Rate Of Protein Biosynthesis Research Articles

Comprehensive translational profiling and STE AI uncover rapid control of protein biosynthesis during cell stress.

Translational control is important in all life, but it remains a challenge to accurately quantify. When ribosomes translate messenger (m)RNA into proteins, they attach to the mRNA in series, forming poly(ribo)somes, and can co-localize. Here, we computationally model new types of co-localized ribosomal complexes on mRNA and identify them using enhanced translation complex profile sequencing (eTCP-seq) based on rapid in vivo crosslinking. We detect long disome footprints outside regions of non-random elongation stalls and show these are linked to translation initiation and protein biosynthesis rates. We subject footprints of disomes and other translation complexes to artificial intelligence (AI) analysis and construct a new, accurate and self-normalized measure of translation, termed stochastic translation efficiency (STE). We then apply STE to investigate rapid changes to mRNA translation in yeast undergoing glucose depletion. Importantly, we show that, well beyond tagging elongation stalls, footprints of co-localized ribosomes provide rich insight into translational mechanisms, polysome dynamics and topology. STE AI ranks cellular mRNAs by absolute translation rates under given conditions, can assist in identifying its control elements and will facilitate the development of next-generation synthetic biology designs and mRNA-based therapeutics.

INFLUENCE OF THE TACA TRANSCRIPTION FACTOR KNOCKOUT ON THE TRANSCRIPTION AND EXPRESSION OF THE CBHI GENE IN THE PENICILLIUM VERRUCULOSUM STRAIN

The tacA gene, encoding the TacA repressor protein, was cloned by “walking the uncloned DNA” method from the genomic DNA of the fungus Penicillium verruculosum B1-221-151. Knockout of the tacA and niaD genes by the CRIS-PR/CAS9 led to the production of a new host strain P. verruculosum ΔniaDΔtacA, characterized by a higher rate of extracellular protein biosynthesis. Analysis of the transcription and expression of the cbhI gene in the original P. verruculosum B1-221-151 strain and in the P. verruculosum ΔniaDΔtacA strain showed a sharp increase in the level of cbhI gene transcription 2 h after the start of induction with cellobiose, cellotriose, gentiobiose, and a mixture of di- and trisaccharides in comparison with the transcription of the cbhI gene in the original strain. The speci c activity of cellobiohydrolase I, the main enzyme of the cellulolytic complex of the fungus P. verruculosum, by 96 h of fermentation of the ΔtacA strain increased 3 times compared to the original strain.

HSP70 and their co-chaperones in the human malaria parasite P. falciparum and their potential as drug targets.

As part of their life-cycle, malaria parasites undergo rapid cell multiplication and division, with one parasite giving rise to over 20 new parasites within the course of 48 h. To support this, the parasite has an extremely high metabolic rate and level of protein biosynthesis. Underpinning these activities, the parasite encodes a number of chaperone/heat shock proteins, belonging to various families. Research over the past decade has revealed that these proteins are involved in a number of essential processes within the parasite, or within the infected host cell. Due to this, these proteins are now being viewed as potential targets for drug development, and we have begun to characterize their properties in more detail. In this article we summarize the current state of knowledge about one particular chaperone family, that of the HSP70, and highlight their importance, function, and potential co-chaperone interactions. This is then discussed with regard to the suitability of these proteins and interactions for drug development.

N1-acetylspermidine is a determinant of hair follicle stem cell fate.

ABSTRACTStem cell differentiation is accompanied by increased mRNA translation. The rate of protein biosynthesis is influenced by the polyamines putrescine, spermidine and spermine, which are essential for cell growth and stem cell maintenance. However, the role of polyamines as endogenous effectors of stem cell fate and whether they act through translational control remains obscure. Here, we investigate the function of polyamines in stem cell fate decisions using hair follicle stem cell (HFSC) organoids. Compared to progenitor cells, HFSCs showed lower translation rates, correlating with reduced polyamine levels. Surprisingly, overall polyamine depletion decreased translation but did not affect cell fate. In contrast, specific depletion of natural polyamines mediated by spermidine/spermine N1-acetyltransferase (SSAT; also known as SAT1) activation did not reduce translation but enhanced stemness. These results suggest a translation-independent role of polyamines in cell fate regulation. Indeed, we identified N1-acetylspermidine as a determinant of cell fate that acted through increasing self-renewal, and observed elevated N1-acetylspermidine levels upon depilation-mediated HFSC proliferation and differentiation in vivo. Overall, this study delineates the diverse routes of polyamine metabolism-mediated regulation of stem cell fate decisions.This article has an associated First Person interview with the first author of the paper.

Component of splicing factor SF3b plays a key role in translational control of polyribosomes on the endoplasmic reticulum

One of the morphological hallmarks of terminally differentiated secretory cells is highly proliferated membrane of the rough endoplasmic reticulum (ER), but the molecular basis for the high rate of protein biosynthesis in these cells remains poorly documented. An important aspect of ER translational control is the molecular mechanism that supports efficient use of targeted mRNAs in polyribosomes. Here, we identify an enhancement system for ER translation promoted by p180, an integral ER membrane protein we previously reported as an essential factor for the assembly of ER polyribosomes. We provide evidence that association of target mRNAs with p180 is critical for efficient translation, and that SF3b4, an RNA-binding protein in the splicing factor SF3b, functions as a cofactor for p180 at the ER and plays a key role in enhanced translation of secretory proteins. A cis-element in the 5' untranslated region of collagen and fibronectin genes is important to increase translational efficiency in the presence of p180 and SF3b4. These data demonstrate that a unique system comprising a p180-SF3b4-mRNA complex facilitates the selective assembly of polyribosomes on the ER.

Evaluating the Nutritional Condition of an Indian Climbing Perch, Anabas Testudineus Fingerlings by the RNA/DNA, Ca/P Ratio and Protein Bio-Synthesis in Liver and Muscle

Early fingerlings of Anabas testudineus were nurtured in the laboratory for six weeks under altered nutritional status (different dietary protein level and source) and feeding regimes (ad libitum, ‘limited’, and ‘starvation’ intervals). Their RNA/DNA, Ca/P ratios and protein biosynthesis were determined with highly sensitive fluorescence technique, spectrophotometer and liquid scintillation system respectively. Well-fed early fingerlings (4-6 weeks age group) having higher RNA/DNA (2.11), Ca/P (1.82) ratio and protein biosynthesis rates both in muscle and liver have significantly differs from fingerlings with ‘limited’ food supply and 'starvation' intervals. Rate of protein biosynthesis >244.33 & 603.24 in muscle & liver respectively (count per minute mg protein-1) when compared with well-fed fingerlings and reared under 'limited' food supply and 'starvation' intervals. At ‘starvation’ interval no significant differences were recorded due to different diet treatment. However, fingerlings showed higher mortalities, decreased growth, RNA/DNA, Ca/P ratio and protein biosynthesis rate that may be caused by malnutrition. Fingerlings (early) sampled after 42 days in nearby areas during late monsoon (September) showed RNA/DNA, Ca/P ratio and protein biosynthesis rate were surprisingly low almost as low as ‘starved’ fingerlings, when judged on the basis of mean body weight as compared to well fed fish. It was also observed under microscope that some of these fingerlings were seriously affected by an intestinal disease and this may be due to the ingestion of unsuitable copepods. The low RNA/DNA, Ca/P ratio and protein biosynthesis rate in field samples and ‘starved’ fish are, therefore, interpreted as a sub-lethal result of this disease and malnutrition respectively.

RNA-binding proteins regulate cell respiration and coenzyme Q biosynthesis by post-transcriptional regulation of COQ7

ABSTRACTCoenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain carrying electrons from complexes I and II to complex III and it is an intrinsic component of the respirasome. CoQ concentration is highly regulated in cells in order to adapt the metabolism of the cell to challenges of nutrient availability and stress stimuli. At least 10 proteins have been shown to be required for CoQ biosynthesis in a multi-peptide complex and COQ7 is a central regulatory factor of this pathway. We found that the first 765 bp of the 3′-untranslated region (UTR) of COQ7 mRNA contains cis-acting elements of interaction with RNA-binding proteins (RBPs) HuR and hnRNP C1/C2. Binding of hnRNP C1/C2 to COQ7 mRNA was found to require the presence of HuR, and hnRNP C1/C2 silencing appeared to stabilize COQ7 mRNA modestly. By contrast, lowering HuR levels by silencing or depriving cells of serum destabilized and reduced the half-life of COQ7 mRNA, thereby reducing COQ7 protein and CoQ biosynthesis rate. Accordingly, HuR knockdown decreased oxygen consumption rate and mitochondrial production of ATP, and increased lactate levels. Taken together, our results indicate that a reduction in COQ7 mRNA levels by HuR depletion causes mitochondrial dysfunction and a switch toward an enhanced aerobic glycolysis, the characteristic phenotype exhibited by primary deficiency of CoQ10. Thus HuR contributes to efficient oxidative phosphorylation by regulating of CoQ10 biosynthesis.

Ribosome-associated ncRNAs: An emerging class of translation regulators

Accumulating recent evidence identified the ribosome as binding target for numerous small and long non-protein-coding RNAs (ncRNAs) in various organisms of all 3 domains of life. Therefore it appears that ribosome-associated ncRNAs (rancRNAs) are a prevalent, yet poorly understood class of cellular transcripts. Since rancRNAs are associated with the arguable most central enzyme of the cell it seems plausible to propose a role in translation control. Indeed first experimental evidence on small rancRNAs has been presented, linking ribosome association with fine-tuning the rate of protein biosynthesis in a stress-dependent manner.

Inhibition of Protein Synthesis Alters Protein Degradation through Activation of Protein Kinase B (AKT)

The homeostasis of protein metabolism is maintained and regulated by the rates of protein biosynthesis and degradation in living systems. Alterations of protein degradation may regulate protein biosynthesis through a feedback mechanism. Whether a change in protein biosynthesis modulates protein degradation has not been reported. In this study, we found that inhibition of protein biosynthesis induced phosphorylation/activation of AKT and led to phosphorylation of AKT target substrates, including FoxO1, GSK3α/β, p70S6K, AS160, and the E3 ubiquitin ligase MDM2. Phosphorylation of ribosomal protein S6 was also modulated by inhibition of protein biosynthesis. The AKT phosphorylation/activation was mediated mainly through the PI3K pathway because it was blocked by the PI3K inhibitor LY294002. The activated AKT phosphorylated MDM2 at Ser(166) and promoted degradation of the tumor suppressor p53. These findings suggest that inhibition of protein biosynthesis can alter degradation of some proteins through activation of AKT. This study reveals a novel regulation of protein degradation and calls for caution in blocking protein biosynthesis to study the half-life of proteins.

Effect of Apolipoprotein A-I Complex with Tetrahydrocortisone on Protein Biosynthesis and Glucose Absorption by Rat Hepatocytes

We studied the effect of apolipoprotein A-I-tetrahydrocortisone complex on (14)C glucose absorption and lactate accumulation and on the rate of protein biosynthesis in isolated rat hepatocytes. The presence of apolipoprotein A-I-tetrahydrocortisone complex in the incubation medium increased absorption of labeled glucose by hepatocytes by 52%, while lactate content in the conditioning medium increased 4-fold. The rate of protein biosynthesis increased by 80% in comparison with control cells. It is hypothesized that the increase in protein biosynthesis rate in hepatocytes under the effect of apolipoprotein A-I-tetrahydrocortisone complex is due to stimulation of energy metabolism, specifically, of its glycolytic component.

Effect of a complex of corticosteroids with apolipoprotein A-I on protein biosynthesis in cultured hepatocytes

A complex of apolipoprotein A-I with steroid hormones containing reduced Δ4, 3-ketogroup in the A ring was shown to increase the rate of protein synthesis in the cultured rat hepatocytes. The biological activity of the hormones depends on the position of the oxygroup of the third carbonic atom and hydrogen at the fifth position of a carbonic atom. The cis-position is more preferable for the biological effect. The oxygroup at the third position of the A-ring may be replaced by the sulfo-group. The complex of dehydroepiandrosterone sulphate with apolipoprotein A-I increases the rate of protein biosynthesis in the cultured rat hepatocytes, which confirms the involvement of this hormone in the regulation of gene expression.

Selective Decrease in Regulatory Iron Response Protein 1 (IRP1) binding to mRNA Iron Response Element (IRE)

In mRNA a family of 5' noncoding structures, iron response elements (IRE), control mRNA translation and protein biosynthesis rates by regulatory protein (IRP) binding when iron concentrations decrease. IRP protein represses translation by binding to the IRE and inhibiting formation of polysomes. We explored the extent to which the phylogentically conserved sequence differences among members of the IRE mRNA family related to differences in signal responses observed in vivo, since the regulatory proteins are the same for each mRNA. We selected two members of the IRE‐RNA family, ferritin and mitochondrial (mt‐) aconitase, to study effects on the 5' UTR IRE‐RNA sequence and IRP1 protein binding because of extensive structural information available, and because the iron responses in vivo range from 4‐fold for mt‐acontitase to more than 100‐fold for ferritin. We report here the binding affinity of IRP1 protein to ferritin and mt‐aconitase mRNA. The relative binding affinities varied dramatically depending on experimental conditions. Selective destabilization of ferritin and mt‐aconitase RNA/protein complexes as reported here explain in part the quantitative differences in signal response to iron in vivo and indicate possible new regulatory interactions.

Characterization of the ubiquitin–proteasome system in bortezomib-adapted cells

Resistance towards the proteasome inhibitor bortezomib is poorly understood. We adapted the HL-60, ARH-77 and AMO-1 cell lines (myeloid leukemia, plasmocytoid lymphoma, myeloma) to bortezomib exceeding therapeutic plasma levels, and compared characteristics of the ubiquitin-proteasome system, alternative proteases and the unfolded protein response (UPR) between adapted cells and parental lines. Adapted cells showed increased transcription rates, activities and polypeptide levels of the bortezomib-sensitive beta5, but also of the beta2 proteasome subunit and consistently retained elevated levels of active beta1/beta5-type proteasome subunits in the presence of therapeutic levels of bortezomib. Bortezomib-adapted HL-60 cells showed increased expression and proteasome association of the 11S proteasome activator, and did not accumulate poly-ubiquitinated protein, activate the UPR or UPR-mediated apoptosis in response to bortezomib. The rate of protein biosynthesis was reduced, and the transcription of chaperone genes downmodulated. We did not observe major changes in the activities of TPPII, cathepsins or deubiquitinating proteases. We conclude that different types of bortezomib-adapted cell lines, including myeloma, show similar patterns of changes in the proteasomal machinery which result in residual proteasome activity in the presence of bortezomib and a quantitative balance between protein biosynthesis and destruction.

Continued Protein Synthesis at Low [ATP] and [GTP] Enables Cell Adaptation during Energy Limitation

One of biology's critical ironies is the need to adapt to periods of energy limitation by using the energy-intensive process of protein synthesis. Although previous work has identified the individual energy-requiring steps in protein synthesis, we still lack an understanding of the dependence of protein biosynthesis rates on [ATP] and [GTP]. Here, we used an integrated Escherichia coli cell-free platform that mimics the intracellular, energy-limited environment to show that protein synthesis rates are governed by simple Michaelis-Menten dependence on [ATP] and [GTP] (K(m)(ATP), 27 +/- 4 microM; K(m)(GTP), 14 +/- 2 microM). Although the system-level GTP affinity agrees well with the individual affinities of the GTP-dependent translation factors, the system-level K(m)(ATP) is unexpectedly low. Especially under starvation conditions, when energy sources are limited, cells need to replace catalysts that become inactive and to produce new catalysts in order to effectively adapt. Our results show how this crucial survival priority for synthesizing new proteins can be enforced after rapidly growing cells encounter energy limitation. A diminished energy supply can be rationed based on the relative ATP and GTP affinities, and, since these affinities for protein synthesis are high, the cells can adapt with substantial changes in protein composition. Furthermore, our work suggests that characterization of individual enzymes may not always predict the performance of multicomponent systems with complex interdependencies. We anticipate that cell-free studies in which complex metabolic systems are activated will be valuable tools for elucidating the behavior of such systems.

On Methionine Restriction, Suppression of Mitochondrial Dysfunction and Aging

Rats and mice, when subjected to methionine restriction (MetR), may live longer with beneficial changes to their mitochondria. Most explanations of these observations have centered on MetR somehow suppressing the effects of oxygen free radicals. It is suggested here that MetR's effects on protein metabolism should also be considered when attempting to explain its apparent anti-aging actions. Methionine is the initiating amino acid in mRNA translation. It is proposed that MetR decreases the protein biosynthesis rate due to methionine limitation, which correspondingly decreases generation of ribosomal-mediated error proteins, which then lowers the total abnormal protein load that cellular proteases and chaperone proteins (mitochondrial and cytoplasmic) must deal with. This will increase protease availability for elimination of proteins damaged postsynthetically and help delay abnormal protein accumulation, the major molecular symptom of aging. The slowed rate of protein synthesis may also alter protein folding, which could also alter polypeptide susceptibility to oxidative attack. MetR will also increase lysosomal proteolysis, including autophagy of dysfunctional mitochondria, and promote mitogenesis. MetR may decrease synthesis of S-adenosyl-methionine (SAM), which could decrease spontaneous O(6)-methylguanine formation in DNA. However decreased SAM may compromise repair of protein isoaspartate residues by protein-isoaspartate methyltransferase (PIMT). Changes in SAM levels may also affect gene silencing. All the above may help explain, at least in part, the beneficial effects of MetR.

Interaction of apolipoproteins A-I and E in the regulation of DNA, RNA, and protein biosynthesis in cultured rat hepatocytes

Experiments on hepatocyte culture showed that apolipoprotein A-I-tetrahydrocortisol complex increases the rate of DNA, RNA, and protein biosynthesis measured by radioactive label incorporation. Apolipoprotein E acted as competitor of the apolipoprotein A-I-tetrahydrocortisol complex and abolished biological activity of the latter. We hypothesize that this mechanism of regulation plays an important role in processes of intracellular regeneration and proliferation.

Effect of complexes of apolipoprotein A-I with tetrahydrocortisol and pregnenolone on protein biosynthesis in rat hepatocytes culture

Complexes of apolipoprotein A-I with tetrahydrocortisol and pregnenolone exhibit high biological activity and increase the rate of protein biosynthesis in the culture of rat hepatocytes. An important role in this process is played by reduced delta44-3-keto group in the A-ring of steroid hormones. A complex of apolipoprotein A-I and pregnenolone modulated the rate of protein biosynthesis in liver cells. Hence, the observed changes are not organ-specific for this steroid. Our results suggest that this mechanism of regulation play an important role in intracellular regeneration and proliferation.

An inhibitor of free radical processes decreases the rate of protein synthesis in gunshot wound tissues and decelerates the general adaptation syndrome

The dynamics of total protein and procollagen biosynthesis was studied in the muscle tissue in gunshot wound during the natural healing and after treatment with antioxidant (AO) 2,6-di-tert-butyl-4-methylphenol (BHT). AO treatment considerably decreased the rate of both total protein and procollagen synthesis in the muscle on the third day of healing. BHT notably decreased protein biosynthesis and inhibited corticosteroid production as measured from the electron spin resonance (ESR) signal of reduced adrenodoxin. AO treatment also decreased the rate of protein biosynthesis in bone marrow, thymus, and spleen. The decreased functional activity of the endocrine and immune systems suggests that AO considerably decelerates the systemic body response (general adaptation syndrome) to acute wound. Lipid peroxide radicals and the products of lipid hydroperoxide degradation are proposed as the primary mediators of the body response to stress.

Effects of environmental hypercapnia on animal physiology: A 13C NMR study of protein synthesis rates in the marine invertebrate Sipunculus nudus

Global climate change is associated with a progressive rise in ocean CO 2 concentrations (hypercapnia) and, consequently, a drop in seawater pH. However, a comprehensive picture of the physiological mechanisms affected by chronic CO 2 stress in marine biota is still lacking. Here we present an analysis of protein biosynthesis rates in isolated muscle of the marine invertebrate Sipunculus nudus, a sediment dwelling worm living at various water depths. We followed the incorporation of 13C-labelled phenylalanine into muscular protein via high-resolution NMR spectroscopy. Protein synthesis decreased by about 60% at a medium pH of 6.70 and a consequently lowered intracellular pH (pHi). The decrease in protein synthesis rates is much stronger than the concomitant suppression of protein degradation (60% versus 10–15%) possibly posing a threat to the cellular homeostasis of structural as well as functional proteins. Considering the progressive rise in ocean CO 2 concentrations, permanent disturbances of cellular protein turnover might seriously affect growth and reproductive performance in many marine organisms with as yet unexplored impacts on species density and composition in marine ecosystems.

Characteristics of the interactions of cortisol-apolipoprotein A-I and tetrahydrocortisol-apolipoprotein A-I complexes with eukaryotic DNA

Primary hepatocyte culture has been used to demonstrate that the cortisol-apolipoprotein A-I complex does not affect the DNA and protein biosynthesis rates, whereas the tetrahydrocortisol-apolipoprotein A-I complex (THC-apoA-I) substantially increases the rates of 3H-thymidine and 14C-leucine incorporation into DNA and protein, respectively. Small-angle X-ray scattering data show that only THC-apoA-I effectively interacts with eukaryotic DNA, which is accompanied by local DNA melting. The (GCC)n repeat, a component of many human and other eukaryotic genes, is the most probable region of the interaction of this complex with DNA. An oligonucleotide (duplex) of this type has been synthesized. Its interaction with THC-apoA-I yields a larger complex, which breaks up, giving rise to complementary oligonucleotide strands. They also interact with THC-apoA-I. The equilibrium kinetics of this multiphase process is described. The interaction of the cortisol-apolipoprotein A-I complex with the duplex is less specific and does not cause its breakup or the formation of complementary oligonucleotides.