Effects of impaired proteostasis on tissue physical properties in a model of bone-like tissue formation

Abstract

Cellular function and viability and thus tissue health and integrity depend on the maintenance of protein homeostasis (proteostasis) by controlled protein degradation. Prolonged dysregulation of proteostasis is associated with age-related diseases such as neurodegenerative disorders, diabetes, and cancer. However, as chronic imbalances rather than acute loss of proteostasis mediate the pathogenesis of these diseases, research models that allow for the study of the complex effects of drugs on tissue properties are almost completely lacking. The question of how impaired proteostasis affects tissues rather than single cells is also of relevance for proteostasis-targeting drugs, which are increasingly used in cancer therapies.

In this project I aimed to determine the functional effects of impaired proteostatic fine-tuning by applying a combination of materials science characterisation techniques to a cell-derived, in vitro model of bone-like tissue formation in which I pharmacologically perturbed protein degradation. I demonstrate that low-level inhibition of VCP/p97 and the proteasome, two major components of the protein degradation machinery, have remarkably different effects on the bone-like material that human bone-marrow derived mesenchymal stromal cells (hMSC) form in vitro. Specifically, whilst proteasome inhibition mildly enhances tissue formation, Raman spectroscopic, atomic force microscopy-based indentation, and electron microscopy imaging reveal that VCP/p97 inhibition induces the formation of bone-like tissue that is softer, contains less protein, appears to have more crystalline mineral, and may involve aberrant micro- and ultra-structural tissue organisation. These observations contrast with findings from conventional osteogenic assays that fail to identify any effect on mineralisation. Taken together, these data suggest that mild proteostatic impairment in hMSC alters the bone-like material they form in ways that could explain some pathologies associated with VCP/p97-related diseases. They also demonstrate the value of quantitative materials science approaches for tackling long-standing questions in biology and medicine and could form the basis for preclinical drug testing platforms to develop therapies for diseases stemming from perturbed proteostasis or for cancer therapies targeting protein degradation. The findings may also have important implications for the field of tissue engineering, as the manufacture of cell-derived biomaterial scaffolds may need to consider proteostasis to effectively replicate native tissues.