Dataset Information

Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.

ABSTRACT: Myristoylation is the attachment of the 14-carbon fatty acid myristate to the N-terminal glycine residue of proteins. Typically a co-translational modification, myristoylation of proapoptotic cysteinyl-aspartyl proteases (caspase)-cleaved Bid and PAK2 was also shown to occur post-translationally and is essential for their proper localization and proapoptotic function. Progress in the identification and characterization of myristoylated proteins has been impeded by the long exposure times required to monitor incorporation of radioactive myristate into proteins (typically 1-3 months). Consequently, we developed a nonradioactive detection methodology in which a bio-orthogonal azidomyristate analog is specifically incorporated co- or post-translationally into proteins at N-terminal glycines, chemoselectively ligated to tagged triarylphosphines and detected by Western blotting with short exposure times (seconds to minutes). This represents over a million-fold signal amplification in comparison to using radioactive labeling methods. Using rational prediction analysis to recognize putative internal myristoylation sites in caspase-cleaved proteins combined with our nonradioactive chemical detection method, we identify 5 new post-translationally myristoylatable proteins (PKC epsilon, CD-IC2, Bap31, MST3, and the catalytic subunit of glutamate cysteine ligase). We also demonstrate that 15 proteins undergo post-translational myristoylation in apoptotic Jurkat T cells. This suggests that post-translational myristoylation of caspase-cleaved proteins represents a novel mechanism widely used to regulate cell death.

SUBMITTER: Martin DD

PROVIDER: S-EPMC2865240 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.

Martin Dale D O DD Vilas Gonzalo L GL Prescher Jennifer A JA Rajaiah Gurram G Falck John R JR Bertozzi Carolyn R CR Berthiaume Luc G LG

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20071010 3

Myristoylation is the attachment of the 14-carbon fatty acid myristate to the N-terminal glycine residue of proteins. Typically a co-translational modification, myristoylation of proapoptotic cysteinyl-aspartyl proteases (caspase)-cleaved Bid and PAK2 was also shown to occur post-translationally and is essential for their proper localization and proapoptotic function. Progress in the identification and characterization of myristoylated proteins has been impeded by the long exposure times require ...[more]

PMID: 17932026

Similar Datasets

Project description:Hydrogels are facile architectures for the controlled presentation of proteins with far-reaching applications, from fundamental biological studies in three-dimensional culture to new regenerative medicine and therapeutic delivery strategies. Here, we demonstrate a versatile approach for spatially-defined presentation of engineered proteins within hydrogels through i) immobilization using bio-orthogonal strain-promoted alkyne-azide click chemistry and ii) dynamic protease-driven protein release using exogenously applied enzyme. Model fluorescent proteins were expressed using nonsense codon replacement to incorporate azide-containing unnatural amino acids in a site-specific manner toward maintaining protein activity: here, cyan fluorescent protein (AzCFP), mCherry fluorescent protein (AzmCh), and mCh decorated with a thrombin cut-site. (AzTMBmCh). Eight-arm poly(ethylene glycol) (PEG) was modified with dibenzylcyclooctyne (DBCO) groups and reacted with azide functionalized PEG in aqueous solution for rapid formation of hydrogels. Azide functionalized full-length fluorescent proteins were successfully incorporated into the hydrogel network by reaction with PEG-DBCO prior to gel formation. Temporal release and removal of select proteins (AzTMBmCh) was triggered with the application of thrombin and monitored in real-time with confocal microscopy, providing a responsive handle for controlling matrix properties. Hydrogels with regions of different protein compositions were created using a layering technique with thicknesses of hundreds of micrometers, affording opportunities for the creation of complex geometries on size scales relevant for controlling cellular microenvironments.Statement of significanceControlling protein presentation within biomaterials is important for modulating interactions with biological systems. For example, native tissues are composed of subunits with different matrix compositions (proteins, stiffness) that dynamically interact with cells, influencing function and fate. Toward mimicking such temporally-regulated and spatially-defined microenvironments, we utilize bio-orthogonal click chemistry and protein engineering to create hydrogels with distinct regions of proteins and modify them over time. Through nonsense codon replacement, we site-specifically functionalize large proteins with i) azides for covalent conjugation and ii) an enzymatic cleavage site for user-defined release from hydrogels. Our results exemplify not only the ability to create unique bio-functionalized hydrogels with controlled mechanical properties, but also the potential for creating interesting interfaces for cell culture and tissue engineering applications.

Dataset Information

Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.

Publications

Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets