scholarly journals Structural basis for autophagy inhibition by the human Rubicon–Rab7 complex

2020 ◽  
Vol 117 (29) ◽  
pp. 17003-17010 ◽  
Author(s):  
Hersh K. Bhargava ◽  
Keisuke Tabata ◽  
Jordan M. Byck ◽  
Maho Hamasaki ◽  
Daniel P. Farrell ◽  
...  
Keyword(s):  
Negative Regulator ◽  
Structural Basis ◽  
Autophagic Flux ◽  
Dimer Interface ◽  
Promising Therapeutic Target ◽  
Switch Regions ◽  
Zinc Clusters ◽  
Rab Effector ◽  
Autophagy Inhibition ◽  
Potent Negative Regulator

Rubicon is a potent negative regulator of autophagy and a potential target for autophagy-inducing therapeutics. Rubicon-mediated inhibition of autophagy requires the interaction of the C-terminal Rubicon homology (RH) domain of Rubicon with Rab7–GTP. Here we report the 2.8-Å crystal structure of the Rubicon RH domain in complex with Rab7–GTP. Our structure reveals a fold for the RH domain built around four zinc clusters. The switch regions of Rab7 insert into pockets on the surface of the RH domain in a mode that is distinct from those of other Rab–effector complexes. Rubicon residues at the dimer interface are required for Rubicon and Rab7 to colocalize in living cells. Mutation of Rubicon RH residues in the Rab7-binding site restores efficient autophagic flux in the presence of overexpressed Rubicon, validating the Rubicon RH domain as a promising therapeutic target.

scholarly journals Structural basis for autophagy inhibition by the human Rubicon-Rab7 complex

2020 ◽  
Author(s):  
Hersh K. Bhargava ◽  
Keisuke Tabata ◽  
Jordan M. Byck ◽  
Maho Hamasaki ◽  
Daniel P. Farrell ◽  
...  
Keyword(s):  
Negative Regulator ◽  
Structural Basis ◽  
Autophagic Flux ◽  
Dimer Interface ◽  
Promising Therapeutic Target ◽  
Switch Regions ◽  
Zinc Clusters ◽  
Rab Effector ◽  
Autophagy Inhibition ◽  
Potent Negative Regulator

AbstractRubicon is a potent negative regulator of autophagy and a potential target for autophagy-inducing therapeutics. Rubicon-mediated inhibition of autophagy requires the interaction of the C-terminal Rubicon homology (RH) domain of Rubicon with Rab7-GTP. Here we report the 2.8 Å crystal structure of the Rubicon RH domain in complex with Rab7-GTP. Our structure reveals a novel fold for the RH domain built around four zinc clusters. The switch regions of Rab7 insert into pockets on the surface of the RH domain in a mode that is distinct from those of other Rab-effector complexes. Rubicon residues at the dimer interface are required for Rubicon and Rab7 to colocalize in living cells. Mutation of Rubicon RH residues in the Rab7 binding site restore efficient autophagic flux in the presence of overexpressed Rubicon, validating the Rubicon RH domain as a promising therapeutic target.

scholarly journals NPHP4, a cilia-associated protein, negatively regulates the Hippo pathway

2011 ◽  
Vol 193 (4) ◽  
pp. 633-642 ◽  
Author(s):  
Sandra Habbig ◽  
Malte P. Bartram ◽  
Roman U. Müller ◽  
Ricarda Schwarz ◽  
Nikolaos Andriopoulos ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cellular Proliferation ◽  
Nuclear Translocation ◽  
Hippo Pathway ◽  
Negative Regulator ◽  
Hippo Signaling ◽  
Transcriptional Coactivator ◽  
Hippo Signaling Pathway ◽  
The Hippo Pathway ◽  
Potent Negative Regulator

The conserved Hippo signaling pathway regulates organ size in Drosophila melanogaster and mammals and has an essential role in tumor suppression and the control of cell proliferation. Recent studies identified activators of Hippo signaling, but antagonists of the pathway have remained largely elusive. In this paper, we show that NPHP4, a known cilia-associated protein that is mutated in the severe degenerative renal disease nephronophthisis, acts as a potent negative regulator of mammalian Hippo signaling. NPHP4 directly interacted with the kinase Lats1 and inhibited Lats1-mediated phosphorylation of the Yes-associated protein (YAP) and TAZ (transcriptional coactivator with PDZ-binding domain), leading to derepression of these protooncogenic transcriptional regulators. Moreover, NPHP4 induced release from 14-3-3 binding and nuclear translocation of YAP and TAZ, promoting TEA domain (TEAD)/TAZ/YAP-dependent transcriptional activity. Consistent with these data, knockdown of NPHP4 negatively affected cellular proliferation and TEAD/TAZ activity, essentially phenocopying loss of TAZ function. These data identify NPHP4 as a negative regulator of the Hippo pathway and suggest that NPHP4 regulates cell proliferation through its effects on Hippo signaling.

scholarly journals Antidepressant Drug Enhanced Trail Receptor-2 Expression and Sensitized Lung Cancer Cells to Trail-Induced Apoptosis via Inhibition of Autophagic Flux

2020 ◽  
Author(s):  
K.M.A. Zinnah ◽  
Jae-Won Seol ◽  
Sang-Youel Park
Keyword(s):  
Lung Cancer ◽  
Cell Death ◽  
Cancer Cells ◽  
Antidepressant Drug ◽  
Lung Cancer Cells ◽  
Autophagic Flux ◽  
Trail Receptor ◽  
Treatment Needs ◽  
Induced Apoptosis ◽  
Autophagy Inhibition

Autophagy, an alternative cell death mechanism, is also termed programmed cell death type II. Autophagy in cancer treatment needs to be regulated. In our study, autophagy inhibition by desipramine or the autophagy inhibitor chloroquine (CQ) enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2 [death receptor (DR5)] expression and subsequently TRAIL-induced apoptosis in TRAIL-resistant A549 lung cancer cells. Genetic inhibition of DR5 substantially reduced desipramine-enhanced TRAIL-mediated apoptosis, proving that DR5 was required to increase TRAIL sensitivity in TRAIL-resistant cancer cells. Desipramine treatment upregulated p62 expression and promoted conversion of light chain 3 (LC3)-I to its lipid-conjugated form, LC3-II, indicating that autophagy inhibition occurred at the final stages of autophagic flux. Transmission electron microscopy analysis showed the presence of condensed autophagosomes, which resulted from the late stages of autophagy inhibition by desipramine. TRAIL, in combination with desipramine or CQ, augmented the expression of apoptosis-related proteins cleaved caspase-8 and cleaved caspase-3. Our results contributed to the understanding of the mechanism underlying the synergistic anti-cancer effect of desipramine and TRAIL and presented a novel mechanism of DR5 upregulation. These findings demonstrated that autophagic flux inhibition by desipramine potentiated TRAIL-induced apoptosis, suggesting that appropriate regulation of autophagy is required for sensitizing TRAIL-resistant cancer cells to TRAIL-mediated apoptosis.

scholarly journals Identification of BMP and Activin Membrane-Bound Inhibitor (BAMBI) as a Potent Negative Regulator of Adipogenesis and Modulator of Autocrine/Paracrine Adipogenic Factors

Diabetes ◽  
2011 ◽  
Vol 61 (1) ◽  
pp. 124-136 ◽  
Author(s):  
X. Luo ◽  
L. J. Hutley ◽  
J. A. Webster ◽  
Y.-H. Kim ◽  
D.-F. Liu ◽  
...  
Keyword(s):  
Negative Regulator ◽  
Membrane Bound ◽  
Potent Negative Regulator

scholarly journals Structural Basis of Rab Effector Specificity

Cell ◽  
1999 ◽  
Vol 96 (3) ◽  
pp. 363-374 ◽  
Author(s):  
Christian Ostermeier ◽  
Axel T. Brunger
Keyword(s):  
Structural Basis ◽  
Rab Effector

scholarly journals Her2-Targeted Therapy Induces Autophagy in Esophageal Adenocarcinoma Cells

2018 ◽  
Vol 19 (10) ◽  
pp. 3069 ◽  
Author(s):  
Félice Janser ◽  
Olivia Adams ◽  
Vanessa Bütler ◽  
Anna Schläfli ◽  
Bastian Dislich ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Esophageal Adenocarcinoma ◽  
Acquired Resistance ◽  
Therapy Resistance ◽  
Her2 Status ◽  
Autophagic Flux ◽  
Cancer Type ◽  
Cancer Resistance ◽  
Her2 Positive ◽  
Autophagy Inhibition

Esophageal adenocarcinoma (EAC) is a highly lethal cancer type with an overall poor survival rate. Twenty to thirty percent of EAC overexpress the human epidermal growth factor receptor 2 (Her2), a transmembrane receptor tyrosine kinase promoting cell growth and proliferation. Patients with Her2 overexpressing breast and gastroesophageal cancer may benefit from Her2 inhibitors. Therapy resistance, however, is well documented. Since autophagy, a lysosome-dependent catabolic process, is implicated in cancer resistance mechanisms, we tested whether autophagy modulation influences Her2 inhibitor sensitivity in EAC. Her2-positive OE19 EAC cells showed an induction in autophagic flux upon treatment with the small molecule Her2 inhibitor Lapatinib. Newly generated Lapatinib-resistant OE19 (OE19 LR) cells showed increased basal autophagic flux compared to parental OE19 (OE19 P) cells. Based on these results, we tested if combining Lapatinib with autophagy inhibitors might be beneficial. OE19 P showed significantly reduced cell viability upon double treatment, while OE19 LR were already sensitive to autophagy inhibition alone. Additionally, Her2 status and autophagy marker expression (LC3B and p62) were investigated in a treatment-naïve EAC patient cohort (n = 112) using immunohistochemistry. Here, no significant correlation between Her2 status and expression of LC3B and p62 was found. Our data show that resistance to Her2-directed therapy is associated with a higher basal autophagy level, which is not per se associated with Her2 status. Therefore, we propose that autophagy may contribute to acquired resistance to Her2-targeted therapy in EAC, and that combining Her2 and autophagy inhibition might be beneficial for EAC patients.

scholarly journals Effect of early-stage autophagy inhibition in BRAFV600E autophagy-dependent brain tumor cells

2019 ◽  
Vol 10 (9) ◽  
Author(s):  
Shadi Zahedi ◽  
Brent E. Fitzwalter ◽  
Andrew Morin ◽  
Sydney Grob ◽  
Michele Desmarais ◽  
...  
Keyword(s):  
Cell Survival ◽  
Tumor Cells ◽  
Late Stage ◽  
Early Stage ◽  
Specific Effect ◽  
Cns Tumors ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Tumor Cell Death ◽  
Autophagy Inhibition

Abstract Autophagy is a multistage process. Progress within the field has led to the development of agents targeting both early (initiation) and late (fusion) stages of this process. The specific stage of autophagy targeted may influence cancer treatment outcomes. We have previously shown that central nervous system (CNS) tumors with the BRAFV600E mutation are autophagy dependent, and late-stage autophagy inhibition improves the response to targeted BRAF inhibitors (BRAFi) in sensitive and resistant cells. Drugs directed toward initiation of autophagy have been shown to reduce tumor cell death in some cancers, but have not been assessed in CNS tumors. We investigated early-stage inhibition for autophagy-dependent CNS tumors. BRAFi-sensitive and resistant AM38 and MAF794 cell lines were evaluated for the response to pharmacologic and genetic inhibition of ULK1 and VPS34, two crucial subunits of the autophagy initiation complexes. Changes in autophagy were monitored by western blot and flow cytometry. Survival was evaluated in short- and long-term growth assays. Tumor cells exhibited a reduced autophagic flux with pharmacologic and genetic inhibition of ULK1 or VPS34. Pharmacologic inhibition reduced cell survival in a dose-dependent manner for both targets. Genetic inhibition reduced cell survival and confirmed that it was an autophagy-specific effect. Pharmacologic and genetic inhibition were also synergistic with BRAFi, irrespective of RAFi sensitivity. Inhibition of ULK1 and VPS34 are potentially viable clinical targets in autophagy-dependent CNS tumors. Further evaluation is needed to determine if early-stage autophagy inhibition is equal to late-stage inhibition to determine the optimal clinical target for patients.

scholarly journals USP7 inhibits Wnt/β-catenin signaling through promoting stabilization of Axin

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Lei Ji ◽  
Bo Lu ◽  
Raffaella Zamponi ◽  
Olga Charlat ◽  
Robert Aversa ◽  
...  
Keyword(s):  
Osteoblast Differentiation ◽  
Adipocyte Differentiation ◽  
Ubiquitin Proteasome System ◽  
Cellular Systems ◽  
Negative Regulator ◽  
Scaffolding Protein ◽  
Complex Stability ◽  
Genetic Ablation ◽  
Ubiquitin Proteasome ◽  
Potent Negative Regulator

Abstract Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/β-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/β-catenin signaling.

scholarly journals Crystal structure of enolase fromDrosophila melanogaster

2017 ◽  
Vol 73 (4) ◽  
pp. 228-234 ◽  
Author(s):  
Congcong Sun ◽  
Baokui Xu ◽  
Xueyan Liu ◽  
Zhen Zhang ◽  
Zhongliang Su
Keyword(s):  
Crystal Structure ◽  
Catalytic Activity ◽  
Structural Basis ◽  
Molecular Replacement ◽  
Biological Processes ◽  
Conserved Residues ◽  
Dimer Interface ◽  
Open Conformation ◽  
Evolutionary Studies ◽  
Important Enzyme

Enolase is an important enzyme in glycolysis and various biological processes. Its dysfunction is closely associated with diseases. Here, the enolase fromDrosophila melanogaster(DmENO) was purified and crystallized. A crystal of DmENO diffracted to 2.0 Å resolution and belonged to space groupR32. The structure was solved by molecular replacement. Like most enolases, DmENO forms a homodimer with conserved residues in the dimer interface. DmENO possesses an open conformation in this structure and contains conserved elements for catalytic activity. This work provides a structural basis for further functional and evolutionary studies of enolase.

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