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.

Xrp1 and Irbp18 trigger a feed-forward loop of proteotoxic stress to induce the loser status

Cell competition induces the elimination of less-fit “loser” cells by fitter “winner” cells. In Drosophila, cells heterozygous mutant in ribosome genes, Rp/+, known as Minutes, are outcompeted by wild-type cells. Rp/+ cells display proteotoxic stress and the oxidative stress response, which drive the loser status. Minute cell competition also requires the transcription factors Irbp18 and Xrp1, but how these contribute to the loser status is partially understood. Here we provide evidence that initial proteotoxic stress in RpS3/+ cells is Xrp1-independent. However, Xrp1 is sufficient to induce proteotoxic stress in otherwise wild-type cells and is necessary for the high levels of proteotoxic stress found in RpS3/+ cells. Surprisingly, Xrp1 is also induced downstream of proteotoxic stress, and is required for the competitive elimination of cells suffering from proteotoxic stress or overexpressing Nrf2. Our data suggests that a feed-forward loop between Xrp1, proteotoxic stress, and Nrf2 drives Minute cells to become losers.

Hyperactivation of the proteasome core particle in Caenorhabditis elegans protects against proteotoxic stress and extends lifespan

Many age-related diseases (ARDs) including virtually all neurodegenerative diseases (NDs) are characterized by the accumulation of proteins that are thought to significantly contribute to disease pathogenesis. One of the cell’s primary systems for the degradation of misfolded/damaged proteins is the Ubiquitin Proteasome System (UPS), and its impairment is implicated in essentially all NDs. Thus, upregulating this system to combat NDs has garnered a great deal of interest in recent years. Various animal models have focused on increasing the total proteasome levels, but thus far, none have focused on intrinsic activation of the proteasome itself. With this in mind, we constructed a, first to our knowledge, animal model that endogenously expresses a hyperactive open-gate proteasome in Caenorhabditis elegans (C. elegans). The gate-destabilizing mutation introduced into the nematode germline created a viable nematode population with substantially enhanced proteasomal peptidase and unstructured protein degradation activity. These CRISPR edited nematodes showed a significantly increased lifespan and substantial resistance to oxidative/proteotoxic stress with surprisingly mild consequential phenotypes. These results show that introducing a constitutively active proteasome into a multicellular organism is feasible and suggests targeting the proteasome gating mechanism as a valid approach for future ARD research efforts in mammals.

Heat Shock Protein Grp78/BiP/HspA5 Binds Directly to TDP-43 and Mitigates Toxicity Associated with Neurodegenerative Disease Pathology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure or effective treatment in which TAR DNA Binding Protein of 43 kDa (TDP-43) abnormally accumulates into misfolded protein aggregates in affected neurons. It is widely accepted that protein misfolding and aggregation promote proteotoxic stress. The molecular chaperones are the body’s primary line of defense against proteotoxic stress and there has been long-standing interest in understanding the relationship between chaperones and aggregated protein in ALS. Of particular interest are the heat shock protein of 70 kDa (Hsp70) family of chaperones; however, defining which of the 13 human Hsp70 isoforms is critical for ALS, has presented many challenges. To gain insight into the specific Hsp70 that modulates TDP-43, we investigated the relationship between TDP-43 and the Hsp70s using proximity-dependent biotin identification (BioID) and discovered several Hsp70 isoforms associated with TDP-43 in the nucleus, raising the possibility of an interaction with native TDP-43. We further found that HspA5 bound specifically to the RNA-binding domain of TDP-43 using recombinantly expressed proteins. HspA5 is increased in prefrontal cortex neurons of ALS patients. Finally, overexpression of HspA5 in Drosophila rescued TDP-43-induced toxicity, suggesting that upregulation of HspA5 may have a compensatory role in ALS pathobiology.

De novo variants in the PSMC3 proteasome AAA-ATPase subunit gene cause neurodevelopmental disorders associated with type I interferonopathies

A critical step in preserving protein homeostasis by the ubiquitin-proteasome system (UPS) is the recognition, binding, unfolding, and translocation of protein substrates by AAA-ATPase proteasome subunits for degradation by 26S proteasomes. Here, we identified fourteen different de novo missense variants in the PSMC3 gene encoding the AAA-ATPase proteasome subunit Rpt5 in twenty-two unrelated heterozygous subjects with an autosomal dominant form of neurodevelopmental delay and intellectual disability. Indeed, depletion of PSMC3 impaired reversal learning capabilities in a Drosophila model. The PSMC3 variants cause proteasome dysfunction in patient-derived cells by disruption of substrate translocation, proteotoxic stress and proteostatic imbalances, as well as alterations in proteins controlling developmental and innate immune programs. Molecular analysis confirmed the induction of cellular stress responses and dysregulated mitophagy along with an elevated type I interferon (IFN) signature. Our data define PSMC3 variants as the genetic cause of proteotoxic stress alerting the innate immune system to mount a type I IFN response and link neurodevelopmental syndromes to interferonopathies.

The Post-Storage Performance of RBCs from Beta-Thalassemia Trait Donors Is Related to Their Storability Profile

Blood donors with beta-thalassemia traits (βThal+) have proven to be good “storers”, since their stored RBCs are resistant to lysis and resilient against oxidative/proteotoxic stress. To examine the performance of these RBCs post-storage, stored βThal+ and control RBCs were reconstituted in plasma donated from transfusion-dependent beta-thalassemic patients and healthy controls, and incubated for 24 h at body temperature. Several physiological parameters, including hemolysis, were evaluated. Moreover, labeled fresh/stored RBCs from the two groups were transfused in mice to assess 24 h recovery. All hemolysis metrics were better in the group of heterozygotes and distinguished them against controls in the plasma environment. The reconstituted βThal+ samples also presented higher proteasome activity and fewer procoagulant extracellular vesicles. Transfusion to mice demonstrated that βThal+ RBCs present a marginal trend for higher recovery, regardless of the recipient’s immune background and the RBC storage age. According to correlation analysis, several of these advantageous post-storage characteristics are related to storage phenotypes, like the cytoskeleton composition, low cellular fragility, and enhanced membrane proteostasis that characterize stored βThal+ RBCs. Overall, it seems that the intrinsic physiology of βThal+ RBCs benefits them in conditions mimicking a recipient environment, and in the circulation of animal models; findings that warrant validation in clinical trials.

Regulation of Hippo pathway by Hsp70-Bag3 complex: Bag3 modulates YAP phosphorylation and its nuclear translocation

Protein abnormalities can accelerate aging causing protein misfolding diseases, various adaptive responses have evolved to relieve proteotoxicity. To trigger these responses, cells must detect the buildup of aberrant proteins. Previously we demonstrated that the Hsp70-Bag3 (HB) complex senses the accumulation of defective ribosomal products, stimulating signaling pathways, such as stress kinases or the Hippo pathway kinase LATS1. Here, we studied how Bag3 regulates the ability for LATS1 to regulate its key downstream target YAP. In naïve cells, Bag3 recruited a complex of LATS1, YAP, and the scaffold AmotL2, which links LATS1 and YAP. Upon inhibition of proteasome, AmotL2 dissociated from Bag3, which prevented phosphorylation of YAP by LATS1 and led to consequent nuclear YAP localization together with Bag3. Mutations in Bag3 that enhanced its translocation into nucleus, also facilitated nuclear translocation of YAP. Interestingly, Bag3 also controlled YAP nuclear localization in response to cell density, indicating broader roles beyond proteotoxic signaling responses for Bag3 in the regulation of YAP. These data implicate Bag3 as a regulator of Hippo pathway signaling, and suggest mechanisms by which proteotoxic stress signals are propagated.

Proteasome Inhibition Is an Effective Treatment Strategy for Microsporidia Infection in Honey Bees

The microsporidia Nosema ceranae is an obligate intracellular parasite that causes honey bee mortality and contributes to colony collapse. Fumagillin is presently the only pharmacological control for N. ceranae infections in honey bees. Resistance is already emerging, and alternative controls are critically needed. Nosema spp. exhibit increased sensitivity to heat shock, a common proteotoxic stress. Thus, we hypothesized that targeting the Nosema proteasome, the major protease removing misfolded proteins, might be effective against N. ceranae infections in honey bees. Nosema genome analysis and molecular modeling revealed an unexpectedly compact proteasome apparently lacking multiple canonical subunits, but with highly conserved proteolytic active sites expected to be receptive to FDA-approved proteasome inhibitors. Indeed, N. ceranae were strikingly sensitive to pharmacological disruption of proteasome function at doses that were well tolerated by honey bees. Thus, proteasome inhibition is a novel candidate treatment strategy for microsporidia infection in honey bees.

Proteotoxic Stress as an Exploitable Vulnerability in Cells with Hyperactive AKT

Hyperactivity of serine-threonine kinase AKT is one of the most common molecular abnormalities in cancer, where it contributes to poor outcomes by facilitating the growth and survival of malignant cells. Despite its well-documented anti-apoptotic effects, hyperactivity of AKT is also known to be stressful to a cell. In an attempt to better elucidate this phenomenon, we observed the signs of proteotoxic stress in cells that harbor hyperactive AKT or have lost its principal negative regulator, PTEN. The activity of HSF1 was predictably elevated under these circumstances. However, such cells proved more sensitive to various regimens of heat shock, including the conditions that were well-tolerated by syngeneic cells without AKT hyperactivity. The sensitizing effect of hyperactive AKT was also seen in HSF1-deficient cells, suggesting that the phenomenon does not require the regulation of HSF1 by this kinase. Notably, the elevated activity of AKT was accompanied by increased levels of XBP1, a key component of cell defense against proteotoxic stress. Interestingly, the cells harboring hyperactive AKT were also more dependent on XBP1 for their growth. Our observations suggest that proteotoxic stress conferred by hyperactive AKT represents a targetable vulnerability, which can be exploited by either elevating the stress above the level tolerated by such cells or by eliminating the factors that enable such tolerance.