Biology subjects

De Luca, V.

Publications and source records attributed to De Luca, V..

7 recordsLinked to original sources

bioRxiv2026

A cinnamyl alcohol dehydrogenase scaffold organizes monoterpenoid indole alkaloid biosynthesis

Biosynthesis of ~3,000 monoterpenoid indole alkaloids (MIAs), including the anticancer drug vinblastine, involves the highly unstable intermediate strictosidine aglycone. Its formation by strictosidine {beta}-glucosidase (SGD) and subsequent conversion by geissoschizine synthase (GS) occur in spatially separated compartments, representing a major biosynthesis bottleneck. Here we discover VinBLAST, a cinnamyl alcohol dehydrogenase-like protein repurposed as a scaffold for efficient processing of this labile intermediate. VinBLAST physically mediates SGD and GS interaction in the nucleus and allosterically enhances GS catalytic efficiency. VinBLAST homologues from diverse plant families enhance biosynthesis of several representative MIAs, with the production of catharanthine increased to ~160 mg L-1 in yeast, nearly 1,000-fold higher than previous studies. Our discovery provides a missing link in organizing MIA biosynthesis and enables scalable bioproduction of geissoschizine-derived therapeutics.

synthetic biology↗

bioRxiv2025

Molecular profiling of glioblastoma-derived extracellular vesicles identifies small nucleolar RNAs as candidate liquid biomarkers for radiation-induced senescence

Radiation-induced senescence (RIS) in glioblastoma (GBM) is an undesirable cell fate that reduces tumor cell death and supports resistance and outgrowth. While senescence-targeting drugs are promising adjuvants, their clinical application will require proper patient selection based on post-treatment RIS burden. Current methods to evaluate senescence, however, are tissue-based, and given GBMs difficult anatomical location, post-treatment biopsies are impractical. Innovative and less invasive biomarkers for RIS are urgently needed. To this end, we aimed to identify candidate extracellular vesicle (EV) liquid biomarkers for RIS by profiling senescence-associated cargo changes within GBM EVs. Using a panel of GBM patient-derived cell cultures, we show that RIS is the primary functional state following radiation exposure and is associated with significant alterations in the cargo of senescent-derived EVs (senEVs). In particular, senEV transcriptomes have an increased abundance of senescence-associated RNA genes and gene sets. Most striking, however, was that senEVs are most differentiated by the significant enrichment of a panel of snoRNAs. This signature was conserved in 4/5 GBM models of RIS and was validated by qRT-PCR. We further confirmed snoRNA enrichment in the senEVs of a breast cancer cell line, as well as the lack of snoRNA enrichment following senescence-independent drug exposure. Analysis by mass spectrometry revealed that snoRNAs are likely co-packaged with their associating proteins, as senEVs had concurrent increases in these binding partners. We examined whether packaging is associated with nucleolar stress during RIS, but found that upregulation in senEVs is likely due to a tightly controlled cellular abundance rather than nucleoli fragmentation and release of nucleolar components to the cytoplasm. Finally, using a preliminary cohort of longitudinal plasma samples from four GBM patients, we determined the feasibility of detecting senescence-associated and snoRNA species in the extracellular vesicles of patient biofluids. Of interest, post-treatment EVs had increased senescence-associated RNAs like CDKN2B and GLB1 and the snoRNA SNORA49. Altogether, this data suggests that senEV RNA species, and particularly snoRNAs, are a promising analyte for RIS-biomarker development. With further study, this work may open avenues for a companion diagnostic for senotherapeutics.

cancer biology↗

bioRxiv2024

Oxidation of Four Monoterpenoid Indole Alkaloid Classes by Three Cytochrome P450 Monooxygenases from Tabernaemontana litoralis

Cytochrome P450 monooxygenases (CYPs) are well known for their ability to catalyze diverse oxidation reactions, playing a significant role in the biosynthesis of various natural products. In the realm of monoterpenoid indole alkaloids (MIAs), one of the largest groups of alkaloids in nature, CYPs are integral to reactions such as hydroxylation, epoxidation, ring opening, ring rearrangement, and aromatization, contributing to the extensive diversification of these compounds. In this study, we investigate the transcriptome, metabolome, and MIA biosynthesis in Tabernaemontana litoralis (milky way tree), a prolific producer of rare pseudoaspidosperma-type MIAs. Alongside known pseudoaspidosperma biosynthetic genes, we identify and characterize three new CYPs that facilitate regio- and stereospecific oxidation of four MIA skeletons: iboga, aspidosperma, pseudoaspidosperma, and quebrachamine. Notably, the tabersonine 14,15-{beta}-epoxidase catalyzes the formation of pachysiphine, the stereoisomer of 14,15--epoxytabersonine (lochnericine) found in Catharanthus roseus (Madagascar periwinkle) roots. The pseudovincadifformine 18-hydroxylase is the first CYP identified to modify a pseudoaspidosperma skeleton. Additionally, we demonstrate that the enzyme responsible for C10-hydroxylation of the iboga MIA coronaridine also catalyzes the same reaction on voaphylline, which bears a quebrachamine skeleton. With the discovery of a new MIA, 11-hydroxypseudovincadifformine, this study provides a comprehensive understanding of MIA biosynthesis and diversification in T. litoralis, highlighting its potential for further exploration.

biochemistry↗

bioRxiv2023

In vivo characterization of a secologanin transporter from Catharanthus roseus

Monoterpenoid indole alkaloid (MIA) biosynthesis in Catharanthus roseus is a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have fully characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1/SLTr. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, and in planta silencing in two varieties of C. roseus resulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. A full panel of in vivo biochemical characterization using the Xenopus laevis oocyte expression system was used to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, rapidly transporting 1 mM of secologanin within 25 min. Notably, the absence of CrMATE1 leads to a transport bottleneck, resulting in the conversion of secologanin to its reduced form, secologanol, both in planta and in the X. laevis system. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of metabolic flux, mediating the balance between anti-herbivory potency and cell homeostasis in planta. MIA and secoiridoid transporters could also be deployed in heterologous systems to guide biosynthetic pathways and improve titers of valuable and life-saving MIAs. SIGNIFICANCEWe have fully characterized CrMATE1, a multidrug and toxic compound extrusion (MATE) family transporter in Catharanthus roseus, as a vacuolar importer of secologanin. The translocation of secologanin into the vacuole is necessary for the first committed step of monoterpenoid indole alkaloid (MIA) biosynthesis.

plant biology↗

bioRxiv2022

Biosynthesis of Kratom Opioids

Mitragyna speciosa (kratom) derived monoterpenoid indole alkaloids (MIAs) such as mitragynine and 7-hydroxymitragynine are a new class of opioids with a corynanthe MIA pharmacophore that is responsible for their significantly reduced side effects and superior safety profiles. While botanical kratom has been historically used for stimulation and pain management in Southeast Asia, the biosynthesis of kratom MIAs is not known. In this study, we identified and characterized 9 reductases bearing various degrees of demethyldihydrocoryanthine/demethylcorynantheidine synthase activity and a new SABATH type methyltransferase that catalyzes highly unusual non-aromatic enol methylation from kratom and several other species, which are required in kratom opioids biosynthesis. With unnatural substrate 4-hydroxytryptamine, we further showed the biosynthesis of mitragynine and its epimer speciogynine using these characterized enzymes. The promiscuity of kratom opioid biosynthetic enzymes suggests that derivatives and analogs of kratom opioids may be manufactured in heterologous systems with appropriate enzymes and substrates.

biochemistry↗

bioRxiv2020

Autoregulatory circuit regulating basolateral cargo export from the TGN: role of the orphan receptor GPRC5A in PKD signaling and cell polarity.

The membrane transport apparatus comprises a series of separate membrane bound compartments, or transport stations, that are responsible for the synthesis, processing, transport, sorting and delivery to their final cellular destinations of most transmembrane and soluble lumenal proteins. Over the last decades the membrane transport system has been shown to be extensively regulated both by environmental inputs and by internal homeostatic signalling systems, or control systems, that operate to maintain the homeostasis and optimal functionality of the main transport stations, such as the endoplasmic reticulum and the Golgi, in the face of internal and external perturbations. The trans-Golgi network (TGN) is a major transport and processing station and the main sorting compartment of the transport apparatus. However, the mechanisms that control cargo export and sorting at the TGN have so far remained elusive. Here we focus on the sorting of basolateral cargo proteins and show that these proteins bind to the TGN localized orphan receptor GPRC5A. The cargo-GPRC5A complex triggers the activation of a signaling pathway that involves the G{beta}{gamma} subunits dependent activation of the phospholipase C beta 3 (PLC{beta}3), which inturn induces diacyl glycerol (DAG) production. DAG recruits and activates protein kinase D (PKD) and the phosphorylation of its substrates. This step results in the formation of basolateral carriers for delivery of these cargoes to the basolateral plasma membrane domain. We term this mechanism "ARTG" (AutoRegulation of TGN export). Remarkably, the impairment of ARTG pathway components, and in particular of GPRC5A, causes defects in the polarized organization of epithelial cells.

cell biology↗

bioRxiv2019

Gcn5 and Ubp8 dependent ubiquitylation affects glycolysis in yeast

Many of the molecular mechanisms affected by ubiquitylation are highly conserved from yeast to humans and are associated to a plethora of diseases including cancers. To elucidate the regulatory role of epigenetic factors such as the catalytic subunits of SAGA complex, KAT-Gcn5 and Ub-protease Ubp8, on ubiquitylation of non-histone proteins we have performed a comprehensive analysis of the Ub-proteome in yeast Saccharomyces cerevisiae in strains disrupted in Gcn5, Ubp8 or both respect to wild type. We found significative alteration of ubiquitylation in proteins belonging to different functional categories with a recurrence of identical proteins in absence of Gcn5 or Ubp8 indicating shared targets and their interlaced function. Among the processes involved we noteworthy identified all major enzymes engaged in energy metabolism and glycolysis such as PFK1, PFK2 and others showing increased ubiquitylation respect to WT. We showed that the higher degree of ubiquitylation found is at post-translational level and does not depend on transcription. Noteworthy, we found in vivo severe defects of growth in poor sugar medium and inability to adaptive switch from fermentative to respiratory growth in strains lacking Gcn5 and Ubp8. Our findings data provide a novel, direct link, between metabolism and epigenetic control with a novel role of DUB-Ubp8 and KAT-Gcn5 on the ubiquitylation marking all the main glycolytic enzymes required for an effective execution of the glycolytic flux. Collectively our experimental results and the proposed model can lead to future research and innovative strategies that by targeting epigenetic modulators might be able to lower sugar utilization also in human cells.\n\nAuthor SummaryMolecular mechanisms dissected in simple yeast might be translated to similar circuitries in human cells for new discoveries in human diseases including cancer. Ubiquitylation of proteins is an evolutionary conserved mechanism required for many biological processes. Different post-translational modifications (PTMs) such as ubiquitylation, acetylation, methylation etc. are reciprocally regulated for deposition or removal. Epigenetic factors writing the PTMs code are often components of multiproteic complexes such as SAGA complex that holds the K-acetyltransferase (KAT) Gcn5 and the Ubiquitin-protease (DUB) Ubp8 highly conserved in Evolution. Cells respond to environment and nutrients by changing metabolism and group of enzymes involved in specific pathways are often coregulated by the deposition of selected PTMs. This study analyses the composition and quantitation of Ub-proteins differentially modified in absence of KAT-Gcn5 and DUB-Ubp8 in yeast. Interstingly, we highlighted the role of Gcn5/Ubp8 dependent ubiquitylation in marking major glycolytic enzymes necessary for glucose utilization. Our study suggests a novel regulatory pathway and, considering that lowering glycolysis is a promising strategy to target tumor metabolism, we propose this study as an interesting perspective to lower enhanced glycolysis in tumors.

molecular biology↗