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1.
Food Chem ; 369: 130942, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34479010

RESUMO

2'-Fucosyllactose (2'-FL) is one of the nutrient ingredients in human milk, which has various beneficial health effects. α-l-fucosidase is a biotechnological tool for 2'-FL preparation. Here, a novel and efficient α-l-fucosidase OUC-Jdch16 from the fucoidan-digesting strain Flavobacterium algicola 12076 was heterologously expressed and applied to produce 2'-FL in vitro. OUC-Jdch16 belongs to glycoside hydrolases (GH) family 29 and exhibits the highest 4-nitrophenyl-α-l-fucopyranoside-hydrolyzing activity at 25 °C and pH 6.0. OUC-Jdch16 could catalyze the synthesis of 2'-FL via transferring the fucosyl residue from pNP-α-fucose to lactose. Under the optimal transfucosylation conditions, the yield of the transfucosylation product reached 84.82% and 92.15% (mol/mol) from pNP-α-fucose within 48 h and 120 h, respectively. Moreover, OUC-Jdch16 was capable of transferring the fucosyl residue to other glycosyl receptors with the generation of novel fucosylated compounds. This study demonstrated that OUC-Jdch16 could be a promising tool to prepare 2'-FL and other novel glycosides.


Assuntos
Oligossacarídeos , alfa-L-Fucosidase , Flavobacterium , Fucose , Humanos , Especificidade por Substrato , Trissacarídeos , alfa-L-Fucosidase/genética , alfa-L-Fucosidase/metabolismo
2.
Anal Chem ; 93(45): 15150-15158, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34738799

RESUMO

Mimicking enzyme specificity via construction of on-demand geometric structures on nanozymes is of great interest in recent years. Although building substrate-specific polymers on nanozymes has achieved great success, polymer-blocked active sites would inevitably lead to decreased activity of nanozymes. Here, we have developed three photoactive metal-organic framework (MOF)-based nanozymes (called 2D-TCPP, 3D-TCPP, and AD-TCPP), which have different geometric structures as well as unshielded active sites. Together with their structural variations and excellent photoresponsive oxidase-like activities, these photoactive nanozymes exhibit structure-dependent specificity for three kinds of substrates (typical oxidase substrates, organic pollutants, and antioxidants). Moreover, AD-TCPP and 3D-TCPP show potential applications for environmental protection and bioanalysis, respectively. This work offers a promising approach to the development of nanozymes with enzyme-like specificity.


Assuntos
Estruturas Metalorgânicas , Nanoestruturas , Catálise , Oxirredutases , Especificidade por Substrato
3.
J Chem Inf Model ; 61(11): 5626-5643, 2021 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-34748335

RESUMO

PlaF is a cytoplasmic membrane-bound phospholipase A1 from Pseudomonas aeruginosa that alters the membrane glycerophospholipid (GPL) composition and fosters the virulence of this human pathogen. PlaF activity is regulated by a dimer-to-monomer transition followed by tilting of the monomer in the membrane. However, how substrates reach the active site and how the characteristics of the active site tunnels determine the activity, specificity, and regioselectivity of PlaF for natural GPL substrates have remained elusive. Here, we combined unbiased and biased all-atom molecular dynamics (MD) simulations and configurational free-energy computations to identify access pathways of GPL substrates to the catalytic center of PlaF. Our results map out a distinct tunnel through which substrates access the catalytic center. PlaF variants with bulky tryptophan residues in this tunnel revealed decreased catalysis rates due to tunnel blockage. The MD simulations suggest that GPLs preferably enter the active site with the sn-1 acyl chain first, which agrees with the experimentally demonstrated PLA1 activity of PlaF. We propose that the acyl chain-length specificity of PlaF is determined by the structural features of the access tunnel, which results in favorable free energy of binding of medium-chain GPLs. The suggested egress route conveys fatty acid (FA) products to the dimerization interface and, thus, contributes to understanding the product feedback regulation of PlaF by FA-triggered dimerization. These findings open up opportunities for developing potential PlaF inhibitors, which may act as antibiotics against P. aeruginosa.


Assuntos
Simulação de Dinâmica Molecular , Pseudomonas aeruginosa , Domínio Catalítico , Dimerização , Humanos , Fosfolipases , Especificidade por Substrato
4.
Proc Natl Acad Sci U S A ; 118(43)2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34635581

RESUMO

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.


Assuntos
Benzotiazóis/farmacologia , COVID-19/tratamento farmacológico , Oligopeptídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/genética , Animais , Benzamidinas/química , Benzotiazóis/farmacocinética , COVID-19/genética , COVID-19/virologia , Linhagem Celular , Desenho de Fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Ésteres/química , Guanidinas/química , Humanos , Pulmão/efeitos dos fármacos , Pulmão/virologia , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Oligopeptídeos/farmacocinética , SARS-CoV-2/patogenicidade , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/ultraestrutura , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
5.
Nature ; 598(7880): 332-337, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34616040

RESUMO

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium1. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization2 and diseases such as inflammatory bowel disease3.


Assuntos
Bacteroides/enzimologia , Colo/metabolismo , Colo/microbiologia , Microbioma Gastrointestinal , Mucinas/metabolismo , Sulfatases/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Animais , Colo/química , Cristalografia por Raios X , Feminino , Galactose/metabolismo , Humanos , Masculino , Camundongos , Modelos Moleculares , Especificidade por Substrato , Sulfatases/química
6.
Nat Commun ; 12(1): 6173, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34702852

RESUMO

The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. These complexes have common catalytic sites, which makes it challenging to determine their distinctive roles in intracellular proteolysis. Here, we chemically synthesize a panel of homogenous ubiquitinated proteins, and use them to compare 20S and 26S proteasomes with respect to substrate selection and peptide-product generation. We show that 20S proteasomes can degrade the ubiquitin tag along with the conjugated substrate. Ubiquitin remnants on branched peptide products identified by LC-MS/MS, and flexibility in the 20S gate observed by cryo-EM, reflect the ability of the 20S proteasome to proteolyze an isopeptide-linked ubiquitin-conjugate. Peptidomics identifies proteasome-trapped ubiquitin-derived peptides and peptides of potential 20S substrates in Hi20S cells, hypoxic cells, and human failing-heart. Moreover, elevated levels of 20S proteasomes appear to contribute to cell survival under stress associated with damaged proteins.


Assuntos
Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Hipóxia Celular , Sobrevivência Celular , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Proteólise , Especificidade por Substrato , Ubiquitina/química , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação
7.
PLoS One ; 16(10): e0256817, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34699529

RESUMO

The glycoside hydrolase 19 (GH19) is a bifunctional family of chitinases and endolysins, which have been studied for the control of plant fungal pests, the recycle of chitin biomass, and the treatment of multi-drug resistant bacteria. The GH19 domain-containing sequences (22,461) were divided into a chitinase and an endolysin subfamily by analyzing sequence networks, guided by taxonomy and the substrate specificity of characterized enzymes. The chitinase subfamily was split into seventeen groups, thus extending the previous classification. The endolysin subfamily is more diverse and consists of thirty-four groups. Despite their sequence diversity, twenty-six residues are conserved in chitinases and endolysins, which can be distinguished by two specific sequence patterns at six and four positions, respectively. Their location outside the catalytic cleft suggests a possible mechanism for substrate specificity that goes beyond the direct interaction with the substrate. The evolution of the GH19 catalytic domain was investigated by large-scale phylogeny. The inferred evolutionary history and putative horizontal gene transfer events differ from previous works. While no clear patterns were detected in endolysins, chitinases varied in sequence length by up to four loop insertions, causing at least eight distinct presence/absence loop combinations. The annotated GH19 sequences and structures are accessible via the GH19 Engineering Database (GH19ED, https://gh19ed.biocatnet.de). The GH19ED has been developed to support the prediction of substrate specificity and the search for novel GH19 enzymes from neglected taxonomic groups or in regions of the sequence space where few sequences have been described yet.


Assuntos
Quitinases/genética , Endopeptidases/genética , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Quitinases/química , Quitinases/metabolismo , Bases de Dados de Proteínas , Endopeptidases/química , Endopeptidases/metabolismo , Evolução Molecular , Fungos/química , Fungos/genética , Fungos/metabolismo , Humanos , Modelos Moleculares , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformação Proteica , Especificidade por Substrato
8.
J Chem Inf Model ; 61(11): 5569-5580, 2021 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-34653331

RESUMO

ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for industrial production of chiral amines. Tailoring ω-TAs to accept non-natural substrates is necessary because of their limited substrate range. We present a computational protocol for predicting the enantioselectivity and catalytic selectivity of an ω-TA from Vibrio fluvialis with different substrates and benchmark it against 62 compounds gathered from the literature. Rosetta-generated complexes containing an external aldimine intermediate of the transamination reaction are used as starting conformations for multiple short independent molecular dynamics (MD) simulations. The combination of molecular docking and MD simulations ensures sufficient and accurate sampling of the relevant conformational space. Based on the frequency of near-attack conformations observed during the MD trajectories, enantioselectivities can be quantitatively predicted. The predicted enantioselectivities are in agreement with a benchmark dataset of experimentally determined ee% values. The substrate-range predictions can be based on the docking score of the external aldimine intermediate. The low computational cost required to run the presented framework makes it feasible for use in enzyme design to screen thousands of enzyme variants.


Assuntos
Simulação de Dinâmica Molecular , Transaminases , Simulação de Acoplamento Molecular , Especificidade por Substrato , Transaminases/metabolismo , Vibrio
9.
Molecules ; 26(19)2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34641578

RESUMO

Choanoflagellates are single-celled eukaryotes with complex signaling pathways. They are considered the closest non-metazoan ancestors to mammals and other metazoans and form multicellular-like states called rosettes. The choanoflagellate Monosiga brevicollis contains over 150 PDZ domains, an important peptide-binding domain in all three domains of life (Archaea, Bacteria, and Eukarya). Therefore, an understanding of PDZ domain signaling pathways in choanoflagellates may provide insight into the origins of multicellularity. PDZ domains recognize the C-terminus of target proteins and regulate signaling and trafficking pathways, as well as cellular adhesion. Here, we developed a computational software suite, Domain Analysis and Motif Matcher (DAMM), that analyzes peptide-binding cleft sequence identity as compared with human PDZ domains and that can be used in combination with literature searches of known human PDZ-interacting sequences to predict target specificity in choanoflagellate PDZ domains. We used this program, protein biochemistry, fluorescence polarization, and structural analyses to characterize the specificity of A9UPE9_MONBE, a M. brevicollis PDZ domain-containing protein with no homology to any metazoan protein, finding that its PDZ domain is most similar to those of the DLG family. We then identified two endogenous sequences that bind A9UPE9 PDZ with <100 µM affinity, a value commonly considered the threshold for cellular PDZ-peptide interactions. Taken together, this approach can be used to predict cellular targets of previously uncharacterized PDZ domains in choanoflagellates and other organisms. Our data contribute to investigations into choanoflagellate signaling and how it informs metazoan evolution.


Assuntos
Coanoflagelados/química , Coanoflagelados/metabolismo , Biologia Computacional/métodos , Domínios PDZ , Ligação Proteica , Sequência de Aminoácidos , Evolução Molecular , Humanos , Filogenia , Conformação Proteica , Transdução de Sinais , Software , Especificidade por Substrato
10.
Enzyme Microb Technol ; 151: 109916, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34649687

RESUMO

Alginate oligosaccharides are enzymolysis products of alginate with versatile bioactivities and their industrial preparation was limited by the insufficient activity and unsatisfying thermostability of alginate lyases. The structure-function information about PL18 alginate lyases was seldom reported since which few positive mutants of PL18 alginate lyases were generated. In present study, a mutant of PL18 alginate lyase E226K was expressed intracellularly and taken as parent for further modification. Site I211 at the lid loop 1 and sites E276, Y292 and R294 at the predicted entrance were chosen as engineering targets based on the E226K-PM4 binding mode in prereaction-state MD simulation and 29 mutants were constructed, from those, the variant E226K/I211T/R294V was screened out as the best mutant (showing 4.78-fold increased catalytic efficiency and the half-time t1/245℃ increased up to 557 min from 89 min). MD simulations indicated that the affinity of E226K/I211T/R294V towards alginate was improved due to the optimized energy distribution of active center, more flexible loops around catalytic cleft and larger substrate entrance. The more efficient proton transmitting endowed E226K/I211T/R294V higher activity and the more complicated intraprotein interactions together with stronger backbone rigidity were responsible for the improved thermostability of E226K/I211T/R294V than E226K. The success in this study enriches the structure-function information of PL18 alginate lyases and provides hints for their further design.


Assuntos
Alginatos , Polissacarídeo-Liases , Catálise , Oligossacarídeos , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Especificidade por Substrato
11.
Enzyme Microb Technol ; 151: 109921, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34649692

RESUMO

ß-Xylosidases are often inhibited by its reaction product xylose or inactivated by high temperature environment, which limited its application in hemicellulosic biomass conversion to fuel and food processing. Remarkably, some ß-xylosidases from GH39 family are tolerant to xylose. Therefore, it is of great significance to elucidate the effect mechanism of xylose on GH39 ß-xylosidases to improve their application. In this paper, based on the homologous model and prediction of protein active pocket constructed by I-TASSA and PyMOL, two putative xylose tolerance relevant sites (283 and 284) were mutated at the bottom of the protein active pocket, where xylose sensitivity and thermostability of Dictyoglomus thermophilum ß-xylosidase Xln-DT were improved by site-directed mutagenesis. The Xln-DT mutant Xln-DT-284ASP and Xln-DT-284ALA showed high xylose tolerance, with the Ki values of 4602 mM and 3708 mM, respectively, which increased by 9-35% compared with the wildtype Xln-DT. The thermostability of mutant Xln-DT-284ASP was significantly improved at 75 and 85 °C, while the activity of the wild enzyme Xln-DT decreased to 40-20%, the activity of the mutant enzyme still remained 100%. The mutant Xln-DT-284ALA showed excellent stability at pH 4.0-7.0, but Xln-DT-284ASP showed slightly decreased activity. Furthermore, in order to explore the key sites and mechanism of xylose's effect on ß-xylosidase activity, the interaction between xylose and enzyme was simulated by molecular docking. Besides binding to the active sites at the bottom of the substrate channel, xylose can also bind to sites in the middle or entrance of the channel with different affinities, which may determine the xylose inhibition of ß-xylosidase. In conclusion, the improved xylose tolerance of mutant enzyme could be more advantageous in the degradation of hemicellulose and the biotransformation of other natural active substances containing xylose. This study supplies new insights into general mechanism of xylose effect on the activity of GH 39 ß-xylosidases as well as related enzymes that modulate their activity via feedback control mechanism.


Assuntos
Xilose , Xilosidases , Bactérias , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Especificidade por Substrato , Xilosidases/genética , Xilosidases/metabolismo
12.
Int J Mol Sci ; 22(19)2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34639118

RESUMO

An α-galactosidase-producing strain named Anoxybacillus vitaminiphilus WMF1, which catalyzed the reverse hydrolysis of d-galactose and glycerol to produce isofloridoside, was isolated from soil. The α-galactosidase (galV) gene was cloned and expressed in Escherichia coli. The galV was classified into the GH36 family with a molecular mass of 80 kDa. The optimum pH and temperature of galV was pH 7.5 and 60 °C, respectively, and it was highly stable at alkaline pH (6.0-9.0) and temperature below 65 °C. The specificity for p-nitrophenyl α-d-galactopyranoside was 70 U/mg, much higher than that for raffinose and stachyose. Among the metals and reagents tested, galV showed tolerance in the presence of various organic solvents. The kinetic parameters of the enzyme towards p-nitrophenyl α-d-galactopyranoside were obtained as Km (0.12 mM), Vmax (1.10 × 10-3 mM s-1), and Kcat/Km (763.92 mM-1 s-1). During the reaction of reverse hydrolysis, the enzyme exhibited high specificity towards the glycosyl donor galactose and acceptors glycerol, ethanol and ethylene glycol. Finally, the isofloridoside was synthesized using galactose as the donor and glycerol as the acceptor with a 26.6% conversion rate of galactose. This study indicated that galV might provide a potential enzyme source in producing isofloridoside because of its high thermal stability and activity.


Assuntos
Anoxybacillus/enzimologia , Galactosídeos/biossíntese , Temperatura Alta , alfa-Galactosidase/metabolismo , Sequência de Aminoácidos , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Peso Molecular , Homologia de Sequência , Especificidade por Substrato , alfa-Galactosidase/química
13.
Int J Mol Sci ; 22(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34639143

RESUMO

Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Hemostáticos/metabolismo , Trombina/metabolismo , Animais , Aptâmeros de Nucleotídeos/química , Sítios de Ligação , Hemostáticos/química , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Especificidade por Substrato , Trombina/química
14.
Nat Commun ; 12(1): 5939, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34642328

RESUMO

Ubiquitin (Ub) and Ub-like proteins (Ubls) such as NEDD8 are best known for their function as covalent modifiers of other proteins but they are also themselves subject to post-translational modifications including phosphorylation. While functions of phosphorylated Ub (pUb) have been characterized, the consequences of Ubl phosphorylation remain unclear. Here we report that NEDD8 can be phosphorylated at S65 - the same site as Ub - and that S65 phosphorylation affects the structural dynamics of NEDD8 and Ub in a similar manner. While both pUb and phosphorylated NEDD8 (pNEDD8) can allosterically activate the Ub ligase Parkin, they have different protein interactomes that in turn are distinct from those of unmodified Ub and NEDD8. Among the preferential pNEDD8 interactors are HSP70 family members and we show that pNEDD8 stimulates HSP70 ATPase activity more pronouncedly than unmodified NEDD8. Our findings highlight the general importance of Ub/NEDD8 phosphorylation and support the notion that the function of pUb/pNEDD8 does not require their covalent attachment to other proteins.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteína NEDD8/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/classificação , Proteínas de Choque Térmico HSP70/genética , Humanos , Cinética , Proteína NEDD8/química , Proteína NEDD8/genética , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Ubiquitina/química , Ubiquitina/genética , Ubiquitinação
15.
Cell Rep ; 37(4): 109892, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34672947

RESUMO

The main viral protease (3CLpro) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CLpro by TAILS substrate-targeted N-terminomics. We identify more than 100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CLpro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CLpro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike in healthy lungs. The 3CLpro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.


Assuntos
COVID-19 , Proteases 3C de Coronavírus/metabolismo , SARS-CoV-2/metabolismo , Humanos , Especificidade por Substrato
16.
Nat Commun ; 12(1): 5969, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645811

RESUMO

The Yersinia outer protein J (YopJ) family effectors are widely deployed through the type III secretion system by both plant and animal pathogens. As non-canonical acetyltransferases, the enzymatic activities of YopJ family effectors are allosterically activated by the eukaryote-specific ligand inositol hexaphosphate (InsP6). However, the underpinning molecular mechanism remains undefined. Here we present the crystal structure of apo-PopP2, a YopJ family member secreted by the plant pathogen Ralstonia solanacearum. Structural comparison of apo-PopP2 with the InsP6-bound PopP2 reveals a substantial conformational readjustment centered in the substrate-binding site. Combining biochemical and computational analyses, we further identify a mechanism by which the association of InsP6 with PopP2 induces an α-helix-to-ß-strand transition in the catalytic core, resulting in stabilization of the substrate recognition helix in the target protein binding site. Together, our study uncovers the molecular basis governing InsP6-mediated allosteric regulation of YopJ family acetyltransferases and further expands the paradigm of fold-switching proteins.


Assuntos
Acetiltransferases/química , Apoproteínas/química , Arabidopsis/microbiologia , Proteínas de Bactérias/química , Ácido Fítico/química , Ralstonia solanacearum/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Regulação Alostérica , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ácido Fítico/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Ralstonia solanacearum/enzimologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Tabaco/microbiologia
17.
Nat Commun ; 12(1): 5963, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645814

RESUMO

P4 ATPases are lipid flippases that are phylogenetically grouped into P4A, P4B and P4C clades. The P4A ATPases are heterodimers composed of a catalytic α-subunit and accessory ß-subunit, and the structures of several heterodimeric flippases have been reported. The S. cerevisiae Neo1 and its orthologs represent the P4B ATPases, which function as monomeric flippases without a ß-subunit. It has been unclear whether monomeric flippases retain the architecture and transport mechanism of the dimeric flippases. Here we report the structure of a P4B ATPase, Neo1, in its E1-ATP, E2P-transition, and E2P states. The structure reveals a conserved architecture as well as highly similar functional intermediate states relative to dimeric flippases. Consistently, structure-guided mutagenesis of residues in the proposed substrate translocation path disrupted Neo1's ability to establish membrane asymmetry. These observations indicate that evolutionarily distant P4 ATPases use a structurally conserved mechanism for substrate transport.


Assuntos
Adenosina Trifosfatases/química , Lisofosfolipídeos/química , Proteínas de Membrana Transportadoras/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Membrana Celular/química , Membrana Celular/enzimologia , Clonagem Molecular , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Lisofosfolipídeos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
18.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638918

RESUMO

Mining of phospholipase D (PLD) with altered acyl group recognition except its head group specificity is also useful in terms of specific acyl size phospholipid production and as diagnostic reagents for quantifying specific phospholipid species. Microbial PLDs from Actinomycetes, especially Streptomyces, best fit this process requirements. In the present studies, a new PLD from marine Streptomyces klenkii (SkPLD) was purified and biochemically characterized. The optimal reaction temperature and pH of SkPLD were determined to be 60 °C and 8.0, respectively. Kinetic analysis showed that SkPLD had the relatively high catalytic efficiency toward phosphatidylcholines (PCs) with medium acyl chain length, especially 12:0/12:0-PC (67.13 S-1 mM-1), but lower catalytic efficiency toward PCs with long acyl chain (>16 fatty acids). Molecular docking results indicated that the different catalytic efficiency was related to the increased steric hindrance of long acyl-chains in the substrate-binding pockets and differences in hydrogen-bond interactions between the acyl chains and substrate-binding pockets. The enzyme displayed suitable transphosphatidylation activity and the reaction process showed 26.18% yield with L-serine and soybean PC as substrates. Present study not only enriched the PLD enzyme library but also provide guidance for the further mining of PLDs with special phospholipids recognition properties.


Assuntos
Proteínas de Bactérias/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipase D/metabolismo , Streptomyces/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Concentração de Íons de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Fosfatidilcolinas/metabolismo , Fosfolipase D/química , Fosfolipase D/genética , Fosfolipídeos/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Água do Mar/microbiologia , Homologia de Sequência de Aminoácidos , Streptomyces/genética , Especificidade por Substrato , Temperatura
19.
Int J Mol Sci ; 22(19)2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34639003

RESUMO

Measuring various biochemical and cellular components in the blood is a routine procedure in clinical practice. Human serum contains hundreds of diverse proteins secreted from all cells and tissues in healthy and diseased states. Moreover, some serum proteins have specific strong interactions with other blood components, but most interactions are probably weak and transient. One of the serum proteins is butyrylcholinesterase (BChE), an enzyme existing mainly as a glycosylated soluble tetramer that plays an important role in the metabolism of many drugs. Our results suggest that BChE interacts with plasma proteins and forms much larger complexes than predicted from the molecular weight of the BChE tetramer. To investigate and isolate such complexes, we developed a two-step strategy to find specific protein-protein interactions by combining native size-exclusion chromatography (SEC) with affinity chromatography with the resin that specifically binds BChE. Second, to confirm protein complexes' specificity, we fractionated blood serum proteins by density gradient ultracentrifugation followed by co-immunoprecipitation with anti-BChE monoclonal antibodies. The proteins coisolated in complexes with BChE were identified by mass spectroscopy. These binding studies revealed that BChE interacts with a number of proteins in the human serum. Some of these interactions seem to be more stable than transient. BChE copurification with ApoA-I and the density of some fractions containing BChE corresponding to high-density lipoprotein cholesterol (HDL) during ultracentrifugation suggest its interactions with HDL. Moreover, we observed lower BChE plasma activity in individuals with severely reduced HDL levels (≤20 mg/dL). The presented two-step methodology for determination of the BChE interactions can facilitate further analysis of such complexes, especially from the brain tissue, where BChE could be involved in the pathogenesis and progression of AD.


Assuntos
Proteínas Sanguíneas/metabolismo , Butirilcolinesterase/metabolismo , Proteínas Sanguíneas/química , Butirilcolinesterase/química , Proteínas de Transporte , Centrifugação com Gradiente de Concentração/métodos , HDL-Colesterol , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Ativação Enzimática , Humanos , Imunoprecipitação , Espectrometria de Massas , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Ligação Proteica , Especificidade por Substrato
20.
Int J Mol Sci ; 22(19)2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34639033

RESUMO

N-terminal acetylation (Nt-acetylation) catalyzed by conserved N-terminal acetyltransferases or NATs embodies a modification with one of the highest stoichiometries reported for eukaryotic protein modifications to date. Comprising the catalytic N-alpha acetyltransferase (NAA) subunit NAA10 plus the ribosome anchoring regulatory subunit NAA15, NatA represents the major acetyltransferase complex with up to 50% of all mammalian proteins representing potential substrates. Largely in consequence of the essential nature of NatA and its high enzymatic activity, its experimentally confirmed mammalian substrate repertoire remained poorly charted. In this study, human NatA knockdown conditions achieving near complete depletion of NAA10 and NAA15 expression resulted in lowered Nt-acetylation of over 25% out of all putative NatA targets identified, representing an up to 10-fold increase in the reported number of substrate N-termini affected upon human NatA perturbation. Besides pointing to less efficient NatA substrates being prime targets, several putative NatE substrates were shown to be affected upon human NatA knockdown. Intriguingly, next to a lowered expression of ribosomal proteins and proteins constituting the eukaryotic 48S preinitiation complex, steady-state levels of protein N-termini additionally point to NatA Nt-acetylation deficiency directly impacting protein stability of knockdown affected targets.


Assuntos
Acetiltransferase N-Terminal A/química , Acetiltransferase N-Terminal A/metabolismo , Acetilação , Catálise , Quinases Ciclina-Dependentes/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Metabolismo dos Lipídeos , Acetiltransferase N-Terminal A/genética , Proteoma , Proteômica/métodos , Especificidade por Substrato
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