Dynamic changes in immunoproteasome assembly and capping with regulatory particles in an invitro model of senescing macrophages

During aging the immune system undergoes a condition leading to a general decline of its functions, called immunosenescence. Molecular mechanisms of cellular senescence in post mitotic immune cells such as macrophages have been poorly investigated. We have developed an in vitro model of macrophage senescence which involves long-term culturing of monocyte-derived human macrophages isolated from buffy coats. After extended culture, macrophages expressed multiple senescent markers: i.e., irreversible cell cycle arrest with increased senescence-associated β-galactosidase activity and p21 and p16 levels, lipofuscin accumulation and enlarged flattened shape. In this model we studied how components of the proteasome network may change during macrophage senescence. We found an alteration in 20S core subunit composition and regulatory particle association: β5i immunoproteasome subunit levels decreased while an increased content of the β5 constitutive counterpart was observed. Up-regulation of β5 subunit occurred at the mRNA and protein levels and was accompanied by activation of transcription factor Nrf1. Native gel analysis of proteasome complexes indicated that the i20S, which is typically capped with the 11S regulator in mature macrophages, decreased over time. Meanwhile increased incorporation of β5 subunit into i20S and capping of the catalytic core particle with the 19S regulator (26S proteasome) was observed. Overall proteasome activity decreased in senescent macrophages and the residual activity was mainly associated to the 26S proteasome. Changes in proteasome/immunoproteasome composition and capping further shade light into the molecular mechanisms involved in immunosenescence which may be relevant to understand macrophage dysfunction in aging.