Food safety evaluation for R-proteins introduced by biotechnology: A case study of VNT1 in late blight protected potatoes.

Resistance genes (R-genes) from wild potato species confer protection against disease and can be introduced into cultivated potato varieties using breeding or biotechnology. The R-gene, Rpi-vnt1, which encodes the VNT1 protein, protects against late blight, caused by Phytophthora infestans. Heterologous expression and purification of active VNT1 in quantities sufficient for regulatory biosafety studies was problematic, making it impractical to generate hazard characterization data. As a case study for R-proteins, a weight-of-evidence, tiered approach was used to evaluate the safety of VNT1. The hazard potential of VNT1 was identified from relevant safety information including history of safe use, bioinformatics, mode of action, expression levels, and dietary intake. From the assessment it was concluded that Tier II hazard characterization was not needed. R-proteins homologous to VNT1 and identified in edible crops, have a history of safe consumption. VNT1 does not share sequence identity with known allergens. Expression levels of R-proteins are generally low, and VNT1 was not detected in potato varieties expressing the Rpi-vnt1 gene. With minimal hazard and negligible exposure, the risks associated with consumption of R-proteins in late blight protected potatoes are exceedingly low. R-proteins introduced into potatoes to confer late blight protection are safe for consumption.

A transcriptomic approach to elucidate the physiological significance of human cytochrome P450 2S1 in bronchial epithelial cells.

BACKGROUND: Cytochrome P450 2S1 (CYP2S1) is an orphan P450 with an unknown biological function. Data from our laboratory and others suggest that CYP2S1 may have an important physiological role in modulating the synthesis and metabolism of bioactive lipids including prostaglandins and retinoids. CYP2S1 expression is elevated in multiple epithelial-derived cancers as well as in the chronic hyperproliferative disease psoriasis. Whether CYP2S1 expression in proliferative disease is protective, detrimental, or neutral to disease progression remains to be determined. Two human bronchial epithelial cells (BEAS-2B) were constructed to represent chronic depletion of CYP2S1 using short-hairpin RNA (shRNA) silencing directed toward the 3'UTR (759) and exon 3 (984) of the CYP2S1 gene and compared with a non-targeting shRNA control (SCRAM). Both CYP2S1 mRNA and protein were depleted by approximately 75% in stable cell lines derived from both targeted shRNA constructs (759 and 984). To elucidate the biological significance of CYP2S1, we analyzed transcriptome alterations in response to CYP2S1 depletion in human lung cells. RESULTS: RNA-sequencing (RNA-seq) analysis was performed to compare the transcriptome of the control (SCRAM) and the CYP2S1-depleted (759) BEAS-2B cell lines. Transcriptomes of the replicates from the two cell lines were found to be distinct populations as determined using Principal Component Analysis and hierarchical clustering. Approximately 1000 genes were differentially expressed in response to CYP2S1 depletion. Consistent with our previous phenotypes, DAVID analysis revealed altered regulation in key pathways implicated in cell proliferation and migration. Transcriptomic profiles were also consistent with the metabolism of proposed endogenous substrates. Pathway analysis also revealed significant expression changes within mTOR signaling, a critical pathway in cell growth. To determine whether these changes manifest as altered cell size, cell diameter and volume were calculated, revealing that CYP2S1 depletion promotes cell growth in BEAS-2B cells. CONCLUSIONS: These data suggest that pathway analysis of sequence-based gene expression is a powerful method to identify pathways and phenotypic alterations in response to changes in orphan enzyme expression. Our results suggest a novel role for CYP2S1-mediated metabolism in modulating BEAS-2B cell size. These findings warrant further studies on CYP2S1 regulated pathways to elucidate potential substrates of CYP2S1.

Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation.

Grb7 is an adaptor molecule mediating signal transduction from multiple cell surface receptors to diverse downstream pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase, ErbB2, are overexpressed in 20-30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, although the participants in these pathways have not been fully determined. In this study, we report the Grb7 protein interacts with Filamin-a, an actin-crosslinking component of the cell cytoskeleton. Additionally, we have demonstrated the interaction between Grb7 and Flna is specific to the RA-PH domains of Grb7, and the immunoglobulin-like repeat 16-19 domains of Flna. We demonstrate that full-length Grb7 and Flna interact in the mammalian cellular environment, as well as in vitro. Immunofluorescent microscopy shows potential co-localization of Grb7 and Flna in membrane ruffles upon epidermal growth factor stimulation. These studies are amongst the first to establish a clear connection between Grb7 signaling and cytoskeletal remodeling.

Shaping cellular form and function by autophagy.

In addition to its familiar role in non-selective bulk degradation of cellular material, autophagy can also bring about specific changes in the structure and function of cells. Autophagy has been proposed to operate in a substrate-selective mode to carry out this function, although evidence to demonstrate selectivity has been lacking. A recent study of synapse formation in the nervous system of the nematode Caenorhabditis elegans now provides experimental evidence for substrate-selective autophagy. Synapses form when presynaptic cells contact their postsynaptic partners during development. This contact induces the assembly of synaptically-localized protein complexes in the postsynaptic cell that contain scaffolding proteins and neurotransmitter receptors. When presynaptic contact was blocked, autophagy in the postsynaptic cell was induced. Substrate selectivity was evident in this system: the gamma-aminobutyric acid type A receptor (GABA(A) receptor), an integral-membrane neurotransmitter receptor, trafficked from the cell surface to autophagosomes. By contrast, the acetylcholine receptor, a structurally-similar neurotransmitter receptor, remained on the cell surface. This result provides experimental support for the idea that autophagy can bring about changes in cell structure and behavior by degrading specific cellular proteins, particularly cell surface receptors that are often important for regulating cell growth, differentiation and function.

Presynaptic terminals independently regulate synaptic clustering and autophagy of GABAA receptors in Caenorhabditis elegans.

Synaptic clustering of GABAA receptors is important for the function of inhibitory synapses, influencing synapse strength and, consequently, the balance of excitation and inhibition in the brain. Presynaptic terminals are known to induce GABAA receptor clustering during synaptogenesis, but the mechanisms of cluster formation and maintenance are not known. To study how presynaptic neurons direct the formation of GABAA receptor clusters, we have investigated GABAA receptor localization in postsynaptic cells that fail to receive presynaptic contacts in Caenorhabditis elegans. Postsynaptic muscles in C. elegans receive acetylcholine and GABA motor innervation, and GABAA receptors cluster opposite GABA terminals. Selective loss of GABA inputs caused GABAA receptors to be diffusely distributed at or near the muscle cell surface, confirming that GABA presynaptic terminals induce GABAA receptor clustering. In contrast, selective loss of acetylcholine innervation had no effect on GABAA receptor localization. However, loss of both GABA and acetylcholine inputs together caused GABAA receptors to traffic to intracellular autophagosomes. Autophagosomes normally transport bulk cytoplasm to the lysosome for degradation. However, we show that GABAA receptors traffic to autophagosomes after endocytic removal from the cell surface and that acetylcholine receptors in the same cells do not traffic to autophagosomes. Thus, autophagy can degrade cell-surface receptors and can do so selectively. Our results show that presynaptic terminals induce GABAA receptor clustering by independently controlling synaptic localization and surface stability of GABAA receptors. They also demonstrate a novel function for autophagy in GABAA receptor degradative trafficking.