scholarly journals Recognition of a glycosylation substrate by the O-GlcNAc transferase TPR repeats

Open Biology ◽  
2017 ◽  
Vol 7 (6) ◽  
pp. 170078 ◽  
Author(s):  
Karim Rafie ◽  
Olawale Raimi ◽  
Andrew T. Ferenbach ◽  
Vladimir S. Borodkin ◽  
Vaibhav Kapuria ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Substrate Specificity ◽  
Active Site ◽  
Target Site ◽  
Post Translational Modification ◽  
Tetratricopeptide Repeats ◽  
Protein Substrates ◽  
Nucleocytoplasmic Proteins ◽  
Glcnac Transferase ◽  
Single Enzyme

O-linked N -acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification found on hundreds of nucleocytoplasmic proteins in metazoa. Although a single enzyme, O-GlcNAc transferase (OGT), generates the entire cytosolic O-GlcNAc proteome, it is not understood how it recognizes its protein substrates, targeting only a fraction of serines/threonines in the metazoan proteome for glycosylation. We describe a trapped complex of human OGT with the C-terminal domain of TAB1, a key innate immunity-signalling O-GlcNAc protein, revealing extensive interactions with the tetratricopeptide repeats of OGT. Confirmed by mutagenesis, this interaction suggests that glycosylation substrate specificity is achieved by recognition of a degenerate sequon in the active site combined with an extended conformation C-terminal of the O-GlcNAc target site.

scholarly journals Protein substrates engage the lumen of O-GlcNac transferase’s tetratricopeptide repeat domain in different ways

2020 ◽  
Author(s):  
Cassandra M. Joiner ◽  
Forrest A. Hammel ◽  
John Janetzko ◽  
Suzanne Walker
Keyword(s):  
Tetratricopeptide Repeat ◽  
Substrate Selection ◽  
Post Translational Modification ◽  
Protein Substrates ◽  
Cytoplasmic Proteins ◽  
C Terminus ◽  
Glycosylation Sites ◽  
Repeat Domain ◽  
Tetratricopeptide Repeat Domain ◽  
Glcnac Transferase

ABSTRACTGlycosylation of nuclear and cytoplasmic proteins is an essential post-translational modification in mammals. O-GlcNAc transferase (OGT), the sole enzyme responsible for this modification, glycosylates over a thousand unique nuclear and cytoplasmic substrates. How OGT selects its substrates is a fundamental question that must be answered to understand OGT’s unusual biology. OGT contains a long tetratricopeptide repeat (TPR) domain that has been implicated in substrate selection, but there is almost no information about how changes to this domain affect glycosylation of individual substrates. Here, we used proteome-wide glycosylation profiling and probed glycosylation of selected purified substrates to show that asparagine and aspartate ladders that extend the full length of OGT’s TPR lumen control substrate glycosylation. We also found that substrates with glycosylation sites close to the C-terminus bypass lumenal binding. Our findings demonstrate that substrates can engage OGT in a variety of different ways for glycosylation.

scholarly journals Dual functionality of O -GlcNAc transferase is required for Drosophila development

Open Biology ◽  
2015 ◽  
Vol 5 (12) ◽  
pp. 150234 ◽  
Author(s):  
Daniel Mariappa ◽  
Xiaowei Zheng ◽  
Marianne Schimpl ◽  
Olawale Raimi ◽  
Andrew T. Ferenbach ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rational Design ◽  
Catalytic Domain ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Tetratricopeptide Repeats ◽  
Sex Combs ◽  
Glcnac Transferase ◽  
Activity Dependent

Post-translational modification of intracellular proteins with O -linked N -acetylglucosamine ( O -GlcNAc) catalysed by O -GlcNAc transferase (OGT) has been linked to regulation of diverse cellular functions. OGT possesses a C-terminal glycosyltransferase catalytic domain and N-terminal tetratricopeptide repeats that are implicated in protein–protein interactions. Drosophila OGT ( Dm OGT) is encoded by super sex combs ( sxc ), mutants of which are pupal lethal. However, it is not clear if this phenotype is caused by reduction of O -GlcNAcylation. Here we use a genetic approach to demonstrate that post-pupal Drosophila development can proceed with negligible OGT catalysis, while early embryonic development is OGT activity-dependent. Structural and enzymatic comparison between human OGT (hOGT) and Dm OGT informed the rational design of Dm OGT point mutants with a range of reduced catalytic activities. Strikingly, a severely hypomorphic OGT mutant complements sxc pupal lethality. However, the hypomorphic OGT mutant-rescued progeny do not produce F2 adults, because a set of Hox genes is de-repressed in F2 embryos, resulting in homeotic phenotypes. Thus, OGT catalytic activity is required up to late pupal stages, while further development proceeds with severely reduced OGT activity.

scholarly journals CRISPR-Cas9-mediated depletion of O-GlcNAc hydrolase and transferase for functional dissection of O-GlcNAcylation in human cells

2020 ◽  
Author(s):  
Andrii Gorelik ◽  
Andrew T. Ferenbach
Keyword(s):  
Rna Interference ◽  
Human Cell ◽  
Compensatory Mechanism ◽  
Human Cell Lines ◽  
Post Translational Modification ◽  
Short Term ◽  
Protein Substrates ◽  
Cytoplasmic Proteins ◽  
Pharmacological Studies ◽  
Glcnac Transferase

AbstractO-GlcNAcylation is an abundant post-translational modification (PTM) on serine and threonine residues of nuclear and cytoplasmic proteins. Although this PTM has been reported on thousands of proteins, O-GlcNAc transferase (OGT) and hydrolase (OGA) are the only two enzymes that perform the respective addition and removal of O-GlcNAc on protein substrates. To examine the consequences of deregulated O-GlcNAcylation, the O-GlcNAc field has mostly relied on the use of RNA interference to knockdown OGT/OGA and inhibitors to block their activities in cells. Here, we describe the first complete CRISPR-Cas9 knockouts of OGA and a knockdown of OGT (with a maximal decrease in expression of over 80%) in two human cell lines. Notably, constitutive depletion of one O-GlcNAc cycling enzyme not only led to a respective increase or decrease in total O-GlcNAcylation levels but also resulted in diminished expression of the opposing enzyme, as a compensatory mechanism, observed in previous short-term pharmacological studies. The OGA knockout system presents a convenient platform to dissect OGA mutations and was used to further characterise the single Ser405 O-GlcNAc site of human OGA using the S-GlcNAc genetic recoding approach, helping to identify an S-GlcNAc-specific antibody which was previously thought to primarily detect O-GlcNAc.

scholarly journals Feedback Regulation of O-GlcNAc Transferase through Translation Control to Maintain Intracellular O-GlcNAc Homeostasis

2021 ◽  
Vol 22 (7) ◽  
pp. 3463
Author(s):  
Chia-Hung Lin ◽  
Chen-Chung Liao ◽  
Mei-Yu Chen ◽  
Teh-Ying Chou
Keyword(s):  
Molecular Mechanisms ◽  
Mrna Level ◽  
Feedback Regulation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cell Physiology ◽  
Hydroxyl Groups ◽  
Post Translational Modification ◽  
Translation Control ◽  
Glcnac Transferase

Protein O-GlcNAcylation is a dynamic post-translational modification involving the attachment of N-acetylglucosamine (GlcNAc) to the hydroxyl groups of Ser/Thr residues on numerous nucleocytoplasmic proteins. Two enzymes are responsible for O-GlcNAc cycling on substrate proteins: O-GlcNAc transferase (OGT) catalyzes the addition while O-GlcNAcase (OGA) helps the removal of GlcNAc. O-GlcNAcylation modifies protein functions; therefore, dysregulation of O-GlcNAcylation affects cell physiology and contributes to pathogenesis. To maintain homeostasis of cellular O-GlcNAcylation, there exists feedback regulation of OGT and OGA expression responding to fluctuations of O-GlcNAc levels; yet, little is known about the molecular mechanisms involved. In this study, we investigated the O-GlcNAc-feedback regulation of OGT and OGA expression in lung cancer cells. Results suggest that, upon alterations in O-GlcNAcylation, the regulation of OGA expression occurs at the mRNA level and likely involves epigenetic mechanisms, while modulation of OGT expression is through translation control. Further analyses revealed that the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) contributes to the downregulation of OGT induced by hyper-O-GlcNAcylation; the S5A/S6A O-GlcNAcylation-site mutant of 4E-BP1 cannot support this regulation, suggesting an important role of O-GlcNAcylation. The results provide additional insight into the molecular mechanisms through which cells may fine-tune intracellular O-GlcNAc levels to maintain homeostasis.

scholarly journals Redesigning the Substrate Specificity of an Enzyme by Cumulative Effects of the Mutations of Non-active Site Residues

1999 ◽  
Vol 274 (4) ◽  
pp. 2344-2349 ◽  
Author(s):  
Shinya Oue ◽  
Akihiro Okamoto ◽  
Takato Yano ◽  
Hiroyuki Kagamiyama
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Cumulative Effects ◽  
Active Site Residues

Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

2022 ◽  
Author(s):  
Jai Krishna Mahto ◽  
Neetu Neetu ◽  
Monica Sharma ◽  
Monika Dubey ◽  
Bhanu Prakash Vellanki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Polyethylene Terephthalate ◽  
Active Site ◽  
Catalytic Domain ◽  
Structural Features ◽  
Comamonas Testosteroni ◽  
Plastic Pollution ◽  
Microbial Utilization ◽  
Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

Substrate Specificity for the Epoxidation of Terpenoids and Active Site Topology of House Fly Cytochrome P450 6A1

1997 ◽  
Vol 10 (2) ◽  
pp. 156-164 ◽  
Author(s):  
John F. Andersen ◽  
Jennifer K. Walding ◽  
Philip H. Evans ◽  
William S. Bowers ◽  
René Feyereisen
Keyword(s):  
Cytochrome P450 ◽  
Substrate Specificity ◽  
Active Site ◽  
House Fly

scholarly journals O-GlcNAc Transferase Links Glucose Metabolism to MAVS-Mediated Antiviral Innate Immunity

2018 ◽  
Vol 24 (6) ◽  
pp. 791-803.e6 ◽  
Author(s):  
Tianliang Li ◽  
Xinghui Li ◽  
Kuldeep S. Attri ◽  
Changhong Liu ◽  
Lupeng Li ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Glucose Metabolism ◽  
Glcnac Transferase ◽  
Antiviral Innate Immunity
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