PKD2 regulates autophagy and forms a protein complex with BECN1 at the primary cilium of hypothalamic neuronal cells.

The primary cilium, hereafter cilium, is an antenna-like organelle that modulates intracellular responses, including autophagy, a lysosomal degradation process essential for cell homeostasis. Dysfunction of the cilium is associated with impairment of autophagy and diseases known as "ciliopathies". The discovery of autophagy-related proteins at the base of the cilium suggests its potential role in coordinating autophagy initiation in response to physiopathological stimuli. One of these proteins, beclin-1 (BECN1), it which is necessary for autophagosome biogenesis. Additionally, polycystin-2 (PKD2), a calcium channel enriched at the cilium, is required and sufficient to induce autophagy in renal and cancer cells. We previously demonstrated that PKD2 and BECN1 form a protein complex at the endoplasmic reticulum in non-ciliated cells, where it initiates autophagy, but whether this protein complex is present at the cilium remains unknown. Anorexigenic pro-opiomelanocortin (POMC) neurons are ciliated cells that require autophagy to maintain intracellular homeostasis. POMC neurons are sensitive to metabolic changes, modulating signaling pathways crucial for controlling food intake. Exposure to the saturated fatty acid palmitic acid (PA) reduces ciliogenesis and inhibits autophagy in these cells. Here, we show that PKD2 and BECN1 form a protein complex in N43/5 cells, an in vitro model of POMC neurons, and that both PKD2 and BECN1 locate at the cilium. In addition, our data show that the cilium is required for PKD2-BECN1 protein complex formation and that PA disrupts the PKD2-BECN1 complex, suppressing autophagy. Our findings provide new insights into the mechanisms by which the cilium controls autophagy in hypothalamic neuronal cells.

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.

Impact of interorganelle coordination between the conventional early secretory pathway and autophagy in cellular homeostasis and stress response.

The conventional early secretory pathway and autophagy are two essential interconnected cellular processes that are crucial for maintaining cellular homeostasis. The conventional secretory pathway is an anabolic cellular process synthesizing and delivering proteins to distinct locations, including different organelles, the plasma membrane, and the extracellular media. On the other hand, autophagy is a catabolic cellular process that engulfs damaged organelles and aberrant cytosolic constituents into the double autophagosome membrane. After fusion with the lysosome and autolysosome formation, this process triggers digestion and recycling. A growing list of evidence indicates that these anabolic and catabolic processes are mutually regulated. While knowledge about the molecular actors involved in the coordination and functional cooperation between these two processes has increased over time, the mechanisms are still poorly understood. This review article summarized and discussed the most relevant evidence about the key molecular players implicated in the interorganelle crosstalk between the early secretory pathway and autophagy under normal and stressful conditions.

How Many Sirtuin Genes Are Out There? Evolution of Sirtuin Genes in Vertebrates With a Description of a New Family Member.

Studying the evolutionary history of gene families is a challenging and exciting task with a wide range of implications. In addition to exploring fundamental questions about the origin and evolution of genes, disentangling their evolution is also critical to those who do functional/structural studies to allow a deeper and more precise interpretation of their results in an evolutionary context. The sirtuin gene family is a group of genes that are involved in a variety of biological functions mostly related to aging. Their duplicative history is an open question, as well as the definition of the repertoire of sirtuin genes among vertebrates. Our results show a well-resolved phylogeny that represents an improvement in our understanding of the duplicative history of the sirtuin gene family. We identified a new sirtuin gene family member (SIRT3.2) that was apparently lost in the last common ancestor of amniotes but retained in all other groups of jawed vertebrates. According to our experimental analyses, elephant shark SIRT3.2 protein is located in mitochondria, the overexpression of which leads to an increase in cellular levels of ATP. Moreover, in vitro analysis demonstrated that it has deacetylase activity being modulated in a similar way to mammalian SIRT3. Our results indicate that there are at least eight sirtuin paralogs among vertebrates and that all of them can be traced back to the last common ancestor of the group that existed between 676 and 615 millions of years ago.

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.

Autophagy in aging-related oral diseases.

Autophagy is an intracellular degradation mechanism that allows recycling of organelles and macromolecules. Autophagic function increases metabolite availability modulating metabolic pathways, differentiation and cell survival. The oral environment is composed of several structures, including mineralized and soft tissues, which are formed by complex interactions between epithelial and mesenchymal cells. With aging, increased prevalence of oral diseases such as periodontitis, oral cancer and periapical lesions are observed in humans. These aging-related oral diseases are chronic conditions that alter the epithelial-mesenchymal homeostasis, disrupting the oral tissue architecture affecting the quality of life of the patients. Given that autophagy levels are reduced with age, the purpose of this review is to discuss the link between autophagy and age-related oral diseases.

Neuronopathic Gaucher disease: Beyond lysosomal dysfunction.

Gaucher disease (GD) is an inherited disorder caused by recessive mutations in the GBA1 gene that encodes the lysosomal enzyme ß-glucocerebrosidase (ß-GC). ß-GC hydrolyzes glucosylceramide (GluCer) into glucose and ceramide in the lysosome, and the loss of its activity leads to GluCer accumulation in different tissues. In severe cases, enzymatic deficiency triggers inflammation, organomegaly, bone disease, and neurodegeneration. Neuronopathic Gaucher disease (nGD) encompasses two different forms of the disease, characterized by chronic or acute damage to the central nervous system (CNS). The cellular and molecular studies that uncover the pathological mechanisms of nGD mainly focus on lysosomal dysfunction since the lysosome is the key organelle affected in GD. However, new studies show alterations in other organelles that contribute to nGD pathology. For instance, abnormal accumulation of GluCer in lysosomes due to the loss of ß-GC activity leads to excessive calcium release from the endoplasmic reticulum (ER), activating the ER-associated degradation pathway and the unfolded protein response. Recent evidence indicates mitophagy is altered in nGD, resulting in the accumulation of dysfunctional mitochondria, a critical factor in disease progression. Additionally, nGD patients present alterations in mitochondrial morphology, membrane potential, ATP production, and increased reactive oxygen species (ROS) levels. Little is known about potential dysfunction in other organelles of the secretory pathway, such as the Golgi apparatus and exosomes. This review focuses on collecting evidence regarding organelle dysfunction beyond lysosomes in nGD. We briefly describe cellular and animal models and signaling pathways relevant to uncovering the pathological mechanisms and new therapeutic targets in GD.

Palmitic acid control of ciliogenesis modulates insulin signaling in hypothalamic neurons through an autophagy-dependent mechanism.

Palmitic acid (PA) is significantly increased in the hypothalamus of mice, when fed chronically with a high-fat diet (HFD). PA impairs insulin signaling in hypothalamic neurons, by a mechanism dependent on autophagy, a process of lysosomal-mediated degradation of cytoplasmic material. In addition, previous work shows a crosstalk between autophagy and the primary cilium (hereafter cilium), an antenna-like structure on the cell surface that acts as a signaling platform for the cell. Ciliopathies, human diseases characterized by cilia dysfunction, manifest, type 2 diabetes, among other features, suggesting a role of the cilium in insulin signaling. Cilium depletion in hypothalamic pro-opiomelanocortin (POMC) neurons triggers obesity and insulin resistance in mice, the same phenotype as mice deficient in autophagy in POMC neurons. Here we investigated the effect of chronic consumption of HFD on cilia; and our results indicate that chronic feeding with HFD reduces the percentage of cilia in hypothalamic POMC neurons. This effect may be due to an increased amount of PA, as treatment with this saturated fatty acid in vitro reduces the percentage of ciliated cells and cilia length in hypothalamic neurons. Importantly, the same effect of cilia depletion was obtained following chemical and genetic inhibition of autophagy, indicating autophagy is required for ciliogenesis. We further demonstrate a role for the cilium in insulin sensitivity, as cilium loss in hypothalamic neuronal cells disrupts insulin signaling and insulin-dependent glucose uptake, an effect that correlates with the ciliary localization of the insulin receptor (IR). Consistently, increased percentage of ciliated hypothalamic neuronal cells promotes insulin signaling, even when cells are exposed to PA. Altogether, our results indicate that, in hypothalamic neurons, impairment of autophagy, either by PA exposure, chemical or genetic manipulation, cause cilia loss that impairs insulin sensitivity.

Clathrin adaptor AP-1-mediated Golgi export of amyloid precursor protein is crucial for the production of neurotoxic amyloid fragments.

One of the hallmarks of Alzheimer's disease is the accumulation of toxic amyloid-ß (Aß) peptides in extracellular plaques. The direct precursor of Aß is the carboxyl-terminal fragment ß (or C99) of the amyloid precursor protein (APP). C99 is detected at elevated levels in Alzheimer's disease brains, and its intracellular accumulation has been linked to early neurotoxicity independently of Aß. Despite this, the causes of increased C99 levels are poorly understood. Here, we demonstrate that APP interacts with the clathrin vesicle adaptor AP-1 (adaptor protein 1), and we map the interaction sites on both proteins. Using quantitative kinetic trafficking assays, established cell lines and primary neurons, we also show that this interaction is required for the transport of APP from the trans-Golgi network to endosomes. In addition, disrupting AP-1-mediated transport of APP alters APP processing and degradation, ultimately leading to increased C99 production and Aß release. Our results indicate that AP-1 regulates the subcellular distribution of APP, altering its processing into neurotoxic fragments.

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.

Palmitic and Stearic Acids Inhibit Chaperone-Mediated Autophagy (CMA) in POMC-like Neurons In Vitro.

The intake of food with high levels of saturated fatty acids (SatFAs) is associated with the development of obesity and insulin resistance. SatFAs, such as palmitic (PA) and stearic (SA) acids, have been shown to accumulate in the hypothalamus, causing several pathological consequences. Autophagy is a lysosomal-degrading pathway that can be divided into macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Previous studies showed that PA impairs macroautophagy function and insulin response in hypothalamic proopiomelanocortin (POMC) neurons. Here, we show in vitro that the exposure of POMC neurons to PA or SA also inhibits CMA, possibly by decreasing the total and lysosomal LAMP2A protein levels. Proteomics of lysosomes from PA- and SA-treated cells showed that the inhibition of CMA could impact vesicle formation and trafficking, mitochondrial components, and insulin response, among others. Finally, we show that CMA activity is important for regulating the insulin response in POMC hypothalamic neurons. These in vitro results demonstrate that CMA is inhibited by PA and SA in POMC-like neurons, giving an overview of the CMA-dependent cellular pathways that could be affected by such inhibition and opening a door for in vivo studies of CMA in the context of the hypothalamus and obesity.

GOLPH3 Regulates EGFR in T98G Glioblastoma Cells by Modulating Its Glycosylation and Ubiquitylation.

Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.

Palmitic acid reduces the autophagic flux in hypothalamic neurons by impairing autophagosome-lysosome fusion and endolysosomal dynamics.

High-fat diet (HFD)-induced obesity is associated with increased cancer risk. Long-term feeding with HFD increases the concentration of the saturated fatty acid palmitic acid (PA) in the hypothalamus. We previously showed that, in hypothalamic neuronal cells, exposure to PA inhibits the autophagic flux, which is the whole autophagic process from the synthesis of the autophagosomes, up to their lysosomal fusion and degradation. However, the mechanism by which PA impairs autophagy in hypothalamic neurons remains unknown. Here, we show that PA-mediated reduction of the autophagic flux is not caused by lysosomal dysfunction, as PA treatment does not impair lysosomal pH or the activity of cathepsin B.Instead, PA dysregulates autophagy by reducing autophagosome-lysosome fusion, which correlates with the swelling of endolysosomal compartments that show areduction in their dynamics. Finally, because lysosomes undergo constant dynamic regulation by the small Rab7 GTPase, we investigated the effect of PA treatment on its activity. Interestingly, we found PA treatment altered the activity of Rab7. Altogether, these results unveil the cellular process by which PA exposure impairs the autophagic flux. As impaired autophagy in hypothalamic neurons promotes obesity, and balanced autophagy is required to inhibit malignant transformation, this could affect tumor initiation, progression, and/or response to therapy of obesity-related cancers.

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.

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.

Pro-Tumoral Functions of Autophagy Receptors in the Modulation of Cancer Progression.

Cancer progression involves a variety of pro-tumorigenic biological processes including cell proliferation, migration, invasion, and survival. A cellular pathway implicated in these pro-tumorigenic processes is autophagy, a catabolic route used for recycling of cytoplasmic components to generate macromolecular building blocks and energy, under stress conditions, to remove damaged cellular constituents to adapt to changing nutrient conditions and to maintain cellular homeostasis. During autophagy, cells form a double-membrane sequestering a compartment termed the phagophore, which matures into an autophagosome. Following fusion with the lysosome, the cargo is degraded inside the autolysosomes and the resulting macromolecules released back into the cytosol for reuse. Cancer cells use this recycling system during cancer progression, however the key autophagy players involved in this disease is unclear. Accumulative evidences show that autophagy receptors, crucial players for selective autophagy, are overexpressed during cancer progression, yet the mechanisms whereby pro-tumorigenic biological processes are modulated by these receptors remains unknown. In this review, we summarized the most important findings related with the pro-tumorigenic role of autophagy receptors p62/SQSTM1, NBR1, NDP52, and OPTN in cancer progression. In addition, we showed the most relevant cargos degraded by these receptors that have been shown to function as critical regulators of pro-tumorigenic processes. Finally, we discussed the role of autophagy receptors in the context of the cellular pathways implicated in this disease, such as growth factors signaling, oxidative stress response and apoptosis. In summary, we highlight that autophagy receptors should be considered important players of cancer progression, which could offer a niche for the development of novel diagnosis and cancer treatment strategies.

KDEL receptor regulates secretion by lysosome relocation- and autophagy-dependent modulation of lipid-droplet turnover.

Inter-organelle signalling has essential roles in cell physiology encompassing cell metabolism, aging and temporal adaptation to external and internal perturbations. How such signalling coordinates different organelle functions within adaptive responses remains unknown. Membrane traffic is a fundamental process in which membrane fluxes need to be sensed for the adjustment of cellular requirements and homeostasis. Studying endoplasmic reticulum-to-Golgi trafficking, we found that Golgi-based, KDEL receptor-dependent signalling promotes lysosome repositioning to the perinuclear area, involving a complex process intertwined to autophagy, lipid-droplet turnover and Golgi-mediated secretion that engages the microtubule motor protein dynein-LRB1 and the autophagy cargo receptor p62/SQSTM1. This process, here named 'traffic-induced degradation response for secretion' (TIDeRS) discloses a cellular mechanism by which nutrient and membrane sensing machineries cooperate to sustain Golgi-dependent protein secretion.

The knocking down of the oncoprotein Golgi phosphoprotein 3 in T98G cells of glioblastoma multiforme disrupts cell migration by affecting focal adhesion dynamics in a focal adhesion kinase-dependent manner.

Golgi phosphoprotein 3 (GOLPH3) is a conserved protein of the Golgi apparatus that in humans has been implicated in tumorigenesis. However, the precise function of GOLPH3 in malignant transformation is still unknown. Nevertheless, clinicopathological data shows that in more than a dozen kinds of cancer, including gliomas, GOLPH3 could be found overexpressed, which correlates with poor prognosis. Experimental data shows that overexpression of GOLPH3 leads to transformation of primary cells and to tumor growth enhancement. Conversely, the knocking down of GOLPH3 in GOLPH3-overexpressing tumor cells reduces tumorigenic features, such as cell proliferation and cell migration and invasion. The cumulative evidence indicate that GOLPH3 is an oncoprotein that promotes tumorigenicity by a mechanism that impact at different levels in different types of cells, including the sorting of Golgi glycosyltransferases, signaling pathways, and the actin cytoskeleton. How GOLPH3 connects mechanistically these processes has not been determined yet. Further studies are important to have a more complete understanding of the role of GOLPH3 as oncoprotein. Given the genetic diversity in cancer, a still outstanding aspect is how in this inherent heterogeneity GOLPH3 could possibly exert its oncogenic function. We have aimed to evaluate the contribution of GOLPH3 overexpression in the malignant phenotype of different types of tumor cells. Here, we analyzed the effect on cell migration that resulted from stable, RNAi-mediated knocking down of GOLPH3 in T98G cells of glioblastoma multiforme, a human glioma cell line with unique features. We found that the reduction of GOLPH3 levels produced dramatic changes in cell morphology, involving rearrangements of the actin cytoskeleton and reduction in the number and dynamics of focal adhesions. These effects correlated with decreased cell migration and invasion due to affected persistence and directionality of cell motility. Moreover, the knocking down of GOLPH3 also caused a reduction in autoactivation of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase that regulates focal adhesions. Our data support a model in which GOLPH3 in T98G cells promotes cell migration by stimulating the activity of FAK.

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.

Functional disruption of the Golgi apparatus protein ARF1 sensitizes MDA-MB-231 breast cancer cells to the antitumor drugs Actinomycin D and Vinblastine through ERK and AKT signaling.

Increasing evidence indicates that the Golgi apparatus plays active roles in cancer, but a comprehensive understanding of its functions in the oncogenic transformation has not yet emerged. At the same time, the Golgi is becoming well recognized as a hub that integrates its functions of protein and lipid biosynthesis to signal transduction for cell proliferation and migration in cancer cells. Nevertheless, the active function of the Golgi apparatus in cancer cells has not been fully evaluated as a target for combined treatment. Here, we analyzed the effect of perturbing the Golgi apparatus on the sensitivity of the MDA-MB-231 breast cancer cell line to the drugs Actinomycin D and Vinblastine. We disrupted the function of ARF1, a protein necessary for the homeostasis of the Golgi apparatus. We found that the expression of the ARF1-Q71L mutant increased the sensitivity of MDA-MB-231 cells to both Actinomycin D and Vinblastine, resulting in decreased cell proliferation and cell migration, as well as in increased apoptosis. Likewise, the combined treatment of cells with Actinomycin D or Vinblastine and Brefeldin A or Golgicide A, two disrupting agents of the ARF1 function, resulted in similar effects on cell proliferation, cell migration and apoptosis. Interestingly, each combined treatment had distinct effects on ERK1/2 and AKT signaling, as indicated by the decreased levels of either phospho-ERK1/2 or phospho-AKT. Our results suggest that disruption of Golgi function could be used as a strategy for the sensitization of cancer cells to chemotherapy.