The Mevalonate Pathway Is a Druggable Target for Vaccine Adjuvant Discovery.

Motivated by the clinical observation that interruption of the mevalonate pathway stimulates immune responses, we hypothesized that this pathway may function as a druggable target for vaccine adjuvant discovery. We found that lipophilic statin drugs and rationally designed bisphosphonates that target three distinct enzymes in the mevalonate pathway have potent adjuvant activities in mice and cynomolgus monkeys. These inhibitors function independently of conventional "danger sensing." Instead, they inhibit the geranylgeranylation of small GTPases, including Rab5 in antigen-presenting cells, resulting in arrested endosomal maturation, prolonged antigen retention, enhanced antigen presentation, and T cell activation. Additionally, inhibiting the mevalonate pathway enhances antigen-specific anti-tumor immunity, inducing both Th1 and cytolytic T cell responses. As demonstrated in multiple mouse cancer models, the mevalonate pathway inhibitors are robust for cancer vaccinations and synergize with anti-PD-1 antibodies. Our research thus defines the mevalonate pathway as a druggable target for vaccine adjuvants and cancer immunotherapies.

The Effect of Sustained Inflammation on Hepatic Mevalonate Pathway Results in Hyperglycemia.

Control of plasma glucose level is essential to organismal survival. Sustained inflammation has been implicated in control of glucose homeostasis in cases of infection, obesity, and type 2 diabetes; however, the precise role of inflammation in these complex disease states remains poorly understood. Here, we find that sustained inflammation results in elevated plasma glucose due to increased hepatic glucose production. We find that sustained inflammation suppresses CYP7A1, leading to accumulation of intermediate metabolites at the branch point of the mevalonate pathway. This results in prenylation of RHOC, which is concomitantly induced by inflammatory cytokines. Subsequent activation of RHO-associated protein kinase results in elevated plasma glucose. These findings uncover an unexpected mechanism by which sustained inflammation alters glucose homeostasis.

Protein prenylation: unique fats make their mark on biology.

The modification of eukaryotic proteins by isoprenoid lipids, which is known as prenylation, controls the localization and activity of a range of proteins that have crucial functions in biological regulation. The roles of prenylated proteins in cells are well conserved across species, underscoring the biological and evolutionary importance of this lipid modification pathway. Genetic suppression and pharmacological inhibition of the protein prenylation machinery have provided insights into several cellular processes and into the aetiology of diseases in which prenylation is involved. The functional dependence of prenylation substrates, such as RAS proteins, on this modification and the therapeutic potential of targeting the prenylation process in pathological conditions accentuate the need to fully understand this form of post-translational modification.

Intracellular lipid surveillance by small G protein geranylgeranylation.

Imbalances in lipid homeostasis can have deleterious effects on health1,2. Yet how cells sense metabolic demand due to lipid depletion and respond by increasing nutrient absorption remains unclear. Here we describe a mechanism for intracellular lipid surveillance in Caenorhabditis elegans that involves transcriptional inactivation of the nuclear hormone receptor NHR-49 through its cytosolic sequestration to endocytic vesicles via geranylgeranyl conjugation to the small G protein RAB-11.1. Defective de novo isoprenoid synthesis caused by lipid depletion limits RAB-11.1 geranylgeranylation, which promotes nuclear translocation of NHR-49 and activation of rab-11.2 transcription to enhance transporter residency at the plasma membrane. Thus, we identify a critical lipid sensed by the cell, its conjugated G protein, and the nuclear receptor whose dynamic interactions enable cells to sense metabolic demand due to lipid depletion and respond by increasing nutrient absorption and lipid metabolism.

ROP GTPases with a geranylgeranylation motif modulate alkaloid biosynthesis in Catharanthus roseus.

Rho of Plant (ROP) GTPases function as molecular switches that control signaling processes essential for growth, development, and defense. However, their role in specialized metabolism is poorly understood. Previously, we demonstrated that inhibition of protein geranylgeranyl transferase (PGGT-I) negatively impacts the biosynthesis of monoterpene indole alkaloids (MIA) in Madagascar periwinkle (Catharanthus roseus), indicating the involvement of prenylated proteins in signaling. Here, we show through biochemical, molecular, and in planta approaches that specific geranylgeranylated ROPs modulate C. roseus MIA biosynthesis. Among the six C. roseus ROP GTPases (CrROPs), only CrROP3 and CrROP5, having a C-terminal CSIL motif, were specifically prenylated by PGGT-I. Additionally, their transcripts showed higher expression in most parts than other CrROPs. Protein-protein interaction studies revealed that CrROP3 and CrROP5, but not ΔCrROP3, ΔCrROP5, and CrROP2 lacking the CSIL motif, interacted with CrPGGT-I. Further, CrROP3 and CrROP5 exhibited nuclear localization, whereas CrROP2 was localized to the plasma membrane. In planta functional studies revealed that silencing of CrROP3 and CrROP5 negatively affected MIA biosynthesis, while their overexpression upregulated MIA formation. In contrast, silencing and overexpression of CrROP2 had no effect on MIA biosynthesis. Moreover, overexpression of ΔCrROP3 and ΔCrROP5 mutants devoid of sequence coding for the CSIL motif failed to enhance MIA biosynthesis. These results implicate that CrROP3 and CrROP5 have a positive regulatory role on MIA biosynthesis and thus shed light on how geranylgeranylated ROP GTPases mediate the modulation of specialized metabolism in C. roseus.

CaaX-motif-adjacent residues influence G protein gamma (Gγ) prenylation under suboptimal conditions.

Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Dysregulation of prenylation contributes to multiple disorders, including cancers and vascular and neurodegenerative diseases. Prenyltransferases tether isoprenoid lipids to proteins via a thioether linkage during prenylation. Pharmacological inhibition of the lipid synthesis pathway by statins is a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential inhibition. We examined the prenylation of carboxy-terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to prenylation and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide, statin sensitivity, and extent of prenylation. Our results also show a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings indicate a plausible mechanism allowing for statins to differentially perturb heterotrimeric G protein signaling in cells depending on their Gγ-subtype composition. Our results may also provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.

GTPase splice variants RAC1 and RAC1B display isoform-specific differences in localization, prenylation, and interaction with the chaperone protein SmgGDS.

Identifying events that regulate the prenylation and localization of small GTPases will help define new strategies for therapeutic targeting of these proteins in disorders such as cancer, cardiovascular disease, and neurological deficits. Splice variants of the chaperone protein SmgGDS (encoded by RAP1GDS1) are known to regulate prenylation and trafficking of small GTPases. The SmgGDS-607 splice variant regulates prenylation by binding preprenylated small GTPases but the effects of SmgGDS binding to the small GTPase RAC1 versus the splice variant RAC1B are not well defined. Here we report unexpected differences in the prenylation and localization of RAC1 and RAC1B and their binding to SmgGDS. Compared to RAC1, RAC1B more stably associates with SmgGDS-607, is less prenylated, and accumulates more in the nucleus. We show that the small GTPase DIRAS1 inhibits binding of RAC1 and RAC1B to SmgGDS and reduces their prenylation. These results suggest that prenylation of RAC1 and RAC1B is facilitated by binding to SmgGDS-607 but the greater retention of RAC1B by SmgGDS-607 slows RAC1B prenylation. We show that inhibiting RAC1 prenylation by mutating the CAAX motif promotes RAC1 nuclear accumulation, suggesting that differences in prenylation contribute to the different nuclear localization of RAC1 versus RAC1B. Finally, we demonstrate RAC1 and RAC1B that cannot be prenylated bind GTP in cells, indicating that prenylation is not a prerequisite for activation. We report differential expression of RAC1 and RAC1B transcripts in tissues, consistent with these two splice variants having unique functions that might arise in part from their differences in prenylation and localization.

Mononuclear phagocyte morphological response to chemoattractants is dependent on geranylgeranyl pyrophosphate.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus.

Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a novel type of protein prenyltransferase, which we named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the ß subunit of RabGGTase. Using a biotinylated geranylgeranyl analogue, we identified the Golgi SNARE protein Ykt6 as a substrate of GGTase-III. GGTase-III transfers a geranylgeranyl group to mono-farnesylated Ykt6, generating doubly prenylated Ykt6. The crystal structure of GGTase-III in complex with Ykt6 provides structural basis for Ykt6 double prenylation. In GGTase-III-deficient cells, Ykt6 remained in a singly prenylated form, and the Golgi SNARE complex assembly was severely impaired. Consequently, the Golgi apparatus was structurally disorganized, and intra-Golgi protein trafficking was delayed. Our findings reveal a fourth type of protein prenyltransferase that generates geranylgeranyl-farnesyl Ykt6. Double prenylation of Ykt6 is essential for the structural and functional organization of the Golgi apparatus.

Broadening the Utility of Farnesyltransferase-Catalyzed Protein Labeling Using Norbornene-Tetrazine Click Chemistry.

Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification, in which one or two 15- or 20-carbon isoprenoid chains are transferred onto cysteine residues near the C-terminus of a target protein. The three main enzymes─protein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II)─that catalyze this process have been shown to tolerate numerous structural modifications in the isoprenoid substrate. This feature has previously been exploited to transfer an array of farnesyl diphosphate analogues with a range of functionalities, including an alkyne-containing analogue for copper-catalyzed bioconjugation reactions. Reported here is the synthesis of an analogue of the isoprenoid substrate embedded with norbornene functionality (C10NorOPP) that can be used for an array of applications, ranging from metabolic labeling to selective protein modification. The probe was synthesized in seven steps with an overall yield of 7% and underwent an inverse electron demand Diels-Alder (IEDDA) reaction with tetrazine-containing tags, allowing for copper-free labeling of proteins. The use of C10NorOPP for the study of prenylation was explored in the metabolic labeling of prenylated proteins in HeLa, COS-7, and astrocyte cells. Furthermore, in HeLa cells, these modified prenylated proteins were identified and quantified using label-free quantification (LFQ) proteomics with 25 enriched prenylated proteins. Additionally, the unique chemistry of C10NorOPP was utilized for the construction of a multiprotein-polymer conjugate for the targeted labeling of cancer cells. That construct was prepared using a combination of norbornene-tetrazine conjugation and azide-alkyne cycloaddition, highlighting the utility of the additional degree of orthogonality for the facile assembly of new protein conjugates with novel structures and functions.

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.

Calmodulin extracts the Ras family protein RalA from lipid bilayers by engagement with two membrane-targeting motifs.

RalA is a small GTPase and a member of the Ras family. This molecular switch is activated downstream of Ras and is widely implicated in tumor formation and growth. Previous work has shown that the ubiquitous Ca2+-sensor calmodulin (CaM) binds to small GTPases such as RalA and K-Ras4B, but a lack of structural information has obscured the functional consequences of these interactions. Here, we have investigated the binding of CaM to RalA and found that CaM interacts exclusively with the C terminus of RalA, which is lipidated with a prenyl group in vivo to aid membrane attachment. Biophysical and structural analyses show that the two RalA membrane-targeting motifs (the prenyl anchor and the polybasic motif) are engaged by distinct lobes of CaM and that CaM binding leads to removal of RalA from its membrane environment. The structure of this complex, along with a biophysical investigation into membrane removal, provides a framework with which to understand how CaM regulates the function of RalA and sheds light on the interaction of CaM with other small GTPases, including K-Ras4B.

Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis.

The guanylate binding protein (GBP) family of interferon-inducible GTPases promotes antimicrobial immunity and cell death. During bacterial infection, multiple mouse Gbps, human GBP2, and GBP5 support the activation of caspase-1-containing inflammasome complexes or caspase-4 which trigger pyroptosis. Whether GBPs regulate other forms of cell death is not known. The apicomplexan parasite Toxoplasma gondii causes macrophage death through unidentified mechanisms. Here we report that Toxoplasma-induced death of human macrophages requires GBP1 and its ability to target Toxoplasma parasitophorous vacuoles through its GTPase activity and prenylation. Mechanistically, GBP1 promoted Toxoplasma detection by AIM2, which induced GSDMD-independent, ASC-, and caspase-8-dependent apoptosis. Identical molecular determinants targeted GBP1 to Salmonella-containing vacuoles. GBP1 facilitated caspase-4 recruitment to Salmonella leading to its enhanced activation and pyroptosis. Notably, GBP1 could be bypassed by the delivery of Toxoplasma DNA or bacterial LPS into the cytosol, pointing to its role in liberating microbial molecules. GBP1 thus acts as a gatekeeper of cell death pathways, which respond specifically to infecting microbes. Our findings expand the immune roles of human GBPs in regulating not only pyroptosis, but also apoptosis.

Cardiac decompensation and promiscuous prenylation of small GTPases in cardiomyocytes in response to local mevalonate pathway disruption.

Investigations of major mevalonate pathway enzymes have demonstrated the importance of local isoprenoid synthesis in cardiac homeostasis. Farnesyl diphosphate synthase (FPPS) synthesizes isoprenoid precursors needed for cholesterol biosynthesis and protein prenylation. Wang, Zhang, Chen et al, in a recently published article in The Journal of Pathology, elegantly elucidated the pathological outcomes of FPPS deficiency in cardiomyocytes, which paradoxically resulted in increased prenylation of the small GTPases Ras and Rheb. Cardiomyocyte FPPS depletion caused severe dilated cardiomyopathy that was associated with enhanced GTP-loading and abundance of Ras and Rheb in lipidated protein-enriched cardiac fractions and robust activation of downstream hypertrophic ERK1/2 and mTOR signaling pathways. Cardiomyopathy and activation of ERK1/2 and mTOR caused by loss of FPPS were ameliorated by inhibition of farnesyltransferase, suggesting that impairment of FPPS activity results in promiscuous activation of Ras and Rheb through non-canonical actions of farnesyltransferase. Here, we discuss the findings and adaptive signaling mechanisms in response to disruption of local cardiomyocyte mevalonate pathway activity, highlighting how alteration in a key branch point in the mevalonate pathway affects cardiac biology and function and perturbs protein prenylation, which might unveil novel strategies and intricacies of targeting the mevalonate pathway to treat cardiovascular diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Stochastic activation and bistability in a Rab GTPase regulatory network.

The eukaryotic endomembrane system is controlled by small GTPases of the Rab family, which are activated at defined times and locations in a switch-like manner. While this switch is well understood for an individual protein, how regulatory networks produce intracellular activity patterns is currently not known. Here, we combine in vitro reconstitution experiments with computational modeling to study a minimal Rab5 activation network. We find that the molecular interactions in this system give rise to a positive feedback and bistable collective switching of Rab5. Furthermore, we find that switching near the critical point is intrinsically stochastic and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, our findings reveal how biochemical signaling networks control vesicle trafficking pathways and how their nonequilibrium properties define the spatiotemporal organization of the cell.

Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association.

Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.

Splice switching an oncogenic ratio of SmgGDS isoforms as a strategy to diminish malignancy.

The chaperone protein SmgGDS promotes cell-cycle progression and tumorigenesis in human breast and nonsmall cell lung cancer. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, facilitate the activation of oncogenic members of the Ras and Rho families of small GTPases through membrane trafficking via regulation of the prenylation pathway. SmgGDS-607 interacts with newly synthesized preprenylated small GTPases, while SmgGDS-558 interacts with prenylated small GTPases. We determined that cancer cells have a high ratio of SmgGDS-607:SmgGDS-558 (607:558 ratio), and this elevated ratio is associated with reduced survival of breast cancer patients. These discoveries suggest that targeting SmgGDS splicing to lower the 607:558 ratio may be an effective strategy to inhibit the malignant phenotype generated by small GTPases. Here we report the development of a splice-switching oligonucleotide, named SSO Ex5, that lowers the 607:558 ratio by altering exon 5 inclusion in SmgGDS pre-mRNA (messenger RNA). Our results indicate that SSO Ex5 suppresses the prenylation of multiple small GTPases in the Ras, Rho, and Rab families and inhibits ERK activity, resulting in endoplasmic reticulum (ER) stress, the unfolded protein response, and ultimately apoptotic cell death in breast and lung cancer cell lines. Furthermore, intraperitoneal (i.p.) delivery of SSO Ex5 in MMTV-PyMT mice redirects SmgGDS splicing in the mammary gland and slows tumorigenesis in this aggressive model of breast cancer. Taken together, our results suggest that the high 607:558 ratio is required for optimal small GTPase prenylation, and validate this innovative approach of targeting SmgGDS splicing to diminish malignancy in breast and lung cancer.

Prenyl Transferases Regulate Secretory Protein Sorting and Parasite Morphology in Toxoplasma gondii.

Protein prenylation is an important protein modification that is responsible for diverse physiological activities in eukaryotic cells. This modification is generally catalyzed by three types of prenyl transferases, which include farnesyl transferase (FT), geranylgeranyl transferase (GGT-1) and Rab geranylgeranyl transferase (GGT-2). Studies in malaria parasites showed that these parasites contain prenylated proteins, which are proposed to play multiple functions in parasites. However, the prenyl transferases have not been functionally characterized in parasites of subphylum Apicomplexa. Here, we functionally dissected functions of three of the prenyl transferases in the Apicomplexa model organism Toxoplasma gondii (T. gondii) using a plant auxin-inducible degron system. The homologous genes of the beta subunit of FT, GGT-1 and GGT-2 were endogenously tagged with AID at the C-terminus in the TIR1 parental line using a CRISPR-Cas9 approach. Upon depletion of these prenyl transferases, GGT-1 and GGT-2 had a strong defect on parasite replication. Fluorescent assay using diverse protein markers showed that the protein markers ROP5 and GRA7 were diffused in the parasites depleted with GGT-1 and GGT-2, while the mitochondrion was strongly affected in parasites depleted with GGT-1. Importantly, depletion of GGT-2 caused the stronger defect to the sorting of rhoptry protein and the parasite morphology. Furthermore, parasite motility was observed to be affected in parasites depleted with GGT-2. Taken together, this study functionally characterized the prenyl transferases, which contributed to an overall understanding of protein prenylation in T. gondii and potentially in other related parasites.

Impact of C-terminal truncations in the Arabidopsis Rab escort protein (REP) on REP-Rab interaction and plant fertility.

Lipid anchors are common post-translational modifications for proteins engaged in signaling and vesicular transport in eukaryotic cells. Rab proteins are geranylgeranylated at their C-termini, a modification which is important for their stable binding to lipid bilayers. The Rab escort protein (REP) is an accessory protein of the Rab geranylgeranyl transferase (RGT) complex and it is obligatory for Rab prenylation. While REP-Rab interactions have been studied by biochemical, structural, and genetic methods in animals and yeast, data on the plant RGT complex are still limited. Here we use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to describe the structural basis of plant REP-Rab binding. The obtained results show that the interaction of REP with Rabs is highly dynamic and involves specific structural changes in both partners. In some cases the Rab and REP regions involved in the interaction are molecule-specific, and in other cases they are common for a subset of Rabs. In particular, the C-terminus of REP is not involved in binding of unprenylated Rab proteins in plants, in contrast to mammalian REP. In line with this, a C-terminal REP truncation does not have pronounced phenotypic effects in planta. On the contrary, a complete lack of functional REP leads to male sterility in Arabidopsis: pollen grains develop in the anthers, but they do not germinate efficiently and hence are unable to transmit the mutated allele. The presented data show that the mechanism of action of REP in the process of Rab geranylgeranylation is different in plants than in animals or yeast.

Combining Isoprenoid Probes with Antibody Markers for Mass Cytometric Analysis of Prenylation in Single Cells.

Protein prenylation is an essential post-translational modification that plays a key role in facilitating protein localization. Aberrations in protein prenylation have been indicated in multiple disease pathologies including progeria, some forms of cancer, and Alzheimer's disease. While there are single-cell methods to study prenylation, these methods cannot simultaneously assess prenylation and other cellular changes in the complex cell environment. Here, we report a novel method to monitor, at the single-cell level, prenylation and expression of autophagy markers. An isoprenoid analogue containing a terminal alkyne, substrate of prenylation enzymes, was metabolically incorporated into cells in culture. Treatment with a terbium reporter containing an azide functional group, followed by copper-catalyzed azide-alkyne cycloaddition, covalently attached terbium ions to prenylated proteins within cells. In addition, simultaneous treatment with a holmium-containing analogue of the reporter, without an azide functional group, was used to correct for non-specific retention at the single-cell level. This procedure was compatible with other mass cytometric sample preparation steps that use metal-tagged antibodies. We demonstrate that this method reports changes in levels of prenylation in competitive and inhibitor assays, while tracking autophagy molecular markers with metal-tagged antibodies. The method reported here makes it possible to track prenylation along with other molecular pathways in single cells of complex systems, which is essential to elucidate the role of this post-translational modification in disease, cell response to pharmacological treatments, and aging.