RESUMO
The homeodomain protein TGIF functions as a negative regulator of multiple classes of transcription factors. Here we report on the characterization of TGIF as an essential component of the tumor necrosis factor alpha (TNF-alpha) cytotoxic program. This proapoptotic role of TGIF does not appear to rely on transcriptional modulation but instead is executed in conjunction with Itch/AIP4, an E3 ubiquitin ligase operating in TNF-alpha-induced apoptosis through its ability to target the caspase antagonist cFlip(L) for degradation. Notably, we found that activation of TNF-alpha signaling induced the association of TGIF with Itch/AIP4, resulting in increased accessibility of cFlip(L) for association and ubiquitination by Itch/AIP4. Moreover, we show that Itch/AIP4 can also stabilize the TGIF protein in response to TNF-alpha by triggering its monoubiquitination at lysine 259, thereby revealing the existence of a functional network that can evolve into a positive feedback loop for ensuring effective execution of the TNF-alpha apoptotic signaling.
Assuntos
Morte Celular/efeitos dos fármacos , Proteínas de Homeodomínio/farmacologia , Proteínas Repressoras/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Lisina/metabolismo , Camundongos , Camundongos Knockout , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The homeodomain protein TGIF functions as a negative modulator for multiple classes of transcription factors. Loss of function mutations in a single copy of TGIF result in holoprosencephaly, a developmental anomaly leading to severe forebrain and craniofacial malformations. However, the mechanisms by which these mutations disrupt the functions of TGIF remain to be elucidated. Here we show that a holoprosencephaly mutation (P63R) interferes with the ability of TGIF to act as a corepressor for c-Jun and Smad2, suggesting that this holoprosencephaly mutation may lead to a general defect in the TGIF protein. In fact, we observed that the P63R mutation affects folding of the TGIF protein, resulting in the disruption of the diffuse nuclear staining pattern characteristic of wild-type (WT) TGIF and the accumulation of TGIF in nuclear aggregates. We also show that the mutant TGIF.P63R is degraded more rapidly when compared with WT TGIF and that this degradation occurs through the ubiquitin-proteasome pathway. Furthermore, we observed that TGIF.P63R homodimerizes with WT TGIF to sequester it into nuclear aggregates and to enhance its ubiquitin-dependent degradation. These results reveal an important mechanism for the degradation of TGIF through the ubiquitin-proteasome pathway, whose deregulation might contribute to the development of human holoprosencephaly.
Assuntos
Holoprosencefalia/metabolismo , Proteínas de Homeodomínio/genética , Mutação , Proteínas Repressoras/genética , Alelos , Substituição de Aminoácidos , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Dimerização , Imunofluorescência , Holoprosencefalia/genética , Holoprosencefalia/patologia , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Modelos Biológicos , Proteínas Mutantes/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Transcrição Gênica , Ubiquitina/metabolismoRESUMO
OBJECTIVE: Aldosterone binds the mineralocorticoid receptor (MR) and is involved in the regulation of ionic transport, mainly sodium retention. MR is encoded by one single gene transcribed into various messengers that are thought to be translated into a unique 107 kDa protein. The aim of this study was to identify and characterize translation initiation variants of the human MR protein. METHODS: For this purpose we analyzed the extreme N-terminal fragment of various MR species, and confronted the predicted pattern of expression with in vitro translation results. Transactivation functions of the identified MR forms obtained by targeted mutagenesis were evaluated by transient transfection assays with different promoter reporter genes. RESULTS: Two major forms of human MR (hMR), named MRA and MRB, generated by alternative initiation of translation from start methionine 1 and 15 respectively, were predicted. Their expected expressions were confirmed by in vitro transcription/translation studies. Interestingly, each form of MR displayed reduced transactivation capacities with wild type (WT)=A+B>MRA>MRB suggesting that the extreme N-terminal fragment of MR has an effect on the transcriptional properties of the receptor. CONCLUSIONS: Altogether these data indicate that MR is expressed, through alternative translation initiation, as distinct protein variants which possess different functional properties. These MR forms could tightly dictate the modulation of aldosterone responsiveness in various tissues or in pathophysiological situations.
