Engineered droplet-forming peptide as photocontrollable phase modulator for fused in sarcoma protein.

The assembly and disassembly of biomolecular condensates are crucial for the subcellular compartmentalization of biomolecules in the control of cellular reactions. Recently, a correlation has been discovered between the phase transition of condensates and their maturation (aggregation) process in diseases. Therefore, modulating the phase of condensates to unravel the roles of condensation has become a matter of interest. Here, we create a peptide-based phase modulator, JSF1, which forms droplets in the dark and transforms into amyloid-like fibrils upon photoinitiation, as evidenced by their distinctive nanomechanical and dynamic properties. JSF1 is found to effectively enhance the condensation of purified fused in sarcoma (FUS) protein and, upon light exposure, induce its fibrilization. We also use JSF1 to modulate the biophysical states of FUS condensates in live cells and elucidate the relationship between FUS phase transition and FUS proteinopathy, thereby shedding light on the effect of protein phase transition on cellular function and malfunction.

A carnivorous mushroom paralyzes and kills nematodes via a volatile ketone.

The carnivorous mushroom Pleurotus ostreatus uses an unknown toxin to rapidly paralyze and kill nematode prey upon contact. We report that small lollipop-shaped structures (toxocysts) on fungal hyphae are nematicidal and that a volatile ketone, 3-octanone, is detected in these fragile toxocysts. Treatment of Caenorhabditis elegans with 3-octanone recapitulates the rapid paralysis, calcium influx, and neuronal cell death arising from fungal contact. Moreover, 3-octanone disrupts cell membrane integrity, resulting in extracellular calcium influx into cytosol and mitochondria, propagating cell death throughout the entire organism. Last, we demonstrate that structurally related compounds are also biotoxic to C. elegans, with the length of the ketone carbon chain being crucial. Our work reveals that the oyster mushroom has evolved a specialized structure containing a volatile ketone to disrupt the cell membrane integrity of its prey, leading to rapid cell and organismal death in nematodes.

A synthetic pregnenolone analog promotes microtubule dynamics and neural development.

BACKGROUND: Pregnenolone (P5) is a neurosteroid that promotes microtubule polymerization. It also reduces stress and negative symptoms of schizophrenia, promotes memory, as well as recovery from spinal cord injury. P5 is the first substance in the steroid-synthetic pathway; it can be further metabolized into other steroids. Therefore, it is difficult to differentiate the roles of P5 versus its metabolites in the brain. To alleviate this problem, we synthesized and screened a series of non-metabolizable P5 derivatives for their ability to polymerize microtubules similar to P5. RESULTS: We identified compound #43 (3-beta-pregnenolone acetate), which increased microtubule polymerization. We showed that compound #43 modified microtubule dynamics in live cells, increased neurite outgrowth and changed growth cone morphology in mouse cerebellar granule neuronal culture. Furthermore, compound #43 promoted the formation of stable microtubule tracks in zebrafish developing cerebellar axons. CONCLUSIONS: We have developed compound #43, a nonmetabolized P5 analog, that recapitulates P5 functions in vivo and can be a new therapeutic candidate for the treatment of neurodevelopmental diseases.

A chemical probe inhibitor targeting STAT1 restricts cancer stem cell traits and angiogenesis in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a worldwide cancer with rising annual incidence. New medications for patients with CRC are still needed. Recently, fluorescent chemical probes have been developed for cancer imaging and therapy. Signal transducer and activator of transcription 1 (STAT1) has complex functions in tumorigenesis and its role in CRC still needs further investigation. METHODS: RNA sequencing datasets in the NCBI GEO repository were analyzed to investigate the expression of STAT1 in patients with CRC. Xenograft mouse models, tail vein injection mouse models, and azoxymethane/dextran sodium sulfate (AOM/DSS) mouse models were generated to study the roles of STAT1 in CRC. A ligand-based high-throughput virtual screening approach combined with SWEETLEAD chemical database analysis was used to discover new STAT1 inhibitors. A newly designed and synthesized fluorescently labeled 4',5,7-trihydroxyisoflavone (THIF) probe (BODIPY-THIF) elucidated the mechanistic actions of STAT1 and THIF in vitro and in vivo. Colonosphere formation assay and chick chorioallantoic membrane assay were used to evaluate stemness and angiogenesis, respectively. RESULTS: Upregulation of STAT1 was observed in patients with CRC and in mouse models of AOM/DSS-induced CRC and metastatic CRC. Knockout of STAT1 in CRC cells reduced tumor growth in vivo. We then combined a high-throughput virtual screening approach and analysis of the SWEETLEAD chemical database and found that THIF, a flavonoid abundant in soybeans, was a novel STAT1 inhibitor. THIF inhibited STAT1 phosphorylation and might bind to the STAT1 SH2 domain, leading to blockade of STAT1-STAT1 dimerization. The results of in vitro and in vivo binding studies of THIF and STAT1 were validated. The pharmacological treatment with BODIPY-THIF or ablation of STAT1 via a CRISPR/Cas9-based strategy abolished stemness and angiogenesis in CRC. Oral administration of BODIPY-THIF attenuated colitis symptoms and tumor growth in the mouse model of AOM/DSS-induced CRC. CONCLUSIONS: This study demonstrates that STAT1 plays an oncogenic role in CRC. BODIPY-THIF is a new chemical probe inhibitor of STAT1 that reduces stemness and angiogenesis in CRC. BODIPY-THIF can be a potential tool for CRC therapy as well as cancer cell imaging.

Sensory cilia as the Achilles heel of nematodes when attacked by carnivorous mushrooms.

Fungal predatory behavior on nematodes has evolved independently in all major fungal lineages. The basidiomycete oyster mushroom Pleurotus ostreatus is a carnivorous fungus that preys on nematodes to supplement its nitrogen intake under nutrient-limiting conditions. Its hyphae can paralyze nematodes within a few minutes of contact, but the mechanism had remained unclear. We demonstrate that the predator-prey relationship is highly conserved between multiple Pleurotus species and a diversity of nematodes. To further investigate the cellular and molecular mechanisms underlying rapid nematode paralysis, we conducted genetic screens in Caenorhabditis elegans and isolated mutants that became resistant to P. ostreatus We found that paralysis-resistant mutants all harbored loss-of-function mutations in genes required for ciliogenesis, demonstrating that the fungus induced paralysis via the cilia of nematode sensory neurons. Furthermore, we observed that P. ostreatus caused excess calcium influx and hypercontraction of the head and pharyngeal muscle cells, ultimately resulting in rapid necrosis of the entire nervous system and muscle cells throughout the entire organism. This cilia-dependent predatory mechanism is evolutionarily conserved in Pristionchus pacificus, a nematode species estimated to have diverged from C. elegans 280 to 430 million y ago. Thus, P. ostreatus exploits a nematode-killing mechanism that is distinct from widely used anthelmintic drugs such as ivermectin, levamisole, and aldicarb, representing a potential route for targeting parasitic nematodes in plants, animals, and humans.