EMBO Journal最新文献

ADP-ribosylation is an important protein post-translational modification catalysed by a family of PARP enzymes in humans and is involved in DNA damage and immunity among other processes. While poly-ADP-ribosylation has been established as a protein degradation signal in several cases, the role of mono-ADP-ribosylation in protein turnover has remained elusive and mostly relies on overexpression systems. Here, we describe a way to visualise high levels of endogenous ADP-ribosylation by inhibiting the ubiquitin pathway. By blocking ubiquitylation/proteasome, we found that ADP-ribosylation by at least three different PARPs (PARP7, PARP1 and TNKS) can be greatly induced. We discovered that specific activation of the aryl hydrocarbon receptor (AHR) pathway in combination with the ubiquitin pathway inhibition promotes quantitative ADP-ribosylation of PARP7 targets, including the mono-ADP-ribosyltransferase PARP7 itself and AHR. We found that DTX2 is the E3 ligase responsible for degrading ADP-ribosylated PARP7, AHR and other PARP7 substrates. This PARP7-DTX2 crosstalk establishes a mechanism to rapidly shut down AHR-mediated transcription by decreasing its protein levels. Taken together, our findings uncover a paradigm where mono-ADP-ribosylation acts as a degradation mark.

Organoid models have significantly enhanced our understanding of adult stem cell function, however, uncovering regulatory mechanisms governing rare and often quiescent stem cells in glandular organs remains challenging. Here, we employ an integrative multi-omics approach, combining single-cell RNA sequencing, bulk ATAC and RNA sequencing, to profile the cellular populations and signaling pathways characterizing a mouse salivary gland organoid model across different temporal stages and after radiation-induced damage. Our findings identify Sox9- and Itgb1/Cd44-expressing cells as primitive adult stem/progenitor populations with a critical migratory role in tissue repair. Notch signaling is a key driver of self-renewal and migration in response to irradiation. Additionally, scRNA-seq analysis of irradiated salivary gland tissue confirms these findings in an in vivo setting. Extending these findings to murine and patient-derived salivary, mammary and thyroid gland organoids, we reveal the conserved role of Notch signaling in coordinating stem/progenitor cell-mediated regeneration across glandular tissues. These insights position Notch signaling as a central regulator of glandular stem cell-like populations and as a promising therapeutic target for enhancing glandular tissue regeneration following cancer therapies.

Plants have evolved an elaborate cell wall integrity (CWI) sensing system to monitor and modify cell wall formation. LRR-extensins (LRXs) are cell wall-anchored proteins that bind RAPID ALKALINIZATION FACTOR (RALF) peptide hormones and induce compaction of cell wall structures. LRXs also form a signaling platform with RALFs and the transmembrane receptor kinase FERONIA (FER) to maintain cell wall integrity. LRX1 of Arabidopsis thaliana is predominantly expressed in root hairs, and lrx1 mutants develop defective root hairs. Here, we identify a regulator of LRX1-RALF-FER signaling as a suppressor of the lrx1 root hair phenotype. The repressor of lrx1_23 (rol23) gene encodes PP2C12, a clade H phosphatase that interacts with FER and dephosphorylates Thr696 in the FER activation loop in vitro. Expression of FER phospho-mimetic and phospho-null mutants in an lrx1 fer-4 background demonstrates that phosphorylation of FER at Thr696 is essential for suppression of lrx1 phenotypes by rol23. The LRX1-related function of PP2Cs appears clade H-specific. Collectively, our data suggest that LRX1 acts upstream of the RALF-FER signaling module and that PP2C12 inhibits FER via activation-loop dephosphorylation.

Bacteria often experience nutrient limitation. While the exponential and stationary growth phases have been characterized in the model bacterium Escherichia coli, little is known about what happens inside individual cells during the transition between these two phases. Through quantitative cell imaging, we found that the positions of nucleoids and cell division sites become increasingly asymmetric during the transition phase. These asymmetries were accompanied by an asymmetric reorganization of protein, ribosome, and RNA probes in the cytoplasm. Results from live-cell imaging experiments, complemented with genetic and ¹³C whole-cell nuclear magnetic resonance spectroscopy studies, show that preferential accumulation of the storage polymer glycogen at the old cell pole leads to the observed rearrangements and asymmetric divisions. Live-cell atomic force microscopy analysis, combined with in vitro biochemical experiments, suggests that these phenotypes are due to the propensity of glycogen to phase-separate into soft condensates in the crowded cytoplasm. Glycogen-associated differences in cell sizes between strains and future daughter cells suggest that glycogen phase-separation allows cells to store large glucose reserves that are not perceived by the cell as cytoplasmic space.

Delivery of antibacterial effector proteins into competitor cells using the Type VI secretion system (T6SS) is a widespread strategy for inter-bacterial competition. While many enzymatic T6SS effectors have been described, relatively few which form pores in target cell membranes have been reported. Here, we describe a widely-occurring family of T6SS-dependent pore-forming effectors, exemplified by Ssp4 of Serratia marcescens Db10. We show in vitro that Ssp4 forms regulated pores with high selectivity for cations, and use structural models and molecular dynamics simulations to predict how these pores conduct ions. Ssp4 has a broader phylogenetic distribution and is active against a wider range of bacterial species than Ssp6, the other pore-forming effector delivered by the same T6SS, with the two effectors displaying distinct ion selectivities and impacts on intoxicated cells. Finally, identification of Ssp4-resistant mutants revealed that a mucA mutant of Pseudomonas fluorescens is protected against T6SS attacks. We propose that deployment of two distinct T6SS-dependent pore-forming toxins is a common strategy to ensure effective de-energisation of closely- and distantly-related competitors.

Plasma C-reactive protein (CRP) is widely used as a biomarker for bacterial infections due to its massive induction during infections. However, the biological function of CRP remains largely undefined. Here we show that CRP enables liver resident macrophages (Kupffer cells) to capture and eliminate a wide range of invasive bacteria from the bloodstream of mice, and thereby provides rapid and sterilizing immunity. Mechanistically, CRP binds to at least 20 capsule types of Gram-positive and -negative pathogens, and shuffles the encapsulated bacteria to Kupffer cells embedded in the lining of the liver sinusoidal vasculatures by the complement-dependent and -independent pathways. The complement-dependent mode involves the activation of complement C3 at the bacterial surface, and the capture of the C3-opsonized bacteria by the CRIg and CR3 complement receptors on Kupffer cells. Cryo-electron microscopy analysis revealed a flexible structural framework for CRP's recognition of structurally diverse capsular polysaccharides. Because human CRP also possesses the broad capsule-binding activities, our findings provide a biological reason for the massive rise of plasma CRP during bacterial infections.

Cells monitor and dynamically regulate the lipid composition and biophysical properties of their plasma membrane (PM). The Target Of Rapamycin complex 2 (TORC2) is a protein kinase that acts as a central regulator of plasma membrane homeostasis, but the mechanisms by which it detects and reacts to membrane stresses are poorly understood. To address this knowledge gap, we characterized a family of amphiphilic molecules that physically perturb plasma membrane organization and in doing so inhibit TORC2 in yeast and mammalian cells. Using fluorescent reporters of various lipids in budding yeast, we show that exposure to these small molecules causes mobilization of PM ergosterol as well as inhibition of TORC2. TORC2 inhibition results in activation of the PM-ER sterol transporters Lam2 and Lam4 and the subsequent rapid removal of accessible ergosterol from the plasma membrane via PM-ER contact sites. This sequence of events, culminating in the reactivation of TORC2, is also observed with several other PM stresses, suggesting that TORC2 acts in a feedback loop to control active sterol levels at the plasma membrane to maintain its homeostasis.

Senescent cells drive age-related tissue dysfunction via the induction of a chronic senescence-associated secretory phenotype (SASP). The cyclin-dependent kinase inhibitors p21^Cip1 and p16^Ink4a have long served as markers of cellular senescence. However, their individual roles remain incompletely elucidated, particularly in vivo. Thus, we conducted a comprehensive examination of multiple single-cell RNA sequencing datasets spanning both murine and human tissues during aging. Our analysis revealed that p21^Cip1 and p16^Ink4a transcripts demonstrate significant heterogeneity across distinct cell types and tissues, frequently exhibiting a lack of co-expression. Moreover, we identified tissue-specific variations in SASP profiles linked to p21^Cip1 or p16^Ink4a expression. Using RNA velocity and pseudotime analyses, we discovered that p21+ and p16+ cells follow independent trajectory dynamics, with no evidence of direct transitions between these two states. Despite this heterogeneity, we identified a limited set of shared "core" SASP factors that may drive common senescence-related functions. Our study underscores the substantial diversity of cellular senescence and the SASP, emphasizing that these phenomena are inherently cell- and tissue-dependent.