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uORF-tools—workflow for the determination of translation-regulatory upstream open reading frames
(2019)
Ribosome profiling (ribo-seq) provides a means to analyze active translation by determining ribosome occupancy in a transcriptome-wide manner. The vast majority of ribosome protected fragments (RPFs) resides within the protein-coding sequence of mRNAs. However, commonly reads are also found within the transcript leader sequence (TLS) (aka 5’ untranslated region) preceding the main open reading frame (ORF), indicating the translation of regulatory upstream ORFs (uORFs). Here, we present a workflow for the identification of translation-regulatory uORFs. Specifically, uORF-Tools uses Ribo-TISH to identify uORFs within a given dataset and generates a uORF annotation file. In addition, a comprehensive human uORF annotation file, based on 35 ribo-seq files, is provided, which can serve as an alternative input file for the workflow. To assess the translation-regulatory activity of the uORFs, stimulus-induced changes in the ratio of the RPFs residing in the main ORFs relative to those found in the associated uORFs are determined. The resulting output file allows for the easy identification of candidate uORFs, which have translation-inhibitory effects on their associated main ORFs. uORF-Tools is available as a free and open Snakemake workflow at https://github.com/Biochemistry1-FFM/uORF-Tools. It is easily installed and all necessary tools are provided in a version-controlled manner, which also ensures lasting usability. uORF-Tools is designed for intuitive use and requires only limited computing times and resources.
Bioactive lipid mediators play a major role in regulating inflammatory processes. Herein, early pro-inflammatory phases are characterized and regulated by prostanoids and leukotrienes, whereas specialized pro-resolving mediators (SPM), including lipoxins, resolvins, protectins, and maresins, dominate during the resolution phase. While pro-inflammatory properties of prostanoids have been studied extensively, their impact on later phases of the inflammatory process has been attributed mainly to their ability to initiate the lipid-mediator class switch towards SPM. Yet, there is accumulating evidence that prostanoids directly contribute to the resolution of inflammation and return to homeostasis. In this mini review, we summarize the current knowledge of the resolution-regulatory properties of prostanoids and discuss potential implications for anti-inflammatory, prostanoid-targeted therapeutic interventions.
The translation inhibitor and tumor suppressor Pdcd4 was reported to be lost in various tumors and put forward as prognostic marker in tumorigenesis. Decreased Pdcd4 protein stability due to PI3K-mTOR-p70S6K1 dependent phosphorylation of Pdcd4 followed by β-TrCP1-mediated ubiquitination, and proteasomal destruction of the protein was characterized as a major mechanism contributing to the loss of Pdcd4 expression in tumors. In an attempt to identify stabilizers of Pdcd4, we used a luciferase-based high-throughput compatible cellular assay to monitor phosphorylation-dependent proteasomal degradation of Pdcd4 in response to mitogen stimulation. Following a screen of approximately 2000 compounds, we identified 1,2-bis(4-chlorophenyl)disulfide as a novel Pdcd4 stabilizer. To determine an initial structure-activity relationship, we used 3 additional compounds, synthesized according to previous reports, and 2 commercially available compounds for further testing, in which either the linker between the aryls was modified (compounds 2–4) or the chlorine residues were replaced by groups with different electronic properties (compounds 5 and 6). We observed that those compounds with alterations in the sulfide linker completely lost the Pdcd4 stabilizing potential. In contrast, modifications in the chlorine residues showed only minor effects on the Pdcd4 stabilizing activity. A reporter with a mutated phospho-degron verified the specificity of the compounds for stabilizing the Pdcd4 reporter. Interestingly, the active diaryl disulfides inhibited proliferation and viability at concentrations where they stabilized Pdcd4, suggesting that Pdcd4 stabilization might contribute to the anti-proliferative properties. Finally, computational modelling indicated that the flexibility of the disulfide linker might be necessary to exert the biological functions of the compounds, as the inactive compound appeared to be energetically more restricted.
Rapid alterations in protein expression are commonly regulated by adjusting translation. In addition to cap-dependent translation, which is e.g. induced by pro-proliferative signaling via the mammalian target of rapamycin (mTOR)-kinase, alternative modes of translation, such as internal ribosome entry site (IRES)-dependent translation, are often enhanced under stress conditions, even if cap-dependent translation is attenuated. Common stress stimuli comprise nutrient deprivation, hypoxia, but also inflammatory signals supplied by infiltrating immune cells. Yet, the impact of inflammatory microenvironments on translation in tumor cells still remains largely elusive. In the present study, we aimed at identifying translationally deregulated targets in tumor cells under inflammatory conditions. Using polysome profiling and microarray analysis, we identified cyp24a1 (1,25-dihydroxyvitamin D3 24-hydroxylase) to be translationally upregulated in breast tumor cells co-cultured with conditioned medium of activated monocyte-derived macrophages (CM). Using bicistronic reporter assays, we identified and validated an IRES within the 5′ untranslated region (5′UTR) of cyp24a1, which enhances translation of cyp24a1 upon CM treatment. Furthermore, IRES-dependent translation of cyp24a1 by CM was sensitive to phosphatidyl-inositol-3-kinase (PI3K) inhibition, while constitutive activation of Akt sufficed to induce its IRES activity. Our data provide evidence that cyp24a1 expression is translationally regulated via an IRES element, which is responsive to an inflammatory environment. Considering the negative feedback impact of cyp24a1 on the vitamin D responses, the identification of a novel, translational mechanism of cyp24a1 regulation might open new possibilities to overcome the current limitations of vitamin D as tumor therapeutic option.
Macrophages constitute a major part of the tumor-infiltrating immune cells. Within the tumor microenvironment, they acquire an alternatively activated, tumor-supporting phenotype. Factors released by tumor cells are crucial for the recruitment of tumor-associated macrophages. In the present project, we aimed to understand the role of hsa-miR-200c-3p (miR-200c) in the interplay between tumor cells and macrophages. To this end, we employed a coculture system of MCF7 breast tumor cells and primary human macrophages and observed the transfer of miR-200c from apoptotic tumor cells to macrophages, which required intact CD36 receptor in macrophages. We further comprehensively determined miR-200c targets in macrophages by mRNA-sequencing and identified numerous migration-associated mRNAs to be downregulated by miR-200c. Consequently, miR-200c attenuated macrophage infiltration into 3-dimensional tumor spheroids. miR-200c-mediated reduction in infiltration further correlated with a miR-200c migration signature comprised of the four miR-200c-repressed, predicted targets PPM1F, RAB11FIB2, RDX, and MSN.
Despite the progress to understand inflammatory reactions, mechanisms causing their resolution remain poorly understood. Prostanoids, especially prostaglandin E2 (PGE2), are well-characterized mediators of inflammation. PGE2 is produced in an inducible manner in macrophages (Mϕ) by microsomal PGE2-synthase-1 (mPGES-1), with the notion that it also conveys pro-resolving properties. We aimed to characterize the role of mPGES-1 during resolution of acute, zymosan-induced peritonitis. Experimentally, we applied the mPGES-1 inhibitor compound III (CIII) once the inflammatory response was established and confirmed its potent PGE2-blocking efficacy. mPGES-1 inhibition resulted in an incomplete removal of neutrophils and a concomitant increase in monocytes and Mϕ during the resolution process. The mRNA-seq analysis identified enhanced C-X3-C motif receptor 1 (CX3CR1) expression in resident and infiltrating Mϕ upon mPGES-1 inhibition. Besides elevated Cx3cr1 expression, its ligand CX3CL1 was enriched in the peritoneal lavage of the mice, produced by epithelial cells upon mPGES-1 inhibition. CX3CL1 not only increased adhesion and survival of Mϕ but its neutralization also completely reversed elevated inflammatory cell numbers, thereby normalizing the cellular, peritoneal composition during resolution. Our data suggest that mPGES-1-derived PGE2 contributes to the resolution of inflammation by preventing CX3CL1-mediated retention of activated myeloid cells at sites of injury.
Inflammation is a crucial part of immune responses towards invading pathogens or tissue damage. While inflammatory reactions are aimed at removing the triggering stimulus, it is important that these processes are terminated in a coordinate manner to prevent excessive tissue damage due to the highly reactive inflammatory environment. Initiation of inflammatory responses was proposed to be regulated predominantly at a transcriptional level, whereas post-transcriptional modes of regulation appear to be crucial for resolution of inflammation. The RNA-binding protein tristetraprolin (TTP) interacts with AU-rich elements in the 3′ untranslated region of mRNAs, recruits deadenylase complexes and thereby facilitates degradation of its targets. As TTP regulates the mRNA stability of numerous inflammatory mediators, it was put forward as a crucial post-transcriptional regulator of inflammation. Here, we summarize the current understanding of the function of TTP with a specific focus on its role in adding to resolution of inflammation.
Clonal hematopoiesis of indeterminate potential (CHIP) is caused by recurrent somatic mutations leading to clonal blood cell expansion. However, direct evidence of the fitness of CHIP-mutated human hematopoietic stem cells (HSCs) in blood reconstitution is lacking. Because myeloablative treatment and transplantation enforce stress on HSCs, we followed 81 patients with solid tumors or lymphoid diseases undergoing autologous stem cell transplantation (ASCT) for the development of CHIP. We found a high incidence of CHIP (22%) after ASCT with a high mean variant allele frequency (VAF) of 10.7%. Most mutations were already present in the graft, albeit at lower VAFs, demonstrating a selective reconstitution advantage of mutated HSCs after ASCT. However, patients with CHIP mutations in DNA-damage response genes showed delayed neutrophil reconstitution. Thus, CHIP-mutated stem and progenitor cells largely gain on clone size upon ASCT-related blood reconstitution, leading to an increased future risk of CHIP-associated complications.
While the importance of the iron-load of lipocalin-2 (Lcn-2) in promoting tumor progression is widely appreciated, underlying molecular mechanisms largely remain elusive. Considering its role as an iron-transporter, we aimed at clarifying iron-loaded, holo-Lcn-2 (hLcn-2)-dependent signaling pathways in affecting renal cancer cell viability. Applying RNA sequencing analysis in renal CAKI1 tumor cells to explore highly upregulated molecular signatures in response to hLcn-2, we identified a cluster of genes (SLC7A11, GCLM, GLS), which are implicated in regulating ferroptosis. Indeed, hLcn-2-stimulated cells are protected from erastin-induced ferroptosis. We also noticed a rapid increase in reactive oxygen species (ROS) with subsequent activation of the antioxidant Nrf2 pathway. However, knocking down Nrf2 by siRNA was not sufficient to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. In contrast, preventing oxidative stress through N-acetyl-l-cysteine (NAC) supplementation was still able to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. Besides an oxidative stress response, we noticed activation of the integrated stress response (ISR), shown by enhanced phosphorylation of eIF-2α and induction of ATF4 after hLcn-2 addition. ATF4 knockdown as well as inhibition of the ISR sensitized hLcn-2-treated renal tumor cells to ferroptosis, thus linking the ISR to pro-tumor characteristics of hLcn-2. Our study provides mechanistic details to better understand tumor pro-survival pathways initiated by iron-loaded Lcn-2.
Bacterial and fungal toll-like receptor activation elicits type I IFN responses in mast cells
(2021)
Next to their role in IgE-mediated allergic diseases and in promoting inflammation, mast cells also have antiinflammatory functions. They release pro- as well as antiinflammatory mediators, depending on the biological setting. Here we aimed to better understand the role of mast cells during the resolution phase of a local inflammation induced with the Toll-like receptor (TLR)-2 agonist zymosan. Multiple sequential immunohistology combined with a statistical neighborhood analysis showed that mast cells are located in a predominantly antiinflammatory microenvironment during resolution of inflammation and that mast cell-deficiency causes decreased efferocytosis in the resolution phase. Accordingly, FACS analysis showed decreased phagocytosis of zymosan and neutrophils by macrophages in mast cell-deficient mice. mRNA sequencing using zymosan-induced bone marrow-derived mast cells (BMMC) revealed a strong type I interferon (IFN) response, which is known to enhance phagocytosis by macrophages. Both, zymosan and lipopolysaccharides (LPS) induced IFN-β synthesis in BMMCs in similar amounts as in bone marrow derived macrophages. IFN-β was expressed by mast cells in paws from naïve mice and during zymosan-induced inflammation. As described for macrophages the release of type I IFNs from mast cells depended on TLR internalization and endosome acidification. In conclusion, mast cells are able to produce several mediators including IFN-β, which are alone or in combination with each other able to regulate the phagocytotic activity of macrophages during resolution of inflammation.