Boosting Immunity by Targeting Post-translational Prenylation of Small GTPases.

Diseases leading to immune activation and autoinflammatory phenotypes may provide a reservoir of potentially druggable pathways for optimizing immune adjuvants or boosting antitumor immune responses. Now, Xia et al. report that lipophilic statins or biphosphonates, targeting the mevalonate pathway, act as efficient vaccine adjuvants and synergize with anti-PD1 against cancer.

Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2.

TOR complex 1 (TORC1) is a potent anabolic regulator of cellular growth and metabolism. When cells have sufficient amino acids, TORC1 is active due to its lysosomal localization mediated via the Rag GTPases. Upon amino acid removal, the Rag GTPases release TORC1, causing it to become cytoplasmic and inactive. We show here that, upon amino acid removal, the Rag GTPases also recruit TSC2 to the lysosome, where it can act on Rheb. Only when both the Rag GTPases and Rheb are inactive is TORC1 fully released from the lysosome. Upon amino acid withdrawal, cells lacking TSC2 fail to completely release TORC1 from the lysosome, fail to completely inactivate TORC1, and fail to adjust physiologically to amino acid starvation. These data suggest that regulation of TSC2 subcellular localization may be a general mechanism to control its activity and place TSC2 in the amino-acid-sensing pathway to TORC1.

14-3-3 proteins interact with a hybrid prenyl-phosphorylation motif to inhibit G proteins.

Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins.

Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway.

p53 is a frequent target for mutation in human tumors, and mutant p53 proteins can actively contribute to tumorigenesis. We employed a three-dimensional culture model in which nonmalignant breast epithelial cells form spheroids reminiscent of acinar structures found in vivo, whereas breast cancer cells display highly disorganized morphology. We found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells to a more acinar-like morphology. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates reveal that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on breast tissue architecture. Mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed sterol biosynthesis genes in human breast tumors. These findings implicate the mevalonate pathway as a therapeutic target for tumors bearing mutations in p53.

Prenylation is essential for the enrichment of cone phosphodiesterase-6 (PDE6) in outer segments and efficient cone phototransduction.

Phosphodiesterase-6 (PDE6) is the key phototransduction effector enzyme residing in the outer segment (OS) of photoreceptors. Cone PDE6 is a tetrameric protein consisting of two inhibitory subunits (γ') and two catalytic subunits (α'). The catalytic subunit of cone PDE6 contains a C-terminus prenylation motif. Deletion of PDE6α' C-terminal prenylation motif is linked to achromatopsia (ACHM), a type of color blindness in humans. However, mechanisms behind the disease and roles for lipidation of cone PDE6 in vision are unknown. In this study, we generated two knock-in mouse models expressing mutant variants of cone PDE6α' lacking the prenylation motif (PDE6α'∆C). We find that the C-terminal prenylation motif is the primary determinant for the association of cone PDE6 protein with membranes. Cones from PDE6α'∆C homozygous mice are less sensitive to light, and their response to light is delayed, whereas cone function in heterozygous PDE6α'∆C/+ mice is unaffected. Surprisingly, the expression level and assembly of cone PDE6 protein were unaltered in the absence of prenylation. Unprenylated assembled cone PDE6 in PDE6α'∆C homozygous animals is mislocalized and enriched in the cone inner segment and synaptic terminal. Interestingly, the disk density and the overall length of cone OS in PDE6α'∆C homozygous mutants are altered, highlighting a novel structural role for PDE6 in maintaining cone OS length and morphology. The survival of cones in the ACHM model generated in this study bodes well for gene therapy as a treatment option for restoring vision in patients with similar mutations in the PDE6C gene.

The cellular function of ROP GTPase prenylation is important for multicellularity in the moss Physcomitrium patens.

A complete picture of how signaling pathways lead to multicellularity is largely unknown. Previously, we generated mutations in a protein prenylation enzyme, GGB, and showed that it is essential for maintaining multicellularity in the moss Physcomitrium patens. Here, we show that ROP GTPases act as downstream factors that are prenylated by GGB and themselves play an important role in the multicellularity of P. patens. We also show that the loss of multicellularity caused by the suppression of GGB or ROP GTPases is due to uncoordinated cell expansion, defects in cell wall integrity and the disturbance of the directional control of cell plate orientation. Expressing prenylatable ROP in the ggb mutant not only rescues multicellularity in protonemata but also results in development of gametophores. Although the prenylation of ROP is important for multicellularity, a higher threshold of active ROP is required for gametophore development. Thus, our results suggest that ROP activation via prenylation by GGB is a key process at both cell and tissue levels, facilitating the developmental transition from one dimension to two dimensions and to three dimensions in P. patens.

A prenyltransferase participates in the biosynthesis of anthraquinones in Rubia cordifolia.

Anthraquinones (AQs) constitute the largest group of natural quinones, which are used as safe natural dyes and have many pharmaceutical applications. In plants, AQs are biosynthesized through two main routes: the polyketide pathway and the shikimate pathway. The latter primarily forms alizarin-type AQs, and the prenylation of 1,4-dihydroxy-2-naphthoic acid (DHNA) is the first pathway-specific step. However, the prenyltransferase (PT) responsible for this key step remains uncharacterized. In this study, the cell suspension culture of Madder (Rubia cordifolia), a plant rich in alizarin-type AQs, was discovered to be capable of prenylating DHNA to form 2-carboxyl-3-prenyl-1,4-naphthoquinone and 3-prenyl-1,4-naphthoquinone. Then, a candidate gene belonging to the UbiA superfamily, R. cordifoliadimethylallyltransferase 1 (RcDT1), was shown to account for the prenylation activity. Substrate specificity studies revealed that the recombinant RcDT1 recognized naphthoic acids primarily, followed by 4-hydroxyl benzoic acids. The prenylation activity was strongly inhibited by 1,2- and 1,4-dihydroxynaphthalene. RcDT1 RNA interference significantly reduced the AQs content in R. cordifolia callus cultures, demonstrating that RcDT1 is required for alizarin-type AQs biosynthesis. The plastid localization and root-specific expression further confirmed the participation of RcDT1 in anthraquinone biosynthesis. The phylogenetic analyses of RcDT1 and functional validation of its rubiaceous homologs indicated that DHNA-prenylation activity evolved convergently in Rubiaceae via recruitment from the ubiquinone biosynthetic pathway. Our results demonstrate that RcDT1 catalyzes the first pathway-specific step of alizarin-type AQs biosynthesis in R. cordifolia. These findings will have profound implications for understanding the biosynthetic process of the anthraquinone ring derived from the shikimate pathway.

Inhibiting Isoprenylation Suppresses FcεRI-Mediated Mast Cell Function and Allergic Inflammation.

IgE-mediated mast cell activation is a driving force in allergic disease in need of novel interventions. Statins, long used to lower serum cholesterol, have been shown in multiple large-cohort studies to reduce asthma severity. We previously found that statins inhibit IgE-induced mast cell function, but these effects varied widely among mouse strains and human donors, likely due to the upregulation of the statin target, 3-hydroxy-3-methylgutaryl-CoA reductase. Statin inhibition of mast cell function appeared to be mediated not by cholesterol reduction but by suppressing protein isoprenylation events that use cholesterol pathway intermediates. Therefore, we sought to circumvent statin resistance by targeting isoprenylation. Using genetic depletion of the isoprenylation enzymes farnesyltransferase and geranylgeranyl transferase 1 or their substrate K-Ras, we show a significant reduction in FcεRI-mediated degranulation and cytokine production. Furthermore, similar effects were observed with pharmacological inhibition with the dual farnesyltransferase and geranylgeranyl transferase 1 inhibitor FGTI-2734. Our data indicate that both transferases must be inhibited to reduce mast cell function and that K-Ras is a critical isoprenylation target. Importantly, FGTI-2734 was effective in vivo, suppressing mast cell-dependent anaphylaxis, allergic pulmonary inflammation, and airway hyperresponsiveness. Collectively, these findings suggest that K-Ras is among the isoprenylation substrates critical for FcεRI-induced mast cell function and reveal isoprenylation as a new means of targeting allergic disease.

Abolishing the prelamin A ZMPSTE24 cleavage site leads to progeroid phenotypes with near-normal longevity in mice.

Prelamin A is a farnesylated precursor of lamin A, a nuclear lamina protein. Accumulation of the farnesylated prelamin A variant progerin, with an internal deletion including its processing site, causes Hutchinson-Gilford progeria syndrome. Loss-of-function mutations in ZMPSTE24, which encodes the prelamin A processing enzyme, lead to accumulation of full-length farnesylated prelamin A and cause related progeroid disorders. Some data suggest that prelamin A also accumulates with physiological aging. Zmpste24-/- mice die young, at ∼20 wk. Because ZMPSTE24 has functions in addition to prelamin A processing, we generated a mouse model to examine effects solely due to the presence of permanently farnesylated prelamin A. These mice have an L648R amino acid substitution in prelamin A that blocks ZMPSTE24-catalyzed processing to lamin A. The LmnaL648R/L648R mice express only prelamin and no mature protein. Notably, nearly all survive to 65 to 70 wk, with ∼40% of male and 75% of female LmnaL648R/L648R mice having near-normal lifespans of 90 wk (almost 2 y). Starting at ∼10 wk of age, LmnaL648R/L648R mice of both sexes have lower body masses than controls. By ∼20 to 30 wk of age, they exhibit detectable cranial, mandibular, and dental defects similar to those observed in Zmpste24-/- mice and have decreased vertebral bone density compared to age- and sex-matched controls. Cultured embryonic fibroblasts from LmnaL648R/L648R mice have aberrant nuclear morphology that is reversible by treatment with a protein farnesyltransferase inhibitor. These novel mice provide a model to study the effects of farnesylated prelamin A during physiological aging.

Heat-shock protein 40 is the key farnesylation target in meristem size control, abscisic acid signaling, and drought resistance.

Protein farnesylation is central to molecular cell biology. In plants, protein farnesyl transferase mutants are pleiotropic and exhibit defective meristem organization, hypersensitivity to the hormone abscisic acid, and increased drought resistance. The precise functions of protein farnesylation in plants remain incompletely understood because few relevant farnesylated targets have been identified. Here, we show that defective farnesylation of a single factor-heat-shock protein 40 (HSP40), encoded by the J2 and J3 genes-is sufficient to confer ABA hypersensitivity, drought resistance, late flowering, and enlarged meristems, indicating that altered function of chaperone client proteins underlies most farnesyl transferase mutant phenotypes. We also show that expression of an abiotic stress-related microRNA (miRNA) regulon controlled by the transcription factor SPL7 requires HSP40 farnesylation. Expression of a truncated SPL7 form mimicking its activated proteolysis fragment of the membrane-bound SPL7 precursor partially restores accumulation of SPL7-dependent miRNAs in farnesyl transferase mutants. These results implicate the pathway directing SPL7 activation from its membrane-bound precursor as an important target of farnesylated HSP40, consistent with our demonstration that HSP40 farnesylation facilitates its membrane association. The results also suggest that altered gene regulation via select miRNAs contributes to abiotic stress-related phenotypes of farnesyl transferase mutants.

Characterization of the N5-dimethylallyl-FMN Intermediate in the Biosynthesis of Prenylated-FMN Catalyzed by UbiX.

Prenylated-FMN (prFMN) is the cofactor used by the UbiD-like family of decarboxylases that catalyzes the decarboxylation of various aromatic and unsaturated carboxylic acids. prFMN is synthesized from reduced FMN and dimethylallyl phosphate (DMAP) by a specialized prenyl transferase, UbiX. UbiX catalyzes the sequential formation of two bonds, the first between N5 of the flavin and C1 of DMAP, and the second between C6 of the flavin and C3 of DMAP. We have examined the reaction of UbiX with both FMN and riboflavin. Although UbiX converts FMN to prFMN, we show that significant amounts of the N5-dimethylallyl-FMN intermediate are released from the enzyme during catalysis. With riboflavin as the substrate, UbiX catalyzes only a partial reaction, resulting in only N5-dimethylallyl-riboflavin being formed. Purification of the N5-dimethylallyl-FMN adduct allowed its structure to be verified by 1H NMR spectroscopy and its reactivity to be investigated. Surprisingly, whereas reduced prFMN oxidizes in seconds to form the stable prFMN semiquinone radical when exposed to air, N5-dimethylallyl-FMN oxidizes much more slowly over several hours; in this case, oxidation is accompanied by spontaneous hydrolysis to regenerate FMN. These studies highlight the important contribution that cyclization of the prenyl-derived ring of prFMN makes to the cofactor's biological activity.

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.

Structural insights into the diverse prenylating capabilities of DMATS prenyltransferases.

Covering: 2009 up to August 2023Prenyltransferases (PTs) are involved in the primary and the secondary metabolism of plants, bacteria, and fungi, and they are key enzymes in the biosynthesis of many clinically relevant natural products (NPs). The continued biochemical and structural characterization of the soluble dimethylallyl tryptophan synthase (DMATS) PTs over the past two decades have revealed the significant promise that these enzymes hold as biocatalysts for the chemoenzymatic synthesis of novel drug leads. This is a comprehensive review of DMATSs describing the structure-function relationships that have shaped the mechanistic underpinnings of these enzymes, as well as the application of this knowledge to the engineering of DMATSs. We summarize the key findings and lessons learned from these studies over the past 14 years (2009-2023). In addition, we identify current gaps in our understanding of these fascinating enzymes.

Surveying the scope of aromatic decarboxylations catalyzed by prenylated-flavin dependent enzymes.

The prenylated-flavin mononucleotide-dependent decarboxylases (also known as UbiD-like enzymes) are the most recently discovered family of decarboxylases. The modified flavin facilitates the decarboxylation of unsaturated carboxylic acids through a novel mechanism involving 1,3-dipolar cyclo-addition chemistry. UbiD-like enzymes have attracted considerable interest for biocatalysis applications due to their ability to catalyse (de)carboxylation reactions on a broad range of aromatic substrates at otherwise unreactive carbon centres. There are now ∼35 000 protein sequences annotated as hypothetical UbiD-like enzymes. Sequence similarity network analyses of the UbiD protein family suggests that there are likely dozens of distinct decarboxylase enzymes represented within this family. Furthermore, many of the enzymes so far characterized can decarboxylate a broad range of substrates. Here we describe a strategy to identify potential substrates of UbiD-like enzymes based on detecting enzyme-catalysed solvent deuterium exchange into potential substrates. Using ferulic acid decarboxylase (FDC) as a model system, we tested a diverse range of aromatic and heterocyclic molecules for their ability to undergo enzyme-catalysed H/D exchange in deuterated buffer. We found that FDC catalyses H/D exchange, albeit at generally very low levels, into a wide range of small, aromatic molecules that have little resemblance to its physiological substrate. In contrast, the sub-set of aromatic carboxylic acids that are substrates for FDC-catalysed decarboxylation is much smaller. We discuss the implications of these findings for screening uncharacterized UbiD-like enzymes for novel (de)carboxylase activity.

Biomimetic and Concise Total Syntheses of Prenylated and Bicyclo[2.2.2]diazaoctane-Containing Indole Alkaloids Including Taichunamide A, Notoamide N and Versicolamide B.

Total syntheses of the title prenylated indole alkaloids together with seven others are reported. Biogenetic considerations have been employed in devising the reaction sequences leading to these targets with, in the opening stages, electrochemically-derived indole-3-carboxaldehyde 15 being subject to an aldol-type condensation reaction involving diketopiperazine derivative 19. This led, after prototopic shifts, intramolecular Diels-Alder cycloaddition and hydrolysis/deprotection steps, to the racemic forms of the bicyclo[2.2.2]diazaoctane-containing natural product stephacidin A (2) and its C6 epimer 3. Epoxidation of the last compound afforded, following rearrangement of the primary oxidation products, a mixture of (±)-taichunamide A [(±)-4] and (±)-versicolamide B [(±)-7]. Related protocols allowed for the conversion of (±)-stephacidin A [(±)-2] into (±)-notoamide B [(±)-5]. Analogous aldol condensation, nucleophilic reduction, and epoxidation steps led to the formation of (-)-notoamide E and its conversion into notoamide C as well as the indole fragmentation product amoenamide E. A late-stage chlorination reaction applied to (±)-stephacidin A provided access to the spirocyclic oxindole (±)-notoamide N [(±)-6].

Advances in the investigation of N6-isopentenyl adenosine i6A RNA modification.

Prenylation (isopentenylation), a key post-transcriptional modification with a hydrophobic prenyl group onto the biomacromolecules such as RNA and proteins, influences their localization and function. Prenyltransferases mediate this process, while cytokinin oxidases degrade the prenylated adenosine in plants. This review summarizes current progress in detecting prenylation modifications in RNA across species and their effects on protein synthesis. Advanced methods have been developed to label and study these modifications in vitro and in vivo, despite challenges posed by the inert chemical properties of prenyl groups. Continued advancements in bioorthogonal chemistry promise new tools for understanding the precise biological functions of prenylated RNA modifications and other related proteins.

Discovery and Heterologous Expression of Functional 4-O-Dimethylallyl-l-tyrosine Synthases from Lichen-Forming Fungi.

Fungal aromatic prenyltransferases are a family of biosynthetic enzymes that catalyze the prenylation of a range of aromatic substrates during the biosynthesis of bioactive indole alkaloids, diketopiperazines, and meroterpenoids. Their broad substrate scope and soluble nature make these enzymes particularly adept for applications in biocatalysis; for example, the enzymatic derivatization of aromatic drugs improves their bioactivity. Here, we investigated four putative aromatic prenyltransferases from lichen-forming fungi, an underexplored group of organisms that produce more than 1,000 unique metabolites. We successfully expressed two enzymes, annotated as dimethylallyltryptophan synthases, from two lichen species in the heterologous host A. oryzae. Based on their in vivo activity, we hypothesize that these enzymes are in fact 4-O-dimethylallyl-l-tyrosine synthases. Our extensive bioinformatic analysis further confirmed that these and related lichen aromatic prenyltransferases are likely not active on indoles but rather on aromatic polyketides and phenylpropanoids, major metabolites in lichens. Overall, our work provides new insights into fungal aromatic prenyltransferases at the family level and enables future efforts aimed at identifying new candidates for biocatalytic transformations of aromatic compounds.

Nitidane: An Irregular Prenylated Diterpene from the Cuticle of the Springtail Heteromurus nitidus.

Collembola are closely related to insects, but our knowledge of their often unique chemistry is limited. Here we report the identification of the epicuticular lipid nitidane, representing a novel class of epicuticular lipids. Nitidane (4) is an irregular terpene consisting of seven isoprene units, made up of a diterpene core that is modified by a geranyl moiety that is itself prenylated. The observed [46+(22+11)1]-terpene structure has not been reported before.

Parallel Evolution of Asco- and Basidiomycete O-Prenyltransferases.

Prenyltransferases (PTs) are involved in the biosynthesis of a multitude of pharmaceutically and agriculturally important plant, bacterial, and fungal compounds. Although numerous prenylated compounds have been isolated from Basidiomycota (mushroom-forming fungi), knowledge of the PTs catalyzing the transfer reactions in this group of fungi is scarce. Here, we report the biochemical characterization of an O- and C-prenylating dimethylallyltryptophan synthase (DMATS)-like enzyme LpTyrPT from the scurfy deceiver Laccaria proxima. This PT transfers dimethylallyl moieties to l-tyrosine at the para-O position and to l-tryptophan at atom C-7 and represents the first basidiomycete l-tyrosine PT described so far. Phylogenetic analysis of PTs in fungi revealed that basidiomycete l-tyrosine PTs have evolved independently from their ascomycete counterparts and might represent the evolutionary origin of PTs acting on phenolic compounds in secondary metabolism.

Biochemical characterization of a multiple prenyltransferase from Tolypocladium inflatum.

Prenylation plays a pivotal role in the diversification and biological activities of natural products. This study presents the functional characterization of TolF, a multiple prenyltransferase from Tolypocladium inflatum. The heterologous expression of tolF in Aspergillus oryzae, coupled with feeding the transformed strain with paxilline, resulted in the production of 20- and 22-prenylpaxilline. Additionally, TolF demonstrated the ability to prenylated the reduced form of paxilline, ß-paxitriol. A related prenyltransferase TerF from Chaunopycnis alba, exhibited similar substrate tolerance and regioselectivity. In vitro enzyme assays using purified recombinant enzymes TolF and TerF confirmed their capacity to catalyze prenylation of paxilline, ß-paxitriol, and terpendole I. Based on previous reports, terpendole I should be considered a native substrate. This work not only enhances our understanding of the molecular basis and product diversity of prenylation reactions in indole diterpene biosynthesis, but also provides insights into the potential of fungal indole diterpene prenyltransferase to alter their position specificities for prenylation. This could be applicable for the synthesis of industrially useful compounds, including bioactive compounds, thereby opening up new avenues for the development of novel biosynthetic strategies and pharmaceuticals. KEY POINTS: • The study characterizes TolF as a multiple prenyltransferase from Tolypocladium inflatum. • TerF from Chaunopycnis alba shows similar substrate tolerance and regioselectivity compared to TolF. • The research offers insights into the potential applications of fungal indole diterpene prenyltransferases.