Cellular Autophagy Machinery Research Articles

Sirtuin 3 governs autophagy-dependent glycolysis during Angiotensin II-induced endothelial-to-mesenchymal transition.

The impairment of autophagy can cause cellular metabolic perturbations involved in endothelial-to-mesenchymal transition (EndoMT). However, the interplay between the cellular autophagy machinery and endothelial metabolism remains elusive. Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, is a major cellular sensor of energy metabolism. The aim of this work was to determine the role of SIRT3-mediated autophagy in cellular metabolism and the process of EndoMT. We demonstrated that Angiotensin II (Ang II) led to defective autophagic flux and high levels of glycolysis in endothelial cells (ECs) accompanied by a loss of mitochondrial SIRT3 during EndoMT. The loss of SIRT3 further induced the hyperacetylation of endogenous autophagy-regulated gene 5 (ATG5), which in turn inhibited autophagosome maturation and increased pyruvate kinase M2 (PKM2) dimer expression. The M2 dimer is the less active form of PKM2, which drives glucose through aerobic glycolysis. Additionally, TEPP-46, a selective PKM2 tetramer activator, produced lower concentrations of lactate and led to the reduction of EndoMT both in vitro and in vivo. In parallel, the blockade of lactate influx from ECs into vascular smooth muscle cells (VSMCs) downregulated synthetic VSMC markers. EC-specific SIRT3 transgenic mice exhibited reduced endothelial cell transition but partial rescue of vascular fibrosis and collagen accumulation. Taken together, these findings reveal that SIRT3 regulates EndoMT by improving the autophagic degradation of PKM2. Pharmacological targeting of glycolysis metabolism may, therefore, represent an effective therapeutic strategy for hypertensive vascular remodeling.

Culprit or Bystander: Defective Mitophagy in Alzheimer's Disease.

Mitophagy is a selective engulfment and degradation of damaged mitochondria through the cellular autophagy machinery, a major mechanism responsible for mitochondrial quality control. Increased accumulation of damaged mitochondria in the Alzheimer’s disease (AD) human brain are evident, although underlying mechanisms largely elusive. Recent studies indicate impaired mitophagy may contribute to the accumulation of damaged mitochondria in cross-species AD animal models and in AD patient iPSC-derived neurons. Studies from AD highlight feed-forward vicious cycles between defective mitophagy, and the principal AD pathological hallmarks, including amyloid-β plaques, tau tangles, and inflammation. The concomitant and intertwined connections among those hallmarks of AD and the absence of a real humanized AD rodent model present a challenge on how to determine if defective mitophagy is an early event preceding and causal of Tau/Aβ proteinopathies. Whilst further studies are required to understand these relationships, targeting defective mitophagy holds promise as a new therapeutic strategy for AD.

COPII Vesicle Transport Is Required for Rotavirus NSP4 Interaction with the Autophagy Protein LC3 II and Trafficking to Viroplasms.

Many viruses that replicate in the cytoplasm dramatically remodel and stimulate the accumulation of host cell membranes for efficient replication by poorly understood mechanisms. For rotavirus, a critical step in virion assembly requires the accumulation of membranes adjacent to virus replication centers called viroplasms. Early electron microscopy studies describe viroplasm-associated membranes as "swollen" endoplasmic reticulum (ER). We previously demonstrated that rotavirus infection initiates cellular autophagy and that membranes containing the autophagy marker protein LC3 and the rotavirus ER-synthesized transmembrane glycoprotein NSP4 traffic to viroplasms, suggesting that NSP4 must exit the ER. This study aimed to address the mechanism of NSP4 exit from the ER and determine whether the viroplasm-associated membranes are ER derived. We report that (i) NSP4 exits the ER in COPII vesicles, resulting in disrupted COPII vesicle transport and ER exit sites; (ii) COPII vesicles are hijacked by LC3 II, which interacts with NSP4; and (iii) NSP4/LC3 II-containing membranes accumulate adjacent to viroplasms. In addition, the ER transmembrane proteins SERCA and calnexin were not detected in viroplasm-associated membranes, providing evidence that the rotavirus maturation process of "budding" occurs through autophagy-hijacked COPII vesicle membranes. These findings reveal a new mechanism for rotavirus maturation dependent on intracellular host protein transport and autophagy for the accumulation of membranes required for virus replication.IMPORTANCE In a morphogenic step that is exceedingly rare for nonenveloped viruses, immature rotavirus particles assemble in replication centers called viroplasms, and bud through cytoplasmic cellular membranes to acquire the outer capsid proteins for infectious particle assembly. Historically, the intracellular membranes used for particle budding were thought to be endoplasmic reticulum (ER) because the rotavirus nonstructural protein NSP4, which interacts with the immature particles to trigger budding, is synthesized as an ER transmembrane protein. This present study shows that NSP4 exits the ER in COPII vesicles and that the NSP4-containing COPII vesicles are hijacked by the cellular autophagy machinery, which mediates the trafficking of NSP4 to viroplasms. Changing the paradigm for rotavirus maturation, we propose that the cellular membranes required for immature rotavirus particle budding are not an extension of the ER but are COPII-derived autophagy isolation membranes.

The role of the unfolded protein response in dengue virus pathogenesis.

Symptomatic dengue virus (DENV) infections range from mild fever to severe haemorrhagic disease and death. Host-viral interactions play a significant role in deciding the fate of the infection. The unfolded protein response (UPR) is a prosurvival cellular reaction induced in response to DENV-mediated endoplasmic reticulum stress. The UPR has complex interactions with the cellular autophagy machinery, apoptosis, and innate immunity. DENV has evolved to manipulate the UPR to facilitate its replication and to evade host immunity. Our knowledge of this intertwined network of events is continuously developing. A better understanding of the UPR mediated antiviral and proviral effects will shed light on dengue disease pathogenesis and may help development of anti-DENV therapeutics. This review summarizes the role of the UPR in viral replication, autophagy, and DENV-induced inflammation to describe how a host response contributes to DENV pathogenesis.

HIV-1 Nef Employs Cellular Autophagy Machinery to Downregulate CD4

During HIV infection, expression of the viral protein, HIV-1 negative regulatory factor (Nef) leads to the lysosomal degradation of CD4 and other surface molecules. In addition, lysosome degradation through autophagy is upregulated. Here, we investigated whether autophagosomes participate in directing CD4 to lysosomes in Nef-expressing cells. Autophagosomes are membrane-bounded structures formed in response to specific stimuli (including starvation and buildup of ubiquitinated protein aggregates). They engulf various cytoplasmic components (i.e., damaged organelles, misfolded proteins and intracellular pathogens), and then fuse with lysosomes for degradation. We found that co-expression of Nef and CD4 in HeLa cells caused significant accumulation of the autophagosomal marker, microtubule-associated protein 1 light chain 3 (LC3) on highly motile subcellular vesicles. This occurred within 2-3 days of Nef expression and could be monitored by imaging of single cells or by flow cytometry in large cell populations. In the absence of Nef, CD4-expressing cells showed very few autophagosomes. LC3-containing vesicles resided in close proximity or overlapped with Nef-containing vesicles in cells co-expressing CD4 and Nef. Since Nef is known to trigger CD4 downregulation, we next looked to see if CD4 was associated with vesicles positive for LC3 and Nef. For this experiment, we inhibited autophagosome fusion with lysosomes using the drugs bafliomycin or chloroquine since this increases autophagosome number within cells. Both drug treatments led to extensive co-localization of CD4 in LC3-positive vesicles. When Nef-expressing cells were treated with 3-methyl adenine to inhibit autophagy, CD4 was still internalized from the plasma membrane but was retained in static endocytic structures rather than being transferred to highly dynamic structures as occurred without the inhibitor. These results suggest that cellular autophagy machinery is involved in transferring CD4 from the endosomal system to lysosomes during Nef-induced downregulation of this protein.

Novel role of VMP1 as modifier of the pancreatic tumor cell response to chemotherapeutic drugs

We hypothesized that inhibiting molecules that mediate the adaptation response to cellular stress can antagonize the resistance of pancreatic cancer cells to chemotherapeutic drugs. Toward this end, here, we investigated how VMP1, a stress-induced autophagy-associated protein, modulate stress responses triggered by chemotherapeutic agents in PDAC. We find that VMP1 is particularly over-expressed in poorly differentiated human pancreatic cancer. Pharmacological studies show that drugs that work, in part, via the endoplasmic reticulum stress response, induce VMP1 expression. Similarly, VMP1 is induced by known endoplasmic reticulum stress activators. Genetic inactivation of VMP1 using RNAi-based antagonize the pancreatic cancer stress response to antitumoral agents. Functionally, we find that VMP1 regulates both autophagy and chemotherapeutic resistance even in the presence of chloroquin, ATG5 or Beclin 1 siRNAs, or a Beclin 1-binding VMP1 mutant. In addition, VMP1 modulates endoplasmic reticulum stress independently of its coupling to the molecular and cellular autophagy machinery. Preclinical studies demonstrate that xenografts expressing an inducible and tractable form of VMP1 show increased resistance to the gemcitabine treatment. These results underscore a novel role for VMP1 as a potential therapeutic target for combinatorial therapies aimed at sensitizing pancreatic cancer cells to chemotherapeutic agents as well as provide novel molecular mechanisms to better understand this phenomenon.

“Strangles” in less regulated sectors of the Irish horse industry

as an important innate defense mechanism, particularly with respect to intracellular bacterial pathogens [1]. Recent studies have demonstrated that specific induction of autophagy can overcome inhibition of phagosomal maturation and lead to suppression of intracellularM. tuberculosis load, indicating promising clinical outcomes by enhancement of innate mechanisms of xenophagy [2]. Following autophagy induction, Beclin-1 and its binding partner class III phosphoinositide 3-kinase (PI3K) are necessary for initiation of autophagosome formation [3], inferred by detection of microtubule-associated protein light chain 3 (LC3), which exists as a cytosolic protein (LC3-I) until induction of autophagy whereupon a lipidated membrane bound protein is formed (LC3-II) [4]. For this study, 1 X 106 cells were seeded in T-25 flasks. Cells were infected with R.equi (moi 5) for 2, 6, 12, 24 and 48 hours. For each time point, duplicates were considered. After the infection, cells were collected and resuspended in 150 ml homogenization buffer, sonicated and centrifuged at 14,000 g for 20minutes at 40C. Following estimation of proteins through Bradford assay, proteins were separated by 10 15% SDS-PAGE. Membranes were incubated with 1:4000 Rabbit polyclonal LC3 (Abcam), 1:1000 Rabbit polyclonal Beclin-1 (Santa Cruz) and Mouse monoclonal 1:5000 b-actin (Sigma) primary antibodies. Later, the membranes were washed with PBS-T and incubated with IRdye 800 conjugated antirabbit (1:5000) and Alexa 680 conjugated anti-mouse (1:15,000) secondary antibodies for an hour. Western blot images were captured and analyzed with an Odyssey IR Imaging system (LICOR). Our data suggest the induction of cellular autophagy machinery in macrophages infected with R.equi. Understanding signaling pathways involved in the regulation of autophagy is important in developing new strategies to abrogate infection with R.equi.

Interplay between the cellular autophagy machinery and positive-stranded RNA viruses.

Autophagy is a conserved cellular process that acts as a key regulator in maintaining cellular homeostasis. Recent studies implicate an important role for autophagy in infection and immunity by removing invading pathogens and through modulating innate and adaptive immune responses. However, several pathogens, notably some positive-stranded RNA viruses, have subverted autophagy to their own ends. In this review, we summarize the current understanding of how viruses with a positive-stranded RNA genome interact with the host autophagy machinery to control their replication and spread. We review the mechanisms underlying the induction of autophagy and discuss the pro- and anti-viral functions of autophagy and the potential mechanisms involved.

Strategies for eliminating PrP c as substrate for prion conversion and for enhancing PrP Sc degradation

Prion diseases are fatal neurodegenerative infectious disorders for which no therapeutic or prophylactic regimens exist. Our work aims to eliminate PrP c as substrate for the conversion into PrP Sc and to increase the cellular clearance capacity of PrP Sc. In order to achieve the first objective, we used chemical compounds which interfere with the subcellular trafficking of PrP c, e.g. by intracellular re-routing. Recently, we found that PrP c requires cholesterol for cell surface localisation. Treatment with mevinolin significantly reduced the amount of cell surface PrP c and led to its accumulation in the Golgi compartment. These data show that cholesterol is essential for the cell surface localisation of PrP c, which is in turn known to be necessary for the formation of PrP Sc. Another anti-prion strategy uses RNA and peptide aptamers directed against PrP c. We have selected peptide aptamers using a constrained peptide library presented on the active site loop of the Escherichia coli protein TrxA in a Y2H screen. Several peptides reproducibly binding to PrP c in several assays were identified. Preliminary data indicate that selected peptide aptamers are able to interfere with prion propagation in prion-infected cells. To obtain additive effects we have tried to clarify cellular mechanisms that enable cells to clear prion infectivity. This goal was achieved by selective interference in intracellular signalling pathways which apparently also increase the cellular autophagy machinery. Finally, we have tried to establish an active auto-vaccination approach directed against PrP, which gave some positive preliminary results in the mouse system. This might open the door to classical immunological interference techniques.

The anticancer drug imatinib induces cellular autophagy

The tyrosine kinase inhibitor imatinib (Gleevec, Novartis Pharmaceuticals Corporation; Basel, Switzerland) is a powerful drug for treatment of chronic myelogenous leukemia (CML) and other malignancies. It selectively targets various tyrosine kinases, thereby leading to growth arrest of respective cancer cells. Given its wide application, it is of high importance to know all related underlying molecular mechanisms. We had previously found that imatinib increases the cellular clearance of intracellular protein aggregates by targeting the abl pathway and thereby upregulating lysosomal activity. Here, we describe that imatinib dose dependently activates the cellular autophagy machinery in mammalian cells, independently of tissue type, species origin or immortalization status of cells. Autophagy is an archetypical cellular degradation mechanism implicated in many physiological and pathophysiological conditions. Our data link for the first time the process of autophagy with the mode of action of imatinib. Induction of autophagy might represent an additional mechanism of imatinib to induce growth arrest, promote apoptosis in cancer cells and eventually even promote tumour regression.

Cellular Autophagy Machinery is not Required for Vaccinia Virus Replication and Maturation

The origin of the primary membrane of vaccinia virus, the double-membrane structure that surrounds the immature virions (IV), is not fully understood. Here we investigated whether the primary membrane is of autophagy origin. Morphologic studies by electron microscopy showed no apparent difference in viral maturation in the autophagy deficient cell lines, the Atg5-/- mouse embryonic fibroblast (MEF) and the Beclin 1 -/- embryonic stem (ES) cells, compared to their isogenic wild-type counterparts. Moreover, quantitative assays indicated that the viruses replicate and maturate in the autophagy deficient cell lines as efficiently as they do in the corresponding isogenic wild type cells. This study indicates that the cellular autophagy machinery is not required for the life-cycle of vaccinia virus, suggesting that the primary vaccinia viral membrane likely does not originate from the autophagic membrane.