Quantitative proteomics screen identifies a substrate repertoire of rhomboid protease RHBDL2 in human cells and implicates it in epithelial homeostasis

This deposit is composed by the main article plus the supplementary materials of the publication.Rhomboids are intramembrane serine proteases conserved in all kingdoms of life. They regulate epidermal growth factor receptor signalling in Drosophila by releasing signalling ligands from their transmembrane tethers. Their functions in mammals are poorly understood, in part because of the lack of endogenous substrates identified thus far. We used a quantitative proteomics approach to investigate the substrate repertoire of rhomboid protease RHBDL2 in human cells. We reveal a range of novel substrates that are specifically cleaved by RHBDL2, including the interleukin-6 receptor (IL6R), cell surface protease inhibitor Spint-1, the collagen receptor tyrosine kinase DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, BCAM and others. We further demonstrate that these substrates can be shed by endogenously expressed RHBDL2 and that a subset of them is resistant to shedding by cell surface metalloproteases. The expression profiles and identity of the substrates implicate RHBDL2 in physiological or pathological processes affecting epithelial homeostasis.Academy of Sciences of the Czech Republic grant: (Purkyne Fellowship); EMBO grant: (Installation Grant no. 2329); Ministry of Education, Youth and Sports of the Czech Republic grants: (projects no. LK11206 and LO1302); Marie Curie Career Integration grant: (project no. 304154); National Subvention for Development of Research Organisations grant: (RVO: 61388963); Institute of Organic Chemistry and Biochemistry; Fundação Calouste Gulbenkian; Worldwide Cancer Research grant: (14–1289); Marie Curie Career Integration grant: (project no. 618769); Fundação para a Ciência e Tecnologica (FCT, PTDC/BEX-BCM/3015/2014); European Crohn’s and Colitis organization (ECCO); COST BM1406; Wellcome Trust grant: (101035/Z/13/Z); Medical Research Council grant: (programme number MC_U105178780).info:eu-repo/semantics/publishedVersio

An N-terminal Peptide Extension Results in Efficient Expression, but not Secretion, of a Synthetic Horseradish Peroxidase Gene in Transgenic Tobacco

BACKGROUND AND AIMS: Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. METHODS: Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. KEY RESULTS: Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. CONCLUSIONS: There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic activity. These will be valuable for exploring the role of these enzymes in stress amelioration and plant development

iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

The apical inflammatory cytokine TNF regulates numerous important biological processes including inflammation and cell death, and drives inflammatory diseases. TNF secretion requires TACE (also called ADAM17), which cleaves TNF from its transmembrane tether. The trafficking of TACE to the cell surface, and stimulation of its proteolytic activity, depends on membrane proteins, called iRhoms. To delineate how the TNF/TACE/iRhom axis is regulated, we performed an immunoprecipitation/mass spectrometry screen to identify iRhom-binding proteins. This identified a novel protein, that we name iTAP (iRhom Tail-Associated Protein) that binds to iRhoms, enhancing the cell surface stability of iRhoms and TACE, preventing their degradation in lysosomes. Depleting iTAP in primary human macrophages profoundly impaired TNF production and tissues from iTAP KO mice exhibit a pronounced depletion in active TACE levels. Our work identifies iTAP as a physiological regulator of TNF signalling and a novel target for the control of inflammation.info:eu-repo/semantics/publishedVersio

Deletion of iRhom2 protects against diet-inducedobesity by increasing thermogenesis

Objective:Obesity is the result of positive energy balance. It can be caused by excessive energy consumption but also by decreased energydissipation, which occurs under several conditions including when the development or activation of brown adipose tissue (BAT) is impaired. Herewe evaluated whether iRhom2, the essential cofactor for the Tumour Necrosis Factor (TNF) sheddase ADAM17/TACE, plays a role in thepathophysiology of metabolic syndrome.Methods:We challenged WT versus iRhom2 KO mice to positive energy balance by chronic exposure to a high fat diet and then compared theirmetabolic phenotypes. We also carried outex vivoassays with primary and immortalized mouse brown adipocytes to establish the autonomy ofthe effect of loss of iRhom2 on thermogenesis and respiration.Results:Deletion of iRhom2 protected mice from weight gain, dyslipidemia, adipose tissue inflammation, and hepatic steatosis and improvedinsulin sensitivity when challenged by a high fat diet. Crucially, the loss of iRhom2 promotes thermogenesis via BAT activation and beigeadipocyte recruitment, enabling iRhom2 KO mice to dissipate excess energy more efficiently than WT animals. This effect on enhanced ther-mogenesis is cell-autonomous in brown adipocytes as iRhom2 KOs exhibit elevated UCP1 levels and increased mitochondrial proton leak.Conclusion:Our data suggest that iRhom2 is a negative regulator of thermogenesis and plays a role in the control of adipose tissue homeostasisduring metabolic diseaseWellcome Trust strategic award (100574/Z/12/Z) and MRC MDU (MC_UU_12012/

Self-concept and Theory of Mind in the classroom

This study investigated the relationship between theory of mind, endorsement of the looking-glass self and aspects of self-concept through a questionnaire measure within a classroom setting. Participants were adolescents from six schools across England (N= 184), with a mean age of 12.06. Endorsement of the looking-glass self was examined; half of the sample supported the model but it was found to relate to poor leadership skills and low popularity. The study also examined whether endorsement of the looking-glass self and better theory of mind were related to self-other agreement, the results suggested that this was the case for the former, with the necessity of further research, but no relationship was found for the latter. The consequences of self-other congruence were explored: incongruence equated to greater aggression and less favourability among classmates. Last, the relationship between theory of mind and current/ideal-self discrepancy was examined; a significant relationship was found but could be explained by co-varying effects of verbal ability. Implications and suggestions for future research are discussed

Use of cell morphology to evaluate the effect of a peroxidase gene on cell death induction thresholds in tobacco

Tobacco suspension cultures were subjected to a range of heat stresses and used to compare morphological aspects of programmed cell death (PCD) and necrosis. Cells undergoing PCD were found to display characteristic death morphology, caused by cytoplasmic retraction of the protoplast, and to have cleaved DNA. We evaluated if the morphological characteristics of PCD could be used to monitor changes in cell death induction thresholds in transgenic cell cultures with high levels of peroxidase activity. Again, using a heat shock assay, we show that tobacco cell cultures with elevated levels of peroxidase have higher cell death induction threshold levels than wild type tobacco cell cultures. Thus, assessing PCD associated morphological changes can report on the effect of altering peroxidase genes on cell death activation in tobacco. This study demonstrates that PCD morphology could routinely be used to monitor the effects of introduced genes on programmed cell death induction thresholds in plants

Altered Activity of Peroxidase and Oxalate Oxidase Influences Lignification in Transgenic Tobacco

Peroxidase and hydrogen peroxide both play important roles in the final stages of the lignification pathway. Peroxidase, in the presence of H2O2 catalyses the oxidation of monolignols to give lignin. In order to examine this process we looked at lignification in transgenic tobacco plants expressing a barley peroxidase gene, HvPrx8, either alone or in combination with a wheat germin gene, g.f 2.8, which encodes oxalate oxidase, thereby providing a source of H2O2. Elevated activity of the antioxidant ascorbate peroxidase was found in plants expressing oxalate peroxidase and was greatly increased by co-expression with the barley peroxidase, although the latter had no effect when expressed alone. An increase was observed in the oxidation of the lignin monomer, syringaldazine in cell lines over-expressing barley peroxidase, while a decrease was observed in double transformants. Plants over-expressing barley peroxidase have elevated levels of lignin deposition compared to that of wild type tobacco plants. Over-expression of the individual enzymes was also shown to enhance heat-induced programmed cell death (PCD) in cell suspension cultures, an effect which was greatly reduced in the double-expressing lines

Transgenic tobacco (Nicotiana tabacum L. cv. Samsun-NN) plants over-expressing a synthetic HRP-C gene are altered in growth, development and susceptibility to abiotic stress

The physiological role of class III peroxidases (EC 1.11.1.7) in controlling plant growth and development has been investigated by overexpression of both native and heterologous peroxidases. However, it has remained an enigma as to why the phenotypes of different peroxidase over-expressing transgenics vary. In order to resolve the conflicting information about the consequences of peroxidase over-expression, we have explored the role of the subcellular targeting of HRP-C in controlling stem growth, root development, axillary branching and abiotic stress tolerance in tobacco (Nicotiana tabacum L.). Altering the sub-cellular targeting of vacuolar HRP-C, such that over-expressed peroxidase accumulates in the cytoplasm and cell wall, induced phenotypic changes that are typically associated with altered auxin homeostasis, and overexpression of cell wall located peroxidases. We conclude that sub-cellular targeting is a determinant of the phenotype of peroxidase over-expressing plants