Autophagy and proteins involved in vesicular trafficking

Amaya, Celina; Fader, Claudio Marcelo; Colombo, María Isabel

doi:10.1016/j.febslet.2015.09.021

Cited by 87 publications

(79 citation statements)

References 141 publications

(178 reference statements)

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“…The importance of monocyte-macrophages as viral reservoirs, vehicles for microbial dissemination, secretors The Journal of Clinical Investigation (59), and such protein rearrangements are autophagy regulated (14,60). Thus, both processes are affected by nanoART and URMC-099.…”

Section: Discussionmentioning

confidence: 99%

“…However, the production of more hydrophobic and protein-bound ARVs provides only a partial solution. Controlling the dynamics of particle trafficking inside the cell and within early, recycling, and late endosomes is necessary to sustain intracellular drug particle depots and limit hepatic and renal metabolism and drug excretion (4,6,11,14,15).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Autophagy facilitates macrophage depots of sustained-release nanoformulated antiretroviral drugs

Gnanadhas¹,

Dash²,

Sillman³

et al. 2017

Journal of Clinical Investigation

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Long-acting anti-HIV products can substantively change the standard of care for patients with HIV/AIDS. To this end, hydrophobic antiretroviral drugs (ARVs) were recently developed for parenteral administration at monthly or longer intervals. While shorter-acting hydrophilic drugs can be made into nanocarrier-encased prodrugs, the nanocarrier encasement must be boosted to establish long-acting ARV depots. The mixed-lineage kinase 3 (MLK-3) inhibitor URMC-099 provides this function by affecting autophagy. Here, we have shown that URMC-099 facilitates ARV sequestration and its antiretroviral responses by promoting the nuclear translocation of the transcription factor EB (TFEB). In monocyte-derived macrophages, URMC-099 induction of autophagy led to retention of nanoparticles containing the antiretroviral protease inhibitor atazanavir. These nanoparticles were localized within macrophage autophagosomes, leading to a 4-fold enhancement of mitochondrial and cell vitality. In rodents, URMC-099 activation of autophagy led to 50-fold increases in the plasma drug concentration of the viral integrase inhibitor dolutegravir. These data paralleled URMC-099-mediated induction of autophagy and the previously reported antiretroviral responses in HIV-1-infected humanized mice. We conclude that pharmacologic induction of autophagy provides a means to extend the action of a long-acting, slow, effective release of antiretroviral therapy. Center for Neural Development and Disease, University of Rochester Medical Center (URMC), Rochester, New York, USA. Conflict of interest:H.A. Gelbard and H.E. Gendelman are members of the scientific advisory board for WavoDyne Therapeutics Inc., which is developing URMC-099 as a firstin-class MLK-3 inhibitor for clinical trials. URMC-099 is owned by URMC (patent nos. US 8,846,909 B2;8,877,772;and 9,181,247 and associated international patents). tion of TFEB mRNA by approximately 4-to 8-fold under different treatment conditions ( Figure 2F). With HIV-1 infection, TFEB mRNA levels were reduced by 2-to 3-fold compared with levels in uninfected controls, and URMC-099 could recover the phenotype. Increased TFEB translocation was delayed and did not reach significant levels until day 7 after URMC-099 treatment (Supplemental Figure 2B). These data confirm that URMC-099 regulates TFEB nuclear translocation in an mTORC1-dependent manner. URMC-099 stimulates autophagy in human MDMs. On the basis of the preceding data sets, we theorized that nuclear translocation of TFEB induces autophagy and lysosomal biogenesis. TFEB serves as the master regulator of both autophagy and lysosomal biogenesis (26), and such regulation could have a profound role in nanoART intracellular sequestration in MDMs. Thus, we examined the autophagosome markers microtubule-associated protein 1 light chain 3 β (LC3B, also known as MAP1LC3B) and beclin 1 (BECN1). Using Western blot assays, we found that each marker was significantly upregulated by URMC-099 ( Figure 3, A-C and Supplemental Figure 3A). URMC-099 increased the expression ...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Autophagy facilitates macrophage depots of sustained-release nanoformulated antiretroviral drugs

Gnanadhas¹,

Dash²,

Sillman³

et al. 2017

Journal of Clinical Investigation

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“…During autophagy, damaged proteins and/or organelles are engulfed within double-membrane vesicles known as autophagosomes and delivered to late endosomes and/or lysosomes for degradation (Amaya et al, 2015). Autophagy is regulated by over 30 identified autophagy-related ( Atg ) genes (Feng et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Sphingosine Kinase 1 Cooperates with Autophagy to Maintain Endocytic Membrane Trafficking

et al. 2016

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Summary SphK1 associates with early endocytic membranes during endocytosis; however, the role of sphingosine or sphingosine-1-phosphate as the critical metabolite in endocytic trafficking has not been established. Here, we demonstrate that the recruitment of SphK1 to sphingosine-enriched endocytic vesicles and the generation of sphingosine-1-phosphate facilitate membrane trafficking along the endosomal pathway. Exogenous sphingosine and sphingosine-based SphK1 inhibitors induce the SphK1-dependent fusion of endosomal membranes to accumulate enlarged late endosomes and amphisomes enriched in sphingolipids. Interestingly, SphK1 also appears to facilitate endosomal fusion independent of its catalytic activity, as catalytically inactive SphK1G82D is recruited to endocytic membranes by sphingosine or sphingosine-based SphK1 inhibitor and promotes membrane fusion. Furthermore, we reveal that the clearance of enlarged endosomes is dependent on the activity of ceramide synthase, lysosomal biogenesis and the restoration of autophagic flux. Collectively, these studies uncover intersecting roles for SphK1, sphingosine and autophagic machinery in endocytic membrane trafficking.

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“…In this context, autophagy is a highly conserved intracellular degradation system that orchestrates the degradation of cellular constituents and the removal of damaged cellular constituents and organelles by action of lysosomes (Amaya et al, 2015;Bolt and Klimecki, 2012). During autophagy, subcellular membranes undergo rearrangement to sequester a portion of the cytoplasm and organelles into an autophagosome, which is then transported to the lysosome for proteolysis of the sequestered materials (Klionsky & Emr, 2000).…”

Section: Introductionmentioning

confidence: 99%

An autophagic process is activated in HepG2 cells to mediate BDE-100-induced toxicity

et al. 2017

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To reduce flammability and meet regulatory requirements, Brominated Flame Retardants (BFRs) are added to a wide variety of consumer products including furniture, textiles, electronics, and construction materials. Exposure to polybrominated phenyl ethers (PBDEs) adversely affects the human health. Bearing in mind that (i) PBDEs are potentially toxic, (ii) the mechanism of PBDE toxicity is unclear, and (iii) the importance of the autophagy to the field of toxicology is overlooked, this study investigates whether an autophagic process is activated in HepG2 cells (human hepatoblastoma cell line) to mediate BDE-100-induced toxicity. HepG2 cells were exposed with BDE-100 at three concentrations (0.1, 5, and 25μM), selected from preliminary toxicity tests, for 24 and 48h. To assess autophagy, immunocytochemistry was performed after exposure of HepG2 cells to BDE-100. Labeling of HepG2 cells with 100nM LysoTracker Red DND-99 aided examination of lysosome distribution. Proteins that are key to the autophagic process (p62 and LC3) were evaluated by western blotting. DNA was isolated and quantified to assess mitochondrial DNA copy number by qPCR on the basis of the number of DNA copies of a mitochondrial encoded gene normalized against a nuclear encoded gene. Conversion of LC3-I to LC3-II increased in HepG2 cells. Pre-addition of 100nM wortmannin decreased the amount of LC3 in the punctuate form and increased nuclear fragmentation (apoptotic feature). HepG2 cells exposed to BDE-100 presented increased staining with the lysosomal dye and had larger LC3 and p62 content after pre-treatment with ammonium chloride. The mitochondrial DNA copy number decreased, which probably constituted an attempt of the cell to manage mitochondrial damage by selective mitochondrial degradation (mitophagy). In conclusion, an autophagic process is activated in HepG2 cells to mediate BDE-100-induced toxicity.

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Autophagy and proteins involved in vesicular trafficking

Cited by 87 publications

References 141 publications

Autophagy facilitates macrophage depots of sustained-release nanoformulated antiretroviral drugs

Autophagy facilitates macrophage depots of sustained-release nanoformulated antiretroviral drugs

Sphingosine Kinase 1 Cooperates with Autophagy to Maintain Endocytic Membrane Trafficking

An autophagic process is activated in HepG2 cells to mediate BDE-100-induced toxicity

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