Background: mTORC1 plays an important role in the regulation of TOP mRNA translation. Results: LA... more Background: mTORC1 plays an important role in the regulation of TOP mRNA translation. Results: LARP1 is a target of mTORC1 that associates with TOP mRNAs via their 5ЈTOP motif to repress their translation. Conclusion: LARP1 represses TOP mRNA translation downstream of mTORC1. Significance: We elucidate an important novel signaling pathway downstream of mTORC1 that controls the production of ribosomes and translation factors in eukaryotic cells. The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factorbinding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1.
Three-dimensional structural model of epoxide hydrolase (PchEHA) from Phanerochaete chrysosporium... more Three-dimensional structural model of epoxide hydrolase (PchEHA) from Phanerochaete chrysosporium was constructed based on X-ray structure of Agrobacterium radiobacter AD1 sEH using SWISS-MODEL server. Conserved residues constituting the active site cavity were identified, of which the functional roles of 14 residues were determined by site-directed mutagenesis. In catalytic triad, Asp105 and His308 play a leading role in alkylation and hydrolysis steps, respectively. Distance between Asp105 and epoxide ring of substrate may determine the regiospecificity in the substrate docking model. Asp277 located at the entrance of substrate tunnel is concerned with catalysis but not essential. D307E had the highest activity and lower enantioselectivity among 14 mutants, suggesting Asp307 may be involved in choice of substrate configuration. Y159F and Y241F almost exhibited no activity, indicating that they are essential to bind substrate and facilitate opening of epoxide ring. Besides, His35-Gly36-Asn37-Pro38, Trp106 and Trp309 surrounding Asp105, may coordinate the integration of active site cavity and influence substrate binding. Especially, W106I reversed the enantioselectivity, perhaps due to more deteriorative impact on the preferred (R)-styrene oxide. Gly65 and Gly67 occurring at β-turns and Gly36 are vital in holding protein conformation. Conclusively, single conserved residue around the active sites has an important impact on catalytic properties.
Like most psychiatric illnesses, major depressive disorder (MDD) is highly heterogenous in regard... more Like most psychiatric illnesses, major depressive disorder (MDD) is highly heterogenous in regard to symptom severity, and response to frontline pharmacological treatments such as serotonin (5-HT) reuptake inhibitors (SSRIs). Although the discovery of SSRIs provided indirect evidence on the involvement of 5-HT in the pathophysiology of depressive disorders, it also became apparent that there were individual differences in the ability of these drugs to relive the depressive symptoms. Some of the factors that contribute to variability in treatment response and disease course are inflammatory mediators know as cytokines. In this book chapter, we review a novel molecular mechanism in the pathophysiology of depressive disorders, mRNA translation, or protein synthesis. We discuss evidence that downregulated signaling pathways impinging on the mRNA translation process, engender behavioral alterations, and 5-HT synaptic alterations, due to dysregulation in inflammatory signaling. Specifical...
Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with the liver-specific micr... more Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with the liver-specific microRNA, miR-122. miR-122 binds to two sites in the 5 untranslated region (UTR) and this interaction promotes HCV RNA accumulation, although the precise role of miR-122 in the HCV life cycle remains unclear. Using biophysical analyses and Selective 2 Hydroxyl Acylation analyzed by Primer Extension (SHAPE) we investigated miR-122 interactions with the 5 UTR. Our data suggests that miR-122 binding results in alteration of nucleotides 1-117 to suppress an alternative secondary structure and promote functional internal ribosomal entry site (IRES) formation. Furthermore, we demonstrate that two hAgo2:miR-122 complexes are able to bind to the HCV 5 terminus simultaneously and SHAPE analyses revealed further alterations to the structure of the 5 UTR to accommodate these complexes. Finally, we present a computational model of the hAgo2:miR-122:HCV RNA complex at the 5 terminus of the viral genome as well as hAgo2:miR-122 interactions with the IRES-40S complex that suggest hAgo2 is likely to form additional interactions with SLII which may further stabilize the HCV IRES. Taken together, our results support a model whereby hAgo2:miR-122 complexes alter the structure of the viral 5 terminus and promote formation of the HCV IRES.
MicroRNAs (miRNAs) exert a broad influence over gene expression by directing effector activities ... more MicroRNAs (miRNAs) exert a broad influence over gene expression by directing effector activities that impinge on translation and stability of mRNAs. We recently discovered that the cap-binding protein 4EHP is a key component of the mammalian miRNA-Induced Silencing Complex (miRISC), which mediates gene silencing. However, little is known about the mRNA repertoire that is controlled by the 4EHP/miRNA mechanism or its biological importance. Here, using ribosome profiling, we identify a subset of mRNAs that are translationally controlled by 4EHP. We show that the Dusp6 mRNA, which encodes an ERK1/2 phosphatase, is translationally repressed by 4EHP and a specific miRNA, miR-145. This promotes ERK1/2 phosphorylation, resulting in augmented cell growth and reduced apoptosis. Our findings thus empirically define the integral role of translational repression in miRNA-induced gene silencing and reveal a critical function for this process in the control of the ERK signaling cascade in mammali...
Translation of messenger RNA (mRNA) into protein (protein synthesis) is a highly regulated proces... more Translation of messenger RNA (mRNA) into protein (protein synthesis) is a highly regulated process that controls gene expression. Various signaling pathways, including the mammalian target of rapamycin (mTOR), control mRNA translation at the initiation step. mTOR is part of a multi-subunit complex that regulates mRNA translation initiation by phosphorylating and inactivating the eukaryotic initiation factor 4E binding proteins (4E-BPs). 4E-BPs are a central mechanism in the control of cap-dependent translation in the brain. This chapter reviews the involvement of the 4E-BPs, particularly 4E-BP2, in brain development and synaptic transmission. Furthermore, it discusses the involvement of 4E-BP2 in autistic-like alterations, learning and memory, circadian rhythm regulation, and its roles in the pathophysiology and treatment of psychiatric (depressive disorders, schizophrenia) and neurodegenerative disorders (Parkinson’s).
Translation of mRNA into protein has a fundamental role in neurodevelopment, plasticity, and memo... more Translation of mRNA into protein has a fundamental role in neurodevelopment, plasticity, and memory formation; however, its contribution in the pathophysiology of depressive disorders is not fully understood. We investigated the involvement of MNK1/2 (MAPK-interacting serine/threonine-protein kinase 1 and 2) and their target, eIF4E (eukaryotic initiation factor 4E), in depression-like behavior in mice. Mice carrying a mutation in eIF4E for the MNK1/2 phosphorylation site (Ser209Ala, Eif4e ki/ki), the Mnk1/2 double knockout mice (Mnk1/2), or mice treated with the MNK1/2 inhibitor, cercosporamide, displayed anxiety- and depression-like behaviors, impaired serotonin-induced excitatory synaptic activity in the prefrontal cortex, and diminished firing of the dorsal raphe neurons. In Eif4e ki/ki mice, brain IκBα, was decreased, while the NF-κB target, TNFα was elevated. TNFα inhibition in Eif4e ki/ki mice rescued, whereas TNFα administration to wild-type mice mimicked the depression-like ...
Highlights d Methylated peptides bind the UBR-box domain with high affinity d Water molecules all... more Highlights d Methylated peptides bind the UBR-box domain with high affinity d Water molecules allow binding of diverse N-terminal residues within a rigid scaffold d Hydrophobic residues are favored in the second position of the N-degron d V122L mutant in Johanson-Blizzard syndrome changes N-degron specificity
Background: mTORC1 plays an important role in the regulation of TOP mRNA translation. Results: LA... more Background: mTORC1 plays an important role in the regulation of TOP mRNA translation. Results: LARP1 is a target of mTORC1 that associates with TOP mRNAs via their 5ЈTOP motif to repress their translation. Conclusion: LARP1 represses TOP mRNA translation downstream of mTORC1. Significance: We elucidate an important novel signaling pathway downstream of mTORC1 that controls the production of ribosomes and translation factors in eukaryotic cells. The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factorbinding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1.
Three-dimensional structural model of epoxide hydrolase (PchEHA) from Phanerochaete chrysosporium... more Three-dimensional structural model of epoxide hydrolase (PchEHA) from Phanerochaete chrysosporium was constructed based on X-ray structure of Agrobacterium radiobacter AD1 sEH using SWISS-MODEL server. Conserved residues constituting the active site cavity were identified, of which the functional roles of 14 residues were determined by site-directed mutagenesis. In catalytic triad, Asp105 and His308 play a leading role in alkylation and hydrolysis steps, respectively. Distance between Asp105 and epoxide ring of substrate may determine the regiospecificity in the substrate docking model. Asp277 located at the entrance of substrate tunnel is concerned with catalysis but not essential. D307E had the highest activity and lower enantioselectivity among 14 mutants, suggesting Asp307 may be involved in choice of substrate configuration. Y159F and Y241F almost exhibited no activity, indicating that they are essential to bind substrate and facilitate opening of epoxide ring. Besides, His35-Gly36-Asn37-Pro38, Trp106 and Trp309 surrounding Asp105, may coordinate the integration of active site cavity and influence substrate binding. Especially, W106I reversed the enantioselectivity, perhaps due to more deteriorative impact on the preferred (R)-styrene oxide. Gly65 and Gly67 occurring at β-turns and Gly36 are vital in holding protein conformation. Conclusively, single conserved residue around the active sites has an important impact on catalytic properties.
Like most psychiatric illnesses, major depressive disorder (MDD) is highly heterogenous in regard... more Like most psychiatric illnesses, major depressive disorder (MDD) is highly heterogenous in regard to symptom severity, and response to frontline pharmacological treatments such as serotonin (5-HT) reuptake inhibitors (SSRIs). Although the discovery of SSRIs provided indirect evidence on the involvement of 5-HT in the pathophysiology of depressive disorders, it also became apparent that there were individual differences in the ability of these drugs to relive the depressive symptoms. Some of the factors that contribute to variability in treatment response and disease course are inflammatory mediators know as cytokines. In this book chapter, we review a novel molecular mechanism in the pathophysiology of depressive disorders, mRNA translation, or protein synthesis. We discuss evidence that downregulated signaling pathways impinging on the mRNA translation process, engender behavioral alterations, and 5-HT synaptic alterations, due to dysregulation in inflammatory signaling. Specifical...
Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with the liver-specific micr... more Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with the liver-specific microRNA, miR-122. miR-122 binds to two sites in the 5 untranslated region (UTR) and this interaction promotes HCV RNA accumulation, although the precise role of miR-122 in the HCV life cycle remains unclear. Using biophysical analyses and Selective 2 Hydroxyl Acylation analyzed by Primer Extension (SHAPE) we investigated miR-122 interactions with the 5 UTR. Our data suggests that miR-122 binding results in alteration of nucleotides 1-117 to suppress an alternative secondary structure and promote functional internal ribosomal entry site (IRES) formation. Furthermore, we demonstrate that two hAgo2:miR-122 complexes are able to bind to the HCV 5 terminus simultaneously and SHAPE analyses revealed further alterations to the structure of the 5 UTR to accommodate these complexes. Finally, we present a computational model of the hAgo2:miR-122:HCV RNA complex at the 5 terminus of the viral genome as well as hAgo2:miR-122 interactions with the IRES-40S complex that suggest hAgo2 is likely to form additional interactions with SLII which may further stabilize the HCV IRES. Taken together, our results support a model whereby hAgo2:miR-122 complexes alter the structure of the viral 5 terminus and promote formation of the HCV IRES.
MicroRNAs (miRNAs) exert a broad influence over gene expression by directing effector activities ... more MicroRNAs (miRNAs) exert a broad influence over gene expression by directing effector activities that impinge on translation and stability of mRNAs. We recently discovered that the cap-binding protein 4EHP is a key component of the mammalian miRNA-Induced Silencing Complex (miRISC), which mediates gene silencing. However, little is known about the mRNA repertoire that is controlled by the 4EHP/miRNA mechanism or its biological importance. Here, using ribosome profiling, we identify a subset of mRNAs that are translationally controlled by 4EHP. We show that the Dusp6 mRNA, which encodes an ERK1/2 phosphatase, is translationally repressed by 4EHP and a specific miRNA, miR-145. This promotes ERK1/2 phosphorylation, resulting in augmented cell growth and reduced apoptosis. Our findings thus empirically define the integral role of translational repression in miRNA-induced gene silencing and reveal a critical function for this process in the control of the ERK signaling cascade in mammali...
Translation of messenger RNA (mRNA) into protein (protein synthesis) is a highly regulated proces... more Translation of messenger RNA (mRNA) into protein (protein synthesis) is a highly regulated process that controls gene expression. Various signaling pathways, including the mammalian target of rapamycin (mTOR), control mRNA translation at the initiation step. mTOR is part of a multi-subunit complex that regulates mRNA translation initiation by phosphorylating and inactivating the eukaryotic initiation factor 4E binding proteins (4E-BPs). 4E-BPs are a central mechanism in the control of cap-dependent translation in the brain. This chapter reviews the involvement of the 4E-BPs, particularly 4E-BP2, in brain development and synaptic transmission. Furthermore, it discusses the involvement of 4E-BP2 in autistic-like alterations, learning and memory, circadian rhythm regulation, and its roles in the pathophysiology and treatment of psychiatric (depressive disorders, schizophrenia) and neurodegenerative disorders (Parkinson’s).
Translation of mRNA into protein has a fundamental role in neurodevelopment, plasticity, and memo... more Translation of mRNA into protein has a fundamental role in neurodevelopment, plasticity, and memory formation; however, its contribution in the pathophysiology of depressive disorders is not fully understood. We investigated the involvement of MNK1/2 (MAPK-interacting serine/threonine-protein kinase 1 and 2) and their target, eIF4E (eukaryotic initiation factor 4E), in depression-like behavior in mice. Mice carrying a mutation in eIF4E for the MNK1/2 phosphorylation site (Ser209Ala, Eif4e ki/ki), the Mnk1/2 double knockout mice (Mnk1/2), or mice treated with the MNK1/2 inhibitor, cercosporamide, displayed anxiety- and depression-like behaviors, impaired serotonin-induced excitatory synaptic activity in the prefrontal cortex, and diminished firing of the dorsal raphe neurons. In Eif4e ki/ki mice, brain IκBα, was decreased, while the NF-κB target, TNFα was elevated. TNFα inhibition in Eif4e ki/ki mice rescued, whereas TNFα administration to wild-type mice mimicked the depression-like ...
Highlights d Methylated peptides bind the UBR-box domain with high affinity d Water molecules all... more Highlights d Methylated peptides bind the UBR-box domain with high affinity d Water molecules allow binding of diverse N-terminal residues within a rigid scaffold d Hydrophobic residues are favored in the second position of the N-degron d V122L mutant in Johanson-Blizzard syndrome changes N-degron specificity
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Papers by Edna Camacho