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1.
Cell ; 187(17): 4586-4604.e20, 2024 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-39137778

RESUMO

Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.


Assuntos
COVID-19 , Influenza Humana , Animais , Humanos , Camundongos , COVID-19/virologia , COVID-19/genética , Influenza Humana/virologia , Replicação Viral , Macrófagos/metabolismo , Macrófagos/virologia , Feminino , Masculino , SARS-CoV-2 , Pulmão/virologia , Pulmão/patologia , Pulmão/metabolismo , Camundongos Endogâmicos C57BL , Ácido Oleico/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Camundongos Knockout , Carga Viral , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/genética , Infecções por Orthomyxoviridae/virologia , Infecções Respiratórias/virologia , Criança
2.
Cell ; 183(6): 1562-1571.e12, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33306955

RESUMO

Ticks transmit a diverse array of microbes to vertebrate hosts, including human pathogens, which has led to a human-centric focus in this vector system. Far less is known about pathogens of ticks themselves. Here, we discover that a toxin in blacklegged ticks (Ixodes scapularis) horizontally acquired from bacteria-called domesticated amidase effector 2 (dae2)-has evolved to kill mammalian skin microbes with remarkable efficiency. Secreted into the saliva and gut of ticks, Dae2 limits skin-associated staphylococci in ticks while feeding. In contrast, Dae2 has no intrinsic ability to kill Borrelia burgdorferi, the tick-borne Lyme disease bacterial pathogen. These findings suggest ticks resist their own pathogens while tolerating symbionts. Thus, just as tick symbionts can be pathogenic to humans, mammalian commensals can be harmful to ticks. Our study underscores how virulence is context-dependent and bolsters the idea that "pathogen" is a status and not an identity.


Assuntos
Bactérias/metabolismo , Fatores Imunológicos/metabolismo , Ixodes/fisiologia , Pele/microbiologia , Simbiose , Animais , Antibacterianos/farmacologia , Biocatálise , Parede Celular/metabolismo , Comportamento Alimentar , Feminino , Trato Gastrointestinal/metabolismo , Interações Hospedeiro-Patógeno , Camundongos , Modelos Moleculares , Peptidoglicano/metabolismo , Filogenia , Saliva/metabolismo , Glândulas Salivares/metabolismo , Staphylococcus epidermidis/fisiologia , Homologia Estrutural de Proteína , Especificidade por Substrato , Regulação para Cima
3.
Cell ; 173(7): 1663-1677.e21, 2018 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-29906447

RESUMO

The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4-containing RNA exosomes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human and show that they unwind structured substrates to promote degradation. We loaded a human exosome with an optimized DNA-RNA chimera that stalls MTR4 during unwinding and determined its structure to an overall resolution of 3.45 Å by cryoelectron microscopy (cryo-EM). The structure reveals an RNA-engaged helicase atop the non-catalytic core, with RNA captured within the central channel and DIS3 exoribonuclease active site. MPP6 tethers MTR4 to the exosome through contacts to the RecA domains of MTR4. EXOSC10 remains bound to the core, but its catalytic module and cofactor C1D are displaced by RNA-engaged MTR4. Competition for the exosome core may ensure that RNA is committed to degradation by DIS3 when engaged by MTR4.


Assuntos
DNA Helicases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Helicases/metabolismo , RNA/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , DNA/genética , DNA/metabolismo , Exorribonucleases/química , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/química , Humanos , Processamento de Imagem Assistida por Computador , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , RNA/genética , RNA Helicases/química , Estabilidade de RNA , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidade por Substrato
4.
Mol Cell ; 2024 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-39321805

RESUMO

Hydrogen sulfide (H2S), a metabolite of the transsulfuration pathway, has been implicated in ferroptosis, a unique form of cell death caused by lipid peroxidation. While the exact mechanisms controlling ferroptosis remain unclear, our study reveals that H2S sensitizes human non-small cell lung cancer (NSCLC) cells to this process, particularly when cysteine levels are low. Combining H2S with cystine depletion significantly enhances the effectiveness of ferroptosis-based cancer therapy. Mechanistically, H2S persulfidates the 195th cysteine on S-adenosyl homocysteine hydrolase (SAHH), reducing its enzymatic activity. This leads to decreased homocysteine levels, subsequently lowering cysteine and glutathione concentrations under cystine depletion conditions. These changes ultimately increase the vulnerability of NSCLC cells to ferroptosis. Our findings establish H2S as a key regulator of homocysteine metabolism and a critical factor in determining NSCLC cell susceptibility to ferroptosis. These results highlight the potential of H2S-based therapies to improve the efficacy of ferroptosis-targeted cancer treatments for NSCLC.

5.
Proc Natl Acad Sci U S A ; 121(8): e2320262121, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38349879

RESUMO

The human malaria parasite Plasmodium falciparum requires exogenous fatty acids to support its growth during the pathogenic, asexual erythrocytic stage. Host serum lysophosphatidylcholine (LPC) is a significant fatty acid source, yet the metabolic processes responsible for the liberation of free fatty acids from exogenous LPC are unknown. Using an assay for LPC hydrolysis in P. falciparum-infected erythrocytes, we have identified small-molecule inhibitors of key in situ lysophospholipase activities. Competitive activity-based profiling and generation of a panel of single-to-quadruple knockout parasite lines revealed that two enzymes of the serine hydrolase superfamily, termed exported lipase (XL) 2 and exported lipase homolog (XLH) 4, constitute the dominant lysophospholipase activities in parasite-infected erythrocytes. The parasite ensures efficient exogenous LPC hydrolysis by directing these two enzymes to distinct locations: XL2 is exported to the erythrocyte, while XLH4 is retained within the parasite. While XL2 and XLH4 were individually dispensable with little effect on LPC hydrolysis in situ, loss of both enzymes resulted in a strong reduction in fatty acid scavenging from LPC, hyperproduction of phosphatidylcholine, and an enhanced sensitivity to LPC toxicity. Notably, growth of XL/XLH-deficient parasites was severely impaired when cultured in media containing LPC as the sole exogenous fatty acid source. Furthermore, when XL2 and XLH4 activities were ablated by genetic or pharmacologic means, parasites were unable to proliferate in human serum, a physiologically relevant fatty acid source, revealing the essentiality of LPC hydrolysis in the host environment and its potential as a target for anti-malarial therapy.


Assuntos
Malária Falciparum , Parasitos , Animais , Humanos , Plasmodium falciparum , Lisofosfatidilcolinas/metabolismo , Lisofosfolipase/genética , Lisofosfolipase/metabolismo , Malária Falciparum/parasitologia , Eritrócitos/metabolismo , Parasitos/metabolismo , Ácidos Graxos/metabolismo , Lipase/metabolismo , Proteínas de Protozoários/metabolismo
6.
Proc Natl Acad Sci U S A ; 121(7): e2314085121, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38330013

RESUMO

Cancer therapy, including immunotherapy, is inherently limited by chronic inflammation-induced tumorigenesis and toxicity within the tumor microenvironment. Thus, stimulating the resolution of inflammation may enhance immunotherapy and improve the toxicity of immune checkpoint inhibition (ICI). As epoxy-fatty acids (EpFAs) are degraded by the enzyme soluble epoxide hydrolase (sEH), the inhibition of sEH increases endogenous EpFA levels to promote the resolution of cancer-associated inflammation. Here, we demonstrate that systemic treatment with ICI induces sEH expression in multiple murine cancer models. Dietary omega-3 polyunsaturated fatty acid supplementation and pharmacologic sEH inhibition, both alone and in combination, significantly enhance anti-tumor activity of ICI in these models. Notably, pharmacological abrogation of the sEH pathway alone or in combination with ICI counter-regulates an ICI-induced pro-inflammatory and pro-tumorigenic cytokine storm. Thus, modulating endogenous EpFA levels through dietary supplementation or sEH inhibition may represent a unique strategy to enhance the anti-tumor activity of paradigm cancer therapies.


Assuntos
Epóxido Hidrolases , Neoplasias , Camundongos , Humanos , Animais , Epóxido Hidrolases/metabolismo , Ácidos Graxos/metabolismo , Inflamação/metabolismo , Neoplasias/terapia , Imunoterapia , Microambiente Tumoral
7.
Annu Rev Microbiol ; 75: 383-406, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34343020

RESUMO

Under stressful growth conditions and nutrient starvation, bacteria adapt by synthesizing signaling molecules that profoundly reprogram cellular physiology. At the onset of this process, called the stringent response, members of the RelA/SpoT homolog (RSH) protein superfamily are activated by specific stress stimuli to produce several hyperphosphorylated forms of guanine nucleotides, commonly referred to as (p)ppGpp. Some bifunctional RSH enzymes also harbor domains that allow for degradation of (p)ppGpp by hydrolysis. (p)ppGpp synthesis or hydrolysis may further be executed by single-domain alarmone synthetases or hydrolases, respectively. The downstream effects of (p)ppGpp rely mainly on direct interaction with specific intracellular effectors, which are widely used throughout most cellular processes. The growing number of identified (p)ppGpp targets allows us to deduce both common features of and differences between gram-negative and gram-positive bacteria. In this review, we give an overview of (p)ppGpp metabolism with a focus on the functional and structural aspects of the enzymes involved and discuss recent findings on alarmone-regulated cellular effectors.


Assuntos
Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Guanosina Pentafosfato/metabolismo , Sistemas do Segundo Mensageiro
8.
Proc Natl Acad Sci U S A ; 120(8): e2219827120, 2023 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-36791107

RESUMO

The discovery of unreported antimicrobial resistance genes (ARGs) remains essential. Here, we report the identification and preliminary characterization of an α/ß-hydrolase that inactivates macrolides. This serine-dependent macrolide esterase co-occurs with emerging ARGs in the environment, animal microbiomes, and pathogens.


Assuntos
Antibacterianos , Macrolídeos , Animais , Antibacterianos/farmacologia , Macrolídeos/farmacologia , Farmacorresistência Bacteriana/genética , Esterases/genética , Serina/genética , Genes Bacterianos
9.
Proc Natl Acad Sci U S A ; 120(8): e2216547120, 2023 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-36800389

RESUMO

Cyanophycin is a bacterial polymer mainly used for nitrogen storage. It is composed of a peptide backbone of L-aspartate residues with L-arginines attached to their side chains through isopeptide bonds. Cyanophycin is degraded in two steps: Cyanophycinase cleaves the polymer into ß-Asp-Arg dipeptides, which are hydrolyzed into free Asp and Arg by enzymes possessing isoaspartyl dipeptide hydrolase activity. Two unrelated enzymes with this activity, isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA) have been shown to degrade ß-Asp-Arg dipeptides, but bacteria which encode cyanophycin-metabolizing genes can lack iaaA and iadA genes. In this study, we investigate a previously uncharacterized enzyme whose gene can cluster with cyanophycin-metabolizing genes. This enzyme, which we name cyanophycin dipeptide hydrolase (CphZ), is specific for dipeptides derived from cyanophycin degradation. Accordingly, a co-complex structure of CphZ and ß-Asp-Arg shows that CphZ, unlike IadA or IaaA, recognizes all portions of its ß-Asp-Arg substrate. Bioinformatic analyses showed that CphZ is found in very many proteobacteria and is homologous to an uncharacterized protein encoded in the "arginine/ornithine transport" (aot) operon of many pseudomonas species, including Pseudomonas aeruginosa. In vitro assays show that AotO is indeed a CphZ, and in cellulo growth experiments show that this enzyme and the aot operon allow P. aeruginosa to take up and use ß-Asp-Arg as a sole carbon and nitrogen source. Together the results establish the novel, highly specific enzyme subfamily of CphZs, suggesting that cyanophycin is potentially used by a much wider range of bacteria than previously appreciated.


Assuntos
Bactérias , Proteínas de Bactérias , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bactérias/metabolismo , Dipeptídeos/genética , Dipeptídeos/metabolismo , Biopolímeros , Nitrogênio/metabolismo , Polímeros
10.
Proc Natl Acad Sci U S A ; 120(26): e2301360120, 2023 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-37339206

RESUMO

Traumatic brain injury (TBI) is a pervasive problem worldwide for which no effective treatment is currently available. Although most studies have focused on the pathology of the injured brain, we have noted that the liver plays an important role in TBI. Using two mouse models of TBI, we found that the enzymatic activity of hepatic soluble epoxide hydrolase (sEH) was rapidly decreased and then returned to normal levels following TBI, whereas such changes were not observed in the kidney, heart, spleen, or lung. Interestingly, genetic downregulation of hepatic Ephx2 (which encodes sEH) ameliorates TBI-induced neurological deficits and promotes neurological function recovery, whereas overexpression of hepatic sEH exacerbates TBI-associated neurological impairments. Furthermore, hepatic sEH ablation was found to promote the generation of A2 phenotype astrocytes and facilitate the production of various neuroprotective factors associated with astrocytes following TBI. We also observed an inverted V-shaped alteration in the plasma levels of four EET (epoxyeicosatrienoic acid) isoforms (5,6-, 8,9-,11,12-, and 14,15-EET) following TBI which were negatively correlated with hepatic sEH activity. However, hepatic sEH manipulation bidirectionally regulates the plasma levels of 14,15-EET, which rapidly crosses the blood-brain barrier. Additionally, we found that the application of 14,15-EET mimicked the neuroprotective effect of hepatic sEH ablation, while 14,15-epoxyeicosa-5(Z)-enoic acid blocked this effect, indicating that the increased plasma levels of 14,15-EET mediated the neuroprotective effect observed after hepatic sEH ablation. These results highlight the neuroprotective role of the liver in TBI and suggest that targeting hepatic EET signaling could represent a promising therapeutic strategy for treating TBI.


Assuntos
Lesões Encefálicas Traumáticas , Fármacos Neuroprotetores , Animais , Camundongos , Fármacos Neuroprotetores/farmacologia , Eicosanoides , Astrócitos , Fígado , Epóxido Hidrolases/genética
11.
J Biol Chem ; 300(2): 105635, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38199576

RESUMO

Microbial epoxide hydrolases, cis-epoxysuccinate hydrolases (CESHs), have been utilized for commercial production of enantiomerically pure L(+)- and D(-)-tartaric acids for decades. However, the stereo-catalytic mechanism of CESH producing L(+)-tartaric acid (CESH[L]) remains unclear. Herein, the crystal structures of two CESH[L]s in ligand-free, product-complexed, and catalytic intermediate forms were determined. These structures revealed the unique specific binding mode for the mirror-symmetric substrate, an active catalytic triad consisting of Asp-His-Glu, and an arginine providing a proton to the oxirane oxygen to facilitate the epoxide ring-opening reaction, which has been pursued for decades. These results provide the structural basis for the rational engineering of these industrial biocatalysts.


Assuntos
Biocatálise , Epóxido Hidrolases , Hidrolases , Epóxido Hidrolases/metabolismo , Hidrolases/química , Hidrolases/genética , Hidrolases/metabolismo , Tartaratos/metabolismo , Modelos Moleculares , Estrutura Terciária de Proteína , Estrutura Quaternária de Proteína
12.
J Biol Chem ; 300(8): 107593, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39032651

RESUMO

KAI2 receptors, classified as plant α/ß hydrolase enzymes, are capable of perceiving smoke-derived butenolide signals and endogenous yet unidentified KAI2-ligands (KLs). While the number of functional KAI2 receptors varies among land plant species, rice has only one KAI2 gene. Rice, a significant crop and representative of grasses, relies on KAI2-mediated Arbuscular mycorrhiza (AM) symbioses to flourish in traditionally arid and nutrient-poor environments. This study presents the first crystal structure of an active rice (Oryza sativa, Os) KAI2 hydrolase receptor. Our structural and biochemical analyses uncover grass-unique pocket residues influencing ligand sensitivity and hydrolytic activity. Through structure-guided analysis, we identify a specific residue whose mutation enables the increase or decrease of ligand perception, catalytic activity, and signal transduction. Furthermore, we investigate OsKAI2-mediated signaling by examining its ability to form a complex with its binding partner, the F-box protein DWARF3 (D3) ubiquitin ligase and subsequent degradation of the target substrate OsSMAX1, demonstrating the significant role of hydrophobic interactions in the OsKAI2-D3 interface. This study provides new insights into the diverse and pivotal roles of the OsKAI2 signaling pathway in the plant kingdom, particularly in grasses.


Assuntos
Oryza , Proteínas de Plantas , Transdução de Sinais , Oryza/metabolismo , Oryza/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Cristalografia por Raios X , Proteínas F-Box/metabolismo , Proteínas F-Box/química , Proteínas F-Box/genética
13.
J Biol Chem ; : 107769, 2024 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-39276930

RESUMO

The bile salt hydrolases (BSHs) are significant constituents of animal microbiomes. An evolving appreciation of their roles in health and disease has established them targets of pharmacological inhibition. These bacterial enzymes belong to the N-terminal nucleophile superfamily and are best known to catalyze the deconjugation of glycine or taurine from bile salts to release bile acid substrates for transformation and or metabolism in the gastrointestinal tract. Here, we identify and describe the BSH from a common member of the Plains bison microbiome, Arthrobacter citreus (BSHAc). Steady-state kinetic analyses demonstrated that the BSHAc is a broad-spectrum hydrolase with a preference for glycine-conjugates and deoxycholic acid (DCA). Second order rate constants (kcat/KM) for BSHAc-catalyzed reactions of relevant bile salts - glyco- and tauro-conjugates of cholic acid and DCA - varied by ∼30-fold and measured between 1.4 x 105 and 4.3 x 106 M-1s-1. Interestingly, a pan-BSH inhibitor named AAA-10 acted as a slow irreversible inhibitor of BSHAc with a rate of inactivation (kinact) of ∼2 h-1 and a second order rate constant (kinact/KI) of ∼24 M-1s-1 for the process. Structural characterization of BSHAc reacted with AAA-10 showed covalent modification of the N-terminal cysteine nucleophile, providing molecular details for an enzyme-stabilized product formed from this mechanism-based inhibitor's α-fluoromethyl ketone warhead. Structural comparison of the BSHs and BSH:inhibitor complexes highlighted the plasticity of the steroid-binding site, including a flexible loop that is variable across well-studied BSHs.

14.
J Biol Chem ; 300(9): 107625, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39122003

RESUMO

Mixed-linkage ß(1,3)/ß(1,4)-glucan (MLG) is abundant in the human diet through the ingestion of cereal grains and is widely associated with healthful effects on metabolism and cholesterol levels. MLG is also a major source of fermentable glucose for the human gut microbiota (HGM). Bacteria from the family Prevotellaceae are highly represented in the HGM of individuals who eat plant-rich diets, including certain indigenous people and vegetarians in postindustrial societies. Here, we have defined and functionally characterized an exemplar Prevotellaceae MLG polysaccharide utilization locus (MLG-PUL) in the type-strain Segatella copri (syn. Prevotella copri) DSM 18205 through transcriptomic, biochemical, and structural biological approaches. In particular, structure-function analysis of the cell-surface glycan-binding proteins and glycoside hydrolases of the S. copri MLG-PUL revealed the molecular basis for glycan capture and saccharification. Notably, syntenic MLG-PULs from human gut, human oral, and ruminant gut Prevotellaceae are distinguished from their counterparts in Bacteroidaceae by the presence of a ß(1,3)-specific endo-glucanase from glycoside hydrolase family 5, subfamily 4 (GH5_4) that initiates MLG backbone cleavage. The definition of a family of homologous MLG-PULs in individual species enabled a survey of nearly 2000 human fecal microbiomes using these genes as molecular markers, which revealed global population-specific distributions of Bacteroidaceae- and Prevotellaceae-mediated MLG utilization. Altogether, the data presented here provide new insight into the molecular basis of ß-glucan metabolism in the HGM, as a basis for informing the development of approaches to improve the nutrition and health of humans and other animals.

15.
J Biol Chem ; 300(9): 107647, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39122010

RESUMO

Curcumin is a plant-derived secondary metabolite exhibiting antitumor, neuroprotective, antidiabetic activities, and so on. We previously isolated Escherichia coli as an enterobacterium exhibiting curcumin-converting activity from human feces, and discovered an enzyme showing this activity (CurA) and named it NADPH-dependent curcumin/dihydrocurcumin reductase. From soil, here, we isolated a curcumin-degrading microorganism (No. 34) using the screening medium containing curcumin as the sole carbon source and identified as Rhodococcus sp. A curcumin-degrading enzyme designated as CurH was purified from this strain and characterized, and compared with CurA. CurH catalyzed hydrolytic cleavage of a carbon-carbon bond in the ß-diketone moiety of curcumin and its analogs, yielding two products bearing a methyl ketone terminus and a carboxylic acid terminus, respectively. These findings demonstrated that a curcumin degradation reaction catalyzed by CurH in the soil environment was completely different from the one catalyzed by CurA in the human microbiome. Of all the curcumin analogs tested, suitable substrates for the enzyme were curcuminoids (i.e., curcumin and bisdemethoxycurcumin) and tetrahydrocurcuminoids. Thus, we named this enzyme curcuminoid hydrolase. The deduced amino acid sequence of curH exhibited similarity to those of members of acetyl-CoA C-acetyltransferase family. Considering results of oxygen isotope analyses and a series of site-directed mutagenesis experiments on our enzyme, we propose a possible catalytic mechanism of CurH, which is unique and distinct from those of enzymes degrading ß-diketone moieties such as ß-diketone hydrolases known so far.

16.
J Biol Chem ; : 107838, 2024 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-39342999

RESUMO

ADP-ribosylation is an ancient posttranslational modification with exceptional versatility in terms of breadth of modification targets including at least seven different amino acid side chains, various moieties on nucleic acids and a variety of small chemical compounds. The spatiotemporal signalling dynamic of the different modification variations is tightly regulated and depends on the writers, erases, and readers of each type. Amongst these, tyrosine ADP-ribosylation (Tyr-ADPr) has been consistently detected as a novel modification type, but systematic analysis of its potential physiological role, modification establishment and reversal are still lacking. Here we present a reanalysis of recent ADP-ribosylome data and show that Tyr-ADPr sites are conserved and enriched amongst ribosome biogenesis and mRNA processing proteins and that these sites are affected by the status of ARH3 ADP-ribose hydrolase. To facilitate the study of Tyr-ADPr, we establish methodologies for the synthesis of well-defined Tyr-ADPr peptides and with these could show that Tyr-ADPr is reversed both by ARH3 and PARG enzymes. Together, our work lays the foundation for the future exploration of the Tyr-ADPr.

17.
J Biol Chem ; 300(5): 107245, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38569940

RESUMO

The IgG-specific endoglycosidases EndoS and EndoS2 from Streptococcus pyogenes can remove conserved N-linked glycans present on the Fc region of host antibodies to inhibit Fc-mediated effector functions. These enzymes are therefore being investigated as therapeutics for suppressing unwanted immune activation, and have additional application as tools for antibody glycan remodeling. EndoS and EndoS2 differ in Fc glycan substrate specificity due to structural differences within their catalytic glycosyl hydrolase domains. However, a chimeric EndoS enzyme with a substituted glycosyl hydrolase from EndoS2 loses catalytic activity, despite high structural homology between the two enzymes, indicating either mechanistic divergence of EndoS and EndoS2, or improperly-formed domain interfaces in the chimeric enzyme. Here, we present the crystal structure of the EndoS2-IgG1 Fc complex determined to 3.0 Å resolution. Comparison of complexed and unliganded EndoS2 reveals relative reorientation of the glycosyl hydrolase, leucine-rich repeat and hybrid immunoglobulin domains. The conformation of the complexed EndoS2 enzyme is also different when compared to the earlier EndoS-IgG1 Fc complex, and results in distinct contact surfaces between the two enzymes and their Fc substrate. These findings indicate mechanistic divergence of EndoS2 and EndoS. It will be important to consider these differences in the design of IgG-specific enzymes, developed to enable customizable antibody glycosylation.


Assuntos
Proteínas de Bactérias , Glicosídeo Hidrolases , Imunoglobulina G , Modelos Moleculares , Streptococcus pyogenes , Humanos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Streptococcus pyogenes/enzimologia , Especificidade por Substrato , Estrutura Quaternária de Proteína
18.
Plant J ; 120(1): 19-28, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39152709

RESUMO

Structural prediction by artificial intelligence can be powerful new instruments to discover novel protein-protein interactions, but the community still grapples with the implementation, opportunities and limitations. Here, we discuss and re-analyse our in silico screen for novel pathogen-secreted inhibitors of immune hydrolases to illustrate the power and limitations of structural predictions. We discuss strategies of curating sequences, including controls, and reusing sequence alignments and highlight important limitations caused by different platforms, sequence depth and computing times. We hope these experiences will support similar interactomic screens by the research community.


Assuntos
Inteligência Artificial , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Mapeamento de Interação de Proteínas , Alinhamento de Sequência
19.
Plant J ; 119(4): 1816-1829, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38899540

RESUMO

Purple carrot accumulates anthocyanins modified with galactose, xylose, glucose, and sinapic acid. Most of the genes associated with anthocyanin biosynthesis have been identified, except for the glucosyltransferase genes involved in the step before the acylation in purple carrot. Anthocyanins are commonly glycosylated in reactions catalyzed by UDP-sugar-dependent glycosyltransferases (UGTs). Although many studies have been conducted on UGTs, the glucosylation of carrot anthocyanins remains unknown. Acyl-glucose-dependent glucosyltransferase activity modifying cyanidin 3-xylosylgalactoside was detected in the crude protein extract prepared from purple carrot cultured cells. In addition, the corresponding enzyme was purified. The cDNA encoding this glucosyltransferase was isolated based on the partial amino acid sequence of the purified protein. The recombinant protein produced in Nicotiana benthamiana leaves via agroinfiltration exhibited anthocyanin glucosyltransferase activity. This glucosyltransferase belongs to the glycoside hydrolase family 3 (GH3). The expression pattern of the gene encoding this GH3-type anthocyanin glucosyltransferase was consistent with anthocyanin accumulation in carrot tissues and cultured cells.


Assuntos
Antocianinas , Daucus carota , Proteínas de Plantas , Daucus carota/genética , Daucus carota/metabolismo , Daucus carota/enzimologia , Antocianinas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/genética , Glucosiltransferases/metabolismo , Glucosiltransferases/genética , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/enzimologia , Glicosilação , Regulação da Expressão Gênica de Plantas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Sequência de Aminoácidos
20.
Plant J ; 118(4): 1218-1231, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38323895

RESUMO

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.


Assuntos
Canfanos , Nudix Hidrolases , Proteínas de Plantas , Pirofosfatases , Pirofosfatases/metabolismo , Pirofosfatases/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Canfanos/metabolismo , Brassicaceae/genética , Brassicaceae/enzimologia , Brassicaceae/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo
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