Elevated Protein Synthesis Research Articles

Shifting Balance of Protein Synthesis and Degradation Sets a Threshold for Larval Growth Under Environmental Stress.

Exogenous environmental factors alter growth rates, yet information remains scant on the biochemical mechanisms and energy trade-offs that underlie variability in the growth of marine invertebrates. Here we study the biochemical bases for differential growth and energy utilization (as adenosine triphosphate [ATP] equivalents) during larval growth of the bivalve Crassostrea gigas exposed to increasing levels of experimental ocean acidification (control, middle, and high pCO2, corresponding to ∼400, ∼800, and ∼1100 µatm, respectively). Elevated pCO2 hindered larval ability to accrete both shell and whole-body protein content. This negative impact was not due to an inability to synthesize protein per se, because size-specific rates of protein synthesis were upregulated at both middle and high pCO2 treatments by as much as 45% relative to control pCO2. Rather, protein degradation rates increased with increasing pCO2. At control pCO2, 89% of cellular energy (ATP equivalents) utilization was accounted for by just 2 processes in larvae, with protein synthesis accounting for 66% and sodium-potassium transport accounting for 23%. The energetic demand necessitated by elevated protein synthesis rates could be accommodated either by reallocating available energy from within the existing ATP pool or by increasing the production of total ATP. The former strategy was observed at middle pCO2, while the latter strategy was observed at high pCO2. Increased pCO2 also altered sodium-potassium transport, but with minimal impact on rates of ATP utilization relative to the impact observed for protein synthesis. Quantifying the actual energy costs and trade-offs for maintaining physiological homeostasis in response to stress will help to reveal the mechanisms of resilience thresholds to environmental change.

Proteasome-associated deubiquitinases and cancer

Maintenance of protein homeostasis is a crucial process for the normal functioning of the cell. The regulated degradation of proteins is primarily facilitated by the ubiquitin proteasome system (UPS), a system of selective tagging of proteins with ubiquitin followed by proteasome-mediated proteolysis. The UPS is highly dynamic consisting of both ubiquitination and deubiquitination steps that modulate protein stabilization and degradation. Deregulation of protein stability is a common feature in the development and progression of numerous cancer types. Simultaneously, the elevated protein synthesis rate of cancer cells and consequential accumulation of misfolded proteins drives UPS addiction, thus sensitizing them to UPS inhibitors. This sensitivity along with the potential of stabilizing pro-apoptotic signaling pathways makes the proteasome an attractive clinical target for the development of novel therapies. Targeting of the catalytic 20S subunit of the proteasome is already a clinically validated strategy in multiple myeloma and other cancers. Spurred on by this success, promising novel inhibitors of the UPS have entered development, targeting the 20S as well as regulatory 19S subunit and inhibitors of deubiquitinating and ubiquitin ligase enzymes. In this review, we outline the manner in which deregulation of the UPS can cause cancer to develop, current clinical application of proteasome inhibitors, and the (pre-)clinical development of novel inhibitors of the UPS.

A Reliable and Reproducible Model for Assessing the Effect of Different Concentrations of α-Solanine on Rat Bone Marrow Mesenchymal Stem Cells.

Αlpha-solanine (α-solanine) is a glycoalkaloid present in potato (Solanum tuberosum). It has been of particular interest because of its toxicity and potential teratogenic effects that include abnormalities of the central nervous system, such as exencephaly, encephalocele, and anophthalmia. Various types of cell culture have been used as experimental models to determine the effect of α-solanine on cell physiology. The morphological changes in the mesenchymal stem cell upon exposure to α-solanine have not been established. This study aimed to describe a reliable and reproducible model for assessing the structural changes induced by exposure of mouse bone marrow mesenchymal stem cells (MSCs) to different concentrations of α-solanine for 24 h. The results demonstrate that nonlethal concentrations of α-solanine (2–6 μM) changed the morphology of the cells, including an increase in the number of nucleoli, suggesting elevated protein synthesis, and the formation of spicules. In addition, treatment with α-solanine reduced the number of adherent cells and the formation of colonies in culture. Immunophenotypic characterization and staining of MSCs are proposed as a reproducible method that allows description of cells exposed to the glycoalkaloid, α-solanine.

Dual abrogation of MNK and mTOR: a novel therapeutic approach for the treatment of aggressive cancers.

Targeting the translational machinery has emerged as a promising therapeutic option for cancer treatment. Cancer cells require elevated protein synthesis and exhibit augmented activity to meet the increased metabolic demand. Eukaryotic translation initiation factor 4E is necessary for mRNA translation, its availability and phosphorylation are regulated by the PI3K/AKT/mTOR and MNK1/2 pathways. The phosphorylated form of eIF4E drives the expression of oncogenic proteins including those involved in metastasis. In this article, we will review the role of eIF4E in cancer, its regulation and discuss the benefit of dual inhibition of upstream pathways. The discernible interplay between the MNK and mTOR signaling pathways provides a novel therapeutic opportunity to target aggressive migratory cancers through the development of hybrid molecules.

Increased signaling by the autism-related Engrailed-2 protein enhances dendritic branching and spine density, alters synaptic structural matching, and exaggerates protein synthesis.

Engrailed 1 (En1) and 2 (En2) code for closely related homeoproteins acting as transcription factors and as signaling molecules that contribute to midbrain and hindbrain patterning, to development and maintenance of monoaminergic pathways, and to retinotectal wiring. En2 has been suggested to be an autism susceptibility gene and individuals with autism display an overexpression of this homeogene but the mechanisms remain unclear. We addressed in the present study the effect of exogenously added En2 on the morphology of hippocampal cells that normally express only low levels of Engrailed proteins. By means of RT-qPCR, we confirmed that En1 and En2 were expressed at low levels in hippocampus and hippocampal neurons, and observed a pronounced decrease in En2 expression at birth and during the first postnatal week, a period characterized by intense synaptogenesis. To address a putative effect of Engrailed in dendritogenesis or synaptogenesis, we added recombinant En1 or En2 proteins to hippocampal cell cultures. Both En1 and En2 treatment increased the complexity of the dendritic tree of glutamatergic neurons, but only En2 increased that of GABAergic cells. En1 increased the density of dendritic spines both in vitro and in vivo. En2 had similar but less pronounced effect on spine density. The number of mature synapses remained unchanged upon En1 treatment but was reduced by En2 treatment, as well as the area of post-synaptic densities. Finally, both En1 and En2 elevated mTORC1 activity and protein synthesis in hippocampal cells, suggesting that some effects of Engrailed proteins may require mRNA translation. Our results indicate that Engrailed proteins can play, even at low concentrations, an active role in the morphogenesis of hippocampal cells. Further, they emphasize the over-regulation of GABA cell morphology and the vulnerability of excitatory synapses in a pathological context of En2 overexpression.

Withaferin A induced impaired autophagy and unfolded protein response in human breast cancer cell-lines MCF-7 and MDA-MB-231

The autophagy-lysosome pathway and the ubiquitin-proteasome systems are the two major routes for eukaryotic intracellular protein clearance. Cancerous cells often display elevated protein synthesis and byproduct disposal, thus, inhibition of the protein degradation pathways became an emerging approach for cancer therapy. The present study revealed that withaferin-A (WA), the biologically active withanolide derived from Withania somnifera, initially induced formation of autophagosomes in human breast cancer cell-lines, MCF-7 and MDA-MB-231. WA treatment elevated the levels of autophagic substrate p62/SQSTM1 (p62) and both LC3-II and LC3-I (microtubule-associated protein 2 light chain 3) and simultaneously reduced the upstream autophagy markers like beclin-1 and ATG5-ATG12 complex, which indicate accumulation of autophagosomes in the cells. WA induced disruption of microtubular network through inhibition of tubulin polymerization and its hyper-acetylation, thus prevent the formation of autolysosome (by merging of autophagosomes with lysosomes) and its recycling process, leading to incomplete autophagy. Further, WA caused ER (Endoplasmic Reticulum) stress, which is evident from the activation of ER-related caspase-4 and increased levels of ER stress marker proteins. Thus, these findings altogether indicate that WA mediated inhibition of proteasomal degradation system and perturbation of autophagy, i.e. suppression of both the intracellular degradation systems caused accumulation of ubiquitinated proteins, which in turn led to unfolded protein response and ER stress mediated proteotoxicity in human breast cancer cell-lines, MCF-7 and MDA-MB-231.

0126 NEUROIMAGING OF CIRCUIT-SPECIFIC PROTEIN SYNTHESIS IN HUMAN SUBJECTS DURING SLEEP-DEPENDENT MEMORY CONSOLIDATION

The notion that memory consolidation depends on protein synthesis is based on demonstrations that protein synthesis inhibitors prevent consolidation. We sought to demonstrate directly that protein synthesis is affected during sleep-dependent memory consolidation. We used L-[1-(11)C]leucine positron emission tomography (PET) to measure circuit-specific brain protein synthesis during a daytime nap opportunity. Subjects completed the texture discrimination task (TDT) before and after the nap opportunity. We randomized 34 subjects to either wakefulness or sleep and counterbalanced training to either left or right visual field. In accord with the retinotopic specificity of the TDT, we considered ipsilateral V1 as the untrained within-subject control. Following training on the TDT, subjects underwent the PET scan with simultaneous polysomnography. V1 was drawn on magnetic resonance volumes and transferred to corresponding PET volumes to compute average rates of protein synthesis in left and right V1. If we disregard visual field trained (left, right), the Condition (wakefulness, sleep) x Local State (untrained, trained) interaction was not statistically significant (F[1,32]=0.07,p=0.79,f^2=0.002). Consideration of visual field trained indicates that the Condition x Visual Field Trained x Local State interaction was statistically significant (F[1,30]=9.08,p=0.005,f^2=0.30). Side-to-side differences were statistically significant only in the sleep condition: protein synthesis in the trained hemisphere was significantly higher if training was in left visual field and significantly lower if training was in right visual field. Another way to view these results is protein synthesis was higher in the right V1 during sleep-dependent memory consolidation regardless of the location of training. If we disregard visual field trained, our study could indicate sleep-dependent memory consolidation depends on processes other than protein synthesis such as synaptic renormalization. Consideration of visual field trained, however, may indicate that each hemisphere has inherent properties requiring either reduced or elevated protein synthesis for memory consolidation to occur during sleep. Intramural Research Program of the National Institute of Mental Health, Intramural Research Program of the National Institute of Neurological Disorders and Stroke, and Military Operational Medicine Research Program of the United States Army Medical Research and Materiel Command.

A potential strategy for counteracting age-related sarcopenia: preliminary evidence of combined exercise training and leucine supplementation.

Previous research has demonstrated the positive effects of concurrent/combined aerobic and resistance exercise or leucine supplementation on skeletal muscle protein synthesis (MPS) and hypertrophy in aging organisms. However, the effects of a multimodal intervention which combines both aerobic and resistance exercise and leucine supplementation has not been fully elucidated. Eighteen month old and 2 month old C57BL/6 mice were assigned to aging control (AC, n = 8), aging and multimodal intervention (AMI, n = 8) and young control (YC, n = 8). Mice in the YC and AC groups were fed an alanine-rich diet (3.4%), and mice in the AMI group received an isonitrogenous leucine-supplemented (5%) diet in combination with combined aerobic (30 minutes swimming) and resistance exercise training (incremental jumping submersed in water with overload corresponding to 40%-50% body weight) for a total of 4 weeks. The gastrocnemius muscles were dissected for western blotting detection (signaling proteins involved in MPS) and the ex vivo determination of protein synthesis and protein content. The muscle strength of the hind limbs was measured pre-experiment and repeated once per week on Sunday for 4 weeks. Mice in the AC and AMI groups showed lower ex vivo protein synthesis, protein content, expression of signaling proteins involved in MPS, maximal grip strength but higher plasma cortisol compared with the YC group post intervention. When compared to AC mice, the multimodal treatment led to lower activity of Sestrin2, higher expression of PI3K III and the phosphorylation of mTOR, p70S6K and 4E-BP1, as well as higher plasma leucine, wet gastrocnemius muscle weight and muscle weight to body weight ratio. Furthermore, the multimodal intervention induced more pronounced anabolic response such as higher ex vivo protein synthesis rate, total protein content, and myofibrillar fractions in gastrocnemius muscle, and greater maximum grip strength. The present research shows that a multimodal intervention including combined both aerobic and resistance exercise training and 5% leucine supplementation has the potential to maintain skeletal muscle protein synthesis and attenuate losses in muscular strength during the aging process.

Getting a "GRiP" on Hypothalamic Endoplasmic Reticulum Stress to Combat Obesity.

The endoplasmic reticulum (ER) coordinates the synthesis, folding, and sorting of proteins for retention in the cell or for entry into the secretory pathway. Certain conditions (e.g., calcium dyshomeostasis, lipotoxicity, abnormally elevated protein synthesis) may result in protein misfolding and aberrant accumulation in the ER. This process, known as ER stress, activates the adaptive unfolded protein response (UPR), a conserved set of molecular pathways that leads to decreased protein synthesis and misfolding and to increased protein degradation and autophagy, overall aiming to restore cellular homeostasis (1,2). However, persistent activation of the UPR triggers cell death pathways. ER stress and the UPR thus play crucial roles in protein folding and cell survival. Several studies further indicate that they have broader functions in the regulation of cell physiology, including growth, differentiation, and cellular metabolism. ER stress can have internal cellular causes (e.g., increased protein synthesis, inefficient protein folding linked to mutations) or may be triggered by factors external to cells. In particular, inflammation caused by chronic overnutrition/unhealthy diet has been proposed to be an inducer of ER stress (3–5). ER stress triggered by such external factors is currently thought to contribute to the etiology of several human diseases. For example, ER stress has emerged as a key mechanism involved in the development of obesity and type 2 diabetes (T2D), as well as in aging and age-related neurodegenerative diseases, including Alzheimer disease (3,6,7). In obesity and T2D, chronic ER stress in peripheral …

Leucine in the Presence of Inflammation Alters BCAA Metabolism in Human Myotubes

RationaleMetabolomics studies suggest a positive association between fasting concentrations of circulating branched chain amino acids (BCAA) and the development of insulin resistance and type II diabetes. Whether BCAAs are a causative factor or a biomarker for the development of insulin resistance remains unclear. Proinflammatory cytokines (e.g., TNFα), which are commonly elevated in obesity, lower the expression of BCAA transport and oxidation genes and reduce leucine influx in differentiated adipocytes. As skeletal muscle accounts for ~65% of whole body BCAA transamination capacity in humans, the effect of inflammation on BCAA metabolism in skeletal muscle is important to understand, but remains unknown.ObjectiveTo test the hypothesis that TNFα will reduce BCAA uptake and metabolism in response to a leucine challenge in differentiated human skeletal muscle cells.MethodsPrimary human progenitor cells (myoblasts) were obtained from the vastus lateralis muscle of young males (n=6). Study 1: Sorted myoblasts were proliferated in growth media (GM) until ~80% confluent, then switched to differentiation media (DM). After 72 h of differentiation, 10 ng/mL of TNFα was added to the DM. Cells were incubated for an additional 48 h. Study 2: Same as Study 1 with the following modification: after 47 h of TNFα treatment, 0.5 mM of leucine (~blood leucine concentration post ingestion of 30g of protein) or 2 mM of leucine (supra‐physiologic dose) was added to the DM+TNFα. Cells from both studies were harvested after 48 h of DM+TNFα with or without 1 h of leucine. Control cells did not receive TNFα or leucine. Real‐time qPCR was used to measure mRNA abundance of genes associated with inflammation (NFκB, IL1β, IL6, TNFα), BCAA transport (Slc7a5), mitochondrial function (PCG1α), and the unfolded protein response (Hspa5). Immunoblotting was used to quantify protein levels of genes associated with BCAA transport (CD98), BCAA metabolism (Bcat2, Bckdhb), and protein synthesis (p70S6K). A colorimetric BCAA assay was used to measure cell culture media levels of BCAAs.ResultsTNFα treatment alone reduced mRNA levels of PCG1α (p<0.05) and increased mRNA levels of NFκB, IL1β, IL6, and endogenous TNFα (p<0.001). TNFα treatment also reduced BCAA levels in culture media (p<0.05), suggesting greater BCAA uptake. The leucine challenge (0.5 and 2 mM), in the presence of TNFα, increased mRNA levels of Slc7a5 and Hspa5 (p<0.01), reduced Bcat2 protein levels (p<0.05), and increased the ratio of phosphorylated p70S6K to total p70S6K (p<0.05). Bckdhb protein levels were lower with the 2mM leucine dose only (p<0.05). Protein levels of CD98 were not significantly different with either leucine dose (p=0.07).ConclusionsContrary to adipocytes and our hypothesis, TNFα increases BCAA uptake through upregulation of BCAA transport in differentiated myoblasts. Further, at a physiologically relevant leucine dose, in the presence of inflammation, protein synthesis and the unfolded protein response are also up‐regulated, while BCAA metabolism and mitochondrial biogenesis and function are down‐regulated. Whether elevated protein synthesis coupled with the unfolded protein response is problematic for the development of insulin resistance is unclear.Support or Funding InformationCornell Start‐up

MTORC1 Coordinates Protein Synthesis and Immunoproteasome Formation via PRAS40 to Prevent Accumulation of Protein Stress

Reduction of translational fidelity often occurs in cells with high rates of protein synthesis, generating defective ribosomal products. If not removed, such aberrant proteins can be a major source of cellular stress causing human diseases. Here, we demonstrate that mTORC1 promotes the formation of immunoproteasomes for efficient turnover of defective proteins and cell survival. mTORC1 sequesters precursors of immunoproteasome β subunits via PRAS40. When activated, mTORC1 phosphorylates PRAS40 to enhance protein synthesis and simultaneously to facilitate the assembly of the β subunits for forming immunoproteasomes. Consequently, the PRAS40 phosphorylations play crucial roles in clearing aberrant proteins that accumulate due to mTORC1 activation. Mutations of RAS, PTEN, and TSC1, which cause mTORC1 hyperactivation, enhance immunoproteasome formation in cells and tissues. Those mutations increase cellular dependence on immunoproteasomes for stress response and survival. These results define a mechanism by which mTORC1 couples elevated protein synthesis with immunoproteasome biogenesis to protect cells against protein stress.

Increased expression of the PI3K catalytic subunit p110δ underlies elevated S6 phosphorylation and protein synthesis in an individual with autism from a multiplex family.

BackgroundDysfunctions in the PI3K/mTOR pathway have gained a lot of attention in autism research. This was initially based on the discovery of several monogenic autism spectrum disorders with mutations or defects in PI3K/mTOR signaling components. Recent genetic studies corroborate that defective PI3K/mTOR signaling might be a shared pathomechanism in autism disorders of so far unknown etiology, but functional molecular analyses in human cells are rare. The goals of this study were to perform a functional screen of cell lines from patients with idiopathic autism for defects in PI3K/mTOR signaling, to test if further functional analyses are suitable to detect underlying molecular mechanisms, and to evaluate this approach as a biomarker tool to identify therapeutic targets.MethodsWe performed phospho-S6- and S6-specific ELISA experiments on 21 lymphoblastoid cell lines from the AGRE collection and on 37 lymphoblastoid cell lines from the Simons Simplex Collection and their healthy siblings. Cell lines from one individual with increased S6 phosphorylation and his multiplex family were analyzed in further detail to identify upstream defects in PI3K signaling associated with autism diagnosis.ResultsWe detected significantly increased S6 phosphorylation in 3 of the 21 lymphoblastoid cell lines from AGRE compared to a healthy control and in 1 of the 37 lymphoblastoid cell lines from the Simons Simplex Collection compared to the healthy sibling. Further analysis of cells from one individual with elevated S6 phosphorylation showed increased expression of the PI3K catalytic subunit p110δ, which was also observed in lymphoblastoid cells from other autistic siblings but not unaffected members in his multiplex family. The p110δ-selective inhibitor IC87114 reduced elevated S6 phosphorylation and protein synthesis in this cell line.ConclusionsOur results suggest that functional analysis of PI3K/mTOR signaling is a biomarker tool to identify disease-associated molecular defects that could serve as therapeutic targets in autism. Using this approach, we discovered impaired signaling and protein synthesis through the PI3K catalytic subunit p110δ as an underlying molecular defect and potential treatment target in select autism spectrum disorders. Increased p110δ activity was recently associated with schizophrenia, and our results suggest that p110δ may also be implicated in autism.Electronic supplementary materialThe online version of this article (doi:10.1186/s13229-015-0066-4) contains supplementary material, which is available to authorized users.

Abstract PR04: HIF-2α dependent lipid storage promotes endoplasmic reticulum homeostasis in clear cell renal cell carcinoma

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common form of renal cancer, defined pathologically by abundant intracellular lipid droplets (LDs) that impart the clear cell phenotype, and molecularly by constitutive activation of the hypoxia inducible factors (HIFs). The primary aim of this study was to identify the mechanisms driving enhanced neutral lipid storage and the function of this phenotype in ccRCC. Our work demonstrates that HIF-2α promotes neutral lipid storage in ccRCC through up-regulation of the LD coat protein PLIN2. Expression profiling in multiple cohorts of primary ccRCC and normal kidney samples revealed that PLIN2 was overexpressed in all stages of disease and correlated with constitutive activation of HIF-2α, but not HIF-1α. Using ccRCC cell lines, 3D tumor spheroids, and tumor xenografts, we showed that HIF-2α dependent PLIN2 expression was required for neutral lipid storage and tumor cell viability. We demonstrated, for the first time, that PLIN2 dependent lipid storage promoted ccRCC tumor growth by maintaining integrity of the endoplasmic reticulum (ER), an organelle that is functionally and physically associated with LDs. Recent work indicates that cellular transformation commits tumors to growth programs that strain ER homeostasis, including elevated protein and lipid synthesis. Such ER stress is exacerbated by conditions of oxygen and nutrient deprivation characteristic of solid tumor microenvironments, which further disrupt cellular protein and lipid homeostasis. Consistent with this theme, we found that the enhanced requirement for PLIN2 in ccRCC was due to heightened ER stress downstream of cellular transformation, including elevated protein synthesis and growth within nutrient and oxygen limited tumor microenvironments. In these settings, PLIN2 depletion elicited irremediable ER stress and triggered a cytotoxic unfolded protein response. Remarkably, suppression of protein synthesis via cycloheximide or pharmacologic inhibitors of mTORC1, which is activated in >80% of ccRCC, prevented ER stress and cell death in PLIN2 depleted cells. On the other hand, inhibition of lipid synthesis was selectively toxic to PLIN2 depleted cells, consistent with an adaptive function of lipid synthesis downstream of ER stress. Our results suggest that PLIN2 is not merely a marker of lipid accumulation in ccRCC. Instead, it promotes lipid storage and maintains integrity of the ER, a vital hub for tumor cell protein and lipid metabolism. Consistent with this model, HIF-2α/PLIN2 dependent lipid storage also protected ccRCC cells against pharmacological ER stress inducing agents, including the proteasome inhibitor Bortezomib (Velcade). These findings reveal a novel function of the well documented clear cell phenotype in ccRCC and identify ER stress as a targetable vulnerability created by HIF-2α/PLIN2 suppression in this common renal malignancy. Citation Format: Bo Qiu, Daniel Ackerman, Danielle J. Sanchez, Bo Li, Joshua D. Ochocki, Alison Grazioli, Ekaterina Bobrovnikova-Marjon, J. Alan Diehl, Brian Keith, M. Celeste Simon. HIF-2α dependent lipid storage promotes endoplasmic reticulum homeostasis in clear cell renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr PR04.

TRN-A-RS Acts As Biomarker for Cancer and Other Diseases

Background: tRNA and ARS, tRN-A-RS, pivot the translational machinery. How their aberrances correlate to diseases, neurological conditions, metabolic disorders, and cancer is the purpose of this study. Methods: Information was retrieved from the literature and from databases, such as OMIM and MITOMAP, related to disease and cancer. Changes of expressions of ARSs were assessed from analyzing NGS data available from TCGA, ENCODE and lately from roadmap epigenomics. Results: A total of 647 tRNAs and 37 ARSs reside in the genomic pool of human, aberrant expressions of some of the nuclear tRNAs and cytoplasmic ARSs correlate predominately to cancer, while anomalous pathways in mitochondria relate more to other diseases. tRNA fragments juggle between tumor suppressor and oncogenic pathways. Brain cancer and neurological disorders seem to gain impetus from translation machinery components. However, investigation of the causes of the diseases particularly cancer due to the aberrations continues to be hamstrung by the lack of deep sequencing reads of tRNAs in different cell lines, and this needs to be addressed. Conclusions: Increased cell proliferation requires elevated protein synthesis levels and makes the correlation between tumour cells and deregulated tRN-A-RS components in translation plausible. We draw the network of tRN-A-RS with cancer and other diseases, and present and expand on some of the hypotheses on the underlying molecular mechanisms, opening up new avenues for research.

Losing p53 loosens up ER-stress.

The endoplasmic reticulum (ER) is the central intracellular organelle responsible for proper folding, localization, and post-translational modifications of proteins in eukaryotic cells. During conditions of cellular stress, incorrectly folded proteins can accumulate in the ER and trigger the unfolded protein response (UPR), which involves the activation of signal transduction pathways designed to restore correct protein folding and recovery from ER stress [1]. In rapidly dividing tumor cells, UPR can become constitutively activated, presumably because higher levels of proliferation require increased protein synthesis [1, 2]. In addition, the tumor micro-environment is often host to additional adverse conditions including nutrient and oxygen deprivation, leading to further perturbations of ER function. Therefore, it may come as no surprise that UPR has been shown to play a crucial cytoprotective role in tumor cells [2]. We have recently unveiled an unexpected role for the tumor suppressor p53 in regulating ER function via the IRE1α/XBP1 pathway [3]. IRE1α is an ER stress sensor that activates the UPR to restore ER function and maintain cellular homeostasis. It was observed that wild-type p53 suppresses IRE1α expression and activation, while the loss of active p53 potentiates the IRE1α/XBP1 pathway, enhancing ER function [3]. This finding suggests a survival benefit for cancer cells that can inactivate p53. Because cancer cells with high protein load on the ER would need enhanced ER function to cope with constant ER stress, losing p53 function would assist tumors to grow in adverse conditions by increasing IRE1α/XBP1 pathway activation. In addition, we found that a p53 target, CDIP1, acts as a proapototic signal mediator in conditions of ER stress [4]. The CDIP1/BAP31 complex was shown to transduce ER stress-mediated apoptotic signaling to the mitochondria and activate the intrinsic apoptosis pathway, suggesting that loss of p53 function may contribute to the survival of cancer cells by enhancing ER function as well as hindering ER stress-mediated apoptosis signaling. The loss of p53 function in relation to ER stress is likely to have important consequences for the sustenance of cancer cell proliferation. Previous studies have demonstrated that the activation of p53 suppresses mTOR activity and consequently inhibits protein translation [5, 6]. The elimination of wild-type p53 function may therefore provide an opportunity for cancer cells to further promote unregulated proliferation, via elevated protein synthesis and a subsequent increase in ER function. We have revealed that wild-type p53 directly interacts with synoviolin (SYVN1) and can thereby stimulate SYVN1-dependent degradation of IRE1α. The loss of p53 disrupts SYVN1-mediated IRE1α downregulation, producing an ER stress-resistant phenotype by increased IRE1α/XBP1 pathway activity [3]. However, although the loss of p53 function facilitates enhanced ER function to manage high ER stress, this also generates an overreliance on IRE1α in cancer cells with p53 mutations. Indeed, we have found that pharmacological inhibition of IRE1α strongly suppresses protein secretion and potently induces cell death in such p53-deficient cancer cells [3]. Importantly, the IRE1α inhibitor exhibited significant inhibitory effects against p53-deficient human tumors in vivo compared to those with wild-type p53. These results illuminate an important proof-of-concept for targeting tumors with mutant p53 via inhibition of the IRE1α/XBP1 pathway. Malignant transformation occurs through a combination of oncogenic pathway activation and the loss of tumor suppression. Understanding the consequences and identifying vulnerabilities caused by such changes can provide promising opportunities to selectively target cancer cells. Mutation in the p53 gene is the most frequent genetic alteration found in human cancer and inactivation of p53 is known as one of the strongest driving factors in cancer development and chemoresistance [7]. The new finding that loss of p53 activates the IRE1α/XBP1 pathway leading to enhanced ER function broadens our understanding of how cancer cells with mutant p53 survive, and also highlights a possibility for the development of novel therapeutic strategies.

Leucine‑induced anabolic‑catabolism: two sides of the same coin.

Leucine is an essential nutrient with potent abilities to stimulate both muscle protein synthesis as well as increased oxidative metabolism supported by mitochondrial biogenesis. Few studies have simultaneously investigated anabolic and catabolic responses following leucine treatment with the exception of master regulator of cellular energetics, AMPK. The unique ability of leucine to stimulate both favorable anabolic and catabolic processes in highly metabolically active tissues suggests leucine consumption/supplementation may provide benefits beyond hypertrophic gains. This review highlights original investigations reporting leucine’s ability to stimulate both anabolic and catabolic processes in muscle. Additionally, this report discusses potential explanations for the simultaneous activation of two seemingly opposing processes, as well as the interplay between them. The role of cellular energetics is of undeniable importance in sustaining increased protein synthesis following leucine administration; however, the dependence of leucine-induced mitochondrial biogenesis on heightened energy expenditure from elevated protein synthesis remains to be defined experimentally.

Aspartic acid racemization reveals a high turnover state in knee compared with hip osteoarthritic cartilage

We investigated tissue turnover in healthy and osteoarthritic cartilage. We challenge long held views that osteoarthritis (OA) is dominated by a similar turnover process in all joints and present evidence that hip and knee cartilage respond very differently to OA. d- and l-Aspartate (Asp) were quantified for whole cartilage, collagen and non-collagenous components of cartilage obtained at the time of joint replacement. We computed the Asp racemization ratio (Asp-RR = d/d + l Asp), reflecting the proportion of old to total protein, for each component. Compared with hip OA, knee OA collagen fibrils (P < 0.0001), collagen (P = 0.007), and non-collagenous proteins (P = 0.0003) had significantly lower age-adjusted mean Asp-RRs consistent with elevated protein synthesis in knee OA. Knee OA collagen had a mean hydroxyproline/proline (H/P) ratio of 1.2 consistent with the presence of type III collagen whereas hip OA collagen had a mean H/P ratio of 0.99 consistent with type II collagen. Based on Asp-RR, the relative age was significantly different in knee and hip OA (P < 0.0005); on average OA knees were estimated to be 30 yrs 'younger', and OA hips 10 yrs 'older' than non-OA. The metabolic response to OA was strikingly different by joint site. Knee OA cartilage evinced an anabolic response that appeared to be absent in hip OA cartilage. These results challenge the long held view that OA cartilage is capable of only minimal repair and that collagen loss is irreversible.

Conserved hippocampal cellular pathophysiology but distinct behavioural deficits in a new rat model of FXS.

Recent advances in techniques for manipulating genomes have allowed the generation of transgenic animals other than mice. These new models enable cross-mammalian comparison of neurological disease from core cellular pathophysiology to circuit and behavioural endophenotypes. Moreover they will enable us to directly test whether common cellular dysfunction or behavioural outcomes of a genetic mutation are more conserved across species. Using a new rat model of Fragile X Syndrome, we report that Fmr1 knockout (KO) rats exhibit elevated basal protein synthesis and an increase in mGluR-dependent long-term depression in CA1 of the hippocampus that is independent of new protein synthesis. These defects in plasticity are accompanied by an increase in dendritic spine density selectively in apical dendrites and subtle changes in dendritic spine morphology of CA1 pyramidal neurons. Behaviourally, Fmr1 KO rats show deficits in hippocampal-dependent, but not hippocampal-independent, forms of associative recognition memory indicating that the loss of fragile X mental retardation protein (FMRP) causes defects in episodic-like memory. In contrast to previous reports from mice, Fmr1 KO rats show no deficits in spatial reference memory reversal learning. One-trial spatial learning in a delayed matching to place water maze task was also not affected by the loss of FMRP in rats. This is the first evidence for conservation across mammalian species of cellular and physiological hippocampal phenotypes associated with the loss of FMRP. Furthermore, while key cellular phenotypes are conserved they manifest in distinct behavioural dysfunction. Finally, our data reveal novel information about the selective role of FMRP in hippocampus-dependent associative memory.

Kinetics of nickel bioaccumulation and its relevance to selected cellular processes in leaves of Elodea canadensis during short-term exposure.

Elodea canadensis is an aquatic macrophyte used widely as a bioindicator for the monitoring of water quality and in the phytoremediation of metal-contaminated waters. This study considers the kinetics of nickel bioaccumulation and changes in accompanying metabolic and stress-related physiological parameters. These include photosynthetic activity, pigment content, the accumulation of thiol-containing compounds, thiobarbituric acid-reactive substance (TBARS) products, and the activity of selected antioxidant enzymes (catalase, glutathione reductase, superoxide dismutase). Elodea leaves accumulated nickel according to pseudo-second-order kinetics, and the protective responses followed a time sequence which was related to the apparent rates of nickel accumulation. The applicability of second-order kinetics to the Ni uptake by Elodea leaves during the first 8 h of exposure to the metal suggested that the passive binding of metal ions (chemisorption) was a rate-limiting step at the initial phase of Ni accumulation. This phase was accompanied by an increase in photosynthetic activity together with elevated photosynthetic pigments and protein synthesis, the enhanced activity of antioxidant enzymes, and increased thiol concentration. In contrast, there was a decrease in metabolic activity upon the accumulation of TBARS, and the decline in enzyme activity was observed in the saturation phase of Ni accumulation (8-24 h). These results show that a correlation exists between the protective response and the apparent kinetic rate of Ni uptake. Thus, the time of exposure to the toxicant is a crucial factor in the activation of specific mechanisms of Ni detoxification and stress alleviation.

R-Baclofen Reverses a Social Behavior Deficit and Elevated Protein Synthesis in a Mouse Model of Fragile X Syndrome.

Background:Fragile X syndrome (FXS) is the most common known inherited form of intellectual disability and the single genomic cause of autism spectrum disorders. It is caused by the absence of a fragile X mental retardation gene (Fmr1) product, FMRP, an RNA-binding translation suppressor. Elevated rates of protein synthesis in the brain and an imbalance between synaptic signaling via glutamate and γ-aminobutyric acid (GABA) are both considered important in the pathogenesis of FXS. In a mouse model of FXS (Fmr1 knockout [KO]), treatment with R-baclofen reversed some behavioral and biochemical phenotypes. A remaining crucial question is whether R-baclofen is also able to reverse increased brain protein synthesis rates.Methods:To answer this question, we measured regional rates of cerebral protein synthesis in vivo with the L-[1-14C]leucine method in vehicle- and R-baclofen–treated wildtype and Fmr1 KO mice. We further probed signaling pathways involved in the regulation of protein synthesis.Results:Acute R-baclofen administration corrected elevated protein synthesis and reduced deficits on a test of social behavior in adult Fmr1 KO mice. It also suppressed activity of the mammalian target of rapamycin pathway, particularly in synaptosome-enriched fractions, but it had no effect on extracellular-regulated kinase 1/2 activity. Ninety min after R-baclofen treatment, we observed an increase in metabotropic glutamate receptor 5 expression in the frontal cortex, a finding that may shed light on the tolerance observed in human studies with this drug.Conclusions:Our results suggest that treatment via activation of the GABA (GABA receptor subtype B) system warrants further study in patients with FXS.