"Protease activity in regurgitant from three replicate tanks were compared using a mixture of 7 internally quenched fluorescent substrates available from CPC Scientific, Sunnyvale, California (Table 1) [AMYD-112, AMYD-114, AMYD-109, MMPS-024, SUBS-017, AMYD-111, CASP-060]. These substrates were chosen based on their diverse sequence composition to enable detection of multiple protease classes."
Abstract
Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites.
SOCIAL MEDIA
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Diao, J., Komura, R., Sano, T., Pantua, H., Storek, K.M., Inaba, H., Ogawa, H., Noland, C.L., Peng, Y., Gloor, S.L. and Yan, D. bioRxiv (2020): 2020-10.
- Departments of Infectious Diseases, Structural Biology, Biochemical and Cellular Pharmacology, Translational Immunology, Pathology, and Early Discovery Biochemistry, Genentech, South San Francisco, CA 94080 USA
The standard assay consists of 6 μL reaction mixture with 3 nM Lgt-DDM, 50 μM phosphatidylglycerol (1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-rac-glycerol), Avanti), 12.5 μM Pal-IAAC peptide substrate derived from the Pal lipoprotein (MQLNKVLKGLMIALPVMAIAACSSNKN, synthesized by CPC Scientific)
Pantua, H., Skippington, E., Braun, M.G., Noland, C.L., Diao, J., Peng, Y., Gloor, S.L., Yan, D., Kang, J., Katakam, A.K. and Reeder, J. MBio 11, no. 5 (2020): 10-1128.
- Departments of Infectious Diseases, OMNI Bioinformatics, Chemistry, Structural Biology, Biochemical and Cellular Pharmacology, Translational Immunology, Pathology, Molecular Biology, Genentech, South San Francisco, California, USA
MQLNKV-L(U13C6,15N)-KGL(U13C6,15N)MIALPVMAIAA-dipalmitoyl2C-SSNKNGG-K-biotin, which upon cleavage by LspA, yields the product peptide dipalmitoyl2C-SSNKNGG-K-biotin. A product standard [dipalmitoyl2C-SSNKNAAK-(NHCH2CH2NH)-biotin; CPC Scientific] was in the reaction mixture as an internal standard for normalization of product quantitation.
Protein-protein and protein-peptide interactions play critical roles in all types of cellular processing. Peptides are natural partners to proteins and, as ligands, bind to proteins with high affinity due to their capacity to adapt to the often flexible protein surface. Despite this, peptides have drawbacks as drug candidates that include low plasma bioavailability, instability from proteolytic enzymes, and poor passive membrane permeability. Some success has been achieved with linear peptides, particularly peptides that maintain α-helical secondary structures. These motifs can be introduced to stabilized α-helical motifs by common “peptide-stapling” approaches, but stapled peptides can suffer from low bioactivity and poor solubility. Another strategy to maintain peptide secondary structure is modification by macrocyclization.
SAN JOSE, CA., Sept 3, 2020 /CPCNewswire/ — CPC Scientific Inc. is pleased to announce that the European Commission (EC) has granted the Conditional Marketing Authorization (CMA) for MYR Pharmaceuticals lead compound HEPCLUDEX®. CPC Scientific serves as a supplier and partner to MYR Pharmaceuticals for the development and manufacturing of bulevirtide (Hepcludex). This drug is […]
Curreli, Francesca, Sofia MB Victor, Shahad Ahmed, Aleksandra Drelich, Xiaohe Tong, Chien-Te K. Tseng, Christopher D. Hillyer, and Asim K. Debnath. Mbio 11, no. 6 (2020): e02451-20.
We have synthesized (CPC Scientific, Inc.) four stapled peptides, as depicted in Figure 2. We also synthesized the linear peptide, NYBSP-C, as a control. Besides, we purchased a linear peptide, SBP1, to use as a control, which was reported recently to bind to SARS-CoV-2 RBD with high affinity (KD = 47nM).
Abdulganiyyu, I. A.; Kaczmarek, K.; Zabrocki, J.; Nachman, R. J.; Marchal, E.; Schellens, S.; Verlinden, H.; Broeck, J. V.; Marco, H.; Jackson, G. E. Insect Biochemistry and Molecular Biology 2020, 103362.
A head-to-tail cyclic, octapeptide analog of Locmi-AKH-I, cycloAKH (cyclo[LNFTPNWG]) was synthesized to severely restrict the conformational freedom of the AKH structure.
Clairfeuille, T., Buchholz, K.R., Li, Q., Verschueren, E., Liu, P., Sangaraju, D., Park, S., Noland, C.L., Storek, K.M., Nickerson, N.N. and Martin, L. Nature 584, no. 7821 (2020): 479-483.
- Departments of Structural Biology, Infectious Diseases, Microchemistry, Proteomics & Lipidomics, Drug Metabolism & Pharmacokinetics, Translational Immunology, BioMolecular Resources, Biochemical & Cellular Pharmacology, Bioinformatics & Computational Biology, Discovery Chemistry Departments, Genentech Inc., South San Francisco, CA, USA.
LAB peptides ([..], CPC Scientific, [..] l, standard solid-phase peptide synthesis) at 10 mM in 50 mM Tris, pH 8, and 100 mM NaCl were diluted in MHB II cation adjusted broth (800 μM top concentration) or LB. Where indicated, EDTA was added to a final concentration of 0.5 mM.
Chan, Leslie W., Melodi N. Anahtar, Ta-Hsuan Ong, Kelsey E. Hern, Roderick R. Kunz, and Sangeeta N. Bhatia. Nature Nanotechnology (2020): 1-9.
HFA-modifed peptides were synthesized by CPC Scientifc (>95% purity). Briefy, the peptide substrate, Ac-CKKK(Cy5)-PEG4-Nle(O-Bzl)-Met(O)2-Oic-Abu-OH, was synthesized on Fmoc-Abu-CTC resin via standard Fmoc solid phase peptide synthesis.