Baohui Zheng

Kinetic Resolution of Indolines by Pd-Catalyzed Asymmetric Allylic Amination

The kinetic resolution of indolines was realized via a Pd-catalyzed allylic substitution reaction by using Trosts chiral ligand L10, affording optically active indolines and N-allylated indolines in high yields and high enantioselectivities with an S factor up to 59, which provided the first example for the kinetic resolution of nucleophiles via a transition-metal-catalyzed allylic substitution reaction.

Ag-Catalyzed diastereo- and enantioselective synthesis of beta-substituted tryptophans from sulfonylindoles.

The asymmetric catalytic synthesis of beta-substituted tryptophan derivatives was realized in high diastereo- and enantioselectivity by the reaction of glycine derivatives with sulfonylindoles in the presence of catalyst derived from AgCl and a commercially available chiral monodentate phosphoramidite ligand. The resulting adduct was readily converted to beta-substituted tryptophan in 95% overall yield for two steps, which presented a highly efficient route to chiral beta-substituted tryptophan.

2‑Bromopalmitate Analogues as Activity-Based Probes To Explore Palmitoyl Acyltransferases

Reversible S-palmitoylation is an important post-translational modification that regulates the trafficking, localization, and activity of proteins. Cysteine-rich Asp-His-His-Cys (DHHC) domain-containing enzymes are evolutionarily conserved protein palmitoyl acyltransferases (PATs). The human genome encodes 23 DHHC-PATs that regulate diverse cellular functions. Although chemical probes and proteomic methods to detect palmitoylated protein substrates have been reported, no probes for direct detection of the activity of PATs are available. Here we report the synthesis and characterization of 2-bromohexadec-15-ynoic acid and 2-bromooctadec-17-ynoic acid, which are analogues of 2-bromopalmitate (2-BP), as activity-based probes for PATs as well as other palmitoylating and 2-BP-binding enzymes. These probes will serve as new chemical tools for activity-based protein profiling to explore PATs, to dissect the functions of PATs in cell signaling and diseases, and to facilitate the identification of their inhibitors.

Autopalmitoylation of TEAD proteins regulates transcriptional output of the Hippo pathway

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.

ZDHHC7-mediated S-palmitoylation of Scribble regulates cell polarity

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.

Intracellular activation of EGFR by fatty acid synthase dependent palmitoylation

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.

Dimethylguanidino valeric acid is a marker of liver fat and predicts diabetes

Unbiased, “nontargeted” metabolite profiling techniques hold considerable promise for biomarker and pathway discovery, in spite of the lack of successful applications to human disease. By integrating nontargeted metabolomics, genetics, and detailed human phenotyping, we identified dimethylguanidino valeric acid (DMGV) as an independent biomarker of CT-defined nonalcoholic fatty liver disease (NAFLD) in the offspring cohort of the Framingham Heart Study (FHS) participants. We verified the relationship between DMGV and early hepatic pathology. Specifically, plasma DMGV levels were correlated with biopsy-proven nonalcoholic steatohepatitis (NASH) in a hospital cohort of individuals undergoing gastric bypass surgery, and DMGV levels fell in parallel with improvements in post-procedure cardiometabolic parameters. Further, baseline DMGV levels independently predicted future diabetes up to 12 years before disease onset in 3 distinct human cohorts. Finally, we provide all metabolite peak data consisting of known and unidentified peaks, genetics, and key metabolic parameters as a publicly available resource for investigations in cardiometabolic diseases.

Chemical Probes to Directly Profile Palmitoleoylation of Proteins

Palmitoleoylation is a unique fatty acylation of proteins in which a monounsaturated fatty acid, palmitoleic acid (C16:1), is covalently attached to a protein. Wnt proteins are known to be palmitoleoylated by cis‐Δ9 palmitoleate at conserved serine residues. O‐palmitoleoylation plays a critical role in regulating Wnt secretion, binding to the receptors, and in the dynamics of Wnt signaling. Therefore, protein palmitoleoylation is important in tissue homeostasis and tumorigenesis. Chemical probes based on saturated fatty acids, such as ω‐alkynyl palmitic acid (Alk‐14 or Alk‐C16), have been used to study Wnt palmitoleoylation. However, such probes require prior conversion to the unsaturated fatty acid by stearoyl‐CoA desaturase (SCD) in cells, significantly decreasing their selectivity and efficiency for studying protein palmitoleoylation. We synthesized and characterized ω‐alkynyl cis‐ and trans‐palmitoleic acids (cis‐ and trans‐Alk‐14:1) as chemical probes to directly study protein palmitoleoylation. We found that cis‐Alk‐14:1 could more efficiently label Wnt proteins in cells. Interestingly, the DHHC family of palmitoyl acyltransferases can charge both saturated and unsaturated fatty acids, potentially using both as acyl donors in protein palmitoylation and palmitoleoylation. Furthermore, proteomic analysis of targets labeled by these probes revealed new cis‐ and trans‐palmitoleoylated proteins. Our studies provided new chemical tools and revealed new insights into palmitoleoylation in cell signaling.

Highly potent visnagin derivatives inhibit Cyp1 and prevent doxorubicin cardiotoxicity

Anthracyclines such as doxorubicin are highly effective chemotherapy agents used to treat many common malignancies. However, their use is limited by cardiotoxicity. We previously identified visnagin as protecting against doxorubicin toxicity in cardiac but not tumor cells. In this study, we sought to develop more potent visnagin analogs in order to use these analogs as tools to clarify the mechanisms of visnagin-mediated cardioprotection. Structure-activity relationship studies were performed in a zebrafish model of doxorubicin cardiomyopathy. Movement of the 5-carbonyl to the 7 position and addition of short ester side chains led to development of visnagin analogs with 1,000-fold increased potency in zebrafish and 250-fold increased potency in mice. Using proteomics, we discovered that doxorubicin caused robust induction of Cytochrome P450 family 1 (CYP1) that was mitigated by visnagin and its potent analog 23. Treatment with structurally divergent CYP1 inhibitors, as well as knockdown of CYP1A, prevented doxorubicin cardiomyopathy in zebrafish. The identification of potent cardioprotective agents may facilitate the development of new therapeutic strategies for patients receiving cardiotoxic chemotherapy. Moreover, these studies support the idea that CYP1 is an important contributor to doxorubicin cardiotoxicity and suggest that modulation of this pathway could be beneficial in the clinical setting.

Auto-fatty acylation of transcription factor RFX3 regulates ciliogenesis

Significance Regulatory Factor X 3 (RFX3) is one of the key transcription factors involved in cilia formation and functions. Our study reveals that auto-fatty acylation is a critical regulatory mechanism for RFX3 transcriptional activities. Fatty acylation of RFX3 is required for its dimerization, leading to transcription of cilia-associated genes. More importantly, fatty acylation of RFX3 regulates ciliogenesis and Hedgehog signaling pathways, which are associated with developmental and degenerative disorders known as ciliopathies. Our results indicate a major role of auto-fatty acylation in the regulation of RFX3 function and ciliogenesis, providing a potential link between deregulation of fatty acid metabolism to ciliopathies and diabetes. Defects in cilia have been associated with an expanding human disease spectrum known as ciliopathies. Regulatory Factor X 3 (RFX3) is one of the major transcription factors required for ciliogenesis and cilia functions. In addition, RFX3 regulates pancreatic islet cell differentiation and mature β-cell functions. However, how RFX3 protein is regulated at the posttranslational level remains poorly understood. Using chemical reporters of protein fatty acylation and mass spectrometry analysis, here we show that RFX3 transcriptional activity is regulated by S-fatty acylation at a highly conserved cysteine residue in the dimerization domain. Surprisingly, RFX3 undergoes enzyme-independent, “self-catalyzed” auto-fatty acylation and displays preferences for 18-carbon stearic acid and oleic acid. The fatty acylation-deficient mutant of RFX3 shows decreased homodimerization; fails to promote ciliary gene expression, ciliogenesis, and elongation; and impairs Hedgehog signaling. Our findings reveal a regulation of RFX3 transcription factor and link fatty acid metabolism and protein lipidation to the regulation of ciliogenesis.