Palmitoylation Sites Research Articles

Abstract 445: A next generation TEAD inhibitor with refined isoform specificity for superior safety & efficacy

Abstract The Hippo pathway is an important regulator of cell proliferation and one of the last major oncogenic pathways not yet extensively targeted in precision oncology. The Hippo pathway is executed by the YAP1/TAZ (WWTR1) co-activators and the TEAD family of transcription factors, which consists of four paralogs (TEAD1-4) with both redundant and unique functions. The transcriptional targets of TEADs include genes playing tumor-promoting roles, including pro-proliferative, immunosuppressive, and anti-apoptotic genes. A growing body of research points to the central importance of the Hippo pathway as a key driver of oncogenesis and as key resistance mechanism to inhibitors of the MAPK pathway or its upstream activators, such as receptor tyrosine kinases. Sporos BioDiscovery has developed novel inhibitors that reversibly bind to the palmitoylation site of TEAD transcription factors to block Hippo pathway activity. Sporos BioDiscovery’s internal bioinformatic insights were leveraged to fine tune the inhibitory activity against the four TEAD paralogs, to maximize efficacy and minimize toxicity. Key characteristics of the SPR1 TEAD inhibitors: (i) low nM, single-agent activity against multiple TEAD-dependent cell lines in vitro in established Hippo-pathway hyperactive mesothelioma cell lines as well as in several non-mesothelioma, cell lines without any obvious lesions in the upstream components of the Hippo pathway. (ii) activity against multiple tumor models in vivo with tumor regression observed even in large established tumors, a feat not previously reported with any other TEAD inhibitor. (iii) strong interactions with inhibitors of the MAPK pathway and inhibitors of its upstream activators, such as RTKs. (iv) favorable ADME profile in rodents, dogs and NHPs, as well as a favorable safety profile. In sum, SPR1 presents monotherapy opportunities in ultra-responder populations based on internal bioinformatic insights, while broader potential exists as an adjuvant for precision oncology targeted therapies, particularly within the MAPK pathway and its upstream activators. Citation Format: Florian Muller, Erkan Baloglu, Andrew D. Morley, Deepavali Chakravarti, Selvi Kunnimalaiyaan, Jeno Gyruis, Sharon Shacham. A next generation TEAD inhibitor with refined isoform specificity for superior safety & efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 445.

Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes

Mammalian voltage-activated L-type Ca2+ channels, such as Ca(v)1.2, control transmembrane Ca2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current-voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca2+ channels in excitable cells.

Protein post-translational modification in SARS-CoV-2 and host interaction.

SARS-CoV-2 can cause lung diseases, such as pneumonia and acute respiratory distress syndrome, and multi-system dysfunction. Post-translational modifications (PTMs) related to SARS-CoV-2 are conservative and pathogenic, and the common PTMs are glycosylation, phosphorylation, and acylation. The glycosylation of SARS-CoV-2 mainly occurs on spike (S) protein, which mediates the entry of the virus into cells through interaction with angiotensin-converting enzyme 2. SARS-CoV-2 utilizes glycans to cover its epitopes and evade the immune response through glycosylation of S protein. Phosphorylation of SARS-CoV-2 nucleocapsid (N) protein improves its selective binding to viral RNA and promotes viral replication and transcription, thereby increasing the load of the virus in the host. Succinylated N and membrane(M) proteins of SARS-CoV-2 synergistically affect virus particle assembly. N protein regulates its affinity for other proteins and the viral genome through acetylation. The acetylated envelope (E) protein of SARS-CoV-2 interacts with bromodomain-containing protein 2/4 to influence the host immune response. Both palmitoylation and myristoylation sites on S protein can affect the virus infectivity. Papain-like protease is a domain of NSP3 that dysregulates host inflammation by deubiquitination and impinges host IFN-I antiviral immune responses by deISGylation. Ubiquitination of ORF7a inhibits host IFN-α signaling by blocking STAT2 phosphorylation. The methylation of N protein can inhibit the formation of host stress granules and promote the binding of N protein to viral RNA, thereby promoting the production of virus particles. NSP3 macrodomain can reverse the ADP-ribosylation of host proteins, and inhibit the cascade immune response with IFN as the core, thereby promoting the intracellular replication of SARS-CoV-2. On the whole, PTMs have fundamental roles in virus entry, replication, particle assembly, and host immune response. Mutations in various SARS-CoV-2 variants, which lead to changes in PTMs at corresponding sites, cause different biological effects. In this paper, we mainly reviewed the effects of PTMs on SARS-CoV-2 and host cells, whose application is to inform the strategies for inhibiting viral infection and facilitating antiviral treatment and vaccine development for COVID-19.

Full-length huntingtin is palmitoylated at multiple sites and post-translationally myristoylated following caspase-cleavage.

Introduction: Huntington disease is an autosomal dominant neurodegenerative disorder which is caused by a CAG repeat expansion in the HTT gene that codes for an elongated polyglutamine tract in the huntingtin (HTT) protein. Huntingtin is subjected to multiple post-translational modifications which regulate its cellular functions and degradation. We have previously identified a palmitoylation site at cysteine 214 (C214), catalyzed by the enzymes ZDHHC17 and ZDHHC13. Reduced palmitoylation level of mutant huntingtin is linked to toxicity and loss of function. Moreover, we have described N-terminal myristoylation by the N-myristoyltransferases of a short fragment of huntingtin (HTT553-586) at glycine 553 (G553) following proteolysis at aspartate 552 (D552). Results: Here, we show that huntingtin is palmitoylated at numerous cysteines: C105, C433, C3134 and C3144. In addition, we confirm that full-length huntingtin is cleaved at D552 and post-translationally myristoylated at G553. Importantly, blocking caspase cleavage at the critical and pathogenic aspartate 586 (D586) significantly increases posttranslational myristoylation of huntingtin. In turn, myristoylation of huntingtin promotes the co-interaction between C-terminal and N-terminal huntingtin fragments, which is also protective. Discussion: This suggests that the protective effect of inhibiting caspase-cleavage at D586 may be mediated through post-translational myristoylation of huntingtin at G553.

Inhibition of acetyl-CoA carboxylase impaired tubulin palmitoylation and induced spindle abnormalities

Tubulin s-palmitoylation involves the thioesterification of a cysteine residue in tubulin with palmitate. The palmitate moiety is produced by the fatty acid synthesis pathway, which is rate-limited by acetyl-CoA carboxylase (ACC). While it is known that ACC is phosphorylated at serine 79 (pSer79) by AMPK and accumulates at the spindle pole (SP) during mitosis, a functional role for tubulin palmitoylation during mitosis has not been identified. In this study, we found that modulating pSer79-ACC level at the SP using AMPK agonist and inhibitor induced spindle defects. Loss of ACC function induced spindle abnormalities in cell lines and in germ cells of the Drosophila germarium, and palmitic acid (PA) rescued the spindle defects in the cell line treated transiently with the ACC inhibitor, TOFA. Furthermore, inhibition of protein palmitoylating or depalmitoylating enzymes also induced spindle defects. Together, these data suggested that precisely regulated cellular palmitate level and protein palmitoylation may be required for accurate spindle assembly. We then showed that tubulin was largely palmitoylated in interphase cells but less palmitoylated in mitotic cells. TOFA treatment diminished tubulin palmitoylation at doses that disrupt microtubule (MT) instability and cause spindle defects. Moreover, spindle MTs comprised of α-tubulins mutated at the reported palmitoylation site exhibited disrupted dynamic instability. We also found that TOFA enhanced the MT-targeting drug-induced spindle abnormalities and cytotoxicity. Thus, our study reveals that precise regulation of ACC during mitosis impacts tubulin palmitoylation to delicately control MT dynamic instability and spindle assembly, thereby safeguarding nuclear and cell division.

Membrane Estrogen Receptor β Is Sufficient to Mitigate Cardiac Cell Pathology.

Estrogen acting through estrogen receptor β (ERβ) has been shown to oppose the stimulation of cardiac myocytes and cardiac fibroblasts that results in cardiac hypertrophy and fibrosis. Previous work has implicated signal transduction from ERβ as being important to the function of estrogen in this regard. Here we address whether membrane ERβ is sufficient to oppose key mechanisms by which angiotensin II (AngII) stimulates cardiac cell pathology. To do this we first defined essential structural elements within ERβ that are necessary for membrane or nuclear localization in cells. We previously determined that cysteine 418 is the site of palmitoylation of ERβ that is required and sufficient for cell membrane localization in mice and is the same site in humans. Here we determined in Chinese hamster ovarian (CHO) cells, and mouse and rat myocytes and cardiac fibroblasts, the effect on multiple aspects of signal transduction by expressing wild-type (WT ) or a C418A-mutant ERβ. To test the importance of the nuclear receptor, we determined a 4-amino acid deletion in the E domain of ERβ that strongly blocked nuclear localization. Using these tools, we expressed WT and mutant ERβ constructs into cardiomyocytes and cardiac fibroblasts from ERβ-deleted mice. We determined the ability of estrogen to mitigate cell pathology stimulated by AngII and whether the membrane ERβ is necessary and sufficient.

Overexpressed Gα13 activates serum response factor through stoichiometric imbalance with Gβγ and mislocalization to the cytoplasm

Gα13, a heterotrimeric G protein α subunit of the G12/13 subfamily, is an oncogenic driver in multiple cancer types. Unlike other G protein subfamilies that contribute to cancer progression via amino acid substitutions that abolish their deactivating, intrinsic GTPase activity, Gα13 rarely harbors such mutations in tumors and instead appears to stimulate aberrant cell growth via overexpression as a wildtype form. It is not known why this effect is exclusive to the G12/13 subfamily, nor has a mechanism been elucidated for overexpressed Gα13 promoting tumor progression. Using a reporter gene assay for serum response factor (SRF)-mediated transcription in HEK293 cells, we found that transiently expressed, wildtype Gα13 generates a robust SRF signal, approximately half the amplitude observed for GTPase-defective Gα13. When epitope-tagged, wildtype Gα13 was titrated upward in cells, a sharp increase in SRF stimulation was observed coincident with a “spillover” of Gα13 from membrane-associated to a soluble fraction. Overexpressing G protein β and γ subunits caused both a decrease in this signal and a shift of wildtype Gα13 back to the membranous fraction, suggesting that stoichiometric imbalance in the αβγ heterotrimer results in aberrant subcellular localization and signalling by overexpressed Gα13. We also examined the acylation requirements of wildtype Gα13 for signalling to SRF. Similar to GTPase-defective Gα13, S-palmitoylation of the wildtype α subunit was necessary for SRF activation but could be replaced functionally by an engineered site for N-terminal myristoylation. However, a key difference was observed between wildtype and GTPase-defective Gα13: whereas the latter protein lacking palmitoylation sites was rescued in its SRF signalling by either an engineered polybasic sequence or a C-terminal isoprenylation site, these motifs failed to restore signalling by wildtype, non-palmitoylated Gα13. These findings illuminate several components of the mechanism in which overexpressed, wildtype Gα13 contributes to growth and tumorigenic signalling, and reveal greater stringency in its requirements for post-translational modification in comparison to GTPase-defective Gα13.

Proteomic analysis of s-acylated proteins in human retinal pigment epithelial cells and the role of palmitoylation of Niemann-Pick type C1 protein in cholesterol transport.

Palmitoylation is a dynamic process that regulates the activity of the modified proteins. Retinal pigment epithelial (RPE) cells play pivotal roles in the visual cycle and maintaining healthy photoreceptor cells. Dysfunctional RPE cells are often associated with degenerative retinal diseases. The aim of the study was to identify potentially palmitoylated proteins in human RPE cells. By using the detergent-resistant membrane, we found 312 potentially palmitoylated peptides which corresponded to 192 proteins in RPE cells, including 55 new candidate proteins which were not reported before. Gene enrichment analysis highlighted significant enrichment of palmitoylated proteins in cell-matrix adhesion, cell-cell recognition, protein cellular localization, and translation, among others. We further studied the effect of 3 potential palmitoylation sites (Cys 799, 900, and 816) of Niemann-Pick type C1 protein (NPC1) on cholesterol accumulation. We found that mutation of any single Cys alone had no significant effect on intracellular cholesterol accumulation while simultaneous mutation of Cys799 and 800 caused significant cholesterol accumulation in the late endosome. No further cholesterol accumulation was observed by adding another mutation at Cys 816. However, the mutation did not alter the cellular localization of the protein. Conclusion: PRE cells have an abundant number of palmitoylated proteins which are involved in cellular processes critical to visual function. The palmitoylation at Cys799 and 800 was needed for cholesterol export, but not the intracellular localization of NPC1.

112 (PB102) - A family of novel TEAD palmitoylation site inhibitors with exceptional pre-clinical anti-neoplastic activity as a monotherapy and in combination with MAPK inhibitors

Background: The Hippo pathway (effected by the YAP/TAZ-TEAD transcriptional complex) is a major regulator of cell density and organ size, and is a major pro-growth, pro-survival pathway yet to be targeted in precision oncology. YAP/TAZ-TEAD transcription is hyperactivated by specific genetic events (e.g. YAP/TAZ oncofusions, NF2 mutations) and has been demonstrated as a key mechanism of resistance acquisition to MAPK pathway inhibition, as well as inhibition of its upstream inputs, such as EGFR and other receptor tyrosine kinases.

Cytoplasmic tail determines the membrane trafficking and localization of SARS-CoV-2 spike protein.

The spike (S) glycoprotein of SARS-CoV-2 mediates viral entry through associating with ACE2 on host cells. Intracellular trafficking and palmitoylation of S protein are required for its function. The short cytoplasmic tail of S protein plays a key role in the intracellular trafficking, which contains the binding site for the host trafficking proteins such as COPI, COPII and SNX27. This cytoplasmic tail also contains the palmitoylation sites of S protein. Protein palmitoylation modification of S protein could be catalyzed by a family of zinc finger DHHC domain-containing protein palmitoyltransferases (ZDHHCs). The intracellular trafficking and membrane location facilitate surface expression of S protein and assembly of progeny virions. In this review, we summarize the function of S protein cytoplasmic tail in transportation and localization. S protein relies on intracellular trafficking pathways and palmitoylation modification to facilitate the life cycle of SARS-CoV-2, meanwhile it could interfere with the host transport pathways. The interplay between S protein and intracellular trafficking proteins could partially explain the acute symptoms or Long-COVID complications in multiple organs of COVID-19 patients.

Integrated analysis to study the interplay between post-translational modifications (PTM) in hepatitis C virus proteins and hepatocellular carcinoma (HCC) development

Many PTMs dysregulation is known to be the major cause of many cancers including HCV induced HCC. PTMs of hepatitis C virus (HCV) regions NS3/4A, NS5A and NS5B are crucial for proper protein functions and replication that directly affect the generation of infectious virus particles and completion of its life cycle. In this study, we have performed comprehensive analysis of PTMs within HCV non-structural proteins (NS3/4A, NS5A and NS5B) through bioinformatics analysis to examine post-translational crosstalk between phosphorylation, palmitoylation, methylation, acetylation and ubiquitination sites in selected viral proteins. Our analysis has revealed many highly putative PTMs sites that are also conserved among major genotypes conferring the importance of these sites. We have also analysed viral 3D structures in their modified and unmodified forms to address extent and signatures of structural changes upon PTM. This study provides evidence that PTMs induce significant conformational changes and make viral proteins more stable. To find the potential role of PTMs in HCV induced HCC, docking analysis between selected viral proteins and p38-MAPK has been performed which also confirms their strong association with HCV induced HCC. The major findings proposed that PTMs at specific sites of HCV viral proteins could dysregulate specific pathways that cause the development of HCC.

S-Palmitoylation of Tyrosinase at Cysteine500 Regulates Melanogenesis

Palmitoylation is a lipid modification involving the attachment of palmitic acid to a cysteine residue, thereby affecting protein function. We investigated the effect of palmitoylation of tyrosinase, the rate-limiting enzyme in melanin synthesis, using a human three-dimensional skin model system and melanocyte culture. The palmitoylation inhibitor, 2-bromopalmitate, increased melanin content and tyrosinase protein levels in melanogenic cells by suppressing tyrosinase degradation. The palmitoylation site was Cysteine500 in the C-terminal cytoplasmic tail of tyrosinase. The nonpalmitoylatable mutant, tyrosinase (C500A), was slowly degraded and less ubiquitinated than wild-type tyrosinase. Screening for the Asp-His-His-Cys (DHHC) family of proteins for tyrosinase palmitoylation suggested that DHHC2, 3, 7, and 15 are involved in tyrosinase palmitoylation. Knockdown of DHHC2, 3, or 15 increased tyrosinase protein levels and melanin content. Determination of their subcellular localization in primary melanocytes revealed that DHHC2, 3, and 15 were localized in the endoplasmic reticulum, Golgi apparatus, and/or melanosomes, whereas only DHHC2 was localized in the melanosomes. Immunoprecipitation showed that DHHC2 and DHHC3 predominantly bind to mature and immature tyrosinase, respectively. Taken together, tyrosinase palmitoylation at Cysteine500 by DHHC2, 3, and/or 15, especially DHHC2 in trans-Golgi apparatus and melanosomes and DHHC3 in the endoplasmic reticulum and cis-Golgi apparatus, regulate melanogenesis by modulating tyrosinase protein levels.

PRDX6: A protein bridging S-palmitoylation and diabetic neuropathy

Diabetic neuropathy is regarded as one of the most debilitating outcomes of diabetes. It can affect both the peripheral and central nervous systems, leading to pain, decreased motility, cognitive decline, and dementia. S-palmitoylation is a reversible posttranslational lipid modification, and its dysregulation has been implicated in metabolic syndrome, cancers, neurological disorders, and infections. However, the role of S-palmitoylation in diabetic neuropathy remains unclear. Here we demonstrate a potential association between activating protein palmitoylation and diabetic neuropathy. We compared the proteomic data of lumbar dorsal root ganglia (DRG) of diabetes mice and palmitoylome profiling data of the HUVEC cell line. The mapping results identified peroxiredoxin-6 (PRDX6) as a novel target in diabetic neuropathy, whose biological mechanism was associated with S-palmitoylation. Bioinformatic prediction revealed that PRDX6 had two palmitoylation sites, Cys47 and Cys91. Immunofluorescence results indicated PRDX6 translocating between the cytoplasm and cell membrane. Protein function analysis proposed that increased palmitoylation could competitively inhibit the formation of disulfide-bond between Cys47 and Cys91 and change the spatial topology of PRDX6 protein. Cl–HCO3- anion exchanger 3 (AE3) was one of the AE family members, which was proved to express in DRG. AE3 activity evoked Cl- influx in neurons which was generally associated with increased excitability and susceptibility to pain. We demonstrated that the S-palmitoylation status of Cys47 could affect the interaction between PRDX6 and the C-terminal domain of AE3, thereby regulating the activity of AE3 anion exchanger enzyme in the nervous system. The results highlight a central role for PRDX6 palmitoylation in protection against diabetic neuropathy.

Palmitoylation of GNAQ/11 is critical for tumor cell proliferation and survival in GNAQ/11-mutant uveal melanoma.

More than 85% of patients with uveal melanoma (UM) carry a GNAQ or GNA11 mutation at a hotspot codon (Q209) that encodes G protein α subunit q/11 polypeptides (Gαq/11). GNAQ/11 relies on palmitoylation for membrane association and signal transduction. Despite the palmitoylation of GNAQ/11 was discovered long before, its implication in UM remains unclear. Here, results of palmitoylation-targeted mutagenesis and chemical interference approaches revealed that the loss of GNAQ/11 palmitoylation substantially affected tumor cell proliferation and survival in UM cells. Palmitoylation inhibition through the mutation of palmitoylation sites suppressed GNAQ/11Q209L-induced malignant transformation in NIH3T3 cells. Importantly, the palmitoylation-deficient oncogenic GNAQ/11 failed to rescue the cell death initiated by the knock down of endogenous GNAQ/11 oncogenes in UM cells, which are much more dependent on Gαq/11 signaling for cell survival and proliferation than other melanoma cells without GNAQ/11 mutations. Furthermore, the palmitoylation inhibitor, 2-bromopalmitate, also specifically disrupted Gαq/11 downstream signaling by interfering with the MAPK pathway and BCL2 survival pathway in GNAQ/11-mutant UM cells and showed a notable synergistic effect when applied in combination with the BCL2 inhibitor, ABT-199, in vitro. The findings validate that GNAQ/11 palmitoylation plays a critical role in UM and may serve as a promising therapeutic target for GNAQ/11-driven UM.

S-palmitoylation of PCSK9 induces sorafenib resistance in liver cancer by activating the PI3K/AKT pathway.

Sorafenib is currently the first-line treatment for advanced hepatocellular carcinoma (HCC). However, sorafenib resistance remains a significant challenge. Aberrant AKT signaling activation is a crucial mechanism driving sorafenib resistance in HCC. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a vital role in antitumor immune responses. In this study, we demonstrate that aberrant PCSK9 upregulation promotes cell proliferation and sorafenib resistance in HCC by inducing AKT-S473 phosphorylation. After palmitoylation at cysteine 600, the binding affinity between PCSK9 and tensin homolog (PTEN) is dramatically increased, inducing lysosome-mediated PTEN degradation and subsequent AKT activation. We identify zinc finger DHHC-type palmitoyltransferase 16 (ZDHHC16) as a palmitoyltransferase that promotes PCSK9 palmitoylation at cysteine 600. We also develop a biologically active PCSK9-derived peptide that competitively inhibits PCSK9 palmitoylation, suppressing AKT phosphorylation and augmenting the antitumor effects of sorafenib in HCC.

Glycosylation and S-palmitoylation regulate SARS-CoV-2 spike protein intracellular trafficking

SummaryPost-translational modifications (PTMs), such as glycosylation and palmitoylation, are critical to protein folding, stability, intracellular trafficking, and function. Understanding regulation of PTMs of SARS-CoV-2 spike (S) protein could help the therapeutic drug design. Herein, the VSV vector was used to produce SARS-CoV-2 S pseudoviruses to examine the roles of the 611LYQD614 and cysteine-rich motifs in S protein maturation and virus infectivity. Our results show that 611LY612 mutation alters S protein intracellular trafficking and reduces cell surface expression level. It also changes S protein glycosylation pattern and decreases pseudovirus infectivity. The S protein contains four cysteine-rich clusters with clusters I and II as the main palmitoylation sites. Mutations of clusters I and II disrupt S protein trafficking from ER-to-Golgi, suppress pseudovirus production, and reduce spike-mediated membrane fusion activity. Taken together, glycosylation and palmitoylation orchestrate the S protein maturation processing and are critical for S protein-mediated membrane fusion and infection.

Membrane Targeting of Constitutively Active Gαq Regulates Inhibition by YM‐254980

Uveal melanoma, the most common cancer of the eye in adults, is driven by an activating glutamine to leucine or proline mutation at residue 209 of the α subunit of G proteins Gq or G11 (Gαq/11QL or Gαq/11QP). This mutation is found in more than 90% of cases and up to 50% of patients experience untreatable metastases. YM‐254980 is a promising inhibitor of constitutively active (CA) Gαq/11, though its mechanism of action is not well understood. Evidence supports that although Gαq/11 has 2 sites of palmitoylation that allow for association with the plasma membrane (PM), these proteins are still found in subcellular locations. We have shown that more strongly targeting GαqQL to membranes by adding an N‐terminal myristoylation site renders it insensitive to YM, suggesting a requirement of Gαq/11 translocating off the membrane to be inhibited. To further understand how PM‐restricted GαqQL loses sensitivity to YM inhibition of signaling, we have generated additional membrane targeting mutants of GαqQL. Although palmitoylation at cysteines 9 and 10 of GαqQL is essential for signaling, preventing palmitoylation by mutating both cysteines 9 and 10 to serines in the context of myristoylated GαqQL does not abolish signaling, as assayed by TEAD‐ and SRE‐dependent luciferase reporter assays in HEK293 q/11 knockout cells. Moreover, signaling by this myristoylated, palmitoylation‐deficient GαqQL mutant remains insensitive to YM, indicating that introduction of a single site for myristoylation, in the absence of any palmitoylation, is sufficient to render GαqQL resistant to YM. To further define the mechanism of YM insensitivity of membrane targeting mutants of GαqQL, we are currently examining other CA Gα subunits, specifically Gα16 (3 sites of palmitoylation) and Gα13 (2 sites of palmitoylation), that have been mutated to introduce a YM binding site. We have generated myristoylated mutants of these Gα subunits, and are testing if they, like myristoylated GαqQL, are resistant to inhibition by YM. These experiments will highlight the requirements of membrane association for signaling by GαqQL and reveal insights into the mechanism of YM inhibition.

YM‐254890 Promotes the Subcellular Redistribution of Gαq

Uveal melanoma (UM), though a rare form of cancer, is the most common primary intraocular cancer in adults. Approximately 50% of patients succumb to metastatic UM for which there are no effective treatments resulting in a survival period of 2‐8 months. Interestingly, most patients with UM harbor a mutually exclusive mutation in Gαq or Gα11. However, no drugs are clinically approved to target these proteins to treat UM. YM‐254890 (YM) and FR‐900359 (FR) have gained much attention due to their ability to inhibit constitutively active Gαq by preventing the release of GDP and thereby inhibiting GDP to GTP exchange. However, their mechanism of action and potential to prevent uveal melanoma progression is not fully understood. While Gα subunits are known to signal at the plasma membrane (PM), some studies also show that these proteins traffic into the cytoplasm or to intracellular organelles particularly upon activation. The objective of this study is to understand the changes in localization of Gαq wild type (WT) and constitutively active GαqQ209L that occur by inhibition with YM. By immunofluorescence microscopy we show that Gαq WT and GαqQ209L display a subcellular redistribution from the PM to the cytoplasm upon YM treatment. We hypothesize that this relocalization is important for YM’s ability to inhibit GαqQ209L. By adding PM‐targeting motifs (myristylation, palmitoylation, or polybasic sites) to constitutively active Gαq we show that the PM‐restricted mutants do not redistribute into the cytoplasm upon YM treatment and, surprisingly, retain their ability to signal upon YM treatment. Interestingly, our signaling assays show that G protein‐coupled receptor (GPCR) activated PM‐restricted Gαq WT remains sensitive to inhibition by YM. Using pull down techniques to determine if YM promotes an inactive conformation of the PM‐restricted GαqQ209L mutants we show that both GαqQ209L and PM‐restricted GαqQ209L mutants display increased binding to Gβγ and decreased binding to RGS2 upon YM treatment, suggesting that YM promotes an inactive conformation in these PM‐restricted mutants in the same manner as with Gαq WT and GαqQ209L. Our studies suggest that an additional way in which YM inhibits GαqQ209L is by promoting the subcellular redistribution of constitutively active Gαq from the PM to the cytoplasm.

Palmitoylation of RGS20 Affects its Cellular Function

Regulator of G protein signaling 20 (RGS20) is a member of the RZ family of the RGS protein superfamily, which serve as key negative regulators of G protein‐mediated signal transduction. Previous studies showed that overexpression of RGS20 is correlated with progression of triple‐negative breast cancer (TNBC) in tumor tissues and contributes to breast cancer cell migration and invasion. However, there is still a large gap relating to the regulation and function of RGS20 due to its late discovery. RGS20 contains a poly cysteine string motif, which is assumed to be heavily palmitoylated. Palmitoylation, an important post‐translational protein modification, plays an important role in cells by changing cellular functions of proteins. Consequently, in this study we determined palmitoylation regulation of RGS20, which could provide more information for new potential treatment strategies for TNBC. We used click chemistry to show that RGS20 is palmitoylated in transfected HEK‐293T cells and MDA‐MB‐231 triple negative breast cancer cells. We also showed that constitutively activated Gαo, GαoQ205L, significantly increases palmitoylation levels of RGS20. Furthermore, palmitoylated RGS20 associates much more with GαoQ205L, while de‐palmitoylated RGS20 lost the association with GαoQ205L. Next, we detected how palmitoylation affects RGS20 GAP activity, as well as cAMP signaling downstream of Gαo. We mapped the palmitoylation site crucial for all of these effects to a conserved cysteine in the RGS domain. Mutating this cysteine residue resulted in a significant decrease in both palmitoylation signal and the association with GαoQ205L. Overall, we showed that palmitoylation affects functions of RGS20 in cells and that a conserved cysteine in the RGS domain is a critical site for its palmitoylation.

A tissue-specific role of membrane-initiated ERα signaling for the effects of SERMs.

Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ER) agonists or antagonists in a tissue-specific manner. ERs exert effects via nuclear actions but can also utilize membrane-initiated signaling pathways. To determine if membrane-initiated ERα (mERα) signaling affects SERM action in a tissue-specific manner, C451A mice, lacking mERα signaling due to a mutation at palmitoylation site C451, were treated with Lasofoxifene (Las), Bazedoxifene (Bza), or estradiol (E2), and various tissues were evaluated. Las and Bza treatment increased uterine weight to a similar extent in C451A and control mice, demonstrating mERα-independent uterine SERM effects, while the E2 effect on the uterus was predominantly mERα-dependent. Las and Bza treatment increased both trabecular and cortical bone mass in controls to a similar degree as E2, while both SERM and E2 treatment effects were absent in C451A mice. This demonstrates that SERM effects, similar to E2 effects, in the skeleton are mERα-dependent. Both Las and E2 treatment decreased thymus weight in controls, while neither treatment affected the thymus in C451A mice, demonstrating mERα-dependent SERM and E2 effects in this tissue. Interestingly, both SERM and E2 treatments decreased the total body fat percent in C451A mice, demonstrating the ability of these treatments to affect fat tissue in the absence of functional mERα signaling. In conclusion, mERα signaling can modulate SERM responses in a tissue-specific manner. This novel knowledge increases the understanding of the mechanisms behind SERM effects and may thereby facilitate the development of new improved SERMs.