MABE343 Sigma-AldrichAnti-Puromycin Antibody, clone 12D10

The Skp1-Cul1-F box complex (SCF) associates with any one of a number of F box proteins, which serve as substrate binding adaptors. The human F box protein βTRCP directs the conjugation of ubiquitin to a variety of substrate proteins, leading to the destruction of the substrate by the proteasome. To identify βTRCP substrates, we employed a recently-developed technique, called Ligase Trapping, wherein a ubiquitin ligase is fused to a ubiquitin-binding domain to "trap" ubiquitinated substrates. 88% of the candidate substrates that we examined were bona fide substrates, comprising twelve previously validated substrates, eleven new substrates and three false positives. One βTRCP substrate, CReP, is a Protein Phosphatase 1 (PP1) specificity subunit that targets the translation initiation factor eIF2α to promote the removal of a stress-induced inhibitory phosphorylation and increase cap-dependent translation. We found that CReP is targeted by βTRCP for degradation upon DNA damage. Using a stable CReP allele, we show that depletion of CReP is required for the full induction of eIF2α phosphorylation upon DNA damage, and contributes to keeping the levels of translation low as cells recover from DNA damage.

Evidence is accumulating that activation of the pancreatic endoplasmic reticulum kinase (PERK) in response to endoplasmic reticulum (ER) stress adapts tumor cells to the tumor microenvironment and enhances tumor angiogenesis by inducing vascular endothelial growth factor A (VEGF-A). Recent studies suggest that VEGF-A can act directly on certain tumor cell types in an autocrine manner, via binding to VEGF receptor 2 (VEGFR2), to promote tumor cell migration and invasion. Although several reports show that PERK activation increases VEGF-A expression in medulloblastoma, the most common solid malignancy of childhood, the role that either PERK or VEGF-A plays in medulloblastoma remains elusive. In this study, we mimicked the moderate enhancement of PERK activity observed in tumor patients using a genetic approach and a pharmacologic approach, and found that moderate activation of PERK signaling facilitated medulloblastoma cell migration and invasion and increased the production of VEGF-A. Moreover, using the VEGFR2 inhibitor SU5416 and the VEGF-A neutralizing antibody to block VEGF-A/VEGFR2 signaling, our results suggested that tumor cell-derived VEGF-A promoted medulloblastoma cell migration and invasion through VEGFR2 signaling, and that both VEGF-A and VEGFR2 were required for the promoting effects of PERK activation on medulloblastoma cell migration and invasion. Thus, these findings suggest that moderate PERK activation promotes medulloblastoma cell migration and invasion through enhancement of VEGF-A/VEGFR2 signaling.

The persistence of experience-dependent changes in brain connectivity requires RNA localization and protein synthesis. Previous studies have demonstrated a role for local translation in altering the structure and function of synapses during synapse formation and experience-dependent synaptic plasticity. In this study, we ask whether in addition to promoting local translation, local stimulation also triggers directed trafficking of RNAs from nucleus to stimulated synapses. Imaging of RNA localization and translation in cultured Aplysia sensory-motor neurons revealed that RNAs were delivered throughout the arbor of the sensory neuron, but that translation was enriched only at sites of synaptic contact and/or synaptic stimulation. Investigation of the mechanisms that trigger local translation revealed a role for calcium-dependent retrograde netrin-1/DCC receptor signaling. Spatially restricting gene expression by regulating local translation rather than by directing the delivery of mRNAs from nucleus to stimulated synapses maximizes the readiness of the entire neuronal arbor to respond to local cues.

Animals are imbued with adaptive mechanisms spanning from the tissue/organ to the cellular scale which insure that processes of homeostasis are preserved in the landscape of size change. However we and others have postulated that the degree of adaptation is limited and that once outside the normal levels of size fluctuations, cells and tissues function in an aberant manner. In this study we examine the function of muscle in the myostatin null mouse which is an excellent model for hypertrophy beyond levels of normal growth and consequeces of acute starvation to restore mass. We show that muscle growth is sustained through protein synthesis driven by Serum/Glucocorticoid Kinase 1 (SGK1) rather than Akt1. Furthermore our metabonomic profiling of hypertrophic muscle shows that carbon from nutrient sources is being channelled for the production of biomass rather than ATP production. However the muscle displays elevated levels of autophagy and decreased levels of muscle tension. We demonstrate the myostatin null muscle is acutely sensitive to changes in diet and activates both the proteolytic and autophagy programmes and shutting down protein synthesis more extensively than is the case for wild-types. Poignantly we show that acute starvation which is detrimental to wild-type animals is beneficial in terms of metabolism and muscle function in the myostatin null mice by normalising tension production.

Endoplasmic reticulum (ER) stress plays an important role in the decline in pancreatic β cell function and mass observed in type 2 diabetes. Here, we developed a novel β cell-based high-throughput screening assay to identify small molecules that protect β cells against ER stress-induced cell death. Mouse βTC6 cells were treated with the ER stressor tunicamycin to induce ER stress, and cell death was measured as a reduction in cellular ATP. A collection of 17600 compounds was screened for molecules that promote β cell survival. Of the approximately 80 positive hits, two selected compounds were able to increase the survival of human primary β cells and rodent β cell lines subjected to ER stressors including palmitate, a free fatty acid of pathological relevance to diabetes. These compounds also restored ER stress-impaired glucose-stimulated insulin secretion responses. We show that the compounds promote β cell survival by reducing the expression of key genes of the unfolded protein response and apoptosis, thus alleviating ER stress. Identification of small molecules that prevent ER stress-induced β cell dysfunction and death may provide a new modality for the treatment of diabetes.

Vanishing white matter disease (VWMD) is an inherited autosomal-recessive hypomyelinating disease caused by mutations in eukaryotic translation initiation factor 2B (eIF2B). eIF2B mutations predominantly affect the brain white matter, and the characteristic features of VWMD pathology include myelin loss and foamy oligodendrocytes. Activation of pancreatic endoplasmic reticulum kinase (PERK) has been observed in oligodendrocytes in VWMD. PERK activation in response to endoplasmic reticulum stress attenuates eIF2B activity by phosphorylating eIF2α, suggesting that impaired eIF2B activity in oligodendrocytes induced by VWMD mutations or PERK activation exploit similar mechanisms to promote selective white matter pathology in VWMD. Using transgenic mice that allow for temporally controlled activation of PERK specifically in oligodendrocytes, we discovered that strong PERK activation in oligodendrocytes during development suppressed eIF2B activity and reproduced the characteristic features of VWMD in mice, including hypomyelinating phenotype, foamy oligodendrocytes, and myelin loss. Notably, impaired eIF2B activity induced by PERK activation in oligodendrocytes of fully myelinated adult mice had minimal effects on morphology or function. Our observations point to a cell-autonomous role of impaired eIF2B activity in myelinating oligodendrocytes in the pathogenesis of VWMD.

Persistent forms of synaptic plasticity are widely thought to require the synthesis of new proteins. This feature of long-lasting forms of plasticity largely has been demonstrated using inhibitors of general protein synthesis, such as either anisomycin or emetine. However, these drugs, which inhibit elongation, cannot address detailed questions about the regulation of translation initiation, where the majority of translational control occurs. Moreover, general protein synthesis inhibitors cannot distinguish between cap-dependent and cap-independent modes of translation initiation. In the present study, we took advantage of two novel compounds, 4EGI-1 and hippuristanol, each of which targets a different component of the eukaryotic initiation factor (eIF)4F initiation complex, and investigated their effects on long-term potentiation (LTP) at CA3-CA1 synapses in the hippocampus. We found that 4EGI-1 and hippuristanol both attenuated long-lasting late-phase LTP induced by two different stimulation paradigms. We also found that 4EGI-1 and hippuristanol each were capable of blocking the expression of newly synthesized proteins immediately after the induction of late-phase LTP. These new pharmacological tools allow for a more precise dissection of the role played by translational control pathways in synaptic plasticity and demonstrate the importance of multiple aspects of eIF4F in processes underlying hippocampal LTP, laying the foundation for future studies investigating the role of eIF4F in hippocampus-dependent memory processes.

Drastic protein degradation occurs during muscle atrophy induced by denervation, fasting, immobility, and various systemic diseases. Although the ubiquitin-proteasome system is highly up-regulated in denervated muscles, the involvement of autophagy and protein synthesis has been controversial. Here, we report that autophagy is rather suppressed in denervated muscles even under autophagy-inducible starvation conditions. This is due to a constitutive activation of mammalian target of rapamycin complex 1 (mTORC1). We further reveal that denervation-induced mTORC1 activation is dependent on the proteasome, which is likely mediated by amino acids generated from proteasomal degradation. Protein synthesis and ribosome biogenesis are paradoxically increased in denervated muscles in an mTORC1-dependent manner, and mTORC1 activation plays an anabolic role against denervation-induced muscle atrophy. These results suggest that denervation induces not only muscle degradation but also adaptive muscle response in a proteasome- and mTORC1-dependent manner.

Autism spectrum disorders (ASDs) are an early onset, heterogeneous group of heritable neuropsychiatric disorders with symptoms that include deficits in social interaction skills, impaired communication abilities, and ritualistic-like repetitive behaviours. One of the hypotheses for a common molecular mechanism underlying ASDs is altered translational control resulting in exaggerated protein synthesis. Genetic variants in chromosome 4q, which contains the EIF4E locus, have been described in patients with autism. Importantly, a rare single nucleotide polymorphism has been identified in autism that is associated with increased promoter activity in the EIF4E gene. Here we show that genetically increasing the levels of eukaryotic translation initiation factor 4E (eIF4E) in mice results in exaggerated cap-dependent translation and aberrant behaviours reminiscent of autism, including repetitive and perseverative behaviours and social interaction deficits. Moreover, these autistic-like behaviours are accompanied by synaptic pathophysiology in the medial prefrontal cortex, striatum and hippocampus. The autistic-like behaviours displayed by the eIF4E-transgenic mice are corrected by intracerebroventricular infusions of the cap-dependent translation inhibitor 4EGI-1. Our findings demonstrate a causal relationship between exaggerated cap-dependent translation, synaptic dysfunction and aberrant behaviours associated with autism.

Rift Valley fever virus (RVFV), an ambisense member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever, an important zoonotic infection in Africa and the Middle East. Phlebovirus proteins are translated from virally transcribed mRNAs that, like host mRNA, are capped but, unlike host mRNAs, are not polyadenylated. Here, we investigated the role of PABP1 during RVFV infection of HeLa cells. Immunofluorescence studies of infected cells demonstrated a gross relocalization of PABP1 to the nucleus late in infection. Immunofluorescence microscopy studies of nuclear proteins revealed costaining between PABP1 and markers of nuclear speckles. PABP1 relocalization was sharply decreased in cells infected with a strain of RVFV lacking the gene encoding the RVFV nonstructural protein S (NSs). To determine whether PABP1 was required for RVFV infection, we measured the production of nucleocapsid protein (N) in cells transfected with small interfering RNAs (siRNAs) targeting PABP1. We found that the overall percentage of RVFV N-positive cells was not changed by siRNA treatment, indicating that PABP1 was not required for RVFV infection. However, when we analyzed populations of cells producing high versus low levels of PABP1, we found that the percentage of RVFV N-positive cells was decreased in cell populations producing physiologic levels of PABP1 and increased in cells with reduced levels of PABP1. Together, these results suggest that production of the NSs protein during RVFV infection leads to sequestration of PABP1 in the nuclear speckles, creating a state within the cell that favors viral protein production.