Biology subjects

Kulyk, H.

Publications and source records attributed to Kulyk, H..

7 recordsLinked to original sources

bioRxiv2026

In vivo analyses reveal rapid and permissive lipid transport between the ER and mitochondria

Interorganelle lipid transport is essential for mitochondrial membrane biogenesis and function, yet its kinetics and substrate selectivity remain poorly understood in vivo. Here, we developed two complementary approaches to quantify lipid trafficking from the endoplasmic reticulum (ER) to mitochondria in yeast. Metabolic labeling combined with organelle fractionation revealed that newly synthesized phospholipids rapidly accumulate in mitochondria, with 20-35% of newly synthesized molecules detected in mitochondrial fractions within minutes of synthesis. To directly quantify lipid flux, we established a synthetic transport assay based on the production of heterologous galactolipids absent from yeast. This approach revealed an ER-to-mitochondria transport flux of approximately 2.6 x 105 lipid molecules per cell per minute. Remarkably, galactolipids were transported with high efficiency despite their absence from fungal membranes, indicating limited substrate selectivity of ER-mitochondria lipid transport pathways. Together, these complementary assays provide quantitative tools to investigate intracellular lipid transport and reveal the rapid and permissive nature of lipid exchange between the ER and mitochondria. SummaryUsing complementary metabolic labeling and synthetic lipid reporter assays, we quantitatively measured ER-mitochondria lipid transport in yeast. Our results reveal rapid lipid exchange, high transport fluxes and limited substrate selectivity, indicating that mitochondrial lipid trafficking efficiently accommodates structurally diverse membrane lipids.

Cell Biology↗

bioRxiv2025

Exploiting Vitamin B6 Dependency: BVL3572S Inhibits HisC and AlaA to Kill Mycobacterium tuberculosis

Tuberculosis remains the leading cause of death from a single infectious agent worldwide, and the growing prevalence of multi-drug resistant Mycobacterium tuberculosis (Mtb) underscores the urgent need for antibiotics with novel mechanisms of action. Here, we characterize BVL3572S, a hydroxamic acid-containing compound that is bactericidal and potently inhibits the growth of both extracellular and intracellular Mtb. Integrated transcriptomic, genetic, and biochemical analyses identified the pyridoxal phosphate (PLP)-dependent aminotransferases HisC (Rv1600) and AlaA (Rv0337c; formerly AspC) as the primary molecular targets of BVL3572S, thereby simultaneously impacting L-histidine and L-alanine biosynthesis. Spontaneous resistance mutants harbored mutations in hisC or alaA. Target engagement was further supported by overexpression studies: AlaA overexpression increased resistance in the presence of L-His whereas HisC overexpression paradoxically increased susceptibility. X-ray crystallography revealed a covalent adduct between PLP and BVL3572S within the HisC active site. The short occupancy of this adduct suggests a futile cycle that sequesters PLP. Isotopic labeling revealed widespread perturbation of amino acid biosynthesis, consistent with PLP starvation. The stepwise resistance observed upon supplementation with L-His and L-Ala together or with PLP alone suggests inhibition of multiple targets. Genome-scale CRISPRi and Tn-seq analyses additionally indicated disruptions in central metabolism, cell envelope integrity, and redox balance, possibly due to PLP depletion cascades. Consistent with its inhibition of AlaA, BVL3572S displayed strong synergy with D-cycloserine, a second-line antitubercular drug targeting D-alanine synthesis and impacting peptidoglycan synthesis, highlighting the potential of this compound in combination therapy. Collectively, our findings establish BVL3572S as a promising lead compound acting through a previously unexploited, multitarget mechanism that induces broad metabolic stress in Mtb, offering a novel therapeutic strategy against drug-resistant tuberculosis.

microbiology↗

bioRxiv2024

The glycosomal ATP-dependent phosphofructokinase of Trypanosoma brucei operates in the gluconeogenic direction in cellulo under in vivo-like conditions

In the glucose-free environment of the midgut of the tsetse fly vector, the procyclic forms of Trypanosoma brucei primarily consume proline to feed its central carbon and energy metabolism. In this context, the parasite produces through gluconeogenesis glucose 6- phosphate (G6P), the precursor of essential metabolic pathways, from proline catabolism. We showed here that the parasite uses three different enzymes to perform the key gluconeogenic reaction producing fructose 6-phosphate (F6P) from fructose 1,6-bisphosphate, (i) fructose-1,6- bisphosphatase (FBPase), the canonical enzyme performing this reaction, (ii) sedoheptulose- 1,7-bisphosphatase (SBPase) and (iii) more surprisingly ATP-dependent phosphofructokinase (PFK), an enzyme considered to irreversibly catalyse the opposite reaction involved in glycolysis. These three enzymes, as well as six other glycolytic/gluconeogenic enzymes, are located in peroxisome-related organelles, named glycosomes. Incorporation of 13C-enriched glycerol (a more effective alternative to proline for monitoring gluconeogenic activity) into F6P and G6P was more affected in the PFK null mutant than in the FBPase null mutant, suggesting the PFK contributes at least as much as FBPase to gluconeogenesis. We also showed that glucose deprivation did not affect the quantities of PFK substrates and products, whereas a 500-fold increase in the substrate/product ratio was expected for PFK to carry out the gluconeogenic reaction. In conclusion, we showed for the first time that ATP-dependent PFK can function in vivo in the gluconeogenic direction, even in the presence of FBPase activity. This particular feature, which precludes loss of ATP through a futile cycle involving PFK and FBPase working simultaneously in the glycolytic and gluconeogenic directions, respectively, is probably due to the supramolecular organisation of the metabolic pathway within glycosomes to overcome thermodynamic barriers through metabolic channelling.

microbiology↗

bioRxiv2024

Combining systems and synthetic biology for in vivo enzymology

Enzymatic parameters are classically determined in vitro, under conditions that are far from those encountered in cells, casting doubt on their physiological relevance. We developed a generic approach combining tools from synthetic and systems biology to measure enzymatic parameters in vivo. In the context of a synthetic carotenoid pathway in Saccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to study in vitro. We designed, built, and analyzed a collection of yeast strains mimicking substantial variations in substrate concentration by strategically manipulating the expression of geranyl-geranyl pyrophosphate (GGPP) synthase. We successfully determined in vivo Michaelis-Menten parameters (KM, Vmax and kcat) for GGPP-converting phytoene synthase from absolute metabolomics, fluxomics and proteomics data, highlighting differences between in vivo and in vitro parameters. Leveraging the versatility of the same set of strains, we then extracted enzymatic parameters for two of the three phytoene desaturases. Our approach demonstrates the feasibility of assessing enzymatic parameters directly in vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.

systems biology↗

bioRxiv2022

E4F1 COORDINATES PYRUVATE METABOLISM AND THE ACTIVITY OF THE ELONGATOR COMPLEX TO ENSURE PROTEIN TRANSLATION FIDELITY DURING NEURONAL DEVELOPMENT

Pyruvate metabolism defects lead to severe neuropathies such as the Leigh syndrome (LS) but the molecular mechanisms underlying neuronal cell death remain poorly understood. Here, we unravel a connection between pyruvate metabolism and the regulation of the epitranscriptome that is relevant to LS pathogenesis. We identified the transcription factor E4F1 as a key coordinator of AcetylCoenzyme A (AcCoA) production by the pyruvate dehydrogenase complex (PDC) and its utilization as an essential co-factor by the Elongator complex to acetylate tRNAs at the wobble position uridine 34 (U34). E4F1-mediated direct transcriptional regulation of Dlat and Elp3, two genes encoding key subunits of the PDC and of the Elongator complex, respectively, ensured proper translation fidelity and cell survival in the central nervous system (CNS) during mouse embryonic development. Furthermore, analysis of PDH-deficient cells highlighted a crosstalk linking the PDC to ELP3 expression that is perturbed in LS patients.

developmental biology↗

bioRxiv2021

The trypanosome UDP-glucose pyrophosphorylase is imported by piggybacking into glycosomes where unconventional sugar nucleotide synthesis takes place

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways usually present in the cytosol of other eukaryotes, such as glycolysis and biosynthesis of sugar nucleotides. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localised in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisomal targeting signal (PTS1 and PTS2). The questions we addressed here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotide functional and (ii) how the PTS-free UGP is imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3-10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knock-down UGP mutant cell line (RNAiUGP) was rescued by expressing a recoded UGP in the organelle (RNAiUGP/EXPrUGP-GPDH). Our conclusion was supported by targeted metabolomic analyses (IC-HRMS) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. ImportanceUnusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localisation of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here we showed that the cytosolic and glycosomal pathways are functional. Like in all other eukaryotes, the cytosolic pathways feed glycosylation reactions, however the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK), which are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

microbiology↗

bioRxiv2019

\"Metabolic contest\", a new way to control carbon source preference

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, allowing to redirect cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism, named \"metabolic contest\", for regulating the use of carbon sources without nutrient sensing and signaling. In contrast to most microorganisms, trypanosomes show a glycerol-to-glucose preference that depends on the combination of three conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-like organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways being both ATP-dependent (glycerol kinase and hexokinase, respectively) and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being ~80-fold higher than the hexokinase activity.

microbiology↗