Effector-mediated subversion of proteasome activator (PA)28αβ enhances lysosomal targeting of Legionella pneumophila within cytokine-activated macrophages

Legionella pneumophila causes Legionnaires' Disease via replication within host macrophages using an arsenal of hundreds of translocated virulence factors termed effector proteins. Effectors are critical for intracellular replication but can also enhance pathogen clearance in mammalian hosts via effector-triggered immunity. The effector LegC4 confers a fitness disadvantage on L. pneumophila within mouse models of Legionnaires' Disease and uniquely potentiates the antimicrobial activity of macrophages activated with either tumor necrosis factor (TNF) or interferon (IFN)-γ. Here, we investigated the mechanism of LegC4 function. We found that LegC4 binds proteasome activator (PA)28α, a subunit of the PA28αβ (11S) proteasome regulator, and that the LegC4 restriction phenotype is abolished within PA28αβ-deficient macrophages. PA28αβ facilitates ubiquitin-independent proteasomal degradation of oxidant-damaged proteins. Impaired proteasome activity results in compensatory upregulation of lysosomal degradation pathways to relieve oxidative proteotoxic stress. We found that LegC4 impairs the resolution of oxidative proteotoxic stress and enhances phagolysosomal fusion with the Legionella-containing vacuole. PA28αβ has been traditionally associated with antigen presentation and adaptive immunity; however, our data support a model whereby suppression of PA28αβ by LegC4 impairs resolution of oxidative proteotoxic stress, which culminates in the lysosomal killing of L. pneumophila within activated macrophages. This work provides a solid foundation on which to evaluate induced proteasome regulators as mediators of cell-autonomous immunity.

PA28γ: New Insights on an Ancient Proteasome Activator

PA28 (also known as 11S, REG or PSME) is a family of proteasome regulators whose members are widely present in many of the eukaryotic supergroups. In jawed vertebrates they are represented by three paralogs, PA28α, PA28β, and PA28γ, which assemble as heptameric hetero (PA28αβ) or homo (PA28γ) rings on one or both extremities of the 20S proteasome cylindrical structure. While they share high sequence and structural similarities, the three isoforms significantly differ in terms of their biochemical and biological properties. In fact, PA28α and PA28β seem to have appeared more recently and to have evolved very rapidly to perform new functions that are specifically aimed at optimizing the process of MHC class I antigen presentation. In line with this, PA28αβ favors release of peptide products by proteasomes and is particularly suited to support adaptive immune responses without, however, affecting hydrolysis rates of protein substrates. On the contrary, PA28γ seems to be a slow-evolving gene that is most similar to the common ancestor of the PA28 activators family, and very likely retains its original functions. Notably, PA28γ has a prevalent nuclear localization and is involved in the regulation of several essential cellular processes including cell growth and proliferation, apoptosis, chromatin structure and organization, and response to DNA damage. In striking contrast with the activity of PA28αβ, most of these diverse biological functions of PA28γ seem to depend on its ability to markedly enhance degradation rates of regulatory protein by 20S proteasome. The present review will focus on the molecular mechanisms and biochemical properties of PA28γ, which are likely to account for its various and complex biological functions and highlight the common features with the PA28αβ paralog.

Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression

Dependence on the 26S proteasome is an Achilles’ heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC. Genome editing or small-molecule mediated inhibition of DYRK2 significantly reduces 26S proteasome activity, bypasses bortezomib resistance, and dramatically delays in vivo tumor growth in MM and TNBC thereby promoting survival. We further characterized the ability of LDN192960, a potent and selective DYRK2-inhibitor, to alleviate tumor burden in vivo. The drug docks into the active site of DYRK2 and partially inhibits all 3 core peptidase activities of the proteasome. Our results suggest that targeting 26S proteasome regulators will pave the way for therapeutic strategies in MM and TNBC.

Regulation of the 20S proteasome by a novel family of inhibitory proteins

The protein degradation machinery plays a critical role in the maintenance of cellular homeostasis, preventing the accumulation of damaged or misfolded proteins and controlling the levels of regulatory proteins. The 20S proteasome degradation machinery is able to cleave any protein with a partially unfolded region, however uncontrolled degradation of the myriad of potential substrates is improbable. Thus, there must exist a regulatory mechanism to control 20S proteasome mediated degradation. Here we have discovered a family of 20S proteasome regulators, named Catalytic Core Regulators (CCRs). They coordinate the function of the 20S proteasome and are involved in the oxidative stress response via Nrf2. The CCRs organize into a feed-forward loop regulatory circuit, with some members stabilizing Nrf2, others being induced by Nrf2, and all of them inhibiting the 20S proteasome. This provides a fine-tuned mechanism to carefully modulate the 20S proteasome, ensuring its proper functioning by controlling the degradative flux.

ZFAND5/ZNF216 is an activator of the 26S proteasome that stimulates overall protein degradation

ZFAND5/ZNF216, a member of the zinc finger AN1-type domain family, is abundant in heart and brain, but is induced in skeletal muscle during atrophy (although not in proteotoxic stress). Because mice lacking ZFAND5 exhibit decreased atrophy, a role in stimulating protein breakdown seemed likely. Addition of recombinant ZFAND5 to purified 26S proteasomes stimulated hydrolysis of ubiquitinated proteins, short peptides, and ATP. Mutating its C-terminal AN1 domain abolished the stimulation of proteasomal peptidase activity. Mutating its N-terminal zinc finger A20 domain, which binds ubiquitin chains, prevented the enhanced degradation of ubiquitinated proteins without affecting peptidase activity. Mouse embryonic fibroblast (MEF) cells lacking ZFAND5 had lower rates of protein degradation and proteasomal activity than WT MEFs. ZFAND5 addition to cell lysates stimulated proteasomal activity and protein degradation. Unlike other proteasome regulators, ZFAND5 enhances multiple 26S activities and overall cellular protein breakdown.