer proliferation, focusing specifically on its ability to target key molecular pathways involved in angiogenesis, epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cells (CSCs), cell cycle arrest, and cancer cell death.

Repeated witness social stress causes cardiomyocyte contractile impairment and intracellular Ca2+ derangement in female rats.

The impact of psychosocial stressors on cardiovascular health in women is of growing interest in both the popular and scientific literature. Rodent models are useful for providing direct experimental evidence of the adverse cardiovascular consequences of psychosocial stressors, yet studies in females are scarce. Here, we investigated the effects of repeated exposure to witness social defeat stress (WS) on cardiomyocyte contractile function and intracellular Ca2+ homeostasis in young adult wild-type Groningen female rats. Female rats bore witness to an aggressive social defeat episode between two males for nine consecutive days or were exposed to a control procedure. Stress-related behaviors were assessed during the first and last WS/control exposure. Twenty-four hours after the last exposure, plasma corticosterone levels were measured, and cardiomyocytes were isolated for analyses of contractile properties and Ca2+ transients, and expression levels of proteins involved in intracellular Ca2+dynamics. The results show an impairment of the intrinsic cardiac mechanical properties and prolonged intracellular Ca2+decay in WS female rats showing social stress-related behavioral (larger amounts of burying behavior) and neuroendocrine (elevated plasma corticosterone levels) phenotypes. Further, the results implicate alterations in the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex in the contractile defects described in cardiomyocytes of WS female rats. In conclusion, this study highlights the utility of the WS model as an ethologically relevant social stressor for investigating pathophysiological processes that occur in the heart of female subjects and may increase vulnerability to social stress-related cardiovascular risk.

In Vivo Treatment with a Standardized Green Tea Extract Restores Cardiomyocyte Contractility in Diabetic Rats by Improving Mitochondrial Function through SIRT1 Activation.

Background. Green tea catechins are known to promote mitochondrial function, and to modulate gene expression and signalling pathways that are altered in the diabetic heart. We thus evaluated the effectiveness of the in vivo administration of a standardized green tea extract (GTE) in restoring cardiac performance, in a rat model of early streptozotocin-induced diabetes, with a focus on the underlying mechanisms. Methods. Twenty-five male adult Wistar rats were studied: the control (n = 9), untreated diabetic animals (n = 7) and diabetic rats subjected to daily GTE administration for 28 days (n = 9). Isolated ventricular cardiomyocytes were used for ex vivo measurements of cell mechanics and calcium transients, and molecular assays, including the analysis of functional protein and specific miRNA expression. Results. GTE treatment induced an almost complete recovery of cardiomyocyte contractility that was markedly impaired in the diabetic cells, by preserving mitochondrial function and energy availability, and modulating the expression of the sarcoplasmic reticulum calcium ATPase and phospholamban. Increased Sirtuin 1 (SIRT1) expression and activity substantially contributed to the observed cardioprotective effects. Conclusions. The data supported the hypothesis that green tea dietary polyphenols, by targeting SIRT1, can constitute an adjuvant strategy for counteracting the initial damage of the diabetic heart, before the occurrence of diabetic cardiomyopathy.

The Potential of Epigallocatechin Gallate (EGCG) in Targeting Autophagy for Cancer Treatment: A Narrative Review.

Autophagy is an evolutionarily conserved process for the degradation of redundant or damaged cellular material by means of a lysosome-dependent mechanism, contributing to cell homeostasis and survival. Autophagy plays a multifaceted and context-dependent role in cancer initiation, maintenance, and progression; it has a tumor suppressive role in the absence of disease and is upregulated in cancer cells to meet their elevated metabolic demands. Autophagy represents a promising but challenging target in cancer treatment. Green tea is a widely used beverage with healthy effects on several diseases, including cancer. The bioactive compounds of green tea are mainly catechins, and epigallocatechin-gallate (EGCG) is the most abundant and biologically active among them. In this review, evidence of autophagy modulation and anti-cancer effects induced by EGCG treatment in experimental cancer models is presented. Reviewed articles reveal that EGCG promotes cytotoxic autophagy often through the inactivation of PI3K/Akt/mTOR pathway, resulting in apoptosis induction. EGCG pro-oxidant activity has been postulated to be responsible for its anti-cancer effects. In combination therapy with a chemotherapy drug, EGCG inhibits cell growth and the drug-induced pro-survival autophagy. The selected studies rightly claim EGCG as a valuable agent in cancer chemoprevention.

Lemur Tyrosine Kinases and Prostate Cancer: A Literature Review.

The members of the Lemur Tyrosine Kinases (LMTK1-3) subfamily constitute a group of three membrane-anchored kinases. They are known to influence a wide variety of key cellular events, often affecting cell proliferation and apoptosis. They have been discovered to be involved in cancer, in that they impact various signalling pathways that influence cell proliferation, migration, and invasiveness. Notably, in the context of genome-wide association studies, one member of the LMTK family has been identified as a candidate gene which could contribute to the development of prostate cancer. In this review, of published literature, we present evidence on the role of LMTKs in human prostate cancer and model systems, focusing on the complex network of interacting partners involved in signalling cascades that are frequently activated in prostate cancer malignancy. We speculate that the modulators of LMTK enzyme expression and activity would be of high clinical relevance for the design of innovative prostate cancer treatment.

Effects of Standardized Green Tea Extract and Its Main Component, EGCG, on Mitochondrial Function and Contractile Performance of Healthy Rat Cardiomyocytes.

We recently showed that the long-term in vivo administration of green tea catechin extract (GTE) resulted in hyperdynamic cardiomyocyte contractility. The present study investigates the mechanisms underlying GTE action in comparison to its major component, epigallocatechin-3-gallate (EGCG), given at the equivalent amount that would be in the entirety of GTE. Twenty-six male Wistar rats were given 40 mL/day of a tap water solution with either standardized GTE or pure EGCG for 4 weeks. Cardiomyocytes were then isolated for the study. Cellular bioenergetics was found to be significantly improved in both GTE- and EGCG-fed rats compared to that in controls as shown by measuring the maximal mitochondrial respiration rate and the cellular ATP level. Notably, the improvement of mitochondrial function was associated with increased levels of oxidative phosphorylation complexes, whereas the cellular mitochondrial mass was unchanged. However, only the GTE supplement improved cardiomyocyte mechanics and intracellular calcium dynamics, by lowering the expression of total phospholamban (PLB), which led to an increase of both the phosphorylated-PLB/PLB and the sarco-endoplasmic reticulum calcium ATPase/PLB ratios. Our findings suggest that GTE might be a valuable adjuvant tool for counteracting the occurrence and/or the progression of cardiomyopathies in which mitochondrial dysfunction and alteration of intracellular calcium dynamics constitute early pathogenic factors.

Flavonoids as Epigenetic Modulators for Prostate Cancer Prevention.

Prostate cancer (PCa) is a multifactorial disease with an unclear etiology. Due to its high prevalence, long latency, and slow progression, PCa is an ideal target for chemoprevention strategies. Many research studies have highlighted the positive effects of natural flavonoids on chronic diseases, including PCa. Different classes of dietary flavonoids exhibit anti-oxidative, anti-inflammatory, anti-mutagenic, anti-aging, cardioprotective, anti-viral/bacterial and anti-carcinogenic properties. We overviewed the most recent evidence of the antitumoral effects exerted by dietary flavonoids, with a special focus on their epigenetic action in PCa. Epigenetic alterations have been identified as key initiating events in several kinds of cancer. Many dietary flavonoids have been found to reverse DNA aberrations that promote neoplastic transformation, particularly for PCa. The epigenetic targets of the actions of flavonoids include oncogenes and tumor suppressor genes, indirectly controlled through the regulation of epigenetic enzymes such as DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC). In addition, flavonoids were found capable of restoring miRNA and lncRNA expression that is altered during diseases. The optimization of the use of flavonoids as natural epigenetic modulators for chemoprevention and as a possible treatment of PCa and other kinds of cancers could represent a promising and valid strategy to inhibit carcinogenesis and fight cancer.

Clusterin Silencing in Prostate Cancer Induces Matrix Metalloproteinases by an NF-κB-Dependent Mechanism.

Clusterin (CLU) is a stress-activated glycoprotein, whose expression is altered both in inflammation and cancer. Previously, we showed that abrogation of CLU expression in cancer-prone mice (TRAMP) results in the enhancement of tumor spreading and homing, concomitant with an enhanced expression of NF-κB. In the present paper, we carried out an extensive experimental work by utilizing microarray gene expression data, as well as in vitro and in vivo models of prostate cancer (PCa). Our results demonstrated that (i) CLU expression is significantly downregulated in human PCa and inversely correlates with the expression of p65 in metastases; (ii) CLU overexpression in PCa cells reduces the Ser536 phosphorylation of p65, inhibits NF-κB nuclear translocation, and reduces the transcription of matrix metalloproteinase-9 and metalloproteinase-2 (MMP-9 and MMP-2). Conversely, CLU silencing promotes NF-κB activation and transcriptional upregulation of MMP-9; and (iii) expression and activity of MMP-2 and MMP-9 are increased in CLU-/- mice (CLUKO) and in TRAMP/CLUKO mice in comparison to their relative Clu+/+ littermates. Taken together, our data support the hypothesis that CLU downregulation, an early and relevant event in PCa onset, may inhibit NF-κB activation and limit the execution of a transcriptional program that favor the disease progression towards a metastatic stage.

Functional interplay between NF-κB-inducing kinase and c-Abl kinases limits response to Aurora inhibitors in multiple myeloma.

Considering that Aurora kinase inhibitors are currently under clinical investigation in hematologic cancers, the identification of molecular events that limit the response to such agents is essential for enhancing clinical outcomes. Here, we discover a NF-κB-inducing kinase (NIK)-c-Abl-STAT3 signaling-centered feedback loop that restrains the efficacy of Aurora inhibitors in multiple myeloma. Mechanistically, we demonstrate that Aurora inhibition promotes NIK protein stabilization via downregulation of its negative regulator TRAF2. Accumulated NIK converts c-Abl tyrosine kinase from a nuclear proapoptotic into a cytoplasmic antiapoptotic effector by inducing its phosphorylation at Thr735, Tyr245 and Tyr412 residues, and, by entering into a trimeric complex formation with c-Abl and STAT3, increases both the transcriptional activity of STAT3 and expression of the antiapoptotic STAT3 target genes PIM1 and PIM2. This consequently promotes cell survival and limits the response to Aurora inhibition. The functional disruption of any of the components of the trimer NIK-c-Abl-STAT3 or the PIM survival kinases consistently enhances the responsiveness of myeloma cells to Aurora inhibitors. Importantly, concurrent inhibition of NIK or c-Abl disrupts Aurora inhibitor-induced feedback activation of STAT3 and sensitizes myeloma cells to Aurora inhibitors, implicating a combined inhibition of Aurora and NIK or c-Abl kinases as potential therapies for multiple myeloma. Accordingly, pharmacological inhibition of c-Abl together with Aurora resulted in substantial cell death and tumor regression in vivo The findings reveal an important functional interaction between NIK, Abl and Aurora kinases, and identify the NIK, c-Abl and PIM survival kinases as potential pharmacological targets for improving the efficacy of Aurora inhibitors in myeloma.

Molecular Targets of Epigallocatechin-Gallate (EGCG): A Special Focus on Signal Transduction and Cancer.

Green tea is a beverage that is widely consumed worldwide and is believed to exert effects on different diseases, including cancer. The major components of green tea are catechins, a family of polyphenols. Among them, epigallocatechin-gallate (EGCG) is the most abundant and biologically active. EGCG is widely studied for its anti-cancer properties. However, the cellular and molecular mechanisms explaining its action have not been completely understood, yet. EGCG is effective in vivo at micromolar concentrations, suggesting that its action is mediated by interaction with specific targets that are involved in the regulation of crucial steps of cell proliferation, survival, and metastatic spread. Recently, several proteins have been identified as EGCG direct interactors. Among them, the trans-membrane receptor 67LR has been identified as a high affinity EGCG receptor. 67LR is a master regulator of many pathways affecting cell proliferation or apoptosis, also regulating cancer stem cells (CSCs) activity. EGCG was also found to be interacting directly with Pin1, TGFR-II, and metalloproteinases (MMPs) (mainly MMP2 and MMP9), which respectively regulate EGCG-dependent inhibition of NF-kB, epithelial-mesenchimal transaction (EMT) and cellular invasion. EGCG interacts with DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), which modulates epigenetic changes. The bulk of this novel knowledge provides information about the mechanisms of action of EGCG and may explain its onco-suppressive function. The identification of crucial signalling pathways that are related to cancer onset and progression whose master regulators interacts with EGCG may disclose intriguing pharmacological targets, and eventually lead to novel combined treatments in which EGCG acts synergistically with known drugs.

Long-Term Oral Administration of Theaphenon-E Improves Cardiomyocyte Mechanics and Calcium Dynamics by Affecting Phospholamban Phosphorylation and ATP Production.

BACKGROUND/AIMS: Dietary polyphenols from green tea have been shown to possess cardio-protective activities in different experimental models of heart diseases and age-related ventricular dysfunction. The present study was aimed at evaluating whether long term in vivo administration of green tea extracts (GTE), can exert positive effects on the normal heart, with focus on the underlying mechanisms. METHODS: The study population consisted of 20 male adult Wistar rats. Ten animals were given 40 mL/day tap water solution of GTE (concentration 0.3%) for 4 weeks (GTE group). The same volume of water was administered to the 10 remaining control rats (CTRL). Then, in vivo and ex vivo measurements of cardiac function were performed in the same animal, at the organ (hemodynamics) and cellular (cardiomyocyte mechanical properties and intracellular calcium dynamics) levels. On cardiomyocytes and myocardial tissue samples collected from the same in vivo studied animals, we evaluated: (1) the intracellular content of ATP, (2) the endogenous mitochondrial respiration, (3) the expression levels of the Sarcoplasmic Reticulum Ca2+-dependent ATPase 2a (SERCA2), the Phospholamban (PLB) and the phosphorylated form of PLB, the L-type Ca2+ channel, the Na+-Ca2+ exchanger, and the ryanodine receptor 2. RESULTS: GTE cardiomyocytes exhibited a hyperdynamic contractility compared with CTRL (the rate of shortening and re-lengthening, the fraction of shortening, the amplitude of calcium transient, and the rate of cytosolic calcium removal were significantly increased). A faster isovolumic relaxation was also observed at the organ level. Consistent with functional data, we measured a significant increase in the intracellular ATP content supported by enhanced endogenous mitochondrial respiration in GTE cardiomyocytes, as well as higher values of the ratios phosphorylated-PLB/PLB and SERCA2/PLB. CONCLUSIONS: Long-term in vivo administration of GTE improves cell mechanical properties and intracellular calcium dynamics in normal cardiomyocytes, by increasing energy availability and removing the inhibitory effect of PLB on SERCA2.

Green Tea Catechins for Prostate Cancer Prevention: Present Achievements and Future Challenges.

Green tea catechins (GTCs) are a family of chemically related compounds usually classified as antioxidant molecules. Epidemiological evidences, supported by interventional studies, highlighted a more than promising role for GTCs in human prostate cancer (PCa) chemoprevention. In the last decades, many efforts have been made to gain new insights into the mechanism of action of GTCs. Now it is clear that GTCs' anticancer action can no longer be simplistically limited to their direct antioxidant/pro-oxidant properties. Recent contributions to the advancement of knowledge in this field have shown that GTCs specifically interact with cellular targets, including cell surface receptors, lipid rafts, and endoplasmic reticulum, modulate gene expression through direct effect on transcription factors or indirect epigenetic mechanisms, and interfere with intracellular proteostasis at various levels. Many of the effects observed in vitro are dose and cell context dependent and take place at concentrations that cannot be achieved in vivo. Poor intestinal absorption together with an extensive systemic and enteric metabolism influence GTCs' bioavailability through still poorly understood mechanisms. Recent efforts to develop delivery systems that increase GTCs' overall bioavailability, by means of biopolymeric nanoparticles, represent the main way to translate preclinical results in a real clinical scenario for PCa chemoprevention.

EGCG antagonizes Bortezomib cytotoxicity in prostate cancer cells by an autophagic mechanism.

The proteasome inhibitors Bortezomib (BZM) and MG132 trigger cancer cell death via induction of endoplasmic reticulum (ER) stress and unfolded protein response. Epigallocatechin gallate (EGCG), the most bioactive green tea polyphenol, is known to display strong anticancer properties as it inhibits proteasome activity and induces ER stress. We investigated whether combined delivery of a proteasome inhibitor with EGCG enhances prostate cancer cell death through increased induction of ER stress. Paradoxically, EGCG antagonized BZM cytotoxicity even when used at low concentrations. Conversely, the MG132 dose-response curve was unaffected by co-administration of EGCG. Moreover, apoptosis, proteasome inhibition and ER stress were inhibited in PC3 cells simultaneously treated with BZM and EGCG but not with a combination of MG132 and EGCG; EGCG enhanced autophagy induction in BZM-treated cells only. Autophagy inhibition restored cytotoxicity concomitantly with CHOP and p-eIF2α up-regulation in cells treated with BZM and EGCG. Overall, these findings demonstrate that EGCG antagonizes BZM toxicity by exacerbating the activation of autophagy, which in turn mitigates ER stress and reduces CHOP up-regulation, finally protecting PC3 cells from cell death.

Distinct promoters, subjected to epigenetic regulation, drive the expression of two clusterin mRNAs in prostate cancer cells.

The human clusterin (CLU) gene codes for several mRNAs characterized by different sequences at their 5' end. We investigated the expression of two CLU mRNAs, called CLU 1 and CLU 2, in immortalized (PNT1a) and tumorigenic (PC3 and DU145) prostate epithelial cells, as well as in normal fetal fibroblasts (WI38) following the administration of the epigenetic drugs 5-aza-2'-deoxycytidine (AZDC) and trichostatin A (TSA) given either as single or combined treatment (AZDC-TSA). Our experimental evidences show that: a) CLU 1 is the most abundant transcript variant. b) CLU 2 is expressed at a low level in normal fibroblasts and virtually absent in prostate cancer cells. c) CLU 1, and to a greater extent CLU 2 expression, increased by AZDC-TSA treatment in prostate cancer cells. d) Both CLU 1 and CLU 2 encode for secreted CLU. e) P2, a novel promoter that overlaps the CLU 2 Transcription Start Site (TSS), drives CLU 2 expression. f) A CpG island, methylated in prostate cancer cells and not in normal fibroblasts, is responsible for long-term heritable regulation of CLU 1 expression. g) ChIP assay of histone tail modifications at CLU promoters (P1 and P2) shows that treatment of prostate cancer cells with AZDC-TSA causes enrichment of Histone3(Lys9)acetylated (H3K9ac) and reduction of Histone3(Lys27)trimethylated (H3K27me3), inducing active transcription of both CLU variants. In conclusion, we show for the first time that the expression of CLU 2 mRNA is driven by a novel promoter, P2, whose activity responds to epigenetic drugs treatment through changes in histone modifications.

Polyphenon E(R), a standardized green tea extract, induces endoplasmic reticulum stress, leading to death of immortalized PNT1a cells by anoikis and tumorigenic PC3 by necroptosis.

Increasing doses of Polyphenon E®, a standardized green tea extract, were given to PNT1a and PC3 prostate epithelial cells mimicking initial and advanced stages of prostate cancer (PCa), respectively. Cell death occurred in both cell lines, with PNT1a being more sensitive [half-maximal inhibitory concentration (IC50) = 35 µg/ml] than PC3 (IC50 = 145 µg/ml) to Polyphenon E®. Cell cycle arrest occurred at G0/G1 checkpoint for PNT1a, and G2/M for PC3 cells. Endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) occurred in both cell lines, with each exhibiting different timing in response to Polyphenon E®. Autophagy was transiently activated in PNT1a cells within 12 h after treatment as a survival response to overcome ERS; then activation of caspases and cleavage of poly (ADP ribose) polymerase 1 occurred, committing cells to anoikis death. Polyphenon E® induced severe ERS in PC3 cells, causing a dramatic enlargement of the ER; persistent activation of UPR produced strong upregulation of GADD153/CHOP, a key protein of ERS-mediated cell death. Thereafter, GADD153/CHOP activated Puma, a BH3-only protein, committing cells to necroptosis, a programmed caspase-independent mechanism of cell death. Our results provide a foundation for the identification of novel targets and strategies aimed at sensitizing apoptosis-resistant cells to alternative death pathways.

A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.

Iron-sulfur (Fe/S) cluster enzymes are crucial to life. Their assembly requires a suite of proteins, some of which are specific for particular subsets of Fe/S enzymes. One such protein is yeast Iba57p, which aconitase and certain radical S-adenosylmethionine enzymes require for activity. Iba57p homologs occur in all domains of life; they belong to the COG0354 protein family and are structurally similar to various folate-dependent enzymes. We therefore investigated the possible relationship between folates and Fe/S cluster enzymes using the Escherichia coli Iba57p homolog, YgfZ. NMR analysis confirmed that purified YgfZ showed stereoselective folate binding. Inactivating ygfZ reduced the activities of the Fe/S tRNA modification enzyme MiaB and certain other Fe/S enzymes, although not aconitase. When successive steps in folate biosynthesis were ablated, folE (lacking pterins and folates) and folP (lacking folates) mutants mimicked the ygfZ mutant in having low MiaB activities, whereas folE thyA mutants supplemented with 5-formyltetrahydrofolate (lacking pterins and depleted in dihydrofolate) and gcvP glyA mutants (lacking one-carbon tetrahydrofolates) had intermediate MiaB activities. These data indicate that YgfZ requires a folate, most probably tetrahydrofolate. Importantly, the ygfZ mutant was hypersensitive to oxidative stress and grew poorly on minimal media. COG0354 genes of bacterial, archaeal, fungal, protistan, animal, or plant origin complemented one or both of these growth phenotypes as well as the MiaB activity phenotype. Comparative genomic analysis indicated widespread functional associations between COG0354 proteins and Fe/S cluster metabolism. Thus COG0354 proteins have an ancient, conserved, folate-dependent function in the activity of certain Fe/S cluster enzymes.

Plant Vacuolar ATP-binding Cassette Transporters That Translocate Folates and Antifolates in Vitro and Contribute to Antifolate Tolerance in Vivo.

The vacuoles of pea (Pisum sativum) leaves and red beet (Beta vulgaris) storage root are major sites for the intracellular compartmentation of folates. In the light of these findings and preliminary experiments indicating that some plant multidrug resistance-associated protein (MRP) subfamily ATP-binding cassette transporters are able to transport compounds of this type, the Arabidopsis thaliana vacuolar MRP, AtMRP1 (AtABCC1), and its functional equivalent(s) in vacuolar membrane vesicles purified from red beet storage root were studied. In so doing, it has been determined that heterologously expressed AtMRP1 and its equivalents in red beet vacuolar membranes are not only competent in the transport of glutathione conjugates but also folate monoglutamates and antifolates as exemplified by pteroyl-l-glutamic acid and methotrexate (MTX), respectively. In agreement with the results of these in vitro transport measurements, analyses of atmrp1 T-DNA insertion mutants of Arabidopsis ecotypes Wassilewskia and Columbia disclose an MTX-hypersensitive phenotype. atmrp1 knock-out mutants are more sensitive than wild-type plants to growth retardation by nanomolar concentrations of MTX, and this is associated with impaired vacuolar antifolate sequestration. The vacuoles of protoplasts isolated from the leaves of Wassilewskia atmrp1 mutants accumulate 50% less [(3)H]MTX than the vacuoles of protoplasts from wild-type plants when incubated in media containing nanomolar concentrations of this antifolate, and vacuolar membrane-enriched vesicles purified from the mutant catalyze MgATP-dependent [(3)H]MTX uptake at only 40% of the capacity of the equivalent membrane fraction from wild-type plants. AtMRP1 and its counterparts in other plant species therefore have the potential for participating in the vacuolar accumulation of folates and related compounds.

Identification of genes encoding the folate- and thiamine-binding membrane proteins in Firmicutes.

Genes encoding high-affinity folate- and thiamine-binding proteins (FolT, ThiT) were identified in the Lactobacillus casei genome, expressed in Lactococcus lactis, and functionally characterized. Similar genes occur in many Firmicutes, sometimes next to folate or thiamine salvage genes. Most thiT genes are preceded by a thiamine riboswitch.

Tomato gamma-glutamylhydrolases: expression, characterization, and evidence for heterodimer formation.

Folates typically have gamma-linked polyglutamyl tails that make them better enzyme substrates and worse transport substrates than the unglutamylated forms. The tail can be shortened or removed by the vacuolar enzyme gamma-glutamyl hydrolase (GGH). It is known that GGH is active only as a dimer and that plants can have several GGH genes whose homodimeric products differ functionally. However, it is not known whether GGH dimers dissociate under in vivo conditions, whether heterodimers form, or how heterodimerization impacts enzyme activity. These issues were explored using the GGH system of tomato (Solanum lycopersicum). Tomato has three GGH genes that, like those in other eudicots, apparently diverged recently. LeGGH1 and LeGGH2 are expressed in fruit and all other organs, whereas LeGGH3 is expressed mainly in flower buds. LeGGH1 and LeGGH2 homodimers differ in bond cleavage preference; the LeGGH3 homodimer is catalytically inactive. Homodimers did not dissociate in physiological conditions. When coexpressed in Escherichia coli, LeGGH1 and LeGGH2 formed heterodimers with an intermediate bond cleavage preference, whereas LeGGH3 formed heterodimers with LeGGH1 or LeGGH2 that had one-half the activity of the matching homodimer. E. coli cells expressing LeGGH2 showed approximately 85% reduction in folate polyglutamates, but cells expressing LeGGH3 did not, confirming that LeGGH2 can function in vivo and LeGGH3 cannot. The formation of LeGGH1-LeGGH2 heterodimers was demonstrated in planta using bimolecular fluorescence complementation. Plant GGH heterodimers thus appear to form wherever different GGH genes are expressed simultaneously and to have catalytic characteristics midway between those of the corresponding homodimers.