RESUMO
As core components of the microRNA-induced silencing complex (miRISC), Argonaute (AGO) proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390) on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.
Assuntos
Proteínas Argonautas/metabolismo , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , MicroRNAs/genética , Proteínas Argonautas/genética , Autoantígenos/metabolismo , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/genética , MicroRNAs/metabolismo , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Since the application of molecular biology in cancer biology, lung cancer research has classically focused on molecular drivers of disease. One such pathway, the hypoxic response pathway, is activated by reduced local oxygen concentrations at the tumor site. Hypoxia-driven gene and protein changes enhance epithelial-to-mesenchymal transition, remodel the extracellular matrix, drive drug resistance, support cancer stem cells and aid evasion from immune cells. However, it is not the tumor cells alone which drive this response to hypoxia, but rather their interaction with a complex milieu of supporting cells. This review will focus on recent advances in our understanding of how these cells contribute to the tumor response to hypoxia in non-small-cell lung cancer.
Assuntos
Carcinogênese , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Pequenas/genética , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/classificação , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Pequenas/classificação , Carcinoma de Células Pequenas/patologia , Hipóxia Celular/genética , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/genética , Matriz Extracelular/patologia , Humanos , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/patologia , Oncogenes/genética , Microambiente Tumoral/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
There are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O(2) tension. In high O(2) tension (normoxia) the PHDs hydroxylate two conserved proline residues on HIF-1α, which leads to binding of the von Hippel-Lindau (VHL) tumour suppressor, the recognition component of a ubiquitin-ligase complex, initiating HIF-1α ubiquitylation and degradation. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIF-1α or as a multiprotein complex. Here we show that the tumour suppressor protein LIMD1 (LIM domain-containing protein) acts as a molecular scaffold, simultaneously binding the PHDs and VHL, thereby assembling a PHD-LIMD1-VHL protein complex and creating an enzymatic niche that enables efficient degradation of HIF-1α. Depletion of endogenous LIMD1 increases HIF-1α levels and transcriptional activity in both normoxia and hypoxia. Conversely, LIMD1 expression downregulates HIF-1 transcriptional activity in a manner depending on PHD and 26S proteasome activities. LIMD1 family member proteins Ajuba and WTIP also bind to VHL and PHDs 1 and 3, indicating that these LIM domain-containing proteins represent a previously unrecognized group of hypoxic regulators.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia , Immunoblotting , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Modelos Biológicos , Poliubiquitina/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Interferência de RNA , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinação , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
Abstract MicroRNAs (miRNAs) comprise a group of small non-coding RNA -21 nucleotides in length. They act as post-transcriptional regulators of gene expression by forming base pairing interactions with target messenger RNA (mRNA). At least 1000 miRNAs are predicted to be expressed in humans and are encoded for in the genome of almost all organisms. Functional studies indicate that every cellular process studied thus far is regulated at some level by miRNAs. Given this expansive role, it is not surprising that disruption of this crucial pathway underlies the initiation of, or in the least, contributes to the development and progression of numerous human diseases and physiological disorders. This review will focus on the latest developments in uncovering the mechanism(s) of miRNA-mediated silencing with specific reference to the function of terminal effector proteins, how translation of target mRNA is inhibited and whether we are moving towards understanding this fundamental gene silencing paradigm.
