Auswahl der wissenschaftlichen Literatur zum Thema „Extra-ribosomal activity“

Proteins belonging to the universal ribosomal protein (rp) uS19 family are constituents of small ribosomal subunits, and their conserved globular parts are involved in the formation of the head of these subunits. The eukaryotic rp uS19 (previously known as S15) comprises a C-terminal extension that has no homology in the bacterial counterparts. This extension is directly implicated in the formation of the ribosomal decoding site and thereby affects translational fidelity in a manner that has no analogy in bacterial ribosomes. Another eukaryote-specific feature of rp uS19 is its essential participance in the 40S subunit maturation due to the interactions with the subunit assembly factors required for the nuclear exit of pre-40S particles. Beyond properties related to the translation machinery, eukaryotic rp uS19 has an extra-ribosomal function concerned with its direct involvement in the regulation of the activity of an important tumor suppressor p53 in the Mdm2/Mdmx-p53 pathway. Mutations in the RPS15 gene encoding rp uS19 are linked to diseases (Diamond Blackfan anemia, chronic lymphocytic leukemia and Parkinson's disease) caused either by defects in the ribosome biogenesis or disturbances in the functioning of ribosomes containing mutant rp uS19, likely due to the changed translational fidelity. Here, we review currently available data on the involvement of rp uS19 in the operation of the translational machinery and in the maturation of 40S subunits, on its extra-ribosomal function, and on relationships between mutations in the RPS15 gene and certain human diseases.

The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co-localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. However, Histone Association Assays indicate that the mutant proteins still bind chromatin. RNA-seq analyses from wing imaginal discs show that Corto represses RPG expression whereas very few genes are deregulated in uL11 mutants. We propose that Corto, by repressing RPG expression, ensures that all ribosomal proteins are present at the correct stoichiometry, and that uL11 fine-tunes its transcriptional regulation of RPGs.

The nucleolus is the site of ribosome biogenesis and has been recently described as important sensor for a variety of cellular stressors. In the last two decades, it has been largely demonstrated that many chemotherapeutics act by inhibiting early or late rRNA processing steps with consequent alteration of ribosome biogenesis and activation of nucleolar stress response. The overall result is cell cycle arrest and/or apoptotic cell death of cancer cells. Our previously data demonstrated that ribosomal protein uL3 is a key sensor of nucleolar stress activated by common chemotherapeutic agents in cancer cells lacking p53. We have also demonstrated that uL3 status is associated to chemoresistance; down-regulation of uL3 makes some chemotherapeutic drugs ineffective. Here, we demonstrate that in colon cancer cells, the uL3 status affects rRNA synthesis and processing with consequent activation of uL3-mediated nucleolar stress pathway. Transcriptome analysis of HCT 116p53−/− cells expressing uL3 and of a cell sub line stably depleted of uL3 treated with Actinomycin D suggests a new extra-ribosomal role of uL3 in the regulation of autophagic process. By using confocal microscopy and Western blotting experiments, we demonstrated that uL3 acts as inhibitory factor of autophagic process; the absence of uL3 is associated to increase of autophagic flux and to chemoresistance. Furthermore, experiments conducted in presence of chloroquine, a known inhibitor of autophagy, indicate a role of uL3 in chloroquine-mediated inhibition of autophagy. On the basis of these results and our previous findings, we hypothesize that the absence of uL3 in cancer cells might inhibit cancer cell response to drug treatment through the activation of cytoprotective autophagy. The restoration of uL3 could enhance the activity of many drugs thanks to its pro-apoptotic and anti-autophagic activity.

In Saccharomyces cerevisiae, the genes coding for the ribosomal protein L2 are present in two copies per haploid genome. The two copies, which encode proteins differing in only a few amino acids, contribute unequally to the L2 mRNA pool: the L2A copy makes 72% of the mRNA, while the L2B copy makes only 28%. Disruption of the L2B gene (delta B strain) did not lead to any phenotypic alteration, whereas the inactivation of the L2A copy (delta A strain) produced a slow-growth phenotype associated with decreased accumulation of 60S subunits and ribosomes. No intergenic compensation occurred at the transcriptional level in the disrupted strains; in fact, delta A strains contained reduced levels of L2 mRNA, whereas delta B strains had almost normal levels. The wild-type phenotype was restored in the delta A strains by transformation with extra copies of the intact L2A or L2B gene. As already shown for other duplicated genes (Kim and Warner, J. Mol. Biol. 165:79-89, 1983; Leeret al., Curr. Genet. 9:273-277, 1985), the difference in expression of the two gene copies could be accounted for via differential transcription activity. Sequence comparison of the rpL2 promoter regions has shown the presence of canonical HOMOL1 boxes which are slightly different in the two genes.

In Saccharomyces cerevisiae, the genes coding for the ribosomal protein L2 are present in two copies per haploid genome. The two copies, which encode proteins differing in only a few amino acids, contribute unequally to the L2 mRNA pool: the L2A copy makes 72% of the mRNA, while the L2B copy makes only 28%. Disruption of the L2B gene (delta B strain) did not lead to any phenotypic alteration, whereas the inactivation of the L2A copy (delta A strain) produced a slow-growth phenotype associated with decreased accumulation of 60S subunits and ribosomes. No intergenic compensation occurred at the transcriptional level in the disrupted strains; in fact, delta A strains contained reduced levels of L2 mRNA, whereas delta B strains had almost normal levels. The wild-type phenotype was restored in the delta A strains by transformation with extra copies of the intact L2A or L2B gene. As already shown for other duplicated genes (Kim and Warner, J. Mol. Biol. 165:79-89, 1983; Leeret al., Curr. Genet. 9:273-277, 1985), the difference in expression of the two gene copies could be accounted for via differential transcription activity. Sequence comparison of the rpL2 promoter regions has shown the presence of canonical HOMOL1 boxes which are slightly different in the two genes.

BackgroundSjögren’s syndrome (SS) can occur alone or in combination with other autoimmune diseases. Systemic lupus erythematosus (SLE) is the most common autoimmune disease associated with SS. The prognosis of SS is generally better than that of SLE. However, the onset of SLE in these patients may be one of the factors that increase mortality.ObjectivesThis study determined the impact of demographic factors, clinical manifestations, disease activity, and serological tests at baseline on future SLE development in Sjögren’s syndrome (SS) patients.MethodsThis retrospective study assessed 1,082 SS patients without other autoimmune diseases at baseline who visited our hospital between January 2012 and March 2021. We analyzed demographic features, extra-glandular manifestations (EGMs), clinical indices, and laboratory values at baseline between the two groups divided per future SLE development (SS/SLE group vs. SS group). The probability and predictors of SLE development in SS patients were estimated using the Kaplan–Meier method and Cox proportional hazards models.ResultsThe median follow-up duration was 1083.5 days. Forty-nine patients (4.5%) developed SLE that met the 2012 Systemic Lupus International Collaborating Clinics or 2019 EULAR/ACR classification criteria. The baseline EULAR SS disease activity index (ESSDAI) score was significantly higher in the SS/SLE group (p<0.001). The SS/SLE group had more lymphadenopathy and renal involvement (p=0.015 andp=0.017, respectively). Shorter SS disease duration (<3 years) (hazard ratio [HR]=2.61,p=0.012), high ESSDAI (HR=3.04,p=0.024), leukopenia (HR=2.20,p=0.017), hypocomplementemia (HR=17.40,p<0.0001), and positive for anti-dsDNA (HR=19.93,p<0.0001), anti-ribonucleoprotein (RNP) (HR=2.96,p=0.025), and anti-ribosomal P (HR=2.74,p=0.048) at baseline were SLE development predictors in SS patients.ConclusionShorter disease duration and higher disease activity of SS at baseline may be risk factors for future SLE development. Serologic predictors of SLE development are hypocomplementemia, leukopenia, and positivity for anti-dsDNA, anti-RNP, and anti-ribosomal P antibodies. If the above factors are observed, close monitoring will be necessary during the follow-up period, considering the possibility of future SLE development.References[1]Lazarus MN and Isenberg DA. Development of additional autoimmune diseases in a population of patients with primary Sjogren’s syndrome.Ann Rheum Dis. 2005; 64: 1062-4.[2]Pasoto SG, Adriano de Oliveira Martins V and Bonfa E. Sjogren’s syndrome and systemic lupus erythematosus: links and risks.Open Access Rheumatol. 2019; 11: 33-45Figure 1.Comparison of disease activity (by ESSDAI score) and extraglandular manifestations (EGMs) at baseline between SS/SLE group and SS group.AcknowledgementsNo acknowledgements to declare.Disclosure of InterestsNone Declared.

Industrial effluent containing textile dyes is regarded as a major environmental concern in the present world. Crystal Violet is one of the vital textile dyes of the triphenylmethane group; it is widely used in textile industry and known for its mutagenic and mitotic poisoning nature. Bioremediation, especially through bacteria, is becoming an emerging and important sector in effluent treatment. This study aimed to isolate and identify Crystal Violet degrading bacteria from industrial effluents with potential use in bioremediation. The decolorizing activity of the bacteria was measured using a photo electric colorimeter after aerobic incubation in different time intervals of the isolates. Environmental parameters such as pH, temperature, initial dye concentration and inoculum size were optimized using mineral salt medium containing different concentration of Crystal Violet dye. Complete decolorizing efficiency was observed in a mineral salt medium containing up to 150 mg/l of Crystal Violet dye by 10% (v/v) inoculums of Enterobacter sp. CV–S1 tested under 72 h of shaking incubation at temperature 35 °C and pH 6.5. Newly identified bacteria Enterobacter sp. CV–S1, confirmed by 16S ribosomal RNA sequencing, was found as a potential bioremediation biocatalyst in the aerobic degradation/de-colorization of Crystal Violet dye. The efficiency of degrading triphenylmethane dye by this isolate, minus the supply of extra carbon or nitrogen sources in the media, highlights the significance of larger-scale treatment of textile effluent.

Members of the genus Streptococcus inhabit a variety of sites in humans and other animals and some species are prolific producers of proteinaceous antibiotics (bacteriocins). As little is known about (i) streptococci inhabiting domestic pets, and (ii) whether novel bacteriocin-producing streptococci can be isolated from domestic pets, the aim of this study is to address these gaps in the research literature. In this study, Streptococcus equinus MDC1, isolated from a healthy dog, was found to exhibit potent antibacterial activity against Micrococcus luteus in a simultaneous antagonism assay, suggesting that strain MDC1 produces a lantibiotic bacteriocin. The inhibitory activity spectrum of S. equinus MDC1, determined using agar-based deferred antagonism assays against >70 indicator strains, was found to be similar to that of nisin U (a lantibiotic produced by Streptococcus uberis). However, the spectra of the two bacteriocins differed by 23 strains, mainly with the MDC1 bacteriocin having no inhibitory activity towards certain streptococci of human origin (e.g., Streptococcus gordonii, Streptococcus anginosus, Streptococcus salivarius). The genome of S. equinus MDC1, which was sequenced completely using single-molecule real-time (SMRT) next-generation DNA sequencing technology, comprises a single 1,936,555-basepair chromosome containing seven copies of the ribosomal RNA operon, 69 tRNA genes and nearly 1900 putative coding sequences. Analysis of the MDC1 genome sequence using the bacteriocin detection algorithms BAGEL4 and antiSMASH revealed the location of a 13,164-basepair 11-gene locus, designated nmd, which encoded a mature nisin E peptide that differed from nisin U by only two amino acids (Ile15→Ala and Leu21→Ile) and an extra C-terminal asparagine residue, and the proteins required for post-translational modification of the bacteriocin, processing, export, and producer immunity. Despite the high homology (90.6% identity, 93.8% similarity) between nisin E and nisin U, there was considerably less homology (47.4–76.3% identity, 68.4–88.8% similarity) between the other proteins encoded by their respective biosynthetic loci. This new natural variant of nisin, called nisin E, represents the first nisin variant to be reported for S. equinus; additionally, its differences with nisin U may provide some insight into the amino acids that influence bacteriocin potency and killing spectrum.

BackgroundIn patients with systemic lupus erythematosus (SLE), renal involvement is associated with high morbidity, and renal flares are major contributors to poor long-term prognosis. Identification of patients at risk of developing renal flares despite immunosuppressant therapy is imperative to optimise management.ObjectivesTo identify predictors of renal flares in patients receiving treatment for active extra-renal SLE.MethodsData from BLISS-52 (NCT00424476), BLISS-76 (NCT00410384), BLISS Northeast Asia (NEA;NCT01345253), and BLISS-SC (NCT01484496) were used (N=3225). The trials included patients with active, seropositive SLE and excluded active severe renal SLE. Participants were assigned to belimumab or placebo, on top of non-biologic standard therapy. We investigated anti-dsDNA, anti-Sm, anti-ribosomal P, and anti-cardiolipin (aCL) antibodies, low C3 and low C4 levels, B cell activating factor (BAFF), serum albumin, serum creatinine, and proteinuria at baseline as potential predictors of renal flares during a 52-week follow-up. We used Cox regression models to evaluate traditional disease features as predictors of renal flares in the pooled trial population. We also performed subgroup analysis of belimumab and placebo recipients. Covariates in the adjusted models included age, sex, ethnicity, body mass index, organ damage, baseline extra-renal activity (SLEDAI-2K score excluding renal and immunological descriptors), current or former renal SLE history (renal BILAG A–D), baseline use of glucocorticoids, antimalarials, and immunosuppressants, and use of belimumab.ResultsThe mean age of the study population was 36.7 years, 94% were women, 54.6% had current or former renal involvement at baseline, and 192 developed a renal flare after a median follow-up time of 197 (IQR: 85–330) days from baseline. Patients with current or former renal involvement at baseline displayed a >9-fold increased hazard to develop a new renal flare (HR: 9.4; 95% CI: 5.0–17.7; P<0.001). In the pooled study population, baseline serum albumin (adjusted HR 0.9; 95% CI: 0.9–0.9; P<0.001), proteinuria (adjusted HR: 1.3; 95% CI: 1.2–1.4; P<0.001), and low C3 levels (adjusted HR: 1.8; 95% CI: 1.3–2.5; P<0.001) were robust determinants of subsequent renal flare occurrence; similar associations were found in the belimumab and placebo subgroups. Furthermore, we observed an association between anti-dsDNA positivity and renal flare development in univariable models (HR: 2.1; 95% CI: 1.4–3.2; P<0.001 in the pooled population), which attenuated in multivariable models (Figure 1). Positive levels of anti-Sm antibodies were associated with renal flare occurrence in the placebo (adjusted HR: 2.9; 95% CI: 1.5–5.6; P=0.002) but not in the belimumab subgroup, whereas anti-ribosomal P antibodies were associated with renal flare development in belimumab-treated (HR: 2.8; 95% CI: 1.5–5.0; P<0.001) but not in placebo-treated patients. Finally, positive levels of aCL antibodies (any isotype) predicted renal flare development in the belimumab group (adjusted HR: 1.8; 95% CI: 1.1–2.8; P=0.020) but yielded a negative association in the placebo group (adjusted HR: 0.4; 95% CI: 0.2–0.9; P=0.028).ConclusionCurrent or former renal involvement, high baseline proteinuria levels, hypoalbuminaemia, and C3 consumption were robust determinants of imminent renal flares. Beyond anti-dsDNA, anti-ribosomal P and aCL antibody positivity may prove valuable early signals of imminent renal flares in patients treated with belimumab. Anti-Sm positivity predicted imminent renal flares in patients treated with non-biological standard therapy, but not in belimumab-treated patients, assumably due to benefit conferred from belimumab in anti-Sm positive individuals, as shown previously [1].Reference[1]Parodis I, et al. Int J Mol Sci. 2020; 21(10):3463Figure 1.Cox regression models evaluating traditional markers as predictors of renal flares in patients with SLE treated with non-biological standard therapy with or without add-on belimumab.AcknowledgementsThe authors would like to thank GlaxoSmithKline for providing data through the CSDR consortium as well as all patients with SLE who participated in the trials.Disclosure of InterestsSandra Jägerback: None declared, Alvaro Gomez: None declared, Ioannis Parodis Grant/research support from: I.P. has received research funding and/or honoraria from Amgen, AstraZeneca, Aurinia Pharmaceuticals, Elli Lilly and Company, Gilead Sciences, GlaxoSmithKline, Janssen Pharmaceuticals, Novartis, Otsuka Pharmaceutical, and F. Hoffmann-La Roche AG.

Abstract Extracellular free calcium (Ca2+) is the most abundant signaling molecule in the hematopoietic stem cell (HSC) niche, and intracellular calcium is an important second messenger controlling critical functions in HSCs. Accordingly, Ca2+ is a master regulator of HSC biology, and nodes of the Ca2+ signaling network are emerging as critical components of the molecular machinery governing HSC regeneration and quiescence. Ca2+ controls many cell functions by forming a complex with Calmodulin (CaM), which binds and activates a family of Calcium/Calmodulin dependent kinase (CaMK) that includes CaMKI, CaMKII, CaMKIV, CaMKK1 and CaMKK2. We have previously investigated the function of CaMKK2 in blood cells and established this protein as a critical component of the calcium signaling pathway that regulates granulopoiesis and macrophage activation. Here, we investigated the function of CaMKK2 in the regenerative response of HSCs. To determine the expression of CaMKK2 under homeostatic conditions, we examined transgenic reporter mice and identified robust reporter activity in primitive long-term hematopoietic stem cells, but reduced signal in the majority of lineage positive blood and bone marrow cells. To establish whether CaMKK2 has a functional role in controlling hematopoiesis and HSC self-renewal, we examined hematopoietic regeneration in mice deficient for CaMKK2. We demonstrate that Camkk2 -/- mice have accelerated bone marrow and peripheral blood regeneration following hematopoietic injury. Camkk2 -/- HSCs showed increased proliferative capacity in vivo and mobilized into the peripheral blood to establish extra-medullary hematopoiesis during regeneration. To identify the proximal target of CaMKK2, we assessed the phosphorylation status of CaMKI, CaMKIV and adenosine monophosphate activated protein kinase (AMPK), which are known targets of CaMKK2. Our results indicate Camkk2 -/- stem and progenitor cells (HSPC) fail to accumulate phospho-AMPK in response to ionizing radiation. Since AMPK is a negative regulator of the mammalian target of rapamycin (mTOR) pathway, we also analyzed the levels of phospho-ribosomal 6 protein (rpS6), which is a distal target of the mTOR cascade that positively regulates cell size and proliferation. We found that irradiated Camkk2 -/- HSPCs accumulate higher levels of phospho-rpS6 compared to wild type cells. This finding implicates CaMKK2 as a critical component of a negative regulatory circuit that links calcium signaling to AMPK activation and cell proliferation in HSPC. Cumulatively, our data demonstrate that CaMKK2 regulates HSC function and suggests CaMKK2 inhibition is a novel approach to stem cell expansion for clinical therapy. Disclosures No relevant conflicts of interest to declare.

Chez la drosophile la protéine ribosomique uL11 est impliquée dans la traduction et régule la transcription. En effet, uL11 triméthylée sur la lysine 3 interagit avec l’Enhancer de Trithorax et Polycomb Corto et co-localise avec lui en de nombreux sites sur les chromosomes polytènes. De plus, l’analyse du transcriptome de disques imaginaux d’ailes surexprimant uL11 ou le chromodomaine de Corto (CortoCD) montre que les gènes codant des protéines importantes pour la biogenèse des ribosomes sont plus exprimés. Nous avons généré 3 mutants d’uL11K3 en utilisant la technologie CRISPR/Cas9 (uL11K3A, uL11K3Y et uL11ΔK3). Seuls les mutants uL11K3A présentent des phénotypes Minute (létalité élevée, retard de développement, soies courtes) corrélés à une diminution du taux global de traduction. Nous avons montré qu’uL11K3A et uL11K3Y n’interagissent pas avec CortoCD. Alors que de manière inattendue, uL11ΔK3 interagit avec CortoCD. L’analyse du transcriptome de disques imaginaux d’ailes uL11K3A et uL11K3Y montre que peu de gènes sont dérégulés : 414 et 84 gènes chez les mutants uL11K3A et uL11K3Y, respectivement. Chez S. cerevisiae, uL11 contrôle la transcription de gènes de la voie PHO dans un milieu pauvre en phosphates. Nous avons donc testé si un milieu nutritionnel pauvre affecterait les mutants uL11. Nous avons étudié les mutants uL11K3Y qui ne présentent pas de phénotypes en conditions normales. Dans ce milieu pauvre, ces mutants ont un développement plus court et des ailes plus petites que les individus sauvages. Ainsi, chez la drosophile comme chez S. cerevisiae, l’activité transcriptionnelle d’uL11 pourrait être importante dans des conditions environnementales défavorables
In drosophila, the ribosomal protein uL11 is involved in translation and regulates transcription. Indeed, uL11 trimethylated on lysine 3 directly interacts with the enhancer of Trithorax and Polycomb Corto and co-localises with it on many sites on polytene chromosomes. Furthermore, RNA-seq analyses of larval wing imaginal discs over-expressing uL11 or the chromodomain of Corto show that many ribosomal biogenesis and ribosomal protein genes are up-regulated. Three mutants of uL11K3 were obtained by CRISPR-Cas9 (uL11K3A, uL11K3Y et uL11ΔK3). Puromycine incorporation showed that only uL11K3A has a decreased translation in agreement with its dominant Minute phenotypes (which include high lethality, longer development, shorter bristles). Co-immunoprecipitation experiments show that uL11K3A and uL11K3Y do not interact with the chromodomain of Corto. Whereas, surprisingly, uL11ΔK3 still interacted with it. RNA-seq analyses of the wing imaginal disc transcriptomes of uL11K3A and uL11K3Y show that few genes are deregulated (414 genes for the former, 84 genes for the latter). As in Saccharomyces cerevisiae, uL11 controls transcription of genes of the PHO pathway in low-phosphate conditions. We next tested whether the uL11 mutants were more affected in restrictive diet conditions. We focused on the uL11K3Y mutants which are unaffected in normal conditions. In a restrictive diet, developmental time of uL11K3Y mutants is longer than the wild-type one, and their wings are smaller. Thus, in drosophila as S. cerevisiae, the transcriptional activity of uL11 might be important in poor environmental conditions