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

Golgi Phosphoprotein 3 Regulates the Physical Association of Glycolipid Glycosyltransferases

Glycolipid glycosylation is an intricate process that mainly takes place in the Golgi by the complex interplay between glycosyltransferases. Several features such as the organization, stoichiometry and composition of these complexes may modify their sorting properties, sub-Golgi localization, enzymatic activity and in consequence, the pattern of glycosylation at the plasma membrane. In spite of the advance in our comprehension about physiological and pathological cellular states of glycosylation, the molecular basis underlying the metabolism of glycolipids and the players involved in this process remain not fully understood. In the present work, using biochemical and fluorescence microscopy approaches, we demonstrate the existence of a physical association between two ganglioside glycosyltransferases, namely, ST3Gal-II (GD1a synthase) and β3GalT-IV (GM1 synthase) with Golgi phosphoprotein 3 (GOLPH3) in mammalian cultured cells. After GOLPH3 knockdown, the localization of both enzymes was not affected, but the fomation of ST3Gal-II/β3GalT-IV complex was compromised and glycolipid expression pattern changed. Our results suggest a novel control mechanism of glycolipid expression through the regulation of the physical association between glycolipid glycosyltransferases mediated by GOLPH3

Human Golgi phosphoprotein 3 is an effector of RAB1A and RAB1B.

Golgi phosphoprotein 3 (GOLPH3) is a peripheral membrane protein localized at the trans-Golgi network that is also distributed in a large cytosolic pool. GOLPH3 has been involved in several post-Golgi protein trafficking events, but its precise function at the molecular level is not well understood. GOLPH3 is also considered the first oncoprotein of the Golgi apparatus, with important roles in several types of cancer. Yet, it is unknown how GOLPH3 is regulated to achieve its contribution in the mechanisms that lead to tumorigenesis. Binding of GOLPH3 to Golgi membranes depends on its interaction to phosphatidylinositol-4-phosphate. However, an early finding showed that GTP promotes the binding of GOLPH3 to Golgi membranes and vesicles. Nevertheless, it remains largely unknown whether this response is consequence of the function of GTP-dependent regulatory factors, such as proteins of the RAB family of small GTPases. Interestingly, in Drosophila melanogaster the ortholog of GOLPH3 interacts with- and behaves as effector of the ortholog of RAB1. However, there is no experimental evidence implicating GOLPH3 as a possible RAB1 effector in mammalian cells. Here, we show that human GOLPH3 interacted directly with either RAB1A or RAB1B, the two isoforms of RAB1 in humans. The interaction was nucleotide dependent and it was favored with GTP-locked active state variants of these GTPases, indicating that human GOLPH3 is a bona fide effector of RAB1A and RAB1B. Moreover, the expression in cultured cells of the GTP-locked variants resulted in less distribution of GOLPH3 in the Golgi apparatus, suggesting an intriguing model of GOLPH3 regulation

GOLPH3 Participates in Mitochondrial Fission and Is Necessary to Sustain Bioenergetic Function in MDA-MB-231 Breast Cancer Cells

In this study, we investigated the inter-organelle communication between the Golgi apparatus (GA) and mitochondria. Previous observations suggest that GA-derived vesicles containing phosphatidylinositol 4-phosphate (PI(4)P) play a role in mitochondrial fission, colocalizing with DRP1, a key protein in this process. However, the functions of these vesicles and potentially associated proteins remain unknown. GOLPH3, a PI(4)P-interacting GA protein, is elevated in various types of solid tumors, including breast cancer, yet its precise role is unclear. Interestingly, GOLPH3 levels influence mitochondrial mass by affecting cardiolipin synthesis, an exclusive mitochondrial lipid. However, the mechanism by which GOLPH3 influences mitochondria is not fully understood. Our live-cell imaging analysis showed GFP-GOLPH3 associating with PI(4)P vesicles colocalizing with YFP-DRP1 at mitochondrial fission sites. We tested the functional significance of these observations with GOLPH3 knockout in MDA-MB-231 cells of breast cancer, resulting in a fragmented mitochondrial network and reduced bioenergetic function, including decreased mitochondrial ATP production, mitochondrial membrane potential, and oxygen consumption. Our findings suggest a potential negative regulatory role for GOLPH3 in mitochondrial fission, impacting mitochondrial function and providing insights into GA–mitochondria communication

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

Different response of APP and C99 to CQ.

<p>(A–B) H4 cells stably expressing GFP-tagged APP-F/P-D/A (A) or C99-F/P-D/A (B) were left untreated or treated for 16 h either with 1 µM DAPT, 100 µM CQ, or with a combination of 1 µM DAPT and 100 µM CQ. Cellular extracts were analyzed by immunoblot with anti-GFP antibody. The positions of molecular mass markers are indicated on the left. (C) Densitometric quantification of the levels of APP and C99 shown in A and B. Bars represent the mean ± SD (APP n = 7; C99 n = 6). *<i>P</i><0.05. (D) Confocal fluorescence microscopy of H4 cells stably expressing GFP-tagged APP-F/P-D/A or C99-F/P-D/A left untreated (Control) or treated with 100 µM CQ for 16 h. Bar, 10 µm.</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