Proteases and proteolysis in the lysosome

Bohley, Peter; Seglen, Per Ottar

doi:10.1007/bf01923508

Cited by 232 publications

(147 citation statements)

References 44 publications

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“…Lysosomes contain many cathepsins that are responsible for the degradation of proteins targeted to these vesicles (24). We previously demonstrated that the protease activity responsible for generating p40 was a cysteine protease (15).…”

Section: Resultsmentioning

confidence: 99%

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Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

Spizz¹,

Blackshear

1997

Journal of Biological Chemistry

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The importance of regulating the cellular concentrations of the myristoylated alanine-rich C kinase substrate (MARCKS), a major cellular substrate of protein kinase C, is indicated by the fact that mice lacking MARCKS exhibit gross abnormalities of central nervous system development and die shortly after birth. We previously identified a novel means of regulating cellular MARCKS concentrations that involved a specific proteolytic cleavage of the protein and implicated a cysteine protease in this process (Spizz, G., and Blackshear, P. J. (1996) J. Biol. Chem. 271, 553-562). Here we show that p40, the carboxyl-terminal fragment resulting from this cleavage of MARCKS, was associated with the mitochondrial/lysosomal pellet fraction of human diploid fibroblasts and that its generation in cells was sensitive to treatment with NH 4 Cl. These data suggest the involvement of lysosomes in the generation and/or stability of p40. The MARCKS-cleaving enzyme (MCE) activity was peripherally associated with a 10,000 ؋ g pellet fraction from bovine liver, and it co-purified with the activity and immunoreactivity of a lysosomal protease, cathepsin B. Cathepsin B catalyzed the generation of p40 from MARCKS in a cell-free system and behaved similarly to the MCE with respect to mutants of MARCKS previously shown to be poor substrates for the MCE. Treatment of fibroblasts with a cell-permeable, specific inhibitor of cathepsin B, CA074-Me, resulted in parallel time-and concentration-dependent inhibition of cathepsin B and MCE activity. Incubation of a synthetic MARCKS phosphorylation site domain peptide with purified cathepsin B resulted in cleavage of the peptide at sites consistent with preferred cathepsin B substrate sites. These data provide evidence for the identity of the MCE as cathepsin B and suggest that this cleavage most likely takes place within lysosomes, perhaps as a result of specific lysosomal targeting sequences within the MARCKS primary sequence. The data also suggest a direct interaction between MARCKS and cathepsin B in cells and leave open the possibility that MARCKS may in some way regulate the protease for which it is a substrate.The myristoylated alanine-rich C kinase substrate (MARCKS) 1 is a prominent cellular substrate for protein kinase C (PKC) (1, 2). Expression of this heat-stable, acidic protein is essential for life, as demonstrated by the perinatal death of mice that are completely deficient in MARCKS (3). Complete lack of expression leads to gross abnormalities of central nervous system development; however, heterozygous mice, which express MARCKS at 50% wild-type levels, appear to be normal.Cellular levels of MARCKS are regulated by both transcriptional and translational mechanisms (4 -14). In addition, we recently demonstrated that the cellular concentrations of MARCKS can also be regulated by a proteolytic event. This proteolytic cleavage results in amino-and carboxyl-terminal fragments of MARCKS that co-exist in cells with the full-length protein. This cleavage of MARCKS was inhibited in intact fi...

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Section: Resultsmentioning

confidence: 99%

“…Consistent with this identity is the MCE's high level expression in spleen, liver, and kidney (15,24,42), stable activity at physiological pH (43), and its inability to bind concanavalin A (17). In addition, purified cathepsin B could generate p40 from MARCKS in a cell-free system.…”

Section: Fig 3 Chromatographic Elution Profiles and Immunoblot Analmentioning

confidence: 86%

Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

Spizz¹,

Blackshear

1997

Journal of Biological Chemistry

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“…45 Autophagy Autophagy, is a lysosomal degradation pathway for cytoplasmic components (for a review, see Klionsky and Emr, 10 Eskelinen 17 and Theos et al 46 ). Unlike the ubiquitin-proteasome degradation system responsible for degradation of short-lived proteins, autophagy is involved in the turnover of long-lived proteins, cellular macromolecules and whole organelles, 13,[47][48][49][50] acting as an essential physiological system for cellular homeostasis. 51 It has also been demonstrated that autophagy has an important role in neurodegenerative diseases, aging, cancer, type-II programmed cell death, antigen presentation, early neonatal stages and bacterial invasion (for a review, see Tanida et al 11 ).…”

Section: Biogenesis Of Mvbsmentioning

confidence: 99%

Autophagy and multivesicular bodies: two closely related partners

2008

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In the majority of cell types, multivesicular bodies (MVBs) are a special kind of late endosomes, crucial intermediates in the internalization of nutrients, ligands and receptors through the endolysosomal system. ESCRT-0, I, II and III (endosomal sorting complex required for transport) are involved in the sorting of proteins into MVBs, generating the intraluminal vesicles. Autophagy is a lysosomal degradation pathway for cytoplasmic components such as proteins and organelles. The autophagosome, a well-characterized structure of the autophagy pathway, can fuse with endocytic structures such as MVBs to generate the amphisome. Finally, the amphisome fuses with the lysosome to degrade the material wrapped inside. Currently, clear evidence suggests that efficient autophagic degradation requires functional MVBs. This review highlights the most recent advances in our understanding of the molecular machinery that participates in MVB biogenesis and regulates the interplay between autophagy and this organelle.

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“…They are potent ubiquitously expressed endopeptidases with broad substrate specificity that work best at acidic pH and reducing redox conditions 8,9 . Cathepsin L-deficient (Ctsl À / À ) mice reproduce normally, although pups showed a slightly increased mortality during the weaning period 10 .…”

mentioning

confidence: 99%

Out-of-frame start codons prevent translation of truncated nucleo-cytosolic cathepsin L in vivo

et al. 2014

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The lysosomal protease cathepsin L has been reported to cleave various functionally important cytosolic or nuclear proteins. To explain nucleo-cytosolic localization of cathepsin L, it has been hypothesized that skipping of the first start codon during translation initiation results in an N-terminally truncated protein lacking the endoplasmic reticulum-import signal. Here we demonstrate that out-of-frame AUGs prevent translation of truncated cathepsin L in cell culture as well as in a new knock-in mouse model. We further evaluate potential roles of nuclear cathepsin L during early embryonic development. Our analysis reveals normal epiblast development of cathepsin L-deficient embryos, but uncovers a pronounced lysosomal storage phenotype in the extra-embryonic tissue of the visceral endoderm. In conclusion, the phenotypes of cathepsin L deficiency can be fully assigned to lack of canonically targeted cathepsin L, while the biogenesis and functionality of nucleo-cytosolic cathepsin L remain elusive.

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Proteases and proteolysis in the lysosome

Cited by 232 publications

References 44 publications

Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

Autophagy and multivesicular bodies: two closely related partners

Out-of-frame start codons prevent translation of truncated nucleo-cytosolic cathepsin L in vivo

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