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.

An essential role of the autophagy activating kinase ULK1 in snRNP biogenesis.

The biogenesis of small uridine-rich nuclear ribonucleoproteins (UsnRNPs) depends on the methylation of Sm proteins catalyzed by the methylosome and the subsequent action of the SMN complex, which assembles the heptameric Sm protein ring onto small nuclear RNAs (snRNAs). In this sophisticated process, the methylosome subunit pICln (chloride conductance regulatory protein) is attributed to an exceptional key position as an 'assembly chaperone' by building up a stable precursor Sm protein ring structure. Here, we show that-apart from its autophagic role-the Ser/Thr kinase ULK1 (Uncoordinated [unc-51] Like Kinase 1) functions as a novel key regulator in UsnRNP biogenesis by phosphorylation of the C-terminus of pICln. As a consequence, phosphorylated pICln is no longer capable to hold up the precursor Sm ring structure. Consequently, inhibition of ULK1 results in a reduction of efficient UsnRNP core assembly. Thus ULK1, depending on its complex formation, exerts different functions in autophagy or snRNP biosynthesis.

The Impact of p70S6 Kinase-Dependent Phosphorylation of Gemin2 in UsnRNP Biogenesis.

The survival motor neuron (SMN) complex is a multi-megadalton complex involved in post-transcriptional gene expression in eukaryotes via promotion of the biogenesis of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). The functional center of the complex is formed from the SMN/Gemin2 subunit. By binding the pentameric ring made up of the Sm proteins SmD1/D2/E/F/G and allowing for their transfer to a uridine-rich short nuclear RNA (UsnRNA), the Gemin2 protein in particular is crucial for the selectivity of the Sm core assembly. It is well established that post-translational modifications control UsnRNP biogenesis. In our work presented here, we emphasize the crucial role of Gemin2, showing that the phospho-status of Gemin2 influences the capacity of the SMN complex to condense in Cajal bodies (CBs) in vivo. Additionally, we define Gemin2 as a novel and particular binding partner and phosphorylation substrate of the mTOR pathway kinase ribosomal protein S6 kinase beta-1 (p70S6K). Experiments using size exclusion chromatography further demonstrated that the Gemin2 protein functions as a connecting element between the 6S complex and the SMN complex. As a result, p70S6K knockdown lowered the number of CBs, which in turn inhibited in vivo UsnRNP synthesis. In summary, these findings reveal a unique regulatory mechanism of UsnRNP biogenesis.

NF90/NFAR (nuclear factors associated with dsRNA) - a new methylation substrate of the PRMT5-WD45-RioK1 complex.

Protein-arginine methylation is a common posttranslational modification, crucial to various cellular processes, such as protein-protein interactions or binding to nucleic acids. The central enzyme of symmetric protein arginine methylation in mammals is the protein arginine methyltransferase 5 (PRMT5). While the methylation reaction itself is well understood, recruitment and differentiation among substrates remain less clear. One mechanism to regulate the diversity of PRMT5 substrate recognition is the mutual binding to the adaptor proteins pICln or RioK1. Here, we describe the specific interaction of Nuclear Factor 90 (NF90) with the PRMT5-WD45-RioK1 complex. We show for the first time that NF90 is symmetrically dimethylated by PRMT5 within the RG-rich region in its C-terminus. Since upregulation of PRMT5 is a hallmark of many cancer cells, the characterization of its dimethylation and modulation by specific commercial inhibitors in vivo presented here may contribute to a better understanding of PRMT5 function and its role in cancer.

Phosphorylation of GAPVD1 Is Regulated by the PER Complex and Linked to GAPVD1 Degradation.

Repressor protein period (PER) complexes play a central role in the molecular oscillator mechanism of the mammalian circadian clock. While the main role of nuclear PER complexes is transcriptional repression, much less is known about the functions of cytoplasmic PER complexes. We found with a biochemical screen for PER2-interacting proteins that the small GTPase regulator GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), which has been identified previously as a component of cytoplasmic PER complexes in mice, is also a bona fide component of human PER complexes. We show that in situ GAPVD1 is closely associated with casein kinase 1 delta (CSNK1D), a kinase that regulates PER2 levels through a phosphoswitch mechanism, and that CSNK1D regulates the phosphorylation of GAPVD1. Moreover, phosphorylation determines the kinetics of GAPVD1 degradation and is controlled by PER2 and a C-terminal autoinhibitory domain in CSNK1D, indicating that the regulation of GAPVD1 phosphorylation is a novel function of cytoplasmic PER complexes and might be part of the oscillator mechanism or an output function of the circadian clock.

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.

Anthraquinones and autophagy - Three rings to rule them all?

In order to overcome therapy resistance in cancer, scientists search in nature for novel lead structures for the development of improved chemotherapeutics. Anthraquinones belong to a class of tricyclic organic natural compounds with promising anti-cancer effects. Anthraquinone derivatives are rich in structural diversity, and exhibit pleiotropic properties, among which the modulation of autophagy seems promising in the context of overcoming cancer-therapy resistance. Among the most promising derivatives in this regard are emodin, aloe emodin, rhein, physcion, chrysophanol and altersolanol A. On the molecular level, these compounds target autophagy via different upstream pathways including the AKT/mTOR-axis and transcription of autophagy-related proteins. The role of autophagy is pro-survival as well as cell death-promoting, depending on derivatives and their cell type specificity. This review summarizes observed effects of anthraquinone derivatives on autophagy and discusses targeted pathways and crosstalks. A cumulative knowledge about this topic paves the way for further research on modes of action, and aids to find a therapeutic window of anthraquinones in cancer-therapy.

Novel meriolin derivatives as rapid apoptosis inducers.

3-(Hetero)aryl substituted 7-azaindoles possessing multikinase inhibitor activity are readily accessed in a one-pot Masuda borylation-Suzuki coupling sequence. Several promising derivatives were identified as apoptosis inducers and, emphasizing the multikinase inhibition potential, as sphingosine kinase 2 inhibitors. Our measurements provide additional insights into the structure-activity relationship of meriolin derivatives, suggesting derivatives bearing a pyridine moiety with amino groups in 2-position as most active anticancer compounds and thus as highly promising candidates for future in vivo studies.

First Results from a Screening of 300 Naturally Occurring Compounds: 4,6-dibromo-2-(2',4'-dibromophenoxy)phenol, 4,5,6-tribromo-2-(2',4'-dibromophenoxy)phenol, and 5-epi-nakijinone Q as Substances with the Potential for Anticancer Therapy.

There is a variety of antineoplastic drugs that are based on natural compounds from ecological niches with high evolutionary pressure. We used two cell lines (Jurkat J16 and Ramos) in a screening to assess 300 different naturally occurring compounds with regard to their antineoplastic activity. The results of the compounds 4,6-dibromo-2-(2',4'-dibromophenoxy)phenol (P01F03), 4,5,6-tribromo-2-(2',4'-dibromophenoxy)phenol (P01F08), and 5-epi-nakijinone Q (P03F03) prompted us to perform further research. Using viability and apoptosis assays on the cell lines of primary human leukemic and normal hematopoietic cells, we found that P01F08 induced apoptosis in the cell lines at IC50 values between 1.61 and 2.95 µM after 72 h. IC50 values of peripheral blood mononuclear cells (PBMNCs) from healthy donors were higher, demonstrating that the cytotoxicity in the cell lines reached 50%, while normal PBMNCs were hardly affected. The colony-forming unit assay showed that the hematopoietic progenitor cells were not significantly affected in their growth by P01F08 at a concentration of 3 µM. P01F08 showed a 3.2-fold lower IC50 value in primary leukemic cells [acute myeloid leukemia (AML)] compared to the PBMNC of healthy donors. We could confirm the antineoplastic effect of 5-epi-nakijinone Q (P03F03) on the cell lines via the induction of apoptosis but noted a similarly strong cytotoxic effect on normal PBMNCs.

Targeting colorectal cancer cell metabolism through development of cisplatin and metformin nano-cubosomes.

BACKGROUND: Colorectal cancer (CRC) remains a leading cause of death worldwide. Utilizing cisplatin in CRC is correlated with severe adverse effects and drug-resistance. Combined anticancer drug-treatment, along with, their enhanced delivery, can effectively kill cancer through multiple pathways. Nano-cubosomes are emerging as nanocarriers for anticancer therapies, hence, we constructed nano-cubosomes bearing cisplatin and cisplatin-metformin combination for investigation on HCT-116 cells. METHODS: Nano-cubosomes bearing either cisplatin alone or cisplatin-metformin combination were formulated using emulsification technique. The loaded nano-cubosomes were characterized in vitro and the optimized formulation was selected. Their cytotoxic effects were investigated by Sulphorhodamine-B (SRB) assay. The AMPK/mTOR metabolic pathway as well as the Akt/mTOR pathway were analyzed using ELISA technique. Colorimetry was used in NADPH oxidase, LDH and caspase-3 activity determination. RESULTS: nano-cubosomal formulations exhibited superior cytotoxic effect compared to unformulated cisplatin. This cytotoxic effect was profound upon incorporation of metformin, an indirect mTOR inhibitor, in cisplatin nano-cubosomes. The induced CRC cell apoptosis was through inhibition of several metabolic pathways, namely, AMPK/mTOR and Akt/mTOR. Drug-loaded nano-cubosomes ensued depletion in glucose and energy levels that led to AMPK activation and thus mTOR inhibition. mTOR was additionally inhibited via suppression of p-Akt (Ser473) levels after nano-cubosomal treatment. Moreover, drug-loaded nano-cubosomes produced a notable escalation in ROS levels, evident as an increase in NADPH oxidase, inhibition of LDH and a consequential upsurge in caspase-3. CONCLUSION: These results demonstrated the influence exerted by cisplatin-loaded nano-cubosomes on CRC cell survival and enhancement of their cytotoxicity upon metformin addition.

Cyclic Cystine-Bridged Peptides from the Marine Sponge Clathria basilana Induce Apoptosis in Tumor Cells and Depolarize the Bacterial Cytoplasmic Membrane.

Investigation of the sponge Clathria basilana collected in Indonesia afforded five new peptides, including microcionamides C (1) and D (2), gombamides B (4), C (5), and D (6), and an unusual amide, (E)-2-amino-3-methyl-N-styrylbutanamide (7), along with 11 known compounds, among them microcionamide A (3). The structures of the new compounds were elucidated by one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of the constituent amino acid residues in 1-7 were determined by Marfey's analysis. Microcionamides A, C, and D (1-3) showed in vitro cytotoxicity against lymphoma (Ramos) and leukemia cell lines (HL-60, Nomo-1, Jurkat J16), as well as against a human ovarian carcinoma cell line (A2780) with IC50 values ranging from 0.45 to 28 µM. Mechanistic studies showed that compounds 1-3 rapidly induce apoptotic cell death in Jurkat J16 and Ramos cells and that 1 and 2 potently block autophagy upon starvation conditions, thereby impairing pro-survival signaling of cancer cells. In addition, microcionamides C and A (1 and 3) inhibited bacterial growth of Staphylococcus aureus and Enterococcus faecium with minimal inhibitory concentrations between 6.2 and 12 µM. Mechanistic studies indicate dissipation of the bacterial membrane potential.

Autophagy signal transduction by ATG proteins: from hierarchies to networks.

Autophagy represents an intracellular degradation process which is involved in both cellular homeostasis and disease settings. In the last two decades, the molecular machinery governing this process has been characterized in detail. To date, several key factors regulating this intracellular degradation process have been identified. The so-called autophagy-related (ATG) genes and proteins are central to this process. However, several additional molecules contribute to the outcome of an autophagic response. Several review articles describing the molecular process of autophagy have been published in the recent past. In this review article we would like to add the most recent findings to this knowledge, and to give an overview of the network character of the autophagy signaling machinery.

The Dok-3/Grb2 protein signal module attenuates Lyn kinase-dependent activation of Syk kinase in B cell antigen receptor microclusters.

Recruitment of the growth factor receptor-bound protein 2 (Grb2) by the plasma membrane-associated adapter protein downstream of kinase 3 (Dok-3) attenuates signals transduced by the B cell antigen receptor (BCR). Here we describe molecular details of Dok-3/Grb2 signal integration and function, showing that the Lyn-dependent activation of the BCR transducer kinase Syk is attenuated by Dok-3/Grb2 in a site-specific manner. This process is associated with the SH3 domain-dependent translocation of Dok-3/Grb2 complexes into BCR microsignalosomes and augmented phosphorylation of the inhibitory Lyn target SH2 domain-containing inositol 5' phosphatase. Hence, our findings imply that Dok-3/Grb2 modulates the balance between activatory and inhibitory Lyn functions with the aim to adjust BCR signaling efficiency.

Application of Cyclic Diaryliodonium Salts in the Synthesis of Axially Chiral Natural Product Analogues.

The application of cyclic diaryliodonium salts in the synthesis of bioactive natural product analogues was demonstrated. Axially chiral biaryls were obtained via the enantioselective ring opening of cyclic diaryliodonium salts. Regioselective borylation was key in accessing both enantiomers of a biphenol key intermediate in eight steps overall. 8,8″-Amino biflavones were synthesized, their bioactivity profiled, and the eutomer identified. The structure-activity relationship was probed.

Novel meriolin derivatives potently inhibit cell cycle progression and transcription in leukemia and lymphoma cells via inhibition of cyclin-dependent kinases (CDKs).

A key feature of cancer is the disruption of cell cycle regulation, which is characterized by the selective and abnormal activation of cyclin-dependent kinases (CDKs). Consequently, targeting CDKs via meriolins represents an attractive therapeutic approach for cancer therapy. Meriolins represent a semisynthetic compound class derived from meridianins and variolins with a known CDK inhibitory potential. Here, we analyzed the two novel derivatives meriolin 16 and meriolin 36 in comparison to other potent CDK inhibitors and could show that they displayed a high cytotoxic potential in different lymphoma and leukemia cell lines as well as in primary patient-derived lymphoma and leukemia cells. In a kinome screen, we showed that meriolin 16 and 36 prevalently inhibited most of the CDKs (such as CDK1, 2, 3, 5, 7, 8, 9, 12, 13, 16, 17, 18, 19, 20). In drug-to-target modeling studies, we predicted a common binding mode of meriolin 16 and 36 to the ATP-pocket of CDK2 and an additional flipped binding for meriolin 36. We could show that cell cycle progression and proliferation were blocked by abolishing phosphorylation of retinoblastoma protein (a major target of CDK2) at Ser612 and Thr82. Moreover, meriolin 16 prevented the CDK9-mediated phosphorylation of RNA polymerase II at Ser2 which is crucial for transcription initiation. This renders both meriolin derivatives as valuable anticancer drugs as they target three different Achilles' heels of the tumor: (1) inhibition of cell cycle progression and proliferation, (2) prevention of transcription, and (3) induction of cell death.

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.

Novel meriolin derivatives activate the mitochondrial apoptosis pathway in the presence of antiapoptotic Bcl-2.

Meriolin derivatives represent a new class of kinase inhibitors with a pronounced cytotoxic potential. Here, we investigated a newly synthesized meriolin derivative (termed meriolin 16) that displayed a strong apoptotic potential in Jurkat leukemia and Ramos lymphoma cells. Meriolin 16 induced apoptosis in rapid kinetics (within 2-3 h) and more potently (IC50: 50 nM) than the previously described derivatives meriolin 31 and 36 [1]. Exposure of Ramos cells to meriolin 16, 31, or 36 for 5 min was sufficient to trigger severe and irreversible cytotoxicity. Apoptosis induction by all three meriolin derivatives was independent of death receptor signaling but required caspase-9 and Apaf-1 as central mediators of the mitochondrial death pathway. Meriolin-induced mitochondrial toxicity was demonstrated by disruption of the mitochondrial membrane potential (ΔΨm), mitochondrial release of proapoptotic Smac, processing of the dynamin-like GTPase OPA1, and subsequent fragmentation of mitochondria. Remarkably, all meriolin derivatives were able to activate the mitochondrial death pathway in Jurkat cells, even in the presence of the antiapoptotic Bcl-2 protein. In addition, meriolins were capable of inducing cell death in imatinib-resistant K562 and KCL22 chronic myeloid leukemia cells as well as in cisplatin-resistant J82 urothelial carcinoma and 2102EP germ cell tumor cells. Given the frequent inactivation of the mitochondrial apoptosis pathway by tumor cells, such as through overexpression of antiapoptotic Bcl-2, meriolin derivatives emerge as promising therapeutic agents for overcoming treatment resistance.

Pro-apoptotic and immunostimulatory tetrahydroxanthone dimers from the endophytic fungus Phomopsis longicolla.

Four tetrahydroxanthone dimers (1-4) and four biogenetically related monomers (5-8), including the new derivatives 4-6, were isolated from the endophyte Phomopsis longicolla. The absolute configurations of 2-4 were established for the first time by TDDFT electronic circular dichroism calculations, and that of phomoxanthone A (1) was revised by X-ray crystallography. Phomoxanthone A (1) showed the strongest pro-apoptotic activity when tested against a panel of human cancer cell lines, including cisplatin-resistant cells, whereas it was up to 100-fold less active against healthy blood cells. It was also the most potent activator of murine T lymphocytes, NK cells, and macrophages, suggesting an activation of the immune system in parallel to its pro-apoptotic activity. This dual effect in combating cancer cells could help in fighting resistance during chemotherapy. Preliminary structure-activity studies of isolated compounds and derivatives obtained by semisynthesis (9a-11) hinted at the location of the biaryl axis and the presence of acetyl groups as important structural elements for the biological activity of the studied tetrahydroxanthones.

Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell.

The spliceosome, responsible for all mature protein-coding transcripts of eukaryotic intron-containing genes, consists of small uridine-rich nuclear ribonucleoproteins (UsnRNPs). The assembly of UsnRNPs depends, on one hand, on the arginine methylation of Sm proteins catalyzed by the PRMT5 complex. On the other hand, it depends on the phosphorylation of the PRMT5 subunit pICln by the Uncoordinated Like Kinase 1 (ULK1). In consequence, phosphorylation of pICln affects the stability of the UsnRNP assembly intermediate, the so-called 6 S complex. The detailed mechanisms of phosphorylation-dependent integrity and subsequent UsnRNP assembly of the 6 S complex in vivo have not yet been analyzed. By using a phospho-specific antibody against ULK1-dependent phosphorylation sites of pICln, we visualize the intracellular distribution of phosphorylated pICln. Furthermore, we detect the colocaliphosphor-pICln1 with phospho-pICln by size-exclusion chromatography and immunofluorescence techniques. We also show that phosphorylated pICln is predominantly present in the 6 S complex. The addition of ULK1 to in vitro produced 6 S complex, as well as the reconstitution of ULK1 in ULK1-deficient cells, increases the efficiency of snRNP biogenesis. Accordingly, inhibition of ULK1 and the associated decreased pICln phosphorylation lead to accumulation of the 6 S complex and reduction in the spliceosomal activity of the cell.