Enhanced Protein Translation Research Articles

TOR and RPS6 transmit light signals to enhance protein translation in deetiolating Arabidopsis seedlings.

Deetiolation is an essential developmental process transforming young plant seedlings into the vegetative phase with photosynthetic activities. Light signals initiate this important developmental process by triggering massive reprogramming of the transcriptome and translatome. Compared with the wealth of knowledge of transcriptional regulation, the molecular mechanism underlying this light-triggered translational enhancement remains unclear. Here we show that light-enhanced translation is orchestrated by a light perception and signaling pathway composed of photoreceptors, CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1), the phytohormone auxin, target of rapamycin (TOR), and ribosomal protein S6 (RPS6). In deetiolating Arabidopsis seedlings, photoreceptors, including phytochrome A and cryptochromes, perceive far-red and blue light to inactivate the negative regulator COP1, which leads to activation of the auxin pathway for TOR-dependent phosphorylation of RPS6. Arabidopsis mutants defective in TOR, RPS6A, or RPS6B exhibited delayed cotyledon opening, a characteristic of the deetiolating process to ensure timely vegetative development of a young seedling. This study provides a mechanistic view of light-triggered translational enhancement in deetiolating Arabidopsis.

MTOR‐dependent selective translation rapidly expands lysosome biogenesis, volume and retention capacity during phagocyte activatio

The molecular mechanisms that govern and adapt the number, size, morphology and activities of organelles to suit the needs of the many cell types and conditions that these cells may encounter are at the frontier of cell biology. Lysosomes are organelles that degrade cargo from various routes including endocytosis, phagocytosis and autophagy. Lysosomes have also emerged as a signalling platform that senses and integrates stress signals such as nutrient deprivation with regulatory kinase hubs like mTOR and AMPK to modulate metabolic activity. For phagocytes and antigen‐presenting cells like macrophages and dendritic cells, lysosomes are a kingpin organelle since they are essential to kill pathogens, and process and ultimately present antigens. During phagocyte activation with lipopolysaccharides, lysosomes undergo a striking reorganization, changing from dozens of globular objects to a tubular network, in a process that requires the phosphatidylinositol‐3‐kinase‐Akt‐mTOR signalling axis. Ultimately, lysosome tubulation is thought to promote pinocytic retention and antigen presentation. Here, we show that lysosome tubulation is accompanied by a rapid boost in lysosome volume and holding capacity of phagocyte activation. We reveal that lysosome expansion depends on biosynthesis of lysosomal proteins but is independent of TFEB and TFE3, transcription factors known to scale up lysosome biogenesis. Instead, we show that lysosome transcripts are selectively targeted for enhanced protein translation in an mTOR‐dependent manner. Indeed, lysosome transcripts were enriched in polyribosome fractions after lipopolysaccharide exposure in an mTOR‐dependent manner, while transcripts encoding non‐lysosomal proteins were unresponsive to LPS and/or mTOR inhibition. Collectively, we identified an example of rapid organelle expansion and remodelling driven by enhanced and selective protein translation during phagocyte activation.Support or Funding InformationCanadian Institutes of Health Research, Canada Research Chair Program, and Ryerson UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation.

SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest.

Protein Translation and Signaling in Human Eosinophils.

We have recently reported that, unlike IL-5 and GM-CSF, IL-3 induces increased translation of a subset of mRNAs. In addition, we have demonstrated that Pin1 controls the activity of mRNA binding proteins, leading to enhanced mRNA stability, GM-CSF protein production and prolonged eosinophil (EOS) survival. In this review, discussion will include an overview of cap-dependent protein translation and its regulation by intracellular signaling pathways. We will address the more general process of mRNA post-transcriptional regulation, especially regarding mRNA binding proteins, which are critical effectors of protein translation. Furthermore, we will focus on (1) the roles of IL-3-driven sustained signaling on enhanced protein translation in EOS, (2) the mechanisms regulating mRNA binding proteins activity in EOS, and (3) the potential targeting of IL-3 signaling and the signaling leading to mRNA binding activity changes to identify therapeutic targets to treat EOS-associated diseases.

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions.

Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5' nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation.IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure.

Enhanced Protein Translation Underlies Improved Metabolic and Physical Adaptations to Different Exercise Training Modes in Young and Old Humans

The molecular transducers of benefits from different exercise modalities remain incompletely defined. Here we report that 12weeks of high-intensity aerobic interval (HIIT), resistance (RT), and combined exercise training enhanced insulin sensitivity and lean mass, but only HIIT and combined training improved aerobic capacity and skeletal muscle mitochondrial respiration. HIIT revealed a more robust increase ingene transcripts than other exercise modalities, particularly in older adults, although little overlap with corresponding individual protein abundance was noted. HIIT reversed many age-related differences in the proteome, particularly of mitochondrial proteins in concert with increased mitochondrial protein synthesis. Both RT and HIIT enhanced proteins involved in translational machinery irrespective of age. Only small changes of methylation of DNA promoter regions were observed. We provide evidence for predominant exercise regulation at the translational level, enhancing translational capacity and proteome abundance to explain phenotypic gains in muscle mitochondrial function and hypertrophy in all ages.

AlkB homolog 3-mediated tRNA demethylation promotes protein synthesis in cancer cells

The mammalian AlkB homolog (ALKBH) family of proteins possess a 2-oxoglutarate- and Fe(II)-dependent oxygenase domain. A similar domain in the Escherichia coli AlkB protein catalyzes the oxidative demethylation of 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) in both DNA and RNA. AlkB homolog 3 (ALKBH3) was also shown to demethylate 1-meA and 3-meC (induced in single-stranded DNA and RNA by a methylating agent) to reverse the methylation damage and retain the integrity of the DNA/RNA. We previously reported the high expression of ALKBH3 in clinical tumor specimens and its involvement in tumor progression. In this study, we found that ALKBH3 effectively demethylated 1-meA and 3-meC within endogenously methylated RNA. Moreover, using highly purified recombinant ALKBH3, we identified N6-methyladenine (N6-meA) in mammalian transfer RNA (tRNA) as a novel ALKBH3 substrate. An in vitro translation assay showed that ALKBH3-demethylated tRNA significantly enhanced protein translation efficiency. In addition, ALKBH3 knockdown in human cancer cells impaired cellular proliferation and suppressed the nascent protein synthesis that is usually accompanied by accumulation of the methylated RNAs. Thus, our data highlight a novel role for ALKBH3 in tumor progression via RNA demethylation and subsequent protein synthesis promotion.

Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5′- and 3′-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5′-UTR and/or downstream 3′-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.

CSIG-25. MiR-338-3p AS A TUMOR SUPPRESSOR THROUGH Pttg MODULATION IN GH3 CELL LINES

MicroRNAs (miRNA) are relatively small nucleotide composed of 20-30 nucleotide which noncoding RNAs that are found in the genomes of animal, plants, and protozoa. miRNAs regulate post-transcriptional expression of target genes predominantly by inhibiting or enhancing protein translation or degrading the target mRNA. Recent studies suggest that aberrant microRNA expression is common in numerous malignancies. Although miR-338-3p has been implicated in several gastrointestinal cancers, its role in pituitary adenoma is unknown. We treated miR-338-3p mimic and inhibitor on rat Growth Hormone (GH) secreting cell line (GH3) to invastigate the biological role of expression level alteration of miR-338-3p. After treatment of mimic and inhibitor, quantitative RT-PCRs(QPCRs) were performed with several targets, that are predicted to affect both hormone secreting system and tumorigenesis. Western blotting was also performed to double-check the results from QPCRs. Evaluating the effect of miR-338-3p on invasiveness, microRNA microarray was performed with 6 patients. The miRNA Microarray System with miRNA Complete Labeling and hybridization Kit (Agilent Technologies) was used according to the manufacturer′s recommended protocol. There were some research about predictive markers of pituitary adenoma behavior, but most of them were restricted to non-functioning pituitary. Although the assumption of prognosis of pituitary adenoma, there was no known about behavior marker for GH secretory adenoma. Lately, microRNA has become of general interest as a cancer biomarker. Our research on several targets, such as prolactin, growth hormone, pttg1, and, gpr101 after mimic and inhibitor of miRNA-338 imply that miRNA-338 could be involved in hormone secretion system and tumorigenesis. FACs data shows good chance of miR-338-3p’s role of cell cycle regulator. In patient sample, mir-338-3p expression level was relatively high, which means it is also related to invasiveness of pituitary adenomas. We need more experiments to form organic connection among our data, for there are no sufficient additional evidences that support them.

Cyfip1 Regulates Presynaptic Activity during Development.

Several developmental brain disorders have been associated with gene duplications and deletions that serve to increase or decrease levels of encoded proteins. Cyfip1 is one such protein, but the role it plays in brain development is poorly understood. We asked whether decreased Cyfip1 levels altered the function of developing synapses. The data show that synapses with reduced Cyfip1 are larger and release neurotransmitter more rapidly. These effects are due to Cyfip1's role in actin polymerization and are reversed by expression of a Cyfip1 mutant protein retaining actin regulatory function or by inhibiting Rac1. Thus, Cyfip1 has a more prominent early role regulating presynaptic activity during a stage of development when activity helps to define neural pathways.

MTOR activation is a biomarker and a central pathway to autoimmune disorders, cancer, obesity, and aging.

The mechanistic target of rapamycin (mTOR) is a ubiquitous serine/threonine kinase, which plays pivotal roles in integrating growth signals on a cellular level. To support proliferation and survival under stress, two interacting complexes that harbor mTOR, mTORC1 and mTORC2, promote the transcription of genes involved in carbohydrate metabolism and lipogenesis, enhance protein translation, and inhibit autophagy. Although rapamycin was originally developed as an inhibitor of T cell proliferation for preventing organ transplant rejection, its molecular target, mTOR, has been subsequently identified as a central regulator of metabolic cues that drive lineage specification in the immune system. Owing to oxidative stress, the activation of mTORC1 has emerged as a central pathway for the pathogenesis of systemic lupus erythematosus and other autoimmune diseases. Paradoxically, mTORC1 has also been identified as a mediator of the Warburg effect that allows cell survival under hypoxia. Rapamycin and new classes of mTOR inhibitors are being developed to block not only transplant rejection and autoimmunity but also to treat obesity and various forms of cancer. Through preventing these diseases, personalized mTOR blockade holds promise to extend life span.

MTOR Silencing in Parkinson's Disease both in vitro and in vivo

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates processes including mRNA translation, proliferation, and survival. As a central element signaling cell growth and enhancing protein translation, (mTOR), when inhibited, induces autophagy. Moreover, as a critical feedback mechanism, reactivation of mTOR terminates autophagy and initiates lysosome reformation

Kaposi's Sarcoma-Associated Herpesvirus ORF45 Mediates Transcriptional Activation of the HIV-1 Long Terminal Repeat via RSK2

Robust activation of human immunodeficiency virus type 1 (HIV-1) gene expression occurs upon superinfection with Kaposi's sarcoma-associated herpesvirus (KSHV), a common AIDS-associated pathogen. Though the mechanisms underlying this phenotype remain unknown, several KSHV-encoded factors have been reported to stimulate HIV-1 long terminal repeat (LTR) activity. Here, we systematically evaluated the ability of KSHV tegument proteins to modulate the activation of an integrated HIV-1 LTR and revealed that the most potent individual activator is ORF45. ORF45 directs an increase in RNA polymerase II recruitment to the HIV-1 LTR, leading to enhanced transcriptional output. ORF45 is a robust activator of the p90 ribosomal S6 kinases (RSK), and we found that this activity is necessary but not sufficient to increase transcription from the LTR. Of the three widely expressed RSK isoforms, RSK2 appears to be selectively involved in LTR stimulation by both KSHV ORF45 and HIV-1 Tat. However, constitutively active RSK2 is unable to stimulate the LTR, suggesting that ORF45 may preferentially direct this kinase to a specific set of targets. Collectively, our findings reveal a novel transcriptional activation function for KSHV ORF45 and highlight the importance of RSK2 in shaping the transcriptional environment during infection. Kaposi's sarcoma-associated herpesvirus (KSHV) is a prominent AIDS-associated pathogen. Previous studies have shown that infection of cells containing human immunodeficiency virus type 1 (HIV-1) with KSHV leads to potent stimulation of HIV-1 gene expression by activating the HIV-1 promoter, termed the long terminal repeat (LTR). Here, we compared the abilities of various KSHV proteins to activate gene expression from the HIV-1 LTR and found that KSHV ORF45 is the most potent activator. ORF45 is known to induce cell signaling through ribosomal S6 kinase (RSK) and enhance protein translation. However, we revealed that the activation of a specific isoform of RSK by ORF45 also leads to increased mRNA synthesis from the LTR by the host RNA polymerase. Collectively, our findings provide new insight into the interviral interactions between KSHV and HIV that may ultimately impact disease.

What Does a Deep Molecular Response Signify?

What Does a Deep Molecular Response Signify?

Abstract 2160: Regulation of DHX33 during oncogenic stress and its crucial role in Ras-initiated tumor formation

Abstract Previously we found that DHX33 is pivotal for RNA polymerase I transcription, changes in DHX33 levels have a dramatic impact on RNA polymerase I activity. In this report, we explore the regulation of DHX33 protein levels by ARF/p53 pathway and Ras signaling. In wild type primary fibroblasts, Ras V12 infection induced a transient increase in DHX33 protein level as well as rRNA transcriptional rate which was eventually overcome by suppression from ARF/p53 pathway. ARF/p53 dependent reduction of DHX33 occurred at the translational level, while Ras activation induced DHX33 levels both at the level of transcription and translation. The induction of DHX33 by Ras activation is sensitive to inhibitors of PI3K, mTOR as well as MAPK. Induction of DHX33 by Ras V12 is pivotal for enhanced rDNA transcription, enhanced protein translation, and thus, the oncogenecity of Ras V12, as DHX33 knockdown abolished the increase in rRNA transcription and protein translation, and completely inhibited the tumorigenecity of Ras V12 both in semisolid agar and xenografts. Our results implicate that DHX33 level could be an important factor to adjust rDNA transcriptional rate in response to ARF induction and Ras activation. Most importantly, we identified DHX33 as a novel downstream target of Ras signaling to regulate rRNA transcription and pointed toward its pivotal role in Ras-initiated tumor formation and therapeutic value to treat Ras mutated cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2160. doi:1538-7445.AM2012-2160

Leucine and mTORC1: a complex relationship

Amino acid availability is a rate-limiting factor in the regulation of protein synthesis. When amino acid supplies become restricted, mammalian cells employ homeostatic mechanisms to rapidly inhibit processes such as protein synthesis, which demands high levels of amino acids. Muscle cells in particular are subject to high protein turnover rates to maintain amino acid homeostasis. Mammalian target of rapamycin complex 1 (mTORC1) is an evolutionary conserved multiprotein complex that coordinates a network of signaling cascades and functions as a key mediator of protein translation, gene transcription, and autophagy. Signal transduction through mTORC1, which is centrally involved in muscle growth through enhanced protein translation, is governed by intracellular amino acid supply. The branched-chain amino acid leucine is critical for muscle growth and acts in part through activation of mTORC1. Recent research has revealed that mTORC1 signaling is coordinated primarily at the lysosomal membranes. This discovery has sparked a wealth of research in this field, revealing several different signaling molecules involved in transducing the amino acid signal to mTORC1, including the Rag GTPases, MAP4K3, and Vps34/ULK1. This review evaluates the current knowledge regarding cellular mechanisms that control and sense the intracellular amino acid pool. We discuss the role of leucine and mTORC1 in the regulation of amino acid transport via the system L and system A transporters such as LAT1 and SNAT2, as well as protein degradation via autophagic and proteasomal pathways. We also describe the complexities of energy homeostasis via AMPK and cell receptor-mediated growth signals that also converge on mTORC1. Leucine is a particularly potent regulator of protein turnover, to the extent where leucine stimulation alone is sufficient to stimulate mTORC1 signal transduction. The significance of leucine in this context is not yet known; however, recent advancements in this area will also be covered within this review.

Does Branched-Chain Amino Acids Supplementation Modulate Skeletal Muscle Remodeling through Inflammation Modulation? Possible Mechanisms of Action

Skeletal muscle protein turnover is modulated by intracellular signaling pathways involved in protein synthesis, degradation, and inflammation. The proinflammatory status of muscle cells, observed in pathological conditions such as cancer, aging, and sepsis, can directly modulate protein translation initiation and muscle proteolysis, contributing to negative protein turnover. In this context, branched-chain amino acids (BCAAs), especially leucine, have been described as a strong nutritional stimulus able to enhance protein translation initiation and attenuate proteolysis. Furthermore, under inflammatory conditions, BCAA can be transaminated to glutamate in order to increase glutamine synthesis, which is a substrate highly consumed by inflammatory cells such as macrophages. The present paper describes the role of inflammation on muscle remodeling and the possible metabolic and cellular effects of BCAA supplementation in the modulation of inflammatory status of skeletal muscle and the consequences on protein synthesis and degradation.

Protein Kinase A Activity at the Endoplasmic Reticulum Surface Is Responsible for Augmentation of Human ether-a-go-go-related Gene Product (HERG)

Human ether-a-go-go-related gene product (HERG) is a cardiac potassium channel commonly implicated in the pathogenesis of the long QT syndrome, type 2 (LQT2). LQT2 mutations typically have incomplete penetrance and affect individuals at various stages of their lives; this may mirror variations in intracellular signaling and HERG regulation. Previous work showed that sustained protein kinase A (PKA) activity augments HERG protein abundance by a mechanism that includes enhanced protein translation. To investigate the subcellular site of this regulation, we generated site-specific probes to the cytoplasmic surface of the endoplasmic reticulum (ER), the presumed locale of channel synthesis. Real-time FRET-based indicators demonstrated both cAMP and PKA activity at the ER. A PKA inhibitor targeted to the ER surface (termed p4PKIg) completely abolished PKA-mediated augmentation of HERG in HEK293 cells as well as rat neonatal cardiomyocytes. Immunofluorescence co-localization, targeted FRET-based PKA biosensors, phospho-specific antibodies, and in vivo phosphorylation experiments confirmed that p4PKIg is preferentially active at the ER surface rather than the plasma membrane. Rerouting this inhibitor to the outer mitochondrial membrane diminishes its ability to block cAMP-dependent HERG induction. Our results support a model where PKA-dependent regulation of HERG synthesis occurs at the ER surface. Furthermore, reagents generated for this study provide novel experimental tools to probe compartmentalized cAMP/PKA signaling within cells.

S1P lyase: a novel therapeutic target for ischemia-reperfusion injury of the heart

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes cardiomyocyte survival and contributes to ischemic preconditioning. S1P lyase (SPL) is a stress-activated enzyme responsible for irreversible S1P catabolism. We hypothesized that SPL contributes to oxidative stress by depleting S1P pools available for cardioprotective signaling. Accordingly, we evaluated SPL inhibition as a strategy for reducing cardiac ischemia-reperfusion (I/R) injury. We measured SPL expression and enzyme activity in murine hearts. Basal SPL activity was low in wild-type cardiac tissue but was activated in response to 50 min of ischemia (n = 5, P < 0.01). Hearts of heterozygous SPL knockout mice exhibited reduced SPL activity, elevated S1P levels, smaller infarct size, and increased functional recovery after I/R compared with littermate controls (n = 5, P < 0.01). The small molecule tetrahydroxybutylimidazole (THI) is a Federal Drug Administration-approved food additive that inhibits SPL. When given overnight at 25 mg/l in drinking water, THI raised S1P levels and reduced SPL activity (n = 5, P < 0.01). THI reduced infarct size and enhanced hemodynamic recovery in response to 50 min of ischemia and to 40 min of reperfusion in ex vivo hearts (n = 7, P < .01). These data correlated with an increase in MAP kinase-interacting serine/threonine kinase 1, eukaryotic translation initiation factor 4E, and ribosomal protein S6 phosphorylation levels after I/R, suggesting that SPL inhibition enhances protein translation. Pretreatment with an S1P₁ and S1P₃ receptor antagonist partially reversed the effects of THI. These results reveal, for the first time, that SPL is an ischemia-induced enzyme that can be targeted as a novel strategy for preventing cardiac I/R injury.

Cell Death–Mediated Cleavage of the Attraction Signal p43 in Human Atherosclerosis

Apoptosis is a key feature of advanced atherosclerotic plaques. Attraction signals such as p43 released from apoptotic cells play a crucial role in the timely removal of the apoptotic remnants by recruiting fresh phagocytes. Here, we sought to determine whether p43 may link apoptosis to inflammation and plaque progression. RT-PCR and immunohistochemistry showed that p43 was abundantly expressed in human plaques compared with nonatherosclerotic mammary arteries and colocalized with splicing factor SC-35. Cell culture experiments indicated that p43 expression was associated with enhanced protein translation. On initiation of apoptosis or necrosis, p43 was cleaved by calpains and released as truncated protein p43(apoptosis-released factor [ARF]). Processing of p43 into endothelial monocyte activating polypeptide II was not observed. Full-length p43, but not p43(ARF) or endothelial monocyte activating polypeptide II, activated THP1 monocytes (upregulation of tumor necrosis factor alpha, interleukin 1 beta, interleukin 8, macrophage inflammatory protein (MIP)-1 alpha, MIP1 beta, MIP2 alpha) and endothelial cells (enhanced synthesis of E-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, tissue factor). The chemotactic activity of p43 or fragments thereof was poor compared with ATP. Treatment of smooth muscle cells with p43 did not induce cell death. p43 is cleaved during apoptosis by calpains and released as a truncated protein that is harmless for the structure of the plaque.