Double deficiency of cathepsins B and L results in massive secretome alterations and suggests a degradative cathepsin-MMP axis - PubMed (original) (raw)

Comparative Study

Double deficiency of cathepsins B and L results in massive secretome alterations and suggests a degradative cathepsin-MMP axis

Stefan Tholen et al. Cell Mol Life Sci. 2014 Mar.

Abstract

Endolysosomal cysteine cathepsins functionally cooperate. Cathepsin B (Ctsb) and L (Ctsl) double-knockout mice die 4 weeks after birth accompanied by (autophago-) lysosomal accumulations within neurons. Such accumulations are also observed in mouse embryonic fibroblasts (MEFs) deficient for Ctsb and Ctsl. Previous studies showed a strong impact of Ctsl on the MEF secretome. Here we show that Ctsb alone has only a mild influence on extracellular proteome composition. Protease cleavage sites dependent on Ctsb were identified by terminal amine isotopic labeling of substrates (TAILS), revealing a prominent yet mostly indirect impact on the extracellular proteolytic cleavages. To investigate the cooperation of Ctsb and Ctsl, we performed a quantitative secretome comparison of wild-type MEFs and Ctsb (-/-) Ctsl (-/-) MEFs. Deletion of both cathepsins led to drastic alterations in secretome composition, highlighting cooperative functionality. While many protein levels were decreased, immunodetection corroborated increased levels of matrix metalloproteinase (MMP)-2. Re-expression of Ctsl rescues MMP-2 abundance. Ctsl and to a much lesser extent Ctsb are able to degrade MMP-2 at acidic and neutral pH. Addition of active MMP-2 to the MEF secretome degrades proteins whose levels were also decreased by Ctsb and Ctsl double deficiency. These results suggest a degradative Ctsl-MMP-2 axis, resulting in increased MMP-2 levels upon cathepsin deficiency with subsequent degradation of secreted proteins such as collagen α-1 (I).

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1

Fig. 1

Ctsb- and Ctsl-deficient MEFs, and to a lesser extent Ctsb single and Ctsl single-deficient MEFs, show accumulations of enlarged acidic Lamp-1-positive vesicles. a Immunofluorescent staining of two wild-type MEF cell lines, two Ctsb-deficient cell lines, two Ctsl-deficient cell lines, and two Ctsb Ctsl double-deficient MEF cell lines with the lysosomal marker Lamp-1 (green). Nuclei are stained with Hoechst (blue). b Visualization of acidic vesicles with acridine orange staining

Fig. 2

Fig. 2

Protein identification and quantification for each biological replicate of the quantitative secretome: comparison of wild-type and Ctsb-deficient cell-conditioned media. a In both biological replicates 1,363 proteins were identified, whereas 199 and 355 proteins were found only in the first or the second replicate, respectively. b Distribution and geometric mean (horizontal bar) of fold change values (log2) of proteins from each replicate comparing the wild-type and Ctsb −_/_− MEF secretome. c Proteins with significantly altered protein abundance. These proteins are identified in both biological replicates, display an ASAPratio p value lower than 0.1 (indicated by arrow bars), show an alteration in abundance of more than 50 % (Fc < −0.58; Fc > 0.58) in both replicates (dark grey bars, replicate 1; light grey bars, replicate 2), and are annotated as secreted or extracellularly localized according to GO or Swissprot annotation

Fig. 3

Fig. 3

N-terminal peptides identified and quantified for each biological replicate in the TAILS experiment comparing wild-type and Ctsb-deficient cell-conditioned media. a Four hundred six peptides (naturally unmodified, chemically dimethylated N-termini) were identified in both biological replicates. b Global specificity pattern upon Ctsb ablation. Graphical presentation of the secretome specificity profile of all N-termini found in the 0–20 quantile and therefore decreased upon Ctsb deletion. Only N-termini annotated as secreted or extracellularly localized according to GO or Swissprot annotation were considered. Positional occurrences are shown as enrichment over natural abundance of murine amino acid abundances as derived from the International Protein Index [74]. TAILS identifies prime-site sequences of proteolytic cleavage sites. The corresponding non-prime sequences are derived bioinformatically by database searches similar to the PICS strategy for protease specificity characterization [52, 54]

Fig. 4

Fig. 4

a A total of 1,826 proteins were identified in both biological replicates of the quantitative proteome comparison of wild-type and Ctsb Ctsl-deficient cell-conditioned media. b Distribution and geometric mean (horizontal bar) of fold change values (log2) of proteins from each replicate comparing the wild-type and Ctsb −_/Ctsl/_− MEF secretome. c Overall connectivity of all extracellular and secreted proteins altered in the quantitative proteome comparison of wild-type and Ctsb Ctsl-deficient MEFs determined by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins). Different line colors represent the types of evidence for each association

Fig. 5

Fig. 5

Western blot analysis of cell-conditioned media of wild-type, Ctsb-deficient, Ctsl-deficient, and Ctsb −_/Ctsl/_− MEFs for collagen α-1 (I), MMP-2, N-cadherin, and periostin. Absence of Ctsb and/or Ctsl was controlled by detection of the Ctsl and Ctsb proform

Fig. 6

Fig. 6

a Western blot analysis of MMP-2 in cell-conditioned media of primary wild-type and Ctsl-deficient MEFs. b Western blot analysis of MMP-2 in cell-conditioned media of DMSO-treated wild-type MEFs and wild-type MEFs treated with 10 μM E64d for 48 h revealed an increase in MMP-2 protein levels upon cysteine cathepsin inhibition. DMSO served as solvent control. c Re-expression of Ctsl in Ctsl-deficient MEFs and overexpression in wild-type MEFs using the retroviral expression vector pMIG. Re-expression of Ctsl results in a decrease of MMP-2 protein levels. d CTSL cleaves human MMP-2 at 5.5 and 7.0. e CTSB cleaves human MMP-2 at 5.5 and 7.0, but with lower efficiency than CTSL. f Western blot analysis of cell-conditioned media of two wild-type MEF cell lines treated with activated pro-MMP-2 (125 ng/ml) compared to untreated cell-conditioned media. Pro-MMP-2 was activated by adding APMA for 3 h (details in “Materials and methods”). Collagen α-1 (I) protein levels decrease upon addition of activated pro-MMP-2 in both wild-type MEF cell lines. ON, overnight

Similar articles

Cited by

References

    1. Turk V, Turk B, Turk D. Lysosomal cysteine proteases: facts and opportunities. EMBO J. 2001;20(17):4629–4633. doi: 10.1093/emboj/20.17.4629. - DOI - PMC - PubMed
    1. Rawlings ND, Barrett AJ, Bateman A. MEROPS: the peptidase database. Nucleic Acids Res. 2010;38:D227–233. - PMC - PubMed
    1. Aronson NN, Jr, Barrett AJ. The specificity of cathepsin B. Hydrolysis of glucagon at the C-terminus by a peptidyldipeptidase mechanism. Biochem J. 1978;171(3):759–765. - PMC - PubMed
    1. Barbarin A, Frade R. Procathepsin L secretion, which triggers tumor progression, is regulated by Rab4A in human melanoma cells. Biochem J. 2011 - PubMed
    1. Joyce JA, Baruch A, Chehade K, Meyer-Morse N, Giraudo E, Tsai FY, Greenbaum DC, Hager JH, Bogyo M, Hanahan D. Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis. Cancer Cell. 2004;5(5):443–453. doi: 10.1016/S1535-6108(04)00111-4. - DOI - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources