Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors.

Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs, and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.

A cascade reaction of cinnamyl azides with vinyl sulfones directly generates dihydro-pyrrolo-pyrazole heterocycles.

This report describes the direct synthesis of dihydro-pyrrolo-pyrazole heterocycles from allylic azides and methyl vinyl sulfone. The product results from a complex cascade reaction that is operationally straightforward, with aromatization being the result of a concomitant elimination step. A variety of azides could participate in this reaction (12 examples) and the isolated yields of the desired product ranged from 51%-72%. Lastly the ethylene sulfone group could be removed by heating the product in pyrrolidine.

Thinking outside the CaaX-box: an unusual reversible prenylation on ALDH9A1.

Protein lipidation is a post-translational modification that confers hydrophobicity on protein substrates to control their cellular localization, mediate protein trafficking, and regulate protein function. In particular, protein prenylation is a C-terminal modification on proteins bearing canonical motifs catalyzed by prenyltransferases. Prenylated proteins have been of interest due to their numerous associations with various diseases. Chemical proteomic approaches have been pursued over the last decade to define prenylated proteomes (prenylome) and probe their responses to perturbations in various cellular systems. Here, we describe the discovery of prenylation of a non-canonical prenylated protein, ALDH9A1, which lacks any apparent prenylation motif. This enzyme was initially identified through chemical proteomic profiling of prenylomes in various cell lines. Metabolic labeling with an isoprenoid probe using overexpressed ALDH9A1 revealed that this enzyme can be prenylated inside cells but does not respond to inhibition by prenyltransferase inhibitors. Site-directed mutagenesis of the key residues involved in ALDH9A1 activity indicates that the catalytic C288 bears the isoprenoid modification likely through an NAD+-dependent mechanism. Furthermore, the isoprenoid modification is also susceptible to hydrolysis, indicating a reversible modification. We hypothesize that this modification originates from endogenous farnesal or geranygeranial, the established degradation products of prenylated proteins and results in a thioester form that accumulates. This novel reversible prenoyl modification on ALDH9A1 expands the current paradigm of protein prenylation by illustrating a potentially new type of protein-lipid modification that may also serve as a novel mechanism for controlling enzyme function.