RESUMEN
The N-degron pathway targets proteins that bear a destabilizing residue at the N terminus for proteasome-dependent degradation1. In yeast, Ubr1-a single-subunit E3 ligase-is responsible for the Arg/N-degron pathway2. How Ubr1 mediates the initiation of ubiquitination and the elongation of the ubiquitin chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here we developed chemical strategies to mimic the reaction intermediates of the first and second ubiquitin transfer steps, and determined the cryo-electron microscopy structures of Ubr1 in complex with Ubc2, ubiquitin and two N-degron peptides, representing the initiation and elongation steps of ubiquitination. Key structural elements, including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, were identified and characterized. These structures provide mechanistic insights into the initiation and elongation of ubiquitination catalysed by Ubr1.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Sitios de Unión , Biocatálisis , Microscopía por Crioelectrón , Lisina/metabolismo , Modelos Moleculares , Proteolisis , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/ultraestructura , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/ultraestructuraRESUMEN
Heterotypic ubiquitin (Ub) chains have emerged as fundamental components in a wide range of cellular processes. The integrative identification of Ub-interacting proteins (readers) and Ub-modifying enzymes (writers and erasers) that selectively recognize and regulate heterotypic ubiquitination may provide crucial insights into these processes. In this study, we employed the bifunctional molecule-assisted (CAET) strategy to develop a type of disulfide bond-activated heterotypic Ub reagents, which allowed to enrich heterotypic Ub-interacting proteins and modifying enzymes simultaneously. The sequential release of readers which are non-covalently bound and writers or erasers which are covalently conjugated by using urea and reductant, respectively, combined with label-free quantitative (LFQ) MS indicated that these heterotypic Ub reagents would facilitate future investigations into functional roles played by heterotypic Ub chains.
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Proteínas , Ubiquitina , Ubiquitina/metabolismo , Indicadores y Reactivos , Ubiquitinación , Proteínas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. In this study, we screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can specifically recognize K29-linked polyubiquitin using chemically synthesized K29-linked diubiquitin. We further determined the crystal structure of this fragment bound to the K29-linked diubiquitin, which revealed the molecular basis of specificity. Using sAB-K29 as a tool, we uncovered that K29-linked ubiquitination is involved in different kinds of cellular proteotoxic stress response as well as cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody and downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.
Asunto(s)
Ubiquitina-Proteína Ligasas/metabolismo , Ciclo Celular , Línea Celular Tumoral , Humanos , Modelos Moleculares , Transducción de Señal , Estrés Fisiológico , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
Ubiquitin (Ub)-like protein ISG15 (interferon-stimulated gene 15) regulates innate immunity and links with the evasion of host response by viruses such as SARS-CoV-2. Dissecting ISGylation pathways recently received increasing attention which can inform related disease interventions, but such studies necessitate the preparation and development of various ISG15 protein tools. Here, we find that the leader protease (Lbpro ) encoded by foot-and-mouth disease virus can promote ligation reactions between recombinant ISG15 and synthetic glycyl compounds, generating protein tools such as ISG15-propargylamide and ISG15-rhodamine110, which are needed for cellular proteomic studies of deISGylases, and the screening and evaluation of inhibitors against SARS-CoV-2 papain-like protease (PLpro). Furthermore, this strategy can be also used to load ISG15 onto the lysine of a synthetic peptide through an isopeptide bond, and prepare Ub and NEDD8 (ubiquitin-like protein Nedd8) protein tools.
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COVID-19 , Péptido Hidrolasas , Animales , Catálisis , Citocinas/metabolismo , Interferones , Lisina , Proteína NEDD8 , Péptido Hidrolasas/metabolismo , Proteómica , SARS-CoV-2 , Ubiquitinas/químicaRESUMEN
Sortase A (SrtA)-mediated ligation, a popular method for protein labeling and semi-synthesis, is limited by its reversibility and dependence on the LPxTG motif, where "x" is any amino acid. Here, we report that SrtA can mediate the efficient and irreversible ligation of a protein/peptide containing a C-terminal thioester with another protein/peptide bearing an N-terminal Gly, with broad tolerance for a wide variety of LPxT-derived sequences. This strategy, the thioester-assisted SrtA-mediated ligation, enabled the expedient preparation of proteins bearing various N- or C-terminal labels, including post-translationally modified proteins such as the Ser139-phosphorylated histone H2AX and Lys9-methylated histone H3, with less dependence on the LPxTG motif. Our study validates the chemical modification of substrates as an effective means of augmenting the synthetic capability of existing enzymatic methods.
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Aminoaciltransferasas , Aminoaciltransferasas/química , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/química , Péptidos/química , Compuestos de AzufreRESUMEN
Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s. Chemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.
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Compuestos de Sulfhidrilo/metabolismo , Ubiquitina-Proteína Ligasas/análisis , Ubiquitina/química , Biocatálisis , Humanos , Estructura Molecular , Compuestos de Sulfhidrilo/química , Ubiquitina/síntesis química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Triazole-based deubiquitylase (DUB)-resistant ubiquitin (Ub) probes have recently emerged as effective tools for the discovery of Ub chain-specific interactors in proteomic studies, but their structural diversity is limited. A new family of DUB-resistant Ub probes is reported based on isopeptide-N-ethylated dimeric or polymeric Ub chains, which can be efficiently prepared by a one-pot, ubiquitin-activating enzyme (E1)-catalyzed condensation reaction of recombinant Ub precursors to give various homotypic and even branched Ub probes at multi-milligram scale. Proteomic studies using label-free quantitative (LFQ) MS indicated that the isopeptide-N-ethylated Ub probes may complement the triazole-based probes in the study of Ub interactome. Our study highlights the utility of modern protein synthetic chemistry to develop structurally and new families of tool molecules needed for proteomic studies.
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Sondas Moleculares/química , Poliubiquitina/química , Enzimas Activadoras de Ubiquitina/química , Ciclina B1/química , Ciclina B1/genética , Células HEK293 , Células HeLa , Histonas/química , Histonas/genética , Humanos , Sondas Moleculares/síntesis química , Mutación , Poliubiquitina/síntesis química , ProteómicaRESUMEN
New synthetic strategies that exploited the strengths of both chemoselective ligation and recombinant protein expression were developed to prepare K27 di-ubiquitins (diUb), which enabled mechanistic studies on the molecular recognition of K27-linked Ubs by single-molecule Förster resonance energy transfer (smFRET) and X-ray crystallography. The results revealed that free K27 diUb adopted a compact conformation, whereas upon binding to UCHL3, K27 diUb was remodeled to an open conformation. The K27 isopeptide bond remained rigidly buried inside the diUb moiety during binding, an interesting unique structural feature that may explain the distinctive biological function of K27 Ub chains.
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Ubiquitina/síntesis química , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Modelos Moleculares , Conformación Proteica , Procesamiento Proteico-Postraduccional , Ubiquitina/químicaRESUMEN
Quasi-racemic crystallography has been used to determine the X-ray structures of K27-linked ubiquitin (Ub) chains prepared through total chemical synthesis. Crystal structures of K27-linked di- and tri-ubiquitins reveal that the isopeptide linkages are confined in a unique buried conformation, which provides the molecular basis for the distinctive function of K27 linkage compared to the other seven Ub chains. K27-linked di- and triUb were found to adopt different structural conformations in the crystals, one being symmetric whereas the other triangular. Furthermore, bioactivity experiments showed that the ovarian tumor family de-ubiquitinase 2 significantly favors K27-linked triUb than K27-linked diUb. K27-linked triUb represents the so-far largest chemically synthesized protein (228 amino acids) that has been crystallized to afford a high-resolution X-ray structure.
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Lisina/química , Poliubiquitina/química , Poliubiquitina/síntesis química , Sitios de Unión , Cristalografía por Rayos X , Endopeptidasas/metabolismo , Lisina/metabolismo , Modelos Moleculares , Poliubiquitina/metabolismo , Conformación Proteica , Tioléster Hidrolasas/metabolismo , UbiquitinaciónRESUMEN
Racemic or quasi-racemic crystallography recently emerges as a useful technology for solution of the crystal structures of biomacromolecules. It remains unclear to what extent the biomacromolecules of opposite handedness can differ from each other in racemic or quasi-racemic crystallography. Here we report a finding that monomeric d-ubiquitin (Ub) has propensity to cocrystallize with different dimers, trimers, and even a tetramer of l-Ub. In these cocrystals the unconnected monomeric d-Ubs can self-assemble to form pseudomirror images of different oligomers of l-Ub. This monomer/oligomer cocrystallization phenomenon expands the concept of racemic crystallography. Using the monomer/oligomer cocrystallization technology we obtained, for the first time the X-ray structures of linear M1-linked tri- and tetra-Ubs and a K11/K63-branched tri-Ub.
Asunto(s)
Multimerización de Proteína , Ubiquitina/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Estructura Cuaternaria de Proteína , EstereoisomerismoRESUMEN
Mambalgins are a class of 57-residue polypeptide toxins isolated from the venom of the African mamba. They exhibit potent analgesic effects by inhibiting the acid-sensing ion channels. Classified as members of the family of three-finger toxins, mambalgins contain four pairs of disulfide bridges that help to stabilize the three-finger scaffold. Here, we report the chemical synthesis of functional mambalgin-1/2/3 by using one-step two-segment hydrazide-based native chemical ligation. The two-segment ligation approach reported here may enable efficient production of mambalgin toxins. These synthetic mambalgins are useful compounds for development of diagnostic or therapeutic reagents. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
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Venenos Elapídicos/síntesis química , Péptidos/síntesis química , Azidas/química , Disulfuros/química , Venenos Elapídicos/química , Modelos Moleculares , Estructura Molecular , Péptidos/químicaRESUMEN
Lysine 2,3-aminomutase (KAM; EC 5.4.3.2) catalyzes the interconversion of l-lysine and l-ß-lysine. The transcription and regulation of the kam locus, including lysine-2,3-aminomutase-encoding genes, in Bacillus thuringiensis were analyzed in this study. Reverse transcription-PCR (RT-PCR) analysis revealed that this locus forms two operons: yodT (yodT-yodS-yodR-yodQ-yodP-kamR) and kamA (kamA-yokU-yozE). The transcriptional start sites (TSSs) of the kamA gene were determined using 5' rapid amplification of cDNA ends (RACE). A typical -12/-24 σ(54) binding site was identified in the promoter PkamA, which is located upstream of the kamA gene TSS. A ß-galactosidase assay showed that PkamA, which directs the transcription of the kamA operon, is controlled by the σ(54) factor and is activated through the σ(54)-dependent transcriptional regulator KamR. The kamA operon is also controlled by σ(K) and regulated by the GerE protein in the late stage of sporulation. kamR and kamA mutants were prepared by homologous recombination to examine the role of the kam locus. The results showed that the sporulation rate in B. thuringiensis HD(ΔkamR) was slightly decreased compared to that in HD73, whereas that in HD(ΔkamA) was similar to that in HD73. This means that other genes regulated by KamR are important for sporulation.
Asunto(s)
Bacillus thuringiensis/genética , Regulación Bacteriana de la Expresión Génica , Transferasas Intramoleculares/biosíntesis , Factor sigma/metabolismo , Transcripción Genética , Fusión Artificial Génica , Sitios de Unión , Genes Reporteros , Transferasas Intramoleculares/genética , Operón , Regiones Promotoras Genéticas , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor sigma/genética , Sitio de Iniciación de la Transcripción , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
To optimize the expression of cry genes in a Bacillus thuringiensis sigK mutant failing in crystal releasing, the transcriptional activity of the cry promoters cry1A, cry3A, cry4A, and cry8E was compared using lacZ gene fusions. A beta-galactosidase assay indicated that the cry8E promoter showed the highest transcriptional activity. A novel Escherichia coli-B. thuringiensis shuttle vector pHT315-8E21b was constructed for cry gene expression using the cry8E promoter and the multiple cloning sites from vector pET21b, based on vector pHT315. SDS-PAGE analysis showed that the expression of the cry1Ac gene directed by the cry8E promoter was increased by approximately 2.4-fold over the expression directed by the cry3A promoter. The cry1Ba gene was expressed in the sigK mutant with the constructed vector pHT315-8E21b. Normal bipyramidal crystals encapsulated in mother cell were observed by transmission electron microscopy (TEM). The encapsulated Cry1Ba protein expressed in the sigK mutant showed activity against Ostrinia furnacalis and Plutella xylostella similar to that of the released Cry1Ba protein expressed in the acrystalliferous strain HD73 and can be protected from inactivation by UV light. All these results suggest that the cry8E promoter can be an efficient transcriptional element for cry gene expression in sigK mutants and can be utilized for the construction of a genetically engineered strain.
Asunto(s)
Bacillus thuringiensis/metabolismo , Regiones Promotoras Genéticas/genética , Animales , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Endotoxinas/genética , Endotoxinas/metabolismo , Endotoxinas/farmacología , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacología , Lepidópteros/efectos de los fármacos , Microscopía Electrónica de TransmisiónRESUMEN
OBJECTIVE: In order to determine the effect of bkdR deletion on Cry protein production. We analyzed the transcriptional regulation of bkd gene cluster and the phenotype of bkdR mutant. METHODS: Sequence of bkd gene cluster in Bacillus thuringiensis was analyzed by sequence alignment. RT-PCRwas used to reveal the transcriptional units of the bkd gene cluster. bkdR insertion mutant was constructed by homologous recombination. Transcriptional activity was analyzed by promoter fusions with lacZ gene. Comparison of the CrylAc protein production was determined by protein quantitation. RESULTS: The bkd gene cluster was composed of eight genes. The ptb-bkdB formed one transcriptional unit. The transcriptional activity of ptb sharply decreased in sigL and bkdR mutants. Deletion of bkdR decreased the motility of cells, but no effect on growth, sporulation efficiency and Cry protein production. The bkd gene cluster is controlled by Sigma 54 and activated by BkdR. Deletion of bkdR has no effect on Cry protein production, but decreased the motility of the cells. CONCLUSION: The bkd gene cluster is controlled by Sigma 54 and activated by BkdR. Deletion of bkdR has nb effect on Cry protein production, but decreased the motility of the cells. It suggested that deletion of bkdR do not affect the Cry protein production the same as sigL mutant. It means decreasing of Cry protein productioninsigL mutant was not caused by only one EBP mutation, but might be multiple roles.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Familia de Multigenes , Transcripción Genética , Bacillus thuringiensis/crecimiento & desarrollo , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Regiones Promotoras GenéticasRESUMEN
OBJECTIVE: To discover new elements for cry gene expression, PexsY, which is the promoter of the exosporium basal layer structural gene exsY, was used to express cry1Ac gene in Bacillus thuringiensis. METHODS: We used be ta- galactosidase assays by promoter-lacZ fusion to analyze the transcriptional activity of exsY promoter and truncated exsY promoter. The cry1Ac gene was directed by the non-cry gene promoter PexsY and was then expressed in Bacillus thuringiensis HD73. Transmission electron microscope (TEM) was used to observe the formation of crystal inclusion. The CrylAc yieldswere evaluated by protein quantification and SDS-PAGE analysis. Bioassays against Ostriniafurnacalis were used for the functional verification. RESULTS: Beta-galactosidase assays showed that the exsY promoter had a strong transcriptional activity in the acrystalliferous mutant strain HD73- on the late sporulation phase. Cry1Ac expression products directed by the PexsY could form diamond crystals. SDS-PAGE analysis showed that the cry1Ac gene directed by the cry8E promoter has the highest protein yield among the four promoters while the cry1Ac gene under the direction of PexsYorcry3A promoters showed similar protein yields. The bioassay results showed that the Cry1Ac protein directed by the PexsY promoter was toxic against Ostrinia furnacalis. CONCLUSION: The cry1Ac gene under the direction ofthe non-cry gene promoter PexsY was able to express the Cry proteins at the late sporulation phase and could form crystal inclusion in a B. thuringiensis strain. Our finding provides applicationpotential for the genetically modification of engineered Bt strains.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Endotoxinas/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Hemolisinas/genética , Regiones Promotoras Genéticas , Animales , Bacillus thuringiensis/crecimiento & desarrollo , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Secuencia de Bases , Endotoxinas/metabolismo , Endotoxinas/farmacología , Ingeniería Genética , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacología , Datos de Secuencia Molecular , Mariposas Nocturnas/efectos de los fármacos , Esporas Bacterianas/genética , Esporas Bacterianas/crecimiento & desarrollo , Esporas Bacterianas/metabolismoRESUMEN
Besides traditional ubiquitin-dependent proteasome degradation, thousands of eukaryotic proteins more than previously appreciated could undergo ubiquitin-independent proteasomal degradation (UbInPD). A pathogen-encoded effector protein SAP05 secreted by phytoplasma, could hijack hostage Rpn10 subunit of proteasome derived from Arabidopsis thaliana and target the degradation of GATA BINDING FACTOR (GATA) or SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors (TFs) without ubiquitin or additional proteasome shuttle factors. To explain how could SAP05 target the degradation bypassing the ubiquitin-dependent pathway, we have determined the crystal structure of the complex between Arabidopsis thaliana Rpn10 and Aster Yellows witches'-broom phytoplasma SAP05 or onion yellow phytoplasma SAP05, which showed a previously unknown recognition interface. Sequence alignment and structural biological evidence showed that this interaction is highly conserved in various SAP05 homologs, suggesting a general mode in plant infection. After docking the complex structure to the plant proteasome, SAP05 was near to the adenosine triphosphatase (ATPase) central pore and enough to submit substrate to degradation process, which suggested a molecular glue-like role to bridge TFs close to the ATPase central pore of proteasomes for the direct degradation.
RESUMEN
AIM: Phosphatidylcholine (PC) is essential for membrane structural integrity and lipid-dependent signaling pathways, and is an essential component required for cancer cell growth. Using hepatocellular carcinoma (HCC) as a tumor model, this study aims to further screen phospholipid biomarkers of the tumor microenvironment and explore the anti-tumor effects and mechanisms of aerobic exercise. MAIN METHODS: The HCC of C57BL/6J mice was induced by the injection of the carcinogen diethylnitrosamine (DEN). Exercise was performed on an ungraded treadmill for weeks. The inflammation-related markers were detected by ELISA, PCR and immunohistochemistry, hepatic metabolic profile was analyzed by GC/MS, and lipid metabolism profile was further detected by lipid-targeted LC/MS. Cell culture was used to verify the anti-inflammatory effect of PC. KEY FINDINGS: Exercise reduced hepatic inflammation, tumor incidence and volume. Metabolomics analysis showed that palmitic acid is a key metabolic marker for exercise to improve tumor microenvironment. Injection of exogenous palmitic acid following exercise impaired the anti-inflammatory and anti-tumor effects of exercise. Lipid metabolomics analysis further showed that metabolites for exercise were enriched in glycerol phospholipid metabolism, including 14 phosphatidylcholines (PCs), 18 phosphatidylethanolamines (PEs), and 6 triglycerides (TGs). These biomarkers contain different lengths of fatty acid chains and different numbers of unsaturated bonds, respectively. Cell culture verified that PC (18:1/18:1) mediated lipopolysaccharide (LPS)-induced inflammation in HepG2 cell. SIGNIFICANCE: Our results suggest that exercise remodels glycerophospholipid metabolism and reduces hepatic palmitic acid loading and PC (18:1/18:1) level, thereby reconstructing a microenvironment that is hostile to HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Ratones , Animales , Carcinoma Hepatocelular/patología , Fosfatidilcolinas , Neoplasias Hepáticas/patología , Microambiente Tumoral , Ácido Palmítico , Ratones Endogámicos C57BL , Fosfolípidos/metabolismo , Inflamación , Antiinflamatorios/uso terapéutico , Ejercicio Físico , BiomarcadoresRESUMEN
It is very important to seek a sustainable improvement in human well-being under a limited resource supply and to promote the scientific and coordinated development of urban economic development, ecological environment protection, and human well-being. This paper constructs a human well-being index that includes economic well-being, culture and education well-being, and social development well-being as factors, and it incorporates the human well-being index into the evaluation system for urban well-being energy eco-efficiency (WEE). It uses the super-slack-based measure (SBM) model, which considers undesirable output, to measure the WEE of 10 prefecture-level cities in Shaanxi Province, China, from 2005 to 2019. The social network analysis (SNA) is used to describe the characteristics of the spatial correlation network of WEE and its spatiotemporal evolutionary trend, and the quadratic assignment procedure (QAP) analysis method is used to identify the driving factors that affect the spatial correlation network. The results show that, first, the WEE in Shaanxi is relatively low as a whole and varies greatly among regions, with the highest level in northern Shaanxi, followed by Guanzhong; the lowest level is in southern Shaanxi. Second, in Shaanxi, WEE has transcended geographical proximity into a complex, multi-threaded spatial correlation network, and Yulin is at the center of the network. Third, the network shows four sectors: the net overflow, main benefit, two-way overflow, and broker. Members in each sector have not fully exploited their advantages, and the whole network can be improved. Fourth, the differences in the economic development level, openness, industrial structure, and population are the main driving factors influencing the formation of the spatial correlation network.
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Desarrollo Económico , Industrias , Humanos , Ciudades , China , EficienciaRESUMEN
Ubiquitination regulates almost every life process of eukaryotes. The study of the ubiquitin (Ub) coupling or decoupling process and the interaction study of Ub-Ub binding protein have always been the central focus. However, such studies are challenging, owing to the transient nature of Ub-coupling enzymes and deubiquitinases in the reactions, as well as the difficulty in preparing large quantities of polyubiquitinated samples. Here we describe a recently developed strategy for the efficient preparation of analogs of Ub chains and analogs for Ub coupling and uncoupling intermediates, which facilitate the study of the ubiquitination process. The strategy includes mainly the following steps: (i) the bifunctional molecule conjugation on the only cysteine (Cys) residue of a target protein (usually a Ub or Ub-conjugating enzyme), exposing an orthogonal reactive site for native chemical ligation; (ii) covalent ligation with a Ub-derived thioester, exposing a free sulfhydryl; and (iii) (optional) a disulfide bond formation with a substrate protein (mainly with only one Cys protein) through nonactivity-based cross-linking or with a deubiquitinase (mainly with several Cys residues) through activity-based cross-linking. When the bifunctional molecule and target proteins are obtained, the final products can be prepared in milligram quantities within 2 weeks.
Asunto(s)
Enzimas Ubiquitina-Conjugadoras , Ubiquitina , Ubiquitinación , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas/metabolismo , Cisteína/metabolismoRESUMEN
OBJECTIVE: Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy. METHODS: The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression. RESULTS: rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity. CONCLUSION: Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.