The androgen receptor (AR) is a member of the steroid hormone group of nuclear receptors (NRs) and the main mediator of androgen signalling. Encoded by the AR gene on the X chromosome, AR is a transcription factor involved in biological functions like cell growth and differentiation, development and homeostasis in many tissues. When inactive, AR is in the cytosol and is associated with chaperones and cochaperones, which form the foldosome complex. Upon testosterone (T) and dihydrotestosterone (DHT) binding, AR dissociates from the heat shock proteins (HSPs) and translocates to the nucleus, where it regulates gene expression. Upon ligand withdrawal, AR is exported back to the cytoplasm where it resides in its inactive state. The foldosome complex assists AR folding in a reversible three-step process that is articulates in the formation of an early, intermediate and late complex. In the early foldosome, AR interacts with HSP40 and HSP70; the intermediate complex is formed with the association of HSP90 and its co-chaperone HOP. At this point, AR can be degraded through the ubiquitination-proteasome system (UPS) by E3 ubiquitin ligases such as STUB1 and MDM2, or can bind the late complex, composed of co-chaperones p23 and HSP90. There is a fine regulation of AR synthesis, maturation, activation and degradation, however the mechanisms controlling it still have to be fully elucidated. Additionally, AR activity is regulated by different post-translational modifications (PTMs); in particular, AR phosphorylation at different sites has been shown to influence AR stability and degradation. Considering this, the central hypothesis of my thesis project is that AR phosphorylation regulates AR interaction with different co-chaperones involved in the balance between AR folding and degradation. The aim is to characterize such protein-protein interactions (PPIs) as well as to identify a potential biological role, which is still poorly understood for some AR interactors. First, we evaluated the interaction in HEK293T cells transfected with AR by immunoprecipitation assays and we found the interaction to be negatively regulated by androgens. Next, to understand whether phosphorylation plays a role, we tested the binding of AR phospho-mimetic mutants to such co-chaperones. The interaction proved to be regulated in a phosphorylation-dependent manner as well as influenced by androgen levels. To test the biological function of these interactions, we performed a transcriptional assay to determine whether the overexpression of these co-chaperones influences the transcriptional activity of AR, either by increasing or decreasing it. Additionally, since AR shuttles between the cytoplasm and the nucleus to exert its function, we looked into potential changes in AR localization in response to the overexpression of cochaperones using nuclear/cytoplasmic fractionation assay. Overall, our results suggest that phosphorylation regulates AR interaction with cochaperones, thus influencing protein-protein interactions and the balance between the folding and the degradation of AR.

The androgen receptor (AR) is a member of the steroid hormone group of nuclear receptors (NRs) and the main mediator of androgen signalling. Encoded by the AR gene on the X chromosome, AR is a transcription factor involved in biological functions like cell growth and differentiation, development and homeostasis in many tissues. When inactive, AR is in the cytosol and is associated with chaperones and cochaperones, which form the foldosome complex. Upon testosterone (T) and dihydrotestosterone (DHT) binding, AR dissociates from the heat shock proteins (HSPs) and translocates to the nucleus, where it regulates gene expression. Upon ligand withdrawal, AR is exported back to the cytoplasm where it resides in its inactive state. The foldosome complex assists AR folding in a reversible three-step process that is articulates in the formation of an early, intermediate and late complex. In the early foldosome, AR interacts with HSP40 and HSP70; the intermediate complex is formed with the association of HSP90 and its co-chaperone HOP. At this point, AR can be degraded through the ubiquitination-proteasome system (UPS) by E3 ubiquitin ligases such as STUB1 and MDM2, or can bind the late complex, composed of co-chaperones p23 and HSP90. There is a fine regulation of AR synthesis, maturation, activation and degradation, however the mechanisms controlling it still have to be fully elucidated. Additionally, AR activity is regulated by different post-translational modifications (PTMs); in particular, AR phosphorylation at different sites has been shown to influence AR stability and degradation. Considering this, the central hypothesis of my thesis project is that AR phosphorylation regulates AR interaction with different co-chaperones involved in the balance between AR folding and degradation. The aim is to characterize such protein-protein interactions (PPIs) as well as to identify a potential biological role, which is still poorly understood for some AR interactors. First, we evaluated the interaction in HEK293T cells transfected with AR by immunoprecipitation assays and we found the interaction to be negatively regulated by androgens. Next, to understand whether phosphorylation plays a role, we tested the binding of AR phospho-mimetic mutants to such co-chaperones. The interaction proved to be regulated in a phosphorylation-dependent manner as well as influenced by androgen levels. To test the biological function of these interactions, we performed a transcriptional assay to determine whether the overexpression of these co-chaperones influences the transcriptional activity of AR, either by increasing or decreasing it. Additionally, since AR shuttles between the cytoplasm and the nucleus to exert its function, we looked into potential changes in AR localization in response to the overexpression of cochaperones using nuclear/cytoplasmic fractionation assay. Overall, our results suggest that phosphorylation regulates AR interaction with cochaperones, thus influencing protein-protein interactions and the balance between the folding and the degradation of AR.

Phosphorylation of androgen receptor dictates protein-protein interactions and function

MENNA, ANGELA
2023/2024

Abstract

The androgen receptor (AR) is a member of the steroid hormone group of nuclear receptors (NRs) and the main mediator of androgen signalling. Encoded by the AR gene on the X chromosome, AR is a transcription factor involved in biological functions like cell growth and differentiation, development and homeostasis in many tissues. When inactive, AR is in the cytosol and is associated with chaperones and cochaperones, which form the foldosome complex. Upon testosterone (T) and dihydrotestosterone (DHT) binding, AR dissociates from the heat shock proteins (HSPs) and translocates to the nucleus, where it regulates gene expression. Upon ligand withdrawal, AR is exported back to the cytoplasm where it resides in its inactive state. The foldosome complex assists AR folding in a reversible three-step process that is articulates in the formation of an early, intermediate and late complex. In the early foldosome, AR interacts with HSP40 and HSP70; the intermediate complex is formed with the association of HSP90 and its co-chaperone HOP. At this point, AR can be degraded through the ubiquitination-proteasome system (UPS) by E3 ubiquitin ligases such as STUB1 and MDM2, or can bind the late complex, composed of co-chaperones p23 and HSP90. There is a fine regulation of AR synthesis, maturation, activation and degradation, however the mechanisms controlling it still have to be fully elucidated. Additionally, AR activity is regulated by different post-translational modifications (PTMs); in particular, AR phosphorylation at different sites has been shown to influence AR stability and degradation. Considering this, the central hypothesis of my thesis project is that AR phosphorylation regulates AR interaction with different co-chaperones involved in the balance between AR folding and degradation. The aim is to characterize such protein-protein interactions (PPIs) as well as to identify a potential biological role, which is still poorly understood for some AR interactors. First, we evaluated the interaction in HEK293T cells transfected with AR by immunoprecipitation assays and we found the interaction to be negatively regulated by androgens. Next, to understand whether phosphorylation plays a role, we tested the binding of AR phospho-mimetic mutants to such co-chaperones. The interaction proved to be regulated in a phosphorylation-dependent manner as well as influenced by androgen levels. To test the biological function of these interactions, we performed a transcriptional assay to determine whether the overexpression of these co-chaperones influences the transcriptional activity of AR, either by increasing or decreasing it. Additionally, since AR shuttles between the cytoplasm and the nucleus to exert its function, we looked into potential changes in AR localization in response to the overexpression of cochaperones using nuclear/cytoplasmic fractionation assay. Overall, our results suggest that phosphorylation regulates AR interaction with cochaperones, thus influencing protein-protein interactions and the balance between the folding and the degradation of AR.
2023
Phosphorylation of androgen receptor dictates protein-protein interactions and function
The androgen receptor (AR) is a member of the steroid hormone group of nuclear receptors (NRs) and the main mediator of androgen signalling. Encoded by the AR gene on the X chromosome, AR is a transcription factor involved in biological functions like cell growth and differentiation, development and homeostasis in many tissues. When inactive, AR is in the cytosol and is associated with chaperones and cochaperones, which form the foldosome complex. Upon testosterone (T) and dihydrotestosterone (DHT) binding, AR dissociates from the heat shock proteins (HSPs) and translocates to the nucleus, where it regulates gene expression. Upon ligand withdrawal, AR is exported back to the cytoplasm where it resides in its inactive state. The foldosome complex assists AR folding in a reversible three-step process that is articulates in the formation of an early, intermediate and late complex. In the early foldosome, AR interacts with HSP40 and HSP70; the intermediate complex is formed with the association of HSP90 and its co-chaperone HOP. At this point, AR can be degraded through the ubiquitination-proteasome system (UPS) by E3 ubiquitin ligases such as STUB1 and MDM2, or can bind the late complex, composed of co-chaperones p23 and HSP90. There is a fine regulation of AR synthesis, maturation, activation and degradation, however the mechanisms controlling it still have to be fully elucidated. Additionally, AR activity is regulated by different post-translational modifications (PTMs); in particular, AR phosphorylation at different sites has been shown to influence AR stability and degradation. Considering this, the central hypothesis of my thesis project is that AR phosphorylation regulates AR interaction with different co-chaperones involved in the balance between AR folding and degradation. The aim is to characterize such protein-protein interactions (PPIs) as well as to identify a potential biological role, which is still poorly understood for some AR interactors. First, we evaluated the interaction in HEK293T cells transfected with AR by immunoprecipitation assays and we found the interaction to be negatively regulated by androgens. Next, to understand whether phosphorylation plays a role, we tested the binding of AR phospho-mimetic mutants to such co-chaperones. The interaction proved to be regulated in a phosphorylation-dependent manner as well as influenced by androgen levels. To test the biological function of these interactions, we performed a transcriptional assay to determine whether the overexpression of these co-chaperones influences the transcriptional activity of AR, either by increasing or decreasing it. Additionally, since AR shuttles between the cytoplasm and the nucleus to exert its function, we looked into potential changes in AR localization in response to the overexpression of cochaperones using nuclear/cytoplasmic fractionation assay. Overall, our results suggest that phosphorylation regulates AR interaction with cochaperones, thus influencing protein-protein interactions and the balance between the folding and the degradation of AR.
Androgen receptor
Phosphorylation
Co-chaperones
PTMs
File in questo prodotto:
File Dimensione Formato  
Menna_Angela.pdf

accesso riservato

Dimensione 2.61 MB
Formato Adobe PDF
2.61 MB Adobe PDF

The text of this website © Università degli studi di Padova. Full Text are published under a non-exclusive license. Metadata are under a CC0 License

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12608/80671