Substrate-mimicking peptides for wild-type or mutant Wnts with a point-mutation, varying in lengths or the number of disulfide bonds were purchased from CPC Scientific (Sunnyvale, CA)..
Abstract
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.
SOCIAL MEDIA
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CPC Scientific Inc., a leading global peptide CRDMO (Contract Research, Development, and Manufacturing Organization) has invested in a new peptide API (Active Pharmaceutical Ingredient) manufacturing site, bringing many new jobs to Rocklin, California. The 41,000 sq ft facility located at 3880 Atherton Rd, Rocklin, CA 95765 will be utilized to manufacture clinical to commercial grade peptide products for increased manufacturing capacity and will diversify CPC Scientific’s supply chain.
CPC Scientific is entering an exciting period of growth and innovation for peptide and oligonucleotide therapeutic development and manufacturing, and we will continue to provide therapeutic APIs to pharmaceutical and biotech companies around the world. We are very pleased to partner with the City of Rocklin, California to bring manufacturing and Life-Science jobs to local American workers,” said Shawn Lee, PhD, CEO.
Ikeda, Z., Kakegawa, K., Kikuchi, F., Itono, S., Oki, H., Yashiro, H., Hiyoshi, H., Tsuchimori, K., Hamagami, K., Watanabe, M. and Sasaki, M. Journal of Medicinal Chemistry 65, no. 12 (2022): 8456-8477.
- Research, Takeda Pharmaceutical Company Limited, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
Subsequently, 5FAM–Abu–Gly–Asp–Asp–Asp–Lys–Ile–Val–Gly–Gly–Lys(CPQ2)–Lys–Lys–NH2 (purity: 97.2%, CPC Scientific, Inc.) was diluted with an assay buffer to prepare a 2.1 μM substrate solution.
The transferred energy from a fluorescent donor is converted into molecular vibrations if the acceptor is a non-fluorescent dye (quencher). When the FRET is terminated (by separating donor and acceptor), an increase of donor fluorescence can be detected. The design and synthesis work at CPC for FRET and TR-FRET peptide substrates include modification of sequences, selection of donor/quencher pairs, improvement of FRET substrate solubility and quenching efficiency.
Genomics research shows that more than a million proteins are encoded by approximately 30,000 human genes. Proteomics, the study of proteins encoded by the genome, includes identifying post-translational modifications, structural analyses, protein localization studies, and protein quantitation. Mass spectroscopybased techniques have evolved as a powerful tool in proteomics. Stable isotope-labeled peptides (SIL peptides) are chemically and physically indistinguishable from their endogenous counterparts concerning retention time, ionization efficiency, and fragmentation pathways.
Peptides play a vital role in the pharmaceutical industry and drug therapeutic development; however, their in vivo applications are sometimes limited due to fast degradation by proteases, poor solubility, antigenic responses, and glomerular filtration in the kidney. The covalent attachment of polyethylene glycol (PEG) chains to peptides is one approach that can reduce immunogenicity, improve solubility, and reduce renal clearance.
Hydrocarbon-stapled peptides are locked into their bioactive alpha-helical conformation through the site-specific introduction of a chemical brace, an all-hydrocarbon staple. The idea of peptide stapling was introduced to overcome the limitations of two broad classes of therapeutic agents (small molecules and protein biologics) in targeting intracellular protein-protein interactions. Small molecules only work on proteins with a specific surface feature, and most protein biologics do not penetrate cells. Because stapled peptides are locked into a stabilized α-helical structure (the most common element of protein secondary structures), they can easily penetrate cells.
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Mogford, Jon E., George E. Davis, Steven H. Platts, and Gerald A. Meininger. Circulation research 79, no. 4 (1996): 821-826.
Locmi-AKH-I, cycloAKH (cyclo[LNFTPNWG]
CPC Scientific is pleased to announce and welcome David A. Godkin as VP of Business Operations! David will be responsible for the growth and development of our HR department and facilities management division. He brings 20+ years of experience in human resources and facility operations from the peptide industry. We are confident David will have […]
Bekdemir, Ahmet, Eden EL Tanner, Jesse Kirkpatrick, Ava P. Soleimany, Samir Mitragotri, and Sangeeta N. Bhatia Advanced Healthcare Materials (2022): 2102685.
Peptides [TDN-qFAM, C-(PEG)2-K(CPQ2)-GGGSRPfGG-5FAM] were obtained from Tufts University Peptide Core Facility or CPC Scientific, Inc (USA)
