NADP+ Binding to the Regulatory Subunit of Methionine Adenosyltransferase II Increases Intersubunit Binding Affinity in the Hetero-Trimer

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.     

Identification of novel deregulated RNA metabolism-related genes in non-small cell lung cancer

Lung cancer is a leading cause of cancer death worldwide. Several alterations in RNA metabolism have been found in lung cancer cells; this suggests that RNA metabolism-related molecules are involved in the development of this pathology. In this study, we searched for RNA metabolism-related genes that exhibit different expression levels between normal and tumor lung tissues. We identified eight genes differentially expressed in lung adenocarcinoma microarray datasets. Of these, seven were up-regulated whereas one was down-regulated. Interestingly, most of these genes had not previously been associated with lung cancer. These genes play diverse roles in mRNA metabolism: three are associated with the spliceosome (ASCL3L1, SNRPB and SNRPE), whereas others participate in RNA-related processes such as translation (MARS and MRPL3), mRNA stability (PCBPC1), mRNA transport (RAE), or mRNA editing (ADAR2, also known as ADARB1). Moreover, we found a high incidence of loss of heterozygosity at chromosome 21q22.3, where the ADAR2 locus is located, in NSCLC cell lines and primary tissues, suggesting that the downregulation of ADAR2 in lung cancer is associated with specific genetic losses. Finally, in a series of adenocarcinoma patients, the expression of five of the deregulated genes (ADAR2, MARS, RAE, SNRPB and SNRPE) correlated with prognosis. Taken together, these results support the hypothesis that changes in RNA metabolism are involved in the pathogenesis of lung cancer, and identify new potential targets for the treatment of this disease.     

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

Background  

Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4⁺ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD).     

Singlet molecular oxygen [O<Subscript>2</Subscript>(<Superscript>1</Superscript>Δ<Subscript>g</Subscript>)]-mediated photodegradation of tyrosine derivatives in the presence of cationic and neutral micellar systems

The kinetics of rose bengal-sensitized photooxidation of tyrosine and several tyrosine-derivatives (tyr-D) named tyrosine methyl ester, tyrosine ethyl ester and tyrosine benzyl ester was studied in buffered pH 11 water, and buffered pH 11 micellar aqueous solutions of 0.01 M cetyltrimethylammonium chloride (CTAC) and 0.01 M-octylphenoxypolyethoxyethanol [triton X100 (TX100)]. Through time-resolved phosphorescence detection of singlet molecular oxygen (O(2)((1)Delta(g))) and polarographic determination of oxygen consumption, the respective bimolecular rate constants for reactive (k(r)) and overall (k(t)) quenching of the oxidative species by tyr-D were evaluated. Both rate constants behave in different fashion depending on the particular reaction medium. k(r)/k(t) values, increase in the sense CTAC相似文献   

Development of Lactococcus lactis encoding fluorescent proteins,GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.     

The realigment of the orchid flora in the mountain range of Guamuahaya,Cienfuegos, Cuba

The present study of the Orchidaceae family was carried out in Guamuahaya’s mountain range, from 2000 to March 2013. Fifteen districts were explored after 33 expeditions in the Province of Cienfuegos. Ninety two plant species were identified in the studied area, taking into account the ecological parameters of the mountainous areas of Cienfuegos and Cumanayagua municipalities.     

Leishmania donovani methionine adenosyltransferase. Role of cysteine residues in the recombinant enzyme.

Methionine adenosyltransferase (MAT, EC 2.5.1.6)-mediated synthesis of S-adenosylmethionine (AdoMet) is a two-step process consisting of the formation of AdoMet and the subsequent cleavage of the tripolyphosphate (PPPi) molecule, a reaction induced, in turn, by AdoMet. The fact that the two activities, AdoMet synthesis and tripolyphosphate hydrolysis, can be measured separately is particularly useful when the site-directed mutagenesis approach is used to determine the functional role of the amino acid residues involved in each. The present report describes the cloning and subsequent functional refolding, using a bacterial expression system, of the MAT gene (GenBank accession number AF179714) from Leishmania donovani, the etiological agent of visceral leishmaniasis. The absolute need to include a sulfhydryl-protection reagent in the refolding buffer for this protein, in conjunction with the rapid inactivation of the functionally refolded protein by N-ethylmaleimide, suggests the presence of crucial cysteine residues in the primary structure of the MAT protein. The seven cysteines in L. donovani MAT were mutated to their isosterical amino acid, serine. The C22S, C44S, C92S and C305S mutants showed a drastic loss of AdoMet synthesis activity compared to the wild type, and the C33S and C47S mutants retained a mere 12% of wild-type MAT activity. C106S mutant activity and kinetics remained unchanged with respect to the wild-type. Cysteine substitutions also modified PPPi cleavage and AdoMet induction. The C22S, C44S and C305S mutants lacked in tripolyphosphatase activity altogether, whereas C33S, C47S and C92S retained low but detectable activity. The behavior of the C92S mutant was notable: its inability to synthesize AdoMet combined with its retention of tripolyphosphatase activity appear to be indicative of the specific involvement of the respective residue in the first step of the MAT reaction.     

Control of box C/D snoRNP assembly by N6‐methylation of adenine

N⁶‐methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N⁶‐methyladenine at a key trans Hoogsteen‐sugar A·G base pair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5‐kDa protein and the induced folding of the RNA. Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N⁶‐methylation of adenine prevents the formation of trans Hoogsteen‐sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson–Crick base pairs) are more susceptible to disruption by N⁶mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction.