Modulation of endogenous opioid signaling by inhibitors of puromycin-sensitive aminopeptidase.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin-sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

[Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy].

Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.

Minnelide Markedly Reduces Proteinuria in Mice with Adriamycin Nephropathy by Protecting Against Podocyte Injury.

Minimal change disease (MCD) is the most common cause of idiopathic nephrotic syndrome in children. The current major therapy is hormones for most steroid-sensitive patients. However, many patients have recurrent relapses of the disease and require long-term immunosuppression, leading to significant morbidity due to the side effects of the drugs. Therefore, better drugs need to be urgently explored to treat nephrotic syndrome while avoiding the side effects of drugs. Minnelide, a water-soluble prodrug of triptolide, has been proved to be effective in treating cancers in many clinical trials. This study aimed to investigate the therapeutic effect of minnelide in mice with adriamycin (ADR) nephropathy, its underlying protection mechanisms, and its reproductive toxicity. Minnelide was administered intraperitoneally to 6-8-week female mice with adriamycin nephropathy for 2 weeks, and the urine, blood, and kidney tissues were taken to analyze the therapeutic effect. In addition, we evaluated reproductive toxicity by measuring the levels of gonadal hormones and observing the histological changes in ovaries and testes. Primary mouse podocytes were exposed to puromycin (PAN) to damage the cytoskeleton and induce apoptosis, and then, triptolide was used to evaluate the therapeutic effect and underlying protection mechanisms in vitro. It was observed that minnelide dramatically alleviated proteinuria and apoptosis in mice with adriamycin nephropathy. In vitro, triptolide ameliorated puromycin-induced cytoskeletal rearrangement and apoptosis via reactive oxygen species-mediated mitochondrial pathway. In addition, minnelide caused no reproductive toxicity to male and female mice. The results suggested that minnelide might be a promising drug for nephrotic syndrome.

Genome-wide gene expression analysis reveals molecular insights into the drug-induced toxicity of nephrotoxic agents.

Drug-induced nephrotoxicity is frequently reported. However, the mechanisms underlying nephrotoxic medications and their overlapping molecular events, which might have therapeutic value, are unclear. We performed a genome-wide analysis of gene expression and a gene set enrichment analysis to identify common and unique pathways associated with the toxicity of colistin, ifosfamide, indomethacin, and puromycin. Rats were randomly allocated into the treatment or control group. The treatment group received a toxic dose once daily of each investigated drug for 1 week. Differentially expressed genes were found in the drug-treated kidney and liver compared to the control, except for colistin in the liver. Upregulated pathways were mainly related to cell death, cell cycle, protein synthesis, and immune response modulation in the kidney. Cell cycle was upregulated by all drugs. Downregulated pathways were associated with carbon metabolism, amino acid metabolism, and fatty acid metabolism. Indomethacin, colistin, and puromycin shared the most altered pathways in the kidney. Ifosfamide and indomethacin affected molecular processes greatly in the liver. Our findings provide insight into the mechanisms underlying the renal and hepatic adverse effects of the four drugs. Further investigation should explore the combinatory drug therapies that attenuate the toxic effects and maximize the effectiveness of nephrotoxic drugs.

A transgenic mouse embryonic stem cell line for puromycin selection of V0V interneurons from heterogenous induced cultures.

BACKGROUND: Spinal interneurons (INs) relay sensory and motor control information between the brain and body. When this relay circuitry is disrupted from injury or disease, it is devastating to patients due to the lack of native recovery in central nervous system (CNS) tissues. Obtaining a purified population of INs is necessary to better understand their role in normal function and as potential therapies in CNS. The ventral V0 (V0V) INs are excitatory neurons involved in locomotor circuits and are thus of interest for understanding normal and pathological spinal cord function. To achieve scalable amounts of V0V INs, they can be derived from pluripotent sources, such as mouse embryonic stem cells (mESCs), but the resultant culture is heterogenous, obscuring the specific role of V0V INs. This study generated a transgenic mESC line to enrich V0V INs from induced cultures to allow for a scalable, enriched population for future in vitro and in vivo studies. METHODS: The transgenic Evx1-PAC mESC line was created by CRISPR-Cas9-mediated insertion of puromycin-N-acetyltransferase (PAC) into the locus of V0V IN marker Evx1. Evx1 and PAC mRNA expression were measured by qPCR. Viability staining helped establish the selection protocol for V0V INs derived from Evx1-PAC mESCs inductions. Immunostaining was used to examine composition of selected inductions. Cultures were maintained up to 30 days to examine maturation by expression of mature/synaptic markers, determined by immunostaining, and functional activity in co-cultures with selected motor neurons (MNs) and V2a INs on microelectrode arrays (MEAs). RESULTS: V0V IN inductions were best selected with 4 µg/mL puromycin on day 10 to 11 and showed reduction of other IN populations and elimination of proliferative cells. Long-term selected cultures were highly neuronal, expressing neuronal nuclear marker NeuN, dendritic marker MAP2, pre-synaptic marker Bassoon, and glutamatergic marker VGLUT2, with some cholinergic VAChT-expressing cells. Functional studies on MEAs showed that co-cultures with MNs or MNs plus V2a INs created neuronal networks with synchronized bursting. CONCLUSIONS: Evx1-PAC mESCs can be used to purify V0V IN cultures for largely glutamatergic neurons that can be used in network formation studies or for rodent models requiring transplanted V0V INs.

Puromycin-based purification of cells with high expression of the cytochrome P450 CYP3A4 gene from a patient with drug-induced liver injury (DILI).

BACKGROUND: Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI). METHODS: To generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations. RESULTS: The hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen. CONCLUSIONS: iPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.

Expansion of the Genetic Code Through the Use of Modified Bacterial Ribosomes.

Biological protein synthesis is mediated by the ribosome, and employs ~20 proteinogenic amino acids as building blocks. Through the use of misacylated tRNAs, presently accessible by any of several strategies, it is now possible to employ in vitro and in vivo protein biosynthesis to elaborate proteins containing a much larger variety of amino acid building blocks. However, the incorporation of this broader variety of amino acids is limited to those species utilized by the ribosome. As a consequence, virtually all of the substrates utilized over time have been L-α-amino acids. In recent years, a variety of structural and biochemical studies have provided important insights into those regions of the 23S ribosomal RNA that are involved in peptide bond formation. Subsequent experiments, involving the randomization of key regions of 23S rRNA required for peptide bond formation, have afforded libraries of E. coli harboring plasmids with the rrnB gene modified in the key regions. Selections based on the use of modified puromycin derivatives with altered amino acids then identified clones uniquely sensitive to individual puromycin derivatives. These clones often recognized misacylated tRNAs containing altered amino acids similar to those in the modified puromycins, and incorporated the amino acid analogues into proteins. In this fashion, it has been possible to realize the synthesis of proteins containing D-amino acids, ß-amino acids, phosphorylated amino acids, as well as long chain and cyclic amino acids in which the nucleophilic amino group is not in the α-position. Of special interest have been dipeptides and dipeptidomimetics of diverse utility.

Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast.

BACKGROUND: Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin. RESULTS: We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways. CONCLUSIONS: We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed.

Mycobacterium tuberculosis puromycin hydrolase displays a prolyl oligopeptidase fold and an acyl aminopeptidase activity.

Puromycin-hydrolizing peptidases have been described as members of the prolyl oligopeptidase peptidase family. These enzymes are present across all domains of life but still little is known of the homologs found in the pathogenic bacterium Mycobacterium tuberculosis. The crystal structure of a M. tuberculosis puromycin hydrolase peptidase has been determined at 3 Angstrom resolution, revealing a conserved prolyl oligopeptidase fold, defined by α/ß-hydrolase and ß-propeller domains with two distinctive loops that occlude access of large substrates to the active site. The enzyme displayed amino peptidase activity with a substrate specificity preference for hydrophobic residues in the decreasing order of phenylalanine, leucine, alanine and proline. The enzyme's active site is lined by residues Glu564 for the coordination of the substrates amino terminal moiety and His561, Val608, Tyr78, Trp306, Phe563 and Ty567 for the accommodation of hydrophobic substrates. The availability of a crystal structure for puromycin hydrolase of M. tuberculosis shall facilitate the development of inhibitors with therapeutic applications.

Acute and chronic effects of Rhaponticum carthamoides and Rhodiola rosea extracts supplementation coupled to resistance exercise on muscle protein synthesis and mechanical power in rats.

BACKGROUND: Owing to its strength-building and adaptogenic properties, Rhaponticum carthamoides (Rha) has been commonly used by elite Soviet and Russian athletes. Rhodiola rosea (Rho) is known to reduce physical and mental fatigue and improve endurance performance. However, the association of these two nutritional supplements with resistance exercise performance has never been tested. Resistance exercise is still the best way to stimulate protein synthesis and induce chronic muscle adaptations. The aim of this study was to investigate the acute and chronic effects of resistance exercise coupled with Rha and Rho supplementation on protein synthesis, muscle phenotype, and physical performance. METHODS: For the acute study, fifty-six rats were assigned to either a trained control group or one of the groups treated with specific doses of Rha and/or Rho. Each rats performed a single bout of climbing resistance exercise. The supplements were administered immediately after exercise by oral gavage. Protein synthesis was measured via puromycin incorporation. For the chronic study, forty rats were assigned to either the control group or one of the groups treated with doses adjusted from the acute study results. The rats were trained five times per week for 4 weeks with the same bout of climbing resistance exercise with additionals loads. Rha + Rho supplement was administered immediately after each training by oral gavage. RESULTS: The findings of the acute study indicated that Rha and Rha + Rho supplementation after resistance exercise stimulated protein synthesis more than resistance exercise alone (p < 0.05). After 4 weeks of training, the mean power performance was increased in the Rha + Rho and Rha-alone groups (p < 0.05) without any significant supplementation effect on muscle weight or fiber cross-sectional area. A tendency towards an increase in type I/ type II fiber ratio was observed in Rha/Rho-treated groups compared to that in the trained control group. CONCLUSION: Rhodiola and Rhaponticum supplementation after resistance exercise could synergistically improve protein synthesis, muscle phenotype and physical performance.

Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes.

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.

Puromycin reactivity does not accurately localize translation at the subcellular level.

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.

Structural studies on dihydrouridine synthase A (DusA) from Pseudomonas aeruginosa.

Dihydrouridination is one of the abundant modifications in tRNA editing. The presence of dihydrouridine is attributed to tRNA stability desired for the efficient gene translation process. The conversion of uridine to dihydrouridine is catalyzed by flavine containing enzyme called dihydrouridine synthase (Dus). We report first ever information about DusA enzyme from Pseudomonas aeruginosa in form of structural and functional studies. The gene coding for DusA from P. aeruginosa (PADusA) was cloned, expressed and purified, using recombinant DNA technology methods. Thermal and chemical stability of PADusA was determined with respect to temperature and urea-induced equilibrium unfolding experiments, with monitoring the change of ellipticity at 200-260 nm by Circular Dichroism (CD) spectroscopy. Unfolding studies revealed that PADusA has acquired a stable tertiary structure fold with a Tm value of 46.2 °C and Cm of 2.7 M for urea. The enzyme contains 43% α-helices and 16% ß-strands. The three dimensional structure of PADusA was modeled using insilico methods. In order to understand the mechanism of substrate recognition and catalysis, tRNA and puromycin were docked on PADusA structure and their binding was analyzed. The structural features suggested that PADusA may also form a novel target for structure based drug design of antimicrobial agents.

Cell-type-specific quantification of protein synthesis in vivo.

Although protein synthesis is a conserved and essential cellular function, it is often regulated in a cell-type-specific manner to influence cell fate, growth and homeostasis. Most methods used to measure protein synthesis depend on metabolically labeling large numbers of cells with radiolabeled amino acids or amino acid analogs. Because these methods typically depend on specialized growth conditions, they have been largely restricted to yeast, bacteria and cell lines. Application of these techniques to investigating protein synthesis within mammalian systems in vivo has been challenging. The synthesis of O-propargyl-puromycin (OP-Puro), an analog of puromycin that contains a terminal alkyne group, has facilitated the quantification of protein synthesis within individual cells in vivo. OP-Puro enters the acceptor site of ribosomes and incorporates into nascent polypeptide chains. Incorporated OP-Puro can be detected through a click-chemistry reaction that links it to a fluorescently tagged azide molecule. In this protocol, we describe how to administer OP-Puro to mice, obtain cells of interest (here, we use bone marrow cells) just 1 h later, and quantify the amount of protein synthesized per hour by flow cytometry on the basis of OP-Puro incorporation. We have used this approach to show that hematopoietic stem cells (HSCs) exhibit an unusually low rate of protein synthesis relative to other hematopoietic cells, and it can be easily adapted to quantify cell-type-specific rates of protein synthesis across diverse mammalian tissues in vivo. Measurement of protein synthesis within bone marrow cells in a cohort of six mice can be achieved in 8-10 h.

Cultured equine satellite cells as a model system to assess leucine stimulated protein synthesis in horse muscle.

Leucine has been shown to stimulate the mammalian/mechanistic target of rapamycin (mTOR) signaling pathway which plays numerous key regulatory roles in cell growth, survival, and metabolism including protein synthesis in a number of species. However, previous work with equine satellite cells has suggested distinct species differences in regards to physiological effects and the magnitude of responses to growth factors and regulators. Because there is limited research available regarding the role of leucine in regulating equine skeletal muscle protein synthesis, the objective of this study was to evaluate the effect of leucine on the mTOR signaling pathway in cultured equine satellite. Protein synthesis was evaluated by measuring the incorporation of [3H] Phenylalanine (3HPhe) in equine satellite cell myotube cultures treated with a leucine titration ranging from 0 to 408 µM. Our results show a 1.8-fold increase (P < 0.02) in protein synthesis at levels slightly greater than those found in the general circulation, 204 and 408 µM when compared to a no leucine control (0 µM). Puromycin incorporation, a nonradioactive surface sensing of translation (SUnSET) methodology, was also measured in cells treated with leucine (LEU; 408 µM), a no-leucine control (CON), and a puromycin-negative vehicle (PURO-). These results demonstrated a 180% increase (P = 0.0056) in puromycin incorporation in LEU compared to CON cultures. To evaluate the mTOR signaling pathway, equine satellite cell myotube cultures were treated with leucine (LEU; 408 µM) or a no-leucine control (CON) in the presence or absence of rapamycin (LR and CR, respectively), an inhibitor of mTOR. The mTOR inhibitor, rapamycin, suppressed phosphorylation of mTOR (P < 0.01) and rS6 (P < 0.01) with an increase in phosphorylation of rS6 in leucine-treated cultures observed when compared to control cultures (P < 0.05). Similarly, there was a 27% increase (P < 0.005) in the hyperphosphorylated γ-form of 4E-BP1 compared to total 4E-BP1 in LEU compared to CON cultures with leucine-induced phosphorylation of 4E-BP1 completely blocked by rapamycin with a smaller decrease observed in CR compared to CON cultures. The major finding of this study was that leucine activated the mTOR translation initiation pathway and increased transcription of global proteins in cultured equine satellite cells. Use of the cell culture system with primary equine muscle cell lines provides the opportunity to distinguish the impact of leucine on muscle and protein synthesis, independent of systemic interactions.

Glomerular filtration drug injury: In vitro evaluation of functional and morphological podocyte perturbations.

The kidney is an organ that plays a major role in the excretion of numerous compounds such as drugs and chemicals. However, a great number of pharmacological molecules are nephrotoxic, affecting the efficiency of the treatment and increasing morbidity or mortality. Focusing on glomerular filtration, we propose in this study a simple and reproducible in vitro human model that is able to bring to light a functional podocyte injury, correlated with morphologic/phenotypic changes after drug exposure. This model was used for the evaluation of paracellular permeability of FITC-dextran molecules as well as FITC-BSA after different treatments. Puromycin aminonucleoside and adriamycin, compounds known to induce proteinuria in vivo and that serve here as positive nephrotoxic drug controls, were able to induce an important increase in fluorescent probe passage through the cell monolayer. Different molecules were then evaluated for their potential effect on podocyte filtration. Our results demonstrated that a drug effect could be time dependent, stable or scalable and relatively specific. Immunofluorescence studies indicated that these functional perturbations were due to cytoskeletal perturbations, monolayer disassembly or could be correlated with a decrease in nephrin expression and/or ZO-1 relocation. Taken together, our results demonstrated that this in vitro human model represents an interesting tool for the screening of the renal toxicity of drugs.

Temporal dynamics of cardiac hypertrophic growth in response to pressure overload.

Hypertension is one of the most important risk factors of heart failure. In response to high blood pressure, the left ventricle manifests hypertrophic growth to ameliorate wall stress, which may progress into decompensation and trigger pathological cardiac remodeling. Despite the clinical importance, the temporal dynamics of pathological cardiac growth remain elusive. Here, we took advantage of the puromycin labeling approach to measure the relative rates of protein synthesis as a way to delineate the temporal regulation of cardiac hypertrophic growth. We first identified the optimal treatment conditions for puromycin in neonatal rat ventricular myocyte culture. We went on to demonstrate that myocyte growth reached its peak rate after 8-10 h of growth stimulation. At the in vivo level, with the use of an acute surgical model of pressure-overload stress, we observed the maximal growth rate to occur at day 7 after surgery. Moreover, RNA sequencing analysis supports that the most profound transcriptomic changes occur during the early phase of hypertrophic growth. Our results therefore suggest that cardiac myocytes mount an immediate growth response in reply to pressure overload followed by a gradual return to basal levels of protein synthesis, highlighting the temporal dynamics of pathological cardiac hypertrophic growth.NEW & NOTEWORTHY We determined the optimal conditions of puromycin incorporation in cardiac myocyte culture. We took advantage of this approach to identify the growth dynamics of cardiac myocytes in vitro. We went further to discover the protein synthesis rate in vivo, which provides novel insights about cardiac temporal growth dynamics in response to pressure overload.

Newly translated proteins are substrates for ubiquitin, ISG15, and FAT10.

The ubiquitin-like modifier, FAT10, is involved in proteasomal degradation and antigen processing. As ubiquitin and the ubiquitin-like modifier, ISG15, cotranslationally modify proteins, we investigated whether FAT10 could also be conjugated to newly synthesized proteins. Indeed, we found that nascent proteins are modified with FAT10, but not with the same preference for newly synthesized proteins as observed for ISG15. Our data show that puromycin-labeled polypeptides are strongly modified by ISG15 and less intensely by ubiquitin and FAT10. Nevertheless, conjugates of all three modifiers copurify with ribosomes. Taken together, we show that unlike ISG15, ubiquitin and FAT10 are conjugated to a similar degree to newly translated and pre-existing proteins.

Essential structural elements in tRNA(Pro) for EF-P-mediated alleviation of translation stalling.

The ribosome stalls on translation of polyproline sequences due to inefficient peptide bond formation between consecutive prolines. The translation factor EF-P is able to alleviate this stalling by accelerating Pro-Pro formation. However, the mechanism by which EF-P recognizes the stalled complexes and accelerates peptide bond formation is not known. Here, we use genetic code reprogramming through a flexible in-vitro translation (FIT) system to investigate how mutations in tRNA(Pro) affect EF-P function. We show that the 9-nt D-loop closed by the stable D-stem sequence in tRNA(Pro) is a crucial recognition determinant for EF-P. Such D-arm structures are shared only among the tRNA(Pro) isoacceptors and tRNA(fMet) in Escherichia coli, and the D-arm of tRNA(fMet) is essential for EF-P-induced acceleration of fMet-puromycin formation. Thus, the activity of EF-P is controlled by recognition elements in the tRNA D-arm.

Detection of O-propargyl-puromycin with SUMO and ubiquitin by click chemistry at PML-nuclear bodies during abortive proteasome activities.

The amino-nucleoside antibiotic, puromycin, acts by covalently linking to elongating polypeptide chains on ribosomes to generate prematurely terminated immature polypeptides. The trafficking of puromycin-conjugated (puromycylated) immature polypeptides within cell has, however, remained elusive. In this study, using O-propargyl-puromycin (OP-Puro), the distribution of puromycylated polypeptides was assessed in HeLa cells by click chemistry. Under standard culture conditions, OP-Puro signals were detected in the cytoplasm and nucleus with the highest concentrations in the nucleolus. Intriguingly, when proteasome activities were aborted using MG132, OP-Puro signals began to accumulate at promyelocytic leukemia nuclear bodies (PML-NBs) in addition to the nucleolus. We also found promiscuous association of OP-Puro signals with SUMO-2/3 and ubiquitin at PML-NBs, but not at the nucleolus, during abortive proteasome activities. This study reveals a previously unknown distribution of OP-Puro that argues for a nuclear function in regulating immature protein homeostasis.