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The construction of co-expression vectors for OGT and GlmM/GlmU. (A) Schematic representation of the proposed pathway involved in UDP-GlcNAc synthesis in E. coli. (B-D) Illustrations for the vector maps of pBAC-MBP-OGT-GlmU, pBAC-MBP-OGT-GlmM and pBAC-MBP-OGT-GlmU-GlmM. (E) The expression of OGT, GlmU and GlmM/GlmU was analyzed by SDS-PAGE followed by Coomassie brilliant blue staining, and the expression of OGT was further confirmed by IB assay using anti-OGT antibody. GlmM: 48 kDa; GlmU: 52 kDa. (F and G) The relative mRNA levels of GlmM (F) and GlmU (G) were analyzed by real-time PCR. (H) The growth curves of the E. coli BL21(DE3) strains transformed with the indicated vectors were examined. The transformants were grown at 37°C until OD 600 reached 0.4-0.5. Then the culture was induced with 0.5 mM IPTG and grown at 25°C. The growth of the culture was examined by spectrophotometer at OD 600 at the indicated time points. O, OGT; U, GlmU; M, GlmM. Mean ± SD (n = 3); ***, P < 0.001.
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O-GlcNAcylation is a ubiquitous and dynamic post-translational modification on serine/threonine residues of nucleocytoplasmic proteins in metazoa, which plays a critical role in numerous physiological and pathological processes. But the O-GlcNAcylation on most proteins is often substoichiometric, which hinders the functional study of the O-GlcNAcyl...
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... that UDP-GlcNAc is the donor substrate of OGT, increas- ing the level of UDP-GlcNAc can improve the O-GlcNAcylation levels of OGT target proteins. There are four successive reactions catalyzed by three enzymes' sequential steps in the biosynthesis of UDP-GlcNAc ( Figure 2A). Previous reports indicated that GlmU and GlmM are crit- ical for the biosynthesis of UDP-GlcNAc in bacteria ( Leiting et al. 1998;Rodriguez-Diaz et al. 2012). ...
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... we tried to overexpress GlmU and/or GlmM to increase the level of UDP-GlcNAc in E. coli. It is known that pBAC has the ability to harbor large DNA fragment, so GlmU and/or GlmM were cloned into the pBAC-MBP-OGT plasmid as illustrated in Figure 2B-D in order to simplify the system. The vec- tors were named pBAC-MBP-OGT-GlmU, pBAC-MBP-OGT-GlmM and pBAC-MBP-OGT-GlmU-GlmM ( Figure 2B-D), respectively. ...
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... is known that pBAC has the ability to harbor large DNA fragment, so GlmU and/or GlmM were cloned into the pBAC-MBP-OGT plasmid as illustrated in Figure 2B-D in order to simplify the system. The vec- tors were named pBAC-MBP-OGT-GlmU, pBAC-MBP-OGT-GlmM and pBAC-MBP-OGT-GlmU-GlmM ( Figure 2B-D), respectively. ...
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... expression of GlmM and GlmU was analyzed by SDS-PAGE. The results showed that GlmU was highly expressed, but the expres- sion level of GlmM was low ( Figure 2E). To detect the possible reason for the low expression of GlmM, RNA was isolated from transfor- mants and real-time PCR was performed after RNA was reverse tran- scribed into cDNA. ...
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... detect the possible reason for the low expression of GlmM, RNA was isolated from transfor- mants and real-time PCR was performed after RNA was reverse tran- scribed into cDNA. The results showed that the mRNA levels of both GlmM and GlmU were significantly increased when GlmM and GlmU were overexpressed ( Figure 2F-G), suggesting the low expression of GlmM might due to its low translational efficiency. ...
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Citations
... The compatible expression system for the production of highly phosphorylation recombinant protein in Escherichia coli was setted up [24][25][26]. Briefly, GST with His-HuR or GST-p-p38 (activated p38) with His-HuR were co-expressed in BL21 cells. ...
... The results showed that GST-p38 interacted with His-HuR under cell-free conditions, indicating that the two proteins interacted directly (Fig. 2F). Furthermore, the phosphorylation of p38 to HuR in vitro was examined by co-expressing GST-p-p38 (activated p38) and HuR in Escherichia coli as described in Materials and Methods [24][25][26]. The results revealed that the GST-p-p38 (GST-p38-T180D-T182D) and not GST induced phosphorylation of His-HuR, confirming that p38 mediates the phosphorylation of HuR directly (Fig. 2G). ...
Post-transcriptional regulation of cytokine/chemokine mRNA turnover is critical for immune processes and contributes to the mammalian cellular response to diverse inflammatory stimuli. The ubiquitous RNA-binding protein human antigen R (HuR) is an integral regulator of inflammation-associated mRNA fate. HuR function is regulated by various post-translational modifications that alter its subcellular localization and ability to stabilize target mRNAs. Both poly (ADP-ribose) polymerase 1 (PARP1) and p38 mitogen-activated protein kinases (MAPKs) have been reported to regulate the biological function of HuR, but their specific regulatory and crosstalk mechanisms remain unclear. In this study, we show that PARP1 acts via p38 to synergistically promote cytoplasmic accumulation of HuR and stabilization of inflammation-associated mRNAs in cells under inflammatory conditions. Specifically, p38 binds to auto-poly ADP-ribosylated (PARylated) PARP1 resulting in the covalent PARylation of p38 by PARP1, thereby promoting the retention and activity of p38 in the nucleus. In addition, PARylation of HuR facilitates the phosphorylation of HuR at the serine 197 site mediated by p38, which then increases the translocation of HuR to the cytoplasm, ultimately stabilizing the inflammation-associated mRNA expression at the post-transcriptional level.
... In this paper, the OBP-tagged strategy was used to successfully improve the O-GlcNAc level of Tau, H2B, and c-Myc proteins. It has been reported that the co-expression of OGT with its target substrates in E. coli could produce O-GlcNAc recombinant proteins using the dual-plasmid system [24,31,32]. However, the transfection efficiency of two plasmids in one system is different, and there is a possibility of plasmid loss. ...
O-GlcNAcylation is a single glycosylation of GlcNAc mediated by OGT, which regulates the function of substrate proteins and is closely related to many diseases. However, a large number of O-GlcNAc-modified target proteins are costly, inefficient, and complicated to prepare. In this study, an OGT binding peptide (OBP)-tagged strategy for improving the proportion of O-GlcNAc modification was established successfully in E. coli. OBP (P1, P2, or P3) was fused with target protein Tau as tagged Tau. Tau or tagged Tau was co-constructed with OGT into a vector expressed in E. coli. Compared with Tau, the O-GlcNAc level of P1Tau and TauP1 increased 4~6-fold. Moreover, the P1Tau and TauP1 increased the O-GlcNAc-modified homogeneity. The high O-GlcNAcylation on P1Tau resulted in a significantly slower aggregation rate than Tau in vitro. This strategy was also used successfully to increase the O-GlcNAc level of c-Myc and H2B. These results indicated that the OBP-tagged strategy was a successful approach to improve the O-GlcNAcylation of a target protein for further functional research.
... To rule out the possibility that the S189A mutation altered enzyme activity by compromising the protein structure, we expressed and purified His-tagged WT and S189A MDH1 from the bacterial host (Escherichia coli) and used a compatible expression system to produce O-GlcNAcylated MDH1 by coexpressing OGT 28 . Upon OGT coexpression, WT MDH1 showed an increased O-GlcNAcylation signal (with a stoichiometry of ~31%) compared with the S189A mutant ( Supplementary Fig. 7a,b). ...
Oncogenic Kras-activated pancreatic ductal adenocarcinoma (PDAC) cells highly rely on an unconventional glutamine catabolic pathway to sustain cell growth. However, little is known about how this pathway is regulated. Here we demonstrate that Kras mutation induces cellular O-linked β-N-acetylglucosamine (O-GlcNAc), a prevalent form of protein glycosylation. Malate dehydrogenase 1 (MDH1), a key enzyme in the glutamine catabolic pathway, is positively regulated by O-GlcNAcylation on serine 189 (S189). Molecular dynamics simulations suggest that S189 glycosylation on monomeric MDH1 enhances the stability of the substrate-binding pocket and strengthens the substrate interactions by serving as a molecular glue. Depletion of O-GlcNAcylation reduces MDH1 activity, impairs glutamine metabolism, sensitizes PDAC cells to oxidative stress, decreases cell proliferation and inhibits tumor growth in nude mice. Furthermore, O-GlcNAcylation levels of MDH1 are elevated in clinical PDAC samples. Our study reveals that O-GlcNAcylation contributes to pancreatic cancer growth by regulating the metabolic activity of MDH1. Kras activation in pancreatic cancer cells induced O-GlcNAc modification of malate dehydrogenase 1, regulating glutamine metabolism and promoting tumor growth.
... This strategy was employed with tau (Yuzwa et al., 2011) and α-synuclein (Zhang et al., 2017b) to address the O-GlcNAc modification pattern of tau and the role of O-GlcNAc in tau and α-synuclein aggregation, respectively. The co-expression OGT/substrate can be optionally accompanied by 1) the co-expression of GlmM and GlmU enzymes that participate to the bacterial UDP-GlcNAc biosynthesis and therefore help protein O-GlcNAcylation by enhancing intracellular UDP-GlcNAc concentration (Gao et al., 2018), and 2) the treatment of bacterial cultures and extracts with PUGNAc, a potent glycosidase inhibitor, since endogenous NagZ glycosidase can hydrolyze O-GlcNAc in exogenous glycosylated proteins, hence reducing the O-GlcNAc modification level (Goodwin et al., 2013). This strategy together with the in vitro incorporation of O-GlcNAc through incubation of the protein substrate with UDP-GlcNAc and expressed OGT (from heterologous expression system) lead to heterogenous, and most importantly, low sub-stoichiometry modification in most cases. ...
Protein aggregation into highly ordered, regularly repeated cross-β sheet structures called amyloid fibrils is closely associated to human disorders such as neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases, or systemic diseases like type II diabetes. Yet, in some cases, such as the HET-s prion, amyloids have biological functions. High-resolution structures of amyloids fibrils from cryo-electron microscopy have very recently highlighted their ultrastructural organization and polymorphisms. However, the molecular mechanisms and the role of co-factors (posttranslational modifications, non-proteinaceous components and other proteins) acting on the fibril formation are still poorly understood. Whether amyloid fibrils play a toxic or protective role in the pathogenesis of neurodegenerative diseases remains to be elucidated. Furthermore, such aberrant protein-protein interactions challenge the search of small-molecule drugs or immunotherapy approaches targeting amyloid formation. In this review, we describe how chemical biology tools contribute to new insights on the mode of action of amyloidogenic proteins and peptides, defining their structural signature and aggregation pathways by capturing their molecular details and conformational heterogeneity. Challenging the imagination of scientists, this constantly expanding field provides crucial tools to unravel mechanistic detail of amyloid formation such as semisynthetic proteins and small-molecule sensors of conformational changes and/or aggregation. Protein engineering methods and bioorthogonal chemistry for the introduction of protein chemical modifications are additional fruitful strategies to tackle the challenge of understanding amyloid formation.
... The closest bacterial counterparts of hOGT may include XcOGT (Xanthomonas campestris), SeOGT (Synechococcus elongatus PCC7942) and TtOGT (Thermobaculum terrenum), all of which belong to family GT-41 (Sokol and Olszewski 2015). Despite that most of the bacterial OGTs (Table I) are cytoplasmic, soluble, single polypeptides and almost all of them belong to pathogenic bacteria (Gao et al. 2018); none of these are characterized for structure and only a few are studied for enzymatic properties (Table I). ...
O-GlcNAcylation is an important post-translational modification of proteins. O-GlcNAcylated proteins have crucial roles in several cellular contexts both in eukaryotes and in bacteria. O-GlcNActransferase (OGT) is the enzyme instrumental in O-GlcNAcylation of proteins. OGT is conserved across eukaryotes. The first bacterial OGT discovered is GmaR in Listeria monocytogenes. GmaR is a GT-2 family bifunctional protein that catalyzes glycosylation of the flagellin protein FlaA and controls transcription of flagellar motility genes in a temperature-dependent manner. Here, we provide methods for heterologous expression and purification of recombinant GmaR and FlaA, in vivo/in vitro glycosylation assays, analysis of the molecular form of recombinant GmaR and detailed enzyme kinetics. We study the structure and functional dynamics of GmaR. Using solution small-angle X-ray scattering and molecular modeling, we show that GmaR adopts an extended shape with two distinctly spaced structural units in the presence of cofactor Mg2+ and with donor UDP-GlcNAc and cofactor combined. Comparisons of restored structures revealed that in-solution binding of Mg2+ ions brings about shape rearrangements and induces structural-rigidity in hyper-variable (HV) regions at the N-terminus of GmaR protein. Taking function and shape data together, we describe that Mg2+ binding enables GmaR to adopt a shape that can bind the substrate. The manuscript provides the first 3D solution structure of a bacterial OGT of GT-2 family and detailed biochemical characterization of GmaR to facilitate its future applications.
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The antimicrobial peptide PMAP-36 is a cationic peptide derived from porcine myeloid. The N-terminally paired lysine of PMAP-36 was substituted with tryptophan, and the C-terminal hydrophobic tail was deleted, thereby obtaining the antimicrobial peptide PRW4. PRW4 is a α-helical antimicrobial peptide with broad-spectrum antimicrobial activity. In this study, PRW4 was fused to the 6× His-Trx, and the fusion protein was successfully expressed in Pichia pastoris GS115 from the vector pPICZαA. The maximal induction of recombinant protein occurred in the presence of 1% methanol after 96 h at pH 6.0. After purification by a Ni-NTA resin column and digestion by enterokinase protease, 15 mg of recombinant PRW4 with a purity of 90% was obtained from 1 L of fermentation culture. The results indicated that recombinant PRW4 had similar antimicrobial activity as synthetic PRW4 against bacteria such as Escherichia coli ATCC 25922, Escherichia coli UB 1005, Salmonella typhimurium C7731, Salmonella typhimurium 7913, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, and Streptococcus faecalis ATCC 29212. We have successfully expressed PRW4 in P. pastoris, and this work provides a reference for the production of modified antimicrobial peptides in P. pastoris.
Protein O-GlcNAcylation is an evolutionary conserved post-translational modification catalysed by the nucleocytoplasmic O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). How site-specific O-GlcNAcylation modulates a diverse range of cellular processes is largely unknown. A limiting factor in studying this is the lack of accessible techniques capable of producing homogeneously O-GlcNAcylated proteins, in high yield, for in vitro studies. Here, we exploit the tolerance of OGT for cysteine instead of serine, combined with a co-expressed OGA to achieve site-specific, highly homogeneous mono-glycosylation. Applying this to DDX3X, TAB1, and CK2α, we demonstrate that near-homogeneous mono-S-GlcNAcylation of these proteins promotes DDX3X and CK2α solubility and enables production of mono-S-GlcNAcylated TAB1 crystals, albeit with limited diffraction. Taken together, this work provides a new approach for functional dissection of protein O-GlcNAcylation.
O-GlcNAc is a common modification found on nuclear and cytoplasmic proteins. Determining the catalytic mechanism of the enzyme O-GlcNAcase (OGA), which removes O-GlcNAc from proteins, enabled the creation of potent and selective inhibitors of this regulatory enzyme. Such inhibitors have served as important tools in helping to uncover the cellular and organismal physiological roles of this modification. In addition, OGA inhibitors have been important for defining the augmentation of O-GlcNAc as a promising disease-modifying approach to combat several neurodegenerative diseases including both Alzheimer’s disease and Parkinson’s disease. These studies have led to development and optimization of OGA inhibitors for clinical application. These compounds have been shown to be well tolerated in early clinical studies and are steadily advancing into the clinic. Despite these advances, the mechanisms by which O-GlcNAc protects against these various types of neurodegeneration are a topic of continuing interest since improved insight may enable the creation of more targeted strategies to modulate O-GlcNAc for therapeutic benefit. Relevant pathways on which O-GlcNAc has been found to exert beneficial effects include autophagy, necroptosis, and processing of the amyloid precursor protein. More recently, the development and application of chemical methods enabling the synthesis of homogenous proteins have clarified the biochemical effects of O-GlcNAc on protein aggregation and uncovered new roles for O-GlcNAc in heat shock response. Here, we discuss the features of O-GlcNAc in neurodegenerative diseases, the application of inhibitors to identify the roles of this modification, and the biochemical effects of O-GlcNAc on proteins and pathways associated with neurodegeneration.
O-GlcNAcylation is a dynamic post-translational modification which affects myriad proteins, cellular functions, and disease states. Its presence or absence modulates protein function via differential protein- and site-specific mechanisms, necessitating innovative techniques to probe the modification in highly selective manners. To this end, a variety of biological and chemical methods have been developed to study specific O-GlcNAc modification events both in vitro and in vivo, each with their own respective strengths and shortcomings. Together, they comprise a potent chemical biology toolbox for the analysis of O-GlcNAcylation (and, in theory, other post-translational modifications) while highlighting the need and space for more facile, generalizable, and biologically authentic techniques.
