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Cells recognize osmotic stress through liquid-liquid phase separation lubricated with poly(ADP-ribose).

ABSTRACT: Cells are under threat of osmotic perturbation; cell volume maintenance is critical in cerebral edema, inflammation and aging, in which prominent changes in intracellular or extracellular osmolality emerge. After osmotic stress-enforced cell swelling or shrinkage, the cells regulate intracellular osmolality to recover their volume. However, the mechanisms recognizing osmotic stress remain obscured. We previously clarified that apoptosis signal-regulating kinase 3 (ASK3) bidirectionally responds to osmotic stress and regulates cell volume recovery. Here, we show that macromolecular crowding induces liquid-demixing condensates of ASK3 under hyperosmotic stress, which transduce osmosensing signal into ASK3 inactivation. A genome-wide small interfering RNA (siRNA) screen identifies an ASK3 inactivation regulator, nicotinamide phosphoribosyltransferase (NAMPT), related to poly(ADP-ribose) signaling. Furthermore, we clarify that poly(ADP-ribose) keeps ASK3 condensates in the liquid phase and enables ASK3 to become inactivated under hyperosmotic stress. Our findings demonstrate that cells rationally incorporate physicochemical phase separation into their osmosensing systems.

SUBMITTER: Watanabe K

PROVIDER: S-EPMC7921423 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Poly(ADP-ribosyl)ation is a common post-translational modification that mediates a wide variety of cellular processes including DNA damage repair, chromatin regulation, transcription, and apoptosis. The difficulty associated with accessing poly(ADP-ribose) (PAR) in a homogeneous form has been an impediment to understanding the interactions of PAR with poly(ADP-ribose) glycohydrolase (PARG) and other binding proteins. Here we describe the chemical synthesis of the ADP-ribose dimer, and we use this compound to obtain the first human PARG substrate-enzyme cocrystal structure. Chemical synthesis of PAR is an attractive alternative to traditional enzymatic synthesis and fractionation, allowing access to products such as dimeric ADP-ribose, which has been detected but never isolated from natural sources. Additionally, we describe the synthesis of an alkynylated dimer and demonstrate that this compound can be used to synthesize PAR probes including biotin and fluorophore-labeled compounds. The fluorescently labeled ADP-ribose dimer was then utilized in a general fluorescence polarization-based PAR-protein binding assay. Finally, we use intermediates of our synthesis to access various PAR fragments, and evaluation of these compounds as substrates for PARG reveals the minimal features for substrate recognition and enzymatic cleavage. Homogeneous PAR oligomers and unnatural variants produced from chemical synthesis will allow for further detailed structural and biochemical studies on the interaction of PAR with its many protein binding partners.

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Project description:Important cellular processes are regulated by poly(ADP-ribosyl)ation. This protein modification is catalyzed mainly by nuclear poly(ADP-ribose) polymerase (PARP) 1 in response to DNA damage. Cytosolic PARP isoforms have been described, whereas the presence of poly(ADP-ribose) (PAR) metabolism in mitochondria is controversial. PAR is degraded by poly(ADP-ribose) glycohydrolase (PARG). Recently, ADP-ribosylhydrolase 3 (ARH3) was also shown to catalyze PAR-degradation in vitro. PARG is encoded by a single, essential gene. One nuclear and three cytosolic isoforms result from alternative splicing. The presence and origin of a mitochondrial PARG is still unresolved. We establish here the genetic background of a human mitochondrial PARG isoform and investigate the molecular basis for mitochondrial poly(ADP-ribose) degradation. In common with a cytosolic 60-kDa human PARG isoform, the mitochondrial protein did not catalyze PAR degradation because of the absence of exon 5-encoded residues. In mice, we identified a transcript encoding an inactive cytosolic 52-kDa PARG lacking the mitochondrial targeting sequence and a substantial portion of exon 5. Thus, mammalian PARG genes encode isoforms that do not catalyze PAR degradation. On the other hand, embryonic fibroblasts from ARH3(-/-) mice lack most of the mitochondrial PAR degrading activity detected in wild-type cells, demonstrating a potential involvement of ARH3 in PAR metabolism.

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