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

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.

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

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Luke A. Perera ◽  
Steffen Preissler ◽  
Nathan R. Zaccai ◽  
Sylvain Prévost ◽  
Juliette M. Devos ◽  
...  

AbstractThe endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP’s ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD’s antagonistic activities, despite a similar mode of engagement of its two substrates — unmodified and AMPylated BiP.


2021 ◽  
Author(s):  
Luke A Perera ◽  
Steffen Preissler ◽  
Nathan R Zaccai ◽  
Sylvain Prevost ◽  
Juliette M Devos ◽  
...  

The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present long-sought crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP's ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD's antagonistic activities, despite a similar mode of engagement of its two substrates - unmodified and AMPylated BiP.


2020 ◽  
Author(s):  
Andrii Gorelik ◽  
Andrew T. Ferenbach

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.


2003 ◽  
Vol 278 (19) ◽  
pp. 17388-17394 ◽  
Author(s):  
Joyce Cheung-Flynn ◽  
Patricia J. Roberts ◽  
Daniel L. Riggs ◽  
David F. Smith

Genetics ◽  
2016 ◽  
Vol 203 (3) ◽  
pp. 1439-1451 ◽  
Author(s):  
Yang Yu ◽  
Zhigang Zhao ◽  
Yanrong Shi ◽  
Hua Tian ◽  
Linglong Liu ◽  
...  

Hepatology ◽  
2020 ◽  
Vol 71 (6) ◽  
pp. 2067-2079 ◽  
Author(s):  
Ranad Shaheen ◽  
Saud Alsahli ◽  
Nour Ewida ◽  
Fatema Alzahrani ◽  
Hanan E. Shamseldin ◽  
...  

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