scholarly journals A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Dick J. H. van den Boomen ◽  
Agata Sienkiewicz ◽  
Ilana Berlin ◽  
Marlieke L. M. Jongsma ◽  
Daphne M. van Elsland ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Ldl Cholesterol ◽  
Ldl Receptor ◽  
Cholesterol Homeostasis ◽  
Cellular Cholesterol ◽  
Genome Wide ◽  
A Genome ◽  
Critical Defect ◽  
Crispr Screen ◽  
And Function

Abstract Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.

scholarly journals A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

2019 ◽  
Author(s):  
Dick J.H. van den Boomen ◽  
Agata Sienkiewicz ◽  
Ilana Berlin ◽  
Marlieke L.M. Jongsma ◽  
Daphne M. van Elsland ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Ldl Cholesterol ◽  
Ldl Receptor ◽  
Cholesterol Homeostasis ◽  
Cellular Cholesterol ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Additional Defect ◽  
And Function

SummaryCholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, using an endogenous cholesterol reporter in a genome-wide CRISPR screen we identify >70 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core component of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical additional defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.

scholarly journals Dengue virus hijacks a noncanonical oxidoreductase function of a cellular oligosaccharyltransferase complex

2017 ◽  
Author(s):  
David L. Lin ◽  
Natalia A. Cherepanova ◽  
Leonia Bozzacco ◽  
Margaret R. Macdonald ◽  
Reid Gilmore ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Mammalian Cells ◽  
Denv Infection ◽  
Oxidoreductase Activity ◽  
Catalytic Function ◽  
Active Site Motif ◽  
Genome Wide ◽  
A Genome ◽  
Close Proximity ◽  
Crispr Screen

AbstractDengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide CRISPR screen to identify host dependency factors required for DENV propagation, and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CxxC active site motif of MAGT1 was necessary for DENV propagation as cells expressing an AxxA MAGT1 mutant were unable to support DENV infection.Interestingly, cells expressing single-cysteine CxxA or AxxC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antivirals targeting DENV.

scholarly journals A genome-wide CRISPR screen identifies UFMylation and TRAMP-like complexes as host factors required for hepatitis A virus infection

Cell Reports ◽  
2021 ◽  
Vol 34 (11) ◽  
pp. 108859
Author(s):  
Jessie Kulsuptrakul ◽  
Ruofan Wang ◽  
Nathan L. Meyers ◽  
Melanie Ott ◽  
Andreas S. Puschnik
Keyword(s):  
Virus Infection ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Host Factors ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

scholarly journals A Genome-wide CRISPR Screen Identifies CDC25A as a Determinant of Sensitivity to ATR Inhibitors

2016 ◽  
Vol 62 (2) ◽  
pp. 307-313 ◽  
Author(s):  
Sergio Ruiz ◽  
Cristina Mayor-Ruiz ◽  
Vanesa Lafarga ◽  
Matilde Murga ◽  
Maria Vega-Sendino ◽  
...  
Keyword(s):  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

scholarly journals Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Rowena DeJesus ◽  
Francesca Moretti ◽  
Gregory McAllister ◽  
Zuncai Wang ◽  
Phil Bergman ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Er Stress Response ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Selection Step ◽  
A Cell ◽  
Cell Type Specific ◽  
Cellular Pathways ◽  
Mtor Signalling

SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.

scholarly journals An ancestral recombination graph of human, Neanderthal, and Denisovan genomes

2021 ◽  
Vol 7 (29) ◽  
pp. eabc0776
Author(s):  
Nathan K. Schaefer ◽  
Beth Shapiro ◽  
Richard E. Green
Keyword(s):  
Incomplete Lineage Sorting ◽  
Simulated Data ◽  
Modern Human ◽  
Ancestral Recombination Graph ◽  
Lineage Sorting ◽  
Human Genomes ◽  
Genome Wide ◽  
A Genome ◽  
Graph Inference ◽  
And Function

Many humans carry genes from Neanderthals, a legacy of past admixture. Existing methods detect this archaic hominin ancestry within human genomes using patterns of linkage disequilibrium or direct comparison to Neanderthal genomes. Each of these methods is limited in sensitivity and scalability. We describe a new ancestral recombination graph inference algorithm that scales to large genome-wide datasets and demonstrate its accuracy on real and simulated data. We then generate a genome-wide ancestral recombination graph including human and archaic hominin genomes. From this, we generate a map within human genomes of archaic ancestry and of genomic regions not shared with archaic hominins either by admixture or incomplete lineage sorting. We find that only 1.5 to 7% of the modern human genome is uniquely human. We also find evidence of multiple bursts of adaptive changes specific to modern humans within the past 600,000 years involving genes related to brain development and function.

scholarly journals Genome-wide CRISPR screen identifies regulators of MAPK and MTOR pathways mediating sorafenib resistance in acute myeloid leukemia

Haematologica ◽  
2020 ◽  
Author(s):  
Alisa Damnernsawad ◽  
Daniel Bottomly ◽  
Stephen E. Kurtz ◽  
Christopher A. Eide ◽  
Shannon K. McWeeney ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Mek Inhibitors ◽  
Genome Wide ◽  
A Genome ◽  
Flt3 Mutations ◽  
Flt3 Inhibitors ◽  
Crispr Screen ◽  
Acute Myeloid

Drug resistance impedes the long-term effect of targeted therapies in acute myeloid leukemia (AML), necessitating the identification of mechanisms underlying resistance. Approximately 25% of AML patients carry FLT3 mutations and develop post-treatment insensitivity to FLT3 inhibitors, including sorafenib. Using a genome-wide CRISPR screen, we identified LZTR1, NF1, TSC1 or TSC2, negative regulators of the MAPK and MTOR pathways, as mediators of sorafenib resistance. Analyses of ex vivo drug sensitivity assays in FLT3-ITD AML patient samples revealed lower expression of LZTR1, NF1, and TSC2 correlated with sorafenib sensitivity. Importantly, MAPK and/or MTOR complex1 (MTORC1) activity were upregulated in AML cells made resistant to several FLT3 inhibitors, including crenolanib, quizartinib, or sorafenib. These cells were sensitive to MEK inhibitors, and the combination of FLT3 and MEK inhibitors showed enhanced efficacy, suggesting its effectiveness in AML patients with FLT3 mutations and those with resistance to FLT3 inhibitors.

Controlling quality and amount of mitochondria by mitophagy: insights into the role of ubiquitination and deubiquitination

2016 ◽  
Vol 397 (7) ◽  
pp. 637-647 ◽  
Author(s):  
Tao Tan ◽  
Marcel Zimmermann ◽  
Andreas S. Reichert
Keyword(s):  
Mammalian Cells ◽  
Mitochondrial Fission ◽  
Baker’S Yeast ◽  
Negative Regulator ◽  
Mitochondrial Outer Membrane ◽  
Baker's Yeast ◽  
Genome Wide ◽  
A Genome ◽  
Autophagy Pathway ◽  
Quantity Control

Abstract Mitophagy is a selective autophagy pathway conserved in eukaryotes and plays an essential role in mitochondrial quality and quantity control. Mitochondrial fission and fusion cycles maintain a certain amount of healthy mitochondria and allow the isolation of damaged mitochondria for their elimination by mitophagy. Mitophagy can be classified into receptor-dependent and ubiquitin-dependent pathways. The mitochondrial outer membrane protein Atg32 is identified as the only known receptor for mitophagy in baker’s yeast, whereas mitochondrial proteins FUNDC1, NIX/BNIP3L, BNIP3 and Bcl2L13 are recognized as mitophagy receptors in mammalian cells. Earlier studies showed that ubiquitination and deubiquitination occurs in yeast, yet there is no direct evidence for an ubiquitin-dependent mitophagy pathway in this organism. In contrast, a ubiquitin-/PINK1-/Parkin-dependent mitophagy pathway was unraveled and was extensively characterized in mammals in recent years. Recently, a quantitative method termed synthetic quantitative array (SQA) technology was developed to identify modulators of mitophagy in baker’s yeast on a genome-wide level. The Ubp3-Bre5 deubiquitination complex was found as a negative regulator of mitophagy while promoting other autophagic pathways. Here we discuss how ubiquitination and deubiquitination regulates mitophagy and other selective forms of autophagy and what argues for using baker’s yeast as a model to study the ubiquitin-dependent mitophagy pathway.

Analysis of MicroRNA Transcriptomes Insights into the miR-148a-3p Inducer of Rumen Development in Goats

2020 ◽  
Author(s):  
Tao Zhong ◽  
Cheng Wang ◽  
Jiangtao Hu ◽  
Xiaoyong Chen ◽  
Lili Niu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Target Genes ◽  
Genetic Regulation ◽  
Mapk Signaling ◽  
Regulation Mechanism ◽  
Ras Signaling ◽  
Genome Wide ◽  
A Genome ◽  
Differentially Expressed Mirnas ◽  
And Function

Abstract Background: Rumen is an important digestive organ of ruminant. From fetal to adult stage, the morphology, structure and function of rumen have changed significantly. But the intrinsic genetic regulation is still limited. We previously reported a genome-wide expression profile of miRNAs in prenatal goat rumens. In the present study, we rejoined analyzed the transcriptomes of rumen miRNAs during prenatal (E60 and E135) and postnatal (D30 and D150) stages.Results: A total of 66 differentially expressed miRNAs (DEMs) were identified in the rumen tissues from D30 and D150 goats. Of these, 17 DEMs were consistently highly expressed in the rumens at the preweaning stages (E60, E135 and D30), while down-regulated at D150. Noteworthy, annotation analysis revealed that the target genes regulated by the DEMs were mainly enriched in MAPK signaling pathway, Jak-STAT signaling pathway and Ras signaling pathway. Interestingly, the expression of miR-148a-3p was significantly high in the embryonic stage and down-regulated at D150. The potential binding sites between miR-148a-3p and QKI were predicted by the TargetScan and verified by the dual luciferase report assay. The co-localization of miR-148a-3p and QKI was observed not in intestinal tracts but in rumen tissues by in situ hybridization. Moreover, the expression of miR-148a-3p in the epithelium was significantly higher than that in the other layers, suggesting that miR-148a-3p involve in the development of rumen epithelial cells by targeting QKI. Subsequently, miR-148a-3p inhibitor was found to induce the proliferation of GES-1 cells.Conclusions: Taken together, these results identified the DEMs involved in the development of rumen and provided an insight into the regulation mechanism of goat rumens during development.

scholarly journals VPS37A directs ESCRT recruitment for phagophore closure

2019 ◽  
Vol 218 (10) ◽  
pp. 3336-3354 ◽  
Author(s):  
Yoshinori Takahashi ◽  
Xinwen Liang ◽  
Tatsuya Hattori ◽  
Zhenyuan Tang ◽  
Haiyan He ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Growth Factor Receptor ◽  
Multivesicular Body ◽  
Regulatory Mechanisms ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Escrt Machinery ◽  
Genome Wide ◽  
A Genome ◽  
Epidermal Growth

The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

Sign in / Sign up

Export Citation Format

Share Document