RESUMO
Vitamin B6 is a critical molecule for metabolism, development, and stress sensitivity in plants. It is a cofactor for numerous biochemical reactions, can serve as an antioxidant, and has the potential to increase tolerance against both biotic and abiotic stressors. Due to the importance of vitamin B6, its biosynthesis is likely tightly regulated. Plants can synthesize vitamin B6 de novo via the concerted activity of Pyridoxine Biosynthesis Protein 1 (PDX1) and PDX2. Previously, PDX proteins have been identified as targets for ubiquitination, indicating they could be marked for degradation by two highly conserved pathways: the Ubiquitin Proteasome Pathway (UPP) and the autophagy pathway. Initial experiments show that PDXs are in fact degraded, but surprisingly, in a ubiquitin-independent manner. Inhibitor studies pointed toward cathepsin B, a conserved lysosomal cysteine protease, which is implicated in both programed cell death and autophagy in humans and plants. In plants, cathepsin Bs are poorly described, and no confirmed substrates have been identified. Here, we present PDX proteins from Arabidopsis thaliana as interactors and substrates of a plant Cathepsin B. These findings not only describe a novel cathepsin B substrate in plants, but also provide new insights into how plants regulate de novo biosynthesis of vitamin B6.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Catepsina B , Vitamina B 6 , Catepsina B/metabolismo , Catepsina B/genética , Arabidopsis/metabolismo , Arabidopsis/genética , Vitamina B 6/metabolismo , Vitamina B 6/biossíntese , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Especificidade por Substrato , Ubiquitinação , Regulação da Expressão Gênica de Plantas , Carbono-Nitrogênio LiasesRESUMO
SUMOylation is a post-translational modification of proteins that regulates these proteins' localization, turnover or function. Aberrant SUMOylation is frequently found in cancers but its origin remains elusive. Using a genome-wide transposon mutagenesis screen in a MYC-driven B-cell lymphoma model, we here identify the SUMO isopeptidase (or deconjugase) SENP6 as a tumor suppressor that links unrestricted SUMOylation to tumor development and progression. Notably, SENP6 is recurrently deleted in human lymphomas and SENP6 deficiency results in unrestricted SUMOylation. Mechanistically, SENP6 loss triggers release of DNA repair- and genome maintenance-associated protein complexes from chromatin thereby impairing DNA repair in response to DNA damages and ultimately promoting genomic instability. In line with this hypothesis, SENP6 deficiency drives synthetic lethality to Poly-ADP-Ribose-Polymerase (PARP) inhibition. Together, our results link SENP6 loss to defective genome maintenance and reveal the potential therapeutic application of PARP inhibitors in B-cell lymphoma.
Assuntos
Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Mutação , Sumoilação/fisiologia , Animais , Biomarcadores Tumorais , Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Cromatina , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Feminino , Instabilidade Genômica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Sumoilação/efeitos dos fármacos , Sumoilação/genética , Mutações Sintéticas Letais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Strictosidine synthase (STR), the gate enzyme for monoterpenoid indole alkaloid biosynthesis, catalyzes the Pictet-Spengler reaction (PSR) of various tryptamine derivatives with secologanin assisted by "indole sandwich" stabilization. Continuous exploration with ß-methyltryptamine (IPA) stereoselectively delivered the C6-methylstrictosidines and C6-methylvincosides by enzymatic and nonenzymatic PSR, respectively. Unexpectedly, the first "nonindole sandwich" binding mode was witnessed by the X-ray structures of STR1-ligand complexes. Site-directed mutagenesis revealed the critical cryptic role of the hydroxyl group of Tyr151 in IPA biotransformation. Further computational calculations demonstrated the adjustable IPA position in STR1 upon the binding of secologanin, and Tyr151-OH facilitates the productive PSR binding mode via an advantageous hydrogen-bond network. Further chemo-enzymatic manipulation of C6-methylvincosides successfully resulted in the discovered antimalarial framework (IC50 = 0.92 µM).
Assuntos
Alcaloides , Carbolinas , Carbono-Nitrogênio Liases , Triptaminas , Humanos , Alcaloides/química , Alcaloides/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Carbolinas/química , Carbolinas/metabolismo , Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Células HL-60 , Modelos Moleculares , Estrutura Molecular , p-Hidroxianfetamina , Ligação Proteica , Conformação Proteica , Triptaminas/química , Triptaminas/metabolismoRESUMO
Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme catalyzing the crosslinking between Gln and Lys residues and involved in various pathophysiological events. Besides this crosslinking activity, TG2 functions as a deamidase, GTPase, isopeptidase, adapter/scaffold, protein disulfide isomerase, and kinase. It also plays a role in the regulation of hypusination and serotonylation. Through these activities, TG2 is involved in cell growth, differentiation, cell death, inflammation, tissue repair, and fibrosis. Depending on the cell type and stimulus, TG2 changes its subcellular localization and biological activity, leading to cell death or survival. In normal unstressed cells, intracellular TG2 exhibits a GTP-bound closed conformation, exerting prosurvival functions. However, upon cell stimulation with Ca2+ or other factors, TG2 adopts a Ca2+-bound open conformation, demonstrating a transamidase activity involved in cell death or survival. These functional discrepancies of TG2 open form might be caused by its multifunctional nature, the existence of splicing variants, the cell type and stimulus, and the genetic backgrounds and variations of the mouse models used. TG2 is also involved in the phagocytosis of dead cells by macrophages and in fibrosis during tissue repair. Here, we summarize and discuss the multifunctional and controversial roles of TG2, focusing on cell death/survival and fibrosis.
Assuntos
Aminoaciltransferases/genética , Carbono-Nitrogênio Liases/genética , Fibrose/enzimologia , Proteínas de Ligação ao GTP/genética , Inflamação/enzimologia , Isomerases de Dissulfetos de Proteínas/genética , Transglutaminases/genética , Processamento Alternativo , Aminoaciltransferases/imunologia , Animais , Cálcio/imunologia , Cálcio/metabolismo , Carbono-Nitrogênio Liases/imunologia , Morte Celular , Sobrevivência Celular , Fibrose/genética , Fibrose/imunologia , Fibrose/patologia , Proteínas de Ligação ao GTP/imunologia , Expressão Gênica , Guanosina Trifosfato/imunologia , Guanosina Trifosfato/metabolismo , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Isoenzimas/genética , Isoenzimas/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Fagocitose/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/imunologiaRESUMO
Isopeptidase activity of proteases plays critical roles in physiological and pathological processes in living organisms, such as protein stability in cancers and protein activity in infectious diseases. However, the kinetics of protease isopeptidase activity has not been explored before due to a lack of methodology. Here, we report the development of novel qFRET-based protease assay for characterizing the isopeptidase kinetics of SENP1. The reversible process of SUMOylation in vivo requires an enzymatic cascade that includes E1, E2, and E3 enzymes and Sentrin/SUMO-specific proteases (SENPs), which can act either as endopeptidases that process the pre-SUMO before its conjugation, or as isopeptidases to deconjugate SUMO from its target substrate. We first produced the isopeptidase substrate of CyPet-SUMO1/YPet-RanGAP1c by SUMOylation reaction in the presence of SUMO E1 and E2 enzymes. Then a qFRET analyses of real-time FRET signal reduction of the conjugated substrate of CyPet-SUMO1/YPet-RanGAP1c to free CyPet-SUMO1 and YPet-RanGAP1c by the SENP1 were able to obtain the kinetic parameters, Kcat, KM, and catalytic efficiency (Kcat/KM) of SENP1. This represents a pioneer effort in isopeptidase kinetics determination. Importantly, the general methodology of qFRET-based protease isopeptidase kinetic determination can also be applied to other proteases.
Assuntos
Carbono-Nitrogênio Liases/química , Carbono-Nitrogênio Liases/metabolismo , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Domínio Catalítico , Cisteína Endopeptidases , Humanos , Cinética , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , SumoilaçãoRESUMO
Pyridoxine (vitamin B6) and its vitamers are used by living organisms both as enzymatic cofactors and as antioxidants. We used Arabidopsis pyridoxine biosynthesis mutant pdx1.3-1 to study the involvement of the PLP-synthase main polypeptide PDX1 in plant responses to ultraviolet radiation of two different qualities, one containing primarily UV-A (315-400 nm) and the other containing both UV-A and UV-B (280-315 nm). The antioxidant capacity and the flavonoid and glucosinolate (GS) profiles were examined. As an indicator of stress, Fv/Fm of photosystem II reaction centers was used. In pdx1.3-1, UV-A + B exposure led to a significant 5% decrease in Fv/Fm on the last day (day 15), indicating mild stress at this time point. The antioxidant capacity of Col-0 wildtype increased significantly (50-73%) after 1 and 3 days of UV-A + B. Instead, in pdx1.3-1, the antioxidant capacity significantly decreased by 44-52% over the same time period, proving the importance of a full complement of functional PDX1 genes for the detoxification of reactive oxygen species. There were no significant changes in the flavonoid glycoside profile under any light condition. However, the GS profile was significantly altered, both with respect to Arabidopsis accession and exposure to UV. The difference in flavonoid and GS profiles reflects that the GS biosynthesis pathway contains at least one pyridoxine-dependent enzyme, whereas no such enzyme is used in flavonoid biosynthesis. Also, there was strong correlation between the antioxidant capacity and the content of some GS compounds. Our results show that vitamin B6 vitamers, functioning both as antioxidants and co-factors, are of importance for the physiological fitness of plants.
Assuntos
Antioxidantes/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Carbono-Nitrogênio Liases/metabolismo , Glucosinolatos/biossíntese , Raios Ultravioleta , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Carbono-Nitrogênio Liases/genética , Cromatografia Líquida de Alta Pressão , Flavonoides/biossíntese , Glucosinolatos/análise , Mutagênese , Complexo de Proteína do Fotossistema II/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismoRESUMO
Experimental assessment of catalytic reaction mechanisms and profiles of radical enzymes can be severely challenging due to the reactive nature of the intermediates and sensitivity of cofactors such as iron-sulfur clusters. Here, we present an enzyme-directed computational methodology for the assessment of thermodynamic reaction profiles and screening for radical stabilization energies (RSEs) for the assessment of catalytic turnovers in radical enzymes. We have applied this new screening method to the radical S-adenosylmethione enzyme 7-carboxy-7-deazaguanine synthase (QueE), following a detailed molecular dynamics (MD) analysis that clarifies the role of both specific enzyme residues and bound Mg2+, Ca2+, or Na+. The MD simulations provided the basis for a statistical approach to sample different conformational outcomes. RSE calculation at the M06-2X/6-31+G* level of theory provided the most computationally cost-effective assessment of enzyme-based energies, facilitated by an initial triage using semiempirical methods. The impact of intermolecular interactions on RSE was clearly established, and application to the assessment of potential alternative substrates (focusing on radical clock type rearrangements) proposes a selection of carbon-substituted analogues that would react to afford cyclopropylcarbinyl radical intermediates as candidates for catalytic turnover by QueE.
Assuntos
Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Carbono-Nitrogênio Liases/química , Metais/metabolismo , Conformação ProteicaRESUMO
Expression profiling for genes involved in Vitamin B6 (VitB6) biosynthesis was undertaken to delineate the involvement of de novo and salvage pathway induced by Bacillus subtilis CBR05 against, Xanthomonas campestris pv. vesicatoria in tomato. Pyridoxine biosynthesis (PDX) genes such as PDX1.2 and PDX1.3, were found to be overexpressed significantly at 72 hpi in B. subtilis and pyridoxine inoculated plants. Most significant upregulation was observed in the transcript profile of PDX1.3, which showed more than 12- fold increase in expression. Unfortunately, salt sensitive overlay4 (SOS4) profiling showed irregular expression which corroborates that SOS4 role in VitB6 biosynthesis needs further studies for deciphering a clear notion about their role in tomato. Antioxidant enzymes i.e., superoxide dismutase, catalase, polyphenol oxidase, and peroxidase activities clearly demonstrate escalation till 48 hpi and gets reduced in 72 hpi. Pot trials also confirm that B. subtilis compared to pyridoxine supplementation alone show plant disease resistance and elongated roots. The present study confirms that B. subtilis, as a versatile agent in eliciting induced systemic resistance regulated by de novo pathway as a model for plant defense against X. campestris pv. vesicatoria substantiated by VitB6 biosynthesis. Nevertheless, the study is preliminary and needs further evidence for affirming this phenomenon.
Assuntos
Vias Biossintéticas/genética , Resistência à Doença/genética , Doenças das Plantas/genética , Solanum lycopersicum/genética , Vitamina B 6/biossíntese , Antibiose , Bacillus subtilis/fisiologia , Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Piridoxal Quinase/genética , Piridoxal Quinase/metabolismo , Xanthomonas vesicatoria/fisiologiaRESUMO
Covalent conjugation of the ubiquitin-related SUMO modifier to lysine residues of cellular proteins (SUMOylation) is a prevalent posttranslational modification. SUMOs are synthesized as precursor proteins that require carboxy-terminal processing prior to conjugation. Subsequently, a multistep enzymatic pathway is used for conjugation to target proteins. SUMOylation generally impacts protein-protein interactions and the assembly of multiprotein complexes. Cellular processes regulated by SUMOylation include DNA damage responses, cell cycle progression, or the control of gene expression. SUMOylation is reversible and commonly only a small fraction of a particular SUMO target is modified at a given time. Deconjugation of SUMO is catalyzed by a group of cysteine proteases termed SUMO proteases or SUMO isopeptidases. In human cells nine SUMO proteases, belonging to three separate families of cysteine proteases have been identified so far. The regulation and target specificity of individual SUMO proteases have not been dissected in detail, but the current view is that each protease controls the modification of subsets of proteins that are functionally and/or physically linked. Importantly, some SUMO proteases/isopeptidases not only function in deconjugation of SUMO from proteins, but also act in C-terminal processing of the SUMO precursors. Here we describe general methods for monitoring SUMO protease/isopeptidase activities in cell or tissue extracts.
Assuntos
Cisteína Proteases/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Carbono-Nitrogênio Liases/metabolismo , Ensaios Enzimáticos/métodos , Humanos , Modelos Moleculares , Especificidade por Substrato , SumoilaçãoRESUMO
MicroRNAs (miRNAs) play important roles in regulating plant responses to various environmental stresses. In our study, overexpression of miR1916 in tomato (OE-1) reduced its tolerance to drought. The miR1916-silenced transgenic plants (ST-1 and Anti-7) significantly increased resistance to drought stress. The transgenic tobacco plants also have a similar result in displaying the tolerance of drought. Physiological analysis revealed that miR1916 affected the osmoregulation and reactive oxygen species (ROS) accumulation. In addition, transcript levels of the miR1916 target genes, histone deacetylases (HDAC) and strictosidine synthase (STR), decreased in miR1916-overexpressing transgenic tobacco plants. Our results suggested that miR1916 is a passive regulator in the plant resistance to drought stress and has potentially impacting on abiotic stress responses in Solanaceae.
Assuntos
Adaptação Fisiológica/genética , Secas , MicroRNAs/farmacologia , Nicotiana/fisiologia , Solanum lycopersicum/fisiologia , Carbono-Nitrogênio Liases/genética , Histona Desacetilases/genética , Osmorregulação/efeitos dos fármacos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismoRESUMO
7-Carboxy-7-deazaguanine synthase, QueE, catalyzes the radical mediated ring contraction of 6-carboxy-5,6,7,8-tetrahydropterin, forming the characteristic pyrrolopyrimidine core of all 7-deazaguanine natural products. QueE is a member of the S-adenosyl-L-methionine (AdoMet) radical enzyme superfamily, which harnesses the reactivity of radical intermediates to perform challenging chemical reactions. Members of the AdoMet radical enzyme superfamily utilize a canonical binding motif, a CX3 CXÏC motif, to bind a [4Fe-4S] cluster, and a partial (ß/α)6 TIM barrel fold for the arrangement of AdoMet and substrates for catalysis. Although variations to both the cluster-binding motif and the core fold have been observed, visualization of drastic variations in the structure of QueE from Burkholderia multivorans called into question whether a re-haul of the defining characteristics of this superfamily was in order. Surprisingly, the structure of QueE from Bacillus subtilis revealed an architecture more reminiscent of the classical AdoMet radical enzyme. With these two QueE structures revealing varying degrees of alterations to the classical AdoMet fold, a new question arises: what is the purpose of these alterations? Here, we present the structure of a third QueE enzyme from Escherichia coli, which establishes the middle range of the spectrum of variation observed in these homologs. With these three homologs, we compare and contrast the structural architecture and make hypotheses about the role of these structural variations in binding and recognizing the biological reductant, flavodoxin. Broader impact statement: We know more about how enzymes are tailored for catalytic activity than about how enzymes are tailored to react with a physiological reductant. Here, we consider structural differences between three 7-carboxy-7-deazaguanine synthases and how these differences may be related to the interaction between these enzymes and their biological reductant, flavodoxin.
Assuntos
Carbono-Nitrogênio Liases/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas Ferro-Enxofre/química , Motivos de Aminoácidos , Cristalografia por Raios X , Flavodoxina , Domínios Proteicos , Especificidade por SubstratoRESUMO
We have previously identified that PPPDE1 is a deubiquitinase (DUB) belonging to a cysteine isopeptidase family. Here we sought to explore the biological significance of PPPDE1 in hepatocellular carcinoma and its underlying molecular mechanism. In the present study, we found that amplification and overexpression of PPPDE1 were associated with poor prognosis in hepatocellular carcinoma (HCC). We also demonstrated that knocking down of PPPDE1 could significantly block the clonal growth and tumorigenicity of human HCC cells, which revealed a critical role for PPPDE1 in HCC development. Furthermore, we proved that PPPDE1 is a key modulator of p53 protein level and its down stream apoptosis pathway. Taken together, these results suggested that PPPDE1 is a putative HCC driver gene and extensive studies should be conducted in the future to investigate the role of PPPDE1 in HCC and other tumors.
Assuntos
Apoptose/genética , Carbono-Nitrogênio Liases/fisiologia , Carcinoma Hepatocelular/patologia , Proliferação de Células/genética , Neoplasias Hepáticas/patologia , Proteína Supressora de Tumor p53/genética , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Estudos de Coortes , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This study aimed to investigate the effects of desumoylating isopeptidase 2 (DESI2) on tumor cell proliferation, apoptosis and invasion of pancreatic cancer, and to assess the signaling pathway involved. Overexpression and silence of DESI2 were designed and the experiments were divided into 5 groups: a normal control group, an interference control group (shRNA-NC); an interference group (sh-DESI2); an overexpression control group (NC), an overexpression group (DESI2). Quantitative real time polymerase chain reaction (qRT-PCR) was used to screen the appropriate interference sequence. The silencing and overexpression of DESI2 were confirmed by qRT-PCR and western blotting. Cell cycling, apoptosis, invasion, and the expression of phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway and caspase 3 were measured. Overexpression and silence of DESI2 were successfully designed in two pancreatic cancer cells, and the interference effect of sh-DESI2-3 showed the best silencing effects. The biological activities of DESI2 were detected in both ASPC-1 and PANCE-1 cells. Our results showed that cell proliferation was significantly increased in the sh-DESI2 group, while decreased in DESI2 group compared with the control group in both cell lines. In ASPC-1 cells, the events in G1 phase decreased and in S phase increased obviously in the sh-DESI2 group, compared with control group. An opposite result was found when DESI2 was overexpressed. In PANCE-1 cells, the events in G2 phase were higher in the sh-DESI2 group, while in the DESI2 group was significantly lower than that in control group. In ASPC-1 and PANCE-1 cells, sh-DESI2 group showed decreased apoptosis, increased cell invasion and increased expression of AKT, p-Akt, PI3K, p-PI3K, p-mTOR and mTOR and decreased caspase 3 expression compared with the control group, while overexpression of DESI2 leaded to increased apoptosis, decreased cell invasion and reduced expression of AKT, p-Akt, PI3K, p-PI3K, p-mTOR and mTOR and increased expression of caspase 3. DESI2 regulates the proliferation and apoptosis of pancreatic cancer cells through PI3K/AKT/mTOR signaling pathway.
Assuntos
Carbono-Nitrogênio Liases/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais/fisiologia , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
7-Carboxy-7-deazaguanine (CDG) is a common intermediate in the biosynthesis of 7-deazapurine-containing natural products. The biosynthesis of CDG from GTP requires three enzymes: GTP cyclohydrolase I, 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) synthase, and CDG synthase (QueE). QueE is a member of the radical S-adenosyl-l-methionine (SAM) superfamily and catalyzes the SAM-dependent radical-mediated ring contraction of CPH4 to generate CDG. This chapter focuses on methods to reconstitute the activity of QueE in vitro.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Produtos Biológicos/metabolismo , Carbono-Nitrogênio Liases/isolamento & purificação , Purinas/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Biocatálise , Carbono-Nitrogênio Liases/metabolismo , Radicais Livres/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismoRESUMO
DESI2 is a novel pro-apoptotic gene. We previously reported that DESI2 overexpression induces S phase arrest and apoptosis by activating checkpoint kinases. This work was to test whether the combination of endostatin, an endogenous antiangiogenic inhibitor, with DESI2 could improve the therapy efficacy in vitro and in vivo. The recombinant plasmid co-expressing DESI2 and endostatin was encapsulated with DOTAP/Cholesterol cationic liposome. Mice bearing CT26 colon carcinoma and LL2 lung cancer were treated with the DNA-liposome complex. We found that, in vitro, the combination of DESI2 and endostatin more efficiently inhibited proliferation of CT26, LL2, HCT116 and A549 cancer cells via apoptosis, as assessed by MTT assay, colony-formation assays, flow cytometric analysis, hoechst staining and activation of caspase-3, respectively. In addition, DESI2 overexpression caused up-regulation of RPS7, a substrate of DESI2 deubiquitination. Furthermore, siRNA targeting RPS7 partially abrogated, whereas RPS7 overexpression enhanced DESI2-induced inhibition of cell proliferation. Importantly, the combination also caused DNA lesions accumulation, which further promotes apoptosis. Mechanistic rationale suggested that endostatin first inhibits DNA-PKcs kinase, and partly abrogated DESI2-induced phosphorylation of DNA-PKcs, leading to increase of DNA damage, then contributes to DESI2-induced apoptosis. In vivo, the combined gene therapy more significantly inhibited tumor growth and efficiently prolonged the survival of tumor bearing mice than mono therapy. The improved antitumor effect was associated with inhibition of cell proliferation via apoptosis, as analyzed by TUNEL assay and PCNA immunostaining. The combination also inhibited angiogenesis, as assessed by alginate-encapsulated tumor cell assay and CD31 staining. Our data suggest that the combined gene therapy of DESI2 and endostatin can significantly enhance the antitumor activity as a DNA lesions accumulator, apoptosis inducer and angiogenesis inhibitor. The present study may provide a novel method for the treatment of cancer.
Assuntos
Carbono-Nitrogênio Liases/genética , Neoplasias do Colo/genética , Endostatinas/genética , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Neoplasias Pulmonares/genética , Plasmídeos/metabolismo , Células A549 , Inibidores da Angiogênese/química , Inibidores da Angiogênese/metabolismo , Animais , Apoptose/genética , Carbono-Nitrogênio Liases/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Proliferação de Células , Colesterol/química , Colesterol/metabolismo , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Fragmentação do DNA , Endostatinas/metabolismo , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/metabolismo , Feminino , Células HCT116 , Humanos , Lipossomos/administração & dosagem , Lipossomos/química , Lipossomos/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/administração & dosagem , Plasmídeos/química , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A subset of viral genes is required for the long-term latent infection of hematopoietic cells by human cytomegalovirus (HCMV). Here, we show that a latency-associated gene product (LUNA) promotes the disruption of cellular PML bodies during latency. Mutation and inhibitor studies reveal that LUNA encodes a deSUMOylase activity responsible for this disruption. Specifically, LUNA encodes a conserved Asp-Cys-Gly motif common to all deSUMOylases. Importantly, mutation of the putative catalytic cysteine is sufficient to reverse LUNA-mediated PML dispersal and markedly reduces the efficiency of viral reactivation. The depletion of PML from cells is sufficient to rescue the reactivation of the LUNA-deficient viruses, arguing that targeting PML is an important biological role of LUNA. Finally, we demonstrate that reactivation of naturally latent HCMV is blocked by deSUMOylase inhibitors. Thus, latent HCMV primes the cellular environment for efficient reactivation via the activity of a virally encoded deSUMOylase.
Assuntos
Citomegalovirus/fisiologia , Proteínas Virais/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Sequência de Aminoácidos , Antígenos CD34/metabolismo , Carbono-Nitrogênio Liases/química , Carbono-Nitrogênio Liases/genética , Domínio Catalítico , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Humanos , Corpos de Inclusão/metabolismo , Mutação/genética , Células THP-1RESUMO
Terpenoid indole alkaloid (TIA) biosynthetic pathway of Catharanthus roseus possesses the major attention in current metabolic engineering efforts being the sole source of highly expensive antineoplastic molecules vinblastine and vincristine. The entire TIA pathway is fairly known at biochemical and genetic levels except the pathway steps leading to biosynthesis of catharanthine and tabersonine. To increase the in-planta yield of these antineoplastic metabolites for the pharmaceutical and drug industry, extensive plant tissue culture-based studies were performed to provide alternative production systems. However, the strict spatiotemporal developmental regulation of TIA biosynthesis has restricted the utility of these cultures for large-scale production. Therefore, the present study was performed to enhance the metabolic flux of TIA pathway towards the biosynthesis of vinblastine by overexpressing two upstream TIA pathway genes, tryptophan decarboxylase (CrTDC) and strictosidine synthase (CrSTR), at whole plant levels in C. roseus. Whole plant transgenic of C. roseus was developed using Agrobacterium tumefaciens LBA1119 strain having CrTDC and CrSTR gene cassette. Developed transgenic lines demonstrated up to twofold enhanced total alkaloid production with maximum ninefold increase in vindoline and catharanthine, and fivefold increased vinblastine production. These lines recorded a maximum of 38-fold and 65-fold enhanced transcript levels of CrTDC and CrSTR genes, respectively.
Assuntos
Alcaloides/metabolismo , Descarboxilases de Aminoácido-L-Aromático/metabolismo , Carbono-Nitrogênio Liases/metabolismo , Catharanthus/metabolismo , Indóis/metabolismo , Terpenos/metabolismo , Catharanthus/enzimologia , Vimblastina/metabolismoRESUMO
Radical SAM enzymes use S-adenosyl-l-methionine as an oxidant to initiate radical-mediated transformations that would otherwise not be possible with Lewis acid/base chemistry alone. These reactions are either redox neutral or oxidative leading to certain expectations regarding the role of SAM as either a reusable cofactor or the ultimate electron acceptor during each turnover. However, these expectations are frequently not realized resulting in fundamental questions regarding the redox handling and movement of electrons associated with these biological catalysts. Herein we provide a focused perspective on several of these questions and associated hypotheses with an emphasis on recently discovered radical SAM enzymes.
Assuntos
Enzimas/química , Enzimas/metabolismo , S-Adenosilmetionina/metabolismo , Alquilação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carbono-Carbono Liases/química , Carbono-Carbono Liases/metabolismo , Carbono-Nitrogênio Liases/química , Carbono-Nitrogênio Liases/metabolismo , Catálise , OxirreduçãoRESUMO
Diaryldienone derivatives with accessible ß-carbons show strong anti-neoplastic properties, related to their ability to make covalent adducts with free thiols by Michael addition, and low toxicity in vivo. Accumulation of poly-ubiquitylated proteins, activation of the unfolded protein response (UPR) and induction of cell death are universal hallmarks of their activities. These compounds have been characterized as inhibitors of isopeptidases, a family of cysteine-proteases, which de-conjugate ubiquitin and ubiquitin-like proteins from their targets. However, it is unclear whether they can also react with additional proteins. In this work, we utilized the biotin-conjugated diaryldienone-derivative named 2c, as a bait to purify novel cellular targets of these small molecules. Proteomic analyses have unveiled that, in addition to isopeptidases, these inhibitors can form stable covalent adducts with different intracellular proteins, thus potentially impacting on multiple functions of the cells, from cytoskeletal organization to metabolism. These widespread activities can explain the ability of diaryldienone derivatives to efficiently trigger different cell death pathways.
Assuntos
Carbono-Nitrogênio Liases/antagonistas & inibidores , Cicloexanonas/metabolismo , Proteômica/métodos , HumanosRESUMO
Monoterpenoid indole alkaloids (MIAs) comprise an important class of molecules for drug discovery, and they have variant carbon skeletons with prominent bioactivities. For instance, in spite of limitations to their use, camptothecins are the only clinically approved topoisomerase I (Top1) inhibitors. The enzyme strictosidine synthase, which is key for MIA biosynthesis, was applied to the enantioselective preparation of three N-substituted (S)-3,14,18,19-tetrahydroangustine (THA) derivatives. These non-camptothecin MIAs were shown to have moderate in vitro HepG2 cytotoxicity and Top1 inhibition activities. The (S)-configured MIAs had stronger cytotoxicity and Top1 inhibition than their chemically synthesized (R)-enantiomers, which aligned with the results of molecular dynamics simulations. A series of N-substituted (S)-THAs were then chemoenzymatically synthesized to investigate structure-activity relationships. The most active analogue observed was the N-(2-Cl benzoyl)-substituted derivative (7i). Insight into the binding mode of 7i and a Top1-DNA covalent complex was investigated by molecular dynamics simulations, which will facilitate future efforts to optimize the Top1 inhibitory activities of non-camptothecin MIAs.