RESUMEN
LARP1 is a key repressor of TOP mRNA translation. It binds the m7Gppp cap moiety and the adjacent 5'TOP motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 controls TOP mRNA translation via LARP1, but the details of the mechanism are unclear. Herein we elucidate the mechanism by which mTORC1 controls LARP1's translation repression activity. We demonstrate that mTORC1 phosphorylates LARP1 in vitro and in vivo, activities that are efficiently inhibited by rapamycin and torin1. We uncover 26 rapamycin-sensitive phospho-serine and -threonine residues on LARP1 that are distributed in 7 clusters. Our data show that phosphorylation of a cluster of residues located proximally to the m7Gppp cap-binding DM15 region is particularly sensitive to rapamycin and regulates both the RNA-binding and the translation inhibitory activities of LARP1. Our results unravel a new model of translation control in which the La module (LaMod) and DM15 region of LARP1, both of which can directly interact with TOP mRNA, are differentially regulated: the LaMod remains constitutively bound to PABP (irrespective of the activation status of mTORC1), while the C-terminal DM15 'pendular hook' engages the TOP mRNA 5'-end to repress translation, but only in conditions of mTORC1 inhibition.
Asunto(s)
Autoantígenos/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Biosíntesis de Proteínas , Ribonucleoproteínas/metabolismo , Secuencias de Aminoácidos , Autoantígenos/química , Células HEK293 , Humanos , Naftiridinas/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica , Ribonucleoproteínas/química , Serina/metabolismo , Sirolimus/farmacología , Treonina/metabolismo , Tirosina/metabolismo , Antígeno SS-BRESUMEN
Periodontitis (PDT) and type 2 diabetes (T2D) have demonstrated a bidirectional relationship and imbalanced oxidative stress linked to mitochondrial dysfunction. Therefore, we investigated mitochondrial and redox (de)regulation in peripheral blood mononuclear cells (PBMCs) in comorbid T2D-PDT, compared to PDT, T2D patients, and control individuals. PBMCs were analyzed for mitochondrial respiration, reactive oxygen species, antioxidant proteins, and expression of Nrf2-target genes. PDT and T2D-PDT patients exhibited altered periodontal clinical markers, while T2D and T2D-PDT patients displayed increased blood HbA1c. Decreased oxygen consumption and ATP production were observed in the PDT patient's PBMCs. PDT and T2D-PDT PBMCs also evidenced increased H2O2 levels and reduced catalase levels (also detected in T2D patients), whereas a compromised glutathione cycle was observed in T2D-PDT patients. PBMCs from both T2D or T2D-PDT patients showed increased Nrf2 protein levels, enhanced GCL activity and GCL-catalytic subunit protein levels, and maintained GCLc, GST, and HO-1 mRNA levels. In contrast, the expressions of Nrf2-target genes were significantly diminished in the PDT patient's PBMCs. Decreased SOD1 and GST mRNA levels were also observed in CD3+CD8+-lymphocytes derived from PDT and T2D-PDT patients. In conclusion, PBMCs from T2D-PDT patients showed major redox changes, while mononuclear cells from PDT patients showed mitochondrial deregulation and reduced expression of Nrf2-target genes.