A phosphate-sensing organelle regulates phosphate and tissue homeostasis.

Inorganic phosphate (Pi) is one of the essential molecules for life. However, little is known about intracellular Pi metabolism and signalling in animal tissues1. Following the observation that chronic Pi starvation causes hyperproliferation in the digestive epithelium of Drosophila melanogaster, we determined that Pi starvation triggers the downregulation of the Pi transporter PXo. In line with Pi starvation, PXo deficiency caused midgut hyperproliferation. Interestingly, immunostaining and ultrastructural analyses showed that PXo specifically marks non-canonical multilamellar organelles (PXo bodies). Further, by Pi imaging with a Förster resonance energy transfer (FRET)-based Pi sensor2, we found that PXo restricts cytosolic Pi levels. PXo bodies require PXo for biogenesis and undergo degradation following Pi starvation. Proteomic and lipidomic characterization of PXo bodies unveiled their distinct feature as an intracellular Pi reserve. Therefore, Pi starvation triggers PXo downregulation and PXo body degradation as a compensatory mechanism to increase cytosolic Pi. Finally, we identified connector of kinase to AP-1 (Cka), a component of the STRIPAK complex and JNK signalling3, as the mediator of PXo knockdown- or Pi starvation-induced hyperproliferation. Altogether, our study uncovers PXo bodies as a critical regulator of cytosolic Pi levels and identifies a Pi-dependent PXo-Cka-JNK signalling cascade controlling tissue homeostasis.

mTORC1-chaperonin CCT signaling regulates m6A RNA methylation to suppress autophagy.

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. Here, we find that the m6A methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in Drosophila and human cells. Furthermore, we show that the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing m6A levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, our study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with m6A RNA methylation and demonstrates their roles in suppressing autophagy.

Tunable Strategy for the Asymmetric Synthesis of Sulfoglycolipids from Mycobacterium tuberculosis To Elucidate the Structure and Immunomodulatory Property Relationships.

We developed a versatile asymmetric strategy to synthesize different classes of sulfoglycolipids (SGLs) from Mycobacterium tuberculosis. The strategy features the use of asymmetrically protected trehaloses, which were acquired from the glycosylation of TMS α-glucosyl acceptors with benzylidene-protected thioglucosyl donors. The positions of the protecting groups at the donors and acceptors can be fine-tuned to obtain different protecting-group patterns, which is crucial for regioselective acylation and sulfation. In addition, a chemoenzymatic strategy was established to prepare the polymethylated fatty acid building blocks. The strategy employs inexpensive lipase as a desymmetrization agent in the preparation of the starting substrate and readily available chiral oxazolidinone as a chirality-controlling agent in the construction of the polymethylated fatty acids. A subsequent investigation on the immunomodulatory properties of each class of SGLs showed how the structures of SGLs impact the host innate immunity response.

Pregnenolone activates CLIP-170 to promote microtubule growth and cell migration.

Pregnenolone (P5) is a neurosteroid that improves memory and neurological recovery. It is also required for zebrafish embryonic development. However, its mode of action is unclear. Here we show that P5 promotes cell migration and microtubule polymerization by binding a microtubule plus end-tracking protein, cytoplasmic linker protein 1 (CLIP-170). We captured CLIP-170 from zebrafish embryonic extract using a P5 photoaffinity probe conjugated to diaminobenzophenone. P5 interacted with CLIP-170 at its coiled-coil domain and changed it into an extended conformation. This increased CLIP-170 interaction with microtubules, dynactin subunit p150(Glued) and LIS1; it also promoted CLIP-170-dependent microtubule polymerization. CLIP-170 was essential for P5 to promote microtubule abundance and zebrafish epiboly cell migration during embryogenesis, and overexpression of the P5-binding region of CLIP-170 delayed this migration. P5 also sustained migration directionality of cultured mammalian cells. Our results show that P5 activates CLIP-170 to promote microtubule polymerization and cell migration.

Zebrafish Establish Female Germ Cell Identity by Advancing Cell Proliferation and Meiosis.

Zebrafish is a popular research model; but its mechanism of sex determination is unclear and the sex of juvenile fish cannot be distinguished. To obtain fish with defined sex, we crossed domesticated zebrafish with the Nadia strain that has a female-dominant W segment. These fish were placed on a ziwi:GFP background to facilitate sorting of fluorescent germ cells for transcriptomic analysis. We analyzed the transcriptomes of germ cells at 10-14 days postfertilization (dpf), when sex dimorphic changes started to appear. Gene ontology showed that genes upregulated in the 10-dpf presumptive females are involved in cell cycles. This correlates with our detection of increased germ cell numbers and proliferation. We also detected upregulation of meiotic genes in the presumptive females at 14 dpf. Disruption of a meiotic gene, sycp3, resulted in sex reversal to infertile males. The germ cells of sycp3 mutants could not reach diplotene and underwent apoptosis. Preventing apoptosis by disrupting tp53 restored female characteristics in sycp3 mutants, demonstrating that adequate germ cells are required for female development. Thus, our transcriptome and gene mutation demonstrate that initial germ cell proliferation followed by meiosis is the hallmark of female differentiation in zebrafish.

Immunomodulatory drug discovery from herbal medicines: Insights from organ-specific activity and xenobiotic defenses.

Traditional herbal medicines, which emphasize a holistic, patient-centric view of disease treatment, provide an exciting starting point for discovery of new immunomodulatory drugs. Progress on identification of herbal molecules with proven single agent activity has been slow, in part because of insufficient consideration of pharmacology fundamentals. Many molecules derived from medicinal plants exhibit low oral bioavailability and rapid clearance, leading to low systemic exposure. Recent research suggests that such molecules can act locally in the gut or liver to activate xenobiotic defense pathways that trigger beneficial systemic effects on the immune system. We discuss this hypothesis in the context of four plant-derived molecules with immunomodulatory activity: indigo, polysaccharides, colchicine, and ginsenosides. We end by proposing research strategies for identification of novel immunomodulatory drugs from herbal medicine sources that are informed by the possibility of local action in the gut or liver, leading to generation of systemic immune mediators.

Colchicine acts selectively in the liver to induce hepatokines that inhibit myeloid cell activation.

Colchicine has served as a traditional medicine for millennia and remains widely used to treat inflammatory and other disorders. Colchicine binds tubulin and depolymerizes microtubules, but it remains unclear how this mechanism blocks myeloid cell recruitment to inflamed tissues. Here we show that colchicine inhibits myeloid cell activation via an indirect mechanism involving the release of hepatokines. We find that a safe dose of colchicine depolymerizes microtubules selectively in hepatocytes but not in circulating myeloid cells. Mechanistically, colchicine triggers Nrf2 activation in hepatocytes, leading to secretion of anti-inflammatory hepatokines, including growth differentiation factor 15 (GDF15). Nrf2 and GDF15 are required for the anti-inflammatory action of colchicine in vivo. Plasma from colchicine-treated mice inhibits inflammatory signalling in myeloid cells in a GDF15-dependent manner, by positive regulation of SHP-1 (PTPN6) phosphatase, although the precise molecular identities of colchicine-induced GDF15 and its receptor require further characterization. Our work shows that the efficacy and safety of colchicine depend on its selective action on hepatocytes, and reveals a new axis of liver-myeloid cell communication. Plasma GDF15 levels and myeloid cell SHP-1 activity may be useful pharmacodynamic biomarkers of colchicine action.

Publisher Correction: Colchicine acts selectively in the liver to induce hepatokines that inhibit myeloid cell activation.

A Correction to this paper has been published: https://doi.org/10.1038/s42255-021-00397-5.

Histone deacetylase inhibitors reduce steroidogenesis through SCF-mediated ubiquitination and degradation of steroidogenic factor 1 (NR5A1).

Histone deacetylase (HDAC) inhibitors such as trichostatin A and valproic acid modulate transcription of many genes by inhibiting the activities of HDACs, resulting in the remodeling of chromatin. Yet this effect is not universal for all genes. Here we show that HDAC inhibitors suppressed the expression of steroidogenic gene CYP11A1 and decreased steroid secretion by increasing the ubiquitination and degradation of SF-1, a factor important for the transcription of all steroidogenic genes. This was accompanied by increased expression of Ube2D1 and SKP1A, an E2 ubiquitin conjugase and a subunit of the E3 ubiquitin ligase in the Skp1/Cul1/F-box protein (SCF) family, respectively. Reducing SKP1A expression with small interfering RNA resulted in recovery of SF-1 levels, demonstrating that the activity of SCF E3 ubiquitin ligase is required for the SF-1 degradation induced by HDAC inhibitors. Overexpression of exogenous SF-1 restored steroidogenic activities even in the presence of HDAC inhibitors. Thus, increased SF-1 degradation is the cause of the reduction in steroidogenesis caused by HDAC inhibitors. The increased SKP1A expression and SCF-mediated protein degradation could be the mechanism underlying the mode of action of HDAC inhibitors.

Nongenomic actions of neurosteroid pregnenolone and its metabolites.

Steroids have been widely used in the clinical setting. They bind and activate nuclear receptors to regulate gene expression. In addition to activating genomic transcription, steroids also exert nongenomic actions. The current article focuses on the nongenomic actions of neurosteroids, including pregnenolone (P5), 7α-hydroxypregnenolone, pregnenolone sulfate and allopregnanolone. Pregnenolone and its derivatives promote neuronal activity by enhancing learning and memory, relieving depression, enhancing locomotor activity, and promoting neuronal cell survival. They exert these effects by activating various target proteins located in the cytoplasm or cell membrane. Pregnenolone and its metabolites bind to receptors such as microtubule-associated proteins and neurotransmitter receptors to elicit a series of reactions including stabilization of microtubules, increase of ion flux into cells, and dopamine release. The wide actions of neurosteroids indicate that pregnenolone derivatives have great potential in future treatment of neurological diseases.

Chemical Inhibition of Human Thymidylate Kinase and Structural Insights into the Phosphate Binding Loop and Ligand-Induced Degradation.

Targeting thymidylate kinase (TMPK) that catalyzes the phosphotransfer reaction for formation of dTDP from dTMP is a new strategy for anticancer treatment. This study is to understand the inhibitory mechanism of a previously identified human TMPK (hTMPK) inhibitor YMU1 (1a) by molecular docking, isothermal titration calorimetry, and photoaffinity labeling. The molecular dynamics simulation suggests that 1a prefers binding at the catalytic site of hTMPK, whereas the hTMPK inhibitors that bear pyridino[d]isothiazolone or benzo[d]isothiazolone core structure in lieu of the dimethylpyridine-fused isothiazolone moiety in 1a can have access to both the ATP-binding and catalytic sites. The binding sites of hTMPK inhibitors were validated by photoaffinity labeling and mass spectrometric studies. Taking together, 1a and its analogues stabilize the conformation of ligand-induced degradation (LID) region of hTMPK and block the catalytic site or ATP-binding site, thus attenuating the ATP binding-induced closed conformation that is required for phosphorylation of dTMP.