Metamorphic proteins at the basis of human autophagy initiation and lipid transfer.

Autophagy is a conserved intracellular degradation pathway that generates de novo double-membrane autophagosomes to target a wide range of material for lysosomal degradation. In multicellular organisms, autophagy initiation requires the timely assembly of a contact site between the ER and the nascent autophagosome. Here, we report the in vitro reconstitution of a full-length seven-subunit human autophagy initiation supercomplex built on a core complex of ATG13-101 and ATG9. Assembly of this core complex requires the rare ability of ATG13 and ATG101 to switch between distinct folds. The slow spontaneous metamorphic conversion is rate limiting for the self-assembly of the supercomplex. The interaction of the core complex with ATG2-WIPI4 enhances tethering of membrane vesicles and accelerates lipid transfer of ATG2 by both ATG9 and ATG13-101. Our work uncovers the molecular basis of the contact site and its assembly mechanisms imposed by the metamorphosis of ATG13-101 to regulate autophagosome biogenesis in space and time.

The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin.

BACKGROUND: The bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified. METHODS: We used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy. RESULTS: We identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound. CONCLUSION: Taken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55. Video Abstract.

Prodigiosin Sensitizes Sensitive and Resistant Urothelial Carcinoma Cells to Cisplatin Treatment.

Cisplatin-based treatment is the standard of care therapy for urothelial carcinomas. However, complex cisplatin resistance mechanisms limit the success of this approach. Both apoptosis and autophagy have been shown to contribute to this resistance. Prodigiosin, a secondary metabolite from various bacteria, exerts different biological activities including the modulation of these two cellular stress response pathways. We analyzed the effect of prodigiosin on protein levels of different autophagy- and apoptosis-related proteins in cisplatin-sensitive and -resistant urothelial carcinoma cells (UCCs). Furthermore, we investigated the effect on cell viability of prodigiosin alone or in combination with cisplatin. We made use of four different pairs of cisplatin-sensitive and -resistant UCCs. We found that prodigiosin blocked autophagy in UCCs and re-sensitized cisplatin-resistant cells to apoptotic cell death. Furthermore, we found that prodigiosin is a potent anticancer agent with nanomolar IC50 values in all tested UCCs. In combination studies, we observed that prodigiosin sensitized both cisplatin-sensitive and -resistant urothelial carcinoma cell lines to cisplatin treatment with synergistic effects in most tested cell lines. These effects of prodigiosin are at least partially mediated by altering lysosomal function, since we detected reduced activities of cathepsin B and L. We propose that prodigiosin is a promising candidate for the therapy of cisplatin-resistant urothelial carcinomas, either as a single agent or in combinatory therapeutic approaches.

In Vitro Assessment of the Genotoxic Hazard of Novel Hydroxamic Acid- and Benzamide-Type Histone Deacetylase Inhibitors (HDACi).

Histone deacetylase inhibitors (HDACi) are already approved for the therapy of leukemias. Since they are also emerging candidate compounds for the treatment of non-malignant diseases, HDACi with a wide therapeutic window and low hazard potential are desirable. Here, we investigated a panel of 12 novel hydroxamic acid- and benzamide-type HDACi employing non-malignant V79 hamster cells as toxicology guideline-conform in vitro model. HDACi causing a ≥10-fold preferential cytotoxicity in malignant neuroblastoma over non-malignant V79 cells were selected for further genotoxic hazard analysis, including vorinostat and entinostat for control. All HDACi selected, (i.e., KSK64, TOK77, DDK137 and MPK77) were clastogenic and evoked DNA strand breaks in non-malignant V79 cells as demonstrated by micronucleus and comet assays, histone H2AX foci formation analyses (γH2AX), DNA damage response (DDR) assays as well as employing DNA double-strand break (DSB) repair-defective VC8 hamster cells. Genetic instability induced by hydroxamic acid-type HDACi seems to be independent of bulky DNA adduct formation as concluded from the analysis of nucleotide excision repair (NER) deficient mutants. Summarizing, KSK64 revealed the highest genotoxic hazard and DDR stimulating potential, while TOK77 and MPK77 showed the lowest DNA damaging capacity. Therefore, these compounds are suggested as the most promising novel candidate HDACi for subsequent pre-clinical in vivo studies.

Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

The targeted elimination of radio- or chemotherapy-induced senescent cells by so-called senolytic substances represents a promising approach to reduce tumor relapse as well as therapeutic side effects such as fibrosis. We screened an in-house library of 178 substances derived from marine sponges, endophytic fungi, and higher plants, and determined their senolytic activities towards DNA damage-induced senescent HCT116 colon carcinoma cells. The Pan-PI3K-inhibitor wortmannin and its clinical derivative, PX-866, were identified to act as senolytics. PX-866 potently induced apoptotic cell death in senescent HCT116, MCF-7 mammary carcinoma, and A549 lung carcinoma cells, independently of whether senescence was induced by ionizing radiation or by chemotherapeutics, but not in proliferating cells. Other Pan-PI3K inhibitors, such as the FDA-approved drug BAY80-6946 (Copanlisib, Aliqopa®), also efficiently and specifically eliminated senescent cells. Interestingly, only the simultaneous inhibition of both PI3K class I alpha (with BYL-719 (Alpelisib, Piqray®)) and delta (with CAL-101 (Idelalisib, Zydelig®)) isoforms was sufficient to induce senolysis, whereas single application of these inhibitors had no effect. On the molecular level, inhibition of PI3Ks resulted in an increased proteasomal degradation of the CDK inhibitor p21WAF1/CIP1 in all tumor cell lines analyzed. This led to a timely induction of apoptosis in senescent tumor cells. Taken together, the senolytic properties of PI3K-inhibitors reveal a novel dimension of these promising compounds, which holds particular potential when employed alongside DNA damaging agents in combination tumor therapies.

Fin56-induced ferroptosis is supported by autophagy-mediated GPX4 degradation and functions synergistically with mTOR inhibition to kill bladder cancer cells.

Ferroptosis is a form of regulated cell death that emerges to be relevant for therapy-resistant and dedifferentiating cancers. Although several lines of evidence suggest that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms remain unclear. Fin56, a type 3 ferroptosis inducer, triggers ferroptosis by promoting glutathione peroxidase 4 (GPX4) protein degradation via a not fully understood pathway. Here, we determined that Fin56 induces ferroptosis and autophagy in bladder cancer cells and that Fin56-triggered ferroptosis mechanistically depends on the autophagic machinery. Furthermore, we found that autophagy inhibition at different stages attenuates Fin56-induced oxidative stress and GPX4 degradation. Moreover, we investigated the effects of Fin56 in combination with Torin 2, a potent mTOR inhibitor used to activate autophagy, on cell viability. We found that Fin56 synergizes with Torin 2 in cytotoxicity against bladder cancer cells. Collectively, our findings not only support the concept that ferroptosis is a type of autophagy-dependent cell death but imply that the combined application of ferroptosis inducers and mTOR inhibitors is a promising approach to improve therapeutic options in the treatment of bladder cancer.

High-throughput screening for natural compound-based autophagy modulators reveals novel chemotherapeutic mode of action for arzanol.

Autophagy is an intracellular recycling pathway with implications for intracellular homeostasis and cell survival. Its pharmacological modulation can aid chemotherapy by sensitizing cancer cells toward approved drugs and overcoming chemoresistance. Recent translational data on autophagy modulators show promising results in reducing tumor growth and metastasis, but also reveal a need for more specific compounds and novel lead structures. Here, we searched for such autophagy-modulating compounds in a flow cytometry-based high-throughput screening of an in-house natural compound library. We successfully identified novel inducers and inhibitors of the autophagic pathway. Among these, we identified arzanol as an autophagy-modulating drug that causes the accumulation of ATG16L1-positive structures, while it also induces the accumulation of lipidated LC3. Surprisingly, we observed a reduction of the size of autophagosomes compared to the bafilomycin control and a pronounced accumulation of p62/SQSTM1 in response to arzanol treatment in HeLa cells. We, therefore, speculate that arzanol acts both as an inducer of early autophagosome biogenesis and as an inhibitor of later autophagy events. We further show that arzanol is able to sensitize RT-112 bladder cancer cells towards cisplatin (CDDP). Its anticancer activity was confirmed in monotherapy against both CDDP-sensitive and -resistant bladder cancer cells. We classified arzanol as a novel mitotoxin that induces the fragmentation of mitochondria, and we identified a series of targets for arzanol that involve proteins of the class of mitochondria-associated quinone-binding oxidoreductases. Collectively, our results suggest arzanol as a valuable tool for autophagy research and as a lead compound for drug development in cancer therapy.

FIP200 controls the TBK1 activation threshold at SQSTM1/p62-positive condensates.

The protein kinase TBK1 is a central regulator of innate immune responses and autophagy, and ablation of either function has been linked to neuroinflammatory or degenerative diseases. Autophagy is an intracellular process that recycles old or damaged proteins and organelles. In recent years, the TBK1-dependent regulation of autophagy pathways has been characterized. However, the autophagy-dependent regulation of TBK1 activity awaits further clarification. Here, we observed that TBK1 is recruited to SQSTM1/p62-containing aggregates via the selective autophagy receptor TAX1BP1. In these aggregates, TBK1 phosphorylates SQSTM1/p62 at serine 403 and thus presumably regulates the efficient engulfment and clearance of these structures. We found that TBK1 activation is strongly increased if FIP200, a component of the autophagy-inducing ULK1 complex, is not present or cannot bind to TAX1BP1. Given our collective findings, we hypothesize that FIP200 ensures the inducible activation of TBK1 at SQSTM1/p62 condensates.