Tumour-specific activation of a tumour-blood transport improves the diagnostic accuracy of blood tumour markers in mice.

BACKGROUND: The accuracy of blood-based early tumour recognition is compromised by signal production at non-tumoral sites, low amount of signal produced by small tumours, and variable tumour production. Here we examined whether tumour-specific enhancement of vascular permeability by the particular tumour homing peptide, iRGD, which carries dual function of binding to integrin receptors overexpressed in the tumour vasculature and is known to promote extravasation via neuropilin-1 receptor upon site-specific cleavage, might be useful to improve blood-based tumour detection by inducing a yet unrecognised vice versa tumour-to-blood transport. METHODS: To detect an iRGD-induced tumour-to-blood transport, we examined the effect of intravenously injected iRGD on blood levels of α-fetoprotein (AFP) and autotaxin in several mouse models of hepatocellular carcinoma (HCC) or in mice with chronic liver injury without HCC, and on prostate-specific antigen (PSA) levels in mice with prostate cancer. FINDINGS: Intravenously injected iRGD rapidly and robustly elevated the blood levels of AFP in several mouse models of HCC, but not in mice with chronic liver injury. The effect was primarily seen in mice with small tumours and normal basal blood AFP levels, was attenuated by an anti-neuropilin-1 antibody, and depended on the concentration gradient between tumour and blood. iRGD treatment was also able to increase blood levels of autotaxin in HCC mice, and of PSA in mice with prostate cancer. INTERPRETATION: We conclude that iRGD induces a tumour-to-blood transport in a tumour-specific fashion that has potential of improving diagnosis of early stage cancer. FUNDING: Deutsche Krebshilfe, DKTK, LOEWE-Frankfurt Cancer Institute.

TBC1D2B undergoes phase separation and mediates autophagy initiation.

Small ubiquitin-like modifiers from the ATG8 family regulate autophagy initiation and progression in mammalian cells. Their interaction with LC3-interacting region (LIR) containing proteins promotes cargo sequestration, phagophore assembly, or even fusion between autophagosomes and lysosomes. Previously, we have shown that RabGAP proteins from the TBC family directly bind to LC3/GABARAP proteins. In the present study, we focus on the function of TBC1D2B. We show that TBC1D2B contains a functional canonical LIR motif and acts at an early stage of autophagy by binding to both LC3/GABARAP and ATG12 conjugation complexes. Subsequently, TBC1D2B is degraded by autophagy. TBC1D2B condensates into liquid droplets upon autophagy induction. Our study suggests that phase separation is an underlying mechanism of TBC1D2B-dependent autophagy induction.

Computational prediction and experimental validation of Salmonella Typhimurium SopE-mediated fine-tuning of autophagy in intestinal epithelial cells.

Macroautophagy is a ubiquitous homeostasis and health-promoting recycling process of eukaryotic cells, targeting misfolded proteins, damaged organelles and intracellular infectious agents. Some intracellular pathogens such as Salmonella enterica serovar Typhimurium hijack this process during pathogenesis. Here we investigate potential protein-protein interactions between host transcription factors and secreted effector proteins of Salmonella and their effect on host gene transcription. A systems-level analysis identified Salmonella effector proteins that had the potential to affect core autophagy gene regulation. The effect of a SPI-1 effector protein, SopE, that was predicted to interact with regulatory proteins of the autophagy process, was investigated to validate our approach. We then confirmed experimentally that SopE can directly bind to SP1, a host transcription factor, which modulates the expression of the autophagy gene MAP1LC3B. We also revealed that SopE might have a double role in the modulation of autophagy: Following initial increase of MAP1LC3B transcription triggered by Salmonella infection, subsequent decrease in MAP1LC3B transcription at 6h post-infection was SopE-dependent. SopE also played a role in modulation of the autophagy flux machinery, in particular MAP1LC3B and p62 autophagy proteins, depending on the level of autophagy already taking place. Upon typical infection of epithelial cells, the autophagic flux is increased. However, when autophagy was chemically induced prior to infection, SopE dampened the autophagic flux. The same was also observed when most of the intracellular Salmonella cells were not associated with the SCV (strain lacking sifA) regardless of the autophagy induction status before infection. We demonstrated how regulatory network analysis can be used to better characterise the impact of pathogenic effector proteins, in this case, Salmonella. This study complements previous work in which we had demonstrated that specific pathogen effectors can affect the autophagy process through direct interaction with autophagy proteins. Here we show that effector proteins can also influence the upstream regulation of the process. Such interdisciplinary studies can increase our understanding of the infection process and point out targets important in intestinal epithelial cell defense.

Selective autophagy controls innate immune response through a TAK1/TAB2/SH3PX1 axis.

Selective autophagy is a catabolic route that turns over specific cellular material for degradation by lysosomes, and whose role in the regulation of innate immunity is largely unexplored. Here, we show that the apical kinase of the Drosophila immune deficiency (IMD) pathway Tak1, as well as its co-activator Tab2, are both selective autophagy substrates that interact with the autophagy protein Atg8a. We also present a role for the Atg8a-interacting protein Sh3px1 in the downregulation of the IMD pathway, by facilitating targeting of the Tak1/Tab2 complex to the autophagy platform through its interaction with Tab2. Our findings show the Tak1/Tab2/Sh3px1 interactions with Atg8a mediate the removal of the Tak1/Tab2 signaling complex by selective autophagy. This in turn prevents constitutive activation of the IMD pathway in Drosophila. This study provides mechanistic insight on the regulation of innate immune responses by selective autophagy.

Exploring selective autophagy in Drosophila: Methods to identify Atg8-interacting proteins.

Autophagy has been described as a catabolic process in which cytoplasmic material is being recycled under various conditions of cellular stress, preventing cell damage and promoting cell survival. Drosophila has been demonstrated to provide an excellent animal model for the study of autophagy. Here, we provide a detailed experimental procedure for the identification of Atg8a interactors, exploiting the iLIR database, followed by the in vitro confirmation of interactions and in situ detection of the respective proteins.

Degradation of arouser by endosomal microautophagy is essential for adaptation to starvation in Drosophila.

Hunger drives food-seeking behaviour and controls adaptation of organisms to nutrient availability and energy stores. Lipids constitute an essential source of energy in the cell that can be mobilised during fasting by autophagy. Selective degradation of proteins by autophagy is made possible essentially by the presence of LIR and KFERQ-like motifs. Using in silico screening of Drosophila proteins that contain KFERQ-like motifs, we identified and characterized the adaptor protein Arouser, which functions to regulate fat storage and mobilisation and is essential during periods of food deprivation. We show that hypomorphic arouser mutants are not satiated, are more sensitive to food deprivation, and are more aggressive, suggesting an essential role for Arouser in the coordination of metabolism and food-related behaviour. Our analysis shows that Arouser functions in the fat body through nutrient-related signalling pathways and is degraded by endosomal microautophagy. Arouser degradation occurs during feeding conditions, whereas its stabilisation during non-feeding periods is essential for resistance to starvation and survival. In summary, our data describe a novel role for endosomal microautophagy in energy homeostasis, by the degradation of the signalling regulatory protein Arouser.

A nuclear role for Atg8-family proteins.

Despite the growing evidence that the macroautophagy/autophagy-related protein LC3 is localized in the nucleus, why and how it is targeted to the nucleus are poorly understood. In our recent study, we found that transcription factor seq (sequoia) interacts via its LIR motif with Atg8a, the Drosophila homolog of LC3, to negatively regulate the transcription of autophagy genes. Atg8a was found to also interact with the nuclear acetyltransferase complex subunit YL-1 and deacetylase Sirt2. Modulation of the acetylation status of Atg8a by YL-1 and Sirt2 affects the interaction between seq and Atg8a, and controls the induction of autophagy. Our work revealed a novel nuclear role for Atg8a, which is linked with the transcriptional regulation of autophagy genes.

Regulation of Expression of Autophagy Genes by Atg8a-Interacting Partners Sequoia, YL-1, and Sir2 in Drosophila.

Autophagy is the degradation of cytoplasmic material through the lysosomal pathway. One of the most studied autophagy-related proteins is LC3. Despite growing evidence that LC3 is enriched in the nucleus, its nuclear role is poorly understood. Here, we show that Drosophila Atg8a protein, homologous to mammalian LC3, interacts with the transcription factor Sequoia in a LIR motif-dependent manner. We show that Sequoia depletion induces autophagy in nutrient-rich conditions through the enhanced expression of autophagy genes. We show that Atg8a interacts with YL-1, a component of a nuclear acetyltransferase complex, and that it is acetylated in nutrient-rich conditions. We also show that Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation to activate autophagy. Our results suggest a mechanism of regulation of the expression of autophagy genes by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1, and Sir2.

Selective autophagic degradation of the IKK complex in Drosophila is mediated by Kenny/IKKγ to control inflammation.

Implication of autophagy in the downregulation of immune signaling pathways through the degradation of their components constitutes an emerging field of investigation. Our work showed that the selective interaction of Drosophila protein Kenny/IKKγ (CG16910) with the autophagic machinery is required for the degradation of the I-kappa B kinase complex. This regulatory mechanism is essential for the downregulation of the immune deficiency (IMD) pathway in response to commensal microbiota to prevent inflammation.

Impact of Autophagy and Aging on Iron Load and Ferritin in Drosophila Brain.

Biometals such as iron, copper, potassium, and zinc are essential regulatory elements of several biological processes. The homeostasis of biometals is often affected in age-related pathologies. Notably, impaired iron metabolism has been linked to several neurodegenerative disorders. Autophagy, an intracellular degradative process dependent on the lysosomes, is involved in the regulation of ferritin and iron levels. Impaired autophagy has been associated with normal pathological aging, and neurodegeneration. Non-mammalian model organisms such as Drosophila have proven to be appropriate for the investigation of age-related pathologies. Here, we show that ferritin is expressed in adult Drosophila brain and that iron and holoferritin accumulate with aging. At whole-brain level we found no direct relationship between the accumulation of holoferritin and a deficit in autophagy in aged Drosophila brain. However, synchrotron X-ray spectromicroscopy revealed an additional spectral feature in the iron-richest region of autophagy-deficient fly brains, consistent with iron-sulfur. This potentially arises from iron-sulfur clusters associated with altered mitochondrial iron homeostasis.

Targeted interplay between bacterial pathogens and host autophagy.

Due to the critical role played by autophagy in pathogen clearance, pathogens have developed diverse strategies to subvert it. Despite previous key findings of bacteria-autophagy interplay, asystems-level insight into selective targeting by the host and autophagy modulation by the pathogens is lacking. We predicted potential interactions between human autophagy proteins and effector proteins from 56 pathogenic bacterial species by identifying bacterial proteins predicted to have recognition motifs for selective autophagy receptors SQSTM1/p62, CALCOCO2/NDP52 and MAP1LC3/LC3. Using structure-based interaction prediction, we identified bacterial proteins capable to modify core autophagy components. Our analysis revealed that autophagy receptors in general potentially target mostly genus-specific proteins, and not those present in multiple genera. The complementarity between the predicted SQSTM1/p62 and CALCOCO2/NDP52 targets, which has been shown for Salmonella, Listeria and Shigella, could be observed across other pathogens. This complementarity potentially leaves the host more susceptible to chronic infections upon the mutation of autophagy receptors. Proteins derived from enterotoxigenic and non-toxigenic Bacillus outer membrane vesicles indicated that autophagy targets pathogenic proteins rather than non-pathogenic ones. We also observed apathogen-specific pattern as to which autophagy phase could be modulated by specific genera. We found intriguing examples of bacterial proteins that could modulate autophagy, and in turn being targeted by autophagy as ahost defense mechanism. We confirmed experimentally an interplay between a Salmonella protease, YhjJ and autophagy. Our comparative meta-analysis points out key commonalities and differences in how pathogens could affect autophagy and how autophagy potentially recognizes these pathogenic effectors. Abbreviations: ATG5: autophagy related 5; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; GST: glutathione S-transferase; LIR: MAP1LC3/LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha; OMV: outer membrane vesicles; SQSTM1/p62: sequestosome 1; SCV: Salmonella containing vesicle; TECPR1: tectonin beta-propeller repeat containing 1; YhjJ: hypothetical zinc-protease.

Assays to Monitor Mitophagy in Drosophila.

Autophagy is a central pathway utilized by many eukaryotic cells in order to recycle intracellular constituents, particularly under periods of nutrient scarcity or cellular damage. The process is evolutionarily conserved from yeast to mammals and can be highly selective with regard to the contents that are targeted for degradation. The availability of Drosophila transgenic lines and fluorophore-labeled autophagic markers allows nowadays for the more effortless visualization of the process within cells. Herein, we provide two protocols to prepare Drosophila samples for confocal and transmission electron microscopy for in vivo monitoring of mitophagy, a specific type of autophagy for the clearance of damaged or superfluous mitochondria from cells.

Assays to Monitor Aggrephagy in Drosophila Brain.

Accumulation of ubiquitinated protein aggregates is a hallmark of most aging-related neurodegenerative disorders. Autophagy has been found to be involved in the selective clearance of these protein aggregates, and this process is called aggrephagy. Here we provide two protocols for the investigation of protein aggregation and their removal by autophagy using western blotting and immunofluorescence techniques in Drosophila brain. Investigating the role of aggrephagy at the cellular and organismal level is important for the development of therapeutic interventions against aging-related diseases.

What We Learned From Big Data for Autophagy Research.

Autophagy is the process by which cytoplasmic components are engulfed in double-membraned vesicles before being delivered to the lysosome to be degraded. Defective autophagy has been linked to a vast array of human pathologies. The molecular mechanism of the autophagic machinery is well-described and has been extensively investigated. However, understanding the global organization of the autophagy system and its integration with other cellular processes remains a challenge. To this end, various bioinformatics and network biology approaches have been developed by researchers in the last few years. Recently, large-scale multi-omics approaches (like genomics, transcriptomics, proteomics, lipidomics, and metabolomics) have been developed and carried out specifically focusing on autophagy, and generating multi-scale data on the related components. In this review, we outline recent applications of in silico investigations and big data analyses of the autophagy process in various biological systems.

Deubiquitinating Enzymes Related to Autophagy: New Therapeutic Opportunities?

Autophagy is an evolutionary conserved catabolic process that allows for the degradation of intracellular components by lysosomes. This process can be triggered by nutrient deprivation, microbial infections or other challenges to promote cell survival under these stressed conditions. However, basal levels of autophagy are also crucial for the maintenance of proper cellular homeostasis by ensuring the selective removal of protein aggregates and dysfunctional organelles. A tight regulation of this process is essential for cellular survival and organismal health. Indeed, deregulation of autophagy is associated with a broad range of pathologies such as neuronal degeneration, inflammatory diseases, and cancer progression. Ubiquitination and deubiquitination of autophagy substrates, as well as components of the autophagic machinery, are critical regulatory mechanisms of autophagy. Here, we review the main evidence implicating deubiquitinating enzymes (DUBs) in the regulation of autophagy. We also discuss how they may constitute new therapeutic opportunities in the treatment of pathologies such as cancers, neurodegenerative diseases or infections.

CHMP1B is a target of USP8/UBPY regulated by ubiquitin during endocytosis.

Integration and down-regulation of cell growth and differentiation signals rely on plasma membrane receptor endocytosis and sorting towards either recycling vesicles or degradative lysosomes via multivesicular bodies (MVB). In this process, the endosomal sorting complex-III required for transport (ESCRT-III) controls membrane deformation and scission triggering intraluminal vesicle (ILV) formation at early endosomes. Here, we show that the ESCRT-III member CHMP1B can be ubiquitinated within a flexible loop known to undergo conformational changes during polymerization. We demonstrate further that CHMP1B is deubiquitinated by the ubiquitin specific protease USP8 (syn. UBPY) and found fully devoid of ubiquitin in a ~500 kDa large complex that also contains its ESCRT-III partner IST1. Moreover, EGF stimulation induces the rapid and transient accumulation of ubiquitinated forms of CHMP1B on cell membranes. Accordingly, CHMP1B ubiquitination is necessary for CHMP1B function in both EGF receptor trafficking in human cells and wing development in Drosophila. Based on these observations, we propose that CHMP1B is dynamically regulated by ubiquitination in response to EGF and that USP8 triggers CHMP1B deubiquitination possibly favoring its subsequent assembly into a membrane-associated ESCRT-III polymer.

Kenny mediates selective autophagic degradation of the IKK complex to control innate immune responses.

Selective autophagy is a catabolic process with which cellular material is specifically targeted for degradation by lysosomes. The function of selective autophagic degradation of self-components in the regulation of innate immunity is still unclear. Here we show that Drosophila Kenny, the homolog of mammalian IKKγ, is a selective autophagy receptor that mediates the degradation of the IκB kinase complex. Selective autophagic degradation of the IκB kinase complex prevents constitutive activation of the immune deficiency pathway in response to commensal microbiota. We show that autophagy-deficient flies have a systemic innate immune response that promotes a hyperplasia phenotype in the midgut. Remarkably, human IKKγ does not interact with mammalian Atg8-family proteins. Using a mathematical model, we suggest mechanisms by which pathogen selection might have driven the loss of LIR motif functionality during evolution. Our results suggest that there may have been an autophagy-related switch during the evolution of the IKKγ proteins in metazoans.

iLIR@viral: A web resource for LIR motif-containing proteins in viruses.

Macroautophagy/autophagy has been shown to mediate the selective lysosomal degradation of pathogenic bacteria and viruses (xenophagy), and to contribute to the activation of innate and adaptative immune responses. Autophagy can serve as an antiviral defense mechanism but also as a proviral process during infection. Atg8-family proteins play a central role in the autophagy process due to their ability to interact with components of the autophagy machinery as well as selective autophagy receptors and adaptor proteins. Such interactions are usually mediated through LC3-interacting region (LIR) motifs. So far, only one viral protein has been experimentally shown to have a functional LIR motif, leaving open a vast field for investigation. Here, we have developed the iLIR@viral database ( http://ilir.uk/virus/ ) as a freely accessible web resource listing all the putative canonical LIR motifs identified in viral proteins. Additionally, we used a curated text-mining analysis of the literature to identify novel putative LIR motif-containing proteins (LIRCPs) in viruses. We anticipate that iLIR@viral will assist with elucidating the full complement of LIRCPs in viruses.

A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila.

BACKGROUND: Lysosomes are the major catabolic compartment within eukaryotic cells, and their biogenesis requires the integration of the biosynthetic and endosomal pathways. Endocytosis and autophagy are the primary inputs of the lysosomal degradation pathway. Endocytosis is specifically needed for the degradation of membrane proteins whereas autophagy is responsible for the degradation of cytoplasmic components. We previously identified the deubiquitinating enzyme UBPY/USP8 as being necessary for lysosomal biogenesis and productive autophagy in Drosophila. Because UBPY/USP8 has been widely described for its function in the endosomal system, we hypothesized that disrupting the endosomal pathway itself may affect the biogenesis of the lysosomes. RESULTS: In the present study, we blocked the progression of the endosomal pathway at different levels of maturation of the endosomes by expressing in fat body cells either dsRNAs or dominant negative mutants targeting components of the endosomal machinery: Shibire, Rab4, Rab5, Chmp1 and Rab7. We observed that inhibition of endosomal trafficking at different steps in vivo is systematically associated with defects in lysosome biogenesis, resulting in autophagy flux blockade. CONCLUSION: Our results show that the integrity of the endosomal system is required for lysosome biogenesis and productive autophagy in vivo.

Using Fluorescent Reporters to Monitor Autophagy in the Female Germline Cells in Drosophila melanogaster.

Oogenesis is a fundamental biological process for the transmission of genetic information to the next generations. Drosophila has proven to be a valuable model for elucidating the molecular and cellular mechanisms involved in this developmental process. It has been shown that autophagy participates in the maturation of the egg chamber. Here we provide a protocol for monitoring and quantification of the autophagic process in the Drosophila germline cells using the fluorescent reporters mCherry-DmAtg8a and GFP-mCherry-DmAtg8a.