Person:

Wu, Xu

Commonly used anesthetic isoflurane has been reported to promote Alzheimer’s disease (AD) neuropathogenesis by inducing caspase-3 activation. However, the up-stream mechanisms of isoflurane’s effects remain largely to be determined. Specifically, there is a lack of a good model/system to elucidate the underlying mechanism of the isoflurane-induced caspase-3 activation. We therefore set out to assess and compare the effects of isoflurane on caspase-3 activation in neural progenitor cells (NPCs) and in primary neurons from wild-type (WT) and AD transgenic (Tg) mice. The NPCs and neurons were obtained, cultured and then treated with either 2% isoflurane or under control condition for 6 h. The NPCs or neurons were harvested at the end of the treatment and were subjected to Western blot analysis. Here we showed for the first time that the isoflurane treatment induced caspase-3 activation in neurons, but not in NPCs, from either WT or AD Tg mice. Consistently, the isoflurane treatment increased cytosol levels of cytochrome c, a potential up-stream mechanism of isoflurane-induced caspase-3 activation in the mice neurons, but not NPCs. Finally, the isoflurane treatment induced a greater casapse-3 activation in the neurons, but not the NPCs, from AD Tg mice as compared to the WT mice. These data demonstrated that investigation and comparison of isoflurane’s effects between mice NPCs and neurons would serve as a model/system to determine the underlying mechanism by which isoflurane induces caspase-3 activation. These findings would promote more research to investigate the effects of anesthetics on AD neuropathogenesis and the underlying mechanisms.

Scribble (SCRIB) is a tumor suppressor protein, playing critical roles in establishing and maintaining epithelial cell polarity. Paradoxically, SCRIB is frequently amplified in human cancers, however, fails to localize properly to cell-cell junctions, suggesting that mislocalization of SCRIB contributes to tumorigenesis. Using chemical reporters, here we showed that SCRIB localization is regulated by S-palmitoylation at conserved cysteine residues. The palmitoylation-deficient mutants of SCRIB are mislocalized, leading to disruption of cell polarity and loss of their tumor suppressive activities to oncogenic YAP, MAPK and PI3K/Akt pathways. We further found that ZDHHC7 is the major palmitoyl acyltransferase regulating SCRIB. Knockout of ZDHHC7 led to SCRIB mislocalization and YAP activation, and disruption of SCRIB’s suppressive activities in HRasV12-induced cell invasion. In summary, we demonstrated that ZDHHC7-mediated SCRIB palmitoylation is critical for SCRIB membrane targeting, cell polarity, and tumor suppression, providing new mechanistic insights of how dynamic protein palmitoylation regulates cell polarity and tumorigenesis.

Epidermal growth factor receptor (EGFR) is an oncogenic receptor tyrosine kinase. Canonically, the tyrosine kinase activity of EGFR is regulated by its extracellular ligands. However, ligand-independent activation of EGFR exists in certain cancer cells, and the underlying mechanism remains to be defined. In this study, using PC3 and A549 cells as a model, we have found that, in the absence of extracellular ligands, a subpopulation of EGFR is constitutively active, which is needed for maintaining cell proliferation. Furthermore, we have found that fatty acid synthase (FASN)-dependent palmitoylation of EGFR is required for EGFR dimerization and kinase activation. Inhibition of FASN or palmitoyl acyltransferases reduced the activity and down-regulated the levels of EGFR, and sensitized cancer cells to EGFR tyrosine kinase inhibitors. It is concluded that EGFR can be activated intracellularly by FASN-dependent palmitoylation. This mechanism may serve as a new target for improving EGFR-based cancer therapy.

TEA domain (TEAD) transcription factors bind to the co-activator YAP/TAZ, and regulate the transcriptional output of Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches fatty acid (palmitate) to cysteine residues, and regulates protein trafficking, membrane localization and signaling activities. Using activity-based chemical probes, we discovered that human TEADs possess intrinsic palmitoylating enzyme-like activities, and undergo autopalmitoylation at evolutionarily conserved cysteine residues under physiological conditions. We determined the crystal structures of lipid-bound TEADs, and found that the lipid chain of palmitate inserts into a conserved deep hydrophobic pocket. Strikingly, palmitoylation is required for TEAD’s binding to YAP/TAZ, but dispensable for the binding to Vgll4 tumor suppressor. In addition, palmitoylation does not alter TEAD’s localization. Moreover, TEAD palmitoylation-deficient mutants impaired TAZ-mediated muscle differentiation in vitro, and Yorkie-mediated tissue overgrowth in Drosophila in vivo. Our study directly linked autopalmitoylation to the transcriptional regulation of Hippo pathway.

The melanocortin-1 receptor (MC1R), a G protein-coupled receptor, plays a crucial role in human and mouse pigmentation1–8. Activation of MC1R in melanocytes by α-melanocyte-stimulating hormone (α-MSH)9 stimulates cAMP signaling and melanin production and enhances DNA repair after UV irradiation (UVR)10–16. Individuals carrying MC1R variants, especially those associated with red hair color, fair skin and poor tanning ability (RHC-variants), are associated with higher risk of melanoma5,17,18,19,20. However, how MC1R activity might be modulated by UV irradiation, why redheads are more prone to developing melanoma, and whether the activity of RHC variants might be restored for therapeutic benefit remain unresolved questions. Here we demonstrate a potential MC1R-targeted intervention strategy to rescue loss-of-function MC1R in MC1R RHC-variants for therapeutic benefit based on activating MC1R protein palmitoylation. Specifically, MC1R palmitoylation, primarily mediated by the protein-acyl transferase (PAT) ZDHHC13, is essential for activating MC1R signaling that triggers increased pigmentation, UVB-induced G1-like cell cycle arrest and control of senescence and melanomagenesis in vitro and in vivo. Using C57BL/6J-MC1Re/eJ mice expressing MC1R RHC-variants we show that pharmacological activation of palmitoylation rescues the defects of MC1R RHC-variants and prevents melanomagenesis. The results highlight a central role for MC1R palmitoylation in pigmentation and protection against melanoma.

The transcriptional coactivator YAP orchestrates a pro-proliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse upon topical administration of drug. Chemical proteomics revealed the relevant target of PY-60 to be Annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase bound, non-phosphorylated, and transcriptionally active form of YAP. This work reveals ANXA2 to be a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale for promoting YAP-dependent regenerative repair in disease.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in regulating cell fate, inflammation and immunity. Cytokines and growth factors activate STAT3 through kinase-mediated tyrosine phosphorylation and dimerization. It remains unknown whether other factors could promote STAT3 activation through different mechanisms. Here we show that STAT3 is posttranslationally S-palmitoylated at the Src Homology 2 (SH2) domain, promoting its dimerization and transcriptional activation. Fatty acids could directly activate STAT3 by enhancing its palmitoylation, in synergy with cytokine stimulation. We further identified ZDHHC19 as a palmitoyl acyltransferase (PAT) regulating STAT3. Cytokine stimulation enhances STAT3 palmitoylation by promoting ZDHHC19–STAT3 association mediated by Grb2 SH3 domain. Silencing ZDHHC19 blocks STAT3 palmitoylation and dimerization, impairing cytokine and fatty acid-induced STAT3 activation. Importantly, ZDHHC19 is frequently amplified in multiple human cancers, including in 39% of lung squamous cell carcinomas (LSCCs). High ZDHHC19 levels correlate with high nuclear STAT3 in patient samples. In addition, ZDHHC19 knockout in LSCC cells significantly blocks STAT3 activity, and inhibits fatty acid-induced tumorsphere formation and high-fat diet (HFD)-induced tumorigenesis in vivo. Taken together, we reveal that fatty acid and ZDHHC19-mediated palmitoylation are additional signals regulating STAT3, linking deregulation of palmitoylation to inflammation and cancer.