Methionine adenosyltransferase from Euglena gracilis (MATX) is a recently discovered member of the MAT family of proteins that synthesize S-adenosylmethionine. Heterologous overexpression of MATX in Escherichia coli rendered the protein mostly in inclusion bodies under all conditions tested. Therefore, a refolding and purification procedure from these aggregates was developed to characterize the enzyme. Maximal recovery was obtained using inclusion bodies devoid of extraneous proteins by washing under mild urea (2M) and detergent (5%) concentrations. Refolding was achieved in two steps following solubilization in the presence of Mg(2+); chaotrope dilution to <1M and dialysis under reducing conditions. Purified MATX is a homodimer that exhibits Michaelis kinetics with a V(max) of 1.46 μmol/min/mg and K(m) values of approximately 85 and 260 μM for methionine and ATP, respectively. The activity is dependent on Mg(2+) and K(+) ions, but is not stimulated by dimethylsulfoxide. MATX exhibits tripolyphosphatase activity that is stimulated in the presence of S-adenosylmethionine. Far-UV circular dichroism revealed β-sheet and random coil as the main secondary structure elements of the protein. The high level of sequence conservation allowed construction of a structural model that preserved the main features of the MAT family, the major changes involving the N-terminal domain.

The threat of antimicrobial-resistant bacteria is ever increasing and over the past-decades development of novel therapeutic counter measurements have virtually come to a halt. This circumstance calls for interdisciplinary approaches to design, evaluate and validate the mode of action of novel antibacterial compounds. Hereby, carbosilane dendritic systems that exhibit antimicrobial properties have the potential to serve as synthetic and rationally designed molecules for therapeutic use. The bow-tie type topology of BDTL049 was recently investigated against the Gram-positive model organism Bacillus subtilis, revealing strong bactericidal properties. In this study, we follow up on open questions concerning the usability of BDTL049. For this, we synthesized a fluorescent-labeled version of BDTL049 that maintained all antimicrobial features to unravel the interaction of the compound and bacterial membrane. Subsequently, we highlight the bacterial sensitivity against BDTL049 by performing a mutational study of known resistance determinants. Finally, we address the cytotoxicity of the compound in human cells, unexpectedly revealing a high sensitivity of the eukaryotic cells upon BDTL049 exposure. The insights presented here further elaborate on the unique features of BDTL049 as a promising candidate as an antimicrobial agent while not precluding that further rounds of rational designing are needed to decrease cytotoxicity to ultimately pave the way for synthetic antibiotics toward clinical applicability.

The rise of drug-resistant fungal pathogens urges for the development of new tools for the discovery of novel antifungal compounds. Polyene antibiotics are potent agents against fungal infections in humans and animals. They inhibit the growth of fungal cells by binding to sterols in the cytoplasmic membrane that subsequently causes pore formation and eventually results in cell death. Many polyenes are produced by Streptomycetes and released into the soil environment, where they can then target fungal hyphae. While not antibacterial, these compounds could nevertheless be also perceived by bacteria sharing the same habitat and serve as signaling molecules. We therefore addressed the question of how polyenes such as amphotericin B are perceived by the soil bacterium, Bacillus subtilis. Global transcriptional profiling identified a very narrow and specific response, primarily resulting in strong upregulation of the lnrLMN operon, encoding an ABC transporter previously associated with linearmycin resistance. Its strong and specific induction prompted a detailed analysis of the lnrL promoter element and its regulation. We demonstrate that the amphotericin response strictly depends on the two-component system LnrJK and that the target of LnrK-dependent gene regulation, the lnrLMN operon, negatively affects LnrJK-dependent signal transduction. Based on this knowledge, we developed a novel whole-cell biosensor, based on a P lnrL -lux fusion reporter construct in a lnrLMN deletion mutant background. This highly sensitive and dynamic biosensor is ready to be applied for the discovery or characterization of novel amphotericin-like polyenes, hopefully helping to increase the repertoire of antimycotic and antiparasitic polyenes available to treat human and animal infections.

Ellis-van Creveld syndrome protein homolog (Evc) was previously shown to mediate expression of Indian hedgehog (Ihh) downstream targets in chondrocytes. Consequently disruption of the Ihh/Pthrp axis was demonstrated in Evc(-/-) mice, but the full extent of Evc involvement in endochondral development was not totally characterized. Herein we have examined further the Evc(-/-) growth plate in a homogeneous genetic background and show that Evc promotes chondrocyte proliferation, chondrocyte hypertrophy and the differentiation of osteoblasts in the perichondrium, hence implicating Evc in both Pthrp-dependent and Pthrp-independent Ihh functions. We also demonstrate that Evc, which localizes to osteoblast primary cilia, mediates Hedgehog (Hh) signaling in the osteoblast lineage. In spite of this, bone collar development is mildly affected in Evc(-/-) mutants. The onset of perichondrial osteoblastogenesis is delayed at the initial stages of endochondral ossification in Evc(-/-) mice, and in later stages, the leading edge of expression of osteoblast markers and Wnt/β-catenin signaling components is located closer to the primary spongiosa in the Evc(-/-) perichondrium owing to impaired osteoblast differentiation. Additionally we have used Ptch1-LacZ reporter mice to learn about the different types of Hh-responsive cells that are present in the perichondrium of normal and Evc(-/-) mice. Evc mediates Hh target gene expression in inner perichondrial cells, but it is dispensable in the external layers of the perichondrium. Finally, we report cranial base defects in Evc(-/-) mice and reveal that Evc is essential for intrasphenoidal synchondrosis development.

Methionine adenosyltransferases MAT I and MAT III (encoded by Mat1a) catalyze S-adenosylmethionine synthesis in normal liver. Major hepatic diseases concur with reduced levels of this essential methyl donor, which are primarily due to an expression switch from Mat1a towards Mat2a. Additional changes in the association state and even in subcellular localization of these isoenzymes are also detected. All these alterations result in a reduced content of the moderate (MAT I) and high Vmax (MAT III) isoenzymes, whereas the low Vmax (MAT II) isoenzyme increases and nuclear accumulation of MAT I is observed. These changes derive in a reduced availability of cytoplasmic S-adenosylmethionine, together with an effort to meet its needs in the nucleus of damaged cells, rendering enhanced levels of certain epigenetic modifications. In this context, the putative role of protein-protein interactions in the control of S-adenosylmethionine synthesis has been scarcely studied. Using yeast two hybrid and a rat liver library we identified PDRG1 as an interaction target for MATα1 (catalytic subunit of MAT I and MAT III), further confirmation being obtained by immunoprecipitation and pull-down assays. Nuclear MATα interacts physically and functionally with the PDRG1 oncogene, resulting in reduced DNA methylation levels. Increased Pdrg1 expression is detected in acute liver injury and hepatoma cells, together with decreased Mat1a expression and nuclear accumulation of MATα1. Silencing of Pdrg1 expression in hepatoma cells alters their steady-state expression profile on microarrays, downregulating genes associated with tumor progression according to GO pathway analysis. Altogether, the results unveil the role of PDRG1 in the control of the nuclear methylation status through methionine adenosyltransferase binding and its putative collaboration in the progression of hepatic diseases.

The PsdRSAB and ApsRSAB detoxification modules, together with the antimicrobial peptides (AMPs)-resistance determinants Dlt system and MprF protein, play major roles in the response to AMPs in Lacticaseibacillus paracasei BL23. Sensitivity assays with a collection of mutants showed that the PsdAB ABC transporter and the Dlt system are the main subtilin resistance determinants. Quantification of the transcriptional response to subtilin indicate that this response is exclusively regulated by the two paralogous systems PsdRSAB and ApsRSAB. Remarkably, a cross-regulation of the derAB, mprF and dlt-operon genes-usually under control of ApsR-by PsdR in response to subtilin was unveiled. The high similarity of the predicted structures of both response regulators (RR), and of the RR-binding sites support this possibility, which we experimentally verified by protein-DNA binding studies. ApsR-P shows a preferential binding in the order PderA > Pdlt > PmprF > PpsdA. However, PsdR-P bound with similar apparent affinity constants to the four promoters. This supports the cross-regulation of derAB, mprF and the dlt-operon by PsdR. The possibility of cross-regulation at the level of RR-promoter interaction allows some regulatory overlap with two RRs controlling the expression of systems involved in maintenance of critical cell membrane functions in response to lantibiotics.

The paradigm of a cytoplasmic methionine cycle synthesizing/eliminating metabolites that are transported into/out of the nucleus as required has been challenged by detection of significant nuclear levels of several enzymes of this pathway. Here, we show betaine homocysteine S-methyltransferase (BHMT), an enzyme that exerts a dual function in maintenance of methionine levels and osmoregulation, as a new component of the nuclear branch of the cycle. In most tissues, low expression of Bhmt coincides with a preferential nuclear localization of the protein. Conversely, the liver, with very high Bhmt expression levels, presents a main cytoplasmic localization. Nuclear BHMT is an active homotetramer in normal liver, although the total enzyme activity in this fraction is markedly lower than in the cytosol. N-terminal basic residues play a role in cytoplasmic retention and the ratio of glutathione species regulates nucleocytoplasmic distribution. The oxidative stress associated with d-galactosamine (Gal) or buthionine sulfoximine (BSO) treatments induces BHMT nuclear translocation, an effect that is prevented by administration of N-acetylcysteine (NAC) and glutathione ethyl ester (EGSH), respectively. Unexpectedly, the hepatic nuclear accumulation induced by Gal associates with reduced nuclear BHMT activity and a trend towards increased protein homocysteinylation. Overall, our results support the involvement of BHMT in nuclear homocysteine remethylation, although moonlighting roles unrelated to its enzymatic activity in this compartment cannot be excluded.

Protein-protein interactions are an important mechanism for the regulation of enzyme function allowing metabolite channeling, crosstalk between pathways or the introduction of post-translational modifications. Therefore, a number of high-throughput studies have been carried out to shed light on the protein networks established under different pathophysiological settings. Surprisingly, this type of information is quite limited for enzymes of intermediary metabolism such as betaine homocysteine S-methyltransferase, despite its high hepatic abundancy and its role in homocysteine metabolism. Here, we have taken advantage of two approaches, affinity purification combined with mass spectrometry and yeast two-hybrid, to further uncover the array of interactions of betaine homocysteine S-methyltransferase in normal liver of Rattus norvegicus. A total of 131 non-redundant putative interaction targets were identified, out of which 20 were selected for further validation by coimmunoprecipitation. Interaction targets validated by two different methods include: S-methylmethionine homocysteine methyltransferase or betaine homocysteine methyltransferase 2, methionine adenosyltransferases α1 and α2, cAMP-dependent protein kinase catalytic subunit alpha, 4-hydroxyphenylpyruvic acid dioxygenase and aldolase b. Network analysis identified 122 nodes and 165 edges, as well as a limited number of KEGG pathways that comprise: the biosynthesis of amino acids, cysteine and methionine metabolism, the spliceosome and metabolic pathways. These results further expand the connections within the hepatic methionine cycle and suggest putative cross-talks with additional metabolic pathways that deserve additional research.

Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP(+) with a 1:1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP(+) binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.