The Combination of Gold and Silver Food Nanoparticles with Gluten Peptides Alters the Autophagic Pathway in Intestinal Crypt-like Cells.

Metallic nanoparticles (mNPs) are widely used as food additives and can interact with gliadin triggering an immune response, but evaluation of the effects on crypts, hypertrophic in celiac subjects, is still lacking. This study evaluated the effects of gold and silver mNPs in combination with gliadin on crypt-like cells (HIEC-6). Transmission electron microscopy (TEM) was used to evaluate gliadin-mNP aggregates in cells. Western blot and immunofluorescence analysis assessed autophagy-related molecule levels (p62, LC3, beclin-1, EGFR). Lysosome functionality was tested with acridine orange (AO) and Magic Red assays. TEM identified an increase in autophagic vacuoles after exposure to gliadin + mNPs, as also detected by significant increments in LC3-II and p62 expression. Immunofluorescence confirmed the presence of mature autophagosomes, showing LC3 and p62 colocalization, indicating an altered autophagic flux, further assessed with EGFR degradation, AO and Magic Red assays. The results showed a significant reduction in lysosomal enzyme activity and a modest reduction in acidity. Thus, gliadin + mNPs can block the autophagic flux inducing a lysosomal defect. The alteration of this pathway, essential for cell function, can lead to cell damage and death. The potential effects of this copresence in food should be further characterized to avoid a negative impact on celiac disease subjects.

Interaction of the sorting nexin 25 homologue Snazarus with Rab11 balances endocytic and secretory transport and maintains the ultrafiltration diaphragm in nephrocytes.

Proper balance of exocytosis and endocytosis is important for the maintenance of plasma membrane lipid and protein homeostasis. This is especially critical in human podocytes and the podocyte-like Drosophila nephrocytes that both use a delicate diaphragm system with evolutionarily conserved components for ultrafiltration. Here, we show that the sorting nexin 25 homologue Snazarus (Snz) binds to Rab11 and localizes to Rab11-positive recycling endosomes in Drosophila nephrocytes, unlike in fat cells where it is present in plasma membrane/lipid droplet/endoplasmic reticulum contact sites. Loss of Snz leads to redistribution of Rab11 vesicles from the cell periphery and increases endocytic activity in nephrocytes. These changes are accompanied by defects in diaphragm protein distribution that resemble those seen in Rab11 gain-of-function cells. Of note, co-overexpression of Snz rescues diaphragm defects in Rab11 overexpressing cells, whereas snz knockdown in Rab11 overexpressing nephrocytes or simultaneous knockdown of snz and tbc1d8b encoding a Rab11 GTPase-activating protein (GAP) leads to massive expansion of the lacunar system that contains mislocalized diaphragm components: Sns and Pyd/ZO-1. We find that loss of Snz enhances while its overexpression impairs secretion, which, together with genetic epistasis analyses, suggest that Snz counteracts Rab11 to maintain the diaphragm via setting the proper balance of exocytosis and endocytosis.

Gemcitabine promotes autophagy and lysosomal function through ERK- and TFEB-dependent mechanisms.

Gemcitabine is a first-line treatment agent for pancreatic ductal adenocarcinoma (PDAC). Contributing to its cytotoxicity, this chemotherapeutic agent is primarily a DNA replication inhibitor that also induces DNA damage. However, its therapeutic effects are limited owing to chemoresistance. Evidence in the literature points to a role for autophagy in restricting the efficacy of gemcitabine. Autophagy is a catabolic process in which intracellular components are delivered to degradative organelles lysosomes. Interfering with this process sensitizes PDAC cells to gemcitabine. It is consequently inferred that autophagy and lysosomal function need to be tightly regulated to maintain homeostasis and provide resistance to environmental stress, such as those imposed by chemotherapeutic drugs. However, the mechanism(s) through which gemcitabine promotes autophagy remains elusive, and the impact of gemcitabine on lysosomal function remains largely unexplored. Therefore, we applied complementary approaches to define the mechanisms triggered by gemcitabine that support autophagy and lysosome function. We found that gemcitabine elicited ERK-dependent autophagy in PDAC cells, but did not stimulate ERK activity or autophagy in non-tumoral human pancreatic epithelial cells. Gemcitabine also promoted transcription factor EB (TFEB)-dependent lysosomal function in PDAC cells. Indeed, treating PDAC cells with gemcitabine caused expansion of the lysosomal network, as revealed by Lysosome associated membrane protein-1 (LAMP1) and LysoTracker staining. More specific approaches have shown that gemcitabine promotes the activity of cathepsin B (CTSB), a cysteine protease playing an active role in lysosomal degradation. We showed that lysosomal function induced by gemcitabine depends on TFEB, the master regulator of autophagy and lysosomal biogenesis. Interfering with TFEB function considerably limited the clonogenic growth of PDAC cells and hindered the capacity of TFEB-depleted PDAC cells to develop orthotopic tumors.

Autophagy in cell fate decisions: knowledge gained from Drosophila.

Autophagy is an important process that maintains adult tissue homeostasis and functions by protecting cells in autonomous and non-cell-autonomous ways. By degrading toxic components or proteins involved in cell signaling pathways, autophagy preserves the balance among stem cells, progenitors, and differentiated cells in various tissues. In this minireview, we discuss recent studies performed in Drosophila that highlight new roles of autophagy in adult cell fate decisions, including quiescence, proliferation, differentiation, and death.

Rab21 in enterocytes participates in intestinal epithelium maintenance.

Membrane trafficking is defined as the vesicular transport of proteins into, out of, and throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways result in impaired function and are linked to diseases. However, how these trafficking pathways regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo system, we investigated enterocyte-specific functions for the early endosomal machinery. We focused on Rab21, which regulates specific steps in early endosomal trafficking. Depletion of Rab21 in enterocytes led to abnormalities in intestinal morphology, with deregulated cellular equilibrium associated with a gain in mitotic cells and increased cell death. Increases in apoptosis and Yorkie signaling were responsible for compensatory proliferation and tissue inflammation. Using an RNA interference screen, we identified regulators of autophagy and membrane trafficking that phenocopied Rab21 knockdown. We further showed that Rab21 knockdown-induced hyperplasia was rescued by inhibition of epidermal growth factor receptor signaling. Moreover, quantitative proteomics identified proteins affected by Rab21 depletion. Of these, we validated changes in apolipoprotein ApoLpp and the trehalose transporter Tret1-1, indicating roles for enterocyte Rab21 in lipid and carbohydrate homeostasis, respectively. Our data shed light on an important role for early endosomal trafficking, and Rab21, in enterocyte-mediated intestinal epithelium maintenance.

Regulation of Endosomal Sorting and Maturation by ER-Endosome Contact Sites.

Endosomes are a heterogeneous population of intracellular organelles responsible for sorting, recycling, or transporting internalized materials for degradation. Endosomal sorting and maturation are controlled by a complex interplay of regulators, with RAB GTPases and phosphoinositides playing key roles. In this decade, another layer of regulation surfaced with the role played by membrane contact sites between the endoplasmic reticulum (ER) and endosomes. Specific regulators of ER-endosome contact sites or proteins localized at these sites are emerging as modulators of this complex endosomal ballet. In particular, lipid transfer or recruitment of various complexes and enzymes at ER-endosome contact sites play an active role in endosome sorting, scission, and maturation. In this short review, we focus on studies describing ER-endosome contact sites in these three endosomal processes.

Snazarus and its human ortholog SNX25 modulate autophagic flux.

Macroautophagy, the degradation and recycling of cytosolic components in the lysosome, is an important cellular mechanism. It is a membrane-mediated process that is linked to vesicular trafficking events. The sorting nexin (SNX) protein family controls the sorting of a large array of cargoes, and various SNXs impact autophagy. To improve our understanding of their functions in vivo, we screened all Drosophila SNXs using inducible RNA interference in the fat body. Significantly, depletion of Snazarus (Snz) led to decreased autophagic flux. Interestingly, we observed altered distribution of Vamp7-positive vesicles with Snz depletion, and the roles of Snz were conserved in human cells. SNX25, the closest human ortholog to Snz, regulates both VAMP8 endocytosis and lipid metabolism. Through knockout-rescue experiments, we demonstrate that these activities are dependent on specific SNX25 domains and that the autophagic defects seen upon SNX25 loss can be rescued by ethanolamine addition. We also demonstrate the presence of differentially spliced forms of SNX14 and SNX25 in cancer cells. This work identifies a conserved role for Snz/SNX25 as a regulator of autophagic flux and reveals differential isoform expression between paralogs.

The FUS gene is dual-coding with both proteins contributing to FUS-mediated toxicity.

Novel functional coding sequences (altORFs) are camouflaged within annotated ones (CDS) in a different reading frame. We show here that an altORF is nested in the FUS CDS, encoding a conserved 170 amino acid protein, altFUS. AltFUS is endogenously expressed in human tissues, notably in the motor cortex and motor neurons. Over-expression of wild-type FUS and/or amyotrophic lateral sclerosis-linked FUS mutants is known to trigger toxic mechanisms in different models. These include inhibition of autophagy, loss of mitochondrial potential and accumulation of cytoplasmic aggregates. We find that altFUS, not FUS, is responsible for the inhibition of autophagy, and pivotal in mitochondrial potential loss and accumulation of cytoplasmic aggregates. Suppression of altFUS expression in a Drosophila model of FUS-related toxicity protects against neurodegeneration. Some mutations found in ALS patients are overlooked because of their synonymous effect on the FUS protein. Yet, we show they exert a deleterious effect causing missense mutations in the overlapping altFUS protein. These findings demonstrate that FUS is a bicistronic gene and suggests that both proteins, FUS and altFUS, cooperate in toxic mechanisms.

A Novel Proteomic Method Reveals NLS Tagging of T-DM1 Contravenes Classical Nuclear Transport in a Model of HER2-Positive Breast Cancer.

The next breakthrough for protein therapeutics is effective intracellular delivery and accumulation within target cells. Nuclear localization signal (NLS)-tagged therapeutics have been hindered by the lack of efficient nuclear localization due to endosome entrapment. Although development of strategies for tagging therapeutics with technologies capable of increased membrane penetration has resulted in proportional increased potency, nonspecific membrane penetration limits target specificity and, hence, widespread clinical success. There is a long-standing idea that nuclear localization of NLS-tagged agents occurs exclusively via classical nuclear transport. In the present study, we modified the antibody-drug conjugate trastuzumab-emtansine (T-DM1) with a classical NLS linked to cholic acid (cell accumulator [Accum]) that enables modified antibodies to escape endosome entrapment and increase nuclear localization efficiency without abrogating receptor targeting. In parallel, we developed a proteomics-based method to evaluate nuclear transport. Accum-modified T-DM1 significantly enhanced cytotoxic efficacy in the human epidermal growth factor receptor 2 (HER2)-positive SKBR3 breast cancer system. We discovered that efficacy was dependent on the nonclassical importin-7. Our evaluation reveals that when multiple classical NLS tagging occurs, cationic charge build-up as opposed to sequence dominates and becomes a substrate for importin-7. This study results in an effective target cell-specific NLS therapeutic and a general approach to guide future NLS-based development initiatives.

Rabs in Signaling and Embryonic Development.

Rab GTPases play key roles in various cellular processes. They are essential, among other roles, to membrane trafficking and intracellular signaling events. Both trafficking and signaling events are crucial for proper embryonic development. Indeed, embryogenesis is a complex process in which cells respond to various signals and undergo dramatic changes in their shape, position, and function. Over the last few decades, cellular studies have highlighted the novel signaling roles played by Rab GTPases, while numerous studies have shed light on the important requirements of Rab proteins at various steps of embryonic development. In this review, we aimed to generate an overview of Rab contributions during animal embryogenesis. We first briefly summarize the involvement of Rabs in signaling events. We then extensively highlight the contribution of Rabs in shaping metazoan development and conclude with new approaches that will allow investigation of Rab functions in vivo.

L-type prostaglandin D synthase regulates the trafficking of the PGD2 DP1 receptor by interacting with the GTPase Rab4.

Accumulating evidence indicates that G protein-coupled receptors (GPCRs) interact with Rab GTPases during their intracellular trafficking. How GPCRs recruit and activate the Rabs is unclear. Here, we report that depletion of endogenous L-type prostaglandin D synthase (L-PGDS) in HeLa cells inhibited recycling of the prostaglandin D2 (PGD2) DP1 receptor (DP1) to the cell surface after agonist-induced internalization and that L-PGDS overexpression had the opposite effect. Depletion of endogenous Rab4 prevented l-PGDS-mediated recycling of DP1, and l-PGDS depletion inhibited Rab4-dependent recycling of DP1, indicating that both proteins are mutually involved in this pathway. DP1 stimulation promoted its interaction through its intracellular C terminus with Rab4, which was increased by l-PGDS. Confocal microscopy revealed that DP1 activation induces l-PGDS/Rab4 co-localization. l-PGDS/Rab4 and DP1/Rab4 co-immunoprecipitation levels were increased by DP1 agonist treatment. Pulldown assays with purified GST-l-PGDS and His6-Rab4 indicated that both proteins interact directly. l-PGDS interacted preferentially with the inactive, GDP-locked Rab4S22N variant rather than with WT Rab4 or with constitutively active Rab4Q67L proteins. Overexpression and depletion experiments disclosed that l-PGDS partakes in Rab4 activation following DP1 stimulation. Experiments with deletion mutants and synthetic peptides revealed that amino acids 85-92 in l-PGDS are involved in its interaction with Rab4 and in its effect on DP1 recycling. Of note, GTPγS loading and time-resolved FRET assays with purified proteins suggested that l-PGDS enhances GDP-GTP exchange on Rab4. Our results reveal how l-PGDS, which produces the agonist for DP1, regulates DP1 recycling by participating in Rab4 recruitment and activation.

Pleiotropic role of Drosophila phosphoribosyl pyrophosphate synthetase in autophagy and lysosome homeostasis.

Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme whose function is important for the biosynthesis of purines, pyrimidines, and pyridines. Importantly, while missense mutations of PRPS1 have been identified in neurological disorders such as Arts syndrome, how they contribute to neuropathogenesis is still unclear. We identified the Drosophila ortholog of PRPS (dPRPS) as a direct target of RB/E2F in Drosophila, a vital cell cycle regulator, and engineered dPRPS alleles carrying patient-derived mutations. Interestingly, while they are able to develop normally, dPRPS mutant flies have a shortened lifespan and locomotive defects, common phenotypes associated with neurodegeneration. Careful analysis of the fat body revealed that patient-derived PRPS mutations result in profound defects in lipolysis, macroautophagy, and lysosome function. Significantly, we show evidence that the nervous system of dPRPS mutant flies is affected by these defects. Overall, we uncovered an unexpected link between nucleotide metabolism and autophagy/lysosome function, providing a possible mechanism by which PRPS-dysfunction contributes to neurological disorders.

Drosophila Snazarus Regulates a Lipid Droplet Population at Plasma Membrane-Droplet Contacts in Adipocytes.

Adipocytes store nutrients as lipid droplets (LDs), but how they organize their LD stores to balance lipid uptake, storage, and mobilization remains poorly understood. Here, using Drosophila fat body (FB) adipocytes, we characterize spatially distinct LD populations that are maintained by different lipid pools. We identify peripheral LDs (pLDs) that make close contact with the plasma membrane (PM) and are maintained by lipophorin-dependent lipid trafficking. pLDs are distinct from larger cytoplasmic medial LDs (mLDs), which are maintained by FASN1-dependent de novo lipogenesis. We find that sorting nexin CG1514 or Snazarus (Snz) associates with pLDs and regulates LD homeostasis at ER-PM contact sites. Loss of SNZ perturbs pLD organization, whereas Snz over-expression drives LD expansion, triacylglyceride production, starvation resistance, and lifespan extension through a DESAT1-dependent pathway. We propose that Drosophila adipocytes maintain spatially distinct LD populations and identify Snz as a regulator of LD organization and inter-organelle crosstalk.

Colorectal cancer cells respond differentially to autophagy inhibition in vivo.

Autophagy has both tumor-promoting and -suppressing effects in cancer, including colorectal cancer (CRC), with transformed cells often exhibiting high autophagic flux. In established tumors, autophagy inhibition can lead to opposite responses resulting in either tumor cell death or hyperproliferation. The functional mechanisms underlying these differences are poorly understood. The present study aimed to investigate the relationship between the autophagic capacities of CRC cells and their sensitivities to autophagy inhibition. All studied CRC cell lines showed high basal autophagic flux. However, only HCT116 and Caco-2/15 cells displayed regulated autophagic flux upon starvation. Knockdown of ATG5 (which disrupts autophagosome elongation) or RAB21 (which decreases autophagosome/lysosome fusion) had little effect on CRC cell proliferation in vitro. Nonetheless, inhibition of autophagy in vivo had a substantial cell line-dependent impact on tumor growth, with some cells displaying decreased (HCT116 and Caco-2/15) or increased (SW480 and LoVo) proliferation. RNA sequencing and Western blot analyses in hyperproliferative SW480 tumors revealed that the mTORC2 and AKT pathways were hyperactivated following autophagy impairment. Inhibition of either mTOR or AKT activities rescued the observed hyperproliferation in autophagy-inhibited SW480 and reduced tumor growth. These results highlight that autophagy inhibition can lead, in specific cellular contexts, to compensatory mechanisms promoting tumor growth.

RAB21 interacts with TMED10 and modulates its localization and abundance.

Membrane trafficking controls vesicular transport of cargo between cellular compartments. Vesicular trafficking is essential for cellular homeostasis and dysfunctional trafficking is linked to several pathologies such as neurodegenerative diseases. Following endocytosis, early endosomes act as sorting stations of internalized materials, routing cargo toward various fates. One important class of membrane trafficking regulators are RAB GTPases. RAB21 has been associated with multiple functions and regulates integrin internalization, endosomal sorting of specific clathrin-independent cargo and autophagy. Although RAB21 is mostly associated with early endosomes, it has been shown to mediate a specific sorting event at the Golgi. From mass spectrometry data, we identified a GTP-favored interaction between RAB21 and TMED10 and 9, essential regulators of COPI and COPII vesicles. Using RAB21 knockout cells, we describe the role of RAB21 in modulating TMED10 Golgi localization. Taken together, our study suggests a new potential function of RAB21 in modulating TMED10 trafficking, with relevance to neurodegenerative disorders.

APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting.

RAB GTPases are central modulators of membrane trafficking. They are under the dynamic regulation of activating guanine exchange factors (GEFs) and inactivating GTPase-activating proteins (GAPs). Once activated, RABs recruit a large spectrum of effectors to control trafficking functions of eukaryotic cells. Multiple proteomic studies, using pull-down or yeast two-hybrid approaches, have identified a number of RAB interactors. However, due to the in vitro nature of these approaches and inherent limitations of each technique, a comprehensive definition of RAB interactors is still lacking. By comparing quantitative affinity purifications of GFP:RAB21 with APEX2-mediated proximity labeling of RAB4a, RAB5a, RAB7a, and RAB21, we find that APEX2 proximity labeling allows for the comprehensive identification of RAB regulators and interactors. Importantly, through biochemical and genetic approaches, we establish a novel link between RAB21 and the WASH and retromer complexes, with functional consequences on cargo sorting. Hence, APEX2-mediated proximity labeling of RAB neighboring proteins represents a new and efficient tool to define RAB functions.

Autophagic Flux Assessment in Colorectal Cancer Cells.

Autophagy protects colorectal cancer cells against therapeutic intervention. Autophagy is a continuous process, and autophagic flux requires both autophagosome synthesis and their subsequent degradation at lysosomes. Hence, cells with elevated autophagic flux display both rapid autophagosome generation and degradation. Here, we describe an immunoblot protocol coupled to pharmaceutical inhibition of autophagosome clearance to monitor autophagic flux levels between colorectal cancer cell lines.

NLS-Cholic Acid Conjugation to IL-5Rα-Specific Antibody Improves Cellular Accumulation and In Vivo Tumor-Targeting Properties in a Bladder Cancer Model.

Receptor-mediated internalization followed by trafficking and degradation of antibody-conjugates (ACs) via the endosomal-lysosomal pathway is the major mechanism for delivering molecular payloads inside target tumor cells. Although a mainstay for delivering payloads with clinically approved ACs in cancer treatment and imaging, tumor cells are often able to decrease intracellular payload concentrations and thereby reduce the effectiveness of the desired application. Thus, increasing payload intracellular accumulation has become a focus of attention for designing next-generation ACs. We developed a composite compound (ChAcNLS) that enables ACs to escape endosome entrapment and route to the nucleus resulting in the increased intracellular accumulation as an interleukin-5 receptor α-subunit (IL-5Rα)-targeted agent for muscle invasive bladder cancer (MIBC). We constructed 64Cu-A14-ChAcNLS, 64Cu-A14-NLS, and 64Cu-A14 and evaluated their performance by employing mechanistic studies for endosome escape coupled to nuclear routing and determining whether this delivery system results in improved 64Cu cellular accumulation. ACs consisting of ∼20 ChAcNLS or NLS moieties per 64Cu-A14 were prepared in good yield, high monomer content, and maintaining high affinity for IL-5Rα. Confocal microscopy analysis demonstrated ChAcNLS mediated efficient endosome escape and nuclear localization. 64Cu-A14-ChAcNLS increased 64Cu cellular accumulation in HT-1376 and HT-B9 cells relative to 64Cu-A14 and 64Cu-A14-NLS. In addition, we tested 64Cu-A14-ChAcNLS in vivo to evaluate its tissue distribution properties and, ultimately, tumor uptake and targeting. A model of human IL-5Rα MIBC was developed by implanting NOD/SCID mice with subcutaneous HT-1376 or HT-B9MIBC tumors, which grow containing high and low IL-5Rα-positive tumor cell densities, respectively. ACs were intravenously injected, and daily blood sampling, biodistribution at 48 and 96 h, and positron emission tomography (PET) at 24 and 48 h were performed. Region of interest (ROI) analysis was also performed on reconstructed PET images. Pharmacokinetic analysis and biodistribution studies showed that 64Cu-A14-ChAcNLS had faster clearance rates from the blood and healthy organs relative to 64Cu-A14. However, 64Cu-A14-ChAcNLS maintained comparable tumor accumulation relative to 64Cu-A14. This resulted in 64Cu-A14-ChAcNLS having superior tumor/normal tissue ratios at both 48 and 96 h biodistribution time points. Visualization of AC distribution by PET and ROI analysis confirmed that 64Cu-A14-ChAcNLS had improved targeting of MIBC tumor relative to 64Cu-A14. In addition, 64Cu-A14 modified with only NLS had poor tumor targeting. This was a result of poor tumor uptake due to extremely rapid clearance. Thus, the overall findings in this model of human IL-5Rα-positive MIBC describe an endosome escape-nuclear localization cholic-acid-linked peptide that substantially enhances AC cellular accumulation and tumor targeting.

Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy.

Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.