Biology subjects

Sladky, V. C.

Publications and source records attributed to Sladky, V. C..

5 recordsLinked to original sources

bioRxiv2025

tRNA thiolation defects disrupt cellular proteostasis and tissue homeostasis in mammals

Sulfur modification of tRNA wobble uridines is an evolutionarily conserved mechanism that ensures efficient protein synthesis. In humans, loss of this anticodon modification due to mutations in CTU2 (cytosolic thiouridylase 2) causes DREAM-PL syndrome, a severe congenital disorder often leading to early postnatal death. However, the mechanisms by which loss of tRNA thiolation drives pathology remain unclear. Here, we show that loss of CTU2 triggers significant cellular proteostasis defects in patient cells and model cell lines. Structural and biochemical analyses reveal that the pathogenic CTU2L63P mutation destabilizes the CTU1/CTU2 complex and abolishes tRNA binding and thiolation. Acute loss of CTU2 caused codon-specific ribosome pausing at A-ending codons decoded by thiolated tRNAs, and decreased ribosome occupancy of A-rich transcripts in a dosage-dependent manner. Codon-biased mRNAs transcribed from genes critical for ciliogenesis are predicted to be most affected, linking their reduced translation to DREAM-PL etiology in humans. Surprisingly, Ctu2L63P mice display severe thiolation defects, but develop normally, are viable and fertile. Our findings highlight the importance of functional tRNA thiolation for organismal health in humans and identify species-specific vulnerabilities during embryonic development in mammals.

cell biology↗

bioRxiv2025

Sequential PIDD1 auto-processing is essential for ploidy control in liver and heart

Polyploidization refers to the balanced increase in gene copy number and is a feature of specialized cells in different mammalian tissues, including the liver and the heart. During organogenesis, hepatocytes and cardiomyocytes undergo scheduled polyploidization events to increase their cellular or nuclear DNA content. This is thought to improve cellular output and enable for rapid genetic adaptation in response to stress. Yet, excessive increases in ploidy can also be disadvantageous and increase the risk of genome instability. Hence, a dedicated machinery, the PIDDosome multi-protein complex, has evolved to prevent exacerbated increases in DNA content. Using targeted mutagenesis in mice, we show that the PIDDosome controls hepatocyte ploidy in a cell-autonomous manner and that sequential and quantitative auto-processing of PIDD1 is key for accurate control of ploidy in postnatal development of liver and heart. Stoichiometric imbalances in bioactive PIDD1-fragments impair p53-dependent and independent cell cycle arrest responses during organogenesis, as well as caspase-2-dependent apoptosis caused by centrosome amplification. Strikingly, targeted mutagenesis of the caspase cleavage motif in the critical E3-ligase controlling p53 protein levels, Mdm2, impairs ploidy control in hepatocytes, but not in cardiomyocytes, indicative of the existence of alternative caspase-2 substrates that help to restrict ploidy in the heart.

cell biology↗

bioRxiv2024

The PIDDosome controls cardiomyocyte polyploidization during postnatal heart development

The adult mammalian heart is characterized by post-mitotic polyploid cardiomyocytes (CMs). Understanding how CMs regulate cell cycle exit and ploidy can help developing new heart regenerative therapies. Here, we uncover that the PIDDosome, a multi-protein complex activating the endopeptidase Caspase-2, helps to implement a CM-specific differentiation program that limits ploidy during postnatal heart development. DNA content analyses show that PIDDosome-loss causes a cell-autonomous increase in nuclear and cellular CM ploidy. Remarkably, increased ploidy does not affect cardiac structure nor function. PIDDosome-imposed ploidy restriction commences at postnatal day 7 (P7), reaching a plateau on P14. PIDDosome activation requires ANKRD26, targeting PIDD1 to mother centrioles. Opposite to prior observations in liver development, the PIDDosome limits CM polyploidization in a p53-independent manner but reliant on p21/Cdkn1a, a notion supported by nuclear RNA sequencing and genetic deletion experiments. Our results provide new insights how proliferation of polyploid CMs is restricted during postnatal heart development.

developmental biology↗

bioRxiv2024

The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice

Studying essential genes required for dynamic processes in live mice is challenging as genetic perturbations are irreversible and limited by slow protein depletion kinetics. The first-generation auxin-inducible-degron (AID) system is a powerful tool for analyzing inducible protein loss in cultured cells. However, auxin administration is toxic to mice, preventing its long-term use in animals. Here, we use an optimized second-generation AID system to achieve the conditional and reversible loss of the essential centrosomal protein CEP192 in live mice. We show that the auxin derivative 5-Ph-IAA is well tolerated over two weeks and drives near-complete CEP192-mAID degradation in less than one hour in vivo. Prolonged CEP192 loss led to cell division failure and cell death in proliferative tissues. Thus, the second-generation AID system is well suited for rapid and/or sustained protein depletion in live mice, offering a valuable new tool for interrogating protein function in vivo.

cell biology↗

bioRxiv2020

PIDDosome-induced p53-activation for ploidy restriction facilitates hepatocarcinogenesis

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. The PIDDosome multi-protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome-deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome-induced p53-activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome-deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro-tumorigenic effect of PIDDosome-mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome-deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts of survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and tumor cell density serves as a novel prognostic marker for recurrence free survival in HCC patients.

cancer biology↗