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

Abstract

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 prenylation motifs catalyzed by prenyltransferases. Such types of proteins have been of interest owing to their potential association 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 reveals 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 indicate 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 on protein prenylation by illustrating a potentially new type of protein-lipid modification that may also serve as a novel mechanism for controlling enzyme function.

In Vivo Prenylomic Profiling in the Brain of a Transgenic Mouse Model of Alzheimer’s Disease Reveals Increased Prenylation of a Key Set of Proteins

“…Those proteins are 4-trimethylaminobutyraldehyde dehydrogenase (Aldh9A1), Ras-related protein Ral-A (Rala), Synaptobrevin homologue Ykt6 (Ykt6), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (Uchl1), Cell division control protein 42 homologue (Cdc42), Ras-related protein Rap-1b;1a,(Rab1b;Rap1a), 2,3-cyclic-nucleotide 3-phosphodiesterase (Cnp), Rab10, Rab11a;b, Rab18, Rab1a;b, Rab2a;b, Rab35, Rab3a, and Rab6a;b (Figure C). While ALDH9A1 is listed here as a farnesylated protein, the identity of the isoprenoid on this protein and the mechanism of its prenylation have not been unambiguously established . Ranking of the enriched proteins by either their average fold change or π-score revealed that Rab18 was the top ranked protein on both lists (SI Figure 4).…”

“…While ALDH9A1 is listed here as a farnesylated protein, the identity of the isoprenoid on this protein and the mechanism of its prenylation have not been unambiguously established. 42 Ranking of the enriched proteins by either their average fold change or π-score revealed that Rab18 was the top ranked protein on both lists (SI Figure 4). 43 Many of these proteins have previously been implicated as key players in the processing and regulation of Aβ peptide, APP, or tau proteins as highlighted in SI Table 1.…”