Abstract: The proteasome is comprised of multiple subunits that catalyze the degradation of proteins to maintain cellular homeostasis. The proteasome targets protein substrates by two different pathways. The ubiquitin‐dependent pathway requires proteins to be labeled with a ubiquitin tag to signal for degradation by the 26S isoform of the proteasome. Protein degradation through this pathway declines during age progression. The ubiquitin‐independent pathway utilizes the 20S proteasome isoform. It can degrade misfolded and intrinsically disordered proteins to decrease cellular stress. Age‐related protein accumulation and aggregation can occur due to the decreased activity and expression of the proteasome. Protein accumulation causes increased cellular stress which can contribute to disease progression. Increasing proteasome activity could serve as a solution to eliminating and preventing protein accumulation. Studies have shown the value of the proteasome as a therapeutic entity to mitigate cellular stress. This perspective explores the link between proteasome activity and cellular stress caused by age‐related misfolded protein accumulation.

Protein homeostasis is a tightly conserved process that is regulated through the ubiquitin proteasome system (UPS) in a ubiquitin-independent or ubiquitin-dependent manner. Over the past two decades, the proteasome has become an excellent therapeutic target through inhibition of the catalytic core particle, inhibition of subunits responsible for recognizing and binding ubiquitinated proteins, and more recently, through targeted protein degradation using proteolysis targeting chimeras (PROTACs). The majority of the developed inhibitors of the proteasome's core particle rely on gaining selectivity through binding interactions within the unprimed substrate channel. Although this has allowed for selective inhibitors and chemical probes to be generated for the different proteasome isoforms, much remains unknown about the interactions that could be harnessed within the primed substrate channel to increase potency or selectivity. Herein, we discuss small molecules that interact with the primed substrate pocket and how their differences may give rise to altered activity. Taking advantage of additional interactions with the primed substrate pocket of the proteasome could allow for the generation of improved chemical tools for perturbing or monitoring proteasome activity.

The immunoproteasome (iCP) can be expressed under inflammatory conditions, such as exposure to interferon-gamma (IFN-γ), that alerts the cell to begin generating iCP preferentially over the standard proteasome (sCP). With the iCP becoming a widely targeted isoform in a variety of diseases, there is a need to understand its activity and expression in cells and in vivo. Activity-based probes for the iCP have been developed but their application has been limited due to their difficult synthesis and cannot be used in tissues or whole animals. Our lab has previously demonstrated we can monitor iCP activity using a 4-mer peptide linked to a fluorophore and a peptoid. This was utilized in the development of the first cell-permeable iCP activity-based probe that did not include a covalent reactive moiety. Here, we demonstrate that this same peptide recognition sequence can be appended to aminoluciferin, caging it, until its interaction with the iCP. This probe should be applicable to monitor iCP activity in animal models where tumor or other tissue has been engineered to produce luciferase. We anticipate it could also be applied to observe iCP activity as tumors are formed in vivo.