NU6300 covalently reacts with cysteine-191 of gasdermin D to block its cleavage and palmitoylation.

Gasdermin D (GSDMD) serves as a vital mediator of inflammasome-driven pyroptosis. In our study, we have identified NU6300 as a specific GSDMD inhibitor that covalently interacts with cysteine-191 of GSDMD, effectively blocking its cleavage while not affecting earlier steps such as ASC oligomerization and caspase-1 processing in AIM2- and NLRC4-mediated inflammation. On the contrary, NU6300 robustly inhibits these earlier steps in NLRP3 inflammasome, confirming a unique feedback inhibition effect in the NLRP3-GSDMD pathway upon GSDMD targeting. Our study reveals a previously undefined mechanism of GSDMD inhibitors: NU6300 impairs the palmitoylation of both full-length and N-terminal GSDMD, impeding the membrane localization and oligomerization of N-terminal GSDMD. In vivo studies further demonstrate the efficacy of NU6300 in ameliorating dextran sodium sulfate-induced colitis and improving survival in lipopolysaccharide-induced sepsis. Overall, these findings highlight the potential of NU6300 as a promising lead compound for the treatment of inflammatory diseases.

Palmitoylation is required for Sept8-204 and Sept5 to form vesicle-like structure and colocalize with synaptophysin.

Sept8 is a vesicle associated protein and there are two typical transcriptional variants (Sept8-204 and Sept8-201) expressed in mice brain. Interestingly, the coexpression of Sept8-204/Sept5 induces the formation of small sized vesicle-like structure, while that of the Sept8-201/Sept5 produces large puncta. Sept8 is previously shown to be palmitoylated. Here it was further revealed that protein palmitoylation is required for Sept8-204/Sept5 to maintain small sized vesicle-like structure and colocalize with synaptophysin, since either the expression of nonpalmitoylated Sept8-204 mutant (Sept8-204-3CA) or inhibiting Sept8-204 palmitoylation by 2-BP with Sept5 produces large puncta, which barely colocalizes with synaptophysin (SYP). Moreover, it was shown that the dynamic palmitoylation of Sept8-204 is controlled by ZDHHC17 and PPT1, loss of ZDHHC17 decreases Sept8-204 palmitoylation and induces large puncta, while loss of PPT1 increases Sept8-204 palmitoylation and induces small sized vesicle-like structure. Together, these findings suggest that palmitoylation is essential for the maintenance of the small sized vesicle-like structure for Sept8-204/Sept5, and may hint their important roles in synaptic functions.

A nanoporous organic polymer using 1, 3-dibromoadamantane as a crosslinker for adsorption/separation of benzene and cyclohexane.

The development of nanoporous organic polymers with cycloaliphatic components for effective benzene (Bz) and cyclohexane (Cy) adsorption/separation poses a significant challenge. This work focuses on synthesizing NOP-Ad-1, a nanoporous organic polymer derived from a Friedel-Crafts reaction between cycloaliphatic 1,3-dibromadantane and aromatic hexaphenylbenzene. At 298 K and P/P0 = 0.95, NOP-Ad-1 can uptake 989 mg g-1 benzene and 441 mg g-1 cyclohexane. Moreover, as the benzene vapor ratio increased from 20% to 80%, the Bz/Cy selectivity of NOP-Ad-1 gradually decreased from 1.75 to 1.24. These findings highlight the potential application of NOP-Ad-1 in the adsorption/separation of Bz/Cy mixtures.

Palmitoylated Sept8-204 modulates learning and anxiety by regulating filopodia arborization and actin dynamics.

Septin proteins are involved in diverse physiological functions, including the formation of specialized cytoskeletal structures. Septin 8 (Sept8) is implicated in spine morphogenesis and dendritic branching through palmitoylation. We explored the role and regulation of a Sept8 variant in human neural-like cells and in the mouse brain. We identified Sept8-204 as a brain-specific variant of Sept8 that was abundant in neurons and modified by palmitoylation, specifically at Cys469, Cys470, and Cys472. Sept8-204 palmitoylation was mediated by the palmitoyltransferase ZDHHC7 and was removed by the depalmitoylase PPT1. Palmitoylation of Sept8-204 bound to F-actin and induced cytoskeletal dynamics to promote the outgrowth of filopodia in N2a cells and the arborization of neurites in hippocampal neurons. In contrast, a Sept8-204 variant that could not be palmitoylated because of mutation of all three Cys residues (Sept8-204-3CA) lost its ability to bind F-actin, and expression of this mutant did not promote morphological changes. Genetic deletion of Sept8, Sept8-204, or Zdhhc7 caused deficits in learning and memory and promoted anxiety-like behaviors in mice. Our findings provide greater insight into the regulation of Sept8-204 by palmitoylation and its role in neuronal morphology and function in relation to cognition.

ALS-linked C9orf72-SMCR8 complex is a negative regulator of primary ciliogenesis.

Massive GGGGCC (G4C2) repeat expansion in C9orf72 and the resulting loss of C9orf72 function are the key features of ~50% of inherited amyotrophic lateral sclerosis and frontotemporal dementia cases. However, the biological function of C9orf72 remains unclear. We previously found that C9orf72 can form a stable GTPase activating protein (GAP) complex with SMCR8 (Smith-Magenis chromosome region 8). Herein, we report that the C9orf72-SMCR8 complex is a major negative regulator of primary ciliogenesis, abnormalities in which lead to ciliopathies. Mechanistically, the C9orf72-SMCR8 complex suppresses the primary cilium as a RAB8A GAP. Moreover, based on biochemical analysis, we found that C9orf72 is the RAB8A binding subunit and that SMCR8 is the GAP subunit in the complex. We further found that the C9orf72-SMCR8 complex suppressed the primary cilium in multiple tissues from mice, including but not limited to the brain, kidney, and spleen. Importantly, cells with C9orf72 or SMCR8 knocked out were more sensitive to hedgehog signaling. These results reveal the unexpected impact of C9orf72 on primary ciliogenesis and elucidate the pathogenesis of diseases caused by the loss of C9orf72 function.

Crosstalk of Synapsin1 palmitoylation and phosphorylation controls the dynamicity of synaptic vesicles in neurons.

The dynamics of synaptic vesicles (SVs) within presynaptic domains are tightly controlled by synapsin1 phosphorylation; however, the mechanism underlying the anchoring of synapsin1 with F-actin or SVs is not yet fully understood. Here, we found that Syn1 is modified with protein palmitoylation, and examining the roles of Syn1 palmitoylation in neurons led us to uncover that Syn1 palmitoylation is negatively regulated by its phosphorylation; together, they manipulate the clustering and redistribution of SVs. Using the combined approaches of electron microscopy and genetics, we revealed that Syn1 palmitoylation is vital for its binding with F-actin but not SVs. Inhibition of Syn1 palmitoylation causes defects in SVs clustering and a reduced number of total SVs in vivo. We propose a model in which SVs redistribution is triggered by upregulated Syn1 phosphorylation and downregulated Syn1 palmitoylation, and they reversibly promote SVs clustering. The crosstalk of Syn1 palmitoylation and phosphorylation thereby bidirectionally manipulates SVs dynamics in neurons.

Protocol to quantify palmitoylation of cysteines in budding yeast.

Palmitoylation is a special kind of lipid modification that targets proteins to membranes. This protocol introduces the acyl-biotin exchange (ABE) assay to determine the palmitoylation of protein cysteines in yeast. Palmitoylation is exchanged by biotinylated compounds so that the palmitoyl proteins can be affinity-purified for downstream assay by western blot. This protocol is easy to perform and can be applied to other biological sources with slight modifications. This protocol is limited to the detection of cysteine-based palmitoylation. For complete details on the use and execution of this profile, please refer to Lei et al. (2021).

GFAP hyperpalmitoylation exacerbates astrogliosis and neurodegenerative pathology in PPT1-deficient mice.

The homeostasis of protein palmitoylation and depalmitoylation is essential for proper physiological functions in various tissues, in particular the central nervous system (CNS). The dysfunction of PPT1 (PPT1-KI, infantile neuronal ceroid lipofuscinosis [INCL] mouse model), which catalyze the depalmitoylation process, results in serious neurodegeneration accompanied by severe astrogliosis in the brain. Endeavoring to determine critical factors that might account for the pathogenesis in CNS by palm-proteomics, glial fibrillary acidic protein (GFAP) was spotted, indicating that GFAP is probably palmitoylated. Questions concerning if GFAP is indeed palmitoylated in vivo and how palmitoylation of GFAP might participate in neural pathology remain unexplored and are waiting to be investigated. Here we show that GFAP is readily palmitoylated in vitro and in vivo; specifically, cysteine-291 is the unique palmitoylated residue in GFAP. Interestingly, it was found that palmitoylated GFAP promotes astrocyte proliferation in vitro. Furthermore, we showed that PPT1 depalmitoylates GFAP, and the level of palmitoylated GFAP is overwhelmingly up-regulated in PPT1-knockin mice, which lead us to speculate that the elevated level of palmitoylated GFAP might accelerate astrocyte proliferation in vivo and ultimately led to astrogliosis in INCL. Indeed, blocking palmitoylation by mutating cysteine-291 into alanine in GFAP attenuate astrogliosis, and remarkably, the concurrent neurodegenerative pathology in PPT1-knockin mice. Together, these findings demonstrate that hyperpalmitoylated GFAP plays critical roles in regulating the pathogenesis of astrogliosis and neurodegeneration in the CNS, and most importantly, pinpointing that cysteine-291 in GFAP might be a valuable pharmaceutical target for treating INCL and other potential neurodegenerative diseases.

Nifurtimox Hampered the Progression of Astroglioma In vivo Via Manipulating the AKT-GSK3ß axis.

BACKGROUND: Astroglioma, one major form of brain tumors, has remained principally tough to handle for decades, due to the complexity of tumor pathology and the poor response to chemo- and radio-therapies. METHODS: Our previous study demonstrated that nifurtimox could regulate the signaling axis of AKT-GSK3ß in various tumor types including the astroglioma U251 cells. Intriguingly, earlier case studies suggested that nifurtimox could possibly permeate the blood brain barrier and arrest neuroblastoma in the brain. These observations jointly encouraged us to explore whether nifurtimox would hinder the growth of astroglioma in vivo. RESULTS: Our results exhibited that nifurtimox could competently hinder the development of astroglioma in the mouse brain as compared to temozolomide, the first line of drug for brain tumors. Meanwhile the surviving rate, as well as the body-weight was dramatically upregulated upon nifurtimox treatment, as compared to that of temozolomide. These findings offered nifurtimox as a better alternative drug in treating astroglioma in vivo. CONCLUSION: Persistently, the manipulation of the signaling axis of AKT-GSK3ß in astroglioma was found in line with earlier findings in neuroblastoma when treated with nifurtimox.

Nifurtimox Inhibits the Progression of Neuroblastoma in vivo.

Neuroblastoma was one of the most life-threatening cancer developed in children, yet the conventional therapies currently used leave an unmet gap for clinical requirements. Temozolomide is the first line of drug in the treatment of neuroblastorma nowadays. Giving the fact that temozolomide treatment offered limited healing effect and patients responded divergently, an alternative beneficial path is urgently requested. Nifurtimox, a drug against Trypanosoma cruzi, was happened to find competent in treating a patient who carried aggressive neuroblastoma. Although in vitro studies demonstrated that nifurtimox has cytotoxic features against tumor cells, a systematic investigation in vivo is generally inadequate. Here we exhibited that nifurtimox could suppress the progression of neuroblastoma in vivo, while maintain the health condition to a great extent. Importantly, as comparing to temozolomide, nifurtimox presented a stronger effect on inhibiting tumor development, strongly suggesting that nifurtimox is a preferential alternative drug in treating neuroblastoma. Additionally, it was shown that Akt-GSK3ß signaling cascade was involved in tumor arrest induced by nifurtimox.

The Effect of Interleukin 38 on Inflammation-induced Corneal Neovascularization.

BACKGROUND: Angiogenesis is tightly linked to inflammation. Cytokines of interleukin 1 (IL-1) family are key mediators in modulating inflammatory responses. METHODS: In this study, we examined the role of IL-38, a member of the IL-1 family, in mediating inflammation-induced angiogenesis. RESULTS: The results showed that the angiogenesis was attenuated by topical administration of IL-38 to the injured corneas in a mouse model of alkali-induced corneal neovascularization (CNV). Further study showed that the expression of inflammatory cytokines TNF-α, IL-6, IL-8 and IL-1ß was decreased in the IL-38-treated corneas. Moreover, the angiogenic activities including the proliferation, migration and tube formation of human retinal endothelial cells were reduced by IL-38 treatment in vitro. CONCLUSION: The data indicate that IL-38 modulates inflammation-induced angiogenesis.

Experimental model of Fusarium solani keratitis in rats.

AIM: To establish a repeatable rat model of Fusarium solani keratitis (F. solani keratitis) that mimicked fungal keratitis in humans. METHODS: Wistar rats' corneas were scratched on the superficial stroma after scraping the unilateral corneal epithelia. Then, the corneal surface was inoculated with different inoculum dose of F. solani spore suspension. Doses ranged from 10(6) to 10(9) colony-forming unit per milliliter (CFU/mL). The treated corneas were covered by contact lenses that were made of Parafilm M membrane. Negative controls were inoculated with sterile phosphate-buffered saline (PBS). For statistical analysis, corneas were evaluated daily on a 12-point scale to check the state of corneal inflammation. Furthermore, the pathological characteristics of this model were investigated. RESULTS: The rat model of F. solani keratitis was established by the combination methods of corneal trauma and parafilm-made contact lens and inoculation of fungus spore suspension. 10(6) and 10(7)CFU/mL of F. solani induced mild corneal infection, while 10(8)CFU/mL of F. solani was sufficient to induce moderate infection that was consistent with human keratomycosis. Dose of 10(9)CFU/mL of F. solani was excessive and led to perforated corneas. CONCLUSION: The rat model of F. solani keratitis, established by the combinational methods of corneal trauma, parafilm-made contact lens and the appropriate dose of inoculum, that imitates the developing processes of F. solani keratitis in human beings and provides a repeatable method of creating a rat model.