LIM domain-wide comprehensive mutagenesis reveals the role of leucine in CSRP3 protein stability

Cardiomyopathies are a severe and chronic cardiovascular burden worldwide, affecting a large cohort in the general population. Cysteine and glycine-rich protein 3 (CSRP3) is one of key proteins implicated in dominant dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). In this study, we device a rapid in-silico screening protocol that creates a mutational landscape map for all possible allowed and disallowed substitutions in the protein of interest. This map provides the structural and functional insights on the stability of LIM domains of CSRP3. Further, the sequence analysis delineates the eukaryotic CSRP3 protein orthologs which complements the mutational map. Next, we also evaluated the effect of HCM/DCM mutations on these domains. One of highly destabilising mutations - L44P (also disease causing) and a neutral mutation - L44M were further subjected to molecular dynamics (MD) simulations. The results establish that L44P substitution affects the LIM domain structure. The present study provides a useful perspective to our understanding of the role of mutations in the CSRP3 LIM domains and their evolution. Experimentally verifying every reported mutation can become challenging both in time and resources used. This study provides a novel screening method for quick identification of key mutation sites for specific protein structures that can reduce the burden on experimental research.

iTRAQ-based Quantitative Proteomic Analysis of Thoracic Aortic from Adult Rats Born to Pre-eclamptic Dams

Background: Preeclampsia and gestational hypertension can cause impaired vascular function in offspring. In our previous work, we described the profiles of protein expression in umbilical artery tissues of patients with preeclampsia. Methods: In order to gain insights into the mechanisms of vascular dysfunction in adult rats born by preeclampsia dams, we present analysis of thoracic aortic tissues by using iTRAQ isobaric tags and 2D nano LC-MS/MS. Results: By using iTRAQ method, we analyzed 1825 proteins in which 106 proteins have shown significantly differential expression in thoracic aortic. Results from ingenuity pathway analysis (IPA) showed majority of proteins with differential expression (DEP)were associated with cardiovascular function. Further analysis indicated that glucose-6-phosphate dehydrogenase (G6PD), which is inhibited by miR-423-5p and activated by TP53, was most affect the cardiovascular function. The expression level of G6PD was up-regulated in thoracic aortic tissues as confirmed by western blot results. Two other vascular function proteins Cysteine and glycine-rich protein 2 (CSRP2) and Tubulin alpha-4A (TUBA4A) were upregulated as demonstrated by mass spectrometry (MS). Conclusions: Although the results need further functional validation, these data provide novel findings relating to impaired vascular function in adult offsprings of preeclamptic mothers.

The Rice GLYCINE-RICH PROTEIN 3 Confers Drought Tolerance by Regulating mRNA Stability of ROS Scavenging-Related Genes

Background Plant glycine-rich proteins are categorized into several classes based on their protein structures. The glycine-rich RNA binding proteins (GRPs) are members of class IV subfamily possessing N-terminus RNA-recognition motifs (RRMs) and proposed to be involved in post-transcriptional regulation of its target transcripts. GRPs are involved in developmental process and cellular stress responses, but the molecular mechanisms underlying these regulations are still elusive. Results Here, we report the functional characterization of rice GLYCINE-RICH PROTEIN 3 (OsGRP3) and its physiological roles in drought stress response. Both drought stress and ABA induce the expression of OsGRP3. Transgenic plants overexpressing OsGRP3 (OsGRP3OE) exhibited tolerance while knock-down plants (OsGRP3KD) were susceptible to drought compared to the non-transgenic control. In vivo, subcellular localization analysis revealed that OsGRP3-GFP was transported from cytoplasm/nucleus into cytoplasmic foci following exposure to ABA and mannitol treatments. Comparative transcriptomic analysis between OsGRP3OE and OsGRP3KD plants suggests that OsGRP3 is involved in the regulation of the ROS related genes. RNA-immunoprecipitation analysis revealed the associations of OsGRP3 with PATHOGENESIS RELATED GENE 5 (PR5), METALLOTHIONEIN 1d (MT1d), 4,5-DOPA-DIOXYGENASE (DOPA), and LIPOXYGENASE (LOX) transcripts. The half-life analysis showed that PR5 transcripts decayed slower in OsGRP3OE but faster in OsGRP3KD, while MT1d and LOX transcripts decayed faster in OsGRP3OE but slower in OsGRP3KD plants. H2O2 accumulation was reduced in OsGRP3OE and increased in OsGRP3KD plants compared to non-transgenic plants (NT) under drought stress. Conclusion OsGRP3 plays a positive regulator in rice drought tolerance and modulates the transcript level and mRNA stability of stress-responsive genes, including ROS-related genes. Moreover, OsGRP3 contributes to the reduction of ROS accumulation during drought stress. Our results suggested that OsGRP3 alleviates ROS accumulation by regulating ROS-related genes’ mRNA stability under drought stress, which confers drought tolerance.

The tomato receptor CuRe1 senses a cell wall protein to identify Cuscuta as a pathogen

Parasitic plants of the genus Cuscuta penetrate shoots of host plants with haustoria and build a connection to the host vasculature to exhaust water, solutes and carbohydrates. Such infections usually stay unrecognized by the host and lead to harmful host plant damage. Here, we show a molecular mechanism of how plants can sense parasitic Cuscuta. We isolated an 11 kDa protein of the parasite cell wall and identified it as a glycine-rich protein (GRP). This GRP, as well as its minimal peptide epitope Crip21, serve as a pathogen-associated molecular pattern and specifically bind and activate a membrane-bound immune receptor of tomato, the Cuscuta Receptor 1 (CuRe1), leading to defense responses in resistant hosts. These findings provide the initial steps to understand the resistance mechanisms against parasitic plants and further offer great potential for protecting crops by engineering resistance against parasitic plants.