Biology subjects

Mihut, A.

Publications and source records attributed to Mihut, A..

6 recordsLinked to original sources

bioRxiv2026

Post-transcriptional glucocorticoid receptor signalling synchronises circadian rhythms

Keeping cellular circadian clocks synchronised is critical for cellular and physiological health. Glucocorticoid (GC) hormones are the most potent systemic timing signal for cellular clocks, acting via the glucocorticoid receptor (GR), a transcription factor conventionally thought to synchronise cells by translocating into the nucleus and activating the transcription of clock genes. Our findings overturn this model: acute changes in GR translocation and nascent transcription are both dispensable for GC-resetting of the circadian clock. Instead, physiological GC pulses act post-transcriptionally to rapidly increase Period1 mRNA through enhanced RNA processing, not synthesis. This elevates PER1 protein production which, alone, is sufficient to reset the clock. Yet each pulse resets only a subset of cells, explaining why, in vivo, repeated daily hormonal cues are required for full re-entrainment after jet lag or shift work. We propose a new model for hormonal signalling to the circadian clock and identify RNA processing kinetics as a general regulatory lever for nuclear receptor signalling.

molecular biology↗

bioRxiv2025

Sustained poly(ADP-ribosyl)ation limits RPA loading and attenuates ATR-CHK1 signaling upon replication fork collapse

Poly(ADP-ribosyl)ation (PARylation) is a transient post-translational modification catalyzed by PARP enzymes and reversed by the glycohydrolase PARG. Inhibition of PARG causes sustained PARylation, a toxic state that could be exploited for cancer therapy. However, the mechanisms by which excessive PARylation drives toxicity and cell death remain incompletely understood. Here, we examined how persistent PARylation influences cellular responses to replication stress and DNA damage. We found that during replication stress, the decline of ATR checkpoint activity marks the transition from fork stalling to fork collapse, a context in which stabilized poly(ADP-ribose) reduces chromatin-bound RPA. This loss of RPA, a key activator of the checkpoint, further reduces ATR signaling, revealing a feedback loop between unrestrained PARylation and checkpoint control. Our results identify a checkpoint-dependent consequence of unregulated PARP activity during fork collapse and highlight the PARylation-RPA axis as a critical determinant of PARG inhibitor cytotoxicity.

cell biology↗

bioRxiv2024

Circadian clocks in human cerebral organoids

Circadian rhythms result from cell-intrinsic timing mechanisms that impact health and disease1,2. To date, however, neural circadian research has largely focused on the hypothalamic circuitry of nocturnal rodents3. Whether circadian rhythms exist in human brain cells is unknown. Here we show bona fide circadian rhythms in human neurons, glia, cerebral organoids, and cerebral organoid slices (ALI-COs)4-8. Human neural circadian rhythms are synchronised by physiological timing cues such as glucocorticoids and daily temperature cycles, and these rhythms are temperature-compensated across the range of normal human brain temperatures9. Astrocyte rhythms are phase-advanced relative to other cultures and they modulate neuronal clock responses to temperature shift. Cerebral organoid rhythms are more robust at physiological brain temperatures; the relative amplitude of these rhythms increases over time in culture and their resetting capacity recapitulates key neurodevelopmental transitions in glucocorticoid signalling10-14. Remarkably, organoid post-transcriptional bioluminescent clock reporter rhythms are retained even when those of their putative transcriptional drivers are indiscernible15, and electrophysiology recordings confirm circadian rhythms in functional activity of monocultures, organoids, and ALI-COs. Around one third of the cerebral organoid proteome and phosphoproteome are circadian-rhythmic, with temporal consolidation of disease-relevant neural processes. Finally, we show that human brain organoid rhythms can be modulated and disrupted by commonly used brain-permeant drugs and mistimed cortisol exposure, respectively. Our results demonstrate that human brain cells and tissues develop their own circadian oscillations and that canonical mechanisms of the circadian clockwork may be inadequate to explain these rhythmic phenomena. 2D and 3D human neural cultures represent complementary and tractable models for exploring the emergence, disruption, and mechanics of the circadian neural clockwork, with important implications for chronobiology, brain function, and brain health.

neuroscience↗

bioRxiv2023

The loss of DNA polymerase epsilon accessory subunits POLE3-POLE4 leads to BRCA1-independent PARP inhibitor sensitivity

The clinical success of PARP1/2 inhibitors prompts the expansion of their applicability beyond homologous recombination deficiency. Here, we demonstrate that the loss of the accessory subunits of DNA polymerase epsilon, POLE3 and POLE4, sensitizes cells to PARP inhibitors. We show that the sensitivity of POLE4 knockouts is not due to a compromised response to DNA damage or homologous recombination deficiency. Instead, POLE4 deletion generates replication stress with the accumulation of single-stranded DNA gaps upon PARP inhibitor treatment. In POLE4 knockouts, replication stress leads to elevated DNA-PK signaling revealing a role of POLE4 in regulating DNA-PK activation. Moreover, POLE4 knockouts show synergistic sensitivity to the co-inhibition of ATR and PARP. Finally, POLE4 loss enhances the sensitivity of BRCA1-deficient cells to PARP inhibitors and counteracts acquired resistance consecutive to restoration of homologous recombination. Altogether, our findings establish POLE4 as a promising target to improve PARP inhibitor driven therapies and hamper acquired PARP inhibitor resistance.

cell biology↗

bioRxiv2023

Thermosensitivity of translation underlies the mammalian nocturnal-diurnal switch

Early mammals were nocturnal until the Cretaceous-Paleogene extinction enabled diurnal niche expansion. Diurnality evolved multiple times independently, but the mechanisms driving this shift remain unclear. We identify a conserved cell-intrinsic signal inversion that facilitates the transition from nocturnality to diurnality. Diurnal and nocturnal mammalian cells respond oppositely to temperature and osmotic cycles, mirroring species activity patterns. Cells exhibit differential global responses to temperature changes, including the phosphoproteome and protein synthesis. mTOR signaling is identified as a central mediator of this inversion, with diurnal mammals converging on modifications to mTOR and WNK pathways during evolution. Reducing mTOR activity induces nocturnal-to-diurnal shifting at cellular, tissue, and organismal levels. Therefore, the mTOR pathway is a cellular nexus that integrates energetic state and environmental signals to determine activity niche.

cell biology↗

bioRxiv2020

Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation and survival

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here we applied kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 were dispensable, 44 protein kinase genes were refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering revealed functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.

microbiology↗