Does protein synthesis occur in the nucleus?

Although it is universally accepted that protein synthesis occurs in the cytoplasm, the possibility that translation can also take place in the nucleus has been hotly debated. Reports have been published claiming to demonstrate nuclear translation, but alternative explanations for these results have not been excluded, and other experiments argue against it. Much of the appeal of nuclear translation is that functional proofreading of newly made mRNAs in the nucleus would provide an efficient way to monitor mRNAs for the presence of premature termination codons, thereby avoiding the synthesis of deleterious proteins. mRNAs that are still in the nucleus-associated fraction of cells are subject to translational proofreading resulting in nonsense-mediated mRNA decay and perhaps nonsense-associated alternate splicing. However, these mRNAs are likely to be in the perinuclear cytoplasm rather than within the nucleus. Therefore, in the absence of additional evidence, we conclude that nuclear translation is unlikely to occur.     

Well-defined protein–polymer conjugates—synthesis and potential applications

During the last decades, numerous studies have focused on combining the unique catalytic/functional properties and structural characteristics of proteins and enzymes with those of synthetic molecules and macromolecules. The aim of such multidisciplinary studies is to improve the properties of the natural component, combine them with those of the synthetic, and create novel biomaterials in the nanometer scale. The specific coupling of polymers onto the protein structures has proved to be one of the most straightforward and applicable approaches in that sense. In this article, we focus on the synthetic pathways that have or can be utilized to specifically couple proteins to polymers. The different categories of well-defined protein–polymer conjugates and the effect of the polymer on the protein function are discussed. Studies have shown that the specific conjugation of a synthetic polymer to a protein conveys its physico-chemical properties and, therefore, modifies the biodistribution and solubility of the protein, making it in certain cases soluble and active in organic solvents. An overview of the applications derived from such bioconjugates in the pharmaceutical industry, biocatalysis, and supramolecular nanobiotechnology is presented at the final part of the article.     

Selective blockade of trypanosomatid protein synthesis by a recombinant antibody anti-Trypanosoma cruzi P2β protein

The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.     

Repression of protein synthesis by miRNAs: how many mechanisms?

MicroRNAs are approximately 21-nucleotide-long regulators of gene expression that gain access to their target mRNAs by complementary base pairing. Recent studies have revealed that animal microRNAs might take diverse routes to repress gene expression, affecting both target mRNA levels and translation. Mechanistic details of microRNA-mediated repression are starting to emerge but a comprehensive picture of the inhibition, and particularly the effects on mRNA translation, is still lacking. Recent data support different microRNA mechanisms and a role for cytoplasmic processing bodies in the degradation and storage of mRNAs targeted by microRNA regulators.     

How is the balance between protein synthesis and degradation achieved?

Unlike most substances that cells manufacture, proteins are not produced and broken down by a common series of chemical reactions, but by completely different (independent and disconnected) mechanisms that possess no intrinsic means of making the rates of the two processes equal and attaining steady state concentrations. Balance between them is achieved extrinsically and is often imagined today to be the result of the actions of chemical feedback agents. But however instantiated, chemical feedback or any similar mechanism can only rectify induced imbalances in a system previously balanced by other means. Those "other means" necessarily involve reversible mass action or equilibrium-based interactions between native and altered forms of protein molecules somewhere in time and space between their synthesis and degradation.     

Is protein synthesis necessary for prostaglandin production by guinea-pig endometrium?

The outputs of prostaglandin (PG) F-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from Day-7 and Day-15 guinea-pig endometrium in culture were reduced by the inclusion of actinomycin D, cycloheximide and puromycin in the culture medium, with the output of PGF-2 alpha from Day-15 endometrium being particularly affected during the first 6 h of culture. The intrauterine administration of actinomycin D on Day 10 decreased the outputs of PGF-2 alpha and PGE-2, but not of 6-keto-PGF-1 alpha, from Day-15 endometrium in culture without affecting PG output from Day-15 myometrium in culture. Actinomycin D, cycloheximide and puromycin did not reduce PG output when superfused over the Day-7 and Day-15 guinea-pig uterus in vitro for 20 min, indicating that these compounds do not have a rapid inhibitory effect on endometrial PG synthesis. In fact, they tended to stimulate PG output during this 20-min period, with cycloheximide having a pronounced effect on PGE-2 output. The synthesis of secreted proteins, but not of cellular proteins, was greater by Day-15 than by Day-7 endometrium in culture. Actinomycin D, cycloheximide and puromycin inhibited the synthesis of secreted and cellular proteins by Day-7 and Day-15 endometrium in culture. Protein synthesis and PG synthesis in the endometrium were both inhibited to a greater extent by cycloheximide and puromycin than by actinomycin D. The intrauterine administration of actinomycin D on Day 10 reduced the syntheses of secreted and cellular proteins by Day-15 endometrium in culture. These findings indicate that the endometrial synthesis of PGs, particularly of PGF-2 alpha towards the end of the oestrous cycle, is dependent upon endometrial protein synthesis.     

The incorporation of the A2 protein to produce novel Qβ virus-like particles using cell-free protein synthesis

Virus-like particles (VLPs) have been employed for a number of nanometric applications because they self-assemble, exhibit a high degree of symmetry, and can be genetically and chemically modified. However, high symmetry does not allow for a single unique modification site on the VLP. Here, we demonstrate the co-expression of the cytotoxic A2 protein and the coat protein of the bacteriophage Qβ to form a nearly monodispersed population of novel VLPs. Cell-free protein synthesis allows for direct access and optimization of protein-synthesis and VLP-assembly. The A2 is shown to be incorporated at high efficiency, approaching a theoretical maximum of one A2 per VLP. This work demonstrates de novo production of a novel VLP, which contains a unique site that has the potential for future nanometric engineering applications.     

Post exercise carbohydrate–protein supplementation: phosphorylation of muscle proteins involved in glycogen synthesis and protein translation

The enzymes Akt, mTOR, p70(S6K), rpS6, GSK3, and glycogen synthase interact in the control of protein and/or glycogen synthesis in skeletal muscle, and each has been found to respond to exercise and nutrient supplementation. In the present study, we tested the hypothesis that nutrient supplementation post exercise, in the form of a carbohydrate-protein (CHO-PRO) supplement, would alter the phosphorylation state of these enzymes in a manner that should increase muscle protein and glycogen synthesis above that produced by exercise alone. After a 45 min cycling session followed by sprints and again 15 min later, the subjects (n = 8) ingested 400 ml of a CHO-PRO drink (7.8% dextrose and 1.8% protein-electrolyte) or a placebo drink, as assigned using a randomized, counter-balanced design with repeated measures. Biopsies of the vastus lateralis were taken before exercise and at 45 min of recovery. At 45 min after supplementation, CHO-PRO treatment yielded greater phosphorylation of Akt (65%), mTOR (86%), rpS6 (85-fold), and GSK3alpha/beta (57%) than pre-exercise levels (p < 0.05). Although p70(S6k) showed an exercise response after 45 min, there were no differences between treatments. Glycogen synthase (GS) phosphorylation was significantly reduced 45 min after exercise for both treatments, but the reduction in phosphorylation was greatest during the CHO-PRO treatment (3-fold decrease; p < 0.05), indicating greater activation of GS following supplementation. No difference between treatments was detected prior to exercise for any of the enzymes. These results suggest that a post exercise CHO-PRO supplement alters the phosporylation levels of the enzymes tested in a manner that should accelerate muscle glycogen synthesis and protein initiation during recovery from cycling exercise.     

Ageing and the regulation of protein synthesis: a balancing act?

Ageing in diverse species ranging from yeast to humans is associated with extensive changes in both general and specific protein synthesis. Accumulating evidence now indicates that these alterations are not simply a corollary of the ageing process but, rather, they have a causative role in senescent decline. Indeed, interfering with mRNA translation significantly influences longevity. Interestingly, the mechanisms that control mRNA translation interface with intricate, conserved signalling pathways and specific conditions that regulate ageing, such as the insulin-insulin growth factor 1 signalling pathway and caloric restriction. This emerging relationship reveals that protein synthesis is a novel determinant of ageing in diverse organisms such as yeast, worms, flies and mice and can thus be considered as a universal component of the ageing process.     

DNA,RNA and protein synthesis in ØLL55-infected Lactobacillus lactis

Lactobacillus lactis cells were infected with the bacteriophage ØLL55. The changes in DNA, RNA and protein synthesis were studied by following a long-term (over 3 h) incorporation of radioactive precursors into acid-insoluble material. Stimulation of DNA synthesis caused by phage occurred 30–35 min after infection and thymidine incorporation continued for about 70 min ceasing 10–20 min before the cells started to lyse. Cumulative (¹⁴C)-uracil incorporation into RNA continued at the level of uninfected cells for 30–40 min before starting to slow up. Protein synthesis in the infected cells followed that of a control culture for 40–50 min before the further incorporation of (¹⁴C)-leucine began to decrease.The additions of antibiotic inhibitors of RNA and protein synthesis (rifampicin and chloramphenicol, respectively) at various times before or during the prereplicative period showed that rifampicin, added up to 15 min after infection and chloramphenicol, added as late as 20–25 min after infection completely prevented the initiation of phage-genome replication. The later addition of these drugs did not prevent the out-burst of thymidine up-take, but promoted, however, a deduction in the initiations of new replication cycles. The results indicate that certain genes of ØLL55 genome must be expressed at the early stages of infection to confirm a proper onset and continuation of phage DNA replication.Abbreviations Rif rifampicin - CAL chloramphenicol - TCA trichloroacetic acid - cpm counts per minute     

Dietary γ-aminobutyric acid affects the brain protein synthesis rate in young rats

Summary. The purpose of this study was to determine whether the γ-aminobutyric acid (GABA) affects the rate of brain protein synthesis in male rats. Two experiments were done on five or three groups of young rats (5 wk) given the diets containing 20% casein administrated 0 mg, 25 mg, 50 mg, 100 mg or 200 mg/100 g body weight GABA dissolved in saline by oral gavage for 1 day (d) (Experiment 1), and given the diets contained 0%, 0.25% or 0.5% GABA added to the 20% casein diet (Experiment 2) for 10 d. The plasma concentration of growth hormone (GH) was the highest in rats administrated 50 mg and 100 mg/100 g body weight GABA. The concentration of serum GABA increased significantly with the supplementation groups. The fractional (Ks) rates of protein synthesis in brain regions, liver and gastrocnemius muscle increased significantly with the 20% casein + 0.25% GABA diet and still more 20% casein + 0.5% GABA compared with the 20% casein diet. In brain regions, liver and gastrocnemius muscle, the RNA activity [g protein synthesized/(g RNA·d)] significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. Our results suggest that the treatment of GABA to young male rats are likely to increase the concentrations of plasma GH and the rate of protein synthesis in the brain, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.     

Is there any relationship between decreased AgNOR protein synthesis and human hair loss?

Argyrophilic nucleolar organizing region associated proteins (AgNORs) play roles in cell proliferation and a variety of diseases. We attempted to determine whether decreased NOR protein synthesis causes human hair loss. We studied 21 healthy males who suffered hair loss on the frontal/vertex portion of the head. Hair root cells from normal and hair loss sites were stained for AgNOR. One hundred nuclei per site were evaluated and the AgNOR number and NORa/TNa proportions of individual cells were determined using a computer program. The cells from normal sites had significantly higher AgNOR counts than those from hair loss sites. Also, the cells from the normal sites had significantly higher NORa/TNa than cells from the hair loss sites. In the normal sites, the cells demonstrated more NOR protein synthesis than cells in hair loss sites. Therefore, decreased NOR protein synthesis appears to be related to hair loss in humans.     

β-Adrenergic receptor blockade blunts postexercise skeletal muscle mitochondrial protein synthesis rates in humans

β-Adrenergic receptor (AR) signaling is a regulator of skeletal muscle protein synthesis and mitochondrial biogenesis in mice. We hypothesized that β-AR blockade blunts postexercise skeletal muscle mitochondrial protein synthesis rates in adult humans. Six healthy men (mean ± SD: 26 ± 6 yr old, 39.9 ± 4.9 ml·kg(-1)·min(-1) peak O(2) uptake, 26.7 ± 2.0 kg/m(2) body mass index) performed 1 h of stationary cycle ergometer exercise (60% peak O(2) uptake) during 1) β-AR blockade (intravenous propranolol) and 2) administration of saline (control). Skeletal muscle mitochondrial, myofibrillar, and sarcoplasmic protein synthesis rates were assessed using [(2)H(5)]phenylalanine incorporation into skeletal muscle proteins after exercise. The mRNA content of signals for mitochondrial biogenesis was determined using real-time PCR. β-AR blockade decreased mitochondrial (from 0.217 ± 0.076 to 0.135 ± 0.031%/h, P < 0.05), but not myofibrillar or sarcoplasmic, protein synthesis rates. Peroxisome proliferator-activated receptor-γ coactivator-1α mRNA was increased ～2.5-fold (P < 0.05) at 5 h compared with 1 h postexercise but was not influenced by β-AR blockade. We conclude that decreased β-AR signaling during cycling can blunt the postexercise increase in mitochondrial protein synthesis rates without affecting mRNA content.     

Can ACG serve as an initiation codon for protein synthesis in eucaryotic cells?

An ACG codon, which replaces the AUG codon used to initiate the synthesis of bacteriophage T7 gene 0.3 protein, was shown to function as a low-efficiency initiation codon in a wheat germ cell-free protein-synthesizing system.     

Androgens on the estrogen receptor II — correlation between nuclear translocation and uterine protein synthesis

In the immature rat uterus, occupation of the androgen and estrogen receptor sites after injection of 5 α dihydrotestosterone (DHT = 17β-hydroxy-5α-androstan-3-one) were compared to the biological responses induced by the androgen on protein synthesis. Injection of 100 μg DHT induced a maximal occupation of androgen receptor sites (RA) but was totally ineffective in translocating the estrogen receptor sites and in increasing general protein synthesis. Conversely, high doses of androgen (? 3 mg) translocated the estrogen receptor (RE) and stimulated general protein synthesis. In addition, these high doses induced a specific uterine protein undistinguishable from that induced by estradiol treatment (IP). These results strongly suggest that the uterotrophic response of the uterus to androgen is correlated with the nuclear translocation of the estrogen receptor and not with that of the androgen receptor which is present in much smaller amounts.