RESUMO
Cesarean scar disorder (CSDi) is a newly recognized cause of secondary infertility. Laparoscopic or hysteroscopic surgery is generally chosen for the surgical treatment of CSDi, depending on the residual myometrial thickness of the cesarean scar. Previously, hysteroscopic transcervical resection for CSDi (TCR-CSDi) has been reported to be a safe procedure, with no cases of postoperative cervical stenosis. Herein, we report a novel case of cervical stenosis after circumferential hysteroscopic TCR-CSDi of an extensive CSDi lesion. Notably, although no cervical stenosis was observed upon postoperative hysteroscopy one month postoperatively, cervical stenosis developed four months after the surgery; therefore, it is important to avoid circumferential resection and cauterization in patients with CSDi, even when abnormal blood vessels are present. Additionally, it is advisable to check for delayed cervical stenosis at least three weeks before embryo transfer in patients who have undergone TCR-CSDi.
RESUMO
Viral proteases, the key enzymes that regulate viral replication and assembly, are promising targets for antiviral drug discovery. Herpesvirus proteases are enzymes with no crystallographically confirmed noncovalent active-site binders, owing to their shallow and polar substrate-binding pockets. Here, we applied our previously reported "Peptide-to-Small Molecule" strategy to generate novel inhibitors of ß-herpesvirus proteases. Rapid selection with a display technology was used to identify macrocyclic peptide 1 bound to the active site of human cytomegalovirus protease (HCMVPro) with high affinity, and pharmacophore queries were defined based on the results of subsequent intermolecular interaction analyses. Membrane-permeable small molecule 19, designed de novo according to this hypothesis, exhibited enzyme inhibitory activity (IC50 = 10-6 to 10-7 M) against ß-herpesvirus proteases, and the design concept was proved by X-ray cocrystal analysis.
RESUMO
BACKGROUND: A uterine diverticulum is defined as the presence of a niche within the inner contour of the uterine myometrial wall. Although secondary uterine diverticula can occur after hysterotomy such as cesarean section, reports of diverticula after myomectomy are extremely rare. CASE PRESENTATION: A 45-year-old nulliparous woman undergoing infertility treatment was referred to our hospital because of abnormal postmenstrual bleeding after myomectomy. Transvaginal sonography and magnetic resonance imaging revealed a diverticulum in the isthmus. Fat-saturated T1 image showed a blood reservoir in the diverticulum. Hysteroscopic surgery was performed to remove the lowed edge of the defect and coagulate the hypervascularized area. Two months after surgery, the abnormal postmenstrual bleeding and chronic endometritis improved. DISCUSSION AND CONCLUSIONS: This report highlights the similarities of the patient's diverticulum to cesarean scar defects in terms of symptoms and pathophysiology. First, this patient developed a diverticulum with hypervascularity after myomectomy and persistent abnormal bleeding. Second, after hysteroscopic surgery, the symptoms of irregular bleeding disappeared. Third, endometrial glands were identified within the resected scar tissue. Fourth, preoperatively identified CD138-positive cells in endometrial tissue spontaneously disappeared after hysteroscopic resection. To the best of our knowledge, this is the first report of symptomatic improvement following hysteroscopic surgery in a patient with an iatrogenic uterine diverticulum with persistent irregular bleeding after myomectomy.
Assuntos
Divertículo , Miomectomia Uterina , Gravidez , Humanos , Feminino , Pessoa de Meia-Idade , Cesárea , Cicatriz , Útero , Divertículo/diagnóstico por imagem , Divertículo/cirurgiaRESUMO
OBJECTIVE: To demonstrate a 5-step approach to accurately identify the extent of resection of a cesarean scar defect (CSD) and perform excision and repair of the lesion. DESIGN: Technical video introducing laparoscopic scar repair using nonperfusion hysteroscopy for patients with a CSD. SETTING: Tertiary referral facility for gynecology. PATIENT(S): A 33-year-old woman who underwent intrauterine insemination for secondary infertility 3 times but did not conceive complained of repeated irregular bleeding caused by a CSD during infertility treatment. INTERVENTION(S): This video presents a systematic 5-step approach to laparoscopic repair of a CSD. Step 1: the lesion was coagulated and marked using a hysteroscope. Step 2: the lesion was thinned by cutting it using the hysteroscope. Step 3: after laparoscopic dissection of the bladder from the lower uterine segment and turning off the laparoscope's light source, the thinned lesion could be identified using light from the hysteroscope. Step 4: an incision was made at the lit-up point from the abdominal cavity side using an ultrasonic coagulation incision device to access the uterus. Step 5: once the uterine lumen was reached, reflux from the hysteroscope was stopped. Intraperitoneal insufflation gas then flowed into the uterus through the uterine wall perforation, and the lesion could be observed without the use of a reflux fluid. This technique is called nonperfusion hysteroscopy. By observing the marked lesion using nonperfusion hysteroscopy, it could be resected laparoscopically along the appropriate incision line. MAIN OUTCOME MEASURE(S): Advantage of performing 5 successive surgical steps to completely resect a CSD using laparoscopic repair and resolve the patient's symptoms. RESULT(S): Laparoscopic repair using nonperfusion hysteroscopy allowed recognition of the upper and lower edges of the lesion from the abdominal cavity side. CONCLUSION(S): The combined use of nonperfusion hysteroscopy allows observation of the uterine lumen without the use of a reflux fluid because pneumoperitoneum gas fills the uterine lumen. Intraoperative monitoring using a hysteroscope and laparoscope allows visualization of the lesion site from both sides while resection is being performed. This 5-step procedure permits precise identification of the lesion area, complete removal of lesions, and prevention of excessive resection that may reduce uterine function and increase perinatal risk.
Assuntos
Cesárea , Laparoscopia , Gravidez , Feminino , Humanos , Adulto , Cesárea/efeitos adversos , Resultado do Tratamento , Histeroscopia/métodos , Cicatriz/diagnóstico , Cicatriz/diagnóstico por imagem , Laparoscopia/efeitos adversos , Laparoscopia/métodosRESUMO
A novel strategy for lead identification that we have dubbed the "Pocket-to-Lead" strategy is demonstrated using HIV-1 protease as a model target. Sometimes, it is difficult to obtain hit compounds because of the difficulties in satisfying the complex pharmacophoric features. In this study, a virtual fragment hit which does not match all of the pharmacophore features but has key interactions and vectors that could grow into remaining pharmacophore features was optimized in silico. The designed compound 9 demonstrated weak but evident inhibitory activity (IC50 = 54 µM), and the design concept was proven by the co-crystal structure. Then, structure-based drug design promptly gave compound 14 (IC50 = 0.0071 µM, EC50 = 0.86 µM), an almost 10,000-fold improvement in activity from 9. The structure of the designed molecules proved to be novel with high synthetic feasibility, indicating the usefulness of this strategy to tackle tough targets with complex pharmacophore.
Assuntos
Inibidores da Protease de HIV , HIV-1 , Desenho de Fármacos , Protease de HIV/metabolismo , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , HIV-1/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Inibidores de ProteasesRESUMO
Ovarian tissue cryopreservation (OTC) and ovarian tissue transplantation (OTT) are fertility preservation options for prepubertal girls or those in whom cancer treatment cannot be delayed. They are important to increasing number of cancer survivors, but there are very few reports on this topic in Japan. This is the first report of a biochemical pregnancy after OTT in Japan. An 18-year-old woman, diagnosed with Ewing sarcoma of the seventh thoracic vertebra, underwent tumor resection. OTC was performed before postoperative chemotherapy. After 7 years, she decided to undergo OTT following the diagnosis of chemotherapy-induced premature ovarian insufficiency. On postoperative day 104, ovarian stimulation was started, yielding one embryo after 3 days. Embryo transfer was performed during a hormone replacement cycle. At 6 weeks and 1 day, the human chorionic gonadotropin level was 81.5 mIU/mL; however, no gestational sac was observed on ultrasonography, indicating a biochemical pregnancy. Our data will be useful for the further development of fertility preservation options in Japan in the future.
Assuntos
Preservação da Fertilidade , Insuficiência Ovariana Primária , Adolescente , Criopreservação , Feminino , Humanos , Japão , GravidezRESUMO
ß-Secretase (BACE1) has an essential role in the production of amyloid ß peptides that accumulate in patients with Alzheimer's disease (AD). Thus, inhibition of BACE1 is considered to be a disease-modifying approach for the treatment of AD. Our hit-to-lead efforts led to a cellular potent 1,3-dihydro-oxazine 6, which however inhibited hERG and showed high P-gp efflux. The close analogue of 5-fluoro-oxazine 8 reduced P-gp efflux; further introduction of electron withdrawing groups at the 6-position improved potency and also mitigated P-gp efflux and hERG inhibition. Changing to a pyrazine followed by optimization of substituents on both the oxazine and the pyrazine culminated in 24 with robust Aß reduction in vivo at low doses as well as reduced CYP2D6 inhibition. On the basis of the X-ray analysis and the QM calculation of given dihydro-oxazines, we reasoned that the substituents at the 6-position as well as the 5-fluorine on the oxazine would stabilize a bioactive conformation to increase potency.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Oxazinas/química , Oxazinas/farmacologia , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Humanos , Simulação de Acoplamento Molecular , Oxazinas/metabolismo , Oxazinas/farmacocinética , Conformação Proteica , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
Both land plants and metazoa have the capacity to reprogram differentiated cells to stem cells. Here we show that the moss Physcomitrella patens Cold-Shock Domain Protein 1 (PpCSP1) regulates reprogramming of differentiated leaf cells to chloronema apical stem cells and shares conserved domains with the induced pluripotent stem cell factor Lin28 in mammals. PpCSP1 accumulates in the reprogramming cells and is maintained throughout the reprogramming process and in the resultant stem cells. Expression of PpCSP1 is negatively regulated by its 3'-untranslated region (3'-UTR). Removal of the 3'-UTR stabilizes PpCSP1 transcripts, results in accumulation of PpCSP1 protein and enhances reprogramming. A quadruple deletion mutant of PpCSP1 and three closely related PpCSP genes exhibits attenuated reprogramming indicating that the PpCSP genes function redundantly in cellular reprogramming. Taken together, these data demonstrate a positive role of PpCSP1 in reprogramming, which is similar to the function of mammalian Lin28.
Assuntos
Bryopsida/fisiologia , Reprogramação Celular/fisiologia , Proteínas e Peptídeos de Choque Frio/fisiologia , Proteínas de Plantas/fisiologia , Células-Tronco/fisiologia , Regiões 3' não Traduzidas/fisiologia , Diferenciação Celular/fisiologia , Proteínas e Peptídeos de Choque Frio/química , Regulação da Expressão Gênica de Plantas/fisiologia , Folhas de Planta/citologia , Folhas de Planta/fisiologia , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , Domínios Proteicos/fisiologiaAssuntos
Ceratite Herpética/diagnóstico , Córnea/virologia , Dermatite Perioral/diagnóstico , Dermatite Perioral/etiologia , Dermatite Perioral/virologia , Herpes Zoster Oftálmico/diagnóstico , Humanos , Ceratite Herpética/complicações , Ceratite Herpética/virologia , Masculino , Meningismo/diagnóstico , Meningismo/etiologia , Gânglio Trigeminal/virologia , Adulto JovemRESUMO
All change at the C terminus: We have established a novel methodology for the ribosomal synthesis of peptides featuring C-terminal cyclization and various modifications, including macrocyclization, by making use of genetic code reprogramming. The C-terminal amide modification of linear and cyclic peptides should enhance their physiological stabilities, and open up the possibility of developing new drug-like peptides. The C terminus of a peptide expressed by the translation apparatus generally ends in a carboxylate group. On the other hand, the C termini of some naturally occurring peptides have amide moieties instead of carboxylates, which are believed to give better biostability. Here, we describe a new strategy for the ribosomal synthesis of peptides featuring C-terminal lactam, thiolactone, and alkylamide units. The method was based on the concept of genetic code reprogramming involving the flexizymes (flexible tRNA acylation ribozymes) and the PURE (peptide synthesis using recombinant elements) system, in which vacant codons are reassigned to nonproteinogenic amino acids; this enabled us to convert the C termini of peptides into the above functionalities. We have also applied this method to the synthesis of a macrocyclic peptide closed by an amide bond formed between a lysine side chain and the peptide C terminus. This method thus offers us new opportunities to express various peptides with C-terminal modifications as well as macrocyclic peptides using the translation apparatus, and potentially to accelerate the discovery of peptidic drugs designed for various therapeutic targets.
Assuntos
Amidas/química , Lactamas/química , Lactonas/química , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Ribossomos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Ciclização , Peptídeos Cíclicos/síntese química , RNA Catalítico/química , RNA Catalítico/metabolismo , RNA de Transferência/metabolismoRESUMO
Here we report a new methodology for the synthesis of bicyclic peptides by using a reconstituted cell-free translation system under the reprogrammed genetic code. Cysteine (Cys) and three different nonproteinogenic amino acids, Cab, Aha, and Pgl, were simultaneously incorporated into a peptide chain. The first cyclization occurred between the chloroacetyl group of Cab and the sulfhydryl group in Cys in situ of translation, and the second cyclization on the side chains of Aha-Pgl via Cu(I)-catalyzed azide-alkyne cycloaddition was performed. This offers us a powerful means of mRNA-programmed synthesis of various peptides with uniform bicyclic scaffolds.
Assuntos
Peptídeos Cíclicos/síntese química , Ribossomos/química , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alcinos/química , Alcinos/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Aminobutiratos/química , Aminobutiratos/metabolismo , Códon , Cisteína/química , Cisteína/metabolismo , Glicina/análogos & derivados , Glicina/química , Glicina/metabolismo , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Moldes GenéticosRESUMO
Here we report a new enabling technology for the synthesis of peptidase-resistant cyclic peptides by means of genetic code reprogramming involving the flexizyme (a tRNA acylation ribozyme) and PURE (a reconstituted cell-free translation) systems. In this work, we have developed a new nonproteinogenic amino acid bearing a chloroacetyl group in the side chain, which forms a physiologically stable thioether bond by intramolecular reaction with the sulfhydryl group of a Cys residue in the peptide chain upon translation. Significantly, this chemistry takes place spontaneously in situ of the translation solution, giving the corresponding cyclic peptides independent of ring sizes. We have used this method to convert human urotensin II, known as a potent vasoconstrictor, to its analogue containing a thioether bond, showing that this new analogue retains biological activity. Moreover, this peptide exhibits remarkable resistance against peptidases under reducing conditions. Thus, this technology offers a new means to accelerate the discovery of therapeutic peptidic drugs.
Assuntos
Endopeptidase K/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ribossomos/metabolismo , Cálcio/metabolismo , Linhagem Celular , Ciclização , Expressão Gênica , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Oxirredução , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The initiation codon dictates that the translation initiation event exclusively begins with methionine. We report here a new technology to reprogram the initiation event, where various amino acids and those bearing N (alpha)-acyl groups can be used as an initiator for peptide synthesis. The technology is built upon the concept of genetic code reprogramming, where methionine is depleted from the translation system and the initiation codon is reassigned to the desired amino acid. We have applied this technology to the synthesis of an antitumor cyclic peptide, G7-18NATE, closed by a physiologically stable bond, and it is also extended to the custom synthesis of its analogues with various ring sizes. Significantly, cyclization occurs spontaneously upon translation of the precursor linear peptides. To demonstrate the practicality of this methodology, we also prepared a small cyclic peptide library designated by 160 distinct mRNAs. Thus, this technology offers a new means to prepare a wide array of in vivo compatible cyclic peptide libraries for the discovery of peptidic drug candidates against various therapeutic targets.
Assuntos
Iniciação Traducional da Cadeia Peptídica , Peptídeos Cíclicos/síntese química , Aminoacil-RNA de Transferência/química , RNA de Transferência de Metionina/metabolismo , Aminoacilação de RNA de Transferência , Aminoacilação , Códon de Iniciação , Ciclização , Estrutura Molecular , Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência de Metionina/química , Ribossomos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizAssuntos
Reprogramação Celular , Código Genético/genética , Peptídeos/síntese química , Engenharia de Proteínas/métodos , Proteínas/síntese química , RNA Catalítico/genética , Desenho de Fármacos , Peptídeos/genética , Processamento de Proteína Pós-Traducional , Proteínas/genética , Aminoacil-RNA de Transferência , Aminoacilação de RNA de TransferênciaRESUMO
Protein-based fluorescent and functional probes are widely used for real-time visualization, purification, and regulation of a variety of biological molecules. The protein-based probes can generally be targeted into subcellular compartments of eukaryotic cells by a particular short peptide sequence. Little is known, however, about the sequence that targets probes into the mitochondrial intermembrane space (IMS). To identify the IMS-targeting sequence, we developed a simple genetic screening method to discriminate the proteins localized in the IMS from those in the mitochondrial matrix, thereby revealing the minimum requisite sequence for the IMS targeting. An IMS-localized protein, Smac/DIABLO, was randomly mutated, and the mitochondrial localization of each mutant was analyzed. We found that the four residues of Ala-Val-Pro-Ile are required for IMS localization, and a sequence of these four residues fused with matrix-targeting signals is sufficient for targeting the Smac/DIABLO into the IMS. The sequence was shown to readily direct three dissimilar proteins of interest to the IMS, which will open avenues to elucidating the functions of the IMS in live cells.
Assuntos
Proteínas Luminescentes/metabolismo , Membranas Mitocondriais/metabolismo , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutagênese , Fragmentos de Peptídeos/metabolismo , Transporte ProteicoRESUMO
The appearance of the premature translation termination codons (PTCs) in the transcript is the major cause of human genetic diseases. PTC-containing transcripts are rapidly degraded through nonsense-mediated decay (NMD) pathway. If such mRNA transcripts were translated in frame like normal transcripts, it would afford not only restoration of the level of full-length protein but also prevention of mRNA degradation by the NMD pathway. Here we describe a novel approach to read through PTC-containing mRNAs using suppressor tRNA that is introduced to cells by transfection. Luciferase reporter gene assay showed that nonsense and four-base codons were suppressed by the corresponding suppressor tRNAs derived from human tRNA(Ser). We also demonstrated that transfection of the suppressor tRNA to Ullrich disease fibroblasts, possessing a frameshift mutation in the collagen VI alpha2 gene, induced the upregulation of the collagen VI alpha2 mRNA and accumulation of the collagen VI protein. PTC suppression potentially provides a novel therapeutic means to rescue various PTC-related diseases.
Assuntos
Códon sem Sentido , Colágeno Tipo VI/genética , RNA de Transferência/química , Supressão Genética , Anticódon/química , Colágeno Tipo VI/biossíntese , Mutação da Fase de Leitura , Genes Reporter , Humanos , Distrofias Musculares/genética , Distrofias Musculares/terapia , Terminação Traducional da Cadeia Peptídica , RNA Mensageiro/biossíntese , RNA de Transferência de Serina/químicaRESUMO
Bioactive peptides isolated from natural sources have diverse acyl groups on the N-terminus. It is difficult to synthesize these peptides in vitro translation system because ribosomal peptide synthesis generally limits the N-terminal group to be N-formylmethionine (fMet). To overcome this restriction, we developed a novel methodology for the ribosomal synthesis of peptides having various terminal N-acyl groups with desired amino acids. In this methodology, two technologies, Flexizyme system consisting of artificial ribozymes and a reconstitute E. coli cell-free translation system (PURE system), were used. First, an amino acid carrying a desired N-acyl group was charged onto an initiation tRNA by the Flexizyme system. The addition of this N-acyl-aminoacyl-tRNA (N-acyl-aa-tRNA) to the PURE system allowed us to initiate the peptide synthesis with the designated N-acyl-amino acid. By means of this methodology, the translation was exclusively initiated by various N-terminal acyl groups as well as amino acids without contamination of N-formylmethionine.
Assuntos
Iniciação Traducional da Cadeia Peptídica , Peptídeos/química , Aminoacil-RNA de Transferência/química , RNA de Transferência de Metionina/metabolismo , Aminoacilação de RNA de Transferência , Aminoacilação , Sistema Livre de Células , Escherichia coli/genética , Fenilalanina/análogos & derivados , RNA Catalítico/metabolismo , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência de Metionina/químicaRESUMO
Here we describe a de novo tRNA acylation system consisting of artificial ribozymes. This system, the flexizyme (Fx) system, allows for the preparation of acyl-tRNAs with almost no limitation on the use of a variety of amino/hydroxy acids and tRNAs. To demonstrate its utility, we have carried out protein synthesis involving site-specific incorporation of nonnatural amino and hydroxy acids via amber or programmed frame-shift suppressions. We have also demonstrated peptide synthesis involving multiple nonnatural amino acids via sense codon suppression. The combination of the Fx system and appropriate cell-free translation is a powerful and flexible tool for mRNA-encoded synthesis of nonnatural polypeptides.
Assuntos
Aminoácidos/química , Biossíntese Peptídica , RNA Catalítico/metabolismo , RNA de Transferência/metabolismo , Aminoácidos/metabolismo , Aminoacilação , Catálise , Hidroxiácidos/metabolismo , Peptídeos/química , Biossíntese de Proteínas , RNA Catalítico/química , RNA de Transferência/química , Especificidade por SubstratoRESUMO
The compartments of eukaryotic cells maintain a distinct protein composition to perform a variety of specialized functions. We developed a new method for identifying the proteins that are transported to the endoplasmic reticulum (ER) in living mammalian cells. The principle is based on the reconstitution of two split fragments of enhanced green fluorescent protein (EGFP) by protein splicing with DnaE from Synechocystis PCC6803. Complementary DNA (cDNA) libraries fused to the N-terminal halves of DnaE and EGFP are introduced in mammalian cells with retroviruses. If an expressed protein is transported into the ER, the N-terminal half of EGFP meets its C-terminal half in the ER, and full-length EGFP is reconstituted by protein splicing. The fluorescent cells are isolated using fluorescence-activated cell sorting and the cDNAs are sequenced. The developed method was able to accurately identify cDNAs that encode proteins transported to the ER. We identified 27 novel proteins as the ER-targeting proteins. The present method overcomes the limitation of the previous GFP- or epitope-tagged methods, using which it was difficult to identify the ER-targeting proteins in a high-throughput manner.
Assuntos
Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/análise , Proteínas de Fluorescência Verde/análise , Proteínas/genética , Proteínas/metabolismo , Animais , Linhagem Celular , Clonagem Molecular , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Biblioteca Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Sinais Direcionadores de Proteínas , Processamento de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNARESUMO
The control of intricate networks within eukaryotic cells relies on differential compartmentalization of proteins. We have developed a method that allows rapid identification of novel proteins compartmentalized in mitochondria by screening large-scale cDNA libraries. The principle is based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing of DnaE derived from Synechocystis sp. PCC6803. The cDNA libraries are expressed in mammalian cells following infection with retrovirus. If a test protein contains a functional mitochondrial targeting signal (MTS), it translocates into the mitochondrial matrix, where EGFP is then formed by protein splicing. The cells harboring this reconstituted EGFP are screened rapidly by fluorescence-activated cell sorting, and the cDNAs are isolated and identified from the cells. The analysis of 258 cDNAs revealed various MTSs, among which we identified new transcripts corresponding to mitochondrial proteins. This method should provide a means to map proteins distributed within intracellular organelles in a broad range of different tissues and disease states.