Novel insights into the non-canonical roles of PSMD14/POH1/Rpn11 in proteostasis and in the modulation of cancer progression.

PSMD14/POH1/Rpn11 plays a crucial role in cellular homeostasis. PSMD14 is a structural subunit of the lid subcomplex of the proteasome 19S regulatory particle with constitutive deubiquitinase activity. Canonically, PSMD14 removes the full ubiquitin chains with K48-linkages by hydrolyzing the isopeptide bond between the substrate and the C-terminus of the first ubiquitin, a crucial step for the entry of substrates into the catalytic barrel of the 20S proteasome and their subsequent degradation, all in context of the 26S proteasome. However, more recent discoveries indicate PSMD14 DUB activity is not only coupled to the translocation of substrates into the core of 20S proteasome. During the assembly of the lid, activity of PSMD14 has been detected in the context of the heterodimer with PSMD7. Additionally, assembly of the lid subcomplex occurs as an independent event of the base subcomplex and 20S proteasome. This feature opens the possibility that the regulatory particle, free lid subcomplex or the heterodimer PSMD14-PSMD7 might play other physiological roles including a positive function on protein stability through deubiquitination. Here we discuss scenarios that could enhance this PSMD14 non-canonical pathway, the potential impact in preventing degradation of substrates by autophagy highlighting the main findings that support this hypothesis. Finally, we discuss why this information should be investigated in biomedicine specifically with focus on cancer progression to design new therapeutic strategies against the lid subcomplex and the heterodimer PSMD14-PSMD7, highlighting PSMD14 as a druggable target for cancer therapy.

Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival.

Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER membrane protein with low expression and known to be degraded by the proteasome under normal conditions. Contrary, under ER stress, levels of HERPUD1 increased rapidly due to a blockage in its proteasomal degradation. Thus, we explored whether HERPUD1 stability could work as a negative regulator of autophagy. In this work, we expressed a version of HERPUD1 with its ubiquitin-like domain (UBL) deleted, which is known to be crucial for its proteasome degradation. In comparison to HERPUD1-WT, we found the UBL-deleted version caused a negative role on basal and induced macroautophagy. Unexpectedly, we found stabilized HERPUD1 promotes ER remodeling independent of unfolded protein response activation observing an increase in stacked-tubular structures resembling previously described tubular ER rearrangements. Importantly, a phosphomimetic S59D mutation within the UBL mimics the phenotype observed with the UBL-deleted version including an increase in HERPUD1 stability and ER remodeling together with a negative role on autophagy. Moreover, we found UBL-deleted version and HERPUD1-S59D trigger an increase in cellular size, whereas HERPUD1-S59D also causes an increased in nuclear size. Interestingly, ER remodeling by the deletion of the UBL and the phosphomimetic S59D version led to an increase in the number and function of lysosomes. In addition, the UBL-deleted version and phosphomimetic S59D version established a tight ER-lysosomal network with the presence of extended patches of ER-lysosomal membrane-contact sites condition that reveals an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival.

The Proteasomal Deubiquitinating Enzyme PSMD14 Regulates Macroautophagy by Controlling Golgi-to-ER Retrograde Transport.

Ubiquitination regulates several biological processes, however the role of specific members of the ubiquitinome on intracellular membrane trafficking is not yet fully understood. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1187 genes of the human "ubiquitinome" using amyloid precursor protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 with Capzimin (CZM) caused a robust increase in APP levels at the Golgi apparatus and the swelling of this organelle. We showed that this phenotype is the result of rapid inhibition of Golgi-to-ER retrograde transport, a pathway implicated in the early steps of the autophagosomal formation. Indeed, we observed that inhibition of PSMD14 with CZM acts as a potent blocker of macroautophagy by a mechanism related to the retention of Atg9A and Rab1A at the Golgi apparatus. As pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and the K63-Ub chains, act as a crucial regulatory factor for macroautophagy by controlling Golgi-to-ER retrograde transport.

Cellular Responses to Proteasome Inhibition: Molecular Mechanisms and Beyond.

Proteasome inhibitors have been actively tested as potential anticancer drugs and in the treatment of inflammatory and autoimmune diseases. Unfortunately, cells adapt to survive in the presence of proteasome inhibitors activating a variety of cell responses that explain why these therapies have not fulfilled their expected results. In addition, all proteasome inhibitors tested and approved by the FDA have caused a variety of side effects in humans. Here, we describe the different types of proteasome complexes found within cells and the variety of regulators proteins that can modulate their activities, including those that are upregulated in the context of inflammatory processes. We also summarize the adaptive cellular responses activated during proteasome inhibition with special emphasis on the activation of the Autophagic-Lysosomal Pathway (ALP), proteaphagy, p62/SQSTM1 enriched-inclusion bodies, and proteasome biogenesis dependent on Nrf1 and Nrf2 transcription factors. Moreover, we discuss the role of IRE1 and PERK sensors in ALP activation during ER stress and the involvement of two deubiquitinases, Rpn11 and USP14, in these processes. Finally, we discuss the aspects that should be currently considered in the development of novel strategies that use proteasome activity as a therapeutic target for the treatment of human diseases.

Interplay Between the Autophagy-Lysosomal Pathway and the Ubiquitin-Proteasome System: A Target for Therapeutic Development in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of age-related dementia leading to severe irreversible cognitive decline and massive neurodegeneration. While therapeutic approaches for managing symptoms are available, AD currently has no cure. AD associates with a progressive decline of the two major catabolic pathways of eukaryotic cells-the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS)-that contributes to the accumulation of harmful molecules implicated in synaptic plasticity and long-term memory impairment. One protein recently highlighted as the earliest initiator of these disturbances is the amyloid precursor protein (APP) intracellular C-terminal membrane fragment ß (CTFß), a key toxic agent with deleterious effects on neuronal function that has become an important pathogenic factor for AD and a potential biomarker for AD patients. This review focuses on the involvement of regulatory molecules and specific post-translational modifications (PTMs) that operate in the UPS and ALP to control a single proteostasis network to achieve protein balance. We discuss how these aspects can contribute to the development of novel strategies to strengthen the balance of key pathogenic proteins associated with AD.

Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein via the MVB/lysosomal pathway.

Brain regions affected by Alzheimer disease (AD) display well-recognized early neuropathologic features in the endolysosomal and autophagy systems of neurons, including enlargement of endosomal compartments, progressive accumulation of autophagic vacuoles, and lysosomal dysfunction. Although the primary causes of these disturbances are still under investigation, a growing body of evidence suggests that the amyloid precursor protein (APP) intracellular C-terminal fragment ß (C99), generated by cleavage of APP by ß-site APP cleaving enzyme 1 (BACE-1), is the primary cause of the endosome enlargement in AD and the earliest initiator of synaptic plasticity and long-term memory impairment. The aim of the present study was to evaluate the possible relationship between the endolysosomal degradation pathway and autophagy on the proteolytic processing and turnover of C99. We found that pharmacologic treatments that either inhibit autophagosome formation or block the fusion of autophagosomes to endolysosomal compartments caused an increase in C99 levels. We also found that inhibition of autophagosome formation by depletion of Atg5 led to higher levels of C99 and to its massive accumulation in the lumen of enlarged perinuclear, lysosomal-associated membrane protein 1 (LAMP1)-positive organelles. In contrast, activation of autophagosome formation, either by starvation or by inhibition of the mammalian target of rapamycin, enhanced lysosomal clearance of C99. Altogether, our results indicate that autophagosomes are key organelles to help avoid C99 accumulation preventing its deleterious effects.-González, A. E., Muñoz, V. C., Cavieres, V. A., Bustamante, H. A., Cornejo, V.-H., Januário, Y. C., González, I., Hetz, C., daSilva, L. L., Rojas-Fernández, A., Hay, R. T., Mardones, G. A., Burgos, P. V. Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein via the MVB/lysosomal pathway.

Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-ß (Aß) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aß species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aß production in vivo.

Turnover of C99 is controlled by a crosstalk between ERAD and ubiquitin-independent lysosomal degradation in human neuroglioma cells.

Alzheimer's disease (AD) is characterized by the buildup of amyloid-ß peptides (Aß) aggregates derived from proteolytic processing of the ß-amyloid precursor protein (APP). Amyloidogenic cleavage of APP by ß-secretase/BACE1 generates the C-terminal fragment C99/CTFß that can be subsequently cleaved by γ-secretase to produce Aß. Growing evidence indicates that high levels of C99/CTFß are determinant for AD. Although it has been postulated that γ-secretase-independent pathways must control C99/CTFß levels, the contribution of organelles with degradative functions, such as the endoplasmic reticulum (ER) or lysosomes, is unclear. In this report, we investigated the turnover and amyloidogenic processing of C99/CTFß in human H4 neuroglioma cells, and found that C99/CTFß is localized at the Golgi apparatus in contrast to APP, which is mostly found in endosomes. Conditions that localized C99/CTFß to the ER resulted in its degradation in a proteasome-dependent manner that first required polyubiquitination, consistent with an active role of the ER associated degradation (ERAD) in this process. Furthermore, when proteasomal activity was inhibited C99/CTFß was degraded in a chloroquine (CQ)-sensitive compartment, implicating lysosomes as alternative sites for its degradation. Our results highlight a crosstalk between degradation pathways within the ER and lysosomes to avoid protein accumulation and toxicity.