Intrinsically disordered proteins (IDPs) encompass signalling and regulatory functions and altered expression of IDPs is associated with many diseases and imbalance in signalling pathways, transcriptional regulation, and splicing. Interest in insulin-like growth factor (IGF) – 1 isoforms on muscle homeostasis, regeneration, differentiation, and diseases has increased significantly. Inclusion or exclusion of exon 5 into the IGF-1 mRNA gives rise to three transcripts, IGF-1Ea, IGF-1Eb and IGF-1Ec, which yield three different C-terminal extensions called Ea, Eb and Ec peptides. Protein-coding sequences of exon 5 showed low rate of synonymous mutations and contain disorder-promoting amino acids, suggesting a regulatory role for these domains (Annibalini et al. 2016). To setup the analysis, the supernatants of HEK293 cells transfected with the specific IGF-1 isoform constructs as described in (De Santi et al. 2016) were studied by limited proteolysis combined with mass spectrometry (MS) using a Q-TOF microTM MS/MS (Micromass, Manchester, UK). Preliminary data showed that the C-terminal region of IGF-1Ea has lower resistance to trypsin digestion compared to the mature IGF-1 demonstrating IDRs in the Ea peptide. MS analyses to the detection of IGF-1E isoforms allowed us to identify both mature IGF-1 and IGF-1Ea isoform in transfected HEK293 cell culture supernatant. Analytical methods to correctly detect and quantify the IGF-1 isoforms are not currently available. In fact, the current existing methods rely on the use of antibodies that primarily recognize the mature IGF-1 peptide, thereby underestimate the isoforms. This finding could provide evidence allowing the detection and identification of the “E-tails” of IGF-1 and targeting these regulatory elements may represent a new strategy to control IGF-1 bioavailability in physio-pathological conditions. 1. Annibalini G, et al. MIR retroposon exonization promotes evolutionary variability and generates species-specific expression of IGF-1 splice variants BBAGRM 2016;1859:757-68. 2. De Santi M, et al. Cell Oncol (Dordr) 2016;39:149-59.
Looking at the role of disordered E-tails of IGF-1
ANNIBALINI, GIOSUE';CONTARELLI, SERENA;GIOACCHINI, ANNA MARIA;VALLORANI, LUCIANA;BRANDI, GIORGIO;PICCOLI, GIOVANNI;STOCCHI, VILBERTO;BARBIERI, ELENA
2016
Abstract
Intrinsically disordered proteins (IDPs) encompass signalling and regulatory functions and altered expression of IDPs is associated with many diseases and imbalance in signalling pathways, transcriptional regulation, and splicing. Interest in insulin-like growth factor (IGF) – 1 isoforms on muscle homeostasis, regeneration, differentiation, and diseases has increased significantly. Inclusion or exclusion of exon 5 into the IGF-1 mRNA gives rise to three transcripts, IGF-1Ea, IGF-1Eb and IGF-1Ec, which yield three different C-terminal extensions called Ea, Eb and Ec peptides. Protein-coding sequences of exon 5 showed low rate of synonymous mutations and contain disorder-promoting amino acids, suggesting a regulatory role for these domains (Annibalini et al. 2016). To setup the analysis, the supernatants of HEK293 cells transfected with the specific IGF-1 isoform constructs as described in (De Santi et al. 2016) were studied by limited proteolysis combined with mass spectrometry (MS) using a Q-TOF microTM MS/MS (Micromass, Manchester, UK). Preliminary data showed that the C-terminal region of IGF-1Ea has lower resistance to trypsin digestion compared to the mature IGF-1 demonstrating IDRs in the Ea peptide. MS analyses to the detection of IGF-1E isoforms allowed us to identify both mature IGF-1 and IGF-1Ea isoform in transfected HEK293 cell culture supernatant. Analytical methods to correctly detect and quantify the IGF-1 isoforms are not currently available. In fact, the current existing methods rely on the use of antibodies that primarily recognize the mature IGF-1 peptide, thereby underestimate the isoforms. This finding could provide evidence allowing the detection and identification of the “E-tails” of IGF-1 and targeting these regulatory elements may represent a new strategy to control IGF-1 bioavailability in physio-pathological conditions. 1. Annibalini G, et al. MIR retroposon exonization promotes evolutionary variability and generates species-specific expression of IGF-1 splice variants BBAGRM 2016;1859:757-68. 2. De Santi M, et al. Cell Oncol (Dordr) 2016;39:149-59.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
