Yoshitaka Umetsu

Central cell-derived peptides regulate early embryo patterning in flowering plants

Tripeptide Maternal Support In flowering plants, fertilization involves multiple gametes. The diploid zygote, which will form the embryonic plant, is surrounded by the often triploid endosperm, which provides a supportive and nourishing function. Working in Arabidopsis, Costa et al. (p. 168; see the Perspective by Bayer) identified a trio of small signaling peptides that derive from the endosperm but that regulate growth of the embryo. RNA interference was used to down-regulate expression of all three peptides. Fertilization was not affected, but seed growth was. The peptides were critical for normal development of the suspensor, which tethers and nourishes the growing embryo. Within plant seeds, signaling functions from the endosperm regulate development of the embryonic plant suspensor. [Also see Perspective by Bayer] Plant embryogenesis initiates with the establishment of an apical-basal axis; however, the molecular mechanisms accompanying this early event remain unclear. Here, we show that a small cysteine-rich peptide family is required for formation of the zygotic basal cell lineage and proembryo patterning in Arabidopsis. EMBRYO SURROUNDING FACTOR 1 (ESF1) peptides accumulate before fertilization in central cell gametes and thereafter in embryo-surrounding endosperm cells. Biochemical and structural analyses revealed cleavage of ESF1 propeptides to form biologically active mature peptides. Further, these peptides act in a non–cell-autonomous manner and synergistically with the receptor-like kinase SHORT SUSPENSOR to promote suspensor elongation through the YODA mitogen-activated protein kinase pathway. Our findings demonstrate that the second female gamete and its sexually derived endosperm regulate early embryonic patterning in flowering plants.

Mesomartoxin, a new Kv1.2-selective scorpion toxin interacting with the channel selectivity filter

Venom-derived neurotoxins are ideal probes for the investigation of structure-function relationship of ion channels and promising scaffolds for the design of ion channel-targeted drug leads as well. The discovery of highly selective toxins against a specific channel subtype facilitates the development of drugs with reduced side effects. Here, we describe the systemic characterization of a new scorpion short-chain K(+) channel blocker from Mesobuthus martensii, termed mesomartoxin (MMTX). MMTX is synthesized as a precursor comprising a signal peptide and a mature peptide of 29 residues. Nuclear magnetic resonance analysis confirmed that recombinant MMTX adopts a typical cysteine-stabilized α-helical and β-sheet fold. Electrophysiological experiments showed that MMTX exhibits high affinity for the Drosophila Shaker K(+) channel but differential selectivity on different members of the rat voltage-gated K(+) channel (Kv) family, with nanomolar affinity (IC50=15.6 nM) for rKv1.2, micromolar affinity for rKv1.3 (IC50=12.5 μM) and no activity on rKv1.1 at >50 μM. Site-directed mutagenesis of the channel pore identified a key site located on the selectivity filter of the pore, which is directly implicated in toxin binding and controls targets selectivity of the toxin. Given a key role of Kv1.2 in epilepsy, MMTX might serve as a potential drug lead for the disease.

Overexpression of an antimicrobial peptide derived from C. elegans using an aggregation-prone protein coexpression system

Antibacterial factor 2 (ABF-2) is a 67-residue antimicrobial peptide derived from the nematode Caenorhabditis elegans. Although it has been reported that ABF-2 exerts in vitro microbicidal activity against a range of bacteria and fungi, the structure of ABF-2 has not yet been solved. To enable structural studies of ABF-2 by NMR spectroscopy, a large amount of isotopically labeled ABF-2 is essential. However, the direct expression of ABF-2 in Escherichia coli is difficult to achieve due to its instability. Therefore, we applied a coexpression method to the production of ABF-2 in order to enhance the inclusion body formation of ABF-2. The inclusion body formation of ABF-2 was vastly enhanced by coexpression of aggregation-prone proteins (partner proteins). By using this method, we succeeded in obtaining milligram quantities of active, correctly folded ABF-2. In addition, 15 N-labeled ABF-2 and a well-dispersed heteronuclear single quantum coherence (HSQC) spectrum were also obtained successfully. Moreover, the effect of the charge of the partner protein on the inclusion body formation of ABF-2 in this method was investigated by using four structurally homologous proteins. We concluded that a partner protein of opposite charge enhanced the formation of an inclusion body of the target peptide efficiently.

C-terminal elongation of growth-blocking peptide enhances its biological activity and micelle binding affinity

Growth-blocking peptide (GBP) is a hormone-like peptide that suppresses the growth of the host armyworm. Although the 23-amino acid GBP (1–23 GBP) is expressed in nonparasitized armyworm plasma, the parasitization by wasp produces the 28-amino acid GBP (1–28 GBP) through an elongation of the C-terminal amino acid sequence. In this study, we characterized the GBP variants, which consist of various lengths of the C-terminal region, by comparing their biological activities and three-dimensional structures. The results of an injection study indicate that 1–28 GBP most strongly suppresses larval growth. NMR analysis shows that these peptides have basically the same tertiary structures and that the extension of the C-terminal region is disordered. However, the C-terminal region of 1–28 GBP undergoes a conformational transition from a random coiled state to an α-helical state in the presence of dodecylphosphocholine micelles. This suggests that binding of the C-terminal region would affect larval growth activity.