Browsing by Subject "Protein Biosynthesis"

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    Arc/Arg3.1 translation is controlled by convergent N-methyl-D-aspartate and Gs-coupled receptor signaling pathways.
    (The Journal of biological chemistry, 2008-01) Bloomer, Wendy AC; VanDongen, Hendrika MA; VanDongen, Antonius MJ
    Arc/Arg3.1 is an immediate early gene whose expression is necessary for the late-phase of long-term potentiation (LTP) and memory consolidation. Whereas pathways regulating Arc transcription have been extensively investigated, less is known about the role of post-transcriptional mechanisms in Arc expression. Fluorescence microscopy experiments in cultured hippocampal neurons revealed that Arc protein level was dramatically increased by activation of the cAMP-dependent protein kinase (PKA) pathway, which is implicated in long-term memory. A PKA-dependent increase in Arc protein level was observed after pharmacological or synaptic activation of N-methyl-D-aspartate (NMDA) receptors, which play a critical role in both LTP induction and learning. Arc protein was also up-regulated by activation of PKA through G(s)-coupled dopamine and beta-adrenergic receptors, which regulate the late-phase of LTP and memory. When agonists for the NMDA and G(s)-coupled receptors were co-applied, they had an additive effect on Arc protein expression. Interestingly, G(s)-coupled receptor stimulation was ineffective in the presence of an NMDA receptor antagonist, suggesting calcium influx through the NMDA receptor plays a gating role in this pathway. Stimulation of the cAMP/PKA pathway did not affect Arc mRNA level or protein stability, identifying translational efficacy as the main determinant of Arc protein expression level. It is concluded that efficient Arc translation requires NMDA receptor activity, whereas a further enhancement can be achieved with activation of G(s)-coupled receptors. These experiments have, therefore, revealed remarkable similarities in the signaling pathways that control Arc expression and those that regulate LTP, learning, and memory.
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    Complementary Roles of GADD34- and CReP-Containing Eukaryotic Initiation Factor 2α Phosphatases during the Unfolded Protein Response.
    (Molecular and cellular biology, 2016-07) Reid, David W; Tay, Angeline SL; Sundaram, Jeyapriya R; Lee, Irene CJ; Chen, Qiang; George, Simi E; Nicchitta, Christopher V; Shenolikar, Shirish
    Phosphorylation of eukaryotic initiation factor 2α (eIF2α) controls transcriptome-wide changes in mRNA translation in stressed cells. While phosphorylated eIF2α (P-eIF2α) attenuates global protein synthesis, mRNAs encoding stress proteins are more efficiently translated. Two eIF2α phosphatases, containing GADD34 and CReP, catalyze P-eIF2α dephosphorylation. The current view of GADD34, whose transcription is stress induced, is that it functions in a feedback loop to resolve cell stress. In contrast, CReP, which is constitutively expressed, controls basal P-eIF2α levels in unstressed cells. Our studies show that GADD34 drives substantial changes in mRNA translation in unstressed cells, particularly targeting the secretome. Following activation of the unfolded protein response (UPR), rapid translation of GADD34 mRNA occurs and GADD34 is essential for UPR progression. In the absence of GADD34, eIF2α phosphorylation is persistently enhanced and the UPR translational program is significantly attenuated. This "stalled" UPR is relieved by the subsequent activation of compensatory mechanisms that include AKT-mediated suppression of PKR-like kinase (PERK) and increased expression of CReP mRNA, partially restoring protein synthesis. Our studies highlight the coordinate regulation of UPR by the GADD34- and CReP-containing eIF2α phosphatases to control cell viability.
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    MicroRNA antagonism of the picornaviral life cycle: alternative mechanisms of interference.
    (PLoS Pathog, 2010-03-19) Kelly, Elizabeth J; Hadac, Elizabeth M; Cullen, Bryan R; Russell, Stephen J
    In addition to modulating the function and stability of cellular mRNAs, microRNAs can profoundly affect the life cycles of viruses bearing sequence complementary targets, a finding recently exploited to ameliorate toxicities of vaccines and oncolytic viruses. To elucidate the mechanisms underlying microRNA-mediated antiviral activity, we modified the 3' untranslated region (3'UTR) of Coxsackievirus A21 to incorporate targets with varying degrees of homology to endogenous microRNAs. We show that microRNAs can interrupt the picornavirus life-cycle at multiple levels, including catalytic degradation of the viral RNA genome, suppression of cap-independent mRNA translation, and interference with genome encapsidation. In addition, we have examined the extent to which endogenous microRNAs can suppress viral replication in vivo and how viruses can overcome this inhibition by microRNA saturation in mouse cancer models.
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    PERK (Protein Kinase RNA-Like ER Kinase) Branch of the Unfolded Protein Response Confers Neuroprotection in Ischemic Stroke by Suppressing Protein Synthesis.
    (Stroke, 2020-05) Wang, Ya-Chao; Li, Xuan; Shen, Yuntian; Lyu, Jingjun; Sheng, Huaxin; Paschen, Wulf; Yang, Wei
    Background and Purpose- Ischemic stroke impairs endoplasmic reticulum (ER) function, causes ER stress, and activates the unfolded protein response. The unfolded protein response consists of 3 branches controlled by ER stress sensor proteins, which include PERK (protein kinase RNA-like ER kinase). Activated PERK phosphorylates eIF2α (eukaryotic initiation factor 2 alpha), resulting in inhibition of global protein synthesis. Here, we aimed to clarify the role of the PERK unfolded protein response branch in stroke. Methods- Neuron-specific and tamoxifen-inducible PERK conditional knockout (cKO) mice were generated by cross-breeding Camk2a-CreERT2 with Perkf/f mice. Transient middle cerebral artery occlusion was used to induce stroke. Short- and long-term stroke outcomes were evaluated. Protein synthesis in the brain was assessed using a surface-sensing-of-translation approach. Results- After tamoxifen-induced deletion of Perk in forebrain neurons was confirmed in PERK-cKO mice, PERK-cKO and control mice were subjected to transient middle cerebral artery occlusion and 3 days or 3 weeks recovery. PERK-cKO mice had larger infarcts and worse neurological outcomes compared with control mice, suggesting that PERK-induced eIF2α phosphorylation and subsequent suppression of translation protects neurons from ischemic stress. Indeed, better stroke outcomes were observed in PERK-cKO mice that received postischemic treatment with salubrinal, which can restore the ischemia-induced increase in phosphorylated eIF2α in these mice. Finally, our data showed that post-treatment with salubrinal improved functional recovery after stroke. Conclusions- Here, we presented the first evidence that postischemic suppression of translation induced by PERK activation promotes recovery of neurological function after stroke. This confirms and further extends our previous observations that recovery of ER function impaired by ischemic stress critically contributes to stroke outcome. Therefore, future research should include strategies to improve stroke outcome by targeting unfolded protein response branches to restore protein homeostasis in neurons.
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    Proteome-wide muscle protein fractional synthesis rates predict muscle mass gain in response to a selective androgen receptor modulator in rats.
    (Am J Physiol Endocrinol Metab, 2016-03-15) Shankaran, Mahalakshmi; Shearer, Todd W; Stimpson, Stephen A; Turner, Scott M; King, Chelsea; Wong, Po-Yin Anne; Shen, Ying; Turnbull, Philip S; Kramer, Fritz; Clifton, Lisa; Russell, Alan; Hellerstein, Marc K; Evans, William J
    Biomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM.
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    Simple and inexpensive ribosome profiling analysis of mRNA translation.
    (Methods (San Diego, Calif.), 2015-12) Reid, David W; Shenolikar, Shirish; Nicchitta, Christopher V
    The development and application of ribosome profiling has markedly advanced our understanding of ribosomes and mRNA translation. The experimental approach, which relies on deep sequencing of ribosome-protected mRNA fragments generated by treatment of polyribosomes with exogenous nucleases, provides a transcriptome-wide assessment of translation. The broad application of ribosome profiling has been slowed by the complexity and expense of the protocol. Here, we provide a simplified ribosome profiling method that uses micrococcal nuclease to generate ribosome footprints in crude cellular extracts, which are then purified simply by size selection via polyacrylamide gel electrophoresis. This simplification removes the laborious or expensive purification of ribosomes that has typically been used. This direct extraction method generates gene-level ribosome profiling data that are similar to a method that includes ribosome purification. This protocol should significantly ease the barrier to entry for research groups interested in employing ribosome profiling.
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    Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase.
    (Cell reports, 2015-06-18) Choy, Meng S; Yusoff, Permeen; Lee, Irene C; Newton, Jocelyn C; Goh, Catherine W; Page, Rebecca; Shenolikar, Shirish; Peti, Wolfgang
    The attenuation of protein synthesis via the phosphorylation of eIF2α is a major stress response of all eukaryotic cells. The growth-arrest- and DNA-damage-induced transcript 34 (GADD34) bound to the serine/threonine protein phosphatase 1 (PP1) is the necessary eIF2α phosphatase complex that returns mammalian cells to normal protein synthesis following stress. The molecular basis by which GADD34 recruits PP1 and its substrate eIF2α are not fully understood, hindering our understanding of the remarkable selectivity of the GADD34:PP1 phosphatase for eIF2α. Here, we report detailed structural and functional analyses of the GADD34:PP1 holoenzyme and its recruitment of eIF2α. The data highlight independent interactions of PP1 and eIF2α with GADD34, demonstrating that GADD34 functions as a scaffold both in vitro and in cells. This work greatly enhances our molecular understanding of a major cellular eIF2α phosphatase and establishes the foundation for future translational work.
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    Targeting phosphorylation of eukaryotic initiation factor-2α to treat human disease.
    (Progress in molecular biology and translational science, 2012-01) Fullwood, Melissa J; Zhou, Wei; Shenolikar, Shirish
    The unfolded protein response, also known as endoplasmic reticulum (ER) stress, has been implicated in numerous human diseases, including atherosclerosis, cancer, diabetes, and neurodegenerative disorders. Protein misfolding activates one or more of the three ER transmembrane sensors to initiate a complex network of signaling that transiently suppresses protein translation while also enhancing protein folding and proteasomal degradation of misfolded proteins to ensure full recovery from ER stress. Gene disruption studies in mice have provided critical insights into the role of specific signaling components and pathways in the differing responses of animal tissues to ER stress. These studies have emphasized an important contribution of translational repression to sustained insulin synthesis and β-cell viability in experimental models of type-2 diabetes. This has focused attention on the recently discovered small-molecule inhibitors of eIF2α phosphatases that prolong eIF2α phosphorylation to reduce cell death in several animal models of human disease. These compounds show significant cytoprotection in cellular and animal models of neurodegenerative disorders, highlighting a potential strategy for future development of drugs to treat human protein misfolding disorders.
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    The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration.
    (Cell, 2009-09-04) Yan, Dong; Wu, Zilu; Chisholm, Andrew D; Jin, Yishi
    Growth cone guidance and synaptic plasticity involve dynamic local changes in proteins at axons and dendrites. The Dual-Leucine zipper Kinase MAPKKK (DLK) has been previously implicated in synaptogenesis and axon outgrowth in C. elegans and other animals. Here we show that in C. elegans DLK-1 regulates not only proper synapse formation and axon morphology but also axon regeneration by influencing mRNA stability. DLK-1 kinase signals via a MAPKAP kinase, MAK-2, to stabilize the mRNA encoding CEBP-1, a bZip protein related to CCAAT/enhancer-binding proteins, via its 3'UTR. Inappropriate upregulation of cebp-1 in adult neurons disrupts synapses and axon morphology. CEBP-1 and the DLK-1 pathway are essential for axon regeneration after laser axotomy in adult neurons, and axotomy induces translation of CEBP-1 in axons. Our findings identify the DLK-1 pathway as a regulator of mRNA stability in synapse formation and maintenance and also in adult axon regeneration.
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    The unfolded protein response triggers selective mRNA release from the endoplasmic reticulum.
    (Cell, 2014-09) Reid, David W; Chen, Qiang; Tay, Angeline S-L; Shenolikar, Shirish; Nicchitta, Christopher V
    The unfolded protein response (UPR) is a stress response program that reprograms cellular translation and gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER). One of the primary means by which the UPR alleviates this stress is by reducing protein flux into the ER via a general suppression of protein synthesis and ER-specific mRNA degradation. We report here an additional UPR-induced mechanism for the reduction of protein flux into the ER, where mRNAs that encode signal sequences are released from the ER to the cytosol. By removing mRNAs from the site of translocation, this mechanism may serve as a potent means to transiently reduce ER protein folding load and restore proteostasis. These findings identify the dynamic subcellular localization of mRNAs and translation as a selective and rapid regulatory feature of the cellular response to protein folding stress.