Rabbit Reticulocyte Lysate System Research Articles

Application of Phosphoramidate ProTide Technology for the Synthesis of 5'-mRNA Cap Analogs Modified on the Exocyclic Amine Group.

Aryloxy triester phosphoramidate methodology, commonly known as ProTide technology, is one of the most widely used prodrug approaches applied to therapeutic nucleosides. This approach has been used extensively by the pharmaceutical industry and researchers in medicinal chemistry. Herein we report our adaptation of this effective method for the synthesis of bioactive 5'-mRNA cap analogues as inhibitors for targeting cap-dependent translation. The synthesis was performed in two main stages: preparation of N2-modified guanosine analogues and their subsequent transformation into prodrugs using phenylethoxy-l-alaninyl phosphorochloridate. The prepared pro-nucleotide cap analogues were tested for their capacity in enzymatic activation, inhibitory properties in a rabbit reticulocyte lysate system, and passive membrane translocation properties.

Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems.

The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, Escherichia coli, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or Escherichia coli. The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation.

Annexin A2 binds the internal ribosomal entry site of c-myc mRNA and regulates its translation

ABSTRACT The expression and localization of the oncoprotein c-Myc is highly regulated at the level of transcription, mRNA transport, translation, as well as stability of the protein. We previously showed that Annexin A2 (AnxA2) binds to a specific localization element in the 3ʹuntranslated region (UTR) of c-myc mRNA and is involved in its localization to the perinuclear region. In the present study, we demonstrate that AnxA2 binds in a Ca 2+ -dependent manner to the internal ribosomal entry site (IRES) containing two pseudo-knots in the 5´UTR of the c-myc mRNA. Here, we employ an in vitro rabbit reticulocyte lysate system with chimeric c-myc reporter mRNAs to demonstrate that binding of AnxA2 to the c-myc IRES modulates the expression of c-Myc. Notably, we show that low levels of AnxA2 appear to increase, while high levels of AnxA2 inhibits translation of the chimeric mRNA. However, when both the AnxA2-binding site and the ribosomal docking site in the c-myc IRES are deleted, AnxA2 has no effect on the translation of the reporter mRNA. Forskolin-treatment of PC12 cells results in upregulation of Ser25 phosphorylated AnxA2 expression while c-Myc expression is down-regulated. The effect of forskolin on c-Myc expression and the level of Ser25 phosphorylated AnxA2 was abolished in the presence of EGTA. These findings indicate that AnxA2 regulates both the transport and subsequent translation of the c-myc mRNA, possibly by silencing the mRNA during its transport. They also suggest that AnxA2 act as a switch to turn off the c-myc IRES activity in the presence of calcium. Abbreviations: AnxA2, Annexin A2; β2--µglob, β2-microglobulin; cpm, counts per minute; hnRNP, heterogenous nuclear ribonucleoprotein; IRES, internal ribosomal entry site; ITAF, IRES trans-acting factor; MM, multiple myeloma; PABP, poly(A)-binding protein; PCBP, poly(rC) binding protein; PSF, PTB-associated splicing factor; PTB, polypyrimidine tract binding protein; RRL, rabbit reticulocyte lysate; UTR, untranslated region; YB, Y-box binding protein.

Richard Jackson (1940–2020) ‐ A towering presence in translation

Richard Jackson (1940–2020) ‐ A towering presence in translation

Backbone and side-chain chemical shift assignments for the ribosome-inactivating protein trichobakin (TBK).

Trichobakin (TBK) is a type-I ribosome-inactivating protein (RIP-I), acting as an extremely potent inhibitor of protein synthesis in the cell-free translation system of rabbit reticulocyte lysate (IC50: 3.5 pM). In this respect, TBK surpasses the well-studied highly homologous RIP-I trichosanthin (IC50: 20-27 pM), therefore creation of recombinant toxins based on it is of great interest. TBK needs to penetrate into cytosol through the cell membrane and specifically bind to α-sarcin/ricin loop of 28S ribosome RNA to perform the function of specific RNA depurination. At the moment, there is no detailed structural-dynamic information in solution about diverse states RIP-I can adopt at different stages on the way to protein synthesis inhibition. In this work, we report a near-complete assignment of 1H, 13C, and 15N TBK (27.3 kDa) resonances and analysis of the secondary structure based on the experimental chemical shifts data. This work will serve as a basis for further investigations of the structure, dynamics and interactions of the TBK with its molecular partners using NMR techniques.

Bovine mRNAs in Cow’s Milk Exosomes Are Bioavailable and Translated into Peptides in Non-bovine Systems (P06-030-19)

ObjectivesExosomes are natural nanoparticles that participate in cell-to-cell communication by transferring regulatory molecules such as microRNAs from donor cells to recipient cells. Previously, we have demonstrated that exosomes and microRNAs are not exclusively obtained from endogenous synthesis but may also be absorbed from milk.We assessed whether 1) exosomes contain mRNAs (i.e., RNAs other than microRNAs), 2) mRNAs are bioavailable in mice, and 3) bovine mRNAs are translated into peptides. MethodsFor mRNA analysis, exosomes were isolated from bovine milk using differential ultracentrifugation, and RNA cargos were analyzed by RNA-sequencing. For bioavailability analysis, a synthetic fragment of fluorophore (IRDye)-labeled bovine CSN3 mRNA was transfected into bovine milk exosomes, which were administered to Balb/c mice by oral gavage. Tissues distribution of CSN3 mRNA was assessed 24 h after gavage by using a LiCor Odyssey CLx imager. For analysis of mRNA translation, mRNA from bovine milk exosomes were translated using the rabbit reticulocyte lysate system and BODIPY-labeled lysine. Peptides were separated by two-dimensional gel electrophoresis and fluorescence was visualized using a Typhoon FLA 7000 scanner. Statistical analysis, NA. ResultsWe detected > 3600 bovine mRNAs in exosomes. Most mRNAs were truncated; 107 mRNAs contained their natural ATG start codons. Thirteen of these mRNAs, including CSN3, encoded bovine proteins and peptides with amino acid sequences distinct from those in human and murine orthologs (Table 1). Mice absorbed CSN3 mRNA encapsulated in milk exosomes, which accumulated primarily in the liver (Fig. 1). Nine bovine peptide spots were detected by two-dimensional electrophoresis (Fig. 2). ConclusionsBovine milk exosomes contain mRNAs which are bioavailable and translated into peptides in non-bovine systems. We speculate that food mRNAs might play a role in food allergies and immune tolerance.Future studies: Identification of peptides by LC/MS-MS is in progress. We will assess the relevance of mRNA translation for allergies and tolerance in animal models. Funding SourcesNIFA, NIH, Gates Foundation, PureTech, Inc. and USDA Hatch and Multistate. J.Z. is a consultant for PureTech. Supporting Tables, Images and/or Graphs▪▪

Identification and characterization of a novel single-chain variable fragment (scFv) antibody against Neisseria meningitidis factor H-binding protein (fHbp)

Purpose. Neisseria meningitidis is the leading global cause of meningitis and sepsis. Detection, followed by identification, of bacterial pathogens is important in medicine and public health. In the present study, we used the ribosome display technique to select single-chain variable fragments (scFv) that are specific to the surface-exposed fHbp antigen of N. meningitidis. Methodology. The recombinant fHbp protein was used as the antigen for the immunization of BALB/c mice. Anti-fHbp VH/k chain ribosome display libraries were assembled by joining VH and k into the VH/k chain with a specially constructed linker by PCR overlap extension. The scFv library was panned against the recombinant fHbp protein by using a single round of the ribosome display method via a rabbit reticulocyte lysate system.Results/Key findings. The selected anti-fHbp antibody exhibited high affinity and specificity in the enzyme-linked immunosorbent assay (ELISA) and the whole bacterial cell enzyme-linked immunosorbent assay (Bact-ELISA).Conclusion. The affinity of the selected scFv was ~8.65×109 M-1. The isolated scFv can provide the basis for developing a diagnostic kit.

Variants of the 5\u2032-terminal region of p53 mRNA influence the ribosomal scanning and translation efficiency

The p53 protein is one of the major cell cycle regulators. The protein is expressed as at least twelve protein isoforms resulting from the use of alternative promoters, alternative splicing or downstream initiation codons. Importantly, there is growing evidence that translation initiation of p53 mRNA may be regulated by the structure and length of the naturally occurring variants of the 5′-terminal region of p53 mRNA transcripts. Here, several mRNA constructs were synthesized with variable length of the p53 5′-terminal regions and encoding luciferase reporter protein, and their translation was monitored continuously in situ in a rabbit reticulocyte lysate system. Moreover, four additional mRNA constructs were prepared. In two constructs, the structural context of AUG1 initiation codon was altered while in the other two constructs, characteristic hairpin motifs present in the p53 5′-terminal region were changed. Translation of the last two constructs was also performed in the presence of the cap analogue to test the function of the 5′-terminal region in cap-independent translation initiation. Superposition of several structural factors connected with the length of the 5′-terminal region, stable elements of the secondary structure, structural environment of the initiation codon and IRES elements greatly influenced the ribosomal scanning and translation efficiency.

Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus

BackgroundIn the present work we described the recombinant production and characterization of heterodimeric construction ZnT8-Arg-Trp325 fused to thioredoxin using a high-performance expression system such as Escherichia coli. In addition, we apply this novel recombinant antigen in a non-radiometric method, with high sensitivity, low operational complexity and lower costs.ResultsZnT8 was expressed in E. coli as a fusion protein with thioredoxin (TrxZnT8). After 3 h for induction, recombinant protein was obtained from the intracellular soluble fraction and from inclusion bodies and purified by affinity chromatography. The expression and purification steps, analyzed by SDS-PAGE and western blot, revealed a band compatible with TrxZnT8 expected theoretical molecular weight (≈ 36.8 kDa). The immunochemical ability of TrxZnT8 to compete with [35S]ZnT8 (synthesized with rabbit reticulocyte lysate system) was assessed qualitatively by incubating ZnT8A positive patient sera in the presence of 0.2–0.3 μM TrxZnT8. Results were expressed as standard deviation scores (SDs). All sera became virtually negative under antigen excess (19.26–1.29 for TrxZnT8). Also, radiometric quantitative competition assays with ZnT8A positive patient sera were performed by adding TrxZnT8 (37.0 pM–2.2 µM), using [35S]ZnT8. All dose–response curves showed similar protein concentration that caused 50% inhibition (14.9–0.15 nM for TrxZnT8). On the other hand, preincubated bridge ELISA for ZnT8A detection was developed. This assay showed 51.7% of sensitivity and 97.1% of specificity.ConclusionsIt was possible to obtain with high-yield purified heterodimeric construction of ZnT8 in E. coli and it was applied in cost-effective immunoassay for ZnT8A detection.

Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems.

A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.

Inhibition of eukaryotic translation by tetratricopeptide-repeat proteins of Orientia tsutsugamushi.

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus. The genome of Orientia tsutsugamushi has revealed multiple ORFs encoding tetratricopeptide-repeat (TPR) proteins. The TPR protein family has been shown to be involved in a diverse spectrum of cellular functions such as cell cycle control, transcription, protein transport, and protein folding, especially in eukaryotic cells. However, little is known about the function of the TPR proteins in O. tsutsugamushi. To investigate the potential role of TPR proteins in host-pathogen interaction, two oriential TPR proteins were expressed in E. coli and applied for GSTpull down assay. DDX3, a DEAD-box containing RNA helicase, was identified as a specific eukaryotic target of the TPR proteins. Since the RNA helicase is involved in multiple RNA-modifying processes such as initiation of translation reaction, we performed in vitro translation assay in the presence of GST-TPR fusion proteins by using rabbit reticulocyte lysate system. The TPR proteins inhibited in vitro translation of a reporter luciferase in a dose dependent manner whereas the GST control proteins did not. These results suggested TPR proteins of O. tsutsugamushi might be involved in the modulation of eukaryotic translation through the interaction with DDX3 RNA helicase after secretion into host cytoplasm.

Prokariotic expression of major autoantigens from Diabetes Mellitus fused to thioredoxin: IA-2 and ZnT8

Prokariotic expression of major autoantigens from Diabetes Mellitus fused to thioredoxin: IA-2 and ZnT8

Activity of core-modified 10-23 DNAzymes against HCV.

The highly conserved untranslated regions of the hepatitis C virus (HCV) play a fundamental role in viral translation and replication and are therefore attractive targets for drug development. A set of modified DNAzymes carrying (2'R)-, (2'S)-2'-deoxy-2'-C-methyl- and -2'-O-methylnucleosides at various positions of the catalytic core were assayed against the 5'-internal ribosome entry site element (5'-IRES) region of HCV. Intracellular stability studies showed that the highest stabilization effects were obtained when the DNAzymes' cores were jointly modified with 2'-C-methyl- and 2'-O-methylnucleosides, yielding an increase by up to fivefold in the total DNAzyme accumulation within the cell milieu within 48 h of transfection. Different regions of the HCV IRES were explored with unmodified 10-23 DNAzymes for accessibility. A subset of these positions was tested for DNAzyme activity using an HCV IRES-firefly luciferase translation-dependent RNA (IRES-FLuc) transcript, in the rabbit reticulocyte lysate system and in the Huh-7 human hepatocarcinoma cell line. Inhibition of IRES-dependent translation by up to 65 % was observed for DNAzymes targeting its 285 position, and it was also shown that the modified DNAzymes are as active as the unmodified one.

Selection of Single Chain Variable Fragments Specific for the Human-Inducible Costimulator Using Ribosome Display

We applied a ribosome display technique to a mouse single chain variable fragment (scFv) library to select scFvs specific for the inducible costimulator (ICOS). mRNA was isolated from the spleens of BALB/c mice immunized with ICOS protein. Heavy and κ chain genes (VH and κ) were amplified separately by reverse transcriptase polymerase chain reaction, and the anti-ICOS VH/κ chain ribosome display library was constructed with a special flexible linker by overlap extension PCR. The VH/κ chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. Then, antibody-ribosome-mRNA complexes were produced and panned against ICOS protein under appropriate conditions. However, in order to isolate specific scFvs for ICOS, negative selection using CD28 was carried out before three rounds of positive selection on ICOS. After three rounds of panning, the selected scFv DNAs were cloned into pET43.1a and detected by SDS-PAGE. Then, enzyme-linked immunosorbent assay showed that we successfully constructed a native ribosome display library, and among seven clones, clone 5 had the highest affinity for the ICOS and low for the CD28. Anti-ICOS scFvs are assessed for binding specificity and affinity and may provide the potential for development of the humanized and acute and chronic allograft rejection.

Cap-dependent translation without base-by-base scanning of an messenger ribonucleic acid

‘Ribosome scanning’ is the generally accepted mechanism for explaining how a ribosome finds an initiation codon located far removed from the ribosome recruiting site (cap structure). However, the molecular characteristics of ribosome scanning along 5′ untranslated regions (UTRs) remain obscure. Herein, using a rabbit reticulocyte lysate (RRL) system and artificial ribonucleic acid (RNA) constructs composed of a capped leader RNA and an uncapped reporter RNA annealed through a double-stranded RNA (dsRNA) bridge, we show that the ribosome can efficiently bypass a stable, dsRNA region without melting the structure. The insertion of an upstream open reading frame in the capped leader RNA impaired the translation of reporter RNA, indicating that a ribosome associated with the 5′-end explores the regions upstream of the dsRNA bridge in search of the initiation codon. These data indicate that a ribosome may skip part(s) of an messenger RNA 5′UTR without thoroughly scanning it.

Abstract LB-414: Study of the kinase activity using NAPPA protein microarray expressed with Human IVTT system

Abstract Protein kinases are highly involved in every type of cancer. They play important roles in all cancer hallmarks, ranging from cell proliferation to metastasis, apoptosis to immortality, to name just a few. The complete understanding of this key class of proteins is important for cancer diagnosis and treatment. In order to study protein kinases we are employing protein microarrays, a solid platform that allows the study of thousand of proteins simultaneously. Our platform of choice is NAPPA (Nucleic Acid Programmable Protein Microarray), which permits the use of fresh expressed proteins for functional assays. On NAPPA, the cDNA coding for the gene of interest, which is fused to a tag, is co-printed on the array with an antibody against the tag. Printed slides are expressed using an in vitro transcription and translation system (IVTT) and the newly synthesized proteins are captured by the anti-tag antibody. Following expression, the slides can be used for a variety of applications including biomarker screening and protein-protein interaction studies. In the past, NAPPA slides have been expressed using Rabbit Reticulocyte (Promega) as the IVTT system. However the main concerns with this system included high background, poor expression of genes from other species including humans, and poor lot-to-lot reproducibility. The recent launch of the first Human IVTT system purified from HeLa cells (ThermoFisher Scientific) has the potential to overcome those shortcomings and expand the number of applications that can be carried out with NAPPA arrays. In this study, we evaluate the use of Human IVTT system in NAPPA arrays to study protein kinases. An initial analysis of protein expression using western blot revealed higher expression of proteins in Human IVTT system compared to rabbit reticulocyte lysate. Further benchmarking between the two expression systems was carried out using protein display on NAPPA slides. Proteins expressed in Human IVTT system were at least 10-fold higher compared to rabbit reticulocyte lysate system. Using Human IVTT system we identified autophosphorylation in many tyrosine kinases, including Abl1 and Src. This autophosphorylation could be selectively repressed by prior addition of specific kinase inhibitors. Further we have demonstrated that dephosphorylated proteins can be phosphosphorylated by addition of human cell extracts and this reaction can be measured using anti-phospho tyrosine antibodies. These results suggest that NAPPA arrays can be used to measure kinase activity in a high throughput format not only in the kinases expressed in vitro but also in the cell extracts that are a source for kinases. Taken together our results suggest that Human IVTT system can be used in the NAPPA platform and this system is suitable to study protein kinases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-414. doi:1538-7445.AM2012-LB-414

Cloning and soluble expression of mature α-luffin from <italic>Luffa cylindrica</italic> and its antitumor activities <italic>in vitro</italic>

Luffin-a, a single-chain Type I ribosome-inactivating protein, which is known to be the most toxic of the luffin family and apparently possesses antitumor activity, was isolated from Luffa cylindrica seeds. In the present study, mature alpha-luffin was cloned from L. cylindrica and it was found that mature alpha-luffin shared 96% amino acid similarity with luffin-a. The recombinant mature alpha-luffin was successfully expressed in a partly soluble form in Escherichia coli after optimization of expression conditions. The effects of the recombinant protein on bacterial growth and its in vitro protein synthesis inhibition activity were tested. Then, its antitumor activities against different human cancer cell lines were evaluated by CCK-8 assay and flow cytometry. The results indicated that the recombinant alpha-luffin was slightly toxic to E. coli. It could inhibit protein synthesis in the rabbit reticulocyte lysate system. At the same time, it inhibited the growth of the tumor cell lines in a dose- and time-dependent manner. Additionally, recombinant alpha-luffin was able to induce cell death by apoptosis. The cytotoxicity of alpha-luffin towards tumor cells makes it a potential antitumor agent.

Antioxidant-induced Phosphorylation of Tyrosine 486 Leads to Rapid Nuclear Export of Bach1 That Allows Nrf2 to Bind to the Antioxidant Response Element and Activate Defensive Gene Expression

Antioxidants cause stabilization and nuclear translocation of NF-E2-related factor 2 (Nrf2), where it binds to the antioxidant response element (ARE) and induces up-regulation of defensive genes that protect cells against oxidative and electrophilic stress. Bach1, the negative regulator of Nrf2, competes with Nrf2 for binding to the ARE in the human NQO1 promoter. In this study, we demonstrate that Bach1 exits the nucleus within 1-2 h upon antioxidant treatment. Genistein, an inhibitor of tyrosine kinases, blocked nuclear export of Bach1. Site-directed mutagenesis and immunoprecipitation assays identified tyrosine 486 that was phosphorylated in response to the antioxidant and was essential for nuclear export of Bach1. Chromatin immunoprecipitation assays revealed a competitive interplay between Bach1 and Nrf2 at 1-2 and 4 h for binding to the human NQO1 ARE. Luciferase and real time PCR assays showed a significant decrease in antioxidant induction of reporter activity and mRNA levels in cells transfected with mutant Bach1 compared with wild type. This decrease was due to the absence of nuclear export of the mutant protein. Bach1 levels inside the nucleus returned to normal at 4 h after antioxidant treatment in the absence but not in the presence of protein synthesis inhibitor cycloheximide. In addition, antioxidant treatment increased the transcription of Bach1 as shown by pulse chase and real time PCR experiments. Taken together, these results indicate that increased synthesis of Bach1 restored its nuclear levels to normal at 4 h. In conclusion, antioxidant-induced tyrosine 486 phosphorylation leads to nuclear exit of Bach1, thus allowing Nrf2 access to the ARE.

Selection of Anti-Sulfadimidine Specific ScFvs from a Hybridoma Cell by Eukaryotic Ribosome Display

BackgroundRibosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes.Methodology/Principal FindingsIn our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM2) by using a ribosome library technology. A DNA library of scFv antibody fragments was constructed for ribosome display, and then mRNA–ribosome–antibody (MRA) complexes were produced by a rabbit reticulocyte lysate system. The synthetic sulfadimidine-ovalbumin (SM2-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning. After four rounds of ribosome display, the expression vector pCANTAB5E containing the selected specific scFv DNA was constructed and transformed into Escherichia coli HB2151. Three positive clones (SAS14, SAS68 and SAS71) were screened from 100 clones and had higher antibody activity and specificity to SM2 by indirect ELISA. The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis.Conclusions/SignificanceThe selection of anti-SM2 specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs.

CAMP response element‐binding protein (CREB) is imported into mitochondria and promotes protein synthesis

The cAMP response element-binding protein (CREB) is a ubiquitous transcription factor in the higher eukaryotes that, once phosphorylated, promotes transcription of cAMP response element-regulated genes. We have studied the mitochondrial import of CREB and its effect on the expression of mtDNA-encoded proteins. [(35)S]Methionine-labelled CREB, synthesized in vitro in the Rabbit Reticulocyte Lysate system using a construct of the human cDNA, was imported into the matrix of isolated rat liver mitochondria by a membrane potential and TOM complex-dependent process. The imported CREB caused cAMP-dependent promotion of the synthesis of mitochondrially encoded subunits of oxidative phosphorylation enzyme complexes. Thus, CREB moves from the cytosol to mitochondria, in addition to the nucleus, and, when phosphorylated by cAMP-dependent protein kinase, promotes the expression of mitochondrial genes.