Inhibition Of Protein Synthesis Research Articles

Differentiation activates mitochondrial OPA1 processing in myoblast cell lines

Mitochondrial optic atrophy-1 (OPA1) plays key roles in adapting mitochondrial structure to bioenergetic function. When transmembrane potential across the inner membrane (Δψm) is intact, long (L-OPA1) isoforms shape the inner membrane through membrane fusion and the formation of cristal junctions. When Δψm is lost, however, OPA1 is cleaved to short, inactive S-OPA1 isoforms by the OMA1 metalloprotease, disrupting mitochondrial structure and priming cellular stress responses such as apoptosis. Previously, we demonstrated that L-OPA1 of H9c2 cardiomyoblasts is insensitive to loss of Δψm via challenge with the protonophore carbonyl cyanide chlorophenyl hydrazone (CCCP), but that CCCP-induced OPA1 processing is activated upon differentiation in media with low serum supplemented with all-trans retinoic acid (ATRA). Here, we show that this developmental induction of OPA1 processing in H9c2 cells is independent of ATRA; moreover, pretreatment of undifferentiated H9c2s with chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, recapitulates the Δψm-sensitive OPA1 processing observed in differentiated H9c2s. L6.C11 and C2C12 myoblast lines display the same developmental and CAP-sensitive induction of OPA1 processing, demonstrating a general mechanism of OPA1 regulation in mammalian myoblast cell settings. Restoration of CCCP-induced OPA1 processing correlates with increased apoptotic sensitivity. Moreover, OPA1 knockdown indicates that intact OPA1 is necessary for effective myoblast differentiation. Taken together, our results indicate that a novel developmental mechanism acts to regulate OMA1-mediated OPA1 processing in myoblast cell lines, in which differentiation engages mitochondrial stress sensing.

Fangchinoline targets human renal cell carcinoma cells through modulation of apoptotic and non‑apoptotic cell deaths

The process of apoptosis is one of the essential processes involved in maintenance of homeostasis in the human body. It can aid to remove misfolded proteins or cellular organelles. This sequence is especially necessary in cancer cells. However, specifically targeting already apoptotic pathways can induce drug resistance in cancer cells and hence drugs can induce cell death by alternative mechanism. We investigated whether fangchinoline (FCN) can target renal carcinoma cells by inducing multiple cell death mechanisms. Both paraptosis, autophagy, and apoptosis were induced by FCN through stimulation of diverse molecular signaling pathways. FCN induced ROS production with GSH/GSSG imbalance, and ER stress. In addition, formation of autophagosome and autophagy related markers were stimulated by FCN. Moreover, FCN induced cell cycle arrest and PARP cleavage. Except for blocking protein synthesis, these three cell death pathways were found to be complementarily working together with each other. FCN also exhibited synergistic effects with paclitaxel in inducing programmed cell death in RCC cells. Our data indicates that FCN could induce apoptotic cell death and non-apoptotic cell death pathways and can be con-tribute to development of novel cancer prevention or therapy.

Auranofin and reactive oxygen species inhibit protein synthesis and regulate the level of the PLK1 protein in Ewing sarcoma cells.

Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.

Potential applications of antofine and its synthetic derivatives in cancer therapy: structural and molecular insights.

Cancer is a major global health challenge, being the second leading cause of morbidity and mortality after cardiovascular disease. The growing economic burden and profound psychosocial impact on patients and their families make it urgent to find innovative and effective anticancer solutions. For this reason, interest in using natural compounds to develop new cancer treatments has grown. In this respect, antofine, an alkaloid class found in Apocynaceae, Lauraceae, and Moraceae family plants, exhibits promising biological properties, including anti-inflammatory, anticancer, antiviral, and antifungal activities. Several molecular mechanisms have been identified underlying antofine anti-cancerous effects, including the inhibition of nuclear factor κB (NF-κB) and AKT/mTOR signaling pathways, epigenetic inhibition of protein synthesis, ribosomal targeting, induction of apoptosis, inhibition of DNA synthesis, and cell cycle arrest. This study discusses the molecular structure, sources, photochemistry, and anticancer properties of antofine in relation to its structure-activity relationship and molecular targets. Then, examine in vitro and in vivo studies and analyze the mechanisms of action underpinning antofine efficacy against cancer cells. This review also discusses multidrug resistance in human cancer and the potential of antofine in this context. Safety and toxicity concerns are also addressed as well as current challenges in antofine research, including the need for clinical trials and bioavailability optimization. This review aims to provide comprehensive information for more effective natural compound-based cancer treatments.

One-Trial Appetitive Learning Tasks for Drug Targeting.

One-trial appetitive learning developed from one-trial passive avoidance learning as a standard test of retrograde amnesia. It consists of one learning trial followed by a retention test, in which physiological manipulations are presented. As in passive avoidance learning, food- or waterdeprived rats or mice finding food or water inside an enclosure are vulnerable to the retrograde amnesia produced by electroconvulsive shock treatment or the injection of various drugs. In one-trial taste or odor learning conducted in rats, birds, snails, bees, and fruit flies, there is an association between a food item or odorant and contextual stimuli or the unconditioned stimulus of Pavlovian conditioning. The odor-related task in bees was sensitive to protein synthesis inhibition as well as cholinergic receptor blockade, both analogous to results found on the passive avoidance response in rodents, while the task in fruit flies was sensitive to genetic modifications and aging, as seen in the passive avoidance response of genetically modified and aged rodents. These results provide converging evidence of interspecies similarities underlying the neurochemical basis of learning.

Bacterial ubiquitin ligases hijack the host deubiquitinase OTUB1 to inhibit MTORC1 signaling and promote autophagy

ABSTRACT Many bacterial pathogens have evolved effective strategies to interfere with the ubiquitination network to evade clearance by the innate immune system. Here, we report that OTUB1, one of the most abundant deubiquitinases (DUBs) in mammalian cells, is subjected to both canonical and noncanonical ubiquitination during Legionella pneumophila infection. The effectors SidC and SdcA catalyze OTUB1 ubiquitination at multiple lysine residues, resulting in its association with a Legionella-containing vacuole. Lysine ubiquitination by SidC and SdcA promotes interactions between OTUB1 and DEPTOR, an inhibitor of the MTORC1 pathway, thus suppressing MTORC1 signaling. The inhibition of MTORC1 leads to suppression of host protein synthesis and promotion of host macroautophagy/autophagy during L. pneumophila infection. In addition, members of the SidE family effectors (SidEs) induce phosphoribosyl (PR)-linked ubiquitination of OTUB1 at Ser16 and Ser18 and block its DUB activity. The levels of the lysine and serine ubiquitination of OTUB1 are further regulated by effectors that function to antagonize the activities of SidC, SdcA and SidEs, including Lem27, DupA, DupB, SidJ and SdjA. Our study reveals an effectors-mediated complicated mechanism in regulating the activity of a host DUB. Abbreviations: BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; DUB: deubiquitinase; Dot/Icm: defective for organelle trafficking/intracellular multiplication; DEPTOR: DEP domain containing MTOR interacting protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; L. pneumophila: Legionella pneumophila; LCV: L egionella-containing vacuole; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTORC1: mechanistic target of rapamycin kinase complex 1; OTUB1: OTU deubiquitinase, ubiquitin aldehyde binding 1; PR-Ub: phosphoribosyl (PR)-linked ubiquitin; PTM: posttranslational modification; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SidEs: SidE family effectors; Ub: ubiquitin.

Acquisition of Resistance to PEGylated branched polyethylenimine Increases Pseudomonas aeruginosa Susceptibility to Aminoglycosides.

PEGylated branched polyethylenimine (PEG-BPEI) has antibacterial and antibiofilm properties. Exposure to PEG-BPEI through serial passage leads to resistant P. aeruginosa strains. The minimum inhibitory concentration (MIC) of 600 Da BPEI and PEGylated 600 Da BPEI (PEG-BPEI) in the wild-type PAO1 strain is 16 µg/ml while, after 15 serial passages, the MIC increased to 1024 µg/mL. An additional 15 rounds ofpassage in the absence of BPEI or PEG-BPEI did not change the 1024 µg/mL MIC. Gentamicin, Neomycin, and Tobramycin, cationic antibiotics that inhibit protein synthesis, have a 16-32 fold reduction of MIC values in PEG350-BPEI resistant strains, suggesting increased permeation. The influx of these antibiotics occurs using a self-mediated uptake mechanism, perhaps due to changes in the outer membrane Data show that resistance causes changes in genes related to outer membrane lipopolysaccharide (LPS) assembly. Mutations were noted in the gene coding for the polymerase Wzy that participates in the assembly of the O-antigen region. Other mutations were noted with wbpE and wbpI of the Wbp pathway responsible for the enzymatic synthesis of ManNAc(3NAc)A in the LPS of P. aeruginosa. These changes suggest thatPEG-BPEI resistance can be countered with antibiotics toprevent the emergence of PEG-BPEI resistant bacterial populations.

Does adjunctive clindamycin have a role in Staphylococcus aureus bacteremia? A protocol for the adjunctive treatment domain of the S. aureus Network Adaptive Platform (SNAP) randomized controlled trial.

The use of adjunctive antibiotics directed against exotoxin production in Staphylococcus aureus bacteremia (SAB) is widespread, and is recommended in many guidelines, but there is limited evidence underpinning this. Existing guidelines are based on the theoretical premise of toxin suppression, as many strains of S. aureus produce toxins such as leucocidins (e.g., Panton-Valentine Leucocidin (PVL), toxic shock syndrome toxin 1 (TSST-1), exfoliative toxins, and various enterotoxins). Many clinicians therefore believe that limiting exotoxin production release by S. aureus could reduce its virulence and improve clinical outcomes. Clindamycin, a protein synthesis inhibitor antibiotic, is commonly used for this purpose. We report the domain-specific protocol, embedded in a large adaptive, platform trial, seeking to definitively answer this question. The Staphylococcus aureus Network Adaptive Platform (SNAP) trial is a pragmatic, randomized, multi-center adaptive platform trial that aims to compare different SAB therapies, simultaneously, for 90-day mortality. The adjunctive treatment domain aims to test the effectiveness of adjunctive antibiotics, initially comparing clindamycin to no adjunctive antibiotic, but future adaptations may include other agents. Individuals will be randomized to receive either five days of adjunctive clindamycin (or lincomycin) or no adjunctive antibiotic therapy alongside standard of care antibiotics. Most participants with SAB (within 72hr of index blood culture and not contraindicated) will be eligible to participate in this domain. Prespecified analyses are defined in the statistical appendix to the core protocol and domain-specific secondary analyses will be adjusted for resistance to clindamycin, disease phenotype (complicated or uncomplicated SAB) and PVL-positive isolate.

The translational inhibitor and amnestic agent emetine also suppresses ongoing hippocampal neural activity similarly to other blockers of protein synthesis.

The consolidation of memory is thought to ultimately depend on the synthesis of new proteins, since translational inhibitors such as anisomycin and cycloheximide adversely affect the permanence of long-term memory. However, when applied directly in brain, these agents also profoundly suppress neural activity to an extent that is directly correlated to the degree of protein synthesis inhibition caused. Given that neural activity itself is likely to help mediate consolidation, this finding is a serious criticism of the strict de novo protein hypothesis of memory. Here, we test the neurophysiological effects of another translational inhibitor, emetine. Unilateral intra-hippocampal infusion of emetine suppressed ongoing local field and multiunit activity at ipsilateral sites as compared to the contralateral hippocampus in a fashion that was positively correlated to the degree of protein synthesis inhibition as confirmed by autoradiography. This suppression of activity was also specific to the circumscribed brain region in which protein synthesis inhibition took place. These experiments provide further evidence that ongoing protein synthesis is necessary and fundamental for neural function and suggest that the disruption of memory observed in behavioral experiments using translational inhibitors may be due, in large part, to neural suppression.

Paraproteinemic neuropathies

Paraproteinemic polyneuropathies (PPN) occur generally infrequently and arise from diseases, associated with formation and accumulation of a pathological protein, commonly known as paraprotein, which is typically an immunoglobulin or its free light chain. Such diseases include the following: monoclonal gammopathy of undetermined significance, multiple myeloma, Waldenström’s macroglobulinemia, AL amyloidosis, POEMS syndrome, etc. Diagnosis of PPN is challenging due to nonspecific, indistinct or atypical clinical manifestation, and it is necessary to apply specialized laboratory methods, including immunochemical analysis of blood and urine. Moreover, medications can cause symptoms of polyneuropathy themselves. Thus, it is important to suspect the first symptoms of the disease in time, determine the mechanism of damage to nerve fibers, carry out differential diagnosis and initiate therapy targeting the suppression of pathological protein synthesis.

Micrococcin cysteine-to-thiazole conversion through transient interactions between the scaffolding protein TclI and the modification enzymes TclJ and TclN.

Thiopeptides are a family of antimicrobial peptides characterized for having sulfur-containing heterocycles and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The thiopeptide micrococcin is synthesized by the ribosome and undergoes several post-translational modifications to acquire its bioactivity. In this study, we identify key interactions within the enzymatic complex that carries out cysteine to thiazole conversion in the biosynthesis of micrococcin.

Strobilurin X acts as an anticancer drug by inhibiting protein synthesis and suppressing mitochondrial respiratory chain activity

PurposeStrobilurins act as antifungal agents by inhibiting the mitochondrial respiratory chain. The cytotoxic activity of strobilurins, focusing on its anticancer activities, has been reported. However, the mechanisms involved in these activities remain unclear.MethodsThe cytotoxic effects of strobilurin X isolated from the mycelium of Mucidula. venosolamellata were examined in human cancer cell lines (A549 and HeLa) and normal fibroblasts (WI-38).ResultsStrobilurin X significantly decreased the viability of A549 and HeLa cells compared to that in the WI-38 cells after 48 h of exposure. The EC50 values for cytotoxicity in the A549, HeLa, and WI-38 cells were 3.4, 5.4, and 16.8 μg/mL, respectively. Strobilurin X inhibited the mitochondrial respiratory chain and enhanced the release of lactate in the A549 cells. The IC50 value of strobilurin X against the mitochondrial respiratory chain complex III activity was 139.8 ng/mL. The cytotoxicity induced by strobilurin X was not completely rescued after adding uridine, methyl pyruvate, or N-acetyl cysteine. Furthermore, pharmacological approaches demonstrated that strobilurin X failed to modulate the mitogen-activated protein kinase family and phosphoinositide 3-kinase-Akt pathways; alternatively, it suppressed protein synthesis independent of uridine.ConclusionStrobilurin X induced cytotoxicity by blocking the mitochondrial respiratory chain and suppressing protein synthesis. These findings may aid in the development of novel anticancer drugs using strobilurins.

Energy stress promotes P-bodies formation via lysine-63-linked polyubiquitination of HAX1.

Energy stress, characterized by the reduction of intracellular ATP, has been implicated in various diseases, including cancer. Here, we show that energy stress promotes the formation of P-bodies in a ubiquitin-dependent manner. Upon ATP depletion, the E3 ubiquitin ligase TRIM23 catalyzes lysine-63 (K63)-linked polyubiquitination of HCLS1-associated protein X-1 (HAX1). HAX1 ubiquitination triggers its liquid‒liquid phase separation (LLPS) and contributes to P-bodies assembly induced by energy stress. Ubiquitinated HAX1 also interacts with the essential P-body proteins, DDX6 and LSM14A, promoting their condensation. Moreover, we find that this TRIM23/HAX1 pathway is critical for the inhibition of global protein synthesis under energy stress conditions. Furthermore, high HAX1 ubiquitination, and increased cytoplasmic localization of TRIM23 along with elevated HAX1 levels, promotes colorectal cancer (CRC)-cell proliferation and correlates with poor prognosis in CRC patients. Our data not only elucidate a ubiquitination-dependent LLPS mechanism in RNP granules induced by energy stress but also propose a promising target for CRC therapy.

A Novel Inhibitor of Translation Initiation Factor eIF5B in Saccharomyces cerevisiae.

The eukaryotic translation initiation factor eIF5B is a bacterial IF2 ortholog that plays an important role in ribosome joining and stabilization of the initiator tRNA on the AUG start codon during the initiation of translation. We identified the fluorophenyl oxazole derivative 2,2-dibromo-1-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)ethanone quinolinol as an inhibitor of fungal protein synthesis using an in vitro translation assay in a fungal system. Mutants resistant to this compound were isolated in Saccharomyces cerevisiae and were demonstrated to contain amino acid substitutions in eIF5B that conferred the resistance. These results suggest that eIF5B is a target of potential antifungal compound and that mutation of eIF5B can confer resistance. Subsequent identification of 16 other mutants revealed that primary mutations clustered mainly on domain 2 of eIF5B and secondarily mainly on domain 4. Domain 2 has been implicated in the interaction with the small ribosomal subunit during initiation of translation. The tested translation inhibitor could act by weakening the functional contact between eIF5B and the ribosome complex. This data provides the basis for the development of a new family of antifungals.

Temperate phage-antibiotic synergy across antibiotic classes reveals new mechanism for preventing lysogeny.

A recent demonstration of synergy between a temperate phage and the antibiotic ciprofloxacin suggested a scalable approach to exploiting temperate phages in therapy, termed temperate phage-antibiotic synergy, which specifically interacted with the lysis-lysogeny decision. To determine whether this would hold true across antibiotics, we challenged Escherichia coli with the phage HK97 and a set of 13 antibiotics spanning seven classes. As expected, given the conserved induction pathway, we observed synergy with classes of drugs known to induce an SOS response: a sulfa drug, other quinolones, and mitomycin C. While some β-lactams exhibited synergy, this appeared to be traditional phage-antibiotic synergy, with no effect on the lysis-lysogeny decision. Curiously, we observed a potent synergy with antibiotics not known to induce the SOS response: protein synthesis inhibitors gentamicin, kanamycin, tetracycline, and azithromycin. The synergy results in an eightfold reduction in the effective minimum inhibitory concentration of gentamicin, complete eradication of the bacteria, and, when administered at sub-optimal doses, drastically decreases the frequency of lysogens emerging from the combined challenge. However, lysogens exhibit no increased sensitivity to the antibiotic; synergy was maintained in the absence of RecA; and the antibiotic reduced the initial frequency of lysogeny rather than selecting against formed lysogens. Our results confirm that SOS-inducing antibiotics broadly result in temperate-phage-specific synergy, but that other antibiotics can interact with temperate phages specifically and result in synergy. This is the first report of a means of chemically blocking entry into lysogeny, providing a new means for manipulating the key lysis-lysogeny decision.IMPORTANCEThe lysis-lysogeny decision is made by most bacterial viruses (bacteriophages, phages), determining whether to kill their host or go dormant within it. With over half of the bacteria containing phages waiting to wake, this is one of the most important behaviors in all of biology. These phages are also considered unusable for therapy because of this behavior. In this paper, we show that many antibiotics bias this behavior to "wake" the dormant phages, forcing them to kill their host, but some also prevent dormancy in the first place. These will be important tools to study this critical decision point and may enable the therapeutic use of these phages.

Neutralization of ricin toxin on building interior surfaces using liquid decontaminants.

Ricin is a highly toxic protein, capable of inhibiting protein synthesis within cells, and is produced from the beans of the Ricinus communis (castor bean) plant. Numerous recent incidents involving ricin have occurred, many in the form of mailed letters resulting in both building and mail sorting facility contamination. The goal of this study was to assess the decontamination efficacy of several commercial off-the-shelf (COTS) cleaners and decontaminants (solutions of sodium hypochlorite [bleach], quaternary ammonium, sodium percarbonate, peracetic acid, and hydrogen peroxide) against a crude preparation of ricin toxin. The ricin was inoculated onto four common building materials (pine wood, drywall joint tape, countertop laminate, and industrial carpet), and the decontaminants were applied to the test coupons using a handheld sprayer. Decontamination efficacy was quantified using an in-vitro cytotoxicity assay to measure the quantity of bioactive ricin toxin extracted from test coupons as compared to the corresponding positive controls (not sprayed with decontaminant). Results showed that decontamination efficacy varied by decontaminant and substrate material, and that efficacy generally improved as the number of spray applications or contact time increased. The solutions of 0.45% peracetic acid and the 20,000-parts per million (ppm) sodium hypochlorite provided the overall best decontamination efficacy. The 0.45% peracetic acid solution achieved 97.8 to 99.8% reduction with a 30-min contact time.

Detection of florfenicol resistance in opportunistic Acinetobacter spp. infections in rural Thailand.

Florfenicol (Ff) is an antimicrobial agent belonging to the class amphenicol used for the treatment of bacterial infections in livestock, poultry, and aquaculture (animal farming). It inhibits protein synthesis. Ff is an analog of chloramphenicol, an amphenicol compound on the WHO essential medicine list that is used for the treatment of human infections. Due to the extensive usage of Ff in animal farming, zoonotic pathogens have developed resistance to this antimicrobial agent. There are numerous reports of resistance genes from organisms infecting or colonizing animals found in human pathogens, suggesting a possible exchange of genetic materials. One of these genes is floR, a gene that encodes for an efflux pump that removes Ff from bacterial cells, conferring resistance against amphenicol, and is often associated with mobile genetic elements and other resistant determinants. In this study, we analyzed bacterial isolates recovered in rural Thailand from patients and environmental samples collected for disease monitoring. Whole genome sequencing was carried out for all the samples collected. Speciation and genome annotation was performed revealing the presence of the floR gene in the bacterial genome. The minimum inhibitory concentration (MIC) was determined for Ff and chloramphenicol. Chromosomal and phylogenetic analyses were performed to investigate the acquisition pattern of the floR gene. The presence of a conserved floR gene in unrelated Acinetobacter spp. isolated from human bacterial infections and environmental samples was observed, suggesting multiple and independent inter-species genetic exchange of drug-resistant determinants. The floR was found to be in the variable region containing various mobile genetic elements and other antibiotic resistance determinants; however, no evidence of HGT could be found. The floR gene identified in this study is chromosomal for all isolates. The study highlights a plausible impact of antimicrobials used in veterinary settings on human health. Ff shares cross-resistance with chloramphenicol, which is still in use in several countries. Furthermore, by selecting for floR-resistance genes, we may be selecting for and facilitating the zoonotic and reverse zoonotic exchange of other flanking resistance markers between human and animal pathogens or commensals with detrimental public health consequences.

Polyamine-Mediated Sensitization of Klebsiella pneumoniae to Macrolides through a Dual Mode of Action.

Chemicals bacteria encounter at the infection site could shape their stress and antibiotic responses; such effects are typically undetected under standard lab conditions. Polyamines are small molecules typically overproduced by the host during infection and have been shown to alter bacterial stress responses. We sought to determine the effect of polyamines on the antibiotic response of Klebsiella pneumoniae, a Gram-negative priority pathogen. Interestingly, putrescine and other natural polyamines sensitized K. pneumoniae to azithromycin, a macrolide protein translation inhibitor typically used for Gram-positive bacteria. This synergy was further potentiated in the physiological buffer, bicarbonate. Chemical genomic screens suggested a dual mechanism, whereby putrescine acts at the membrane and ribosome levels. Putrescine permeabilized the outer membrane of K. pneumoniae (NPN and β-lactamase assays) and the inner membrane (Escherichia coli β-galactosidase assays). Chemically and genetically perturbing membranes led to a loss of putrescine-azithromycin synergy. Putrescine also inhibited protein synthesis in an E. coli-derived cell-free protein expression assay simultaneously monitoring transcription and translation. Profiling the putrescine-azithromycin synergy against a combinatorial array of antibiotics targeting various ribosomal sites suggested that putrescine acts as tetracyclines targeting the 30S ribosomal acceptor site. Next, exploiting the natural polyamine-azithromycin synergy, we screened a polyamine analogue library for azithromycin adjuvants, discovering four azithromycin synergists with activity starting from the low micromolar range and mechanisms similar to putrescine. This work sheds light on the bacterial antibiotic responses under conditions more reflective of those at the infection site and provides a new strategy to extend the macrolide spectrum to drug-resistant K. pneumoniae.

Skeletal muscle ULK1 and ULK2 modulate protein synthesis impacting fiber size

Maintenance of skeletal muscle homeostasis is largely influenced by the processes of protein synthesis and degradation. However, the regulation of these processes remains incompletely understood particularly in a tissue or cell type-specific manner. We have previously demonstrated that Unc-51 like autophagy activating kinases 1 and 2 (Ulk1/2) are among the top 5 mostly enriched putative autophagy genes in skeletal muscle when compared to >90 other mouse tissues and cell lines (Fuqua et al., FASEB J, 2019). However, because protein kinases commonly have several downstream targets, here we hypothesized that skeletal muscle ULK1 and ULK2 redundantly modulate other important cellular processes. To begin to address this question, we generated mice with skeletal muscle-specific knockout of ULK1 and ULK2 (i.e., ULK1/2 skmDKO) and studied those at different ages. Muscle basal autophagy flux was impaired in ULK1/2 skmDKO mice. However, contrary to other models of autophagy impairment, which commonly leads to muscle atrophy, adult ULK1/2 skmDKO mice had increased muscle size (15-25%, P≤0.01). Hypertrophy occurred in all fiber types (6-20% increase in fiber diameter, P≤0.05) and in response to an acute (4-week) deficiency of ULK1/2 (via electroporations of plasmids encoding ULK1/2miR), where TA fiber diameter increased by 9% (p<0.05). Further studies using D2O labeling and cellular fractionation revealed that ULK1/2 skmDKO mice have increased myofibrillar protein synthesis rates (~5%/day, p<0.05). Examination of the molecular mechanisms involved suggests that ULK1/2 skmDKO mice have enhanced mTORC1-dependent signaling, particularly in the fed state. Altogether, our findings reveal for the first time that ULK1 and ULK2 collectively stimulate protein degradation and inhibit protein synthesis in skeletal muscle potentially impacting muscle mass and function in conditions of muscle atrophy and hypertrophy. Supported by the University of Iowa Fraternal Order of Eagles Diabetes Research Center (UIowa FOEDRC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The E3 ubiquitin-protein ligase synoviolin (Syvn1/Hrd1) promotes adaptive decreases in cardiac myocyte protein synthesis via eIF2α/ATF4 pathway activation

As the heart undergoes pathologic hypertrophy in response to many disease conditions, an imbalance between protein synthesis and degradation in cardiac myocytes, due to the increases in protein synthesis relative to protein degradation, happens, which challenges protein folding and endoplasmic reticulum (ER) proteostasis. This leads to ER stress and the activation of the ER stress response. Synoviolin (Syvn1), also known as HMG-CoA reductase degradation protein 1 (Hrd1), is an ER-localized protein that is upregulated in cardiac myocytes during ER stress. We previously found that Syvn1 mediates the ubiquitylation and consequent degradation of toxic misfolded proteins and preserved cardiac function in a model of pressure overload-induced cardiac pathology. The objective of this study was to examine whether Syvn1 is involved in regulating protein synthesis, and if so, what its function is during hypertrophic growth of cardiac myocytes. Given that cardiac hypertrophy is caused by increases in protein synthesis, we hypothesized that Syvn1 provides an adaptive response in part through regulation of protein synthesis and consequent decrease in cardiac hypertrophic response. We examined the effects of Syvn1 on ventricular myocyte growth in vitro and found that overexpression of Syvn1 decreased cardiac myocyte surface area as well as protein synthesis when treated with phenylephrine (PE), a treatment which normally leads to increased protein synthesis and increased cardiac myocyte size, mimicking pathological cardiac hypertrophy. We next focused on identifying the mechanism of Syvn1-mediated growth inhibition and found that increases in Syvn1 lead to activation of the PERK (protein kinase RNA (PKR)-like ER kinase) branch of the ER stress response, which leads to increased phosphorylation of the α-subunit of translation initiation factor 2 (eIF2α) at Ser51, thereby inhibiting global translational initiation. PERK activation also results in selective translation of a subset of mRNAs, including activating transcription factor 4 (ATF4), which activates the transcription of a wide range of genes involved in adaptation to stress conditions. We found increased ATF4 levels and increased ATF4 target gene expression with Syvn1 overexpression during PE treatment, compared to control PE treatment. Inhibition of the PERK pathway blocked Syvn1-mediated decrease in cardiac myocyte size and protein synthesis as well as PERK activation and ATF4 target gene induction. Additionally, we found that an adeno-associated virus encoding Syvn1, administered in a therapeutically-relevant manner after the onset of pathological cardiac hypertrophy induced by transverse-aortic constriction in mice, blunted pathological remodeling and preserved cardiac function. We posit that the Syvn1/eIF2α/ATF4 pathway constitutes a newly-discovered mechanism for balancing the proteome in cardiac myocytes. Here we found that Syvn1 upregulation blunts maladaptive increase in cardiac myocyte size during pathologic hypertrophy. In conclusion, these findings suggest that Syvn1 is a critical regulator of cardiac myocyte protein synthesis and subsequent growth and that increases in Syvn1 lead to the activation of the PERK branch of the ER stress response, which results in inhibition of global protein synthesis, and activation of ATF4. This work was supported by the National Institutes of Health (R01HL157027) and the University of Arizona Health Sciences Career Development Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.