A Novel Anti-diabetic Metabolite from Plants: Biosynthesis, Gene Discovery, and Metabolic Engineering of Montbretin A

Plant specialized metabolites (i.e. secondary metabolites) have been employed by humans for centuries in traditional and modern medicine. They remain an important source for the discovery of new pharmaceuticals and nutraceuticals. Montbretin A (MbA) is a complex acylated flavonoid glycoside discovered in the below-ground storage organs (corms) of the ornamental plant montbretia (Crocosmia x crocosmiiflora). MbA a highly potent and selective inhibitor of the human pancreatic α-amylase (HPA), a key enzyme in starch degradation. MbA is being tested for the treatment of type-2 diabetes. However, due to low abundance of MbA in montbretia plants and due the complex chemical structure of MbA, natural product extraction and chemical synthesis are insufficient for MbA production. Our goal is to develop a heterologous plant production system or a microbial production system for MbA. This requires knowledge of the genes, enzymes and regulating factors of the MbA biosynthetic system in montbretia. We achieved the discovery of the complete biosynthetic pathway of MbA using an approach that combined knowledge of montbretia biology, metabolite profiling, differential transcriptome analysis, cDNA cloning, heterologous gene expression in E. coli, yeast and tobacco, and enzyme biochemistry. This includes the discovery of five new UDP-sugar dependent glycosyltransferases (UGTs) and a BAHD-acyltransferases (AT) which together catalyze the complete assembly of MbA from its different building blocks. To reconstruct MbA production in tobacco (Nicotiana benthamiana) we enhanced the biosynthesis of flavonol precursors using genes for myricetin biosynthesis and transcription factors from montbtretia, which were stacked with genes of the MbA assembly pathway. We will highlight both challenges and opportunities of exploring novel biosynthetic systems of plant specialized metabolites for the development of new drugs, and bioproducts in general

Interactions between the Nse3 and Nse4 Components of the SMC5-6 Complex Identify Evolutionarily Conserved Interactions between MAGE and EID Families

The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6-8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins.We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins

The serum zinc concentration as a potential biological marker in patients with major depressive disorder

Despite many clinical trials assessing the role of zinc in major depressive disorder (MDD), the conclusions still remain ambiguous. The aim of the present clinical study was to determine and comparison the zinc concentration in the blood of MDD patients (active stage or remission) and healthy volunteers (controls), as well as to discuss its potential clinical usefulness as a biomarker of the disease. In this study 69 patients with current depressive episode, 45 patients in remission and 50 controls were enrolled. The zinc concentration was measured by electrothermal atomic absorption spectrometry (ET AAS). The obtained results revealed, that the zinc concentration in depressed phase were statistically lower than in the healthy volunteers [0.89 vs. 1.06 mg/L, respectively], while the zinc level in patients achieve remission was not significantly different from the controls [1.07 vs. 1.06 mg/L, respectively]. Additionally, among the patients achieve remission a significant differences in zinc concentration between group with and without presence of drug-resistance in the previous episode of depression were observed. Also, patients in remission demonstrated correlation between zinc level and the average number of depressive episodes in the last year. Serum zinc concentration was not dependent on atypical features of depression, presence of psychotic symptoms or melancholic syndrome, age, age of onset or duration of disease, number of episodes in the life time, duration of the episode/remission and severity of depression measured by the Hamilton Rating Scale for Depression (HDRS), and the Montgomery-Asberg Depression Rating Scale (MADRS). Concluding, our findings confirm the correlation between zinc deficit present in the depressive episode, and are consistent with the majority of previous studies. These results may also indicate that serum zinc concentration might be considered as a potential biological marker of MDD

The SUMO Isopeptidase Ulp2p Is Required to Prevent Recombination-Induced Chromosome Segregation Lethality following DNA Replication Stress

SUMO conjugation is a key regulator of the cellular response to DNA replication stress, acting in part to control recombination at stalled DNA replication forks. Here we examine recombination-related phenotypes in yeast mutants defective for the SUMO de-conjugating/chain-editing enzyme Ulp2p. We find that spontaneous recombination is elevated in ulp2 strains and that recombination DNA repair is essential for ulp2 survival. In contrast to other SUMO pathway mutants, however, the frequency of spontaneous chromosome rearrangements is markedly reduced in ulp2 strains, and some types of rearrangements arising through recombination can apparently not be tolerated. In investigating the basis for this, we find DNA repair foci do not disassemble in ulp2 cells during recovery from the replication fork-blocking drug methyl methanesulfonate (MMS), corresponding with an accumulation of X-shaped recombination intermediates. ulp2 cells satisfy the DNA damage checkpoint during MMS recovery and commit to chromosome segregation with similar kinetics to wild-type cells. However, sister chromatids fail to disjoin, resulting in abortive chromosome segregation and cell lethality. This chromosome segregation defect can be rescued by overproducing the anti-recombinase Srs2p, indicating that recombination plays an underlying causal role in blocking chromatid separation. Overall, our results are consistent with a role for Ulp2p in preventing the formation of DNA lesions that must be repaired through recombination. At the same time, Ulp2p is also required to either suppress or resolve recombination-induced attachments between sister chromatids. These opposing defects may synergize to greatly increase the toxicity of DNA replication stress

Structural Maintenance of Chromosomes (SMC) Proteins Promote Homolog-Independent Recombination Repair in Meiosis Crucial for Germ Cell Genomic Stability

In meiosis, programmed DNA breaks repaired by homologous recombination (HR) can be processed into inter-homolog crossovers that promote the accurate segregation of chromosomes. In general, more programmed DNA double-strand breaks (DSBs) are formed than the number of inter-homolog crossovers, and the excess DSBs must be repaired to maintain genomic stability. Sister-chromatid (inter-sister) recombination is postulated to be important for the completion of meiotic DSB repair. However, this hypothesis is difficult to test because of limited experimental means to disrupt inter-sister and not inter-homolog HR in meiosis. We find that the conserved Structural Maintenance of Chromosomes (SMC) 5 and 6 proteins in Caenorhabditis elegans are required for the successful completion of meiotic homologous recombination repair, yet they appeared to be dispensable for accurate chromosome segregation in meiosis. Mutations in the smc-5 and smc-6 genes induced chromosome fragments and dismorphology. Chromosome fragments associated with HR defects have only been reported in mutants, which have disrupted inter-homolog crossover. Surprisingly, the smc-5 and smc-6 mutations did not disrupt the formation of chiasmata, the cytologically visible linkages between homologous chromosomes formed from meiotic inter-homolog crossovers. The mutant fragmentation defect appeared to be preferentially enhanced by the disruptions of inter-homolog recombination but not by the disruptions of inter-sister recombination. Based on these findings, we propose that the C. elegans SMC-5/6 proteins are required in meiosis for the processing of homolog-independent, presumably sister-chromatid-mediated, recombination repair. Together, these results demonstrate that the successful completion of homolog-independent recombination is crucial for germ cell genomic stability

Simulation data for Modelling DNA-strand displacement reactions in the presence of base-pair mismatches

Raw and processed simulation data from the paper Modelling DNA-strand displacement reactions in the presence of base-pair mismatches (J. Am. Chem. Soc.)Raw and processed simulation data from the paper Modelling DNA-strand displacement reactions in the presence of base-pair mismatches (J. Am. Chem. Soc.)