Biology subjects

San Martin, A.

Publications and source records attributed to San Martin, A..

14 recordsLinked to original sources

bioRxiv2026

DECAPPING PROTEIN 5 regulates the circadian clock in Arabidopsis

The Arabidopsis transcriptome must be continuously reshaped to integrate endogenous and environmental cues, including the circadian clock and ambient light. This reprogramming relies on the coordinated synthesis and degradation of mRNAs, yet the contribution of RNA decay factors to circadian regulation remains poorly characterized. Here, we show that nearly all core clock genes are high-amplitude clock-controlled genes (HA-CCGs). A meta-analysis of transcriptome-wide mRNA decay datasets revealed that HA-CCGs have consistently shorter half-lives across multiple studies. We further identify the mRNA decapping activator DECAPPING PROTEIN 5 (DCP5) as a key regulator of circadian RNA stability. Using iCLIP, we show that DCP5 binds preferentially to HA-CCG transcripts at AU-rich motifs in their 3' UTRs, thereby modulating their stability. Accordingly, the dcp5 mutant displays an extended circadian period at both physiological and molecular levels. Our findings establish mRNA decay as a post-transcriptional regulatory layer that fine-tunes the decay of multiple key transcripts within the circadian system.

plant biology↗

bioRxiv2026

Opportunistic neuronal fuelling

Neurons can utilize either glucose or lactate, yet fuel selection during periods of activity remains unresolved. By combining optical monitoring of cytosolic pyruvate and NADH with mathematical modelling, we systematically evaluated mitochondrial fuelling in cultured neurons and acute brain slices. Under substrate concentrations typical of resting brain tissue, neurons rely almost exclusively on glucose, with a minor contribution from pyruvate. However, when extracellular lactate rises to levels mimicking tissue activity, glycolysis is inhibited and lactate becomes a major substrate, irrespective of ongoing neuronal activity. Ultimately, neuronal fuel selection is dictated not by internal energy demand, but by extracellular lactate availability, which fluctuates with local glial metabolism and systemic states such as exercise. These findings redefine our understanding of short-term metabolic flexibility in the brain and underscore the significant yet understudied role of extracellular pyruvate.

physiology↗

bioRxiv2025

MPC is a facultative pyruvate / lactate exchanger

The mitochondrial uptake of pyruvate is mediated by the MPC. In the present study, we investigated the functional properties of the carrier using genetically encoded fluorescent sensors. Mitochondrial lactate efflux was inhibited by >80% by both pharmacological and genetic MPC ablation. MPC-dependent pyruvate influx accelerated the efflux of lactate, a fingerprint of shared transport. The non-metabolized pyruvate analog oxamate, which was found to enter mitochondria solely through the MPC, was also able to trans-accelerate lactate. Reportedly, mitochondria can produce lactate under reductive stress; our findings indicate that the MPC participates in this energy/redox venting by exchanging pyruvate by lactate.

cell biology↗

bioRxiv2025

A cell-based method to assess the activity of the human sodium-coupled citrate transporter

Increasing evidence supports the human sodium-coupled citrate transporter (hNaCT) as a potential therapeutic target for metabolic syndrome, while genetic polymorphisms in hNaCT have been associated with neurological disorder. Isotopic tracers remain the primary method for evaluating citrate transport, which are labor-intensive, require dedicated equipment, and demand highly skilled personnel. Herein, we develop and validate a robust, live-cell-compatible fluorescent method to evaluate citrate transport activity mediated by hNaCT at single-cell and high-throughput levels. This method utilizes a baculoviral vector to modify HEK293 cells to co-express a genetically encoded citrate sensor (Citron1) and the hNaCT. This cell-based platform enabled real-time monitoring of citrate transport using fluorescent microscopy and standard multiwell plate reader. The present method enables the functional characterization of hNaCT gain- and loss-of-function mutation, as well as the evaluation of pharmacological inhibitors targeting citrate transport. These results demonstrate that the cell-based method reliably assesses citrate transport at a single-cell resolution and is compatible with high-throughput screening, to identify novel lead compounds for the therapeutic modulation of hNaCT.

cell biology↗

bioRxiv2025

Metabolic Adaptations of Prostate Cancer Cells Under Chronic Fructose Stimulation

BackgroundDietary sugars, particularly fructose, are increasingly implicated in cancer progression through their impact on tumor metabolism. However, the specific metabolic adaptations driven by fructose in prostate cancer (PCa) remain unexplored. MethodsWe investigated the metabolic consequences of chronic fructose exposure in androgen-sensitive (LNCaP) and androgen-independent (PC-3) PCa cell lines. We evaluated the expression, localization, and function of the fructose transporter Glut-5 and assessed metabolic fluxes, enzyme expression, lipid accumulation, and global metabolite profiles using molecular, imaging, and metabolomic approaches. ResultsGlut-5 was primarily localized to early endosomes under basal and fructose-stimulated conditions, suggesting a non-canonical role potentially consistent with transceptor function. Chronic fructose exposure significantly upregulated Glut-5 expression and enhanced fructose uptake, but did not alter substantially its subcellular localization. Functionally, fructose reduced lactate production and mitochondrial ATP output, indicating a metabolic shift away from glycolysis and oxidative phosphorylation. In LNCaP cells, fructose induced robust activation of de novo lipogenesis (DNL), evidenced by upregulation of FASN and G6PD, increased lipid droplet accumulation, and enhanced levels of key fatty acid metabolites (e.g., TG, EPA, DHA). In contrast, PC-3 cells exhibited a distinct metabolic response, characterized by increased ceramide and amino acid metabolites. Notably, pharmacological inhibition of lipid metabolism using etomoxir abrogated proliferation in both cell lines under fructose stimulation. ConclusionsThese findings reveal that fructose promotes a metabolic reprogramming in PCa cells that is cell type- and AR-dependent, enhancing lipogenesis and potentially contributing to tumor progression. Our study identifies Glut-5-mediated fructolysis and lipid metabolic pathways as key vulnerabilities in PCa, offering potential avenues for metabolic intervention.

cell biology↗

bioRxiv2025

Two-Point Calibration Protocol for the FRET Indicator Pyronic in Neurons

SignificancePyruvate is a nodal intermediate in cellular metabolism, positioned at the crossroads between glycolysis and fermentative metabolism. It is exchanged between the intracellular and extracellular compartments through the proton-coupled monocarboxylate transporters and between the cytosol and mitochondria through the mitochondrial pyruvate carrier, where it serves as a primary carbon source for respiration. AimOur goal is to present a detailed protocol for quantifying cytosolic pyruvate concentration in neurons at single-cell resolution using a minimally invasive, two-point calibration approach with the FRET-based genetically-encoded fluorescent indicator Pyronic. ApproachThis protocol is based on a non-invasive pharmacological two-point calibration approach, where Pyronics dynamic range ({Delta}RMAX) is established by using trans-acceleration exchange to deplete intracellular pyruvate (RMIN), and by inducing Pyronic saturation (RMAX) through the combination of inhibition of pyruvate export, stimulation of its production, and blockade of its mitochondrial consumption. The protocol also incorporates the previously published KD values for Pyronic obtained from in vitro experiments. This procedure does not require the use of detergents to permeabilize the cells. ResultsImplementing this protocol enables the measurement of absolute cytosolic pyruvate concentrations. This quantitative parameter facilitates comparisons of pyruvate metabolism across different cells, samples and experimental batches, thereby enabling the comparison between a plethora of experimental conditions. ConclusionsThe FRET-based fluorescent indicator Pyronic can be reliably calibrated using a minimally invasive, pharmacology-based two-point calibration protocol in neurons, thus providing a robust and quantitative method to study pyruvate metabolism under various physiological and pathological scenarios.

neuroscience↗

bioRxiv2025

Acute stimulation of glucose metabolism by H2O2 sustains theNADPH steady-state under oxidative stress

Oxidative stress reprograms metabolic flux from glycolysis to the pentose phosphate pathway. Recently, it has been proposed that NADPH acts as a key molecule in pentose phosphate pathway regulation by exerting negative feedback through tonic inhibition of glucose-6-phosphate dehydrogenase. Interestingly, recent studies show that NADPH levels remain stable during acute exposure to hydrogen peroxide in the presence of glucose, ruling out NADPH-dependent feedback inhibition. We hypothesize that hydrogen peroxide triggers a feedforward activation mechanism, increasing NADPH production even before any detectable NADPH depletion. To probe this hypothesis, we used a panel of genetically encoded fluorescent indicators to monitor glucose, NADPH, F1,6BP, and pyruvate in single cells with high temporal resolution. Our results reveal that hydrogen peroxide rapidly activates glucose transport and consumption rates, enabling cells to preserve NADPH steady-state levels during early oxidative stress. Notably, this response precedes NADPH depletion, implying an anticipatory phenomenon that boosts NADPH production prior to its consumption. Furthermore, hydrogen peroxide induced an acute perturbation of F1,6BP steady-state and an increase of pyruvate accumulation. The pharmacological inhibition of the PPPs gateway enzymes, glucose-6-phosphate dehydrogenase and transketolase, abolished the hydrogen peroxide-dependent alterations in F1,6BP steady-state levels and pyruvate accumulation, respectively. These findings suggest that a substantial fraction of glucose-derived carbon flux is diverted to the pentose phosphate pathway under oxidative stress, underscoring the importance of feedforward control in maintaining redox balance.

biochemistry↗

bioRxiv2025

The activation of pentose phosphate pathway flux by hydrogen peroxide is not regulated by NADPH-mediated feedback inhibition

Background and PurposeOxidative stress induces a rerouting of metabolic flux from glycolysis to the pentose phosphate pathway. One proposed mechanism involves negative feedback via tonic inhibition of glucose-6-phosphate dehydrogenase by NADPH. However, recent evidence shows that NADPH levels do not decrease five seconds after hydrogen peroxide (H2O2) treatment. This finding is inconsistent with the canonical model wherein feedback inhibition loop is modulated by NADPH-depletion. This inconsistency prompts us to test the involvement of feedback inhibition at high temporal resolution. Experimental ApproachWe employed genetically encoded fluorescent indicators for H2O2 (HyPerRed) and NADPH (iNap1) expressed in epithelial HEK293 cells. These tools enable simultaneous real-time, single-cell monitoring of NADPH and H2O2. Key ResultsGlucose sustains NADPH levels under acute oxidative stress in the first seconds following H2O2 exposure. This result contradicts the reported feedback inhibition, which is considered one of the fundamental mechanisms to explain the acute rerouting of glycolysis to PPP. Furthermore, pharmacological inhibition of G6PDH suggests that the PPP is the primary source of cytosolic NADPH under oxidative stress. Monitoring NADPH levels following G6PDH inhibition allows for the assessment of the NADPH consumption flux. This parameter is low under baseline conditions, but rises dramatically under oxidative stress. Conclusion and ImplicationsOur results support an anticipatory phenomenon that maintains NADPH levels under acute H2O2 exposure, thereby discarding the proposed feedback inhibition loop. This work offers a new perspective on the regulatory nuances of a metabolic pathway implicated in aging, cancer and a plethora of pathological conditions associated with the deleterious consequences of oxidative stress.

cell biology↗

bioRxiv2024

Arabidopsis PRMT5 Buffers Pre-mRNA Splicing and Development Against Genetic Variation in Donor Splice Sites

Genetic variation at splice site signals significantly influences alternative splicing, leading to transcriptomic and proteomic diversity that enhances phenotypic plasticity and adaptation. However, novel splice variants can negatively impact gene expression and developmental stability. Canalization--the ability of an organism to maintain a consistent phenotype despite genetic or environmental variations--helps balance the effects of genetic variation on development and evolution. Protein arginine methyltransferase 5 (PRMT5) is a key splicing regulator in plants and animals. Most splicing changes in prmt5 mutants are linked to weak donor splice sites, suggesting that PRMT5 may buffer splicing against genetic variation. We examined PRMT5s effects on splicing and development in two genetically divergent Arabidopsis thaliana accessions with different single nucleotide polymorphisms (SNPs) affecting donor splice sites. While PRMT5 inactivation similarly affected splicing in both backgrounds, it significantly increased splicing and phenotypic differences between the accessions. Our findings suggest that PRMT5 contributes to canalization, mitigating the impact of splice site polymorphisms and facilitating the evolution of adaptive splicing patterns.

molecular biology↗

bioRxiv2024

Lactate-carried Mitochondrial Energy Overflow

We addressed the question of mitochondrial lactate metabolism using genetically-encoded sensors. The organelle was found to contain a dynamic lactate pool that leads to dose- and time-dependent protein lactylation. In neurons, mitochondrial lactate reported blood lactate levels with high fidelity. The exchange of lactate across the inner mitochondrial membrane was found to be mediated by a high affinity H+-coupled transport system involving the mitochondrial pyruvate carrier MPC. Assessment of electron transport chain activity and determination of lactate flux showed that mitochondria are tonic lactate producers, a phenomenon driven by energization and stimulated by hypoxia. We conclude that an overflow mechanism caps the redox level of mitochondria, while saving energy in the form of lactate. One Sentence SummaryMitochondrial lactate production

biochemistry↗

bioRxiv2023

A Systematic Identification of RBPs Driving Aberrant Splicing in Cancer

Alternative Splicing (AS) is a post-transcriptional process by which a single RNA can lead to different mRNA and, in some cases, several proteins. Various processes (probably many of them yet to be discovered) are involved in the regulation of alternative splicing. This work focuses on the regulation by RNA-binding proteins (RBPs). In addition to splicing regulation, these proteins are related to cancer prognosis and are emerging therapeutic targets for cancer treatment. CLIP-seq experiments target selected RBPs and result in uncovering the loci of the nascent transcriptome to where the RBP binds to. The presence of changes in the splicing status surrounding these loci is a good starting point to establishing a causal relationship. The selection of the specific RBP(s) to target in the CLIP-seq experiment is not straightforward; in many cases, this selection is driven by apriori hypotheses. In this work, we have developed an algorithm to detect RBPs that are likely related to the splicing changes between conditions. To do this we have integrated several databases of CLIP-seq experiments with an algorithm that detects differential splicing events to discover RBPs that are especially enriched in these events. This is a follow-up of a previous work that is refined by 1) improving the algorithm to predict the splicing events and 2) testing different enrichment statistics, and 3) performing additional validation experiments. As a result, the new method provides more accurate predictions, and it is also included in the Bioconductor package EventPointer. We tested the algorithm in four different experiments where seven different RBPs were knocked down. The algorithm accurately states the statistical significance of these RBPs using only the alterations in splicing. We also applied the algorithm to study sixteen cancer types from The Cancer Genome Atlas (TCGA). We found relationships between RBPs and several cancer types like CREBBP and MBNL2 alterations in adenocarcinomas of the lung, liver, prostate, rectum, stomach, and colon cancer. Some of these relationships have been validated in the literature but other ones are novel. AvailabilityThis method is integrated EventPointer, an available Bioconductor R package.

bioinformatics↗

bioRxiv2021

Characterization of Poldip2 knockout mice: avoiding incorrect gene targeting

POLDIP2 is a multifunctional protein whose roles are only partially understood. Our laboratory previously reported physiological studies performed using a mouse gene trap model, which suffered from two limitations: perinatal lethality in homozygotes and constitutive Poldip2 inactivation. To overcome these limitations, we developed a new conditional floxed Poldip2 model. The first part of the present study shows that our initial floxed mice were affected by an unexpected mutation, which was not readily detected by Southern blotting and traditional PCR. It consisted of a 305 kb duplication around Poldip2 with retention of the wild type allele and could be traced back to the original targeted ES cell clone. We offer simple suggestions to rapidly detect similar accidents, which may affect genome editing using both traditional and CRISPR-based methods. In the second part of the present study, correctly targeted floxed Poldip2 mice were generated and used to produce a new constitutive knockout line by crossing with a Cre deleter. In contrast to the gene trap model, many homozygous knockout mice were viable, in spite of having no POLDIP2 expression. To further characterize the effects of Poldip2 ablation in the vasculature, an RNA-seq experiment was performed in constitutive knockout carotid arteries. Results support the involvement of POLDIP2 in multiple cellular processes and provide new opportunities for future in-depth study of its functions.

genetics↗

bioRxiv2020

Highly responsive single-fluorophoreindicator to explore lactate dynamics in high calcium environments

Lactate is an energy substrate and intercellular signaling molecule with multiple bodily functions. Lactate has physiological roles in neurogenesis, axon integrity, memory consolidation, immune response, exercise, adipose tissue lipolysis, etc, and is involved in inflammation, cancer and neurodegeneration. The FRET lactate indicator Laconic has been instrumental in the discovery of mechanisms involved in neurometabolic coupling, and has advanced the understanding of lactate transport, glycolysis and mitochondrial physiology. However, the low fluorescent response and the complex saturation kinetics of Laconic limit its use for high-throughput screening and quantitation. Using the bacterial periplasmic binding protein TTHA0766 from Thermus thermophilus, we have now developed the first single-fluorophore indicator for lactate. The sensor exhibited an intensiometric fluorescence increase of {Delta}F/F0 3.0 and a single binding site with a KD of 293 M. The fluorescence is not affected by other monocarboxylates or pH. However, it is sensitive to Ca2+ in the nanomolar range. Targeting of the sensor to the endoplasmic reticulum revealed that this organelle presents a high permeability for lactate. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. This indicator, which we have termed CanlonicSF, is well suited to explore lactate dynamics in environments with micromolar Ca2+ or higher, such as the endoplasmic reticulum and the extracellular space.

cell biology↗

bioRxiv2019

A highly responsive pyruvate sensor reveals pathway-regulatory role of the mitochondrial pyruvate carrier MPC

Mitochondria generate ATP and building blocks for cell growth and regeneration, using pyruvate as the main substrate. Here we introduce PyronicSF, a user-friendly GFP-based sensor of improved dynamic range that enables real-time subcellular quantitation of mitochondrial pyruvate transport, concentration and flux. We report that cultured mouse astrocytes maintain mitochondrial pyruvate in the low micromolar range, below cytosolic pyruvate, which means that the mitochondrial pyruvate carrier MPC controls the decision between respiration and anaplerosis/gluconeogenesis in an ultrasensitive fashion. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. Mitochondrial subpopulations are known to coexist within a given cell, which differ in their morphology, mobility, membrane potential, and vicinity to other organelles. The present tool can be used to investigate how mitochondrial diversity relates to metabolism, to study the role of MPC in disease, and to screen for small-molecule MPC modulators.

cell biology↗