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Title: Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice

Abstract

Cathepsin B (CTSB) and cathepsin L (CTSL) are two widelyexpressed cysteine proteases thought to predominantly reside withinlysosomes. Functional analysis of CTSL in humans is complicated by theexistence of two CTSL-like homologues (CTSL and CTSL2), in contrast tomice which contain only one CTSL enzyme. Thus transgenic expression ofhuman CTSL in CTSL deficient mice provides an opportunity to study the invivo functions of this human protease without interference by its highlyrelated homologue. While mice with single gene deficiencies for murineCTSB or CTSL survive without apparent neuromuscular impairment, murineCTSB/CTSL double deficient mice display degeneration of cerebellarPurkinje cells and neurons of the cerebral cortex, resulting in severehypotrophy, motility defects, and lethality during their third to fourthweek of life. Here we show that expression of human CTSL through agenomic transgene results in widespread expression of human CTSL in themouse which is capable of rescuing the lethality found in CTSB/CTSLdouble-deficient animals. Human CTSL is expressed in the brain of thesecompound mutants predominantly in neurons of the cerebral cortex and inPurkinje cells of the cerebellum, where it appears to prevent neuronalcell death.

Authors:
; ; ;
Publication Date:
Research Org.:
COLLABORATION - Institut fur Molekulare Medizinund Zellforschung, Albert-Ludwigs-Universitat Freiburg,Germany
OSTI Identifier:
927243
Report Number(s):
LBNL-59312
R&D Project: GHPG4B; TRN: US200811%%65
DOE Contract Number:
DE-AC02-05CH11231
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biological Chemistry; Journal Volume: 387; Journal Issue: 7; Related Information: Journal Publication Date: 07/2006
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ANIMALS; BRAIN; CATHEPSINS; CEREBELLUM; CEREBRAL CORTEX; CYSTEINE; DEATH; DEFECTS; FUNCTIONAL ANALYSIS; GENES; IN VIVO; LYSOSOMES; MICE; MUTANTS; NERVE CELLS; Cathepsin, Neurodegeneration, Purkinje cell

Citation Formats

Sevenich, Lisa, Pennacchio, Len A., Peters, Christoph, and Reinheckel, Thomas. Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice. United States: N. p., 2006. Web. doi:10.1515/BC.2006.112.
Sevenich, Lisa, Pennacchio, Len A., Peters, Christoph, & Reinheckel, Thomas. Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice. United States. doi:10.1515/BC.2006.112.
Sevenich, Lisa, Pennacchio, Len A., Peters, Christoph, and Reinheckel, Thomas. Mon . "Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice". United States. doi:10.1515/BC.2006.112. https://www.osti.gov/servlets/purl/927243.
@article{osti_927243,
title = {Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice},
author = {Sevenich, Lisa and Pennacchio, Len A. and Peters, Christoph and Reinheckel, Thomas},
abstractNote = {Cathepsin B (CTSB) and cathepsin L (CTSL) are two widelyexpressed cysteine proteases thought to predominantly reside withinlysosomes. Functional analysis of CTSL in humans is complicated by theexistence of two CTSL-like homologues (CTSL and CTSL2), in contrast tomice which contain only one CTSL enzyme. Thus transgenic expression ofhuman CTSL in CTSL deficient mice provides an opportunity to study the invivo functions of this human protease without interference by its highlyrelated homologue. While mice with single gene deficiencies for murineCTSB or CTSL survive without apparent neuromuscular impairment, murineCTSB/CTSL double deficient mice display degeneration of cerebellarPurkinje cells and neurons of the cerebral cortex, resulting in severehypotrophy, motility defects, and lethality during their third to fourthweek of life. Here we show that expression of human CTSL through agenomic transgene results in widespread expression of human CTSL in themouse which is capable of rescuing the lethality found in CTSB/CTSLdouble-deficient animals. Human CTSL is expressed in the brain of thesecompound mutants predominantly in neurons of the cerebral cortex and inPurkinje cells of the cerebellum, where it appears to prevent neuronalcell death.},
doi = {10.1515/BC.2006.112},
journal = {Biological Chemistry},
number = 7,
volume = 387,
place = {United States},
year = {Mon Jan 09 00:00:00 EST 2006},
month = {Mon Jan 09 00:00:00 EST 2006}
}
  • We have isolated a human genomic DNA cosmid clone while screening for the cathepsin L gene that, when sequenced, revealed close similarity with but significant differences from cDNA sequences that have been reported for cathepsin L (CTSL). The clone bears a novel sequence that shows 88% identity to the coding regions of the cathepsin L gene and a similar exon arrangement. We have called this sequence the {open_quotes}human cathepsin L-like gene 1{close_quotes} (CTSLL1). Translating putative exon sequences reveals a single premature stop codon; therefore no functional products are likely to arise from this gene. Fluorescence in situ hybridization (FISH) studiesmore » mapped the clone to chromosome 10q. Somatic cell hybrid mapping confirmed the location of CTSLL1 to human chromosome 10 distinct from the cathepsin L locus (CTSL) on chromosome 9. Furthermore, the FISH mapping studies show that a family of at least three related sequences exists on chromosome 10q, similar to the pattern of duplicated glutamate dehydrogenase (GLUD) gene loci reported on 10q. Using PCR and sequencing with genomic DNA samples, we have identified two additional novel related sequences (CTSLL2 and CTSLL3), and by PCR analysis of cDNA samples we have identified corresponding transcripts. Comparison of changes between our CTSLL1 sequence and the cathepsin L gene at mutation insensitive sites suggests that the two sequences arose from a duplication event 40-50 million years ago, and therefore at the time of divergence of early primates. 41 refs., 9 figs., 1 tab.« less
  • No abstract prepared.
  • Cathepsin L plays a key role in many pathophysiological conditions including rheumatoid arthritis, tumor invasion and metastasis, bone resorption and remodeling. Here we report the crystal structures of two analogous dipeptidyl inhibitor complexes which inhibit human cathepsin L in reversible and irreversible modes, respectively. To-date, there are no crystal structure reports of complexes of proteases with their glyoxal inhibitors or complexes of cathepsin L and their diazomethylketone inhibitors. These two inhibitors - inhibitor 1, an {alpha}-keto-{beta}-aldehyde and inhibitor 2, a diazomethylketone, have different groups in the S1 subsite. Inhibitor 1 [Z-Phe-Tyr (OBut)-COCHO], with a Ki of 0.6 nM, is themore » most potent, reversible, synthetic peptidyl inhibitor of cathepsin L reported to-date. The structure of the inhibitor 1 complex was refined up to 2.2 {angstrom} resolution. The structure of the complex of the inhibitor 2 [Z-Phe-Tyr (t-Bu)-diazomethylketone], an irreversible inhibitor that can inactivate cathepsin L at {micro}M concentrations, was refined up to 1.76 {angstrom} resolution. These two inhibitors have substrate-like interactions with the active site cysteine (Cys25). Inhibitor 1 forms a tetrahedral hemithioacetal adduct, whereas the inhibitor 2 forms a thioester with Cys25. The inhibitor 1 {beta}-aldehyde group is shown to make a hydrogen bond with catalytic His163, whereas the ketone carbonyl oxygen of the inhibitor 2 interacts with the oxyanion hole. tert-Butyl groups of both inhibitors are found to make several non-polar contacts with S' subsite residues of cathepsin L. These studies, combined with other complex structures of cathepsin L, reveal the structural basis for their potency and selectivity.« less
  • No abstract prepared.
  • The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose–response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed tomore » their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. - Highlights: • z-FA-CMK is toxic and induce cell death in the human T cells. • z-FA-CMK toxicity requires the CMK group, alanine and the benzyloxycarbonyl group. • z-FA-CMK induced apoptosis at low concentration and necrosis at high concentration.« less