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The AKT signaling pathway plays critical roles in the resolution of inflammation. However, the underlying mechanisms of anti-inflammatory regulation and signal coordination remain unclear. Here, we report that anti-inflammatory AKT signaling is coordinated by glutamyl-prolyl-tRNA synthetase 1 (EPRS1). Upon inflammatory activation, AKT specifically phosphorylates Ser999 of EPRS1 in the cytoplasmic multi-tRNA synthetase complex, inducing release of EPRS1. EPRS1 compartmentalizes AKT to early endosomes via selective binding to the endosomal membrane lipid phosphatidylinositol 3-phosphate and assembles an AKT signaling complex specific for anti-inflammatory activity. These events promote AKT activation-mediated GSK3β phosphorylation, which increase anti-inflammatory cytokine production. EPRS1-deficient macrophages do not assemble the early endosomal complex and consequently exacerbate inflammation, decreasing the survival of EPRS1-deficient mice undergoing septic shock and ulcerative colitis. Collectively, our findings show that the housekeeping protein EPRS1 acts as a mediator of inflammatory homeostasis by coordinating compartment-specific AKT signaling.

Copper levels are known to be elevated in inflamed and malignant tissues. But the mechanism underlying this selective enrichment has been elusive. In this study, we report a axis by which inflammatory cytokines, such as IL-17, drive cellular copper uptake via the induction of a metalloreductase, STEAP4. IL-17-induced elevated intracellular copper level leads to the activation of an E3-ligase, XIAP, which potentiates IL-17-induced NFκB activation and suppresses the caspase 3 activity. Importantly, this IL-17-induced STEAP4-dependent cellular copper uptake is critical for colon tumor formation in a murine model of colitis-associated tumorigenesis and STEAP4 expression correlates with IL-17 level and XIAP activation in human colon cancer. In summary, this study reveals a IL-17-STEAP4-XIAP axis through which the inflammatory response induces copper uptake, promoting colon tumorigenesis.

Expression of inflammatory genes is determined in part by post-transcriptional regulation of mRNA metabolism but how stimulus- and transcript-dependent nuclear export influence is poorly understood. Here, we report a novel pathway in which LPS/TLR4 engagement promotes nuclear localization of IRAK2 to facilitate nuclear export of a specific subset of inflammation-related mRNAs for translation in murine macrophages. IRAK2 kinase activity is required for LPS-induced RanBP2-mediated IRAK2 sumoylation and subsequent nuclear translocation. Array analysis showed that an SRSF1-binding motif is enriched in mRNAs dependent on IRAK2 for nuclear export. Nuclear IRAK2 phosphorylates SRSF1 to reduce its binding to target mRNAs, which promotes the RNA binding of the nuclear export adaptor ALYREF and nuclear export receptor Nxf1 loading for the export of the mRNAs. In summary, LPS activates a nuclear function of IRAK2 that facilitates the assembly of nuclear export machinery to export selected inflammatory mRNAs to the cytoplasm for translation.

The mammalian cytoplasmic multi-tRNA synthetase complex (MSC) is a depot system that regulates non-translational cellular functions. Here we found that the MSC component glutamyl-prolyl-tRNA synthetase (EPRS) switched its function following viral infection and exhibited potent antiviral activity. Infection-specific phosphorylation of EPRS at Ser990 induced its dissociation from the MSC, after which it was guided to the antiviral signaling pathway, where it interacted with PCBP2, a negative regulator of mitochondrial antiviral signaling protein (MAVS) that is critical for antiviral immunity. This interaction blocked PCBP2-mediated ubiquitination of MAVS and ultimately suppressed viral replication. EPRS-haploid (Eprs+/-) mice showed enhanced viremia and inflammation and delayed viral clearance. This stimulus-inducible activation of MAVS by EPRS suggests an unexpected role for the MSC as a regulator of immune responses to viral infection.

Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors. [ABSTRACT FROM AUTHOR]

Background: Platinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers. Methods: Protein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing. Results: CD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2. Conclusions: For the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry. [ABSTRACT FROM AUTHOR]

Hypomyelinating leukodystrophy (HLD) is an autosomal recessive disorder characterized by defective central nervous system myelination. Exome sequencing of two siblings with severe cognitive and motor impairment and progressive hypomyelination characteristic of HLD revealed homozygosity for a missense single-nucleotide variant (SNV) in EPRS1 (c.4444 C > A; p.Pro1482Thr), encoding glutamyl-prolyl-tRNA synthetase, consistent with HLD15. Patient lymphoblastoid cell lines express markedly reduced EPRS1 protein due to dual defects in nuclear export and cytoplasmic translation of variant EPRS1 mRNA. Variant mRNA exhibits reduced METTL3 methyltransferase-mediated writing of N6-methyladenosine (m6A) and reduced reading by YTHDC1 and YTHDF1/3 required for efficient mRNA nuclear export and translation, respectively. In contrast to current models, the variant does not alter the sequence of m6A target sites, but instead reduces their accessibility for modification. The defect was rescued by antisense morpholinos predicted to expose m6A sites on target EPRS1 mRNA, or by m6A modification of the mRNA by METTL3-dCas13b, a targeted RNA methylation editor. Our bioinformatic analysis predicts widespread occurrence of SNVs associated with human health and disease that similarly alter accessibility of distal mRNA m6A sites. These results reveal a new RNA-dependent etiologic mechanism by which SNVs can influence gene expression and disease, consequently generating opportunities for personalized, RNA-based therapeutics targeting these disorders. [ABSTRACT FROM AUTHOR]

Low-grade systemic inflammation is often associated with metabolic syndrome, which plays a critical role in the development of the obesity-associated inflammatory diseases, including insulin resistance and atherosclerosis. Here, we investigate how Toll-like receptor-MyD88 signaling in myeloid and endothelial cells coordinately participates in the initiation and progression of high fat diet-induced systemic inflammation and metabolic inflammatory diseases. MyD88 deficiency in myeloid cells inhibits macrophage recruitment to adipose tissue and their switch to an M1-like phenotype. This is accompanied by substantially reduced diet-induced systemic inflammation, insulin resistance, and atherosclerosis. MyD88 deficiency in endothelial cells results in a moderate reduction in diet-induced adipose macrophage infiltration and M1 polarization, selective insulin sensitivity in adipose tissue, and amelioration of spontaneous atherosclerosis. Both in vivo and ex vivo studies suggest that MyD88-dependent GM-CSF production from the endothelial cells might play a critical role in the initiation of obesity-associated inflammation and development of atherosclerosis by priming the monocytes in the adipose and arterial tissues to differentiate into M1-like inflammatory macrophages. Collectively, these results implicate a critical MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases.

Viruses are obligate, intracellular parasites that co-opt host cell machineries for propagation. Critical among these machineries are those that translate RNA into protein and their mechanisms of control. Most regulatory mechanisms effectuate their activity by targeting sequence or structural features at the RNA termini, i.e., at the 5′ or 3′ ends, including the untranslated regions (UTRs). Translation of most eukaryotic mRNAs is initiated by 5′ cap-dependent scanning. In contrast, many viruses initiate translation at internal RNA regions at internal ribosome entry sites (IRESs). Eukaryotic mRNAs often contain upstream open reading frames (uORFs) that permit condition-dependent control of downstream major ORFs. To offset genome compression and increase coding capacity, some viruses take advantage of out-of-frame overlapping uORFs (oORFs). Lacking the essential machinery of protein synthesis, for example, ribosomes and other translation factors, all viruses utilize the host apparatus to generate virus protein. In addition, some viruses exhibit RNA elements that bind host regulatory factors that are not essential components of the translation machinery. SARS-CoV-2 is a paradigm example of a virus taking advantage of multiple features of eukaryotic host translation control: the virus mimics the established human GAIT regulatory element and co-opts four host aminoacyl tRNA synthetases to form a stimulatory binding complex. Utilizing discontinuous transcription, the elements are present and identical in all SARS-CoV-2 subgenomic RNAs (and the genomic RNA). Thus, the virus exhibits a post-transcriptional regulon that improves upon analogous eukaryotic regulons, in which a family of functionally related mRNA targets contain elements that are structurally similar but lacking sequence identity. This "thrifty" virus strategy can be exploited against the virus since targeting the element can suppress the expression of all subgenomic RNAs as well as the genomic RNA. Other 3′ end viral elements include 3′-cap-independent translation elements (3′-CITEs) and 3′-tRNA-like structures. Elucidation of virus translation control elements, their binding proteins, and their mechanisms can lead to novel therapeutic approaches to reduce virus replication and pathogenicity. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (aaRSs) are ancient enzymes that serve a foundational role in the efficient and accurate translation of genetic information from messenger RNA to proteins. These proteins play critical, non-canonical functions in a multitude of cellular processes. Multiple viruses are known to hijack the functions of aaRSs for proviral outcomes, while cells modify antiviral responses through non-canonical functions of certain synthetases. Recent findings have revealed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronaviral disease 19 (COVID-19), utilizes canonical and non-canonical functions of aaRSs, establishing a complex interplay of viral proteins, cellular factors and host aaRSs. In a striking example, an unconventional multi-aaRS complex consisting of glutamyl-prolyl-, lysyl-, arginyl- and methionyl-tRNA synthetases interact with a previously unknown RNA-element in the 30-end of SARSCoV-2 genomic and subgenomic RNAs. This review aims to highlight the aaRS-SARSCoV-2 interactions identified to date, with possible implications for the biology of host aaRSs in SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, generates multiple protein-coding, subgenomic RNAs (sgRNAs) from a longer genomic RNA, all bearing identical termini with poorly understood roles in regulating viral gene expression. Insulin and interferon-gamma, two host-derived, stress-related agents, and virus spike protein, induce binding of glutamyl-prolyl-tRNA synthetase (EPRS1), within an unconventional, tetra-aminoacyl-tRNA synthetase complex, to the sgRNA 3′-end thereby enhancing sgRNA expression. We identify an EPRS1-binding sarbecoviral pan-end activating RNA (SPEAR) element in the 3′-end of viral RNAs driving agonist-induction. Translation of another co-terminal 3′-end feature, ORF10, is necessary for SPEAR-mediated induction, independent of Orf10 protein expression. The SPEAR element enhances viral programmed ribosomal frameshifting, thereby expanding its functionality. By co-opting noncanonical activities of a family of essential host proteins, the virus establishes a post-transcriptional regulon stimulating global viral RNA translation. A SPEAR-targeting strategy markedly reduces SARS-CoV-2 titer, suggesting a pan-sarbecoviral therapeutic modality. Here, Khan et al. identify a pan-genome-end sarbecoviral RNA element in SARS-CoV-2 that recruits an unconventional host multiprotein-complex to enhance viral translation and assigns a new function to ORF10. Antisense targeting of the element points to a potential novel therapeutic modality. [ABSTRACT FROM AUTHOR]

Recent studies have indicated that high-density lipoproteins (HDLs) and their major structural protein, apolipoprotein A1 (apoA1), recovered from human atheroma are dysfunctional and are extensively oxidized by myeloperoxidase (MPO). In [...]

Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein--associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2009.03.004 Byline: Rupak Mukhopadhyay (1), Jie Jia (1), Abul Arif (1), Partho Sarothi Ray (1)(2), Paul L. Fox (1) Abstract: Functionally related genes are coregulated by specific RNA-protein interactions that direct transcript-selective translational control. In myeloid cells, interferon (IFN)-[gamma] induces formation of the heterotetrameric, IFN-[gamma]-activated inhibitor of translation (GAIT) complex comprising glutamyl-prolyl tRNA synthetase (EPRS), NS1-associated protein 1 (NSAP1), ribosomal protein L13a and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This complex binds defined 3' untranslated region elements within a family of inflammatory mRNAs and suppresses their translation. IFN-[gamma]-dependent phosphorylation, and consequent release of EPRS and L13a from the tRNA multisynthetase complex and 60S ribosomal subunit, respectively, regulates GAIT complex assembly. EPRS recognizes and binds target mRNAs, NSAP1 negatively regulates RNA binding, and L13a inhibits translation initiation by binding eukaryotic initiation factor 4G. Repression of a post-transcriptional regulon by the GAIT system might contribute to the resolution of chronic inflammation. Author Affiliation: (1) Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, NC10, Cleveland, OH, 44195, USA (2) Department of Biology, Indian Institute of Science Education and Research, Kolkata 700106, India

Author(s): Abul Arif [1]; Fulvia Terenzi [1]; Alka A. Potdar [2]; Jie Jia [1]; Jessica Sacks [1]; Arnab China [1]; Dalia Halawani [1]; Kommireddy Vasu [1]; Xiaoxia Li [3]; J. [...]

Multi-component, macromolecular complexes perform essential cellular functions that require spatial or temporal coordination of activities. Complexes also facilitate co-regulation of protein amounts and cellular localization of individual components. We propose a novel function of multi-component complexes as depots for regulatory proteins that, upon release, acquire new auxiliary functions. We further propose that component release is inducible and context-dependent. We describe two cases in which multi-component assemblies--the ribosome and tRNA multi-synthetase complex--function as depots. Both complexes have crucial roles in supporting protein synthesis but they also release regulatory proteins for inflammation-responsive, transcript-specific translational control. Recent evidence indicates that other macromolecular assemblies might be sources for proteins with auxiliary functions, and the depot mechanism might be widespread in nature.