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

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

© FASEB. Brain regions affected by Alzheimer disease (AD) displaywell-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 b (C99), generated by cleavage of APP by b-site APP cleaving enzyme 1 (BACE-1), is the primary cause of the endosome enlargement inADand the earliest initiator of synaptic plasticity and long-termmemory impairment. The aimof 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 autophagosomeformationorblock the fusionof autophagosomes to

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

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 β (CTFβ), 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

Proposed processing and turnover routes of C99.

<p>(A) (<i>i</i>) A small fraction of newly-synthesized APP in the endoplasmic reticulum (ER) can be a substrate of BACE1 that generates C99. Ubiquitinated (Ub) C99 can be a substrate of the endoplasmic reticulum-associated protein degradation (ERAD) pathway to ultimately be degraded by the proteasome. (<i>ii</i>) En route through the secretory pathway, a fraction of APP at the Golgi apparatus can also be a substrate of BACE1 that generates C99, which subsequently can be a substrate of γ-secretase (γ-sec) activity that generates Aβ peptides and cytosolic AICDγ, a proteolytic processing that can be inhibited by DAPT. (<i>iii</i>) Finally, within endo/lysosomal compartments APP can be degraded by acid hydrolases. (B) (<i>i</i>) Upon MG132 inhibition, ubiquitinated C99 accumulates within the ER. Ubiquitinated C99 can exit the ER and reach the Golgi apparatus. (<i>ii</i>) Both ubiquitinated C99 and C99 generated from APP can be cleaved at the Golgi apparatus by γ-secretase activity. Upon Brefeldin A (BFA) treatment, C99 can be relocated from the Golgi apparatus to the ER where it can be also cleaved by γ-secretase activity. (<i>iii</i>) Both APP and the excess of C99 can be degraded by acid hydrolases. (C) (<i>i</i>) Upon MG132 treatment, and (<i>ii</i>) the generation of an excess of C99 at the Golgi apparatus, (<i>iii</i>) chloroquine (CQ) treatment results in accumulation of both APP and C99 within endo/lysosomal compartments. For simplicity, other APP metabolites, such as sAPPβ, which is the other product of BACE1 activity on APP, or the C31 fragment, are not depicted.</p

C99 is degraded after redistribution to the endoplasmic reticulum.

<p>H4 cells stably expressing GFP-tagged C99-F/P-D/A were treated as follows: (A) with increasing concentrations of MG132 for 4 h; (B) left untreated or treated either with 1 µM DAPT for 16 h, 1 µM MG132 for 4 h, or 1 µM DAPT for 12 h followed by a combination of 1 µM DAPT and 1 µM MG132 for 4 h; (C) left untreated or treated for 4 h either with 5 µg/ml BFA, 1 µM MG132, or a combination of 1 µM MG132 and 5 µg/ml BFA; or (D) pretreated with 5 µg/ml BFA without or with 1 µM MG132 for 4 h followed by CHX-chase for 0–60 min without or with 1 µM MG132. (E) H4 cells stably expressing GFP-tagged APP-F/P-D/A were left untreated or treated for 4 h either with 5 µg/ml BFA, or a combination of 5 µg/ml BFA and 1 µM MG132. Cellular extracts were analyzed by immunoblot with anti-GFP antibody (A–E), or WO2 monoclonal antibody to detect C99 in cells expressing GFP-tagged APP-F/P-D/A (E). Immunoblot with anti-β-actin antibody was used as loading control. The positions of molecular mass markers are indicated on the left. (F) Densitometric quantification of the levels of C99 shown in E. Bars represent the mean ± SD (n = 4). *<i>P</i><0.05.</p

Accumulation of C99 in response to MG132, CQ and lack of its cytosolic tyrosine residues.

<p>(A) Schematic representation of GFP-tagged C99 indicating its topological domains, the position of the Aβ peptide, the γ-secretase cleavage site, the AICDγ fragment, and the sequence of the cytosolic tail highlighting the substitutions in its three tyrosine residues (bold underline). (B) Immunoblot analysis of H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-3Y/A-F/P-D/A (C99-3Y/A). Cells were left untreated or treated with 1 µM DAPT for 16 h and subsequently analyzed by immunoblot with anti-GFP antibody. Immunoblot with anti-β-actin was used as loading control. The positions of molecular mass markers are indicated on the left. (C) H4 cells stably expressing C99-3Y/A were left untreated or treated for 16 h either with 1 µM MG132, 100 µM CQ, or with a combination of 1 µM MG132 and 100 µM CQ. Cells were biotinylated on the cell surface with Sulfo-NHS-LC-Biotin and soluble extracts pulled down with NeutrAvidin-agarose. Total and biotinylated proteins were analyzed by immunoblot with anti-GFP antibody. Immunoblot with anti-β-actin or anti-transferrin receptor (TfR) antibodies was used as loading control for total or biotinylated proteins, respectively. The positions of molecular mass markers are indicated on the left. (D) Densitometric quantification of C99-3Y/A left untreated or treated for 16 h either with 100 µM CQ, 1 µM MG132, or with a combination of 100 µM CQ and 1 µM MG132. Bars represent the mean ± SD (n = 3). *<i>P</i><0.05; **<i>P</i><0.01.</p

C99 is proteolytically cleaved in different sites.

<p>(A) Schematic representation of GFP-tagged C99, C83 and C31 indicating their topological domains, and the position of the Aβ peptide, the p3 peptide, the proteolytic cleavage sites (α, γ and caspase), and the AICDγ fragment. (B) H4 cells transiently expressing wild-type C99-GFP (WT) or C99-GFP with either the D87A mutation, the F38P mutation, or both (F/P-D/A), were left untreated or treated with 1 µM DAPT for 16 h. Cellular extracts were analyzed by immunoblot with anti-GFP antibody. The positions of molecular mass markers are indicated on the left.</p

Intracellular localization and proteolytic processing of C99.

<p>(A) Schematic representation of GFP-tagged APP and C99 indicating their topological domains and the position of the HA tag, the Aβ peptide, the proteolytic cleavage sites (α, β and γ), and the AICDγ fragment. (B) Fluorescence microscopy analysis of H4 human neuroglioma cells transiently expressing APP-GFP or C99-GFP. Bar, 10 µm. (C–E) H4 cells transiently expressing C99-GFP were left untreated or treated for 16 h with 1 µM DAPT, labeled for 4 hr at 20°C with 1 mCi/ml [³⁵S]-methionine-cysteine, and chased at 37°C for the indicated times. C99 and Aβ species were immunoprecipitated from cell lysates with anti-GFP antibody (C), or from the culture medium with 6E10 antibody (E), respectively. Proteins were analyzed on 10%–20% Tricine gels and fluorography. The positions of molecular mass markers are indicated on the left. (D) Densitometric quantification of the levels of C99, C83, and AICDγ shown in C.</p