Genetically encoded protein sulfation in mammalian cells

Italia, James S.; Peeler, Jennifer C.; Hillenbrand, Christen M.; Latour, Christopher; Weerapana, Eranthie; Chatterjee, Abhishek

doi:10.1038/s41589-020-0493-1

Cited by 65 publications

(65 citation statements)

References 26 publications

(42 reference statements)

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“…The evolved sTyrRS displayed good efficiency in comparison to its parental wild-type EcTyrRS and to a well-established AzFRS. While a side-by-side evaluation has not been conducted with evolved sTyrRSs from this and Chatterjee's work 28 , comparable activities are expected since they have similar mutations, including Leu71Val and Asp182Gly. The Chatterjee's sTyrRS does not have the Trp129Phe mutation as the sTyrRS in this report, but does contain a unique Leu186Met mutation.…”

Section: Discussionmentioning

confidence: 75%

“…However, the reported Mj tRNA Tyr -sTyrRS pair is limited to E. coli host and cannot be used in mammalian cells where protein sulfation happens and plays important biological roles. While our manuscript was under preparation, Chatterjee et al published an elegant work on the genetic incorporation of sTyr in mammalian cells 28 .…”

Section: Introductionmentioning

confidence: 99%

“…Here we report the synthesis of site-specifically sulfated proteins in both live yeast and mammalian cells through the genetic incorporation of sTyr using an engineered tyrosyl-tRNA synthetase derived from E. coli ( Ec TyrRS). Unlike the recently published work by Chatterjee et al 28 , our engineering of Ec TyrRS is conducted by using yeast as the selection host. Although E. coli is a more widely adopted platform in the directed evolution of aminoacyl-tRNA synthetases (aaRSs), yeast has the translational machinery that is more similar to that of the mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

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Functional genetic encoding of sulfotyrosine in mammalian cells

Chen

Beltran

et al. 2020

Nat Commun

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Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

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Section: Discussionmentioning

confidence: 75%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Functional genetic encoding of sulfotyrosine in mammalian cells

Chen

Beltran

et al. 2020

Nat Commun

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“…3B and C). The first co-evolved tRNA Tyr /TyrRS pairs suffered from limited orthogonality (Wang et al 2001;Chin et al 2003), but further developments now allow archaeal or modified bacterial tRNA/aaRS pairs to be employed for both rapid selection in bacteria and application of the pairs in mammalian cells (Mukai et al 2008;Neumann et al 2008;Yanagisawa et al 2008;Chen et al 2009;Italia et al 2018Italia et al , 2020. An overview of the most common tRNA/aaRS pairs is provided in Chin (2017), while the selection process for synthetases is reviewed in Davis & Chin (2012) and Chin (2014).…”

Section: Nonsense Suppression With Co-evolved Trna and Aminoacyl-trnamentioning

confidence: 99%

“…2009; Italia et al . 2018, 2020). An overview of the most common tRNA/aaRS pairs is provided in Chin (2017), while the selection process for synthetases is reviewed in Davis & Chin (2012) and Chin (2014).…”

Section: Genetic Modification Of Proteinsmentioning

confidence: 99%

The current chemical biology tool box for studying ion channels

Braun

Sheikh

Pless

2020

The Journal of Physiology

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Ion channels play important roles in human physiology and their dysfunction is linked to a variety of diseases. This has sparked considerable interest in their molecular function and pharmacology and generated a need to manipulate them with great precision. The use of high-sensitivity electrophysiological methods allows for the implementation of chemical biology manipulations, as even minute protein amounts can be studied. For example, modification of solvent-accessible cysteines is a powerful tool to site-selectively modify proteins through the introduction of charged moieties or those with fluorescent properties. This has been harnessed to study ion conduction pathways and monitor conformational dynamics. In ligand-directed chemistry, a high-affinity ligand is used to modify an ion channel with a chemical probe via a reactive linker. While these approaches are typically limited to extracellular positions, genetic code expansion provides a means to introduce non-canonical amino acids in any position of the protein. This enables the insertion of subtle analogues of naturally occurring side chains or the protein backbone, as well as amino acids with fluorescent, cross-linking or photo-switchable properties. Finally, protein semi-synthesis enables the simultaneous insertion of multiple modifications, including those that would not be tolerated by the ribosomal translation machinery. Collectively, these chemical biology tools have overcome various shortcomings of conventional mutagenesis and vastly expanded the scope of possible modifications and the type of ion channels they can be Nina Braun studied biochemistry at the University of Bayreuth and Stockholm University before discovering her interest in ion channels and non-canonical amino acids while working on her Masters thesis with Stephan A. Pless at the University of Copenhagen. She completed her PhD in the Pless lab and is currently working on high-throughput assessment of non-canonical amino acid incorporation using photocrosslinkers in acid-sensing ion channels, with the goal of studying protein-protein interactions. Zeshan P. Sheikh completed his MSc degree at the University of Copenhagen, where he discovered his passion for ion channels, mainly focusing on recombinant expression and purification. He embarked on a PhD in the Pless lab pursuing his interest in ion channel biophysics. Here, he gained particular interest in the application of chemical biological techniques for modifying ion channels. His current research priority is applying split inteins to modify acid-sensing ion channels to gain insights into their structural dynamics.

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Genetic Incorporation of ϵ‐N‐Benzoyllysine by Engineering Methanomethylophilus alvus Pyrrolysyl‐tRNA Synthetase

Cao

Ghelichkhani

et al. 2021

ChemBioChem

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Post‐translational modifications regulate protein structure and function. Lysine benzoylation is a newly discovered histone modification with unique physiological relevance. To construct proteins with this modification site‐specifically, we generated orthogonal tRNAPyl‐MaBzKRS pairs by engineering Methanomethylophilus alvus pyrrolysyl‐tRNA synthetase, allowing the genetic incorporation of ϵ‐N‐benzoyllysine (BzK) into proteins with high efficiency in E. coli and mammalian cells. Two types of MaBzKRS were identified to incorporate BzK using mutations located at different positions of the amino acid binding pocket. These MaBzKRS are small in size and highly expressed, which will afford broad utilities in studying the biological effects of lysine benzoylation.

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Genetically encoded protein sulfation in mammalian cells

Cited by 65 publications

References 26 publications

Functional genetic encoding of sulfotyrosine in mammalian cells

Functional genetic encoding of sulfotyrosine in mammalian cells

The current chemical biology tool box for studying ion channels

Genetic Incorporation of ϵ‐N‐Benzoyllysine by Engineering Methanomethylophilus alvus Pyrrolysyl‐tRNA Synthetase

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