scholarly journals O-GlcNAc Transferase Recognizes Protein Substrates Using an Asparagine Ladder in the Tetratricopeptide Repeat (TPR) Superhelix

2018 ◽  
Vol 140 (10) ◽  
pp. 3510-3513 ◽  
Author(s):  
Zebulon G. Levine ◽  
Chenguang Fan ◽  
Michael S. Melicher ◽  
Marina Orman ◽  
Tania Benjamin ◽  
...  
Keyword(s):  
Tetratricopeptide Repeat ◽  
Protein Substrates ◽  
Glcnac Transferase

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 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 What Curves α-Solenoids?

2002 ◽  
Vol 277 (51) ◽  
pp. 49791-49798 ◽  
Author(s):  
Andrey V. Kajava
Keyword(s):  
Structural Model ◽  
Quaternary Structure ◽  
Tetratricopeptide Repeat ◽  
Protein Substrates ◽  
Unfolded Protein ◽  
Curved Structures ◽  
Repeat Proteins ◽  
Pattern Characteristic ◽  
The Common ◽  
Tetratricopeptide Repeat Proteins

The α-helical solenoid proteins adopt a variety of elongated curved structures. They have been examined to identify the interactions that determine their curvature. A sequence pattern characteristic for strongly curved α-helical solenoids has been constructed and was found to match protein sequences containing the proteasome/cyclosome repeats. Based on this, a structural model of the repeat-containing domains of the Rpn1/S2 and Rpn2/S1 proteins, which represent the largest subunits of the 26 S proteasome, has been proposed. The model has a novel architecture resembling an α-helical toroid. Molecular modeling shows that these toroids have a central pore that would allow passage of an unfolded protein substrate through it. This implies that the Rpn1 and Rpn2 toroids are aligned along the common axial pores of the ATPase hexamer and form an “antechamber” of the 26 S proteasome. The proposed quaternary structure agrees with the available experimental data. It is suggested that the function of this antechamber is assistance to the ATPases in the unfolding of protein substrates prior to proteolysis. An evolutionary link between the PC repeat-containing proteins and tetratricopeptide repeat proteins is proposed.

scholarly journals Overview of the Assays to Probe O-Linked β-N-Acetylglucosamine Transferase Binding and Activity

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1037
Author(s):  
Cyril Balsollier ◽  
Roland J. Pieters ◽  
Marko Anderluh
Keyword(s):  
Posttranslational Modification ◽  
Phase 1 ◽  
Medical Community ◽  
Biological Processes ◽  
Degenerative Diseases ◽  
Protein Substrates ◽  
Inhibitor Discovery ◽  
Glcnac Transferase ◽  
Regulation Of Enzyme Activity ◽  
Complex Expression

O-GlcNAcylation is a posttranslational modification that occurs at serine and threonine residues of protein substrates by the addition of O-linked β-d-N-acetylglucosamine (GlcNAc) moiety. Two enzymes are involved in this modification: O-GlcNac transferase (OGT), which attaches the GlcNAc residue to the protein substrate, and O-GlcNAcase (OGA), which removes it. This biological balance is important for many biological processes, such as protein expression, cell apoptosis, and regulation of enzyme activity. The extent of this modification has sparked interest in the medical community to explore OGA and OGT as therapeutic targets, particularly in degenerative diseases. While some OGA inhibitors are already in phase 1 clinical trials for the treatment of Alzheimer’s disease, OGT inhibitors still have a long way to go. Due to complex expression and instability, the discovery of potent OGT inhibitors is challenging. Over the years, the field has grappled with this problem, and scientists have developed a number of techniques and assays. In this review, we aim to highlight assays and techniques for OGT inhibitor discovery, evaluate their strength for the field, and give us direction for future bioassay methods.

scholarly journals Protein Substrates Engage the Lumen of O-GlcNAc Transferase’s Tetratricopeptide Repeat Domain in Different Ways

Biochemistry ◽  
2021 ◽  
Vol 60 (11) ◽  
pp. 847-853
Author(s):  
Cassandra M. Joiner ◽  
Forrest A. Hammel ◽  
John Janetzko ◽  
Suzanne Walker
Keyword(s):  
Tetratricopeptide Repeat ◽  
Protein Substrates ◽  
Repeat Domain ◽  
Tetratricopeptide Repeat Domain

scholarly journals The conserved threonine-rich region of the HCF-1PRO repeat activates promiscuous OGT:UDP-GlcNAc glycosylation and proteolysis activities

2018 ◽  
Vol 293 (46) ◽  
pp. 17754-17768 ◽  
Author(s):  
Vaibhav Kapuria ◽  
Ute F. Röhrig ◽  
Patrice Waridel ◽  
Fabienne Lammers ◽  
Vladimir S. Borodkin ◽  
...  
Keyword(s):  
Host Cell ◽  
Tetratricopeptide Repeat ◽  
Rich Region ◽  
Repeat Sequences ◽  
Regulatory Strategies ◽  
Activation Mechanisms ◽  
Proteolytic Activities ◽  
Glcnac Transferase ◽  
Host Cell Factor 1 ◽  
The Relationship

O-Linked GlcNAc transferase (OGT) possesses dual glycosyltransferase–protease activities. OGT thereby stably glycosylates serines and threonines of numerous proteins and, via a transient glutamate glycosylation, cleaves a single known substrate—the so-called HCF-1PRO repeat of the transcriptional co-regulator host-cell factor 1 (HCF-1). Here, we probed the relationship between these distinct glycosylation and proteolytic activities. For proteolysis, the HCF-1PRO repeat possesses an important extended threonine-rich region that is tightly bound by the OGT tetratricopeptide-repeat (TPR) region. We report that linkage of this HCF-1PRO-repeat, threonine-rich region to heterologous substrate sequences also potentiates robust serine glycosylation with the otherwise poor Rp-αS-UDP-GlcNAc diastereomer phosphorothioate and UDP-5S-GlcNAc OGT co-substrates. Furthermore, it potentiated proteolysis of a non-HCF-1PRO-repeat cleavage sequence, provided it contained an appropriately positioned glutamate residue. Using serine- or glutamate-containing HCF-1PRO-repeat sequences, we show that proposed OGT-based or UDP-GlcNAc–based serine-acceptor residue activation mechanisms can be circumvented independently, but not when disrupted together. In contrast, disruption of both proposed activation mechanisms even in combination did not inhibit OGT-mediated proteolysis. These results reveal a multiplicity of OGT glycosylation strategies, some leading to proteolysis, which could be targets of alternative molecular regulatory strategies.

scholarly journals Cryo-EM structure provides insights into the dimer arrangement of the O-linked β-N-acetylglucosamine transferase OGT

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Richard W. Meek ◽  
James N. Blaza ◽  
Jil A. Busmann ◽  
Matthew G. Alteen ◽  
David J. Vocadlo ◽  
...  
Keyword(s):  
Repeat Unit ◽  
Structural Basis ◽  
Tetratricopeptide Repeat ◽  
Dimer Interface ◽  
X Ray ◽  
X Ray Crystallography ◽  
Protein Ligands ◽  
Repeat Units ◽  
Unit Region ◽  
Glcnac Transferase

AbstractThe O-linked β-N-acetylglucosamine modification is a core signalling mechanism, with erroneous patterns leading to cancer and neurodegeneration. Although thousands of proteins are subject to this modification, only a single essential glycosyltransferase catalyses its installation, the O-GlcNAc transferase, OGT. Previous studies have provided truncated structures of OGT through X-ray crystallography, but the full-length protein has never been observed. Here, we report a 5.3 Å cryo-EM model of OGT. We show OGT is a dimer, providing a structural basis for how some X-linked intellectual disability mutations at the interface may contribute to disease. We observe that the catalytic section of OGT abuts a 13.5 tetratricopeptide repeat unit region and find the relative positioning of these sections deviate from the previously proposed, X-ray crystallography-based model. We also note that OGT exhibits considerable heterogeneity in tetratricopeptide repeat units N-terminal to the dimer interface with repercussions for how OGT binds protein ligands and partners.

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