Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.380
Filtrar
1.
Life Sci Alliance ; 5(12)2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35926874

RESUMO

The HECT-type UPL3 ligase plays critical roles in plant development and stress protection, but understanding of its regulation remains limited. Here, the multi-omics analyses of ubiquitinated proteins in upl3 mutants were performed. A landscape of UPL3-dependent ubiquitinated proteins is constructed: Preferential ubiquitination of proteins related to carbon fixation represented the largest set of proteins with increased ubiquitination in the upl3 plant, including most of carbohydrate metabolic enzymes, BRM, and variant histone, whereas a small set of proteins with reduced ubiquitination caused by the upl3 mutation were linked to cysteine/methionine synthesis, as well as hexokinase 1 (HXK1) and phosphoenolpyruvate carboxylase 2 (PPC2). Notably, ubiquitin hydrolase 12 (UBP12), BRM, HXK1, and PPC2 were identified as the UPL3-interacting partners in vivo and in vitro. Characterization of brm, upl3, ppc2, gin2, and ubp12 mutant plants and proteomic and transcriptomic analysis suggested that UPL3 fine-tunes carbohydrate metabolism, mediating cellular senescence by interacting with UBP12, BRM, HXK1, and PPC2. Our results highlight a regulatory pattern of UPL3 with UBP12 as a hub of regulator on proteolysis-independent regulation and proteolysis-dependent degradation.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Adenosina Trifosfatases/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Endopeptidases/metabolismo , Ligases/genética , Ligases/metabolismo , Senescência Vegetal , Proteômica , Proteínas Ubiquitinadas/metabolismo
2.
Acta Biochim Biophys Sin (Shanghai) ; 54(7): 1-9, 2022 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-35880565

RESUMO

Intervertebral disc degeneration (IDD) is featured as enhanced catabolism of extracellular matrix (ECM) in the nucleus pulposus (NP), in which tumor necrosis factor-alpha (TNF-α)-related cell senescence is involved. Chromobox homolog protein 4 (CBX4) exhibits anti-inflammatory effects and shows promising therapeutic potential. Thus, in the present study, we explore the role of CBX4 in IDD. Immunohistochemistry staining reveals that CBX4 expression is decreased in severe degenerative NP tissues compared to mild degenerative tissues, and real-time PCR and western blot analysis results show that CBX4 expression is downregulated under TNF-α stimulation in NP cells. siRNA and adenoviruses are used to knockdown or overexpress CBX4, respectively. The results demonstrate that CBX4 knockdown augments the catabolism of ECM in human NP cells, while CBX4 overexpression in rat NP cells restores the ECM degradation induced by TNF-α, as illustrated by immunofluorescence and western blot analysis. In addition, transcriptome sequencing results reveal the regulatory effect of CBX4 on the cell cycle, and further western blot analysis and senescence-associated ß-galactosidase staining assay indicate that CBX4 overexpression alleviates cell senescence in the presence of TNF-α. Moreover, the phosphorylation of p65, which indicates the activation of NF-κB signaling, is measured by western blot analysis and immunofluorescence assay, and the results reveal that CBX4 overexpression reduces the TNF-α-induced increase in the p-p65/p65 ratio. In addition, the effect of CBX4 overexpression in NP cells is suppressed by NF-κB agonist. In summary, our results indicate that CBX4 overexpression can suppress TNF-α-induced matrix catabolism and cell senescence in the NP by inhibiting NF-κB activation. This study may provide new approaches for preventing and treating IDD.


Assuntos
Degeneração do Disco Intervertebral , Ligases/metabolismo , Núcleo Pulposo , Complexo Repressor Polycomb 1/metabolismo , Animais , Humanos , Degeneração do Disco Intervertebral/patologia , NF-kappa B/metabolismo , Núcleo Pulposo/patologia , Proteínas do Grupo Polycomb/metabolismo , Ratos , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
4.
Appl Microbiol Biotechnol ; 106(13-16): 5153-5165, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35821431

RESUMO

As a novel protein post-translational modification (PTM), lysine succinylation is widely involved in metabolism regulation by altering the activity of catalytic enzymes. Inactivating succinyl-CoA synthetase in Saccharopolyspora erythraea HL3168 E3 was proved significantly inducing the global protein hypersuccinylation. To investigate the effects, succinylome of the mutant strain E3ΔsucC was identified by using a high-resolution mass spectrometry-based proteomics approach. PTMomics analyses suggested the important roles of succinylation on protein biosynthesis, carbon metabolism, and antibiotics biosynthesis in S. erythraea. Enzymatic experiments in vivo and in vitro were further conducted to determine the succinylation regulation in the TCA cycle. We found out that the activity of aconitase (SACE_3811) was significantly inhibited by succinylation in E3ΔsucC, which probably led to the extracellular accumulation of pyruvate and citrate during the fermentation. Enzyme structural analyses indicated that the succinylation of K278 and K373, conservative lysine residues locating around the protein binding pocket, possibly affects the activity of aconitase. To alleviate the metabolism changes caused by succinyl-CoA synthetase inactivation and protein hypersuccinylation, CRISPR interference (CRISPRi) was applied to mildly downregulate the transcription level of gene sucC in E3. The erythromycin titer of the CRISPRi mutant E3-sucC-sg1 was increased by 54.7% compared with E3, which was 1200.5 mg/L. Taken together, this work not only expands our knowledge of succinylation regulation in the TCA cycle, but also validates that CRISPRi is an efficient strategy on the metabolic engineering of S. erythraea. KEY POINTS: • We reported the first systematic profiling of the S. erythraea succinylome. • We found that the succinylation regulation on the activity of aconitase. • We enhanced the production of erythromycin by using CRISPRi to regulate the transcription of gene sucC.


Assuntos
Eritromicina , Saccharopolyspora , Aconitato Hidratase/genética , Aconitato Hidratase/metabolismo , Acil Coenzima A , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ligases/genética , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Saccharopolyspora/genética , Saccharopolyspora/metabolismo
5.
Molecules ; 27(14)2022 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-35889224

RESUMO

The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an l-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides.


Assuntos
Acetato Quinase , Dipeptídeos , Trifosfato de Adenosina , Biocatálise , Dipeptídeos/química , Ligases/metabolismo
6.
Sci Rep ; 12(1): 12484, 2022 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-35864126

RESUMO

Enzymes with dedicated cofactor preference are essential for advanced biocatalysis and biomanufacturing, especially when employing nonnatural nicotinamide cofactors in redox reactions. However, directed evolution of an enzyme to switch its cofactor preference is often hindered by the lack of efficient and affordable method for screening as the cofactor per se or the substrate can be prohibitively expensive. Here, we developed a growth-based selection platform to identify nonnatural cofactor-dependent oxidoreductase mutants. The growth of bacteria depended on the nicotinamide cytosine dinucleotide (NCD) mediated conversion of non-metabolizable phosphite into phosphate. The strain BW14329 lacking the ability to oxidize phosphite was suitable as host, and NCD-dependent phosphite dehydrogenase (Pdh*) is essential to the selection platform. Previously confirmed NCD synthetase with NCD synthesis capacity and NCD-dependent malic enzyme were successfully identified by using the platform. The feasibility of this strategy was successfully demonstrated using derived NCD-active malic enzyme as well as for the directed evolution of NCD synthetase in Escherichia coli. A phosphite-based screening platform was built for identification of enzymes favoring nonnatural cofactor NCD. In the future, once Pdh variants favoring other biomimetic or nonnatural cofactors are available this selection platform may be readily redesigned to attain new enzyme variants with anticipated cofactor preference, providing opportunities to further expand the chemical space of redox cofactors in chemical biology and synthetic biology.


Assuntos
Enzimas , Fosfitos , Enzimas/química , Escherichia coli/genética , Ligases , NAD , Niacinamida/química , Oxirredução
7.
Int J Mol Sci ; 23(14)2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35887358

RESUMO

SUMOylation is a post-translational modification that has emerged in recent decades as a mechanism involved in controlling diverse physiological processes and that is essential in vertebrates. The SUMO pathway is regulated by several enzymes, proteases and ligases being the main actors involved in the control of sumoylation of specific targets. Dysregulation of the expression, localization and function of these enzymes produces physiological changes that can lead to the appearance of different types of cancer, depending on the enzymes and target proteins involved. Among the most studied proteases and ligases, those of the SENP and PIAS families stand out, respectively. While the proteases involved in this pathway have specific SUMO activity, the ligases may have additional functions unrelated to sumoylation, which makes it more difficult to study their SUMO-associated role in cancer process. In this review we update the knowledge and advances in relation to the impact of dysregulation of SUMO proteases and ligases in cancer initiation and progression.


Assuntos
Ligases , Neoplasias , Animais , Endopeptidases/metabolismo , Humanos , Ligases/metabolismo , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo
8.
EMBO Rep ; 23(7): e53855, 2022 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-35642598

RESUMO

The retrovirus HIV-1 integrates into the host genome and establishes a latent viral reservoir that escapes immune surveillance. Molecular mechanisms of HIV-1 latency have been studied extensively to achieve a cure for the acquired immunodeficiency syndrome (AIDS). Latency-reversing agents (LRAs) have been developed to reactivate and eliminate the latent reservoir by the immune system. To develop more promising LRAs, it is essential to evaluate new therapeutic targets. Here, we find that CBX4, a component of the Polycomb Repressive Complex 1 (PRC1), contributes to HIV-1 latency in seven latency models and primary CD4+ T cells. CBX4 forms nuclear bodies with liquid-liquid phase separation (LLPS) properties on the HIV-1 long terminal repeat (LTR) and recruits EZH2, the catalytic subunit of PRC2. CBX4 SUMOylates EZH2 utilizing its SUMO E3 ligase activity, thereby enhancing the H3K27 methyltransferase activity of EZH2. Our results indicate that CBX4 acts as a bridge between the repressor complexes PRC1 and PRC2 that act synergistically to maintain HIV-1 latency. Dissolution of phase-separated CBX4 bodies could be a potential intervention to reactivate latent HIV-1.


Assuntos
Infecções por HIV , HIV-1 , Linfócitos T CD4-Positivos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , HIV-1/genética , Humanos , Ligases , Corpos Nucleares , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb/genética , Latência Viral/genética
9.
Microb Cell Fact ; 21(1): 106, 2022 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-35643562

RESUMO

BACKGROUND: Capsaicinoids are produced by plants in the Capsicum genus and are the main reason for the pungency of chili pepper fruits. They are strong agonists of TRPV1 (the transient receptor potential cation channel subfamily V member 1) and used as active ingredients in pharmaceuticals for the treatment of pain. The use of bioengineered microorganisms in a fermentation process may be an efficient route for their preparation, as well as for the discovery of (bio-)synthetic capsaicinoids with improved or novel bioactivities. RESULTS: Saccharomyces cerevisiae was engineered to over-express a selection of amide-forming N-acyltransferase and CoA-ligase enzyme cascades using a combinatorial gene assembly method, and was screened for nonivamide production from supplemented vanillylamine and nonanoic acid. Data from this work demonstrate that Tyramine N-hydroxycinnamoyl transferase from Capsicum annuum (CaAT) was most efficient for nonivamide formation in yeast, outcompeting the other candidates including AT3 (Pun1) from Capsicum spp. The CoA-ligase partner with highest activity from the ones evaluated here were from Petunia hybrida (PhCL) and Spingomonas sp. Ibu-2 (IpfF). A yeast strain expressing CaAT and IpfF produced 10.6 mg L-1 nonivamide in a controlled bioreactor setup, demonstrating nonivamide biosynthesis by S. cerevisiae for the first time. CONCLUSIONS: Baker's yeast was engineered for production of nonivamide as a model capsaicinoid, by expressing N-acyltransferases and CoA-ligases of plant and bacterial origin. The constructed yeast platform holds potential for in vivo biocatalytic formation of capsaicinoids and could be a useful tool for the discovery of novel drugs.


Assuntos
Capsicum , Saccharomyces cerevisiae , Aciltransferases/genética , Capsaicina/análogos & derivados , Capsicum/genética , Coenzima A , Frutas , Ligases , Saccharomyces cerevisiae/genética
10.
Int J Mol Sci ; 23(11)2022 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-35682945

RESUMO

E3 ligases promote protein ubiquitination and degradation, which regulate every aspect of eukaryotic life. The Ariadne (ARI) proteins of RBR (ring between ring fingers) protein subfamily has been discovered as a group of potential E3 ubiquitin ligases. Only a few available research studies show their role in plant adaptations processes against the external environment. Presently, the functions of ARI proteins are largely unknown in plants. Therefore, in this study, we performed genome-wide analysis to identify the ARI gene family and explore their potential importance in B. napus. A total of 39 ARI genes were identified in the B. napus genome and were classified into three subfamilies (A, B and C) based on phylogenetic analysis. The protein-protein interaction networks and enrichment analysis indicated that BnARI genes could be involved in endoreduplication, DNA repair, proteasome assembly, ubiquitination, protein kinase activity and stress adaptation. The transcriptome data analysis in various tissues provided us an indication of some BnARI genes' functional importance in tissue development. We also identified potential BnARI genes that were significantly responsive towards the abiotic stresses. Furthermore, eight BnARI genes were identified as candidate genes for multiple agronomic traits through association mapping analysis in B. napus; among them, BnaA02g12100D, which is the ortholog of AtARI8, was significantly associated with ten agronomic traits. This study provided useful information on BnARI genes, which could aid targeted functional research and genetic improvement for breeding in B. napus.


Assuntos
Brassica napus , Brassica napus/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Ligases/metabolismo , Filogenia , Melhoramento Vegetal
11.
J Am Chem Soc ; 144(23): 10230-10240, 2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35647706

RESUMO

Lanthipeptide synthetases construct macrocyclic peptide natural products by catalyzing an iterative cascade of post-translational modifications. Class II lanthipeptide synthetases (LanM enzymes) catalyze multiple rounds of peptide dehydration and thioether macrocycle formation in a manner that guides precursor peptide maturation to the biologically active final product with high fidelity. The mechanistic details underlying the contradictory phenomena of substrate flexibility coupled with high biosynthetic fidelity have proven challenging to illuminate. In this work, we employ mass spectrometry to investigate how the structure of a maturing precursor lanthipeptide (HalA2) influences the local and global structure of its cognate lanthipeptide synthetase (HalM2). Using enzymatically synthesized HalA2 peptides that contain sets of native thioether macrocycles, we employ ion mobility mass spectrometry (IM-MS) to show that HalA2 macrocyclization alters the conformational landscape of the HalM2 enzyme in a systematic manner. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies show that local HalM2 structural dynamics also change in response to HalA2 post-translational modification. Notably, deuterium uptake in a critical HalM2 α-helical region depends on the number of thioether macrocycles present in the HalA2 core peptide. Binding of the isolated leader and core peptide portions of the modular HalA2 precursor led to a synergistic structuring of this α-helical region, providing evidence for distinct leader and core peptide binding sites that independently alter the dynamics of this functionally critical α-helix. The data support a mechanistic model where the sequential post-translational modification of HalA2 alters the conformational dynamics of HalM2 in regions of the enzyme that are known to be functionally critical.


Assuntos
Bacteriocinas , Ligases , Bacteriocinas/química , Deutério , Ligases/metabolismo , Peptídeos , Sulfetos
12.
Plant Sci ; 322: 111357, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35718335

RESUMO

Sumoylation is a posttranslational modification (PTM) in which SUMO (small ubiquitin-like modifier) is covalently conjugated to protein substrates via a range of enzymes. SUMO E3 ligase SIZ1 is involved in mediating several essential or nonessential element-responsive SUMO conjugations in Arabidopsis. However, whether SIZ1 is involved in the cadmium (Cd) response remains to be identified. In this study, we found that SIZ1 positively regulates plant Cd tolerance. The loss-of-function siz1-2 mutant exhibited impaired resistance to Cd exposure and accumulated more reactive oxygen species (ROS). Moreover, the transcription of GSH1, GSH2, PCS1, and PCS2 was suppressed while the accumulation of Cd was enhanced in the siz1-2 mutant under Cd exposure. Further analysis revealed that the higher Cd sensitivity of the siz1-2 mutant was partially rescued by the overexpression of GSH1. Consistently, Cd stress stimulated the accumulation of SUMO1 conjugates in wild-type plants but not in the siz1-2 mutant. Together, our results demonstrate that Cd-induced SIZ1 activates GSH- and PC synthesis-related gene expression to increase the synthesis of GSH- and PCs, thereby leading to higher Cd tolerance in plants.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cádmio/metabolismo , Cádmio/toxicidade , Regulação da Expressão Gênica de Plantas , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Ligases/genética , Ligases/metabolismo , Fitoquelatinas/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
13.
Methods Mol Biol ; 2530: 177-193, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35761050

RESUMO

Peptidyl Asx-specific ligases (PALs) effect peptide ligation by catalyzing transpeptidation reactions at Asn/Asp-peptide bonds. Owing to their high efficiency and mild aqueous reaction conditions, these ligases have emerged as powerful biotechnological tools for protein manipulation in recent years. PALs are enzymes of the asparaginyl endopeptidase (AEP) superfamily but have predominant transpeptidase activity as opposed to typical AEPs which are predominantly hydrolases. Butelase-1 and VyPAL2, two PALs discovered by our teams, have been used successfully in a wide range of applications, including macrocyclization of synthetic peptides and recombinant proteins, protein N- or C-terminal modification, and cell-surface labeling. As shown in numerous reports, PAL-mediated ligation is highly efficient at Asn junctions. Although considerably less efficient, Asp-specific ligation has also been shown to be practically useful under suitable conditions. Herein, we describe the methods of using VyPAL2 for protein macrocyclization and labeling at an Asp residue as well as for protein dual labeling through orthogonal Asp- and Asn-directed ligations. We also describe a method for cell-surface protein modification using butelase-1, demonstrating its advantageous features over previous methods.


Assuntos
Ligases , Proteínas de Plantas , Ligases/química , Peptídeos/química , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo
14.
Biomed Pharmacother ; 152: 113199, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35653888

RESUMO

Sepsis-induced cardiac dysfunction is a leading cause of mortality in intensive care units. However, the molecular mechanisms underlying septic cardiomyopathy remain elusive. Irisin is a cleaved product of fibronectin type III domain-containing protein 5 (FNDC5) that protects the heart from ischemia/reperfusion injury through upregulation of mitochondrial ubiquitin ligase (MITOL). Gasdermin D (GSDMD)-dependent pyroptosis plays a pivotal role in septic cardiomyopathy by regulating mitochondrial homeostasis. However, whether irisin can regulate MITOL to inhibit GSDMD-dependent pyroptosis in septic cardiomyopathy is yet to be investigated. Thus, this study was designed to explore the role of irisin in septic cardiomyopathy and its underlying molecular mechanisms. Our results demonstrate that irisin improves cardiac function against sepsis-induced cardiac dysfunction by reducing cardiac inflammation and myocardial pyroptosis. Using MITOL siRNA in vitro, the results revealed that the protective role of irisin against lipopolysaccharide (LPS)-induced cell injury was mediated by MITOL activation and the resulting inhibition of GSDMD-dependent pyroptosis. Moreover, irisin alleviated LPS-induced H9c2 cell injury by suppressing IL-1ß expression and reducing serum LDH and CK-MB concentrations in a MITOL/GSDMD-dependent manner. Collectively, our data suggest that irisin treatment ameliorates cardiac dysfunction in septic cardiomyopathy by activating MITOL and inhibiting GSDMD-dependent pyroptosis. These findings highlight the clinical relevance and therapeutic potential of irisin and MITOL for the management of sepsis-induced cardiac dysfunction.


Assuntos
Cardiomiopatias , Cardiopatias , Sepse , Cardiomiopatias/etiologia , Fibronectinas , Humanos , Inflamação , Ligases , Lipopolissacarídeos/metabolismo , Piroptose/fisiologia , Sepse/complicações , Sepse/metabolismo , Ubiquitinas
15.
Med Oncol ; 39(9): 125, 2022 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-35716322

RESUMO

Dysfunction at any regulatory point along the apoptotic signaling pathway is closely related to many diseases including cancers. The apoptotic protein expression level is an important cause of cancer-related death, and the correct degradation of apoptotic proteins is involved in tumor development. Therefore, understanding of a regulatory point that underlying cancer-related death may help the development of new strategies to overcome the clinical challenges. Here, proteasome inhibitor Bortezomib and calpain inhibitor ALLN were examined on protein levels of caspase-3, caspase-9, XIAP, and E3-ligase PARC in HEK293T cells overexpressing XIAP and caspase-9. ATP depletion and caspase-3 activation were as a consequence of Bortezomib and ALLN function. Higher numbers of PI-stained cells provided evidence of cell death by both inhibitors. Western blotting analysis showed that both ALLN and Bortezomib equally inhibited degradation of XIAP, but only ALLN was effective at inhibiting caspase proteolytic degradation. Moreover, treatment of cells with both types of inhibitors significantly increased the level of E3-ligase PARC. Our findings showed that inhibition of proteasome and calpains enhanced the level of anti-apoptotic, XIAP and PARC, and pro-apoptotic, caspase-9 and 3 proteins, which totally promote cell death significantly.


Assuntos
Neoplasias , Complexo de Endopeptidases do Proteassoma , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/farmacologia , Bortezomib/farmacologia , Calpaína/metabolismo , Calpaína/farmacologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Caspase 9/farmacologia , Morte Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Ligases/metabolismo , Ligases/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/farmacologia
16.
Int J Mol Sci ; 23(11)2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35682582

RESUMO

As a common abiotic stress, drought severely impairs the growth, development, and even survival of plants. Here we report a transcription factor, Caragana korshinskii REVOLUTA(CkREV), which can bidirectionally regulate the expression of the critical enzyme gene CkYUC5 in auxin synthesis according to external environment changes, so as to control the biosynthesis of auxin and further enhance the drought resistance of plants. Quantitative analysis reveals that the expression level of both CkYUC5 and AtYUC5 is down-regulated after C. korshinskii and Arabidopsis thaliana are exposed to drought. Functional verification of CkREV reveals that CkREV up-regulates the expression of AtYUC5 in transgenic A. thaliana under common conditions, while down-regulating it under drought conditions. Meanwhile, the expression of CkYUC5 is also down-regulated in C. korshinskii leaves instantaneously overexpressing CkREV. We apply a dual-luciferase reporter system to discover that CkREV can bind to the promoter of CkYUC5 to regulate its expression, which is further proved by EMSA and Y1H esxperiments. Functional verification of CkREV in C. korshinskii and transgenic A. thaliana shows that CkREV can regulate the expression of CkYUC5 and AtYUC5 in a contrary way, maintaining the equilibrium of plants between growth and drought resisting. CkREV can positively regulate the expression of CkYUC5 to promote auxin synthesis in favor of growth under normal development. However, CkREV can also respond to external signals and negatively regulate the expression of CkYUC5, which inhibits auxin synthesis in order to reduce growth rate, lower water demands, and eventually improve the drought resistance of plants.


Assuntos
Arabidopsis , Caragana , Arabidopsis/genética , Arabidopsis/metabolismo , Caragana/genética , Secas , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Ligases/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Estresse Fisiológico/genética
17.
J Am Chem Soc ; 144(22): 9938-9948, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35617676

RESUMO

Phosphonate natural products are potent inhibitors of cellular metabolism with an established record of commercialization in medicine and biotechnology. Although genome mining has emerged as an accelerated method for the discovery of new phosphonates, a robust framework of their metabolism is needed to identify the pathways most likely to yield compounds with desired activities. Here we expand our understanding of these natural products by reporting the complete biosynthetic pathway for valinophos, a phosphonopeptide natural product containing the unusual (R)-2,3-dihydroxypropylphosphonate (DHPPA) scaffold. The pathway was defined by several enzymatic transformations and intermediates previously unknown to phosphonate natural products. A dedicated dehydrogenase served as a new phosphoenolpyruvate mutase coupling enzyme. Notably, its reduction of phosphonopyruvate to phosphonolactate defined a new early branchpoint in phosphonate biosynthesis. Functionally interconnected kinase and reductase enzymes catalyzed reactions reminiscent of glycolysis and arginine biosynthesis to produce a transient, but essential, phosphonolactaldehyde intermediate. We demonstrate esterification of l-valine onto DHPPA as a new biochemical activity for ATP-Grasp ligase enzymes. Unexpectedly, a second amino acid ligase then adjoined additional amino acids at the valinyl moiety to produce a suite of DHPPA-dipeptides. The genes for DHPPA biosynthesis were discovered among genomes of bacteria from wide-ranging habitats, suggesting a wealth of unknown compounds that may originate from this core pathway. Our findings establish new biosynthetic principles for natural products and provide definition to unexplored avenues for bioactive phosphonate genome mining.


Assuntos
Produtos Biológicos , Organofosfonatos , Bactérias/metabolismo , Produtos Biológicos/química , Vias Biossintéticas , Ligases/metabolismo , Organofosfonatos/metabolismo
18.
Front Immunol ; 13: 866087, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35634293

RESUMO

Anti-synthetase syndrome (ASSD) is an autoimmune disease characterized by the presence of autoantibodies targeting one of several aminoacyl t-RNA synthetases (aaRSs) along with clinical features including interstitial lung disease, myositis, Raynaud's phenomenon, arthritis, mechanic's hands, and fever. The family of aaRSs consists of highly conserved cytoplasmic and mitochondrial enzymes, one for each amino acid, which are essential for the RNA translation machinery and protein synthesis. Along with their main functions, aaRSs are involved in the development of immune responses, regulation of transcription, and gene-specific silencing of translation. During the last decade, these proteins have been associated with cancer, neurological disorders, infectious responses, and autoimmune diseases including ASSD. To date, several aaRSs have been described to be possible autoantigens in different diseases. The most commonly described are histidyl (HisRS), threonyl (ThrRS), alanyl (AlaRS), glycyl (GlyRS), isoleucyl (IleRS), asparaginyl (AsnRS), phenylalanyl (PheRS), tyrosyl (TyrRS), lysyl (LysRS), glutaminyl (GlnRS), tryptophanyl (TrpRS), and seryl (SerRS) tRNA synthetases. Autoantibodies against the first eight autoantigens listed above have been associated with ASSD while the rest have been associated with other diseases. This review will address what is known about the function of the aaRSs with a focus on their autoantigenic properties. We will also describe the anti-aaRSs autoantibodies and their association to specific clinical manifestations, and discuss their potential contribution to the pathogenesis of ASSD.


Assuntos
Aminoacil-tRNA Sintetases , Ligases , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Autoanticorpos , Autoantígenos , RNA
19.
Biochem J ; 479(10): 1103-1119, 2022 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-35608338

RESUMO

Apoptosis, pyroptosis, and necroptosis are distinct forms of programmed cell death that eliminate infected, damaged, or obsolete cells. Many proteins that regulate or are a part of the cell death machinery undergo ubiquitination, a post-translational modification made by ubiquitin ligases that modulates protein abundance, localization, and/or activity. For example, some ubiquitin chains target proteins for degradation, while others function as scaffolds for the assembly of signaling complexes. Deubiquitinases (DUBs) are the proteases that counteract ubiquitin ligases by cleaving ubiquitin from their protein substrates. Here, we review the DUBs that have been found to suppress or promote apoptosis, pyroptosis, or necroptosis.


Assuntos
Inflamação , Ubiquitina , Morte Celular , Enzimas Desubiquitinantes/metabolismo , Humanos , Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação
20.
Front Endocrinol (Lausanne) ; 13: 882906, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35574028

RESUMO

Objective: We aimed to retrospectively collect pathologically identified pheochromocytoma and paraganglioma (PPGL) tumor tissues from our center and investigate the expression of apelin and succinyl-CoA synthetase subunit beta (SUCLG2), human epidermal growth factor receptor-2 (HER2 or ERBB-2), contactin 4 (CNTN4), chromogranin B (CHGB), and succinate dehydrogenase B (SDHB) in metastatic and non-metastatic PPGLs, for exploring their roles in the diagnosis of metastatic PPGLs. Methods: A total of 369 patients with pathologically and surgically confirmed PPGLs at Xiangya Hospital, Central South University, between June 2010 and June 2020 were retrospectively included. Sixty patients-12 patients with metastatic PPGLs and 48 patients with non-metastatic PPGLs-were selected through propensity score matching (1:4) to reduce the effect of PPGL type, sex, and age. We observed and quantified the expression of apelin, SDHB, CHGB, ERBB-2, CNTN4, and SUCLG2 in paraffin-embedded samples using immunohistochemical staining. Results: No significant differences were observed between the metastatic group and non-metastatic group with respect to the expression of CNTN4 and SUCLG2. The expression of apelin, SDHB, CHGB, and ERBB-2 was significantly different between the two groups. The expression of apelin, SDHB, and CHGB was significantly lower in the metastatic group than that in the non-metastatic group (P < 0.001). ERBB-2 expression was significantly higher in the metastatic group than in the non-metastatic group (P = 0.042). Kaplan-Meier analysis revealed that patients with negative expression of apelin, SDHB, and CHGB showed significantly lower metastasis-free survival than those with positive expression. Multivariate Cox analysis revealed that SDHB and CHGB levels were independently associated with metastasis-free survival. Conclusion: The expression levels of apelin, CHGB, SDHB, and ERBB-2 may be predictive biomarkers for the diagnosis of metastatic PPGLs. Patients with negative expression of apelin, CHGB, and SDHB should be subjected to frequent postoperative follow-up procedures.


Assuntos
Neoplasias das Glândulas Suprarrenais , Neuroblastoma , Paraganglioma , Feocromocitoma , Acil Coenzima A , Neoplasias das Glândulas Suprarrenais/diagnóstico , Neoplasias das Glândulas Suprarrenais/patologia , Apelina/metabolismo , Cromogranina B/metabolismo , Contactinas/metabolismo , Humanos , Ligases/metabolismo , Paraganglioma/patologia , Feocromocitoma/patologia , Estudos Retrospectivos , Succinato Desidrogenase/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...