Porcupine Activity Research Articles

In vitro reconstitution of Wnt acylation reveals structural determinants of substrate recognition by the acyltransferase human Porcupine

Wnt proteins regulate a large number of processes, including cellular growth, differentiation, and tissue homeostasis, through the highly conserved Wnt signaling pathway in metazoans. Porcupine (PORCN) is an endoplasmic reticulum (ER)-resident integral membrane enzyme that catalyzes posttranslational modification of Wnts with palmitoleic acid, an unsaturated lipid. This unique form of lipidation with palmitoleic acid is a vital step in the biogenesis and secretion of Wnt, and PORCN inhibitors are currently in clinical trials for cancer treatment. However, PORCN-mediated Wnt lipidation has not been reconstituted in vitro with purified enzyme. Here, we report the first successful purification of human PORCN and confirm, through in vitro reconstitution with the purified enzyme, that PORCN is necessary and sufficient for Wnt acylation. By systematically examining a series of substrate variants, we show that PORCN intimately recognizes the local structure of Wnt around the site of acylation. Our in vitro assay enabled us to examine the activity of PORCN with a range of fatty acyl-CoAs with varying length and unsaturation. The selectivity of human PORCN across a spectrum of fatty acyl-CoAs suggested that the kink in the unsaturated acyl chain is a key determinant of PORCN-mediated catalysis. Finally, we show that two putative PORCN inhibitors that were discovered with cell-based assays indeed target human PORCN. Together, these results provide discrete, high-resolution biochemical insights into the mechanism of PORCN-mediated Wnt acylation and pave the way for further detailed biochemical and structural studies.

Indian crested porcupineHystrix indicaKerr (Rodentia:Hystricidae): an emerging threat to saffron cultivation in Jammu and Kashmir

The Indian crested porcupine, Hystrix indica Kerr (Rodentia: Hystricidae) is emerging as a serious pest of saffron. Porcupine is largest rodent, herbivorous, noctranal and fossorial animal. A survey conducted for two years (2008 and 2009) in saffron growing districts, i.e. Pulwama and Budgam indicated that seven villages, i.e. Samboora, Chandhara, Lethpora, Kakpora, Shar-Mandekpal, Gundbal, Ladhoo of district Pulwama and one village, i.e. Hyatpura in District Badgam revealed that saffron and fruit orchards are badly infested by porcupine activity in the month of February to April causing damage to the planted saffron corms and standing fruit trees. Around four thousand families residing in the targeted blocks are directly or indirectly dependent on horticulture and allied activities. Only 3% damage to saffron by this pest can lead to the loss estimated to the tune of 17.4 million rupees annually which will prove to be major setback to the state economy in near future.

Porcupine-dependent Wnt activity within the uterine epithelium is essential for fertility.

The secretion of mammalian Wnt ligands within the cell is dependent on the activity of Porcupine, a gene located on the X-chromosome that encodes for a membrane-bound O-acyl transferase. Here, we report that postnatal ablation of Porcupine in the uterine luminal epithelium alone results in the decrease in endometrial gland number. Despite having uterine glands, mutant females are completely infertile. Epithelial ablation of Porcupine causes defects in timely apposition of the lumen, along with failure to respond to artificial decidual induction. Interestingly, progesterone supplementation was able to rescue the initiation of decidualization, but the decidua was not maintained and subsequently resorbed. Transcriptome analysis demonstrated that deletion of Porcupine in the epithelium resulted in the stromal dysregulation of members of the Wnt signaling pathway (Lef1, Wnt4, and Wnt16), dysregulation of receptors and ligands in the Notch signaling pathway (Notch1, Notch4, and Dll4) as well as Hoxa10. Our results demonstrate the crucial requirement of Wnt signaling in the epithelium for fertility and demonstrate that epithelial Wnts regulate stromal Wnt gene expression as well as regulating the expression of essential signaling factors and effectors required for successful embryo implantation.

An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine

Wnt proteins are a family of secreted signaling proteins that play key roles in regulating cell proliferation in both embryonic and adult tissues. Production of active Wnt depends on attachment of palmitoleate, a monounsaturated fatty acid, to a conserved serine by the acyltransferase Porcupine (PORCN). Studies of PORCN activity relied on cell-based fatty acylation and signaling assays as no direct enzyme assay had yet been developed. Here, we present the first in vitro assay that accurately recapitulates PORCN-mediated fatty acylation of a Wnt substrate. The critical feature is the use of a double disulfide-bonded Wnt peptide that mimics the two-dimensional structure surrounding the Wnt acylation site. PORCN-mediated Wnt acylation was abolished when the Wnt peptide was treated with DTT, and did not occur with a linear (non-disulfide-bonded) peptide, or when the double disulfide-bonded Wnt peptide contained Ala substituted for the Ser acylation site. We exploited this in vitro Wnt acylation assay to provide direct evidence that the small molecule LGK974, which is in clinical trials for managing Wnt-driven tumors, is a bona fide PORCN inhibitor whose IC50 for inhibition of Wnt fatty acylation in vitro closely matches that for inhibition of Wnt signaling. Side-by-side comparison of PORCN and Hedgehog acyltransferase (HHAT), two enzymes that attach 16-carbon fatty acids to secreted proteins, revealed that neither enzyme will accept the other's fatty acyl-CoA or peptide substrates. These findings illustrate the unique enzyme-substrate selectivity exhibited by members of the membrane-bound O-acyl transferase family.

Pines and porcupines: a tree-ring analysis of browsing and dynamics of an overmature pine forest

Past feeding activities of the North American porcupine (Erethizon dorsatum L.) in an overmature forest were reconstructed using tree-ring dated feeding scars on jack pine (Pinus banksiana Lamb.) trees in Parc national du Bic (Quebec). Following a long fire-free period, most pines that regenerated after the 1847 fire are currently senescent, moribund, or dead, and the youngest trees are nearly 100 years old. Balsam fir (Abies balsamea (L.) Mill.), the most abundant species in the regeneration layer, is predicted to replace jack pine given the absence of recent fire and present fire protection practices in the park. According to the age structure of feeding scars, porcupine activity has been particularly high during the 1980s, which is coincident with the creation of the park. Porcupine activity was lower before this decade and in the late 20th century and absent in this century (2000–2015). The recent decrease in the number of feeding scars is consistent with the decline of the porcupine population, as shown by censuses of active dens and inventories of individuals. Observed tree death is linked with tree senescence rather than excessive feeding. In the absence of fire, it is probable that the shift from the overmature pine stand to a balsam fir stand will profoundly affect porcupine activity. This research shows how the study of tree rings can unravel some of the ecological relationships structuring a forest ecosystem through fire, succession, and animal browsing.

Abstract 4449: A novel Porcupine inhibitor is effective in the treatment of cancers with RNF43 mutations

Abstract Various mutations in the Wnt pathway contribute to aberrant activation of Wnt signaling, which is implicated in multiple cancers. Besides the mutation of Adenomatous polyposis coli (APC) or beta-catenin which causes the continuous activation of Wnt signaling by stabilizing beta-catenin, there are mutations of other genes such as RNF43 that regulate Wnt signaling at the level of the ligand or cell surface receptor. Inhibition of the secretion of all human Wnts by blocking their palmitoleoylation by the O-acyltransferase, Porcupine (PORCN), could be an alternative therapeutic approach. Here we have developed a compound with a novel pharmacophore that can inhibit PORCN activity and hence Wnt signaling in nanomolar concentration. In vivo efficacy study demonstrated that the compound is highly efficacious in preventing tumor growth in genetic modification model MMTV-WNT1 mouse. We have identified a subset of pancreatic cancer, cholangiocarcinoma and mucinous ovarian cancer cell lines that are sensitive to the compound. Most of these cell lines harbor mutations in RNF43. Xenograft tumor models derived from the cancer cell lines were found to be sensitive to the compound. Our results demonstrate that inhibiting the Wnt/beta-catenin pathway by targeting PORCN with small-molecule inhibitors in a subset of cancers with mutation in RNF43 is a feasible and nontoxic strategy to overcome the problem of redundancy of Wnts, thereby, providing new option for therapy in diseases with up regulated Wnt expression. Citation Format: Zhiyuan Ke, Babita Madan, Shermaine Q.y. Lim, Sifang Wang, Jenefer Alam, Soo Yei Ho, Duraiswamy Athisayamani Jeyaraj, Kakaly Ghosh, Yun Shan Chew, Li Jun Ding, Vithya Monoharan, Vishal Pendharkar, Esther Ong, Jeffrey Hill, Kanda Sangthongpitag, Thomas Keller, May Ann Lee, David M. Virshup. A novel Porcupine inhibitor is effective in the treatment of cancers with RNF43 mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4449. doi:10.1158/1538-7445.AM2015-4449

Precise Regulation of Porcupine Activity Is Required for Physiological Wnt Signaling

Gradients of diverse Wnt proteins regulate development, renewal, and differentiation. Porcupine (PORCN) is a membrane-bound O-acyltransferase that is required for post-translational modification of all Wnts to enable their transport, secretion, and activity. Mutations in PORCN are associated with focal dermal hypoplasia (FDH), whereas gene deletion causes embryonic lethality in mice. To study the protein in more detail, zinc finger nucleases were used to edit the PORCN genomic locus, establishing two HT1080 fibrosarcoma clones null for PORCN activity that facilitate the study of PORCN structure and function. We establish that PORCN is a key non-redundant node for the regulation of global Wnt signaling because PORCN null cells are completely incapable of autocrine Wnt signaling. The strength of Wnt signaling is exquisitely sensitive to PORCN expression, with a dynamic range of at least 3 orders of magnitude, suggesting that PORCN activity is a key modulator of all Wnt ligand activity. Consistent with this, we find that multiple FDH-associated mutants have only subtle alterations in enzyme activity yet are associated with a severe FDH phenotype. These studies support an essential regulatory role of PORCN in shaping Wnt signaling gradients.

Déchiffrer l’histoire taphonomique de l’assemblage osseux du Paléolithique supérieur de la Grotte Zouhrah/El Harhoura 1, maroc

Overlap in the use of caves between hominids and animals has frequently been documented in the prehistoric record. In the Maghreb, Zouhrah Cave, also called El Harhoura 1 (Temara, Morocco), has yielded a few Aterian lithic tools and an abundance of fauna. This faunal assemblage is dominated by gazelles (41% of identified remains) and many carnivore species, including remains of both spotted and striped hyena. Extensive porcupine activity is also evident. The observation of numerous coprolites, in combination with the study of ungulate mortality profiles, skeletal element representation and pattern of bone modifications, all demonstrate that the cave functioned more as a carnivore den than an anthropogenic site even during the Neolithic. Our analysis reveals that the Zouhrah Cave faunal assemblage was created by multiple agents: human, hyena and porcupine. We suggest that this may be a typical signature for Upper Pleistocene cave sites in the Mediterranean region.

Small molecule–mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer

SUMMARYThe pervasive influence of secreted Wnt signaling proteins in tissue homeostasis and tumorigenesis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. By screening a diverse synthetic chemical library, we have discovered two novel classes of small molecules that disrupt Wnt pathway responses - whereas one class inhibits the activity of Porcupine (Porcn), a membrane-bound acyltransferase that is essential to the production of Wnt proteins, the other abrogates destruction of Axin proteins, suppressors of Wnt/β-catenin pathway activity. With these small molecules we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue. We achieve transient, reversible suppression of Wnt/β-catenin pathway response in vivo, and establish a mechanism-based approach to target cancerous cell growth. The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.

Porcupine-mediated lipid-modification regulates the activity and distribution of Wnt proteins in the chick neural tube

A long-term goal of developmental biology is to understand how morphogens establish gradients that promote proper tissue patterning. A number of reports describe the formation of the Wg (Wnt1) gradient in Drosophila and have shown that Porcupine, a predicted membrane-bound O-acyl transferase, is required for the correct distribution of Wg protein. The discovery that Wnts are palmitoylated on a conserved cysteine residue suggests that porcupine activity and Wnt palmitoylation are important for the generation of Wnt gradients. To establish the role of porcupine in Wnt gradient formation in vertebrates, we tested the role of porcupine/Wnt palmitoylation in human embryonic kidney 293T cells and in the chick neural tube. Our results lead us to conclude that: (1) vertebrate Wnt1 and Wnt3a possess at least one additional site for porcupine-mediated lipid-modification; (2) porcupine-mediated lipid-modification of Wnt proteins promotes their activity in 293T cells and in the chick neural tube; and (3) porcupine-mediated lipid-modification reduces the range of activity of Wnt1 and Wnt3a in the chick neural tube. These findings highlight the importance of porcupine-mediated lipid modifications in the formation of vertebrate Wnt activity gradients.

Impact of feeding damage by the porcupine on western hemlock – Sitka spruce forests of north-coastal British Columbia: 15-year results

American porcupine (Erithizon dorsatum L.) feed on the cambial tissues of several tree species. This study assessed feeding by porcupine over a 15-year period on midrotation age western hemlock (Tsuga heterophylla (Raf.) Sarg.) dominated stands on the north coast of British Columbia, Canada. Although four coniferous hosts were available, hemlock was, by far, the most preferred and most seriously impacted. Of 398 hemlock studied, 260 were wounded by porcupine feeding and 86 died by 2000. Porcupine attacked two-thirds of the dead trees, over 82% of which were dominant or codominant, while almost all unattacked dead trees were intermediate or suppressed. This loss resulted in a 14.6% reduction in the proportion of volume per hectare represented by hemlock and an increase in the representation by other species, all of which suffered little or no feeding damage. Results indicate that the final merchantable volume of hemlock expected at rotation age will be lower than projected and will consist mainly of damaged trees of poor form and quality due to decay fungi, such as Stereum sanguinolentum (A. & S. ex Fr.), entering through feeding wounds. Porcupine activity has led to a shift in species composition and volume from a condition where a single species, western hemlock, dominates to a mixed forest consisting of amabilis fir (Abies amabilis (Dougl. ex Loud.) Dougl. ex J. Forbes), Sitka spruce (Picea sitchensis (Bong.) Carrière), and western hemlock.

Observations on the feeding habits of the Indian crested porcupine (Hystrix indica) and the distribution of some hemicryptophytes and geophytes in the Negev desert highlands

More than 17 geophytes and hemicryptophytes are consumed by porcupines or inhabit porcupine diggings during the year. By consuming subterranean bulbs and leaves, diggings are created, sometimes as many as 2 or 3/m2. Under desert conditions each digging accumulates organic matter, seeds and runoff water. In the flat areas covered with loess it is possible for a digging to remain for longer than 5 years. The plants which show the greatest interaction between porcupine activity and their own survival mechanisms are four species of geophytes and hemicryptophytes. Of these, porcupines consume only part of the plant—the undamaged part renews itself and develops later in conditions improved by the digging. There are four other groups: (a) Three species were totally consumed, and the porcupine digging became a suitable microhabitat for germination for at least two of them. Another 10 species of plants were: (b) either consumed, and inhabit diggings, (c) possibly not eaten by porcupines but inhabit diggings, (d) consumed from time to time, but not found in diggings.