Biology subjects

Distefano, M. D.

Publications and source records attributed to Distefano, M. D..

3 recordsLinked to original sources

bioRxiv2022

Mevalonate metabolites contribute to granulocyte chemotaxis and mortality in murine endotoxemia

Statins appear to dramatically increase sepsis survival but only when treatment is initiated prior to hospitalization. This implies that statins exhibit a delayed-onset pro-survival benefit in septic patients that results in clinical futility of statin-therapy for patients already diagnosed with sepsis. Identification of mechanisms that contribute to increased sepsis-survival following statin-pretreatment may reveal novel therapeutic targets that do not suffer similar delayed onset benefits. Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. This indirectly inhibits the de novo synthesis of not only cholesterol but also isoprenoids that are involved in prenylation, the post-translational lipid modification of proteins. Mirroring clinical observations, chronic but not acute treatment with simvastatin significantly increased survival in a murine endotoxemia model. This was associated with reduced systemic granulocyte chemotaxis that occurred in a cell-intrinsic manner. In vitro modeling showed that simvastatin abolished chemoattractant responses and that this could be reversed by restoring geranylgeranyl pyrophosphate (GGPP) but not farnesyl pyrophosphate (FPP) nor cholesterol. Treatment with prenyltransferase inhibitors showed that chemoattractant responses were dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Given the kinetic problems with initiating statin treatment after sepsis onset, prenyltransferases and geranylgeranylated proteins, such as RAC1 and GNG2, are promising interventional candidates for sepsis and critical inflammatory illness.

immunology↗

bioRxiv2022

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 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.

biochemistry↗

bioRxiv2022

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

Dysregulation of protein prenylation has been implicated in many diseases, including Alzheimers disease (AD). Prenylomic analysis, the combination of metabolic incorporation of an isoprenoid analogue (C15AlkOPP) into prenylated proteins with a bottom-up proteomic analysis, has allowed identification of prenylated proteins in various cellular models. Here, transgenic AD mice were administered with C15AlkOPP through intracerebroventricular (ICV) infusion over 13 days. Using prenylomic analysis, 36 prenylated proteins were enriched in the brains of AD mice. Importantly, the prenylated forms of 15 proteins were consistently upregulated in AD mice compared to non-transgenic wild-type controls. These results highlight the power of this in vivo metabolic labeling approach to identify multiple post-translationally modified proteins that may serve as potential therapeutic targets for a disease that has proved refractory to treatment thus far. Moreover, this method should be applicable to many other types of protein modifications, significantly broadening its scope.

biochemistry↗