The Human CLN2 Protein/Tripeptidyl-Peptidase I Is a Serine Protease That Autoactivates at Acidic pH

Lin, Lan; Sohár, István; Lackland, Henry; Lobel, Peter

doi:10.1074/jbc.m008562200

Cited by 131 publications

(149 citation statements)

References 27 publications

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“…2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate was from Applied Biosystems. Recombinant human TPP I proenzyme containing a C-terminal hexahistidine tag was expressed and purified as described (7). All peptide synthesis and HPLC solvents were purchased from Aldrich.…”

Section: Methodsmentioning

confidence: 99%

“…Fluorescence-based Enzyme Assay-The conditions for the enzyme kinetic assay were described previously (7,13). Briefly, purified proenzyme of TPP I (1 mg/ml, 16.6 M) was 10-fold diluted into 0.1 M sodium formate buffer, 0.15 M NaCl, 0.1% Triton X-100, pH 3.5, and incubated overnight at room temperature to allow for complete activation.…”

Section: Methodsmentioning

confidence: 99%

“…This enzyme is a serine protease (7) that, based on the structure of the bacterial homologs, has an unusual Ser, Asp, and Glu catalytic triad, allowing activity at acidic pH (8,9). TPP I has two proteolytic activities, a weak endopeptidase activity with a pH optimum of 3.0 (10) that may be important for the low pH-triggered intramolecular autoactivation and autoprocessing of the 66-kDa inactive proenzyme to the 46-kDa mature enzyme (7,11), and a stronger exopeptidase activity with a pH optimum of 4.5. The exopeptidase activity of TPP I enables the cleavage of tripeptides sequentially from unsubstituted N termini of polypeptides or proteins.…”

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confidence: 99%

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Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates

Tian¹,

Sohár²,

Taylor

et al. 2006

Journal of Biological Chemistry

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Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant k cat /K M . In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1 and P2 residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates

Tian¹,

Sohár²,

Taylor

et al. 2006

Journal of Biological Chemistry

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“…16,[18][19][20] Third, a significant fraction of newly synthesized TPP-I protein is secreted as pro-TPP-I, a 563 amino-acid inactive form which can crosscorrect nearby nontransduced cells through mannose 6-phosphate receptor-mediated uptake and subsequent activation in lysosomes to the 367 amino-acid mature form. 21,22 This cross-correction between transduced cells and neighboring cells suggests that it will not be necessary to transfer the normal CLN2 cDNA to all the cells in the CNS, thus extending the effective range of gene transfer to broader regions of the brain. 17 Finally, comparisons of genotype and phenotype of children with CLN2 mutations reveal that expression as low as 5% of normal TPP-I levels lead to a much milder form of the disease with a delayed age of onset, less severe symptoms, and a prolonged lifespan, suggesting that this level of enzyme activity is a reasonable target for therapy.…”

Section: Introductionmentioning

confidence: 99%

AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL

et al. 2005

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“…3 This is, to our knowledge, the first report documenting a possible implication of TPP1 in TNF-induced apoptosis. Human TPP1 is a lysosomal serine protease with an acidic pH optimum that cleaves off tripeptides from the free N termini of small polypeptides (68,69). However, its natural substrates are unknown.…”

Section: Discussionmentioning

confidence: 99%

Mannose 6-Phosphorylated Proteins Are Required for Tumor Necrosis Factor-induced Apoptosis

Tardy¹,

Autefage

Garcia

et al. 2004

Journal of Biological Chemistry

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Whereas caspases are essential components in apoptosis, other proteases seem to be involved in programmed cell death. This study investigated the role of lysosomal mannose 6-phosphorylated proteins in tumor necrosis factor (TNF)-induced apoptosis. We report that fibroblasts isolated from patients affected with inclusion-cell disease (ICD), having a deficient activity of almost all lysosomal hydrolases, are resistant to the toxic effect of TNF. These mutant cells exhibited a defect in TNF-induced caspase activation, Bid cleavage, and release of cytochrome c. In contrast, TNF-induced p42/ p44 MAPK activation and CD54 expression remained unaltered. Human ICD lymphoblasts and fibroblasts derived from mice nullizygous for Igf2 and the two mannose 6-phosphate (M6P) receptors, Mpr300 and Mpr46, which develop an ICD-like phenotype, were also resistant to CD95 ligand and TNF, respectively. Moreover, correction of the lysosomal enzyme defect of ICD fibroblasts, using a medium enriched in M6P-containing proteins, enabled restoration of sensitivity to TNF. This effect was blocked by exogenous M6P but not by cathepsin B or L inhibitors. Altogether, these findings suggest that some M6P-bearing glycoproteins modulate the susceptibility to TNF-induced apoptosis. As a matter of fact, exogenous tripeptidyl peptidase 1, a lysosomal carboxypeptidase, could sensitize ICD fibroblasts to TNF. These observations highlight the hitherto unrecognized role of some mannose 6-phosphorylated proteins such as tripeptidyl peptidase 1 in the apoptotic cascade triggered by TNF.

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The Human CLN2 Protein/Tripeptidyl-Peptidase I Is a Serine Protease That Autoactivates at Acidic pH

Cited by 131 publications

References 27 publications

Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates

Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates

AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL

Mannose 6-Phosphorylated Proteins Are Required for Tumor Necrosis Factor-induced Apoptosis

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