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1.
Dev Cell ; 56(20): 2790-2807.e8, 2021 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-34599882

RESUMO

SARS-CoV-2 virions are surrounded by a lipid bilayer that contains membrane proteins such as spike, responsible for target-cell binding and virus fusion. We found that during SARS-CoV-2 infection, spike becomes lipid modified, through the sequential action of the S-acyltransferases ZDHHC20 and 9. Particularly striking is the rapid acylation of spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics, and biochemical approaches, we show that this massive lipidation controls spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid-rich lipid nanodomains in the early Golgi, where viral budding occurs. Finally, S-acylation of spike allows the formation of viruses with enhanced fusion capacity. Our study points toward S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.


Assuntos
Acilação/fisiologia , COVID-19/tratamento farmacológico , Lipídeos de Membrana/metabolismo , SARS-CoV-2/patogenicidade , Aciltransferases/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Humanos , Montagem de Vírus/fisiologia
2.
mBio ; 12(5): e0234221, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34700373

RESUMO

The recent emergence and spread of zoonotic viruses highlights that animal-sourced viruses are the biggest threat to global public health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an HKU2-related bat coronavirus that was spilled over from Rhinolophus bats to swine, causing large-scale outbreaks of severe diarrhea disease in piglets in China. Unlike other porcine coronaviruses, SADS-CoV possesses broad species tissue tropism, including primary human cells, implying a significant risk of cross-species spillover. To explore host dependency factors for SADS-CoV as therapeutic targets, we employed genome-wide CRISPR knockout library screening in HeLa cells. Consistent with two independent screens, we identified the zinc finger DHHC-type palmitoyltransferase 17 (ZDHHC17 or ZD17) as an important host factor for SADS-CoV infection. Through truncation mutagenesis, we demonstrated that the DHHC domain of ZD17 that is involved in palmitoylation is important for SADS-CoV infection. Mechanistic studies revealed that ZD17 is required for SADS-CoV genomic RNA replication. Treatment of infected cells with the palmitoylation inhibitor 2-bromopalmitate (2-BP) significantly suppressed SADS-CoV infection. Our findings provide insight on SADS-CoV-host interactions and a potential therapeutic application. IMPORTANCE The recent emergence of deadly zoonotic viral diseases, including Ebola virus and SARS-CoV-2, emphasizes the importance of pandemic preparedness for the animal-sourced viruses with potential risk of animal-to-human spillover. Over the last 2 decades, three significant coronaviruses of bat origin, SARS-CoV, MERS-CoV, and SARS-CoV-2, have caused millions of deaths with significant economy and public health impacts. Lack of effective therapeutics against these coronaviruses was one of the contributing factors to such losses. Although SADS-CoV, another coronavirus of bat origin, was only known to cause fatal diarrhea disease in piglets, the ability to infect cells derived from multiple species, including human, highlights the potential risk of animal-to-human spillover. As part of our effort in pandemic preparedness, we explore SADS-CoV host dependency factors as targets for host-directed therapeutic development and found zinc finger DHHC-type palmitoyltransferase 17 is a promising drug target against SADS-CoV replication. We also demonstrated that a palmitoylation inhibitor, 2-bromopalmitate (2-BP), can be used as an inhibitor for SADS-CoV treatment.


Assuntos
Aciltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Alphacoronavirus/patogenicidade , Proteínas do Tecido Nervoso/metabolismo , Aciltransferases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Alphacoronavirus/efeitos dos fármacos , Animais , COVID-19/metabolismo , Células HeLa , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Proteínas do Tecido Nervoso/genética , Palmitatos/farmacologia , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/patogenicidade , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade , Suínos
3.
Nat Commun ; 12(1): 5872, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620861

RESUMO

Glucose transporter GLUT1 is a transmembrane protein responsible for the uptake of glucose into the cells of many tissues through facilitative diffusion. Plasma membrane (PM) localization is essential for glucose uptake by GLUT1. However, the mechanism underlying GLUT1 PM localization remains enigmatic. We find that GLUT1 is palmitoylated at Cys207, and S-palmitoylation is required for maintaining GLUT1 PM localization. Furthermore, we identify DHHC9 as the palmitoyl transferase responsible for this critical posttranslational modification. Knockout of DHHC9 or mutation of GLUT1 Cys207 to serine abrogates palmitoylation and PM distribution of GLUT1, and impairs glycolysis, cell proliferation, and glioblastoma (GBM) tumorigenesis. In addition, DHHC9 expression positively correlates with GLUT1 PM localization in GBM specimens and indicates a poor prognosis in GBM patients. These findings underscore that DHHC9-mediated GLUT1 S-palmitoylation is critical for glucose supply during GBM tumorigenesis.


Assuntos
Aciltransferases/metabolismo , Carcinogênese/metabolismo , Glioblastoma/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glicólise/fisiologia , Aciltransferases/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose , Transportador de Glucose Tipo 1/genética , Glicólise/genética , Xenoenxertos , Humanos , Lipoilação , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Pessoa de Meia-Idade , Processamento de Proteína Pós-Traducional
4.
J Biomed Sci ; 28(1): 72, 2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34706729

RESUMO

BACKGROUND: During autophagy defense against invading microbes, certain lipid types are indispensable for generating specialized membrane-bound organelles. The lipid composition of autophagosomes remains obscure, as does the issue of how specific lipids and lipid-associated enzymes participate in autophagosome formation and maturation. Helicobacter pylori is auxotrophic for cholesterol and converts cholesterol to cholesteryl glucoside derivatives, including cholesteryl 6'-O-acyl-α-D-glucoside (CAG). We investigated how CAG and its biosynthetic acyltransferase assist H. pylori to escape host-cell autophagy. METHODS: We applied a metabolite-tagging method to obtain fluorophore-containing cholesteryl glucosides that were utilized to understand their intracellular locations. H. pylori 26695 and a cholesteryl glucosyltransferase (CGT)-deletion mutant (ΔCGT) were used as the standard strain and the negative control that contains no cholesterol-derived metabolites, respectively. Bacterial internalization and several autophagy-related assays were conducted to unravel the possible mechanism that H. pylori develops to hijack the host-cell autophagy response. Subcellular fractions of H. pylori-infected AGS cells were obtained and measured for the acyltransferase activity. RESULTS: The imaging studies of fluorophore-labeled cholesteryl glucosides pinpointed their intracellular localization in AGS cells. The result indicated that CAG enhances the internalization of H. pylori in AGS cells. Particularly, CAG, instead of CG and CPG, is able to augment the autophagy response induced by H. pylori. How CAG participates in the autophagy process is multifaceted. CAG was found to intervene in the degradation of autophagosomes and reduce lysosomal biogenesis, supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Furthermore, we performed the enzyme activity assay of subcellular fractions of H. pylori-infected AGS cells. The analysis showed that the acyltransferase is mainly distributed in autophago-lysosomal compartments. CONCLUSIONS: Our results support the idea that the acyltransferase is mainly distributed in the subcellular compartment consisting of autophagosomes, late endosomes, and lysosomes, in which the acidic environment is beneficial for the maximal acyltransferase activity. The resulting elevated level of CAG can facilitate bacterial internalization, interfere with the autophagy flux, and causes reduced lysosomal biogenesis.


Assuntos
Aciltransferases/metabolismo , Colesterol/análogos & derivados , Infecções por Helicobacter/fisiopatologia , Helicobacter pylori/fisiologia , Lisossomos/fisiologia , Animais , Colesterol/biossíntese , Infecções por Helicobacter/enzimologia , Infecções por Helicobacter/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organismos Livres de Patógenos Específicos
5.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502081

RESUMO

Eggplant berries are rich in anthocyanins like delphinidin-3-rutinoside (D3R) and nasunin (NAS), which are accumulated at high amounts in the peel. NAS is derived by D3R through acylation and glycosylation steps. The presence of D3R or NAS is usually associated with black-purple or lilac fruit coloration of the most cultivated varieties, respectively. Building on QTL mapping position, a candidate gene approach was used to investigate the involvement of a BAHD anthocyanin acyltransferase (SmelAAT) in determining anthocyanin type. The cDNA sequence comparison revealed the presence of a single-base deletion in D3R-type line '305E40' (305E40_aat) with respect to the NAS-type reference line '67/3'. This is predicted to cause a frame shift mutation, leading to a loss of SmelAAT function and, thus, D3R retention. RT-qPCR analyses confirmed SmelAAT and 305E40_aat expression during berry maturation. In D3R-type lines, '305E40' and 'DR2', overexpressing the functional SmelAAT allele from '67/3', the transcript levels of the transgene correlated with the accumulation of NAS in fruit peel. Furthermore, it was also found a higher expression of the transcript for glucosyltransferase Smel5GT1, putatively involved with SmelAAT in the last steps of anthocyanin decoration. Finally, an indel marker matching with anthocyanin type in the '305E40' × '67/3' segregating population was developed and validated in a wide number of accessions, proving its usefulness for breeding purposes.


Assuntos
Aciltransferases/genética , Antocianinas/metabolismo , Proteínas de Plantas/genética , Solanum melongena/genética , Aciltransferases/metabolismo , Antocianinas/genética , Frutas/genética , Frutas/metabolismo , Mutação , Pigmentação , Proteínas de Plantas/metabolismo , Solanum melongena/metabolismo
6.
Molecules ; 26(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34500646

RESUMO

Arachidonylethanolamide (anandamide) acts as an endogenous ligand of cannabinoid receptors, while other N-acylethanolamines (NAEs), such as palmitylethanolamide and oleylethanolamide, show analgesic, anti-inflammatory, and appetite-suppressing effects through other receptors. In mammalian tissues, NAEs, including anandamide, are produced from glycerophospholipid via N-acyl-phosphatidylethanolamine (NAPE). The ɛ isoform of cytosolic phospholipase A2 (cPLA2) functions as an N-acyltransferase to form NAPE. Since the cPLA2 family consists of six isoforms (α, ß, γ, δ, ɛ, and ζ), the present study investigated a possible involvement of isoforms other than ɛ in the NAE biosynthesis. Firstly, when the cells overexpressing one of the cPLA2 isoforms were labeled with [14C]ethanolamine, the increase in the production of [14C]NAPE was observed only with the ɛ-expressing cells. Secondly, when the cells co-expressing ɛ and one of the other isoforms were analyzed, the increase in [14C]N-acyl-lysophosphatidylethanolamine (lysoNAPE) and [14C]NAE was seen with the combination of ɛ and γ isoforms. Furthermore, the purified cPLA2γ hydrolyzed not only NAPE to lysoNAPE, but also lysoNAPE to glycerophospho-N-acylethanolamine (GP-NAE). Thus, the produced GP-NAE was further hydrolyzed to NAE by glycerophosphodiesterase 1. These results suggested that cPLA2γ is involved in the biosynthesis of NAE by its phospholipase A1/A2 and lysophospholipase activities.


Assuntos
Etanolaminas/metabolismo , Fosfolipases A2/metabolismo , Isoformas de Proteínas/metabolismo , Aciltransferases/metabolismo , Amidas/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Linhagem Celular , Endocanabinoides/metabolismo , Etanolamina/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Oleicos/metabolismo , Ácidos Palmíticos/metabolismo , Fosfatidiletanolaminas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Alcamidas Poli-Insaturadas/metabolismo
7.
Front Immunol ; 12: 661202, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34557182

RESUMO

Protein S-palmitoylation is a covalent and reversible lipid modification that specifically targets cysteine residues within many eukaryotic proteins. In mammalian cells, the ubiquitous palmitoyltransferases (PATs) and serine hydrolases, including acyl protein thioesterases (APTs), catalyze the addition and removal of palmitate, respectively. The attachment of palmitoyl groups alters the membrane affinity of the substrate protein changing its subcellular localization, stability, and protein-protein interactions. Forty years of research has led to the understanding of the role of protein palmitoylation in significantly regulating protein function in a variety of biological processes. Recent global profiling of immune cells has identified a large body of S-palmitoylated immunity-associated proteins. Localization of many immune molecules to the cellular membrane is required for the proper activation of innate and adaptive immune signaling. Emerging evidence has unveiled the crucial roles that palmitoylation plays to immune function, especially in partitioning immune signaling proteins to the membrane as well as to lipid rafts. More importantly, aberrant PAT activity and fluctuations in palmitoylation levels are strongly correlated with human immunologic diseases, such as sensory incompetence or over-response to pathogens. Therefore, targeting palmitoylation is a novel therapeutic approach for treating human immunologic diseases. In this review, we discuss the role that palmitoylation plays in both immunity and immunologic diseases as well as the significant potential of targeting palmitoylation in disease treatment.


Assuntos
Doenças do Sistema Imunitário/metabolismo , Sistema Imunitário/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Aciltransferases/metabolismo , Imunidade Adaptativa , Animais , Humanos , Sistema Imunitário/imunologia , Doenças do Sistema Imunitário/imunologia , Imunidade Inata , Lipoilação , Proteínas/imunologia , Transdução de Sinais
8.
Elife ; 102021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34491895

RESUMO

Inducible regulatory T (iTreg) cells play a central role in immune suppression. As iTreg cells are differentiated from activated T (Th0) cells, cell metabolism undergoes dramatic changes, including a shift from fatty acid synthesis (FAS) to fatty acid oxidation (FAO). Although the reprogramming in fatty acid metabolism is critical, the mechanism regulating this process during iTreg differentiation is still unclear. Here we have revealed that the enzymatic activity of ATP-citrate lyase (ACLY) declined significantly during iTreg differentiation upon transforming growth factor ß1 (TGFß1) stimulation. This reduction was due to CUL3-KLHL25-mediated ACLY ubiquitination and degradation. As a consequence, malonyl-CoA, a metabolic intermediate in FAS that is capable of inhibiting the rate-limiting enzyme in FAO, carnitine palmitoyltransferase 1 (CPT1), was decreased. Therefore, ACLY ubiquitination and degradation facilitate FAO and thereby iTreg differentiation. Together, we suggest TGFß1-CUL3-KLHL25-ACLY axis as an important means regulating iTreg differentiation and bring insights into the maintenance of immune homeostasis for the prevention of immune diseases.


Assuntos
ATP Citrato (pro-S)-Liase/metabolismo , Aciltransferases/metabolismo , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proteínas Culina/metabolismo , Ácidos Graxos/metabolismo , Ubiquitinação , ATP Citrato (pro-S)-Liase/genética , Aciltransferases/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Técnicas de Reprogramação Celular , Colite/patologia , Proteínas Culina/genética , Ácidos Graxos/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL
9.
Molecules ; 26(18)2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34576938

RESUMO

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.


Assuntos
Aciltransferases/química , Aciltransferases/genética , Aciltransferases/metabolismo , Aminoácidos/genética , Aciltransferases/isolamento & purificação , Microscopia Crioeletrônica , Detergentes/química , Células HEK293 , Humanos , Mutação , Conformação Proteica , Subunidades Proteicas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445771

RESUMO

The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and potential to increase the production of L-lysine. The effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in a high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3 ± 4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening the tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189 ± 8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35 ± 0.05 g/(g·h) in a 5-L jar fermenter. The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.


Assuntos
Corynebacterium glutamicum/metabolismo , Ácido Diaminopimélico/metabolismo , Lisina/metabolismo , Transdução de Sinais/fisiologia , Aciltransferases/metabolismo , Compostos de Amônio/metabolismo , Oxirredutases/metabolismo
11.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360902

RESUMO

Acyl-CoA:lysophosphatidylethanolamine acyltransferases (LPEATs) are known as enzymes utilizing acyl-CoAs and lysophospholipids to produce phosphatidylethanolamine. Recently, it has been discovered that they are also involved in the growth regulation of Arabidopsis thaliana. In our study we investigated expression of each Camelina sativa LPEAT isoform and their behavior in response to temperature changes. In order to conduct a more extensive biochemical evaluation we focused both on LPEAT enzymes present in microsomal fractions from C. sativa plant tissues, and on cloned CsLPEAT isoforms expressed in yeast system. Phylogenetic analyses revealed that CsLPEAT1c and CsLPEAT2c originated from Camelina hispida, whereas other isoforms originated from Camelina neglecta. The expression ratio of all CsLPEAT1 isoforms to all CsLPEAT2 isoforms was higher in seeds than in other tissues. The isoforms also displayed divergent substrate specificities in utilization of LPE; CsLPEAT1 preferred 18:1-LPE, whereas CsLPEAT2 preferred 18:2-LPE. Unlike CsLPEAT1, CsLPEAT2 isoforms were specific towards very-long-chain fatty acids. Above all, we discovered that temperature strongly regulates LPEATs activity and substrate specificity towards different acyl donors, making LPEATs sort of a sensor of external thermal changes. We observed the presented findings not only for LPEAT activity in plant-derived microsomal fractions, but also for yeast-expressed individual CsLPEAT isoforms.


Assuntos
Aciltransferases/metabolismo , Camellia/enzimologia , Camellia/genética , Fosfatidiletanolaminas/metabolismo , Proteínas de Plantas/metabolismo , Sementes/enzimologia , Temperatura , Acil Coenzima A/metabolismo , Aciltransferases/genética , Camellia/classificação , Camellia/crescimento & desenvolvimento , Resposta ao Choque Frio , DNA de Plantas/genética , Ativação Enzimática , Resposta ao Choque Térmico , Isoenzimas/genética , Microssomos/enzimologia , Filogenia , Proteínas de Plantas/genética , Sementes/crescimento & desenvolvimento , Especificidade por Substrato
12.
Phytochemistry ; 191: 112912, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34450419

RESUMO

The esterification of carotenoids has been associated with high-level accumulation, greater stability and potentially improved dietary bioavailability. Engineering the formation of ketocarotenoids into tomato fruit has resulted in the esterification of these non-endogenous metabolites. A genotype of tomato was created that contains; (i) the mutant pale yellow petal (pyp)1-1 allele, which is responsible for the absence of carotenoid esters in tomato flowers and (ii) the heterologous enzymes for ketocarotenoid formation. Analysis of the resulting progeny showed altered quantitative and qualitative differences in esterified carotenoids. For example, in ripe fruit tissues, in the presence of the pyp mutant allele, non-endogenous ketocarotenoid esters were absent while their free forms accumulated. These data demonstrate the involvement of the PYP gene product in the esterification of diverse xanthophylls.


Assuntos
Lycopersicon esculentum , Aciltransferases/metabolismo , Esterificação , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Proteínas de Plantas/metabolismo , Plastídeos/metabolismo , Xantofilas/metabolismo
13.
Nat Plants ; 7(9): 1288-1300, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34354261

RESUMO

Plant lignification exhibits notable plasticity. Lignin in many species, including Populus spp., has long been known to be decorated with p-hydroxybenzoates. However, the molecular basis for such structural modification remains undetermined. Here, we report the identification and characterization of a Populus BAHD family acyltransferase that catalyses monolignol p-hydroxybenzoylation, thus controlling the formation of p-hydroxybenzoylated lignin structures. We reveal that Populus acyltransferase PHBMT1 kinetically preferentially uses p-hydroxybenzoyl-CoA to acylate syringyl lignin monomer sinapyl alcohol in vitro. Consistently, disrupting PHBMT1 in Populus via CRISPR-Cas9 gene editing nearly completely depletes p-hydroxybenzoates of stem lignin; conversely, overexpression of PHBMT1 enhances stem lignin p-hydroxybenzoylation, suggesting PHBMT1 functions as a prime monolignol p-hydroxybenzoyltransferase in planta. Altering lignin p-hydroxybenzoylation substantially changes the lignin solvent dissolution rate, indicative of its structural significance on lignin physiochemical properties. Identification of monolignol p-hydroxybenzoyltransferase offers a valuable tool for tailoring lignin structure and physiochemical properties and for engineering the industrially important platform chemical in woody biomass.


Assuntos
Aciltransferases/genética , Aciltransferases/metabolismo , Hidroxibenzoatos/metabolismo , Lignina/biossíntese , Lignina/genética , Populus/genética , Populus/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Plantas Geneticamente Modificadas/metabolismo
14.
J Biol Chem ; 297(4): 101112, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34428449

RESUMO

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of ß-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators.


Assuntos
COVID-19/patologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Acilação , Aciltransferases/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , COVID-19/virologia , Cisteína/metabolismo , Células HEK293 , Humanos , Lipoilação , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus
15.
Sci Rep ; 11(1): 15770, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349203

RESUMO

Berberine (BBR) is an isoquinoline alkaloid from plants known to improve cardiac mitochondrial function in gestational diabetes mellitus (GDM) offspring but the mechanism is poorly understood. We examined the role of the mitochondrial phospholipid cardiolipin (CL) in mediating this cardiac improvement. C57BL/6 female mice were fed either a Lean-inducing low-fat diet or a GDM-inducing high-fat diet for 6 weeks prior to breeding. Lean and GDM-exposed male offspring were randomly assigned a low-fat, high-fat, or high-fat diet containing BBR at weaning for 12 weeks. The content of CL was elevated in the heart of GDM offspring fed a high fat diet containing BBR. The increase in total cardiac CL was due to significant increases in the most abundant and functionally important CL species, tetralinoleoyl-CL and this correlated with an increase in the expression of the CL remodeling enzyme tafazzin. Additionally, BBR treatment increased expression of cardiac enzymes involved in fatty acid uptake and oxidation and electron transport chain subunits in high fat diet fed GDM offspring. Thus, dietary BBR protection from cardiac dysfunction in GDM exposed offspring involves improvement in mitochondrial function mediated through increased synthesis of CL.


Assuntos
Berberina/farmacologia , Cardiolipinas/metabolismo , Diabetes Gestacional/etiologia , Dieta Hiperlipídica/efeitos adversos , Cardiopatias/etiologia , Cardiopatias/prevenção & controle , Troca Materno-Fetal/fisiologia , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Berberina/administração & dosagem , Transporte de Elétrons/efeitos dos fármacos , Ácidos Graxos/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Oxirredução/efeitos dos fármacos , Gravidez
16.
Sheng Wu Gong Cheng Xue Bao ; 37(6): 1887-1899, 2021 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-34227283

RESUMO

Plant serine carboxypeptidase-like acyltransferases (SCPL-AT) have similar structural characteristics and high homology compared to the serine carboxypeptidase. They can transfer the acyl from acyl glucose esters to many natural products, participate in the acylation modification of plant secondary metabolites, enrich the structural diversity of natural products, and improve the physicochemical properties such as water solubility and stability of compounds. This review summarizes the structural characteristics, catalytic mechanism, functional characterization, and biocatalytic applications of SCPL-AT from plants. This will help to promote the functional characterization of these acyltransferase genes and the biosynthesis of useful plant secondary metabolites by synthetic biotechnology.


Assuntos
Aciltransferases , Carboxipeptidases , Plantas/enzimologia , Acilação , Aciltransferases/genética , Aciltransferases/metabolismo , Carboxipeptidases/genética , Carboxipeptidases/metabolismo
17.
Nat Commun ; 12(1): 4254, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253723

RESUMO

Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis.


Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Membrana Celular/metabolismo , Lipoproteínas/biossíntese , Acilação , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Sequência Conservada , Cisteína/metabolismo , Análise Mutacional de DNA , Processamento de Proteína Pós-Traducional , Relação Estrutura-Atividade , Especificidade por Substrato
18.
Cancer Sci ; 112(10): 4303-4316, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34289205

RESUMO

Yes-associated protein 1 (YAP1) and its paralogue PDZ-binding motif (TAZ) play pivotal roles in cell proliferation, migration, and invasion, and abnormal activation of these TEAD transcriptional coactivators is found in diverse cancers in humans and mice. Targeting YAP1/TAZ signaling is thus a promising therapeutic avenue but, to date, few selective YAP1/TAZ inhibitors have been effective against cancer cells either in vitro or in vivo. We screened chemical libraries for potent YAP1/TAZ inhibitors using a highly sensitive luciferase reporter system to monitor YAP1/TAZ-TEAD transcriptional activity in cells. Among 29 049 low-molecular-weight compounds screened, we obtained nine hits, and the four of these that were the most effective shared a core structure with the natural product alantolactone (ALT). We also tested 16 other structural derivatives of ALT and found that natural ALT was the most efficient at increasing ROS-induced LATS kinase activities and thus YAP1/TAZ phosphorylation. Phosphorylated YAP1/TAZ proteins were subject to nuclear exclusion and proteosomic degradation such that the growth of ALT-treated tumor cells was inhibited both in vitro and in vivo. Our data show for the first time that ALT can be used to target the ROS-YAP pathway driving tumor cell growth and so could be a potent anticancer drug.


Assuntos
Aciltransferases/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Lactonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sesquiterpenos de Eudesmano/farmacologia , Aciltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Auranofina/farmacologia , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular , Proteínas de Ligação a DNA/metabolismo , Descoberta de Drogas , Feminino , Inula/química , Luciferases , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Neoplasias da Língua/induzido quimicamente , Neoplasias da Língua/prevenção & controle , Fatores de Transcrição/metabolismo , Ativação Transcricional
19.
Angew Chem Int Ed Engl ; 60(35): 19139-19143, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34219345

RESUMO

A bioassay-guided fractionation led to the isolation of hangtaimycin (HTM) from Streptomyces spectabilis CCTCC M2017417 and the discovery of its hepatoprotective properties. Structure elucidation by NMR suggested the need for a structural revision. A putative HTM degradation product was also isolated and its structure was confirmed by total synthesis. The biosynthetic gene cluster was identified and resembles a hybrid trans-AT PKS/NRPS biosynthetic machinery whose first PKS enzyme contains an internal dehydrating bimodule, which is usually found split in other trans-AT PKSs. The mechanisms of such dehydrating bimodules have often been proposed, but have never been deeply investigated. Here we present in vivo mutations and in vitro enzymatic experiments that give first and detailed mechanistic insights into catalysis by dehydrating bimodules.


Assuntos
Aciltransferases/metabolismo , Policetídeos/metabolismo , Estrutura Molecular , Policetídeos/química , Streptomyces/química , Streptomyces/metabolismo
20.
J Biol Chem ; 297(3): 101005, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34314685

RESUMO

Barth syndrome (BTHS) is an X-linked disorder of mitochondrial phospholipid metabolism caused by pathogenic variants in TAFFAZIN, which results in abnormal cardiolipin (CL) content in the inner mitochondrial membrane. To identify unappreciated pathways of mitochondrial dysfunction in BTHS, we utilized an unbiased proteomics strategy and identified that complex I (CI) of the mitochondrial respiratory chain and the mitochondrial quality control protease presenilin-associated rhomboid-like protein (PARL) are altered in a new HEK293-based tafazzin-deficiency model. Follow-up studies confirmed decreased steady state levels of specific CI subunits and an assembly factor in the absence of tafazzin; this decrease is in part based on decreased transcription and results in reduced CI assembly and function. PARL, a rhomboid protease associated with the inner mitochondrial membrane with a role in the mitochondrial response to stress, such as mitochondrial membrane depolarization, is increased in tafazzin-deficient cells. The increased abundance of PARL correlates with augmented processing of a downstream target, phosphoglycerate mutase 5, at baseline and in response to mitochondrial depolarization. To clarify the relationship between abnormal CL content, CI levels, and increased PARL expression that occurs when tafazzin is missing, we used blue-native PAGE and gene expression analysis to determine that these defects are remediated by SS-31 and bromoenol lactone, pharmacologic agents that bind CL or inhibit CL deacylation, respectively. These findings have the potential to enhance our understanding of the cardiac pathology of BTHS, where defective mitochondrial quality control and CI dysfunction have well-recognized roles in the pathology of diverse forms of cardiac dysfunction.


Assuntos
Aciltransferases/genética , Cardiolipinas/metabolismo , Mitocôndrias/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Aciltransferases/metabolismo , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Células HEK293 , Humanos , Lipidômica , Proteômica
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