Biology subjects

Titov, D. V.

Publications and source records attributed to Titov, D. V..

9 recordsLinked to original sources

bioRxiv2026

Metabolic glues as a means of purine sensing and chemotherapeutic response

Molecular glues stabilize weak interactions to impart novel functionalities onto complexes. While plant hormones or drugs are known molecular glues, it is still unknown whether this modality provides endogenous regulation in human cells. Here, we show that purine nucleotides are molecular glues that tether the rate-limiting enzyme of purine biosynthesis, phosphoribosyl-pyrophosphate-amidotransferase (PPAT), to its inhibitor NUDT5. This mechanism allows cells to sense purine levels and establish essential feedback control of their synthesis. Thiopurine chemotherapeutics, in clinical use since the 1950s, act as molecular glues of the same complex, but adopt unique orientations for enhanced function. Distinct from the recognition of many therapeutic glues, metabolic glue pockets can adjust their conformation to significant compound alterations and thereby enable increasing glue potency without sacrificing specificity. Our findings therefore identify endogenous metabolic glues as a mode of nutrient sensing that can be exploited to obtain compounds that rewire metabolic pathways for therapeutic benefit.

biochemistry↗

bioRxiv2026

F26BP enables control of glycolysis rate independent of energy state

Glycolysis is a conserved metabolic pathway that produces ATP and biosynthetic precursors. Multiple allosteric regulators control glycolytic enzymes in vitro. For example, phosphofructokinase (PFK) is allosterically regulated by fructose-2,6-bisphosphate (F26BP), ATP, ADP, AMP, citrate, acyl-CoA, and inorganic phosphate. It is not well understood which properties of homeostasis are enabled by each of these regulators, and whether they perform redundant or distinct functions. Using mathematical modeling and experiments with human cells lacking F26BP, we demonstrate that F26BP alters glycolytic rate independent of cellular ATP demand-a unique function not shared by other regulators. We also identified several downstream glycolytic intermediates as novel regulators of F26BP levels. Our findings clarify the role of F26BP as a unique regulator that controls the glycolytic rate independently of the cellular energy state in response to hormone and biosynthetic precursor levels. The F26BP regulatory circuit enables respiratory fuel selection and biosynthesis from glycolytic intermediates.

biochemistry↗

bioRxiv2025

Glycolytic ATP production enables rapid mammalian cell growth

Cells use ATP to fuel growth and maintenance. Surprisingly, much of the ATP in rapidly growing cells is produced through glycolytic fermentation rather than the more efficient process of respiration, a puzzling phenomenon known as the Warburg Effect. One proposed explanation is that glycolysis produces ATP faster than respiration, thereby enabling faster growth rates. But whether this explains the Warburg Effect is uncertain, as the ATP costs of mammalian cell growth have not been rigorously estimated. Here, we perform over 7500 measurements to estimate the ATP costs of growth and maintenance in mammalian cells across a range of growth rates and under perturbations to ATP production and demand. We find that respiration alone cannot meet the ATP demands of cells doubling faster than every 30 hours, roughly one-third of the maximal mammalian cell growth rate, demonstrating that the Warburg Effect is required to sustain the ATP demands of rapid cell growth.

systems biology↗

bioRxiv2025

Genetically encoded tool for manipulation of ATP/ADP ratio in human cells

The ability of cells to power energy-demanding processes depends on maintaining the ATP hydrolysis reaction a billion-fold away from equilibrium. Cells respond to changes in energy state by sensing changes in ATP, ADP, AMP, and inorganic phosphate. A key barrier to a better understanding of the maintenance of energy homeostasis is a lack of tools for direct manipulation of energy state in living cells. Here, we report the development of ATPGobble-a genetically encoded tool for controlling cellular ATP hydrolysis rate. We validated ATPGobble by showing that it doubles the energy demand, decreases [ATP]/[ADP] and [ATP]/[AMP] ratios, and activates AMPK activity in human cells. We then used ATPGobble to systematically characterize the proteome and phosphoproteome changes caused by direct manipulation of the energy state. Our results establish ATPGobble as a powerful approach for dissecting the regulatory roles of energy state in human cells, opening new opportunities to study how cellular energy state governs physiology, stress responses, and disease processes.

systems biology↗

bioRxiv2025

SRS microscopy identifies inhibition of vitellogenesis as a mediator of lifespan extension by caloric restriction in C. elegans

The molecular mechanisms of aging are not fully understood. Here, we used label-free Stimulated Raman scattering (SRS) microscopy to investigate changes in proteins and lipids throughout the lifespan of C. elegans. We observed a dramatic buildup of proteins within the body cavity or pseudocoelom of aged adults that was blunted by interventions that extend lifespan: caloric restriction (CR) and the reduced insulin/insulin-like growth factor signaling (IIS) pathway. Using a combination of microscopy, proteomic analysis, and validation with mutant strains, we identified vitellogenins as the key molecular components of the protein buildup in the pseudocoelom. Vitellogenins shuttle nutrients from intestine to embryos and are homologous to human apolipoprotein B, the causal driver of cardiovascular disease. We then showed that CR and knockdown of vitellogenins both extend lifespan by >60%, but their combination has no additional effect on lifespan, suggesting that CR extends the lifespan of C. elegans in part by inhibiting vitellogenesis. The extensive dataset of more than 12,000 images stitched into over 350 whole-animal SRS images of C. elegans at different ages and subjected to different longevity intervention will be a valuable resource for researchers interested in aging.

cell biology↗

bioRxiv2023

Genetically encoded tool for manipulation of Δ{Psi}m identifies the latter as the driver of integrative stress response induced by ATP Synthase dysfunction

Mitochondrial membrane potential ({Delta}{Psi}m) is one of the key parameters controlling cellular bioenergetics. Investigation of the role of {Delta}{Psi}m in live cells is complicated by a lack of tools for its direct manipulation without off-target effects. Here, we adopted the uncoupling protein UCP1 from brown adipocytes as a genetically encoded tool for direct manipulation of {Delta}{Psi}m. We validated the ability of exogenously expressed UCP1 to induce uncoupled respiration and lower {Delta}{Psi}m in mammalian cells. UCP1 expression lowered {Delta}{Psi}m to the same extent as chemical uncouplers but did not inhibit cell proliferation, suggesting that it manipulates {Delta}{Psi}m without the off-target effects of chemical uncouplers. Using UCP1, we revealed that elevated {Delta}{Psi}m is the driver of the Integrated Stress Response induced by ATP synthase inhibition in mammalian cells.

biochemistry↗

bioRxiv2022

Data-driven model of glycolysis identifies the role of allostery in maintaining ATP homeostasis

Glycolysis is a conserved metabolic pathway that produces ATP and biosynthetic precursors. Here, we use mathematical modeling to investigate how the control of mammalian glycolytic enzymes through allostery and mass action accomplishes various tasks of ATP homeostasis, such as controlling the rate of ATP production, maintaining high and stable ATP levels, and ensuring that ATP hydrolysis generates a net excess of energy. Our model uses data-derived enzyme rate equations, recapitulates the key tasks of glycolytic ATP homeostasis, and accurately predicts absolute concentrations of glycolytic intermediates and isotope tracing kinetics in live cells. We find that allosteric regulation of hexokinase (HK) and phosphofructokinase (PFK) by ATP, ADP, inorganic phosphate and glucose-6-phosphate (G6P), the surplus of lower glycolysis enzymes, and a large non-adenine phosphate pool are essential to robustly maintain high ATP levels and to prevent uncontrolled accumulation of phosphorylated intermediates of upper glycolysis. Meanwhile, mass action alone is sufficient to control ATP production rate and maintain high energy of ATP hydrolysis. Our results suggest a revision of the textbook view that the function of allosteric regulation of HK, PFK and PK is to control the net flux through glycolysis in response to variable ATP demand.

systems biology↗

bioRxiv2022

The Warburg Effect is the result of faster ATP production by glycolysis than respiration

Many prokaryotic and eukaryotic cells metabolize glucose to organism-specific byproducts instead of fully oxidizing it to carbon dioxide and water-a phenomenon referred to as the Warburg Effect. The benefit to a cell has been unclear, given that partial metabolism of glucose yields an order of magnitude less ATP per molecule of glucose than complete oxidation. We show that glycolysis produces ATP faster per gram of pathway protein than respiration in E. coli, S. cerevisiae, and mammalian cells. A simple mathematical model that uses yield, rate, and proteome occupancy of glycolysis and respiration as the only parameters accurately predicts absolute rates of glycolysis and respiration in all three organisms under diverse conditions. Our study suggests that the Warburg Effect is a consequence of the optimization of the rate of energy generation under the constraint of finite proteome space. One-Sentence SummaryThe Warburg Effect is a manifestation by which cells across kingdoms of life optimize the rate of energy production.

systems biology↗

bioRxiv2021

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1,000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX rescued this phenotype. These studies show that NAD+ regeneration, rather than oxidative phosphorylation, represents the primary role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

microbiology↗