Protective Effects of Human and Mouse Soluble Scavenger-Like CD6 Lymphocyte Receptor in a Lethal Model of Polymicrobial Sepsis

Sepsis still constitutes an unmet clinical need, which could benefit from novel adjunctive strategies to conventional antibiotic therapy. The soluble form of the scavenger-like human CD6 lymphocyte receptor (shCD6) binds to key pathogenic components from Gram-positive and -negative bacteria and shows time- and dose-dependent efficacy in mouse models of monobacterial sepsis. The objective of the present work was to demonstrate the effectiveness of infusing mouse and human sCD6 by different systemic routes, either alone or as adjunctive therapy to gold standard antibiotics, in a lethal model of polymicrobial sepsis. To this end, C57BL/6 mice undergoing high-grade septic shock induced by cecal ligation and puncture (CLP; ≥90% lethality) were infused via the intraperitoneal (i.p.) or intravenous (i.v.) route with shCD6 at different doses and time points, either alone or in combination with imipenem/cilastatin (I/C) at a dose of 33 mg/kg of body weight every 8 h. Significantly reduced mortality and proinflammatory cytokine levels were observed by i.p. infusion of a single shCD6 dose (1.25 mg/kg) 1 h pre- or post-CLP. When using the i.v. route, mice survival was significantly extended by starting shCD6 infusion at later time points post-CLP (up to 6 h after CLP). Significant adjunctive effects on mouse survival were observed by i.p. or i.v. infusion of shCD6 in combination with i.p. I/C post-CLP. Similar results were obtained in mice expressing high sustained levels (5 to 10 μg/ml) of mouse sCD6 in serum by means of transduction with hepatotropic adeno-associated virus (AAV). Taken together, the data support the conserved antibacterial effects of human and mouse sCD6 and their use as adjunctive therapy in experimental models of complex and severe polymicrobial sepsis.Ministerio de Economía y Competitividad (España) SAF2013-46151-R PCIN-2015-070Instituto de Salud Carlos III RD12/0015/0018European Development Regional Fund RD12/0015/0018Fundació La Marató TV3 201319-30-3

Trypanosoma brucei PRMT1 Is a Nucleic Acid Binding Protein with a Role in Energy Metabolism and the Starvation Stress Response.

In Trypanosoma brucei and related kinetoplastid parasites, transcription of protein coding genes is largely unregulated. Rather, mRNA binding proteins, which impact processes such as transcript stability and translation efficiency, are the predominant regulators of gene expression. Arginine methylation is a posttranslational modification that preferentially targets RNA binding proteins and is, therefore, likely to have a substantial impact on T. brucei biology. The data presented here demonstrate that cells depleted of T. brucei PRMT1 (TbPRMT1), a major type I protein arginine methyltransferase, exhibit decreased virulence in an animal model. To understand the basis of this phenotype, quantitative global proteomics was employed to measure protein steady-state levels in cells lacking TbPRMT1. The approach revealed striking changes in proteins involved in energy metabolism. Most prominent were a decrease in glycolytic enzyme abundance and an increase in proline degradation pathway components, changes that resemble the metabolic remodeling that occurs during T. brucei life cycle progression. The work describes several RNA binding proteins whose association with mRNA was altered in TbPRMT1-depleted cells, and a large number of TbPRMT1-interacting proteins, thereby highlighting potential TbPRMT1 substrates. Many proteins involved in the T. brucei starvation stress response were found to interact with TbPRMT1, prompting analysis of the response of TbPRMT1-depleted cells to nutrient deprivation. Indeed, depletion of TbPRMT1 strongly hinders the ability of T. brucei to form cytoplasmic mRNA granules under starvation conditions. Finally, this work shows that TbPRMT1 itself binds nucleic acids in vitro and in vivo, a feature completely novel to protein arginine methyltransferases.IMPORTANCETrypanosoma brucei infection causes human African trypanosomiasis, also known as sleeping sickness, a disease with a nearly 100% fatality rate when untreated. Current drugs are expensive, toxic, and highly impractical to administer, prompting the community to explore various unique aspects of T. brucei biology in search of better treatments. In this study, we identified the protein arginine methyltransferase (PRMT), TbPRMT1, as a factor that modulates numerous aspects of T. brucei biology. These include glycolysis and life cycle progression signaling, both of which are being intensely researched toward identification of potential drug targets. Our data will aid research in those fields. Furthermore, we demonstrate for the first time a direct association of a PRMT with nucleic acids, a finding we believe could translate to other organisms, including humans, thereby impacting research in fields as distant as human cancer biology and immune response modulation. Copyright © 2018 Kafková et al

Decapping complex is essential for functional P-body formation and is buffered by nuclear localization

mRNA decay is a key step in regulating the cellular proteome. Cytoplasmic mRNA is largely turned over in processing bodies (P-bodies). P-body units assemble to form P-body granules under stress conditions. How this assembly is regulated, however, remains still poorly understood. Here, we show that the translational repressor Scd6 and the decapping stimulator Edc3 act partially redundantly in P-body assembly by capturing the Dcp1/2 decapping complex and preventing it from becoming imported into the nucleus by the karyopherin ß Kap95. Nuclear Dcp1/2 does not drive mRNA decay and might be stored there as a ready releasable pool, indicating a dynamic equilibrium between cytoplasmic and nuclear Dcp1/2. Cytoplasmic Dcp1/2 is linked to Dhh1 via Edc3 and Scd6. Functional P-bodies are present at the endoplasmic reticulum where Dcp2 potentially acts to increase the local concentration of Dhh1 through interaction with Scd6 and Edc3 to drive phase separation and hence P-body formation

Regulation of processing body formation in the budding yeast "Saccharomyces cerevisiae"

The life cycle of mRNA, from transcription to decay, is a tightly regulated biological process. mRNA is an essential molecule that the cell utilizes to alter gene expression. Degradation is one process through which the cell can regulate mRNA levels. P-bodies constitute the main mRNA decay pathway in yeast cells and are induced in response to various stresses. The presented work aimed to create a better understanding of P-body localization and regulation and to uncover the mRNAs contained within them. A former graduate student in the lab examined P-body formation in secretory mutants and how this stress differed from other stresses such as starvation. Surprisingly, she found numerous P-bodies were induced in secretory mutants. Similarly, the addition of Ca2+ was able to phenocopy the increased P-body induction as seen in secretory mutants. The calcium-binding protein calmodulin, as well as the core P-body proteins Scd6 and Pat1, were required for the formation of these P-bodies. In addition, different pathways appeared to control P-body number. Lastly, we observed that in yeast, P-bodies occur in close proximity to the ER, implying that the ER may play a role in post-transcriptional regulation. This finding that P-bodies were associated with the ER led us to examine proteins that may regulate the formation of P-bodies. Through a crosslinking tandem affinity purification approach we were able to identify several P-body components as well as two polysome-associated ER localized proteins: Scp160 and Bfr1. We went on to show that Scp160 is able to associate with P-body components in an mRNA dependent manner at polysomes. Loss of either BFR1 or SCP160 caused the formation of many Dcp2 positive foci under normal growth conditions, suggesting that Bfr1 and Scp160 may be inhibiting the formation of P-bodies. Furthermore, in ?scp160 cells P-bodies failed to properly assemble, indicating the Scp160 is required for P-body assembly. Despite the increase in P-body number under normal growth conditions, general translation was unaffected, thus, uncoupling P-body formation and translation attenuation. Taken together, our results suggest that Bfr1 and Scp160 inhibit P-body formation under normal growth conditions, possibly by limiting the mRNA that is passed from polysomes to P-bodies. Lastly, we modified a published protocol to uncover the mRNAs that are contained within P-bodies under various stresses. Several rounds of trials and changes were performed and although these modifications were not sufficient to produce a library for deep sequencing, our group has further developed the method and now has a reliably working protocol

Physical Interactions With Bacteria and Protozoan Parasites Establish the Scavenger Receptor SSC4D as a Broad-Spectrum Pattern Recognition Receptor

Since the pioneering discoveries, by the Nobel laureates Jules Hoffmann and Bruce Beutler, that Toll and Toll-like receptors can sense pathogenic microorganisms and initiate, in vertebrates and invertebrates, innate immune responses against microbial infections, many other families of pattern recognition receptors (PRRs) have been described. One of such receptor clusters is composed by, if not all, at least several members of the scavenger receptor cysteine-rich (SRCR) superfamily. Many SRCR proteins are plasma membrane receptors of immune cells; however, a small subset consists of secreted receptors that are therefore in circulation. We here describe the first characterization of biological and functional roles of the circulating human protein SSC4D, one of the least scrutinized members of the family. Within leukocyte populations, SSC4D was found to be expressed by monocytes/macrophages, neutrophils, and B cells, but its production was particularly evident in epithelial cells of several organs and tissues, namely, in the kidney, thyroid, lung, placenta, intestinal tract, and liver. Similar to other SRCR proteins, SSC4D shows the capacity of physically binding to different species of bacteria, and this opsonization can increase the phagocytic capacity of monocytes. Importantly, we have uncovered the capacity of SSC4D of binding to several protozoan parasites, a singular feature seldom described for PRRs in general and here demonstrated for the first time for an SRCR family member. Overall, our study is pioneer in assigning a PRR role to SSC4D.This work was funded by National Funds through FCT– Fundação para a Ciência e a Tecnologia, I.P., under the projects SRecognite Infect-ERA/0003/2015 and UIDB/04293/ 2020. Individual funding to JT was provided by FCT through CEECIND/02362/2017. MC, RS, and MS were recipients of studentships from FCT, respectively, SFRH/BD/116791/2016, SFRH/BD/110691/2015, and SFRH/BD/133485/2017. This paper is dedicated to our colleague and friend Rui Appelberg (1960-2020). The authors acknowledge the support of the i3S Scientific Platform BioSciences Screening, member of the national infrastructure PPBI–Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122) and PT-OPENSCREEN. Tissue sections were kindly provided by Amaro Frutuoso, Department of Complementary Means of Diagnosis and Therapy, Service of Pathology, Hospital Pedro Hispano, Matosinhos

The Scd6/Lsm14 protein xRAPB has properties different from RAP55 in selecting mRNA for early translation or intracellular distribution in Xenopus oocytes

© 2015 Elsevier B.V. Oocytes accumulate mRNAs in the form of maternal ribonucleoprotein (RNP) particles, the protein components of which determine the location and stability of individual mRNAs prior to translation. Scd6/Lsm14 proteins, typified by RAP55, function in a wide range of eukaryotes in repressing translation and relocating mRNPs to processing bodies and stress granules. In Xenopus laevis, the RAP55 orthologue xRAPA fulfils these functions. Here we describe the properties of a variant of xRAPA, xRAPB, which is a member of the Lsm14B group. xRAPB differs from xRAPA in various respects: it is expressed at high concentration earlier in oogenesis; it interacts specifically with the DDX6 helicase Xp54; it is detected in polysomes and stalled translation initiation complexes; its over-expression leads to selective binding to translatable mRNA species without evidence of translation repression or mRNA degradation. Since both Xp54 and xRAPA are repressors of translation, activation appears to be effected through targeting of xRAPB/Xp54

Dcp2 C-terminal Cis-Binding Elements Control Selective Targeting of the Decapping Enzyme by Forming Distinct Decapping Complexes [preprint]

A single Dcp1-Dcp2 decapping enzyme targets diverse classes of yeast mRNAs for decapping-dependent 5’ to 3’ decay, but the molecular mechanisms controlling selective mRNA targeting by the enzyme remain elusive. Through extensive genetic analyses we uncover cis-regulatory elements in the Dcp2 C-terminal domain that control selective targeting of the decapping enzyme by forming distinct decapping complexes. Two Upf1-binding motifs target the decapping enzyme to NMD substrates, and a single Edc3-binding motif targets both Edc3 and Dhh1 substrates. Pat1-binding leucine-rich motifs target Edc3 and Dhh1 substrates under selective conditions. Although it functions as a unique targeting component of specific complexes, Edc3 is a common component of multiple complexes. Xrn1 also has a specific Dcp2 binding site, allowing it to be directly recruited to decapping complexes. Collectively, our results demonstrate that Upf1, Edc3, and Pat1 function as regulatory subunits of the holo-decapping enzyme, controlling both its targeting specificity and enzymatic activation

Commentary: The Scavenger Receptor SSc5D Physically Interacts with Bacteria through the SRCR-Containing N-Terminal Domain

The recently published article by Bessa Pereira et al. reports that the human SSc5D receptor physically interacts with some bacterial species (1), thus basically confirming previous available information on its mouse homolog (S5D-SRCRB) (2). The interspecies conservation of such a basic innate immune function (bacterial binding) has been noticed for other members of the scavenger receptor cysteine-rich superfamily (SRCR-SF) (e.g., human Spα and its mouse homolog AIM/Api6/CD5L) (3, 4). This advocates for its functional physiological relevance in innate defense of body surfaces as it has been proposed for the urogenital tract (5). A substantive part of the work by Bessa Pereira et al. is also devoted to explore putative qualitative and/or quantitative differences on the bacterial-binding properties of SSc5D with other human SRCR-SF proteins, namely, CD5, Spα, and CD6 by using conventional protein-bacteria binding assays and surface plasmon resonance-based assays. They were chosen based on previously reported information showing that Spα (4) and CD6 (6-8) but not CD5 (9) exhibit broad bacterial-binding properties. While the authors confirmed the work on Spα and CD5, they were unable to replicate that on CD6. Exclusively based on a single experimental evidence, the authors cast doubt on the well-documented bacterial-binding properties of CD6 (6-8). These properties were unveiled by using a recombinant soluble form of human CD6 (rshCD6) encompassing from D25 to M400 and, indistinctly, produced in different mammalian cell expression systems (NSO, HEK293-EBNA, and CHO cells). Further confirmation was obtained by demonstrating similar properties displayed by a natural soluble CD6 form isolated from human serum, as well as by Jurkat cell transfectants expressing a membrane-bound full-length form of CD6 (6). Accordingly, it was later reported that rshCD6 infusion significantly reduces mouse mortality following septic shock induced by intraperitoneal monobacterial infection of Gram-positive (S. aureus) or Gram-negative (A. baumannii) origin (7). More recently, new evidence shows that not only rshCD6 but also adenovirally expressed mouse sCD6 have protective survival effects on polymicrobial septic shock induced by cecal ligation and puncture (8), the gold standard model for experimental sepsis