An immune-based tool platform for in vivo cell clearance.

Immunological targeting of pathological cells has been successful in oncology and is expanding to other pathobiological contexts. Here, we present a flexible platform that allows labeling cells of interest with the surface-expressed model antigen ovalbumin (OVA), which can be eliminated via either antigen-specific T cells or newly developed OVA antibodies. We demonstrate that hepatocytes can be effectively targeted by either modality. In contrast, pro-fibrotic fibroblasts associated with pulmonary fibrosis are only eliminated by T cells in initial experiments, which reduced collagen deposition in a fibrosis model. This new experimental platform will facilitate development of immune-based approaches to clear potential pathological cell types in vivo.

Modulating TRADD to restore cellular homeostasis and inhibit apoptosis.

Cell death in human diseases is often a consequence of disrupted cellular homeostasis. If cell death is prevented without restoring cellular homeostasis, it may lead to a persistent dysfunctional and pathological state. Although mechanisms of cell death have been thoroughly investigated1-3, it remains unclear how homeostasis can be restored after inhibition of cell death. Here we identify TRADD4-6, an adaptor protein, as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. TRADD deficiency inhibits RIPK1-dependent extrinsic apoptosis and proteasomal stress-induced intrinsic apoptosis. We also show that the small molecules ICCB-19 and Apt-1 bind to a pocket on the N-terminal TRAF2-binding domain of TRADD (TRADD-N), which interacts with the C-terminal domain (TRADD-C) and TRAF2 to modulate the ubiquitination of RIPK1 and beclin 1. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). We conclude that pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.

Regulation of a distinct activated RIPK1 intermediate bridging complex I and complex II in TNFα-mediated apoptosis.

Stimulation of cells with TNFα can promote distinct cell death pathways, including RIPK1-independent apoptosis, necroptosis, and RIPK1-dependent apoptosis (RDA)-the latter of which we still know little about. Here we show that RDA involves the rapid formation of a distinct detergent-insoluble, highly ubiquitinated, and activated RIPK1 pool, termed "iuRIPK1." iuRIPK1 forms after RIPK1 activation in TNF-receptor-associated complex I, and before cytosolic complex II formation and caspase activation. To identify regulators of iuRIPK1 formation and RIPK1 activation in RDA, we conducted a targeted siRNA screen of 1,288 genes. We found that NEK1, whose loss-of-function mutations have been identified in 3% of ALS patients, binds to activated RIPK1 and restricts RDA by negatively regulating formation of iuRIPK1, while LRRK2, a kinase implicated in Parkinson's disease, promotes RIPK1 activation and association with complex I in RDA. Further, the E3 ligases APC11 and c-Cbl promote RDA, and c-Cbl is recruited to complex I in RDA, where it promotes prodeath K63-ubiquitination of RIPK1 to lead to iuRIPK1 formation. Finally, we show that two different modes of necroptosis induction by TNFα exist which are differentially regulated by iuRIPK1 formation. Overall, this work reveals a distinct mechanism of RIPK1 activation that mediates the signaling mechanism of RDA as well as a type of necroptosis.

Synergistic effect of a novel autophagy inhibitor and Quizartinib enhances cancer cell death.

Drug combinations have been increasingly applied in chemotherapy as a strategy to enhance the efficacy of anti-cancer treatment. The appropriate drug combinations may achieve synergistic effects beyond monotherapies alone. AC220 (Quizartinib), an FLT3 receptor tyrosine kinase inhibitor, developed for the treatment of AML, has been tested in phase II human clinical trials. However, AC220 as a monotherapy is not efficacious enough. In this study, we performed a small-molecule screening of 12 640 compounds in order to find a compound that increase the AC220 efficacy in chemotherapy. We identified that TAK-165, a HER2 inhibitor, even when used at low nanomolar doses in combination with AC220, was able to induce cell death in different cancer cells, but not in non-cancer cell lines. We showed that TAK-165 and AC220 act synergistically to downregulate key signaling pathways and potently induce cancer cell death. Furthermore, we demonstrated that TAK-165 inhibited autophagy in a HER2-independent manner. Finally, we showed that the combination of TAK-165 and AC220 induced cell death in cancer cells through the activation of chaperone-mediated autophagy. Overall, these findings support the strategy for using AC220 and an autophagy inhibitor such as TAK-165 in a combinatorial treatment to enhance the efficacy of cancer therapies.

Regulation of RIPK1 activation by TAK1-mediated phosphorylation dictates apoptosis and necroptosis.

Stimulation of TNFR1 by TNFα can promote three distinct alternative mechanisms of cell death: necroptosis, RIPK1-independent and -dependent apoptosis. How cells decide which way to die is unclear. Here, we report that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this critical decision. Using phospho-Ser321 as a marker, we show that the transient phosphorylation of RIPK1 intermediate domain induced by TNFα leads to RIPK1-independent apoptosis when NF-κB activation is inhibited by cycloheximide. On the other hand, blocking Ser321 phosphorylation promotes RIPK1 activation and its interaction with FADD to mediate RIPK1-dependent apoptosis (RDA). Finally, sustained phosphorylation of RIPK1 intermediate domain at multiple sites by TAK1 promotes its interaction with RIPK3 and necroptosis. Thus, absent, transient and sustained levels of TAK1-mediated RIPK1 phosphorylation may represent distinct states in TNF-RSC to dictate the activation of three alternative cell death mechanisms, RDA, RIPK1-independent apoptosis and necroptosis.TNFα can promote three distinct mechanisms of cell death: necroptosis, RIPK1-independent and dependent apoptosis. Here the authors show that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this decision.

Direct quantification of autophagic flux by a single molecule-based probe.

Macroautophagy/autophagy remains a rapidly advancing research topic, and there continues to be a need for constantly evolving methodology to investigate this pathway at each individual step. Accordingly, new assays to measure autophagic flux in a robust and reliable manner are essential to understand the mechanism and physiological roles of autophagy. Kaizuka et al. recently reported a new fluorescence probe, GFP-LC3-RFP-LC3ΔG to directly demonstrate autophagic flux without being combined with lysosomal inhibitors (see the Kaizuka et al. punctum in this issue of the journal). When expressed in cells, the probe is cleaved into a degradable/quenchable part, GFP-LC3, and stable/cytosolic part, RFP-LC3ΔG. The latter serves as an internal control and a decrease of the GFP:RFP ratio indicates the occurrence of autophagy. Since the key index of this probe is the degradation of GFP-LC3, it can be used to measure the cumulative effect of basal autophagy. The assay is applicable to high-throughput drug discovery as well as in vivo analysis.

RIPK1 mediates axonal degeneration by promoting inflammation and necroptosis in ALS.

Mutations in the optineurin (OPTN) gene have been implicated in both familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of this protein in the central nervous system (CNS) and how it may contribute to ALS pathology are unclear. Here, we found that optineurin actively suppressed receptor-interacting kinase 1 (RIPK1)-dependent signaling by regulating its turnover. Loss of OPTN led to progressive dysmyelination and axonal degeneration through engagement of necroptotic machinery in the CNS, including RIPK1, RIPK3, and mixed lineage kinase domain-like protein (MLKL). Furthermore, RIPK1- and RIPK3-mediated axonal pathology was commonly observed in SOD1(G93A) transgenic mice and pathological samples from human ALS patients. Thus, RIPK1 and RIPK3 play a critical role in mediating progressive axonal degeneration. Furthermore, inhibiting RIPK1 kinase may provide an axonal protective strategy for the treatment of ALS and other human degenerative diseases characterized by axonal degeneration.

Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death.

Hexokinase II (HK2), a key enzyme involved in glucose metabolism, is regulated by growth factor signaling and is required for initiation and maintenance of tumors. Here we show that metabolic stress triggered by perturbation of receptor tyrosine kinase FLT3 in non-acute myeloid leukemia cells sensitizes cancer cells to autophagy inhibition and leads to excessive activation of chaperone-mediated autophagy (CMA). Our data demonstrate that FLT3 is an important sensor of cellular nutritional state and elucidate the role and molecular mechanism of CMA in metabolic regulation and mediating cancer cell death. Importantly, our proteome analysis revealed that HK2 is a CMA substrate and that its degradation by CMA is regulated by glucose availability. We reveal a new mechanism by which excessive activation of CMA may be exploited pharmacologically to eliminate cancer cells by inhibiting both FLT3 and autophagy. Our study delineates a novel pharmacological strategy to promote the degradation of HK2 in cancer cells.

G-protein-coupled receptors regulate autophagy by ZBTB16-mediated ubiquitination and proteasomal degradation of Atg14L.

Autophagy is an important intracellular catabolic mechanism involved in the removal of misfolded proteins. Atg14L, the mammalian ortholog of Atg14 in yeast and a critical regulator of autophagy, mediates the production PtdIns3P to initiate the formation of autophagosomes. However, it is not clear how Atg14L is regulated. In this study, we demonstrate that ubiquitination and degradation of Atg14L is controlled by ZBTB16-Cullin3-Roc1 E3 ubiquitin ligase complex. Furthermore, we show that a wide range of G-protein-coupled receptor (GPCR) ligands and agonists regulate the levels of Atg14L through ZBTB16. In addition, we show that the activation of autophagy by pharmacological inhibition of GPCR reduces the accumulation of misfolded proteins and protects against behavior dysfunction in a mouse model of Huntington's disease. Our study demonstrates a common molecular mechanism by which the activation of GPCRs leads to the suppression of autophagy and a pharmacological strategy to activate autophagy in the CNS for the treatment of neurodegenerative diseases.

Activation of necroptosis in multiple sclerosis.

Multiple sclerosis (MS), a common neurodegenerative disease of the CNS, is characterized by the loss of oligodendrocytes and demyelination. Tumor necrosis factor α (TNF-α), a proinflammatory cytokine implicated in MS, can activate necroptosis, a necrotic cell death pathway regulated by RIPK1 and RIPK3 under caspase-8-deficient conditions. Here, we demonstrate defective caspase-8 activation, as well as activation of RIPK1, RIPK3, and MLKL, the hallmark mediators of necroptosis, in the cortical lesions of human MS pathological samples. Furthermore, we show that MS pathological samples are characterized by an increased insoluble proteome in common with other neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Finally, we show that necroptosis mediates oligodendrocyte degeneration induced by TNF-α and that inhibition of RIPK1 protects against oligodendrocyte cell death in two animal models of MS and in culture. Our findings demonstrate that necroptosis is involved in MS and suggest that targeting RIPK1 may represent a therapeutic strategy for MS.

Discovery of type II inhibitors of TGFß-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2).

We developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure-activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16 and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. A 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.

SNARE proteins are required for macroautophagy.

Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.

The Golgi as a potential membrane source for autophagy.

In macroautophagy (hereafter autophagy), a morphological hallmark is the formation of double-membrane vesicles called autophagosomes that sequester and deliver cytoplasmic components to the lysosome/vacuole for degradation. This process begins with an initial sequestering compartment, the phagophore, which expands into the mature autophagosome. A tremendous amount of work has been carried out to elucidate the mechanism of how the autophagosome is formed. However, an important missing piece in this puzzle is where the membrane comes from. Independent lines of evidence have shown that preexisting organelles may continuously supply lipids to support autophagosome formation. In our analysis, we identified several components of the late stage secretory pathway that may redirect Golgi-derived membrane to autophagosome formation in response to starvation conditions.

Post-Golgi Sec proteins are required for autophagy in Saccharomyces cerevisiae.

In eukaryotic cells, autophagy mediates the degradation of cytosolic contents in response to environmental change. Genetic analyses in fungi have identified over 30 autophagy-related (ATG) genes and provide substantial insight into the molecular mechanism of this process. However, one essential issue that has not been resolved is the origin of the lipids that form the autophagosome, the sequestering vesicle that is critical for autophagy. Here, we report that two post-Golgi proteins, Sec2 and Sec4, are required for autophagy. Sec4 is a Rab family GTPase, and Sec2 is its guanine nucleotide exchange factor. In sec2 and sec4 conditional mutant yeast, the anterograde movement of Atg9, a proposed membrane carrier, is impaired during starvation conditions. Similarly, in the sec2 mutant, Atg8 is inefficiently recruited to the phagophore assembly site, which is involved in autophagosome biogenesis, resulting in the generation of fewer autophagosomes. We propose that following autophagy induction the function of Sec2 and Sec4 are diverted to direct membrane flow to autophagosome formation.

Positive or negative roles of different cyclin-dependent kinase Pho85-cyclin complexes orchestrate induction of autophagy in Saccharomyces cerevisiae.

As a major intracellular degradation pathway, autophagy is tightly regulated to prevent cellular dysfunction in all eukaryotic cells. The rapamycin-sensitive Tor kinase complex 1 is a major regulator of autophagy. Several other nutrient-sensory kinases also play critical roles to precisely modulate autophagy; however, the network of regulatory mechanisms remains largely elusive. We used genetic analyses to elucidate the mechanism by which the stress-responsive, cyclin-dependent kinase Pho85 and its corresponding cyclin complexes antagonistically modulate autophagy in Saccharomyces cerevisiae. When complexed with cyclins Pho80 and Pcl5, Pho85 negatively regulates autophagy through downregulating the protein kinase Rim15 and the transcription factors Pho4 and Gcn4. The cyclins Clg1, Pcl1, and Pho80, in concert with Pho85, positively regulate autophagy through promoting the degradation of Sic1, a negative regulator of autophagy that targets Rim15. Our results suggest a model in which Pho85 and its cyclin complexes have opposing roles in autophagy regulation.

Determining Atg protein stoichiometry at the phagophore assembly site by fluorescence microscopy.

In eukaryotic cells, autophagy is a lysosomal/ vacuolar degradative pathway necessary for the turnover of different macromolecules. Autophagy is under precise regulation, not only qualitatively but also quantitatively, and excess or reduced levels of autophagy may lead to various human diseases. In yeast, genetic screens led to the identification of more than 30 autophagy-related (ATG) genes, and most of the gene products reside at the phagophore assembly site (PAS). However, our attempt to understand the quantitative properties of autophagy is usually hampered, because traditional methods of analysis cannot provide stoichiometric information. We have recently used a fluorescence microscopy-based method to study the stoichiometry of Atg proteins at the PAS, trying to explain the mechanism of how the vesicle formation process is precisely regulated. This article describes a practical guide on this method. Its application and further analysis will improve our understanding of the quantitative properties of autophagy.

A genomic screen for yeast mutants defective in selective mitochondria autophagy.

Mitophagy is the process of selective mitochondrial degradation via autophagy, which has an important role in mitochondrial quality control. Very little is known, however, about the molecular mechanism of mitophagy. A genome-wide yeast mutant screen for mitophagy-defective strains identified 32 mutants with a block in mitophagy, in addition to the known autophagy-related (ATG) gene mutants. We further characterized one of these mutants, ylr356wDelta that corresponds to a gene whose function has not been identified. YLR356W is a mitophagy-specific gene that was not required for other types of selective autophagy or macroautophagy. The deletion of YLR356W partially inhibited mitophagy during starvation, whereas there was an almost complete inhibition at post-log phase. Accordingly, we have named this gene ATG33. The new mutants identified in this analysis will provide a useful foundation for researchers interested in the study of mitochondrial homeostasis and quality control.

Indirect estimation of the area density of Atg8 on the phagophore.

Atg8 is a ubiquitin-like protein that controls the expansion of the phagophore during autophagosome formation. It is recruited to the phagophore during the expansion stage and released upon the completion of the autophagosome. One possible model explaining the function of Atg8 is that it acts as an adaptor of a coat complex. Here, we tested the coat-adaptor model by estimating the area density of Atg8 molecules on the phagophore. We developed a computational process to simulate the random sectioning of vesicles heterogeneous in size. This method can be applied to estimate the original sizes of intracellular vesicles from sizes of their random sections obtained through transmission electron microscopy. Using this method, we found that the estimated area density of Atg8 is comparable with that of proteins that form the COPII coat.

Quantitative regulation of vesicle formation in yeast nonspecific autophagy.

In eukaryotic cells, autophagy is a degradative pathway necessary for the turnover of bulk cytoplasm. In yeast, this pathway also mediates the specific transport of a vacuolar hydrolase zymogen, precursor aminopeptidase (prApe1), from the cytoplasm to the vacuole. Autophagy is under precise regulation, not only qualitatively but also quantitatively, especially in the steps involved in the vesicle formation process. We have recently used a fluorescence microscopy-based method to study the stoichiometry of autophagy-related (Atg) proteins during different conditions. This analysis shows that increased expression of Atg11 in the cytoplasm to vacuole targeting (Cvt) pathway increases the amount of this protein localized at the phagophore assembly site (PAS). In turn, under nutrient-rich conditions, the increased level of Atg11 causes the recruitment of higher than normal levels of Atg8 and Atg9 to the PAS, resulting in the formation of more Cvt vesicles, whereas the vesicle size is not affected. Combined with results from previous studies in starvation conditions, in this addendum we discuss the possible role of Atg8 and Atg9 in quantitatively regulating the vesicle formation process.