9 generic fluorogenic substrates (CPC Scientific) (Figure S4) were added to the final concentration of 20 μM. Fluorescence was measured at Excitation/Emission=330/390 nm on BioTek Synergy H1 microplate reader and proteolytic activity was calculated as a change in relative fluorescence units per sec using the slope of the linear range for this signal.
Abstract
Malassezia form the dominant eukaryotic microbial community on the human skin. The Malassezia genus possesses a repertoire of secretory hydrolytic enzymes involved in protein and lipid metabolism which alter the external cutaneous environment. The exact role of most Malassezia secreted enzymes, including those in interaction with the epithelial surface, are not well characterized. In this study, we explored the functional roles of Malassezia aspartyl proteases in skin health, we generated a knockout mutant of the predominant aspartyl protease in the genetically tractable Malassezia furfur. We observed the loss of MFSAP1 resulted in dramatic changes in the cell adhesion and dispersal in both culture and a human 3D reconstituted epidermis model. In a murine model of Malassezia colonization, we further demonstrated Mfsap1 contributes to inflammation as observed by reduced edema and inflammatory cell infiltration with the knockout mutant versus wildtype. Taken together, we show that this dominant secretory Malassezia aspartyl protease has an important role in enabling a planktonic cellular state that can potentially aid in colonization and additionally as a virulence factor in barrier-compromised skin, highlighting the importance of considering the contextual relevance when evaluating the functions of secreted microbial enzymes.
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Hao, Liangliang, Renee T. Zhao, Nicole L. Welch, Edward Kah Wei Tan, Qian Zhong, Nour Saida Harzallah, Chayanon Ngambenjawong et al. Nature Nanotechnology (2023): 1-10.
All peptides and oligonucleotides were synthesized and HPLC purified by CPC Scientific and Integrated DNA Technologies (IDT), respectively. Peptide–oligonucleotide conjugates were generated by copper-free click chemistry.
Kikuchi, F., Ikeda, Z., Kakegawa, K., Nishikawa, Y., Sasaki, S., Fukuda, K., Takami, K., Banno, Y., Nishikawa, H., Taya, N. and Nakahata, T. Bioorganic & Medicinal Chemistry 93 (2023): 117462.
- Research, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
- Pharmaceutical Sciences, Takeda Pharmaceutical Company Ltd., 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
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 5.4 μM substrate solution.
Senotherapeutic peptide treatment reduces biological age and senescence burden in human skin models.
Zonari, A.; Brace, L. E.; Al-Katib, K.; Porto, W. F.; Foyt, D.; Guiang, M.; Cruz, E. A. O.; Marshall, B.; Gentz, M.; Guimaraes, G. R.; Franco, O. L.; Oliveira, C. R.; Boroni, M.; Carvalho, J. L., NPJ Aging 2023, 9 (1), 10.
The top hit peptides selected (Pep 14, 144, 156, 195, and 393) from the screening and the fluorescence labeled peptide (5FAM-PEG2-Pep 14) were purchased from CPC Scientific Inc. (USA), which synthesized the peptide by solid phase (Fmoc) on a Rink amide resin, with >95% purity, in the form of acetate salt.
Synthetic oligonucleotides constitute an important class of therapeutics developed to treat a variety of indications. Two main synthetic approaches exist for the conjugation of a peptide to an oligonucleotide: parallel and linear. The primary benefit of the linear approach is the one-pot solid-phase assembly and compatibility with machine automation. However, in cases where poor compatibility of peptide and oligo chemistries exist or long peptide and oligo fragments are required, preparing both components separately and linking both compounds together may offer the simplest solution.
Solid-phase peptide synthesis (SPPS) approaches require that the side chains of certain amino acids be protected from undesired reactivity during synthesis. The installation and removal of these protection groups results in a lower atom economy in the production process. Removal of the protection groups often requires large volumes of trifluoroacetic acid (TFA) or other strong acids which can result in lower yields and pose a significant risk to the environment.
Schiemer, J., Maxwell, A., Horst, R., Liu, S., Uccello, D.P., Borzilleri, K., Rajamohan, N., Brown, M.F. and Calabrese, M.F. Nature Communications 14, no. 1 (2023): 1189.
- Discovery Sciences, Pfizer Worldwide Research and Development, Groton, CT, USA
After 3 h the resin was washed with binding buffer until no protein was detected, followed by elution with 0.2 mg ml−1 FLAG peptide DYKDDDDK (CPC Scientific Peptide company)
Järveläinen, Harri A., Christian Schmithals, Maike von Harten, Bianca Kakoschky, Thomas J. Vogl, Stephen Harris, Claire Henson, Gemma Bullen-Clerkson, and Albrecht Piiper. International Journal of Molecular Sciences 24, no. 6 (2023): 5700.
The cyclic iRGD/CEND-1 peptide [sequence: CRGDKGPDC] and RGD control peptide (CRGDDGPKC) for pre-clinical use was sourced either from GenScript (Piscataway, NJ, USA) or CPC Scientific (Hangzhou, China).
Lin, Wilson, Eduardo Aluicio-Sarduy, Hailey A. Houson, Todd E. Barnhart, Volkan Tekin, Justin J. Jeffery, Ashley M. Weichmann, Kendall E. Barrett, Suzanne E. Lapi, and Jonathan W. Engle. Nuclear Medicine and Biology) 118 (2023): 108329.
Lyophilized NOTA-NT-20.3 (Ac-Lys(NOTA)-Pro-NMeArg-Arg-Pro-Tyr-Tle-Leu, 1383.6 g/mol, “NT”, CPC Scientific) was diluted to 1 mg/mL in ultrapure water (Milli-Q, >18.2 MΩ-cm) and used without further purification.