Cytoplasmic Protein Synthesis Research Articles

Post-Translational Regulation of a Bidomain Glycerol-3-Phosphate Dehydrogenase Catalyzing Glycerol Synthesis under Salinity Stress in Chlamydomonas reinhardtii

Core chlorophytes possess glycerol-3-phosphate dehydrogenases (GPDs) with an unusual bidomain structure, consisting of a glycerol-3-phosphate phosphatase (GPP) domain fused to canonical GPD domains. These plastid-localized enzymes have been implicated in stress responses, being required for the synthesis of glycerol under high salinity and triacylglycerols under nutrient deprivation. However, their regulation under varying environmental conditions is poorly understood. C. reinhardtii transgenic strains expressing constitutively bidomain GPD2 did not accumulate glycerol or triacylglycerols in the absence of any environmental stress. Although the glycerol contents of both wild type and transgenic strains increased significantly upon exposure to high salinity, cycloheximide, an inhibitor of cytoplasmic protein synthesis, abolished this response in the wild type. In contrast, GPD2 transgenic strains were still capable of glycerol accumulation when cultured in medium containing cycloheximide and NaCl. Thus, the pre-existing GPD2 protein appears to become activated for glycerol synthesis upon salt stress. Interestingly, staurosporine, a non-specific inhibitor of protein kinases, prevented this post-translational GPD2 protein activation. Structural modeling analyses suggested that substantial conformational rearrangements, possibly triggered by high salinity, may characterize an active GPD2 GPP domain. Understanding this mechanism(s) may provide insights into the rapid acclimation responses of microalgae to osmotic/salinity stress.

Effects of desiccation rate and inhibition of protein synthesis and the violaxanthin cycle on the rewetting recovery of Porella platyphylla

Following 5 different rates of 1-week desiccation to which Porella platyphylla (a desiccation-tolerant leafy liverwort) was subjected, physiological recovery upon rehydration (after 1 h, 24 h, 48 h) in light was monitored by cytological, photosynthetic (e-transport rate, photoprotective mechanisms), and other metabolic parameters using nuclear- and chloroplast-encoded protein synthesis inhibitors and a violaxanthin cycle inhibitor (DTT: dithiothreitol). Desiccation tolerance is mainly constitutive in P. platyphylla, allowing survival of rapid drying, and employs an active rehydration-induced repair and recovery mechanism. Following 24 hours of rehydration after the one-week dehydration in laboratory air (∼35% relative humidity /RH/), the chloroplasts had an irregular round shape. The recovery within 1 h of the thylakoid-function-related photosynthetic processes was extremely fast and independent of protein synthesis, while the overall recovery (except plants dried up at 76% RH, which depended on protein synthesis only to a limited extent) required de novo protein synthesis during rewetting. During the course of rehydration protein synthesis affects rehydrin expression, osmolite and general carbohydrate metabolism, the main repair and antioxidant mechanisms. Recovery was the best in samples dried up at 76% RH. Membrane permeability increased upon rehydration following 1-week dehydration. Normal membrane function recovered after 24 h (max. 48 h), except in plants that suffered drastic dehydration. Total protein content of plants dried under different conditions and of samples taken during recovery was generally lower than in the control plants at full turgor. The xanthophyll cycle has a great importance during the recovery in light; we confirmed the existence of a greater zeaxanthin-dependent and a smaller zeaxanthin-insensitive NPQ (nonphotochemical quenching). Plants dried under more favorable conditions had better light protection. The highest values of the osmotic potential belonged to the plants subjected to intense dehydration. Total soluble carbohydrate content was well-balanced and seemed to be unchanged following the different treatments. There was a significant increase in proline, while total fructan showed lower values at different degrees of 1-week desiccation. The cytoplasmic protein synthesis is likely to be involved in the change of the fructan content during rehydration.

The Marine Product Elatol Is a Novel Inhibitor of Mitochondrial Translation Triggering the Integrated Stress Response and Apoptosis in Leukemia and Lymphoma Cells

Background: Leukemia stem cells (LSCs) show unique, targetable dependence on activity of mitochondrial ribosomes (mitoribosomes). In addition, aggressive B-cell lymphomas may have gene expression signatures of increased dependence on oxidative phosphorylation (OXPHOS) and show sensitivity to mitoribosome inhibitors such as the FDA approved antibiotic tigecycline. Discovery and optimization of new mitoribosome inhibitors, however, is relatively unexplored in drug development for hematologic malignancies despite demonstrated therapeutic potential. Here, we assess the marine natural product elatol as a novel mitochondrial translation inhibitor. Methods and Results: We have previously reported that elatol, the major compound from the red alga Laurencia microcladia, is effective against several non-Hodgkin lymphomas and primary chronic lymphoid leukemia (CLL) cells. In vitro studies showed elatol inhibits eIF4A1 helicase activity, suppressing cytoplasmic cap-dependent translation initiation. Further assessments using 35S-methionine incorporation in newly synthesized polypeptides in HEK293T cells with or without single-digit micromolar concentrations of elatol for short time periods revealed strong attenuation of mitochondrial protein synthesis with no effect on mitochondrial transcription. Based on previously published work highlighting the effectiveness of tigecycline in leukemia and our data supporting elatol cytotoxicity in hematologic malignancies, we assessed elatol cytotoxicity and mitochondrial translation inhibition in several leukemia and lymphoma cell lines in comparison to tigecycline. All but one line decreased viability by 50% or more (compared to vehicle control) after elatol incubation with EC50 values near 1 μM. This potency was 10-40x higher than for tigecycline in side-by-side comparisons across several lines when compared at 72h. The compound cooperated to activate a heightened integrated stress response (ISR) through DELE1 cleavage by the mitochondrial stress-activated protease OMA1. This in turn suppressed glycolytic capacity, resulting in adenosine triphosphate (ATP) depletion and subsequent cell death. We found elatol does not affect integrity of small and large mitoribosome subunits through sedimentation property analysis using sucrose gradients. Although the specific target on the mitochondrial translation apparatus remains elusive, we found that its mechanism of action differs from that of chloramphenicol, which inhibits translation elongation. Conclusions: We have performed proof-of-concept studies using HEK293T and HeLa cell lines, isolated mitochondria from HEK293T, and leukemia and lymphoma cell lines to reveal that elatol is a potent inhibitor of mitochondrial protein synthesis at concentrations that do not affect cytoplasmic protein synthesis and that this mechanism differs from chloramphenicol. Elatol triggers the OMA1-DELE1-ATF4 mitochondrial stress pathway and apoptosis of leukemia and lymphoma cells. Tigecycline's compelling preclinical data in combination with kinase inhibitors informed design of a pending clinical trial (NCT02883036). Elatol's greatly improved potency provides a potential starting point for further optimization of this paradigm.

Saporin Toxin Delivered by Engineered Colloidal Nanoparticles Is Strongly Effective against Cancer Cells.

Ribosome-inactivating proteins, including Saporin toxin, have found application in the search for innovative alternative cancer therapies to conventional chemo- and radiotherapy. Saporin’s main mechanism of action involves the inhibition of cytoplasmic protein synthesis. Its strong theoretical efficacy is counterbalanced by negligible cell uptake and diffusion into the cytosol. In this work, we demonstrate that by immobilizing Saporin on iron oxide nanoparticles coated with an amphiphilic polymer, which promotes nanoconjugate endosomal escape, a strong cytotoxic effect mediated by ribosomal functional inactivation can be achieved. Cancer cell death was mediated by apoptosis dependent on nanoparticle concentration but independent of surface ligand density. The cytotoxic activity of Saporin-conjugated colloidal nanoparticles proved to be selective against three different cancer cell lines in comparison with healthy fibroblasts.

Deletion of the N-Terminal Domain of Yeast Eukaryotic Initiation Factor 4B Reprograms Translation and Reduces Growth in Urea.

The yeast eukaryotic initiation factor 4B binds the 40S subunit in translation preinitiation complexes (PICs), promoting mRNA recruitment. Recent evidence indicates yeast mRNAs have variable dependence on eIF4B under optimal growth conditions. Given the ability of eIF4B to promote translation as a function of nutrient conditions in mammalian cells, we wondered if eIF4B activities in translation could alter phenotypes in yeast through differential mRNA selection for translation. Here we compared the effects of disrupting yeast eIF4B RNA- and 40S-binding motifs under ∼1400 growth conditions. The RNA-Recognition Motif (RRM) was dispensable for stress responses, but the 40S-binding N-terminal Domain (NTD) promoted growth in response to stressors requiring robust cellular integrity. In particular, the NTD conferred a strong growth advantage in the presence of urea, which may be important for pathogenesis of related fungal species. Ribosome profiling indicated that similar to complete eIF4B deletion, deletion of the NTD dramatically reduced translation, particularly of those mRNAs with long and highly structured 5-prime untranslated regions. This behavior was observed both with and without urea exposure, but the specific mRNA pool associated with ribosomes in response to urea differed. Deletion of the NTD led to relative increases in ribosome association of shorter transcripts with higher dependence on eIF4G, as was noted previously for eIF4B deletion. Gene ontology analysis indicated that proteins encoded by eIF4B NTD-dependent transcripts were associated with the cellular membrane system and the cell wall, while NTD-independent transcripts encoded proteins associated with cytoplasmic proteins and protein synthesis. This analysis highlighted the difference in structure content of mRNAs encoding membrane versus cytoplasmic housekeeping proteins and the variable reliance of specific gene ontology classes on various initiation factors promoting otherwise similar functions. Together our analyses suggest that deletion of the eIF4B NTD prevents cellular stress responses by affecting the capacity to translate a diverse mRNA pool.

GTP binding to translation factor eIF2B stimulates its guanine nucleotide exchange activity

SummaryeIF2B is the guanine nucleotide exchange factor (GEF) required for cytoplasmic protein synthesis initiation in eukaryotes and its regulation within the integrated stress response (ISR). It activates its partner factor eIF2, thereby promoting translation initiation. Here we provide evidence through biochemical and genetic approaches that eIF2B can bind directly to GTP and this can enhance its rate of GEF activity toward eIF2–GDP in vitro. GTP binds to a subcomplex of the eIF2Bγ and ε subunits. The eIF2Bγ amino-terminal domain shares structural homology with hexose sugar phosphate pyrophosphorylase enzymes that bind specific nucleotides. A K66R mutation in eIF2Bγ is especially sensitive to guanine or GTP in a range of functional assays. Taken together, our data suggest eIF2Bγ may act as a sensor of purine nucleotide availability and thus modulate eIF2B activity and protein synthesis in response to fluctuations in cellular nucleotide levels.

Inhibition of Mitochondrial Translation By the Marine Natural Product Elatol Shows Potent Antileukemia Activity

Targeted signaling inhibitors for hematologic malignancies may lead to limited clinical efficacy due to the outgrowth of subpopulations with alternative pathways independent of the drug target. Relapse/refractory disease that results from treatment with targeted signaling inhibitors is a major hurdle in obtaining curative responses. Interestingly, work over the past decade or more has shown that chronic myelogenous leukemia (CML) stem cells (CD34+CD38-) are resistant to targeted signaling inhibitors, such as the BCR-ABL kinase class of inhibitors, often a problematic source of resistance leading to residual disease that may precipitate later progression (Hamilton et al., 2012).Recent studies have shown that some forms of lymphoma and leukemia cell have an energy metabolism highly dependent on mitochondrial oxidative phosphorylation (Ashton et al., 2018). Tigecycline, a US FDA approved antibiotic, has been shown to inhibit synthesis of mitochondrion-encoded proteins due to the similarity of bacterial and mitochondrial ribosomes, leading to selective lethality in hematologic malignancies reliant on enhanced oxidative phosphorylation (Norberg et al., 2017). Indeed, it was established that CML stem cells are reliant on upregulated oxidative phosphorylation, and combination treatment with the tyrosine-kinase inhibitor (TKI) imatinib and tigecycline eradicated therapy-resistant CML, both in vitro and in animal models (Kuntz et al., 2017).We have previously reported that elatol, the major compound from the red alga Laurencia microcladia, is effective against several non-Hodgkin lymphomas and primary chronic myelogenous leukemia cells (Peters et al., 2018). In vitro studies showed that elatol inhibits eIF4A1 helicase activity, suppressing cytoplasmic cap-dependent translation initiation. Further assessments using 35-S-methionine incorporation in HEK293T cells with or without single-digit micromolar concentrations of elatol for short time periods revealed strong downregulation of mitochondrion-encoded proteins as in Figure 1, (with no effect on mitochondrial transcription). This was confirmed in CML and acute lymphoblastic leukemia (ALL) cell lines whose 24-hour elatol LD50 ranged from high nanomolar to low micromolar concentrations. This potency was 10-40x higher than for tigecycline in side-by-side comparisons across several leukemia cell lines when compared at 72h. Additionally, we established that elatol does not affect integrity of small and large mitochondrial ribosomal units through sedimentation property analysis using sucrose gradients. Although the specific target on the mitochondrial translation apparatus remains elusive, we have uncovered that its mechanism of action differs from that of chloramphenicol, which inhibits translation elongation.In summary, we have performed proof-of-concept studies using HEK293T and HeLa cell lines, isolated mitochondria from HEK293T, and CML and ALL cell lines to reveal that elatol is a potent inhibitor of mitochondrial protein synthesis at concentrations that do not affect cytoplasmic protein synthesis and that this mechanism differs from chloramphenicol. Tigecycline's compelling preclinical data in combination with TKI informed design of a pending clinical trial (NCT02883036). Elatol's greatly improved potency provide a potential starting point for further optimization of this paradigm. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Protein expression profiling of Staphylococcus aureus in response to the bacteriocin bovicin HC5.

Alternative strategies to antibiotic treatment are required to inhibit pathogens, including Staphylococcus aureus. Bacteriocins, such as the lantibiotic bovicin HC5, have shown potential to control pathogens. This study aims to evaluate the stress response of S. aureus to bovicin HC5 using a proteomic approach. Sublethal concentrations of the bacteriocin repressed the synthesis of 62 cytoplasmic proteins, whereas 42 proteins were induced in S. aureus COL. Specifically, synthesis of several proteins involved in amino acid biosynthesis, mainly products of ilv-leu operon, and DNA metabolism, such as DNA polymerase I, decreased following bovicin treatment while proteins involved in catabolism, mainly tricarboxylic acid cycle metabolism, and chaperones were over-expressed. The levels of CodY and CcpA, important regulators involved in the stationary phase adaptation and catabolite repression, respectively, also increased in the presence of the bacteriocin. These results indicate that stress caused by the sublethal concentration of bovicin HC5 in the cell membrane results in growth reduction, reduced protein synthesis, and, at the same time, enhanced the levels of chaperones and enzymes involved in energy-efficient catabolism in an attempt to restore energy and cell homeostasis. These results bring relevant information to amplify the knowledge concerning the bacterial physiological changes in response to the stress caused by the cell exposition to bovicin HC5. New potential targets for controlling this pathogen can also be determined from the new protein expression pattern presented. KEY POINTS: • Bovicin HC5 changed the synthesis of cytoplasmic proteins of S. aureus. • Bovicin HC5 interfered in the synthesis of proteins of amino acids biosynthesis. • Synthesis of chaperones enhanced in the presence of sublethal dosage of bovicin HC5.

Mitochondrial Proteostasis Requires Genes Encoded in a Neurodevelopmental Syndrome Locus.

Eukaryotic cells maintain proteostasis through mechanisms that require cytoplasmic and mitochondrial translation. Genetic defects affecting cytoplasmic translation perturb synapse development, neurotransmission, and are causative of neurodevelopmental disorders, such as Fragile X syndrome. In contrast, there is little indication that mitochondrial proteostasis, either in the form of mitochondrial protein translation and/or degradation, is required for synapse development and function. Here we focus on two genes deleted in a recurrent copy number variation causing neurodevelopmental disorders, the 22q11.2 microdeletion syndrome. We demonstrate that SLC25A1 and MRPL40, two genes present in the microdeleted segment and whose products localize to mitochondria, interact and are necessary for mitochondrial ribosomal integrity and proteostasis. Our Drosophila studies show that mitochondrial ribosome function is necessary for synapse neurodevelopment, function, and behavior. We propose that mitochondrial proteostasis perturbations, either by genetic or environmental factors, are a pathogenic mechanism for neurodevelopmental disorders.SIGNIFICANCE STATEMENT The balance between cytoplasmic protein synthesis and degradation, or cytoplasmic proteostasis, is required for normal synapse function and neurodevelopment. Cytoplasmic and mitochondrial ribosomes are necessary for two compartmentalized, yet interdependent, forms of proteostasis. Proteostasis dependent on cytoplasmic ribosomes is a well-established target of genetic defects that cause neurodevelopmental disorders, such as autism. Here we show that the mitochondrial ribosome is a neurodevelopmentally regulated organelle whose function is required for synapse development and function. We propose that defective mitochondrial proteostasis is a mechanism with the potential to contribute to neurodevelopmental disease.

GTP Binding to Translation Factor eIF2B Stimulates Its Guanine Nucleotide Exchange Activity

eIF2B is the guanine nucleotide exchange factor (GEF) required for cytoplasmic protein synthesis initiation in eukaryotes and its regulation within the integrated stress response (ISR). It activates its partner factor eIF2 thereby promoting translation initiation. Here we provide evidence through biochemical and genetic approaches that eIF2B can bind directly to GTP and this can enhance its rate of GEF activity towards eIF2–GDP in vitro. GTP binds to a subcomplex of the eIF2Bγ and e subunits. The eIF2Bγ amino-terminal domain shares structural homology with hexose sugar phosphate pyrophosphorylase enzymes that bind specific nucleotides. A K66R mutation in eIF2Bγ is especially sensitive to guanine or GTP in a range of functional assays. Taken together, our data suggest eIF2Bγ may act as a sensor of purine nucleotide availability and thus modulate eIF2B activity

Cadmium exposure alters expression of protective enzymes and protein processing genes in venom glands of the wolf spider Pardosa pseudoannulata.

Cadmium (Cd) pollution is currently the most serious type of heavy metal pollution throughout the world. Previous studies have shown that Cd elevates the mortality of paddy field spiders, but the lethal mechanism remains to be explored profoundly. In the present study, we measured the activities of protective enzymes (acetylcholinesterase, glutathione peroxidase, phenol oxidase) and a heavy metal chelating protein (metallothionein) in the pond wolf spider Pardosa pseudoannulata after Cd exposure. The results indicated that Cd initially increased the enzyme activities and protein concentration of the spider after 10- and 20-day exposure before inhibiting them at 30-day exposure. Further analysis showed that the enzyme activities in the cephalothorax were inhibited to some extent. Since the cephalothorax region contains important venom glands, we performed transcriptome sequencing (RNA-seq) analysis of the venom glands collected from the spiders after long-term Cd exposure. RNA-seq yielded a total of 2826 differentially expressed genes (DEGs), and most of the DEGs were annotated into the process of protein synthesis, processing and degradation. Furthermore, a mass of genes involved in protein recognition and endoplasmic reticulum (ER) -associated protein degradation were down-regulated. The reduction of protease activities supports the view that protein synthesis and degradation in organelles and cytoplasm were dramatically inhibited. Collectively, our outcomes illustrate that Cd poses adverse effects on the expression of protective enzymes and protein, which potentially down-regulates the immune function in the venom glands of the spiders via the alteration of protein processing and degradation in the ER.

CReP mediates selective translation initiation at the endoplasmic reticulum.

Eukaryotic protein synthesis control at multiple levels allows for dynamic, selective responses to diverse conditions, but spatial organization of translation initiation machinery as a regulatory principle has remained largely unexplored. Here we report on a role of constitutive repressor of eIF2α phosphorylation (CReP) in translation of poliovirus and the endoplasmic reticulum (ER)-resident chaperone binding immunoglobulin protein (BiP) at the ER. Functional, proximity-dependent labeling and cell fractionation studies revealed that CReP, through binding eIF2α, anchors translation initiation machinery at the ER and enables local protein synthesis in this compartment. This ER site was protected from the suppression of cytoplasmic protein synthesis by acute stress responses, e.g., phosphorylation of eIF2α(S51) or mTOR blockade. We propose that partitioning of translation initiation machinery at the ER enables cells to maintain active translation during stress conditions associated with global protein synthesis suppression.

Effects of Prolonged Energy Restriction and Dietary Protein on Muscle Protein Synthesis and Proteome Dynamics in Obese Zucker Rats

Abstract Objectives High protein (HP) diets during short-term energy restriction (ER) attenuate energy-mediated reductions in muscle protein synthesis (MPS). MPS-adaptive responses to HP diets during prolonged ER are not well described. This study examined the effects of prolonged ER and HP on MPS and the synthesis rates of numerous individual muscle proteins. Methods Female 6-wk-old obese Zucker (leprfa+/fa+, n = 48) rats were randomized to one of four diet groups for 10 weeks: ad libitum-standard protein (AL-SP; 14% protein), AL-HP (35% protein), ER-SP, and ER-HP (both fed 60% of intake of AL-SP). At the start of week 10, D2O was administered by intraperitoneal injection and isotopic equilibrium was maintained daily by providing D2O in drinking water. Rats were euthanized after 1 week of labeling, and mixed-MPS (gastrocnemius), absolute mixed-MPS (mixed-MPS x muscle protein content), proteome dynamics, and protein half-lives [rate/d (k) = –ln(1-f)/d, where f is mixed-MPS and t is time in days; t1/2 (days) = ln(2)/k] were quantified. Results Mixed-MPS was not altered by energy status and protein intake. Gastrocnemius mass was lower (P < 0.001) in ER-fed rats than AL-fed rats and higher (P = 0.034) for AL-HP than AL-SP. As a result, absolute mixed-MPS was lower (P < 0.005) in ER than AL, regardless of dietary protein. Absolute synthesis in 24 of 26 myofibrillar, 32 of 61 mitochondrial, and 55 of 60 cytoplasmic measured proteins were lower in ER than AL (P < 0.05), regardless of dietary protein. The difference in absolute synthesis of myofibrillar, mitochondrial, and cytoplasmic proteins due to ER compared to AL was 28%, 16%, and 27%, respectively. Comparison of HP and SP within each energy state revealed lower turnover rates and prolonged half-lives for a majority of measured muscle proteins in HP than in SP in both ER and AL conditions (P < 0.001). Conclusions Prolonged ER in obese Zucker rats exerted a strong suppressive effect on myofibrillar, mitochondrial, and cytoplasmic MPS, suggesting reduced protein accretion contributed to lower gastrocnemius mass in ER-fed rats. Lower turnover rates of most muscle proteins in HP-fed rats without reductions in protein pool size (i.e., tissue mass) suggests prolonged HP intake, independent of energy, may prolong muscle protein lifespan of in obese Zucker rats. Funding Sources Supported by USAMRDC; authors’ views not official U.S. Army or DoD policy.

Inhibition of Yeast Growth by Tryptamine and Recovery with Tryptophan

Background:Tryptamine, a biogenic monoamine that is present in trace levels in the mammalian central nervous system, has probable roles as a neurotransmitter and/or a neuromodulator and may be associated with various neuropsychiatric disorders. One of the ways tryptamine may affect the body is by the competitive inhibition of the attachment of tryptophan to tryptophanyl tRNA synthetases.Methods:This study has explored the effects of tryptamine on growth of six yeast species (Saccharomyces cerevisiae, Candida glabrata, C. krusei, C. dubliniensis, C. tropicalis and C. lusitaniae) in media with glucose or ethanol as the carbon source, as well as recovery of growth inhibition by the addition of tryptophan.Results:Tryptamine was found to have an inhibitory effect on respiratory growth of all yeast species when grown with ethanol as the carbon source. Tryptamine also inhibited fermentative growth of Saccharomyces cerevisiae, C. krusei and C. tropicalis with glucose as the carbon source. In most cases the inhibitory effects were reduced by added tryptophan.Conclusion:The results obtained in this study are consistent with tryptamine competing with tryptophan to bind mitochondrial and cytoplasmic tryptophanyl tRNA synthetases in yeast: effects on mitochondrial and cytoplasmic protein synthesis can be studied as a function of growth with glucose or ethanol as a carbon source. Of the yeast species tested, there is variation in the sensitivity to tryptamine and the rescue by tryptophan. The current study suggests appropriate yeast strains and approaches for further studies.

Alternative strategies for the application of aminoglycoside antibiotics against the biofilm-forming human pathogenic bacteria.

Aminoglycosides are one of the common classes of antibiotics that have been widely used for treating infections caused by pathogenic bacteria. The mechanism of bactericidal action by aminoglycosides is well-known, by which it terminates the cytoplasmic protein synthesis. However, the potentials of aminoglycosides become hindered when facing the evolution of bacterial resistance mechanisms. Among multiple resistance mechanisms displayed by bacteria against antibiotics, the formation of biofilm is the mechanism that provides a barrier for antibiotics to reach the cellular level. Bacteria present in the biofilm also get protection against the impact of host immune responses, harsh environmental conditions, and other antimicrobial treatments. Hence, with the multifaceted resistance developed by biofilm-forming pathogenic bacteria, antibiotics are therefore discontinued for further applications. However, the recent research developed several alternative strategies such as optimization of the active concentration, modification of the environmental conditions, modification of the chemical structure, combinatorial application with other active agents, and formulation with biocompatible carrier materials to revitalize and exploit the new potential of aminoglycosides. The present review article describes the above mentioned multiple approaches and possible mechanisms for the application of aminoglycosides to treat biofilm-associated infections.

Quadriceps miR-542-3p and -5p are elevated in COPD and reduce function by inhibiting ribosomal and protein synthesis.

Reduced physical performance reduces quality of life in patients with chronic obstructive pulmonary disease (COPD). Impaired physical performance is, in part, a consequence of reduced muscle mass and function, which is accompanied by mitochondrial dysfunction. We recently showed that miR-542-3p and miR-542-5p were elevated in a small cohort of COPD patients and more markedly in critical care patients. In mice, these microRNAs (miRNAs) promoted mitochondrial dysfunction suggesting that they would affect physical performance in patients with COPD, but we did not explore the association of these miRNAs with disease severity or physical performance further. We therefore quantified miR-542-3p/5p and mitochondrial rRNA expression in RNA extracted from quadriceps muscle of patients with COPD and determined their association with physical performance. As miR-542-3p inhibits ribosomal protein synthesis its ability to inhibit protein synthesis was also determined in vitro. Both miR-542-3p expression and -5p expression were elevated in patients with COPD (5-fold P < 0.001) and the degree of elevation associated with impaired lung function (transfer capacity of the lung for CO in % and forced expiratory volume in 1 s in %) and physical performance (6-min walk distance in %). In COPD patients, the ratio of 12S rRNA to 16S rRNA was suppressed suggesting mitochondrial ribosomal stress and mitochondrial dysfunction and miR-542-3p/5p expression was inversely associated with mitochondrial gene expression and positively associated with p53 activity. miR-542-3p suppressed RPS23 expression and maximal protein synthesis in vitro. Our data show that miR-542-3p and -5p expression is elevated in COPD patients and may suppress physical performance at least in part by inhibiting mitochondrial and cytoplasmic ribosome synthesis and suppressing protein synthesis.NEW & NOTEWORTHY miR-542-3p and -5p are elevated in the quadriceps muscle of patients with chronic obstructive pulmonary disease (COPD) in proportion to the severity of their lung disease. These microRNAs inhibit mitochondrial and cytoplasmic protein synthesis suggesting that they contribute to impaired exercise performance in COPD.

Mitochondrial ubiquinone-mediated longevity is marked by reduced cytoplasmic mRNA translation.

Mutations in the clk-1 gene impair mitochondrial ubiquinone biosynthesis and extend lifespan in C. elegans. We demonstrate here that this life extension is linked to the repression of cytoplasmic mRNA translation, independent of the alleged nuclear form of CLK-1. Clk-1 mutations inhibit polyribosome formation similarly to daf-2 mutations that dampen insulin signaling. Comparisons of total versus polysomal RNAs in clk-1(qm30) mutants reveal a reduction in the translational efficiencies of mRNAs coding for elements of the translation machinery and an increase in those coding for the oxidative phosphorylation and autophagy pathways. Knocking down the transcription initiation factor TAF-4, a protein that becomes sequestered in the cytoplasm during early embryogenesis to induce transcriptional silencing, ameliorates the clk-1 inhibition of polyribosome formation. These results underscore a prominent role for the repression of cytoplasmic protein synthesis in eukaryotic lifespan extension and suggest that mutations impairing mitochondrial function are able to exploit this repression similarly to reductions of insulin signaling. Moreover, this report reveals an unexpected role for TAF-4 as a repressor of polyribosome formation when ubiquinone biosynthesis is compromised.

Concerted regulation of mitochondrial and nuclear non-coding RNAs by a dual-targeted RNase Z.

The molecular roles of the dually targeted ElaC domain protein 2 (ELAC2) during nuclear and mitochondrial RNA processing in vivo have not been distinguished. We generated conditional knockout mice of ELAC2 to identify that it is essential for life and its activity is non-redundant. Heart and skeletal muscle-specific loss of ELAC2 causes dilated cardiomyopathy and premature death at 4 weeks. Transcriptome-wide analyses of total RNAs, small RNAs, mitochondrial RNAs, and miRNAs identified the molecular targets of ELAC2 in vivo We show that ELAC2 is required for processing of tRNAs and for the balanced maintenance of C/D box snoRNAs, miRNAs, and a new class of tRNA fragments. We identify that correct biogenesis of regulatory non-coding RNAs is essential for both cytoplasmic and mitochondrial protein synthesis and the assembly of mitochondrial ribosomes and cytoplasmic polysomes. We show that nuclear tRNA processing is required for the balanced production of snoRNAs and miRNAs for gene expression and that 3' tRNA processing is an essential step in the production of all mature mitochondrial RNAs and the majority of nuclear tRNAs.

PTCD1 Is Required for 16S rRNA Maturation Complex Stability and Mitochondrial Ribosome Assembly

The regulation of mitochondrial RNA life cycles and their roles in ribosome biogenesis and energy metabolism are not fully understood. We used CRISPR/Cas9 to generate heart- and skeletal-muscle-specific knockout mice of the pentatricopeptide repeat domain protein 1, PTCD1, and show that its loss leads to severe cardiomyopathy and premature death. Our detailed transcriptome-wide and functional analyses of these mice enabled us to identify the molecular role of PTCD1 as a 16S rRNA-binding protein essential for its stability, pseudouridylation, and correct biogenesis of the mitochondrial large ribosomal subunit. We show that impaired mitoribosome biogenesis can have retrograde signaling effects on nuclear gene expression through the transcriptional activation of the mTOR pathway and upregulation of cytoplasmic protein synthesis and pro-survival factors in the absence of mitochondrial translation. Taken together, our data show that impaired assembly of the mitoribosome exerts itsconsequences via differential regulation of mitochondrial and cytoplasmic protein synthesis.

A novel system to monitor mitochondrial translation in yeast.

The mitochondrial genome is responsible for the production of a handful of polypeptides that are core subunits of the membrane-bound oxidative phosphorylation system. Until now the mechanistic studies of mitochondrial protein synthesis inside cells have been conducted with inhibition of cytoplasmic protein synthesis to reduce the background of nuclear gene expression with the undesired consequence of major disturbances of cellular signaling cascades. Here we have generated a system that allows direct monitoring of mitochondrial translation in unperturbed cells. A recoded gene for superfolder GFP was inserted into the yeast (Saccharomyces cerevisiae) mitochondrial genome and enabled the detection of translation through fluorescence microscopy and flow cytometry in functional mitochondria. This novel tool allows the investigation of the function and regulation of mitochondrial translation during stress signaling, aging and mitochondrial biogenesis.