The Insulin-like growth factor-1 (IGF-1) is a polypeptide growth factor with essential roles in physio-pathological conditions, including cellular growth and differentiation. Secretion of IGF-1 required proper post-translational modifications of IGF-1 prohormones (proIGF-1s), which include furin cleavage and N-glycosylation. This complex mechanism of IGF-1 production determines the presence of an IGF-1 pool composed of several IGF-1 protein variants: unglycosylated and glycosylated proIGF-1s, mature IGF-1, and E-peptides. Chapter I of the Thesis provides a description of the complex molecular mechanism regulating the production of the proIGF-1Es and the currently available methods for detecting and quantifying these IGF-1 protein variants. Particular attention was paid to the recent evidence showing that proIGF-1s are stable intermediate of IGF-1 processing and have specific biological activities. Subsequently, we describe a system to produce a relatively high amount of the glycosylated proIGF-1Ea (glyc_proIGF-1Ea) based on HEK-293 cells stably expressing the human IGF-1Ea isoform. Recombinant components of the IGF-1 pool, were characterized by High Resolution Mass Spectrometry (HRMS). Moreover, we used HRMS to detect the proGF-1Ea and Ea-peptide in the cell culture supernatants of HEK-293 cells overexpressing the IGF-1Ea isoform. The different strategies adopted to increase the glyc_proIGf-1Ea yield and the protein stability were also described. The synthesis of new protein standards corresponding to different IGF-1 isoforms, combined with the use of HRMS technology, may provide a useful tool to characterize and quantify the different components of the IGF-1 poll in complex biological matrices. The II chapter describes the development of a fluorescent- based Western Blot (WB) for the simultaneous detection of the three different proIGF-1s and E-peptides. Specifically, we described the use of two different primary antibodies, which recognize different epitopes of proIGF-1 sequences, and two secondary antibodies conjugated to different fluorophores to develop a multiplex fluorescent WB able to discriminate between the different IGF-1 protein variants. Our results demonstrate the feasibility of simultaneously detecting different isoforms of the same protein using different fluorescence filter combinations. Furthermore, combining antibodies to two different epitopes of the same target protein increased the specificity and reliability of protein detection. In the III chapter, we analyzed the molecular mechanisms regulating the 1 production of proIGF-1Ea and the biological effect of IGF-1 on 3D Breast Cancer (BC) spheroids formation. We describe different cell-culture conditions that favor the MCF-7 and MDA-2B-231 spheroids formation and the effects of IGF-1 on spheroids growth, compactness, viability, and gene expression patterns of metabolic and epithelial to mesenchymal transition (EMT) markers. Our results highlight the value of 3D spheroid models to better understand the role of IGF-1 on multiple aspects of tumor progression, including cancer dormancy and EMT. The IV chapter describes the impact of N-glycosylation inhibition on IGF-1 production and IGF-1 receptor (IGF1R) signalling pathway activation in the context of diseases associated with aberrant N-glycosylation such as Congenital Disorder of Glycosylation (CDG). Using muscle cellular models and mice models we demonstrated that N-glycosylation inhibition reduces myoblast fusion and impairs the early stage of the myogenic program in vitro and decreased myogenic markers in mice muscles. Finally, muscle gly_proIGF-1Ea production and IGF1R signalling pathway activation were markedly inhibited after N-glycosylation inhibition. Our results offer new insights that increase understanding of possible impairments of the myogenic differentiation capacity in the pathological context of disorders of N-glycosylation.

The complexity of insulin-like growth factor 1 (IGF-1): development of innovative bio-molecular methods for detection and quantification of IGF-1 protein variants and study of their biological roles in physio-pathological conditions

BOCCONCELLI, MATTEO
2023

Abstract

The Insulin-like growth factor-1 (IGF-1) is a polypeptide growth factor with essential roles in physio-pathological conditions, including cellular growth and differentiation. Secretion of IGF-1 required proper post-translational modifications of IGF-1 prohormones (proIGF-1s), which include furin cleavage and N-glycosylation. This complex mechanism of IGF-1 production determines the presence of an IGF-1 pool composed of several IGF-1 protein variants: unglycosylated and glycosylated proIGF-1s, mature IGF-1, and E-peptides. Chapter I of the Thesis provides a description of the complex molecular mechanism regulating the production of the proIGF-1Es and the currently available methods for detecting and quantifying these IGF-1 protein variants. Particular attention was paid to the recent evidence showing that proIGF-1s are stable intermediate of IGF-1 processing and have specific biological activities. Subsequently, we describe a system to produce a relatively high amount of the glycosylated proIGF-1Ea (glyc_proIGF-1Ea) based on HEK-293 cells stably expressing the human IGF-1Ea isoform. Recombinant components of the IGF-1 pool, were characterized by High Resolution Mass Spectrometry (HRMS). Moreover, we used HRMS to detect the proGF-1Ea and Ea-peptide in the cell culture supernatants of HEK-293 cells overexpressing the IGF-1Ea isoform. The different strategies adopted to increase the glyc_proIGf-1Ea yield and the protein stability were also described. The synthesis of new protein standards corresponding to different IGF-1 isoforms, combined with the use of HRMS technology, may provide a useful tool to characterize and quantify the different components of the IGF-1 poll in complex biological matrices. The II chapter describes the development of a fluorescent- based Western Blot (WB) for the simultaneous detection of the three different proIGF-1s and E-peptides. Specifically, we described the use of two different primary antibodies, which recognize different epitopes of proIGF-1 sequences, and two secondary antibodies conjugated to different fluorophores to develop a multiplex fluorescent WB able to discriminate between the different IGF-1 protein variants. Our results demonstrate the feasibility of simultaneously detecting different isoforms of the same protein using different fluorescence filter combinations. Furthermore, combining antibodies to two different epitopes of the same target protein increased the specificity and reliability of protein detection. In the III chapter, we analyzed the molecular mechanisms regulating the 1 production of proIGF-1Ea and the biological effect of IGF-1 on 3D Breast Cancer (BC) spheroids formation. We describe different cell-culture conditions that favor the MCF-7 and MDA-2B-231 spheroids formation and the effects of IGF-1 on spheroids growth, compactness, viability, and gene expression patterns of metabolic and epithelial to mesenchymal transition (EMT) markers. Our results highlight the value of 3D spheroid models to better understand the role of IGF-1 on multiple aspects of tumor progression, including cancer dormancy and EMT. The IV chapter describes the impact of N-glycosylation inhibition on IGF-1 production and IGF-1 receptor (IGF1R) signalling pathway activation in the context of diseases associated with aberrant N-glycosylation such as Congenital Disorder of Glycosylation (CDG). Using muscle cellular models and mice models we demonstrated that N-glycosylation inhibition reduces myoblast fusion and impairs the early stage of the myogenic program in vitro and decreased myogenic markers in mice muscles. Finally, muscle gly_proIGF-1Ea production and IGF1R signalling pathway activation were markedly inhibited after N-glycosylation inhibition. Our results offer new insights that increase understanding of possible impairments of the myogenic differentiation capacity in the pathological context of disorders of N-glycosylation.
5-dic-2023
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11576/2725874
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