共查询到20条相似文献,搜索用时 15 毫秒
1.
Yuwei Zhu Jianxin Dai Tiancheng Zhang Xu Li Pengfei Fang Huajing Wang Yongliang Jiang Xiaojie Yu Tian Xia Liwen Niu Yajun Guo Maikun Teng 《The Journal of biological chemistry》2013,288(35):25165-25172
Ricin belongs to the type II ribosome-inactivating proteins that depurinate the universally conserved α-sarcin loop of rRNA. The RNA N-glycosidase activity of ricin also largely depends on the ribosomal proteins that play an important role during the process of rRNA depurination. Therefore, the study of the interaction between ricin and the ribosomal elements will be better to understand the catalysis mechanism of ricin. The antibody 6C2 is a mouse monoclonal antibody exhibiting unusually potent neutralizing ability against ricin, but the neutralization mechanism remains unknown. Here, we report the 2.8 Å crystal structure of 6C2 Fab in complex with the A-chain of ricin (RTA), which reveals an extensive antigen-antibody interface that contains both hydrogen bonds and van der Waals contacts. The complementarity-determining region loops H1, H2, H3, and L3 form a pocket to accommodate the epitope on the RTA (residues Asp96–Thr116). ELISA results show that Gln98, Glu99, Glu102, and Thr105 (RTA) are the key residues that play an important role in recognizing 6C2. With the perturbation of the 6C2 Fab-RTA interface, 6C2 loses its neutralization ability, measured based on the inhibition of protein synthesis in a cell-free system. Finally, we propose that the neutralization mechanism of 6C2 against ricin is that the binding of 6C2 hinders the interaction between RTA and the ribosome and the surface plasmon resonance and pulldown results confirm our hypothesis. In short, our data explain the neutralization mechanism of mAb 6C2 against ricin and provide a structural basis for the development of improved antibody drugs with better specificity and higher affinity. 相似文献
2.
Vaheh Oganesyan Li Peng Robert M. Woods Herren Wu William F. Dall'Acqua 《The Journal of biological chemistry》2015,290(24):14979-14985
We report the three-dimensional structure of human interferon α-2A (IFN-α2A) bound to the Fab fragment of a therapeutic monoclonal antibody (sifalimumab; IgG1/κ). The structure of the corresponding complex was solved at a resolution of 3.0 Å using molecular replacement and constitutes the first reported structure of a human type I IFN bound to a therapeutic antibody. This study revealed the major contribution made by the first complementarity-determining region in each of sifalimumab light and heavy chains. These data also provided the molecular basis for sifalimumab mechanism of action. We propose that its interferon-neutralizing properties are the result of direct competition for IFN-α2A binding to the IFN receptor subunit 1 (IFNAR1) and do not involve inhibiting IFN-α2A binding to the IFN receptor subunit 2 (IFNAR2). 相似文献
3.
G. A. Efimov J. M. H. Raats R. G. S. Chirivi J. W. G. van Rosmalen S. A. Nedospasov 《Molecular Biology》2017,51(6):921-926
Tumor necrosis factor (TNF) is a proinflammatory cytokine implicated in pathogenesis of multiple autoimmune and inflammatory diseases. Anti-TNF therapy has revolutionized the therapeutic paradigms of autoimmune diseases and became one of the most successful examples of the clinical use of monoclonal antibodies. Currently, anti-TNF therapy is used by millions of patients worldwide. At the moment, fully human anti-TNF antibody Adalimumab is the best-selling anti-cytokine drug in the world. Here, we present a story about a highly potent anti-TNF monoclonal antibody initially characterized more than 20 years ago and further developed into chimeric and humanized versions. We present comparative analysis of this antibody with Infliximab and Adalimumab. 相似文献
4.
Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9) variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with KD of 1.63 nM, comparable to its parental murine D9 (2.55 nM). In a mouse model, intraperitoneal (i.p.) administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning. 相似文献
5.
6.
Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction. 相似文献
7.
Manuel Covarrubias Annika?F. Barber Vincenzo Carnevale Werner Treptow Roderic?G. Eckenhoff 《Biophysical journal》2015,109(10):2003-2011
General anesthesia is a relatively safe medical procedure, which for nearly 170 years has allowed life saving surgical interventions in animals and people. However, the molecular mechanism of general anesthesia continues to be a matter of importance and debate. A favored hypothesis proposes that general anesthesia results from direct multisite interactions with multiple and diverse ion channels in the brain. Neurotransmitter-gated ion channels and two-pore K+ channels are key players in the mechanism of anesthesia; however, new studies have also implicated voltage-gated ion channels. Recent biophysical and structural studies of Na+ and K+ channels strongly suggest that halogenated inhalational general anesthetics interact with gates and pore regions of these ion channels to modulate function. Here, we review these studies and provide a perspective to stimulate further advances. 相似文献
8.
Bai M Pang X Lou J Zhou Q Zhang K Ma J Li J Sun F Hsu VW 《The Journal of biological chemistry》2012,287(34):28675-28685
Coat complexes sort protein cargoes into vesicular transport pathways. An emerging class of coat components has been the GTPase-activating proteins (GAPs) that act on the ADP-ribosylation factor (ARF) family of small GTPases. ACAP1 (ArfGAP with coiled-coil, ankyrin repeat, and PH domains protein 1) is an ARF6 GAP that also acts as a key component of a recently defined clathrin complex for endocytic recycling. Phosphorylation by Akt has been shown to enhance cargo binding by ACAP1 in explaining how integrin recycling is an example of regulated transport. We now shed further mechanistic insights into how this regulation is achieved at the level of cargo binding by ACAP1. We initially defined a critical sequence in the cytoplasmic domain of integrin β1 recognized by ACAP1 and showed that this sequence acts as a recycling sorting signal. We then pursued a combination of structural, modeling, and functional studies, which suggest that phosphorylation of ACAP1 relieves a localized mechanism of autoinhibition in regulating cargo binding. Thus, we have elucidated a key regulatory juncture that controls integrin recycling and also advanced the understanding of how regulated cargo binding can lead to regulated transport. 相似文献
9.
Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex
《Journal of molecular biology》2019,431(19):3647-3661
Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as Pseudomonas putida CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N-demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial N-demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial N1- and N3-specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper N-demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during N-demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications. 相似文献
10.
Stoichiometry of Monoclonal Antibody Neutralization of T-Cell Line-Adapted Human Immunodeficiency Virus Type 1 下载免费PDF全文
Kristian Schnning Ole Lund Ole Sgaard Lund John-Erik Stig Hansen 《Journal of virology》1999,73(10):8364-8370
In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb neutralization gradually increased. Virus neutralization by virion aggregation was minimal, as MAb binding to HIV-1 Env did not interfere with an AMLV Env-mediated infection by HIV-1(AMLV/HIV-1) pseudotypes of CD4(-) HEK293 cells. MAb neutralization of chimeric virions could be described as a third-order function of the proportion of Env antigen refractory to MAb binding. This scenario is consistent with the Env oligomer constituting the minimal functional unit and neutralization occurring incrementally as each Env oligomer binds MAb. Alternatively, the data could be fit to a sigmoid function. Thus, these data could not exclude the existence of a threshold for neutralization. However, results from MAb neutralization of chimeric virus containing wild-type Env and Env defective in CD4 binding was readily explained by a model of incremental MAb neutralization. In summary, the data indicate that MAb neutralization of T-cell line-adapted HIV-1 is incremental rather than all or none and that each MAb binding an Env oligomer reduces the likelihood of infection. 相似文献
11.
12.
Kelvin B. Luther Hermann Schindelin Robert S. Haltiwanger 《The Journal of biological chemistry》2009,284(5):3294-3305
The Notch receptor is critical for proper development where it orchestrates
numerous cell fate decisions. The Fringe family of
β1,3-N-acetylglucosaminyltransferases are regulators of this
pathway. Fringe enzymes add N-acetylglucosamine to O-linked
fucose on the epidermal growth factor repeats of Notch. Here we have analyzed
the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis
strategy for Lfng was guided by a multiple sequence alignment of Fringe
proteins and solutions from docking an epidermal growth factor-like
O-fucose acceptor substrate onto a homology model of Lfng. We
targeted three main areas as follows: residues that could help resolve where
the fucose binds, residues in two conserved loops not observed in the
published structure of Manic Fringe, and residues predicted to be involved in
UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a
kinetic analysis of mutant enzyme activity toward the small molecule acceptor
substrate 4-nitrophenyl-α-l-fucopyranoside to judge their
effect on Lfng activity. Our results support the positioning of
O-fucose in a specific orientation to the catalytic residue. We also
found evidence that one loop closes off the active site coincident with, or
subsequent to, substrate binding. We propose a mechanism whereby the ordering
of this short loop may alter the conformation of the catalytic aspartate.
Finally, we identify several residues near the UDP-GlcNAc-binding site, which
are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases,
including multiple sclerosis
(1), several forms of cancer
(2-4),
cerebral autosomal dominant arteriopathy with sub-cortical infarcts and
leukoencephalopathy (5), and
spondylocostal dysostosis
(SCD)3
(6-8).
The transmembrane Notch signaling receptor is activated by members of the DSL
(Delta, Serrate, Lag2) family of ligands
(9,
10). In the endoplasmic
reticulum, O-linked fucose glycans are added to the epidermal growth
factor-like (EGF) repeats of the Notch extracellular domain by protein
O-fucosyltransferase 1
(11-13).
These O-fucose monosaccharides can be elongated in the Golgi
apparatus by three highly conserved
β1,3-N-acetylglucosaminyltransferases of the Fringe family
(Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals)
(14-16).
The formation of this GlcNAc-β1,3-Fuc-α1,
O-serine/threonine disaccharide is necessary and sufficient for
subsequent elongation to a tetrasaccharide
(15,
19), although elongation past
the disaccharide in Drosophila is not yet clear
(20,
21). Elongation of
O-fucose by Fringe is known to potentiate Notch signaling from Delta
ligands and inhibit signaling from Serrate ligands
(22). Delta ligands are termed
Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate
are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on
Drosophila Notch can be recapitulated in Notch ligand in
vitro binding assays using purified components, suggesting that the
elongation of O-fucose by Fringe alters the binding of Notch to its
ligands (21). Although Fringe
also appears to alter Notch-ligand interactions in mammals, the effects of
elongation of the glycan past the O-fucose monosaccharide is more
complicated and appears to be cell type-, receptor-, and ligand-dependent (for
a recent review see Ref.
23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate
UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc
disaccharide
(14-16).
They belong to the GT-A-fold of inverting glycosyltransferases, which includes
N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase
I (17,
18). The mechanism is presumed
to proceed through the abstraction of a proton from the acceptor substrate by
a catalytic base (Asp or Glu) in the active site. This creates a nucleophile
that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its
configuration from α (on the nucleotide sugar) to β (in the
product) (24,
25). The enzyme then releases
the acceptor substrate modified with a disaccharide and UDP. The Mfng
structure (26) leaves little
doubt as to the identity of the catalytic residue, which in all likelihood is
aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe
throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc
soaked into the crystals (26)
showed density only for the UDP portion of the nucleotide-sugar donor and no
density for two loops flanking either side of the active site. The presence of
flexible loops that become ordered upon substrate binding is a common
observation with glycosyltransferases in the GT-A fold family
(18,
25). Density for the entire
donor was observed in the structure of rabbit
N-acetylglucosaminyltransferase I
(27). In this case, ordering
of a previously disordered loop upon UDP-GlcNAc binding may have contributed
to increased stability of the donor. In the case of bovine
β1,4-galactosyltransferase I, a section of flexible random coil from the
apo-structure was observed to change its conformation to α-helical upon
donor substrate binding (28).
Both loops in Lfng are highly conserved, and we have mutated a number of
residues in each to test the hypothesis that they interact with the
substrates. The mutagenesis strategy was also guided by docking of an
EGF-O-fucose acceptor substrate into the active site of the Lfng
model as well as comparison of the Lfng model with a homology model of the
β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on
thrombospondin type 1 repeats
(29,
30). The β3GlcT is
predicted to be a GT-A fold enzyme related to the Fringe family
(17,
18,
29). 相似文献
13.
Alexandre Chigaev Anna Waller Or Amit Liliana Halip Cristian G. Bologa Larry A. Sklar 《The Journal of biological chemistry》2009,284(21):14337-14346
Integrins are heterodimeric adhesion receptors that regulate immune cell
adhesion. Integrin-dependent adhesion is controlled by multiple conformational
states that include states with different affinity to the ligand, states with
various degrees of molecule unbending, and others. Affinity change and
molecule unbending play major roles in the regulation of cell adhesion. The
relationship between different conformational states of the integrin is
unclear. Here we have used conformationally sensitive antibodies and a small
LDV-containing ligand to study the role of the inside-out signaling through
formyl peptide receptor and CXCR4 in the regulation of
α4β1 integrin conformation. We found that in
the absence of ligand, activation by formyl peptide or SDF-1 did not result in
a significant exposure of HUTS-21 epitope. Occupancy of the ligand binding
pocket without cell activation was sufficient to induce epitope exposure.
EC50 for HUTS-21 binding in the presence of LDV was identical to a
previously reported ligand equilibrium dissociation constant at rest and after
activation. Furthermore, the rate of HUTS-21 binding was also related to the
VLA-4 activation state even at saturating ligand concentration. We propose
that the unbending of the integrin molecule after guanine nucleotide-binding
protein-coupled receptor-induced signaling accounts for the enhanced rate of
HUTS-21 binding. Taken together, current results support the existence of
multiple conformational states independently regulated by both inside-out
signaling and ligand binding. Our data suggest that VLA-4 integrin hybrid
domain movement does not depend on the affinity state of the ligand binding
pocket.In the bloodstream circulating leukocytes respond to inflammatory signals
by rapid changes of cell adhesive properties. These include cell tethering,
rolling, arrest, and firm adhesion, all of which are well described steps of
leukocyte recruitment to the sites of inflammation
(1). Leukocyte arrest and firm
adhesion are mediated exclusively by integrin receptors
(2). At the same time integrins
can also mediate tethering and rolling
(3). These largely diverse cell
adhesive properties are achieved by sophisticated conformational regulation;
multiple states of the same molecule with different affinity for its ligand
and different degrees of molecular unbending are attributed to various types
of “cellular behavior.” It is proposed that the low affinity bent
state translates into a non-adhesive resting cell, the low affinity unbent or
extended state of integrin results in cell rolling, and the high affinity
state promotes cell arrest (4,
5). However, the exact sequence
of conformational events and the relationship between integrin conformational
and functional activity remain key questions
(6).Integrin conformation is regulated through G-protein-coupled receptors by a
signaling pathway which is initiated by ligand binding to a
GPCR,3 propagated
inside the cell, and results in the binding of signaling proteins (such as
talin and others) to cytoplasmic domains of integrin subunits. This binding
leads to a separation of the integrin cytoplasmic domains and inside-out
activation (6). Chemokines
(chemotactic cytokines) as well as “classical” chemoattractants
(such as formyl peptide) preferentially signal through heterotrimeric
G-proteins coupled to the Gαi subunit
(1). Activation by these
ligands results in up-regulation of integrin affinity and/or conformational
unbending (extension) of the integrin molecule. These conformational changes
lead to cell arrest and firm adhesion. G-protein receptors coupled to
Gαs-coupled subunit (adenylyl cyclase/cAMP signaling pathway)
can actively down-regulate the affinity state of the ligand binding pocket
without changing integrin conformational unbending. This provides an
anti-adhesive signal and results in cell de-adhesion
(7). Thus, interaction of
multiple G-protein-coupled receptors on a single cell creates a plethora of
conformational states. Understanding of the relationship between inside-out
signaling through GPCRs and integrin conformational regulation will provide
valuable insight into the dynamic regulation of cell adhesion.One technique to study conformational changes of integrins uses
conformationally sensitive mAbs that bind to epitopes which are hidden in one
conformation and exposed under certain conditions. Lately, it has been
accepted that integrins exhibit two major conformations, resting and
activated. A number of mAbs for “activated” integrins have been
described, and the epitopes have been mapped. Together with mapping of these
epitopes into three-dimensional structures of integrin
(8), epitope exposure can
provide helpful information about integrin conformational changes upon
signaling. Moreover, because integrin inside-out activation through different
signaling pathways can result in different activation states, the use of
previously mapped mAbs can help dissect conformational changes upon
activation.Although it is clear that inside-out activation results in a conformational
rearrangement of the integrin molecule, the relationship between affinity
state of the ligand binding pocket and overall molecule conformation is still
debated. Currently, two contrasting models of integrin inside-out integrin
activation are described. The “switchblade” model implies that an
open head structure with swung-out β-hybrid domain represents the high
(or at least intermediate) affinity state. A feature of this model is that
integrin extension provides space for hybrid domain swing. The
“deadbolt” model proposes that the movement of β-hybrid
domain is not related to the inside-out signal. Ligand binding by itself can
provide the energy for the hybrid domain swing out (for details, see Ref.
9 and references therein).
Because these two models assign different roles to the hybrid domain motion,
we evaluated the exposure of VLA-4 hybrid domain epitopes upon activation
through two Gαi-coupled GPCRs (FPR and CXCR4) and ligand
binding using the conformationally sensitive HUTS-21 mAb with an epitope
mapped to the hybrid domain of β1-integrin
(10).We found that contrary to previous reports, where these mAbs were reported
to bind or used for the detection of activated integrin
(10–13),
formyl peptide or SDF-1 treatment alone did not result in any significant
exposure of HUTS-21 epitope despite the fact that the VLA-4 affinity
up-regulation was detected in parallel on the same batch of cells.
Quantitative analysis of mAb binding in real time on live cells suggests that
for both the low (resting) and high affinity (induced by inside-out pathway)
states, occupancy of the ligand binding pocket rather than inside-out
signaling by itself causes the conformational change. Thus, these data support
the idea that the hybrid domain movement, which results in the exposure of the
mAb epitope, and the high affinity state of the binding pocket are regulated
separately and independently of each other, a feature of the deadbolt model of
inside-out activation. 相似文献
14.
15.
16.
《Journal of molecular biology》2019,431(17):3139-3145
Recently, it has been discovered that different DNA-(cytosine C5)-methyltransferases including DNMT3A generate low levels of 3mC [Rosic et al. (2018), Nat. Genet., 50, 452–459]. This reaction resulted in the co-evolution of DNMTs and ALKB2 DNA repair enzymes, but its mechanism remained elusive. Here, we investigated the catalytic mechanism of DNMT3A for cytosine N3 methylation. We generated several DNMT3A variants with mutated catalytic residues and measured their activities in 5mC and 3mC generation by liquid chromatography linked to tandem mass spectrometry. Our data suggest that the methylation of N3 instead of C5 is caused by an inverted binding of the flipped cytosine target base into the active-site pocket of the DNA methyltransferase, which is partially compatible with the arrangement of catalytic amino acid residues. Given that all DNA-(cytosine C5)-methyltransferases have a common catalytic mechanism, it is likely that other enzymes of this class generate 3mC following the same mechanism. 相似文献
17.
Tomohiko Murase Ruixiang Blake Zheng Maju Joe Yu Bai Sandra L. Marcus Kenneth K.S. Ng 《Journal of molecular biology》2009,392(2):381-3000
Mycobacteria are major human pathogens responsible for such serious and widespread diseases as tuberculosis and leprosy. Among the evolutionary adaptations essential for pathogenicity in mycobacteria is a complex carbohydrate-rich cell-wall structure that contains as a major immunomodulatory molecule the polysaccharide lipoarabinomannan (LAM). We report here crystal structures of three fragments from the non-reducing termini of LAM in complex with a murine antibody Fab fragment (CS-35Fab). These structures reveal for the first time the three-dimensional structures of key components of LAM and the molecular basis of LAM recognition at between 1.8- and 2.0-Å resolution. The antigen-binding site of CS-35Fab forms three binding pockets that show a high degree of complementarity to the reducing end, the branch point and one of the non-reducing ends of the Y-shaped hexasaccharide moiety found at most of the non-reducing termini of LAM. Structures of CS-35Fab bound to two additional tetrasaccharides confirm the general mode of binding seen in the hexasaccharide and indicate how different parts of LAM are recognized. Altogether, these structures provide a rational basis for understanding the overall architecture of LAM and identify the key elements of an epitope that may be exploited for the development of novel and more effective anti-mycobacterial vaccines. Moreover, this study represents the first high-resolution X-ray crystallographic investigation of oligofuranoside-protein recognition. 相似文献
18.
Marie F. Soulière Jean-Pierre Perreault Martin Bisaillon 《Nucleic acids research》2010,38(21):7599-7610
Decapping enzymes are required for the removal of the 5′-end m7GpppN cap of mRNAs to allow their decay in cells. While many cap-binding proteins recognize the cap structure via the stacking of the methylated guanosine ring between two aromatic residues, the precise mechanism of cap recognition by decapping enzymes has yet to be determined. In order to get insights into the interaction of decapping enzymes with the cap structure, we studied the vaccinia virus D10 decapping enzyme as a model to investigate the important features for substrate recognition by the enzyme. We demonstrate that a number of chemically modified purines can competitively inhibit the decapping reaction, highlighting the molecular features of the cap structure that are required for recognition by the enzyme, such as the nature of the moiety at positions 2 and 6 of the guanine base. A 3D structural model of the D10 protein was generated which suggests amino acids implicated in cap binding. Consequently, we expressed 17 mutant proteins with amino acid substitutions in the active site of D10 and found that eight are critical for the decapping activity. These data underscore the functional features involved in the non-canonical cap-recognition by the vaccinia virus D10 decapping enzyme. 相似文献
19.
Zhiqun Li Longfa Xu Delei He Lisheng Yang Che Liu Yixin Chen James Wai Kuo Shih Jun Zhang Qinjian Zhao Tong Cheng Ningshao Xia 《PloS one》2014,9(10)
Enterovirus 71 (EV71) is a neurotropic virus capable of inducing severe neurological symptoms and death. No direct targeting antivirals are useful in the treatment of severe EV71 infection. Because of low toxicity and good specificity, monoclonal antibodies (MAb) are a potential candidate for the treatment of viral infections. Therefore, we developed an EV71-specific conformational MAb with high in vitro cross-neutralization activity to heterologous EV71 subgenotypes. The in vivo treatment experiment at different days post-infection indicated that a single treatment of MAb CT11F9 within day 3 post-infection fully protected mice from morbidity and mortality (0% PBS vs. 100% at 10 µg/g per body weight ***P<0.0001). Immunohistochemical and histological analysis confirmed that CT11F9 significantly prohibited EV71 VP1 expression in various tissues and prevented EV71-induced myonecrosis. Moreover, thrice-treatment at day 4, 5, 6 post-infection was associated with an increased survival rate (18.2% single vs. 50% thrice at 20 µg/g per body weight), and the mice recovered from limb paralysis. Competitive ELISA also confirmed that CT11F9-recognized epitopes were immunodominant in humans. In conclusion, MAb CT11F9 is an ideal candidate to be humanized and used in severe EV71 infection. 相似文献