RESUMO
The canine anti-tumor necrosis factor-alpha (TNF-α) monoclonal antibody is a potential therapeutic option for treating canine arthritis. The current treatments for arthritis in dogs have limitations due to side effects, emphasizing the need for safer and more effective therapies. The crystal structure of canine TNF-α (cTNF-α) was successfully determined at a resolution of 1.85 Å, and the protein was shown to assemble as a trimer, with high similarity to the functional quaternary structure of human TNF-α (hTNF-α). Adalimumab (Humira), a known TNF-α inhibitor, effectively targets and neutralizes TNF-α to reduce inflammation and has been used to manage autoimmune conditions such as rheumatoid arthritis. By comparing the structure of cTNF-α with the complex structure of hTNF-α and adalimumab-Fab, the epitope of adalimumab on cTNF-α was identified. The significant structural similarities of epitopes in cTNF-α and hTNF-α indicate the potential of using adalimumab to target cTNF-α. Therefore, a canine/human chimeric antibody, Humivet-R1, was created by grafting the variable domain of adalimumab onto a canine antibody framework derived from ranevetmab. Humivet-R1 exhibits potent neutralizing ability (IC50 = 0.05 nM) and high binding affinity (EC50 = 0.416 nM) to cTNF-α, comparable to that of adalimumab for both hTNF-α and cTNF-α. These results strongly suggest that Humivet-R1 has the potential to provide effective treatment for canine arthritis with reduced side effects. Here, we propose a structure-guided antibody design for the use of a chimeric antibody to treat canine inflammatory disease. Our successful development strategy can speed up therapeutic antibody discovery for animals and has the potential to revolutionize veterinary medicine.
Assuntos
Artrite Reumatoide , Fator de Necrose Tumoral alfa , Cães , Animais , Humanos , Adalimumab/farmacologia , Adalimumab/uso terapêutico , Anticorpos Monoclonais , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologiaRESUMO
Translational readthrough of UGA stop codons by selenocysteine-specific tRNA (tRNASec) enables the synthesis of selenoproteins. Seryl-tRNA synthetase (SerRS) charges tRNASec with serine, which is modified into selenocysteine and delivered to the ribosome by a designated elongation factor (eEFSec in eukaryotes). Here we found that components of the human selenocysteine incorporation machinery (SerRS, tRNASec, and eEFSec) also increased translational readthrough of non-selenocysteine genes, including VEGFA, to create C-terminally extended isoforms. SerRS recognizes target mRNAs through a stem-loop structure that resembles the variable loop of its cognate tRNAs. This function of SerRS depends on both its enzymatic activity and a vertebrate-specific domain. Through eCLIP-seq, we identified additional SerRS-interacting mRNAs as potential readthrough genes. Moreover, SerRS overexpression was sufficient to reverse premature termination caused by a pathogenic nonsense mutation. Our findings expand the repertoire of selenoprotein biosynthesis machinery and suggest an avenue for therapeutic targeting of nonsense mutations using endogenous factors.
Assuntos
Biossíntese de Proteínas , Serina-tRNA Ligase , Humanos , Códon sem Sentido , Códon de Terminação , RNA Mensageiro/metabolismo , Selenocisteína/genética , Selenocisteína/metabolismo , Selenoproteínas/genética , Serina-tRNA Ligase/genéticaRESUMO
Class II histone deacetylases (HDACs) are considered as potential targets to treat Alzheimer's disease (AD). Previously, C-3 substituted phenothiazine-containing compounds with class II HDAC-inhibiting activities was found to promote neurite outgrowth. This study replaced phenothiazine moiety with phenoxazine that contains many C-3 and C-4 substituents. Some resulting compounds bearing the C-4 substituent on a phenoxazine ring displayed potent class II HDAC inhibitory activities. Structure-activity relationship (SAR) of these compounds that inhibited HDAC isoenzymes was disclosed. Molecular modelling analysis demonstrates that the potent activities of C-4 substituted compounds probably arise from π-π stacked interactions between these compounds and class IIa HDAC enzymes. One of these, compound 7d exhibited the most potent class II HDAC inhibition (IC50= 3-870 nM). Notably, it protected neuron cells from H2O2-induced neuron damage at sub-µM concentrations, but with no significant cytotoxicity. These findings show that compound 7d is a lead compound for further development of anti-neurodegenerative agents.
Assuntos
Antineoplásicos , Ácidos Hidroxâmicos , Ácidos Hidroxâmicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Peróxido de Hidrogênio/farmacologia , Relação Estrutura-Atividade , Histona Desacetilases/metabolismo , Antineoplásicos/farmacologia , Histona Desacetilase 1/farmacologia , Proliferação de CélulasRESUMO
TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor-binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support.
Assuntos
5-Metilcitosina , Dioxigenases , 5-Metilcitosina/metabolismo , Animais , Domínio Catalítico , Fenômenos Fisiológicos Celulares , DNA , Dioxigenases/genética , Dioxigenases/metabolismo , Mamíferos/genéticaRESUMO
A specific α-oxoamine synthase (VsAOS-2) and an oxidoreductase (VsOR) identified from marine Vibrio sp. QWI-06 were involved in the decarboxylative condensation of l-tyrosine to lauroyl-CoA following the reduction of the ketone group to form vitroprocine-type compound 1. The intermediates and products were characterized through HR-MS and their MS/MS fragmentations. This study reveals the biosynthetic pathway of vitroprocines and provides a useful model for elucidating the reaction mechanism underlying the production of amino acid-polyketide derivatives in microorganisms.
Assuntos
Policetídeos , Vibrio , Oxirredutases/metabolismo , Policetídeos/metabolismo , Espectrometria de Massas em TandemRESUMO
BACKGROUND: K1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. pneumoniae infection. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge. METHODS: We analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. pneumoniae-infected mice with the wild-type and mutant enzymes. RESULTS: We found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1-3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric ß-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also demonstrate that the enzyme is capable of protecting mice from K1 K. pneumoniae infection, even against a high challenge dose. CONCLUSIONS: Our results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism.
Assuntos
Bacteriófagos , Infecções por Klebsiella , Liases , Animais , Cápsulas Bacterianas/genética , Bacteriófagos/genética , Humanos , Cinética , Klebsiella pneumoniae , CamundongosRESUMO
Specific antibody interactions with short peptides have made epitope tagging systems a vital tool employed in virtually all fields of biological research. Here, we present a novel epitope tagging system comprised of a monoclonal antibody named GD-26, which recognises the TD peptide (GTGATPADD) derived from Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant. The crystal structure of the antigen-binding fragment (Fab) of GD-26 complexed with the TD peptide was determined to a resolution of 1.45 Å. The TD peptide was found to adopt a 310 helix conformation within the binding cleft, providing a characteristic peptide structure for recognition by GD-26 Fab. Based on the structure information, polar and nonpolar forces collectively contribute to the strong binding. Attempts to engineer the TD peptide show that the proline residue is crucial for the formation of the 310 helix in order to fit into the binding cleft. Isothermal calorimetry (ITC) reported a dissociation constant KD of 12 ± 2.8 nm, indicating a strong interaction between the TD peptide and GD-26 Fab. High specificity of GD-26 IgG to the TD peptide was demonstrated by western blotting, ELISA and immunofluorescence as only TD-tagged proteins were detected, suggesting the effectiveness of the GD-26/TD peptide tagging system. In addition to already-existing epitope tags such as the FLAG tag and the ALFA tag adopting either extended or α-helix conformations, the unique 310 helix conformation of the TD peptide together with the corresponding monoclonal antibody GD-26 offers a novel tagging option for research.
Assuntos
Anticorpos Monoclonais/imunologia , Bacteriorodopsinas/imunologia , Epitopos/imunologia , Peptídeos/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/ultraestrutura , Especificidade de Anticorpos/genética , Bacteriorodopsinas/genética , Bacteriorodopsinas/ultraestrutura , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/ultraestrutura , Haloarcula marismortui/imunologia , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Peptídeos/genéticaRESUMO
The pathogenesis of Alzheimer's disease (AD) has been associated with dysregulation of histone deacetylases (HDACs). Previously, acridine-based HDAC inhibitors have shown potential in ameliorating HDAC activity and enhancing neurite outgrowth. In this study, the acridine ring was modified using various phenothiazine derivatives. Several resulting compounds exhibited potent enzyme-inhibiting activity towards class II HDACs when compared to the clinically approved HDAC inhibitor SAHA. Compound 4f demonstrated the highest class II HDAC inhibition (IC50 = 4.6-600 nM), as well as promotion of neurite outgrowth. Importantly, compound 4f displayed no cytotoxicity against neuron cells. Compound 4f was further evaluated for cellular effects. Altogether, these findings show a potential strategy in HDAC inhibition for treatment of the neurological disease.
Assuntos
Inibidores de Histona Desacetilases/síntese química , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Fenotiazinas/química , Acetilação/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Sítios de Ligação , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Simulação de Acoplamento Molecular , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Fenotiazinas/metabolismo , Fenotiazinas/farmacologia , Fenotiazinas/uso terapêutico , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismoRESUMO
The formation of succinimide in proteins has attracted considerable attention in protein aging and biopharmaceutical research. The succinimide formation occurs spontaneously in proteins and is prone to hydrolysis to yield aspartate and isoaspartate, resulting in altered protein functions. Herein, we demonstrated that the coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) can mediate intramolecular cyclization of aspartic acid to form succinimide efficiently in the LL37-derived short antimicrobial peptide KR12. The formation of succinimide in KR12 was confirmed by liquid chromatography tandem mass spectrometry and nuclear magnetic resonance. Moreover, the succinimide-containing KR12 displayed decreased antimicrobial activity, helicity, and serum stability in comparison with unmodified KR12. The succinimide formation usually changes the protein structure and function, and only in rare cases, it can help to maintain the protein stability. In addition to succinimide, DMTMM can also mediate intraresidue cyclization of N-terminal glutamate to form pyroglutamate. Our work thus provides a convenient and efficient method for preparation of succinimide/pyroglutamate-containing peptides, which can be used for studying their impact on peptide/protein function.
RESUMO
Proteins with sequence or structure similar to those of di-Zn exopeptidases are usually classified as the M28-family enzymes, including the mammalian-type glutaminyl cyclases (QCs). QC catalyzes protein N-terminal pyroglutamate formation, a posttranslational modification important under many physiological and pathological conditions, and is a drug target for treating neurodegenerative diseases, cancers and inflammatory disorders. Without functional characterization, mammalian QCs and their orthologs remain indistinguishable at the sequence and structure levels from other M28-family proteins, leading to few reported QCs. Here, we show that a low-barrier carboxylic-acid hydrogen-bond network (CAHBN) is required for QC activity and discriminates QCs from M28-family peptidases. We demonstrate that the CAHBN-containing M28 peptidases deposited in the PDB are indeed QCs. Our analyses identify several thousands of QCs from the three domains of life, and we enzymatically and structurally characterize several. For the first time, the interplay between a CAHBN and the binuclear metal-binding center of mammalian QCs is made clear. We found that the presence or absence of CAHBN is a key discriminator for the formation of either the mono-Zn QCs or the di-Zn exopeptidases. Our study helps explain the possible roles of QCs in life.
Assuntos
Aminoaciltransferases/metabolismo , Ácidos Carboxílicos/metabolismo , Família Multigênica , Animais , Proteínas Arqueais/metabolismo , Bases de Dados de Proteínas , Humanos , Ligação de Hidrogênio , Íons , Cinética , Mamíferos/metabolismo , Metais/farmacologia , FilogeniaRESUMO
Isoprenoids, also known as terpenes or terpenoids, represent a large family of natural products composed of five-carbon isopentenyl diphosphate or its isomer dimethylallyl diphosphate as the building blocks. Isoprenoids are structurally and functionally diverse and include dolichols, steroid hormones, carotenoids, retinoids, aromatic metabolites, the isoprenoid side-chain of ubiquinone, and isoprenoid attached signaling proteins. Productions of isoprenoids are catalyzed by a group of enzymes known as prenyltransferases, such as farnesyltransferases, geranylgeranyltransferases, terpenoid cyclase, squalene synthase, aromatic prenyltransferase, and cis- and trans-prenyltransferases. Because these enzymes are key in cellular processes and metabolic pathways, they are expected to be potential targets in new drug discovery. In this review, six distinct subsets of characterized prenyltransferases are structurally and mechanistically classified, including (1) head-to-tail prenyl synthase, (2) head-to-head prenyl synthase, (3) head-to-middle prenyl synthase, (4) terpenoid cyclase, (5) aromatic prenyltransferase, and (6) protein prenylation. Inhibitors of those enzymes for potential therapies against several diseases are discussed. Lastly, recent results on the structures of integral membrane enzyme, undecaprenyl pyrophosphate phosphatase, are also discussed.
Assuntos
Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/metabolismo , Inibidores Enzimáticos/farmacologia , Animais , Catálise , Dimetilaliltranstransferase/antagonistas & inibidores , Humanos , Conformação ProteicaAssuntos
Bioquímica/educação , COVID-19 , Cooperação Internacional , Biologia Molecular/educação , Pandemias , Ensino , Humanos , EstudantesRESUMO
Vibrio cholerae is the causative agent of the diarrheal disease cholera, for which biofilm communities are considered to be environmental reservoirs. In endemic regions, and after algal blooms, which may result from phosphate enrichment following agricultural runoff, the bacterium is released from biofilms resulting in seasonal disease outbreaks. However, the molecular mechanism by which V. cholerae senses its environment and switches lifestyles from the biofilm-bound state to the planktonic state is largely unknown. Here, we report that the major biofilm scaffolding protein RbmA undergoes autocatalytic proteolysis via a phosphate-dependent induced proximity activation mechanism. Furthermore, we show that RbmA mutants that are defective in autoproteolysis cause V. cholerae biofilms to grow larger and mechanically stronger, correlating well with the observation that RbmA stability directly affects microbial community homeostasis and rheological properties. In conclusion, our biophysical study characterizes a novel phosphate-dependent breakdown pathway of RbmA, while microbiological data suggest a new, sensory role of this biofilm scaffolding element.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Compostos de Magnésio/farmacologia , Fosfatos/farmacologia , Proteólise , Vibrio cholerae/metabolismo , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/crescimento & desenvolvimentoRESUMO
Bacterial wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising the transmembrane and ATPase subunits TagG and TagH. Here the dimeric structure of the N-terminal domain of TagH (TagH-N) was solved by single-wavelength anomalous diffraction using a selenomethionine-containing crystal, which shows an ATP-binding cassette (ABC) architecture with RecA-like and helical subdomains. Besides significant structural differences from other ABC transporters, a prominent patch of positively charged surface is seen in the center of the TagH-N dimer, suggesting a potential binding site for the glycerol phosphate chain of WTA. The ATPase activity of TagH-N was inhibited by clodronate, a bisphosphonate, in a non-competitive manner, consistent with the proposed WTA-binding site for drug targeting.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Cristalografia por Raios X , Sistemas de Liberação de Medicamentos , Hidrolases/química , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Difosfonatos/farmacologia , Hidrolases/antagonistas & inibidores , Hidrolases/metabolismo , Cinética , Modelos MolecularesRESUMO
Interleukin-1ß (IL-1ß) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1ß regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1ß to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1ß initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1ß neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1ß. We validated IgG26AW-neutralizing antibodies specific for IL-1ß in vivo to prevent human IL-1ß-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1ß also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1ß complex structure. Meanwhile, SPR experiments showed that IL-1ß bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1ß/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1ß, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1ß is critical to pathogenesis.
Assuntos
Anticorpos Bloqueadores/química , Anticorpos Monoclonais/química , Proteína Acessória do Receptor de Interleucina-1/química , Interleucina-1beta/química , Modelos Moleculares , Conformação Proteica , Receptores Tipo I de Interleucina-1/química , Animais , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Humanos , Imunoglobulina G/química , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Modelos Biológicos , NF-kappa B/metabolismo , Biblioteca de Peptídeos , Ligação Proteica/efeitos dos fármacos , Receptores Tipo I de Interleucina-1/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Two systems of antibody-drug conjugates (ADCs), noncleavable H32-DM1 and cleavable H32-VCMMAE, were developed by using different linkers and drugs attached to the anti-HER2 antibody H32, which is capable of cell internalization. Activated functional groups, including an N-hydroxysuccinimidyl (NHS) ester and a maleimide, were utilized to make the ADCs. Mass spectrometry, hydrophobic interaction chromatography, polyacrylamide gel electrophoresis, and in vitro cell assays were performed to analyze and optimize the ADCs. Several H32-VCMMAE ADCs were established with higher DARs and greater synthetic yields without compromising potency. The anticancer efficacy of H32-DM1 was 2- to 8-fold greater than that of Kadcyla®. The efficacy of H32-VCMMAE was in turn better than that of H32-DM1. The anticancer efficacy of these ADCs against N87, SK-BR-3 and BT474 cells was in the following order: H32-VCMMAE series > H32-DM1 series > Kadcyla®. The optimal DAR for H32-VCMMAE was found to be 6.6, with desirable attributes including good cell penetration, a releasable payload in cancer cells, and high potency. Our results demonstrated the potential of H32-VCMMAE as a good ADC candidate.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Neoplasias da Mama/metabolismo , Imunoconjugados/farmacologia , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Ado-Trastuzumab Emtansina/química , Ado-Trastuzumab Emtansina/farmacologia , Anticorpos Monoclonais Humanizados/química , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Imunoconjugados/química , Concentração Inibidora 50 , Oligopeptídeos/química , Oligopeptídeos/farmacologiaRESUMO
The undecaprenyl pyrophosphate phosphatase (UppP) is an integral membrane pyrophosphatase. In bacteria, UppP catalyzes the dephosphorylation of undecaprenyl pyrophosphate (C55-pp) to undecaprenyl phosphate (C55-P) in the periplasmic space, which is an essential step for the isoprenyl lipid carrier to reenter the peptidoglycan synthesis cycle. Besides bacteria, the UppP homologs are widely distributed in archaea genome. However, all archaea lack peptidoglycan structure in their cell wall components, and the major archaeal lipid carriers are dolichol phosphate (Dol-p) and dolichol pyrophosphate (Dol-pp), so the functions of the UppP homolog in archaea remain unclear. Here, we purified a recombinant polyisoprenyl pyrophosphatase of a thermoacidophilic archaeon, Saccharolobus solfataricus (SsUppP), and characterized its enzymatic properties. Two isoprenyl pyrophosphate, farnesyl pyrophosphate (Fpp) and geranylgeranyl pyrophosphate (Ggpp), were used as the surrogate substrates, simulating the bacterial and archaeal lipid carriers. SsUppP dephosphorylated Fpp and Ggpp at 37⯰C, but retained the phosphatase activity at high temperatures. The optimal condition for the enzymatic activity was found to be at pH 7 and 70⯰C. The thermostability of SsUppP was also supported by molecular dynamics simulation studies. Our results indicated that the archaeal SsUppP can dephosphorylate isoprenyl pyrophosphates at the natural environment of high temperature, and the possibility to catalyze the dephosphorylation of archaeal lipid carriers.
Assuntos
Archaea/enzimologia , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Proteínas Arqueais/metabolismo , Membrana Celular/metabolismo , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Sesquiterpenos/metabolismoRESUMO
Linker design is crucial to the success of antibody-drug conjugates (ADCs). In this work, we developed a modular linker format for attaching molecular cargos to antibodies based on strand pairing between complementary oligonucleotides. We prepared antibody-oligonucleotide conjugates (AOCs) by attaching 18-mer oligonucleotides to an anti-HER2 antibody through thiol-maleimide chemistry, a method generally applicable to any immunoglobulin with interchain disulfide bridges. The hybridization of drug-bearing complementary oligonucleotides to our AOCs was rapid, stoichiometric, and sequence-specific. AOCs loaded with cytotoxic payloads were able to selectively target HER2-overexpressing cell lines such as SK-BR-3 and N87, with in vitro potencies similar to that of the marketed ADC Kadcyla (T-DM1). Our results demonstrated the potential of utilizing AOCs as a highly versatile and modular platform, where a panel of well-characterized AOCs bearing DNA, RNA, or various nucleic acid analogs, such as peptide nucleic acids, could be easily paired with any cargo of choice for a wide range of diagnostic or therapeutic applications.
Assuntos
Imunoconjugados/química , Oligonucleotídeos/química , Complexo Antígeno-Anticorpo , Antineoplásicos/química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoglobulina G/imunologia , Maitansina/química , Receptor ErbB-2/imunologiaRESUMO
BACKGROUND: Polyethylene glycol (PEG) is widely used in industry and medicine. Anti-PEG antibodies have been developed for characterizing PEGylated drugs and other applications. However, the underlying mechanism for specific PEG binding has not been elucidated. METHODS: The Fab of two cognate anti-PEG antibodies 3.3 and 2B5 were each crystallized in complex with PEG, and their structures were determined by X-ray diffraction. The PEG-Fab interactions in these two crystals were analyzed and compared with those in a PEG-containing crystal of an unrelated anti-hemagglutinin 32D6-Fab. The PEG-binding stoichiometry was examined by using analytical ultracentrifuge (AUC). RESULTS: A common PEG-binding mode to 3.3 and 2B5 is seen with an S-shaped core PEG fragment bound to two dyad-related Fab molecules. A nearby satellite binding site may accommodate parts of a longer PEG molecule. The core PEG fragment mainly interacts with the heavy-chain residues D31, W33, L102, Y103 and Y104, making extensive contacts with the aromatic side chains. At the center of each half-circle of the S-shaped PEG, a water molecule makes alternating hydrogen bonds to the ether oxygen atoms, in a similar configuration to that of a crown ether-bound lysine. Each satellite fragment is clamped between two arginine residues, R52 from the heavy chain and R29 from the light chain, and also interacts with several aromatic side chains. In contrast, the non-specifically bound PEG fragments in the 32D6-Fab crystal are located in the elbow region or at lattice contacts. The AUC data suggest that 3.3-Fab exists as a monomer in PEG-free solution but forms a dimer in the presence of PEG-550-MME, which is about the size of the S-shaped core PEG fragment. CONCLUSIONS: The differing amino acids in 3.3 and 2B5 are not involved in PEG binding but engaged in dimer formation. In particular, the light-chain residue K53 of 2B5-Fab makes significant contacts with the other Fab in a dimer, whereas the corresponding N53 of 3.3-Fab does not. This difference in the protein-protein interaction between two Fab molecules in a dimer may explain the temperature dependence of 2B5 in PEG binding, as well as its inhibition by crown ether.