RESUMO
Androgen receptor (AR) acts as a ligand-regulated transcription factor that conveys the message of both natural and synthetic androgens directly to the level of gene programs. Reporter gene assays provide a convenient and quantitative way to measure the transcriptional activity of AR and the functionality of its binding sites (AREs) in DNA. Many reporter genes and different transfection methods can be used for this purpose. In this chapter, we describe the use of firefly luciferase gene-based reporters and transfection protocols for the measurement of AR activity in heterologous COS-1 cells cotransfected with an AR expression vector and in VCaP prostate cancer cells expressing endogenous AR. We also discuss the suitability of different reporter constructs and transfection methods for different cell types and how reporter gene assays can be employed to complement chromatin immunoprecipitation assays.
Assuntos
Genes Reporter , Receptores Androgênicos/metabolismo , Transcrição Gênica , Animais , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Técnicas Genéticas , Vetores Genéticos/genética , Humanos , Masculino , TransfecçãoRESUMO
Androgen receptor (AR) is a ligand-controlled transcription factor that is deregulated and therefore targeted in prostate cancer. In addition to androgens, AR is regulated by post-translational modifications (PTMs). SUMOylation, conjugation of small ubiquitin-related modifier (SUMO) protein 1, 2, or 3, is a bulky PTM regulating several important physiological processes. We have shown that AR is modified by SUMO-1 at two conserved lysine residues in its N-terminal domain. This agonist-enhanced modification represses the transcriptional activity of the receptor in a reversible and target gene-selective fashion. Acceptor sites for SUMOs are also found in several other nuclear receptors. Since the cellular steady-state level of SUMO modifications of most substrates, including AR, is very low, transfection- and SUMO overexpression-based protocols are often needed to render the modifications clearly detectable. This chapter describes protocols for analyzing AR SUMOylation in cultured cells by immunoblotting, gel mobility shift assays, and immunoprecipitation. These methodologies are generally applicable for determining whether a particular protein is SUMOylated and for identifying the lysine residue(s) modified.
Assuntos
Receptores Androgênicos/metabolismo , Sumoilação , Animais , Western Blotting , Células COS , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoprecipitação , Tubulina (Proteína)/metabolismoRESUMO
Small ubiquitin-related modifiers (SUMOs) are important regulator proteins. Caenorhabditis elegans contains a single SUMO ortholog, SMO-1, necessary for the reproduction of C. elegans. In this study, we constructed transgenic C. elegans strains expressing human SUMO-1 under the control of pan-neuronal (aex-3) or pan-muscular (myo-4) promoter and SUMO-2 under the control of myo-4 promoter. Interestingly, muscular overexpression of SUMO-1 or -2 resulted in morphological changes of the posterior part of the nematode. Movement, reproduction and aging of C. elegans were perturbed by the overexpression of SUMO-1 or -2. Genome-wide expression analyses revealed that several genes encoding components of SUMOylation pathway and ubiquitin-proteasome system were upregulated in SUMO-overexpressing nematodes. Since muscular overexpression of SMO-1 also brought up reproductive and mobility perturbations, our results imply that the phenotypes were largely due to an excess of SUMO, suggesting that a tight control of SUMO levels is important for the normal development of multicellular organisms.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Proteína SUMO-1/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Proteína SUMO-1/química , Proteína SUMO-1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Ubiquitina/metabolismo , UbiquitinaçãoAssuntos
Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Movimento Celular , Lisina/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Inibidoras de STAT Ativados/fisiologia , Estrutura Terciária de Proteína , Sumoilação , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1alpha) is a key coordinator of gene programs in metabolism and energy homeostasis in mammals. It is highly responsive to changes in the cellular environment and physiological status of mammals and regulated by post-translational modifications: acetylation, phosphorylation, and methylation. Here, we show that PGC-1alpha is covalently modified by small ubiquitin-like modifier (SUMO) 1 protein, an important regulator of signaling and transcription. Conserved lysine residue 183 located in the activation domain of PGC-1alpha was identified as the major site of SUMO conjugation. Interestingly, the same Lys residue is also a target for acetylation. Therefore, the E185A mutation disrupting the SUMOylation consensus sequence was utilized to show that SUMOylation plays a role in the regulation of PGC-1alpha function. Our results show that SUMOylation does not have an apparent effect on the subcellular localization or the stability of PGC-1alpha, but it attenuates the transcriptional activity of the coactivator, probably by enhancing the interaction of PGC-1alpha with corepressor RIP140. Mutation that abolished the SUMOylation augments the activity of PGC-1alpha also in the context of PPARgamma-dependent transcription. Thus, our findings showing that reversible SUMOylation can adjust the activity of PGC-1alpha add a novel layer to the regulation of the coactivator.
Assuntos
Proteínas de Choque Térmico/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteína SUMO-1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Mutação de Sentido Incorreto , PPAR gama/genética , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Estrutura Terciária de Proteína/fisiologia , Proteína SUMO-1/genética , Fatores de Transcrição/genéticaRESUMO
The interactions and functions of protein inhibitors of activated STAT (PIAS) proteins are not restricted to the signal transducers and activators of transcription (STATs), but PIAS1, -2, -3 and -4 interact with and regulate a variety of distinct proteins, especially transcription factors. Although the majority of PIAS-interacting proteins are prone to modification by small ubiquitin-related modifier (SUMO) proteins and the PIAS proteins have the capacity to promote the modification as RING-type SUMO ligases, they do not function solely as SUMO E3 ligases. Instead, their effects are often independent of their Siz/PIAS (SP)-RING finger, but dependent on their capability to noncovalently interact with SUMOs or DNA through their SUMO-interacting motif and scaffold attachment factor-A/B, acinus and PIAS domain, respectively. Here, we present an overview of the cellular regulation by PIAS proteins and propose that many of their functions are due to their capability to mediate and facilitate SUMO-linked protein assemblies.
Assuntos
Proteínas Inibidoras de STAT Ativados/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , DNA/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Proteínas/metabolismoRESUMO
The acceptor sites for small ubiquitin-like modifier (SUMO) are conserved in the N-terminal domains of several nuclear receptors. Here, we show that androgens induce rapid and dynamic conjugation of SUMO-1 to androgen receptor (AR). Nuclear import of AR is not sufficient for SUMOylation, because constitutively nuclear apo-ARs or antagonist-bound ARs are only very weakly modified by SUMO-1 in comparison with agonist-bound ARs. Of the SUMO-specific proteases (SENP)-1, -2, -3, -5, and -6, only SENP1 and SENP2 are efficient in cleaving AR-SUMO-1 conjugates in intact cells and in vitro. Both SENP1 and -2 are nuclear and found at sites proximal to AR. Their expression promotes AR-dependent transcription, but in a promoter-selective fashion. SENP1 and -2 stimulated the activity of holo-AR on compound androgen response element-containing promoters. The effects of SENP1 and -2 on AR-dependent transcription were dependent on catalytic activity and required intact SUMO acceptor sites in AR, indicating that their coactivating effects are mainly due to their direct isopeptidase activity on holo-AR. In prostate cancer cells, ectopic expression of SENP1, but not that of SENP2, increased the transcription activity of endogenous AR. Silencing of SENP1 attenuated the expression of several AR target genes and blunted androgen-stimulated growth of LNCaP cells. Our results indicate that SENP1 reverses the ligand-induced SUMOylation of AR and helps fine tune the cellular responses to androgens in a target promoter-selective manner.
Assuntos
Androgênios/farmacologia , Endopeptidases/fisiologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Proteína SUMO-1/metabolismo , Acetilação/efeitos dos fármacos , Animais , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Cisteína Endopeptidases , Regulação Neoplásica da Expressão Gênica , Humanos , Lisina/metabolismo , Masculino , Fosforilação/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico , Receptores Androgênicos/química , Receptores Androgênicos/fisiologia , Células Tumorais CultivadasRESUMO
RIP140/NRIP1 (receptor-interacting protein 140) functions as a corepressor of nuclear receptors. It plays an important role in the transcriptional control of energy metabolism and female fertility. RIP140 contains four distinct repression domains (RD1-RD4), and the repressive activity of RIP140 involves complex mechanisms. The function of both RD1 and RD2 is linked to recruitment of histone deacetylases and C-terminal binding protein, respectively, but the mechanism of repression for RD3 and RD4 has remained elusive. Because covalent modification by small ubiquitin-like modifiers (SUMO-1, -2, and -3; SUMOylation) is often associated with transcriptional repression, we studied whether SUMOylation is involved in the repressive activity of RIP140. We show that two conserved lysines, Lys(756) and Lys(1154), located in RD3 and RD4, respectively, are subject to reversible SUMOylation, with SUMO-1 being more efficiently conjugated than SUMO-2. Interestingly, mutations of the RIP140 SUMOylation sites compromised the transcription repressor function of RIP140 and blunted its capacity to repress estrogen receptor alpha-dependent transcription. Conjugation of SUMO-1 also influenced the subnuclear distribution pattern of RIP140. In sum, our demonstration that the function of RIP140 repression domains 3 and 4 can be modulated by reversible SUMO modification thus adds a novel level to the regulation of RIP140 activity, which may have ramifications in the control of gene networks exerted by RIP140.