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1.
J Vis Exp ; (167)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33491672

RESUMO

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials. One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Maleimidas/química , Compostos de Sulfidrila/química , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/química , Neoplasias/tratamento farmacológico , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Domínios Proteicos , Engenharia de Proteínas
2.
Nucleic Acids Res ; 49(1): 479-490, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330934

RESUMO

The mammalian Ate1 gene encodes an arginyl transferase enzyme with tumor suppressor function that depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. We report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1-R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore RNA structure revert splicing to that of the wild type. In the endogenous Ate1 pre-mRNA, blocking the competing base pairings by LNA/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of R2R5 interaction changes the ratio of MXE. That is, Ate1 splicing is controlled by two independent, dynamically interacting, and functionally distinct RNA structure modules. Exon 7a becomes more included in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted, indicating that exon 7a/7b ratio depends on co-transcriptional RNA folding. In sum, these results demonstrate that splicing is coordinated both in time and in space over very long distances, and that the interaction of these components is mediated by RNA structure.


Assuntos
Processamento Alternativo/genética , Aminoaciltransferases/genética , Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Dobramento de RNA , Precursores de RNA/genética , RNA Mensageiro/genética , Células A549 , Sequência de Bases , Linhagem Celular Tumoral , Sequência Conservada , Éxons/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/genética , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/genética , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Elongação da Transcrição Genética
3.
Chemosphere ; 262: 128361, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33182109

RESUMO

Although Cd is threatening to the environment, animal, and human, the eco-friendly approach to mitigate the Cd-toxicity in alfalfa was barely studied. Therefore, this study aims at elucidating the role of S, a crucial macroelement, in alleviating Cd toxicity in alfalfa plants. The supplementation of S in Cd-stressed alfalfa reversed the detrimental effect on plant biomass, chlorophyll synthesis, and protein concentration. Interestingly, S surplus restored the photosynthetic kinetics, such as Fv/Fm, Pi_ABS, and Mo values in leaves of Cd-stressed alfalfa. Further, Cd-induced adverse effect on membrane stability, cell viability, and redox status was restored due to S under Cd stress. The exogenous S not only increased S status and the expression of sulfate transporters (MsSULRT1;2 and MsSULTR1;3), but also decreased the Cd concentration in the shoot by retaining elevated Cd in root tissue. Further analysis revealed the upregulation of MsGS (glutathione synthetase) and MsPCS1 (phytochelatin synthase) genes along with the increased concentration of glutathione and phytochelatin, predominantly in roots subjected to S surplus under Cd stress. The subcellular Cd analysis showed elevated Cd in the cell wall but not in the vacuole. It suggests that S-induced elevated glutathione enables the phytochelatin to bind with excess Cd leading to subcellular sequestration in the cell wall of roots. Also, S stimulates the S-metabolites and GR enzyme that coordinately counteracts Cd-induced oxidative damage. These findings can be utilized to popularize the application of S and to perform breeding/transgenic experiments to develop Cd-free forage crops.


Assuntos
Cádmio/toxicidade , Glutationa/metabolismo , Medicago sativa/fisiologia , Fitoquelatinas/metabolismo , Poluentes do Solo/toxicidade , Enxofre/toxicidade , Aminoaciltransferases , Cádmio/metabolismo , Parede Celular/metabolismo , Medicago sativa/metabolismo , Oxirredução , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Poluentes do Solo/metabolismo , Enxofre/metabolismo
4.
Ecotoxicol Environ Saf ; 205: 111323, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32956864

RESUMO

Using an ideal parental line to breed hybrid rice with low cadmium (Cd) accumulation in grain is an environmental-friendly approach to reduce the risk of Cd contamination in field. A grain low-Cd rice line YaHui2816 has stably low Cd in grain but strong Cd accumulation in straw, revealing specific pattern of its straw-grain Cd remobilization is beneficial to effectively breed hybrid rice for safe production as well as phytoremediation. In this study, a pot experiment was conducted to investigate Cd partitioning of YaHui2816 at different stages by comparison with a common rice C268A. The Cd from lower nodes and leaves was restricted in nodeⅡ, this Cd retention led to weak Cd transport from straw to ear in YaHui2816. Cd concentration in nodeⅡ of YaHui2816 was 1.56-fold and 7.36-fold higher than C268A at filling and mature stages. Thus, glutathione (GSH) and phytochelatin (PC) concentration, PC synthase (OsPCS1), GSH S-Transferase (OsGST) and Cd-remobilization associated genes were measured at filling stage. OsHMA2 and OsCCX2 were up-regulated in nodeⅡ of YaHui2816, relative expression of them were significantly higher than C268A. PCs participated in Cd remobilization process, remarkable PC increase in nodeⅡ of YaHui2816 was found in response to Cd treatment under regulation of OsPCS1 and OsGST of which PC2 was dominant form. Taken as a whole, the Cd retention in nodeⅡ of YaHui2816 acts as a 'firewall' to restrain Cd transport to grain. This work provides more insight to understand possible function of alleles for reducing Cd concentration in grain as well as strengthening Cd accumulation in straw of YaHui2816.


Assuntos
Cádmio/metabolismo , Oryza/fisiologia , Poluentes do Solo/metabolismo , Aminoaciltransferases , Grão Comestível/química , Oryza/metabolismo , Fitoquelatinas/metabolismo , Folhas de Planta/metabolismo , Poluentes do Solo/análise
5.
Ecotoxicol Environ Saf ; 205: 111175, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32836161

RESUMO

Mangroves are susceptible to contamination due to their proximity to shores and human activities. Exposure to excessive trace metals can disturb their physiological functions and may eventually lead to death. Rhizophora mucronata is a common species growing in the mangrove forests of Thailand. Previous studies have shown that seedlings of R. mucronata are tolerant of trace metal and that they accumulate a large metal content in their root tissue. However, knowledge of their tolerance mechanisms is still lacking. To elicit the role of metal detoxification and sequestration by phytochelatins (PC) in the roots of R. mucronata seedlings, the impacts of Cu and Zn exposure were assessed on 1) physiological characteristics 2) the concentration of glutathione (GSH), a precursor of PC and 3) the level of the transcripts encoding phytochelatin synthase (PCS), the key enzyme for PC biosynthesis. Seedlings of R. mucronata were exposed to Cu and Zn in a hydroponic experiment (200 mg Cu or Zn/L in 1/4× Hoagland solution containing 8‰ NaCl, single addition). We found that both trace metals were largely accumulated in the roots. Only Cu-treated seedlings showed a decrease in the photosynthetic efficiency, in line with observed toxicity symptoms (i.e. bent stems and slight wilting of leaves). Metal accumulation, however, did not induce oxidative stress in the roots as indicated by similar level of total reactive species and lipid peroxidation across treatments. The GSH content in the roots exposed to Cu was significantly reduced while no change was observed in Zn-exposed roots. Coordinated semi-quantitative PCR and RT-qPCR revealed pcs down-regulation in Cu-treated roots, whereas Zn-treated roots showed a down-regulation on day 1 and a subsequent recovery on day 5. Failure of detoxification and sequestration of excess Cu due to GSH limitation and down-regulation of pcs may lead to the phytotoxic effects observed in Cu-treated plants. Our results suggest that both GSH and PC play an important role in trace metal tolerance in R. mucronata seedlings.


Assuntos
Aminoaciltransferases/genética , Cobre/toxicidade , Glutationa/metabolismo , Rhizophoraceae/efeitos dos fármacos , Oligoelementos/metabolismo , Zinco/toxicidade , Adaptação Fisiológica/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Rhizophoraceae/genética , Rhizophoraceae/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(20): 10778-10788, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32366662

RESUMO

The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal (Nt) residues. If a substrate bears, for example, Nt-Asn, its targeting involves deamidation of Nt-Asn, arginylation of resulting Nt-Asp, binding of resulting (conjugated) Nt-Arg to the UBR1-RAD6 E3-E2 ubiquitin ligase, ligase-mediated synthesis of a substrate-linked polyubiquitin chain, its capture by the proteasome, and substrate's degradation. We discovered that the human Nt-Asn-specific Nt-amidase NTAN1, Nt-Gln-specific Nt-amidase NTAQ1, arginyltransferase ATE1, and the ubiquitin ligase UBR1-UBE2A/B (or UBR2-UBE2A/B) form a complex in which NTAN1 Nt-amidase binds to NTAQ1, ATE1, and UBR1/UBR2. In addition, NTAQ1 Nt-amidase and ATE1 arginyltransferase also bind to UBR1/UBR2. In the yeast Saccharomyces cerevisiae, the Nt-amidase, arginyltransferase, and the double-E3 ubiquitin ligase UBR1-RAD6/UFD4-UBC4/5 are shown to form an analogous targeting complex. These complexes may enable substrate channeling, in which a substrate bearing, for example, Nt-Asn, would be captured by a complex-bound Nt-amidase, followed by sequential Nt modifications of the substrate and its polyubiquitylation at an internal Lys residue without substrate's dissociation into the bulk solution. At least in yeast, the UBR1/UFD4 ubiquitin ligase interacts with the 26S proteasome, suggesting an even larger Arg/N-degron-targeting complex that contains the proteasome as well. In addition, specific features of protein-sized Arg/N-degron substrates, including their partly sequential and partly nonsequential enzymatic modifications, led us to a verifiable concept termed "superchanneling." In superchanneling, the synthesis of a substrate-linked poly-Ub chain can occur not only after a substrate's sequential Nt modifications, but also before them, through a skipping of either some or all of these modifications within a targeting complex.


Assuntos
Proteólise , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação , Amidoidrolases/metabolismo , Aminoaciltransferases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
7.
Sci Rep ; 10(1): 8520, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444661

RESUMO

Sortase enzymes are attractive antivirulence drug targets that attach virulence factors to the surface of Staphylococcus aureus and other medically significant bacterial pathogens. Prior efforts to discover a useful sortase inhibitor have relied upon an in vitro activity assay in which the enzyme is removed from its native site on the bacterial surface and truncated to improve solubility. To discover inhibitors that are effective in inactivating sortases in vivo, we developed and implemented a novel cell-based screen using Actinomyces oris, a key colonizer in the development of oral biofilms. A. oris is unique because it exhibits sortase-dependent growth in cell culture, providing a robust phenotype for high throughput screening (HTS). Three molecules representing two unique scaffolds were discovered by HTS and disrupt surface protein display in intact cells and inhibit enzyme activity in vitro. This represents the first HTS for sortase inhibitors that relies on the simple metric of cellular growth and suggests that A. oris may be a useful platform for discovery efforts targeting sortase.


Assuntos
Actinomyces/crescimento & desenvolvimento , Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Biofilmes/crescimento & desenvolvimento , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Actinomyces/efeitos dos fármacos , Actinomyces/enzimologia , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Células Cultivadas
8.
PLoS One ; 15(4): e0226661, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240171

RESUMO

CD47 is an immune checkpoint protein that downregulates both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα. Biologics, including humanized CD47 monoclonal antibodies and decoy SIRPα receptors, that block the SIRPα-CD47 interaction, are currently being developed as cancer immunotherapy agents. However, adverse side effects and limited penetration of tumor tissue associated with their structure and large size may impede their clinical application. We recently developed a quantitative high throughput screening assay platform to identify small molecules that disrupt the binding of SIRPα and CD47 as an alternative approach to these protein-based therapeutics. Here, we report on the development and optimization of a cell-based binding assay to validate active small molecules from our biochemical screening effort. This assay has a low volume, high capacity homogenous format that relies on laser scanning cytometry (LSC) and associated techniques to enhance signal to noise measurement of cell surface binding. The LSC assay is specific, concentration dependent, and validated for the two major human SIRPα variants (V1 and V2), with results that parallel those of our biochemical data as well as published studies. We also utilized the LSC assay to confirm published studies showing that the inhibition of amino-terminal pyroglutamate formation on CD47 using the glutaminyl cyclase inhibitor SEN177 disrupts SIRPα binding. The SIRPα-CD47 interaction could be quantitatively measured in live and fixed tumor cells. Use of fixed cells reduces the burden of cell maintenance and provides stable cell standards to control for inter- and intra-assay variations. We also demonstrate the utility of the assay to characterize the activity of the first reported small molecule antagonists of the SIRPα-CD47 interaction. This assay will support the screening of thousands of compounds to identify or validate active small molecules as hits, develop structure activity relationships and assist in the optimization of hits to leads by a typical iterative medicinal chemistry campaign.


Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Antígenos de Diferenciação/genética , Antígeno CD47/genética , Neoplasias/tratamento farmacológico , Receptores Imunológicos/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Imunidade Adaptativa/genética , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/química , Antígenos de Diferenciação/química , Antígeno CD47/química , Desenvolvimento de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunoterapia/métodos , Células Jurkat , Citometria de Varredura a Laser , Ligantes , Oncologia/tendências , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Fagocitose/efeitos dos fármacos , Mapas de Interação de Proteínas/genética , Receptores Imunológicos/química , Bibliotecas de Moléculas Pequenas/química
9.
Ecotoxicol Environ Saf ; 194: 110402, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32151867

RESUMO

Sulfur (S) application in pakchoi (Brassica chinensis L.) cultivation is vital for reducing cadmium (Cd) accumulation in the plants. However, the mechanism of S application on Cd uptake and translocation in pakchoi is unclear. In this study, a hydroponic experiment was performed to investigate the effects of S application on Cd accumulation in pakchoi at one Cd concentration (50 µM, in comparison to the control condition, 0 µM) and three S levels (0, 2, 4 mM). The results showed that excessive S application (4 mM) reduced Cd accumulation and alleviated pakchoi growth inhibition caused by Cd stress in shoots and roots. With increased S application, the proportion of Cd in the vacuolar fraction and the proportion of NaCl-extractable Cd increased in roots. Additionally, S application increased the content of glutathione (GSH) and phytochelatins (PCs). The reduced Cd uptake and accumulation in pakchoi shoots could have been due to increased Cd chelation and vacuolar sequestration in roots. In addition, sufficient S application (2 mM) increased the expression of γ-glutamylcysteine synthetase (GSH1) and nicotinamide synthase (NAS) in roots, and excessive S application upregulated the expression of ATP sulfurylase (ATPS) and phytochelatin synthase (PCs). This study provides evidence for the mechanism of mitigating Cd toxicity in pakchoi and will be helpful for developing strategies to reduce Cd accumulation in the edible parts of pakchoi through S fertilizer application.


Assuntos
Brassica/efeitos dos fármacos , Cádmio/metabolismo , Poluentes do Solo/metabolismo , Sulfatos/farmacologia , Aminoaciltransferases/metabolismo , Transporte Biológico , Brassica/crescimento & desenvolvimento , Brassica/metabolismo , Cádmio/toxicidade , Fertilizantes/análise , Glutationa/metabolismo , Hidroponia , Modelos Teóricos , Fitoquelatinas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Poluentes do Solo/toxicidade , Sulfato Adenililtransferase/metabolismo , Sulfatos/metabolismo
10.
Chem Commun (Camb) ; 56(28): 3943-3946, 2020 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-32195505

RESUMO

Sortase is one of the most widely used enzymes for covalent protein conjugation that links protein and protein/small molecules together in a site-specific way. It typically recognizes the "GGG" and "LPXTG" peptide sequences and conjugates them into an "LPXTGGG" linker. As a non-natural linker with several flexible glycine residues, it is unknown whether it affects the properties of the conjugated protein. To verify the use of sortase for protein-protein conjugation, we combined single-molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations to characterize sortase-conjugated polyprotein I27 with three different linkers. We found that the I27 with classic linkers "LPETGGG" and "LPETG" from sortase ligation were of normal stability. However, a protein with a longer artificial linker "LPETGGGG" showed a 15% lower unfolding force. MD simulations revealed that the 4G linker showed a high probability of a closed conformation, in which the adjacent monomer has transient protein-protein interaction. Thus, we verify the use of sortase for protein conjugation, and a longer linker with a higher glycine content should be used with caution.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Poliproteínas/química , Simulação de Dinâmica Molecular , Imagem Individual de Molécula
11.
Sci Rep ; 10(1): 598, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31953451

RESUMO

Myocardial hypertrophy, an inflammatory condition of cardiac muscles is a maladaptive response of the heart to biomechanical stress, hemodynamic or neurohormonal stimuli. Previous studies indicated that knockout of Arginyltransferase (ATE1) gene in mice and embryos leads to contractile dysfunction, defective cardiovascular development, and impaired angiogenesis. Here we found that in adult rat model, downregulation of ATE1 mitigates cardiac hypertrophic, cardiac fibrosis as well as apoptosis responses in the presence of cardiac stress i.e. renal artery ligation. On contrary, in wild type cells responding to renal artery ligation, there is an increase of cellular ATE1 protein level. Further, we have shown the cardioprotective role of ATE1 silencing is mediated by the interruption of TAK1 activity-dependent JNK1/2 signaling pathway. We propose that ATE1 knockdown in presence of cardiac stress performs a cardioprotective action and the inhibition of its activity may provide a novel approach for the treatment of cardiac hypertrophy.


Assuntos
Aminoaciltransferases/genética , Cardiomegalia/genética , MAP Quinase Quinase Quinases/metabolismo , Miócitos Cardíacos/citologia , Transdução de Sinais , Animais , Apoptose , Cardiomegalia/patologia , Linhagem Celular , Modelos Animais de Doenças , Fibrose , Técnicas de Silenciamento de Genes , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Ratos Wistar
12.
Biochem Biophys Res Commun ; 523(2): 548-553, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31932034

RESUMO

Phytochelatin synthases (PCSs) are activated by toxic metals/metalloids such as cadmium and arsenic and synthesize phytochelatins for detoxification of toxic elements. Rice (Oryza sativa L.) has two PCSs (OsPCS1 and OsPCS2), and we previously revealed that OsPCS1 has a higher responsiveness to arsenic than to cadmium, while OsPCS2 has a higher responsiveness to cadmium than to arsenic. Moreover, we found that the specific responsiveness of OsPCS1 to arsenic at rice nodes is a key factor in reducing arsenic in rice grains. However, the molecular characteristics of two PCSs in rice that contribute to the responsiveness to arsenic or heavy metals, including Cd, remain unclear. Here, we experimentally demonstrate that the C-terminal region in PCSs determines the responsiveness to arsenic or cadmium. We constructed chimeric proteins between OsPCS1 and OsPCS2 and performed an in vitro phytochelatin synthesis assay. A chimeric protein in which the 183 C-terminal amino acids of OsPCS2 were replaced with the 185 C-terminal amino acids of OsPCS1 showed higher responsiveness to arsenite than to cadmium, similar to OsPCS1. Contrary to expectations, mutations of cysteine residues that are unique to OsPCS1 or OsPCS2 had little influence on the responsiveness, although cysteine residues are reported to be representative of sites that interact with metals/metalloids. These results would enable the development of a breeding technology for reducing arsenic in rice grains by improving the arsenic-dependent activation of PCSs.


Assuntos
Aminoaciltransferases/metabolismo , Arsênico/toxicidade , Metais Pesados/toxicidade , Oryza/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Aminoaciltransferases/química , Aminoaciltransferases/genética , Cisteína/genética , Mutação , Oryza/metabolismo , Fitoquelatinas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
J Biol Chem ; 295(9): 2664-2675, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31974162

RESUMO

Engineering and bioconjugation of proteins is a critically valuable tool that can facilitate a wide range of biophysical and structural studies. The ability to orthogonally tag or label a domain within a multidomain protein may be complicated by undesirable side reactions to noninvolved domains. Furthermore, the advantages of segmental (or domain-specific) isotopic labeling for NMR, or deuteration for neutron scattering or diffraction, can be realized by an efficient ligation procedure. Common methods-expressed protein ligation, protein trans-splicing, and native chemical ligation-each have specific limitations. Here, we evaluated the use of different variants of Staphylococcus aureus sortase A for a range of ligation reactions and demonstrate that conditions can readily be optimized to yield high efficiency (i.e. completeness of ligation), ease of purification, and functionality in detergents. These properties may enable joining of single domains into multidomain proteins, lipidation to mimic posttranslational modifications, and formation of cyclic proteins to aid in the development of nanodisc membrane mimetics. We anticipate that the method for ligating separate domains into a single functional multidomain protein reported here may enable many applications in structural biology.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Engenharia de Proteínas/métodos , Staphylococcus aureus/enzimologia , Domínios Proteicos
14.
Emerg Microbes Infect ; 9(1): 169-179, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31969071

RESUMO

Staphylococcus aureus (S. aureus), especially methicillin-resistant Staphylococcus aureus (MRSA), is a major cause of pneumonia, resulting in severe morbidity and mortality in adults and children. Sortase A (SrtA), which mediates the anchoring of cell surface proteins in the cell wall, is an important virulence factor of S. aureus. Here, we found that salvianolic acid A (Sal A), which is a natural product that does not affect the growth of S. aureus, could inhibit SrtA activity (IC50 = 5.75 µg/ml) and repress the adhesion of bacteria to fibrinogen, the anchoring of protein A to cell wall, the biofilm formation, and the ability of S. aureus to invade A549 cells. Furthermore, in vivo studies demonstrated that Sal A treatment reduced inflammation and protected mice against lethal pneumonia caused by MRSA. More significantly, full protection (a survival rate of 100%) was achieved when Sal A was administered in combination with latamoxef. Together, these results indicate that Sal A could be developed into a promising therapeutic drug to combat MRSA infections while limiting resistance development.


Assuntos
Ácidos Cafeicos/administração & dosagem , Lactatos/administração & dosagem , Staphylococcus aureus Resistente à Meticilina/fisiologia , Moxalactam/administração & dosagem , Pneumonia/prevenção & controle , Infecções Estafilocócicas/prevenção & controle , Aminoaciltransferases/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Sinergismo Farmacológico , Feminino , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/microbiologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia
15.
Int J Mol Sci ; 21(3)2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31991788

RESUMO

Transglutaminase 2 (TG2) is a Ca2+-dependent enzyme, which regulates various cellular processes by catalyzing protein crosslinking or polyamination. Intracellular TG2 is activated and inhibited by Ca2+ and GTP binding, respectively. Although aberrant TG2 activation has been implicated in the pathogenesis of diverse diseases, including cancer and degenerative and fibrotic diseases, the structural basis for the regulation of TG2 by Ca2+ and GTP binding is not fully understood. Here, we produced and analyzed a Ca2+-containing TG2 crystal, and identified two glutamate residues, E437 and E539, as Ca2+-binding sites. The enzymatic analysis of the mutants revealed that Ca2+ binding to these sites is required for the transamidase activity of TG2. Interestingly, we found that magnesium (Mg2+) competitively binds to the E437 and E539 residues. The Mg2+ binding to these allosteric sites enhances the GTP binding/hydrolysis activity but inhibits transamidase activity. Furthermore, HEK293 cells transfected with mutant TG2 exhibited higher transamidase activity than cells with wild-type TG2. Cells with wild-type TG2 showed an increase in transamidase activity under Mg2+-depleted conditions, whereas cells with mutant TG2 were unaffected. These results indicate that E437 and E539 are Ca2+-binding sites contributing to the reciprocal regulation of transamidase and GTP binding/hydrolysis activities of TG2 through competitive Mg2+ binding.


Assuntos
Aminoaciltransferases/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Magnésio/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/química , Ligação Competitiva , Cálcio/química , Ativação Enzimática , Proteínas de Ligação ao GTP/química , Guanosina Trifosfato/química , Humanos , Hidrólise , Magnésio/química , Modelos Biológicos , Conformação Molecular , Ligação Proteica , Relação Estrutura-Atividade , Transglutaminases/química
16.
Biochim Biophys Acta Gen Subj ; 1864(2): 129419, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31449838

RESUMO

Structural study of multidomain proteins using NMR is an emerging issue for understanding biological functions. To this end, domain-specific labeling is expected to be a key technology for facilitating the NMR-assignment process and for collecting distance information via spin labeling. To obtain domain-specific labeled samples, use of sortase A as a protein ligation tool is a viable approach. Sortase A enables ligation of separately expressed proteins (domains) through the Leu-Pro-X-Thr-Gly linker. However, the ligation reaction mediated by sortase A is not efficient. Poor yield and long reaction times hamper large-scale preparation using sortase A. Here we report the application of highly active sortases to NMR analyses. Optimal yields can be achieved within several hours when the ligation reaction are mediated by highly active sortases at 4 °C. We propose that this protocol can contribute to structural analyses of multidomain proteins by NMR.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Staphylococcus aureus/enzimologia , Escherichia coli , Hidrólise , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Temperatura , Vinculina/química
17.
Cell Mol Life Sci ; 77(1): 81-91, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31728578

RESUMO

The compaction of DNA and the continuous action of DNA transactions, including transcription and DNA replication, create complex DNA topologies that require Type IIA Topoisomerases, which resolve DNA topological strain and control genome dynamics. The human TOP2 enzymes catalyze their reactions via formation of a reversible covalent enzyme DNA-protein crosslink, the TOP2 cleavage complex (TOP2cc). Spurious interactions of TOP2 with DNA damage, environmental toxicants and chemotherapeutic "poisons" perturbs the TOP2 reaction cycle, leading to an accumulation of DNA-protein crosslinks, and ultimately, genomic instability and cell death. Emerging evidence shows that TOP2-DNA protein crosslink (DPC) repair entails multiple strand break repair activities, such as removal of the poisoned TOP2 protein and rejoining of the DNA ends through homologous recombination (HR) or non-homologous end joining (NHEJ). Herein, we discuss the molecular mechanisms of TOP2-DPC resolution, with specific emphasis on the recently uncovered ZATTZnf451-licensed TDP2-catalyzed TOP2-DPC reversal mechanism.


Assuntos
Quebras de DNA , Reparo do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Animais , DNA/química , DNA/genética , DNA Topoisomerases Tipo II/química , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/química , Conformação Proteica , Sumoilação , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
18.
Chemosphere ; 240: 124902, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31563721

RESUMO

Eisenia fetida earthworm is an ecotoxicologically important test species to monitor various pollutants. However, there is a little knowledge about the effects of cadmium (Cd) on earthworms at the transcriptional level. Firstly, we exposed E. fetida to soils supplemented with different concentrations (10, 30, 60 mg/kg soil) of Cd. Moreover, we depicted the characterization of gene expressions with E. fetida using high-throughput profiling of gene expression. In addition, a comparison of the gene expression profiles between each Cd treatment group and the control group suggested that differential expressional genes (DEGs) mainly enriched in enzyme activity, metabolism, oxidative stress, regeneration and apoptosis pathways. 8 DEGs from these pathways had been selected randomly to confirm the data of RNA-seq. Among these DEGs, six genes (metallothionein-2, phytochelatin synthase 1a, CuZn superoxide dismutase, sex determining region Y-box 2, sex determining region Y-box 4b, TP53-regulated inhibitor of apoptosis 1-like) up-regulated and 2 genes (beta-1,4-endoglucanase, apoptosis-stimulating of p53 protein 2-like) down-regulated in response to Cd exposure. The alteration of them indicated that earthworms could reduce the toxicity and bioavailability of Cd in polluted soil ecosystems through different pathways. This work lays an important foundation for linking earthworm transcriptional level with the ecological risk of Cd in soil ecosystem.


Assuntos
Cádmio/toxicidade , Oligoquetos/fisiologia , Poluentes do Solo/toxicidade , Aminoaciltransferases , Animais , Disponibilidade Biológica , Cádmio/análise , Ecossistema , Perfilação da Expressão Gênica , Metalotioneína/genética , Metalotioneína/metabolismo , Oligoquetos/efeitos dos fármacos , Oligoquetos/genética , Estresse Oxidativo/efeitos dos fármacos , Solo , Poluentes do Solo/análise , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
19.
Biochem J ; 476(24): 3835-3847, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31815278

RESUMO

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A-F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.


Assuntos
Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/enzimologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Cisteína Endopeptidases/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Concentração de Íons de Hidrogênio , Filogenia , Especificidade por Substrato , Temperatura
20.
Future Med Chem ; 11(24): 3179-3194, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31838899

RESUMO

A diverse range of N-terminally truncated and modified forms of amyloid-ß (Aß) oligomers have been discovered in Alzheimer's disease brains, including the pyroglutamate-Aß (AßpE3). AßpE3 species are shown to be more neurotoxic when compared with the full-length Aß peptide. Findings visibly suggest that glutaminyl cyclase (QC) catalyzed the generation of cerebral AßpE3, and therapeutic effects are achieved by reducing its activity. In recent years, efforts to effectively develop QC inhibitors have been pursued worldwide. The inhibitory activity of current QC inhibitors is mainly triggered by zinc-binding groups that coordinate Zn2+ ion in the active site and other common features. Herein, we summarized the current state of discovery and evolution of QC inhibitors as a potential Alzheimer's disease-modifying strategy.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Aminoaciltransferases/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Descoberta de Drogas/métodos , Inibidores Enzimáticos/uso terapêutico , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Inibidores Enzimáticos/química , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular
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