Heterologous expression and purification of native and mutated low molecular mass glutenin subunits from durum wheat

Targeting the Urotensin II/UT G Protein-Coupled Receptor to Counteract Angiogenesis and Mesenchymal Hypoxia/Necrosis in Glioblastoma

“…The urantide-inhibitory function raises the possibility of a biased function negatively regulating the UT-signaling cascades, e.g., partially Gq and internalization as previously shown (Brulé et al, 2014), specifically involved in tubulogenesis. Diebold et al (2012) demonstrated a NOX2-containing NADPH oxidase as a source of ROS responsible for UII-induced angiogenesis (Patacchini et al, 2003), supporting the idea that UII recruits NOX2 through a mechanism involving at least in part Gq and/or internalization of UT. To dissect the involvement of the urotensinergic system in GBM growth and angiogenesis, U87 heterotopic xenografts in Nude mice were injected with UT "agonists" (UII or URP or hUII 4−11 ), biased ligand (urantide) or antagonist (palosuran) alone, or in combination with UII.…”

“…Both URP and hUII 4−11 were used as lead sequence for the design of UT synthetic drugs including antagonists. The first "peptide antagonists" of rodent UT were obtained by focusing on Lys8 of UII to develop urantide (Table 1; Patacchini et al, 2003) exhibiting on CHO or HEK cells expressing recombinant human UT residual agonist (Camarda et al, 2004) or biased activity (Brulé et al, 2014). Here, when urantide was directly tested on EC-forming tubulogenesis, a marked constitutive inhibition is observed whereas the antagonist palosuran (Table 1) remained inactive on tube formation, both preventing UII-induced angiogenesis.…”

“…By comparison, the most well-studied primate UT antagonist palosuran only delayed gliomagenesis and animal death. It can be explained by the low antagonist behavior of palosuran toward rodent UT present in host cells, compared with the antagonist activity of urantide toward murine UT (Patacchini et al, 2003) combined with its biased activity on human UT (Brulé et al, 2014). As recently validated with the design of a radiolabeled DOTA-urantide binding UT injectable in vivo (Poret et al, 2020), UT expressed in GBM cells as well as in vascular/myeloid compartments would constitute a key pharmacological target for the design of therapeutic molecules based on the structure of urantide.…”

Expression, Purification, and Functional Analysis of Three Low‐Molecular‐Weight Glutenin Subunits from Wheat Cultivar Cheyenne

“…The results of SDS-PAGE showed molecular weights of approximately 50,000, 42,000, and 45,000 for LMW-CND1, LMW-CND2, and LMW-CND3, respectively, which were approximately 3,000-5,000 larger than the calculated values from their amino acid sequences. Such behavior of glutenin subunits is caused by their unusual structural characteristics, including the formation of the repetitive domain and the intermolecular disulfide bonds formed between cysteines, which lead to anomalous mobility on the gel (Lafiandra et al 1993;Patacchini et al 2003).…”

“…Because of the large number of LMW-GS gene copies with frequent mutation, as well as the close overlap with gliadins on SDS-PAGE (Payne and Corfield 1979), it is rather difficult to separate single LMW-GS from combined LMW-GS and gliadins by using traditional methods. Nevertheless, research on heterologous expression of wheat gluten proteins showed that the expression systems consisting of pET vectors and Escherichia coli hosts are practicable for most situations (Maruyama et al 1998;Dowd and Bekes 2002;Patacchini et al 2003), providing high-level and rapid expression. The proven prokaryotic expression technique makes it possible to produce highly purified individual LMW-GS efficiently, so as to provide satisfactory materials for defining the function of individual LMW-GS (Tamás and Shewry 2006;Li et al 2010).…”