Global Identification and Characterization of C2 Domain-Containing Proteins Associated with Abiotic Stress Response in Rice (Oryza sativa L.).

C2 domain-containing proteins (C2DPs) have been identified in different genomes that contain single or multiple C2 domains in their C- or N-terminal. It possesses higher functional activity in the transmembrane regions. The identification of C2 domains were reported in a previous study, such as multiple C2 domains and transmembrane-region proteins (MCTPs) and N-terminal-TM-C2 domain proteins (NTMC2s) of rice, Arabidopsis thaliana, and cotton, whereas the C2DP gene family in rice has not been comprehensively studied, and the role of the C2DP gene in rice in response to abiotic stress is not yet fully understood. In this study, we identified 82 C2DPs in the rice genome and divided them into seven groups through phylogenetic analysis. The synteny analysis revealed that duplication events were either exhibited within the genome of rice or between the genomes of rice and other species. Through the analysis of cis-acting elements in promoters, expression profiles, and qRT-PCR results, the functions of OsC2DPs were found to be widely distributed in diverse tissues and were extensively involved in phytohormones-related and abiotic stresses response in rice. The prediction of the microRNA (miRNA) targets of OsC2DPs revealed the possibility of regulation by consistent miRNAs. Notably, OsC2DP50/51/52 as a co-tandem duplication exhibited similar expression variations and involved the coincident miRNA-regulation pathway. Moreover, the results of the genotypic variation and haplotype analysis revealed that OsC2DP17, OsC2DP29, and OsC2DP49 were associated with cold stress responses. These findings provided comprehensive insights for characterizations of OsC2DPs in rice as well as for their roles for abiotic stress.

The C2 domain of calpain 5 contributes to enzyme activation and membrane localization.

The enzymatic characteristics of the ubiquitous calpain 5 (CAPN5) remain undescribed despite its high expression in the central nervous system and links to eye development and disease. CAPN5 contains the typical protease core domains but lacks the C terminal penta-EF hand domain of classical calpains, and instead contains a putative C2 domain. This study used the SH-SY5Y neuroblastoma cell line stably transfected with CAPN5-3xFLAG variants to assess the potential roles of the CAPN5 C2 domain in Ca2+ regulated enzyme activity and intracellular localization. Calcium dependent autoproteolysis of CAPN5 was documented and characterized. Mutation of the catalytic Cys81 to Ala or addition of EGTA prevented autolysis. Eighty µM Ca2+ was sufficient to stimulate half-maximal CAPN5 autolysis in cellular lysates. CAPN5 autolysis was inhibited by tri-leucine peptidyl aldehydes, but less effectively by di-Leu aldehydes, consistent with a more open conformation of the protease core relative to classical calpains. In silico modeling revealed a type II topology C2 domain including loops with the potential to bind calcium. Mutation of the acidic amino acid residues predicted to participate in Ca2+ binding, particularly Asp531 and Asp589, resulted in a decrease of CAPN5 membrane association. These residues were also found to be invariant in several genomes. The autolytic fragment of CAPN5 was prevalent in membrane-enriched fractions, but not in cytosolic fractions, suggesting that membrane association facilitates the autoproteolytic activity of CAPN5. Together, these results demonstrate that CAPN5 undergoes Ca2+-activated autoproteolytic processing and suggest that CAPN5 association with membranes enhances CAPN5 autolysis.

A structure model explaining the binding between a ubiquitous unconventional G-protein (OsYchF1) and a plant-specific C2-domain protein (OsGAP1) from rice.

The unconventional G-protein OsYchF1 plays regulatory roles in plant defense and abiotic stress responses. We have previously resolved the crystal structures of OsYchF1 and its plant-specific regulator, OsGAP1, and determined the residues on OsGAP1 that are essential for its binding to OsYchF1. In this study, we employed site-directed mutagenesis to identify four critical residues on the TGS domain of OsYchF1 that are critical for its binding to OsGAP1. We also generated a docking model of the OsYchF1 : OsGAP1 complex to dissect the molecular basis of their interactions. Our finding not only reveals the roles of the key interacting residues controlling the binding between OsYchF1 and OsGAP1, but also provides a working model on the potential regulatory mechanism mediated by a TGS domain, particularly in the class of GTPase of the OBG family.

Structural and functional characterization of Solanum lycopersicum phosphatidylinositol 3-kinase C2 domain.

Phosphatidylinositol 3-kinases (PI3Ks) are characterized by the presence of a C2 domain at the N-terminal end (class I, III); or at both the N-terminal and C-terminal ends (class II), sometimes including a Plextrin homology domain and/or a Ras domain. Plant PI3Ks are analogous to the class III mammalian PI3K. An N-terminal fragment (~170 aa) of the tomato PI3K regulatory domain including the C2 domain, was cloned and expressed in a bacterial system. This protein was purified to homogeneity and its physicochemical properties analyzed. The purified protein showed strong binding with monophosphorylated phosphatidylinositols, and the binding was dependent on calcium ion concentration and pH. In the overall tertiary structure of PI3K, C2 domain showed unique characteristics, having three antiparallel beta-sheets, hydrophobic regions, acidic as well as alkaline motifs, that can enable its membrane binding upon activation. To elucidate the functional significance of C2 domain, transgenic tobacco plants expressing the C2 domain of PI3K were generated. Transgenic plants showed defective pollen development and disrupted seed set. Flowers from the PI3K-C2 transgenic plants showed delayed wilting, and a decrease in ethylene production. It is likely that introduction of the PI3K-C2 segment may have interfered with the normal binding of PI3K to the membrane, delaying the onset of membrane lipid catabolism that lead to senescence.