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An extracellular proteasome releases endostatin from human collagen XVIII

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Abstract

Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.

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Acknowledgements

We thank Dr. Marilene Demasi and Dr. Claudio Masuda for helpful discussions and for the donation of reagents. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP—Project—12/50191-4R), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq—Projects—471340/2011-1 and 470388/2010-2).

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Authors and Affiliations

  1. Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

    Maria L. V. Reiss-Pistilli, Madalena M. S. Barroso & Tatiana Coelho-Sampaio

  2. Institute of Translational Immunology and Research Center for Immunotherapy (FZI), University Medical Center, Johannes Gutenberg-University, Mainz, Germany

    Detlef Schuppan & Michael Bauer

  3. Institute of Microbiology Paulo de Goes, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

    Iranaia Assunção-Miranda

  4. Department of Biophysics, Escola Paulista de Medicina, Federal University of São Paulo, São Paulo, SP, Brazil

    Shirley Farias, Luiz Juliano & Maria A. Juliano

  5. Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

    Letícia Lery

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  1. Maria L. V. Reiss-Pistilli
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Correspondence to Tatiana Coelho-Sampaio.

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Supplementary Figure 1

A protease isolated from human glioblastoma produces endostatin in cultures of human umbilical vein endothelial cells. A protein fraction was isolated from a primary culture of human glioblastoma exactly as done to produce F6 out of EOMA (see Materials and Methods). Such fraction (100 μl) was incubated with a confluent culture of HUVEC in a six-well plate (9.6 cm2) for 2 h at 37°C in growth medium (199 plus 20% FBS). Culture supernatant was analyzed in SDS-PAGE 12% followed by western blot using a polyclonal anti-endostatin antibody (Millipore, AB1878). A band of 20 kDa was detected, whereas such band was strongly reduced when the experiment was carried out in the presence of 10 μM AEBSF. (DOCX 95 kb)

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Reiss-Pistilli, M.L.V., Schuppan, D., Barroso, M.M.S. et al. An extracellular proteasome releases endostatin from human collagen XVIII. Angiogenesis 20, 125–137 (2017). https://doi.org/10.1007/s10456-016-9533-6

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