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Detection of Lysosomal Membrane Permeabilization
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Anne-Marie Ellegaard, Line Groth-Pedersen, Marja Jäättelä
Already in the 1970s, Christian de Duve initiated an intensive search for compounds that destabilize lysosomal membranes for the treatment of cancer. As a result, amines with long hydrophobic chains and high pK values were identified as lysosomotropic detergents with potential applications in cancer therapy (Firestone et al., 1979). One of the best-studied lysosomotropic detergents is L-leucyl-L-leucine methyl ester (LLOMe). It is endocytosed and first activated inside the lysosomes, where cathepsin C cleaves it to generate the active detergent that effectively permeabilizes the lysosomal membrane (Uchimoto et al., 1999). Due to the relatively low levels of cathepsin C in most cancer cells, LLOMe is not suitable for cancer therapy, whereas high cathepsin C levels in white blood cells has encouraged its development as a treatment for bone marrow transplantation-associated graft versus host disease (Charley et al., 1986). A recent report suggests, however, that LLOMe-induced cell death is not mediated by the leakage of active cathepsins but rather by the inactivation of cathepsins and subsequent loss of lysosomal function (Repnik et al., 2017).
Protease-Catalyzed Oligomerization
Published in Willi Kullmann, Enzymatic Peptide Synthesis, 1987
Protease-catalyzed oligomerization of natural or synthetic peptide monomers, not possessing classic plastein forming activity, was reported by Fruton and co-workers.912 These authors observed that cathepsin C., a thiol protease also known as “dipeptidyl aminotransferase”, which possesses a chymotrypsin-like specificity, catalyzed the oligomerization of the following dipeptide amides in the pH-range of 6.6 to 7.8: H-Gly-Phe-NH2, H-Gly-Tyr-NH2, H-Gly-Trp-NH2, H-Ala-Phe-NH2, H-Ala-Tyr-NH2, H-Gly-Tyr(CH3)-NH2, H-Lys(ϵ-Ac)-Phe-NH2. The sparingly soluble products were present mainly as octa- or decapeptide amides. However, oligomers were not formed either when amino acid amides, tri- or tetrapeptide amides were used as reactants, or when dipeptidyl amides were incubated at pH 5.2, under which conditions the proteolytic activity of cathepsin C is optimal. Successful chain propagation proceeded by stepwise addition, via consecutive transamidation reactions, of dipeptidyl units to the carboxyl end of the growing chain.13
Granzyme B as a therapeutic target for wound healing
Published in Expert Opinion on Therapeutic Targets, 2019
Christopher T. Turner, Sho Hiroyasu, David J. Granville
The human granzyme B protein is a 247 amino acid polypeptide consisting of two 6-stranded β sheets and 3 trans-domain segments [13]. The granzyme B gene (GZMB), located on chromosome 14q.11.2,5, is approximately 3,500 base pairs long and contains five exons and four introns [14]. Immune and non-immune cells express granzyme B, with this occurring at both the transcriptional and translational level, regulated by the same factors responsible for immune cell activation [15]. Synthesized with a signal sequence that facilitates traffic to the endoplasmic reticulum, granzyme B is then cleaved to produce an inactive pro-enzyme. Once in the Golgi, granzyme B is tagged with mannose-6-phosphate (M6P), which when bound to the M6P receptor, is targeted to the acidified lytic granule [16]. Cathepsin C then activates granzyme B by removing the N-terminal di-peptide, which becomes stored on a serglycin proteoglycan scaffold [17]. Within the acidic granule the proteolytic activity of granzyme B remains low due to having a neutral pH optima. However, when secreted, the 32 kDa granzyme B becomes activated, predominantly cleaving peptides adjacent to aspartate (Asp) residues at the P1 position [18]. The active site contains an arginine (Arg) residue positioned adjacent to the active site pocket, providing granzyme B with its substrate specificity [18].
Update on: proteome analysis in thyroid pathology – part II: overview of technical and clinical enhancement of proteomic investigation of the thyroid lesions
Published in Expert Review of Proteomics, 2018
Isabella Piga, Stefano Casano, Andrew Smith, Silvia Tettamanti, Davide Leni, Giulia Capitoli, Angela Ida Pincelli, Marcella Scardilli, Stefania Galimberti, Fulvio Magni, Fabio Pagni
FNA samples from a wider cohort of patients (more than 200) with follicular, medullary and papillary carcinoma were investigated by Ciregia et al. by 2DE and nano-liquid chromatography electrospray ionization tandem mass spectrometry (NanoLC-ESI-MS/MS) [42]. They highlighted 25 proteins with altered expression and 8 of them (ANXA1, MSN, CRNN, ENO1, MDH, LDH, DJ1, and SOD) were validated in FNA specimens by ELISA and WB. In particular, Annexin A1 (ANXA1) and Cornulin (CRNN) correlated well with clinical parameters (presurgical thyroid volume) of patients. ANXA1 is involved in inflammatory reactions, cell proliferation and promotes apoptosis whilst CRNN is a calcium-binding protein present in the upper layer of squamous epithelia and it has been shown to play a role in the clonogenicity of squamous esophageal epithelium cell lines, attenuating deoxycholic acid (DCA)-induced apoptotic cell death. Recently, the group of Wu CC et al. [39] investigated FNAs specimens after immunodepletion of the 14 most abundant serum proteins by GeLC-MS/MS to identify new markers for PTC. They considered seven benign lesions, five PTC samples and the secretome of two PTC cell lines (BHP 7–13 and CGTHW3). Higher tumor tissue expressions of two proteins, agrin (AGRN) and cathepsin C (CTSC), were observed to be significantly correlated with lymph node metastasis, distant metastasis and poor prognosis of PTC patients. AGRN could be involved in the tumor vascularization [43] whilst CTSC appears to have a role in various tumorigenic processes such as angiogenesis, proliferation, apoptosis and invasion.
Epithelial damage in the cystic fibrosis lung: the role of host and microbial factors
Published in Expert Review of Respiratory Medicine, 2022
Arlene M. A. Glasgow, Catherine M. Greene
Cysteine cathepsins are a group of cysteine proteases that are located in lysosomes and work most effectively at acidic pH, with the exception of cathepsin S and cathepsin C which can function at neutral to alkaline pH [91]. Although they have many intracellular functions such as lysosomal degradation of pathogen proteins and antigen presentation, they can also be released extracellularly from a range of cell types including macrophages, neutrophils, mast cells, fibroblasts, and lung epithelial and endothelial cells. Here, they can potently degrade ECM proteins such as elastin, collagen, fibronectin, and laminin [91].