Biology subjects

Distefano, M. D.

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

5 recordsLinked to original sources

bioRxiv2026

Cell-type resolved prenylomics in mouse liver enabled by a low-input proteomics workflow

Protein prenylation is a lipid modification crucial for membrane association and cellular signaling, but its dysregulation is linked to cancer and neurodegenerative diseases. Typical "prenylomic" workflows rely on metabolic labeling with bio-orthogonal isoprenoid probes followed by streptavidin enrichment; however, they generally require milligram-scale protein inputs (15-25 million cells), preventing the study of scarce primary or sorted cell populations. To overcome this limitation, we developed a highly sensitive, low-input prenylomic workflow combining optimized streptavidin enrichment with library-free data-independent acquisition (DIA) mass spectrometry. This method enables deep prenylomic coverage from just 50 ug of total protein input, a 20-fold reduction compared to prior methods. In COS7 cells, this workflow identified 91 prenylated protein groups, outperforming TMT-DDA methods that yielded 81 groups from 2 mg of input, using the same bio-orthogonal probe for enrichment, while coefficient of variation (CV) analysis demonstrated superior quantitative precision and reproducibility. Applied in vivo in mice following subcutaneous probe administration, the low-input methodology overcame sample input limitations to profile cell-type-specific prenylomes from dissociated liver tissue, identifying 29 enriched prenylated proteins in hepatocytes and 18 in macrophages. Pathway enrichment analysis showed that the identified proteins are consistent with the biology of the respective cell-types. This robust methodology significantly lowers the sample threshold for lipid proteomics, expanding the accessibility of prenylomics to limited primary cells and sorted tissue populations to study cell-type-specific prenylation dynamics in disease contexts.

biochemistry↗

bioRxiv2024

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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/583211v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@193cfe1org.highwire.dtl.DTLVardef@134edccorg.highwire.dtl.DTLVardef@1e81deforg.highwire.dtl.DTLVardef@56406_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

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↗