Huang, S.-S. | Search bioRxiv

bioRxiv2023

The NPH3-domain protein NRL5 is a plant specific type of GTPase essential for drought resistance

The mechanisms of plant resistance to low water potential ({psi}w) during drought are unclear but may involve signaling and trafficking at the plasma membrane as well as metabolic reprogramming, including proline accumulation. Forward genetic screening using a Proline Dehydrogenase 1 (ProDH1) promoter:reporter line identified a mutant with extreme low {psi}w hypersensitivity due to a single amino acid substitution (P335L) in the Non-Phototrophic Hypocotyl3 (NPH3) domain of NPH3/RPT2-Like5 (NRL5)/Naked Pins in Yucca8 (NPY8). Further experiments found that NRL5, and other NPH3-domain proteins, are GTPases. NRL5 interacted with RAB small GTPases and the SNARE proteins VAMP721/722 and had polar localization. NRL5P335L had greatly reduced GTPase activity, impaired RAB and VAMP721/722 interaction and disrupted polar localization. These data demonstrate that NRL5-mediated restraint of proline catabolism is required for drought resistance and also more broadly define unexpected functions of the NPH3 domain such that the role of NPH3-domain proteins in signaling, trafficking, and cellular polarity can be critically re-evaluated. One-Sentence SummaryA protein containing the plant-specific NPH3-domain has GTPase activity, trafficking interaction and drought resistance function.

plant biology↗

bioRxiv2020

Structure-based functional study of a peptide of an ecdysozoan superfamily: reveling a common molecular architecture and receptor-interacting residues

A neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs and increased significantly after animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy revealing that it has an N-terminal tail, three -helices (2, Gly9-Asn28; 3, His34-Gly38; 5, Glu62-Arg72), and a {pi}-helix ({pi}4, Cys43-Tyr53) and is structurally constrained by a pattern of disulfide bonds (Cys7-Cys43, Cys23-Cys39, Cys26-Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 [A]. Ten residues of Sco-CHH-L were chosen for alanine-substituted and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, G73) are located at either end of the sequence, which are sterically close to each other and presumably constitutes the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily revealing a molecular architecture, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand-receptor binding specificity.

biochemistry↗