Publications by authors named "Peter K Jackson"

114 Publications

The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D.

Nature 2021 Apr 14;592(7856):794-798. Epub 2021 Apr 14.

Department of Pediatrics, Stanford University, Stanford, CA, USA.

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer. However, the mechanisms that regulate levels of cyclin D are incompletely understood. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of  the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.
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http://dx.doi.org/10.1038/s41586-021-03474-7DOI Listing
April 2021

Connecting autoimmune disease to Bardet-Biedl syndrome and primary cilia.

EMBO Rep 2021 Feb 28;22(2):e52180. Epub 2021 Jan 28.

Baxter Laboratory, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA, USA.

Bardet-Biedl syndrome (BBS) is a genetic disorder caused by the dysfunction of the primary cilium. To date, immunological defects in the disease have not been systematically assessed. In this issue, Tsyklauri and colleagues find, through detailed analysis of BBS mutant animals, that B-cell development is altered in mutant mice (Tsyklauri et al, 2021). The authors further report that BBS patients are more susceptible to autoimmune disorders. This study sheds new light on the potential role of primary cilia in controlling immune function in disease.
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http://dx.doi.org/10.15252/embr.202052180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857418PMC
February 2021

Ethacridine inhibits SARS-CoV-2 by inactivating viral particles in cellular models.

bioRxiv 2020 Oct 28. Epub 2020 Oct 28.

More than a million people have now died from COVID-19, because of infection with the SARS-CoV-2 coronavirus. Currently, the FDA has approved remdesivir, an inhibitor of SARS-CoV-2 replication, to treat COVID-19, though very recent data from WHO showed little if any COVID19 protective effect. Here we report that ethacridine, a safe and potent antiseptic use in humans, effectively inhibits SARS-CoV-2, at very low concentrations (EC ~ 0.08 M). Ethacridine was identified through a high-throughput screening of an FDA-approved drug library in living cells using a fluorescent assay. Interestingly, the main mode of action of ethacridine is to inactivate virus particles, preventing binding to the host cells. Thus, our work has identified a potent drug with a distinct mode of action against SARS-CoV-2.
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http://dx.doi.org/10.1101/2020.10.28.359042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605555PMC
October 2020

ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs.

Nat Commun 2020 10 28;11(1):5453. Epub 2020 Oct 28.

Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine, Stanford, CA, USA.

The coronavirus SARS-CoV-2 is the causative agent of the ongoing severe acute respiratory disease pandemic COVID-19. Tissue and cellular tropism is one key to understanding the pathogenesis of SARS-CoV-2. We investigate the expression and subcellular localization of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), within the upper (nasal) and lower (pulmonary) respiratory tracts of human donors using a diverse panel of banked tissues. Here, we report our discovery that the ACE2 receptor protein robustly localizes within the motile cilia of airway epithelial cells, which likely represents the initial or early subcellular site of SARS-CoV-2 viral entry during host respiratory transmission. We further determine whether ciliary ACE2 expression in the upper airway is influenced by patient demographics, clinical characteristics, comorbidities, or medication use, and show the first mechanistic evidence that the use of angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARBs) does not increase susceptibility to SARS-CoV-2 infection through enhancing the expression of ciliary ACE2 receptor. These findings are crucial to our understanding of the transmission of SARS-CoV-2 for prevention and control of this virulent pathogen.
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http://dx.doi.org/10.1038/s41467-020-19145-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595232PMC
October 2020

cAMP Signaling in Nanodomains.

Authors:
Peter K Jackson

Cell 2020 09;182(6):1379-1381

Baxter Laboratory, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Pathology, Stanford University, Stanford, CA 94305, USA. Electronic address:

Cyclic-3',5'-adenosine monophosphate (cAMP) is an ancient second messenger but organizing signaling selectivity on the nanoscale is poorly understood. Examining transport of a new fluorescent cAMP probe, Bock and coworkers observe "buffered diffusion" and establish phosphodiesterase activity can organize cAMP nanodomains, while Zhao and coworkers find that protein kinase A regulatory subunits assemble liquid droplets to further localize cAMP signaling.
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http://dx.doi.org/10.1016/j.cell.2020.08.041DOI Listing
September 2020

Combined Proteomic and Genetic Interaction Mapping Reveals New RAS Effector Pathways and Susceptibilities.

Cancer Discov 2020 Dec 29;10(12):1950-1967. Epub 2020 Jul 29.

Baxter Laboratory, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, California.

Activating mutations in RAS GTPases drive many cancers, but limited understanding of less-studied RAS interactors, and of the specific roles of different RAS interactor paralogs, continues to limit target discovery. We developed a multistage discovery and screening process to systematically identify genes conferring RAS-related susceptibilities in lung adenocarcinoma. Using affinity purification mass spectrometry, we generated a protein-protein interaction map of RAS interactors and pathway components containing hundreds of interactions. From this network, we constructed a CRISPR dual knockout library targeting 119 RAS-related genes that we screened for -dependent genetic interactions (GI). This approach identified new RAS effectors, including the adhesion controller RADIL and the endocytosis regulator RIN1, and >250 synthetic lethal GIs, including a potent -dependent interaction between and Many GIs link specific paralogs within and between gene families. These findings illustrate the power of multiomic approaches to uncover synthetic lethal combinations specific for hitherto untreatable cancer genotypes. SIGNIFICANCE: We establish a deep network of protein-protein and genetic interactions in the RAS pathway. Many interactions validated here demonstrate important specificities and redundancies among paralogous RAS regulators and effectors. By comparing synthetic lethal interactions across -dependent and -independent cell lines, we identify several new combination therapy targets for RAS-driven cancers..
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http://dx.doi.org/10.1158/2159-8290.CD-19-1274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710624PMC
December 2020

Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells.

Cancer Cell 2020 07 11;38(1):129-143.e7. Epub 2020 Jun 11.

Department of Pediatrics, Stanford University, 265 Campus Drive, Stanford, CA 94305-5457, USA; Department of Genetics, Stanford University, Stanford, CA 94305, USA. Electronic address:

Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.
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http://dx.doi.org/10.1016/j.ccell.2020.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363571PMC
July 2020

Robust ACE2 protein expression localizes to the motile cilia of the respiratory tract epithelia and is not increased by ACE inhibitors or angiotensin receptor blockers.

medRxiv 2020 May 12. Epub 2020 May 12.

Department of Otorhinolaryngology, China Medical University Hospital, Taichung, Taiwan.

We investigated the expression and subcellular localization of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), within the upper (nasal) and lower (pulmonary) respiratory tracts of healthy human donors. We detected ACE2 protein expression within the cilia organelle of ciliated airway epithelial cells, which likely represents the initial or early subcellular site of SARS-CoV-2 viral entry during respiratory transmission. We further determined whether ACE2 expression in the cilia of upper respiratory cells was influenced by patient demographics, clinical characteristics, co-morbidities, or medication use, and found no evidence that the use of angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARBs) increases ACE2 protein expression.
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http://dx.doi.org/10.1101/2020.05.08.20092866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273284PMC
May 2020

CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.

Nature 2020 04 11;580(7801):136-141. Epub 2020 Mar 11.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.

Cancer genomics studies have identified thousands of putative cancer driver genes. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.
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http://dx.doi.org/10.1038/s41586-020-2099-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368463PMC
April 2020

Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy.

Mol Biol Cell 2020 03 2;31(7):619-639. Epub 2020 Jan 2.

Baxter Laboratory, Department of Microbiology and Immunology and Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305.

Primary cilia in many cell types contain a periaxonemal subcompartment called the inversin compartment. Four proteins have been found to assemble within the inversin compartment: INVS, ANKS6, NEK8, and NPHP3. The function of the inversin compartment is unknown, but it appears to be critical for normal development, including left-right asymmetry and renal tissue homeostasis. Here we combine superresolution imaging of human RPE1 cells, a classic model for studying primary cilia in vitro, with a genetic dissection of the protein-protein binding relationships that organize compartment assembly to develop a new structural model. We observe that INVS is the core structural determinant of a compartment composed of novel fibril-like substructures, which we identify here by three-dimensional single-molecule superresolution imaging. We find that NEK8 and ANKS6 depend on INVS for localization to these fibrillar assemblies and that ANKS6-NEK8 density within the compartment is regulated by NEK8. Together, NEK8 and ANKS6 are required downstream of INVS to localize and concentrate NPHP3 within the compartment. In the absence of these upstream components, NPHP3 is redistributed within cilia. These results provide a more detailed structure for the inversin compartment and introduce a new example of a membraneless compartment organized by protein-protein interactions.
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http://dx.doi.org/10.1091/mbc.E19-09-0499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202064PMC
March 2020

Omega-3 Fatty Acids Activate Ciliary FFAR4 to Control Adipogenesis.

Cell 2019 11 21;179(6):1289-1305.e21. Epub 2019 Nov 21.

Baxter Laboratory, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Adult mesenchymal stem cells, including preadipocytes, possess a cellular sensory organelle called the primary cilium. Ciliated preadipocytes abundantly populate perivascular compartments in fat and are activated by a high-fat diet. Here, we sought to understand whether preadipocytes use their cilia to sense and respond to external cues to remodel white adipose tissue. Abolishing preadipocyte cilia in mice severely impairs white adipose tissue expansion. We discover that TULP3-dependent ciliary localization of the omega-3 fatty acid receptor FFAR4/GPR120 promotes adipogenesis. FFAR4 agonists and ω-3 fatty acids, but not saturated fatty acids, trigger mitosis and adipogenesis by rapidly activating cAMP production inside cilia. Ciliary cAMP activates EPAC signaling, CTCF-dependent chromatin remodeling, and transcriptional activation of PPARγ and CEBPα to initiate adipogenesis. We propose that dietary ω-3 fatty acids selectively drive expansion of adipocyte numbers to produce new fat cells and store saturated fatty acids, enabling homeostasis of healthy fat tissue.
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http://dx.doi.org/10.1016/j.cell.2019.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332222PMC
November 2019

E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family.

Nat Commun 2019 07 3;10(1):2939. Epub 2019 Jul 3.

Department of Pediatrics, 300 Pasteur Drive, Stanford University, Stanford, CA, 94305, USA.

E2F transcription factors are central regulators of cell division and cell fate decisions. E2F4 often represents the predominant E2F activity in cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. This role for E2F4 is independent of the RB family. Furthermore, E2F4 functionally interacts with chromatin regulators associated with gene activation and we observed decreased histone acetylation at the promoters of cell cycle genes and E2F targets upon loss of E2F4 in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that provide insights into the biology of rapidly dividing cells.
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http://dx.doi.org/10.1038/s41467-019-10901-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610666PMC
July 2019

Oligomeric self-association contributes to E2A-PBX1-mediated oncogenesis.

Sci Rep 2019 03 20;9(1):4915. Epub 2019 Mar 20.

Department of Pathology, Stanford University School of Medicine, Stanford, CA, 94305, USA.

The PBX1 homeodomain transcription factor is converted by t(1;19) chromosomal translocations in acute leukemia into the chimeric E2A-PBX1 oncoprotein. Fusion with E2A confers potent transcriptional activation and constitutive nuclear localization, bypassing the need for dimerization with protein partners that normally stabilize and regulate import of PBX1 into the nucleus, but the mechanisms underlying its oncogenic activation are incompletely defined. We demonstrate here that E2A-PBX1 self-associates through the PBX1 PBC-B domain of the chimeric protein to form higher-order oligomers in t(1;19) human leukemia cells, and that this property is required for oncogenic activity. Structural and functional studies indicate that self-association facilitates the binding of E2A-PBX1 to DNA. Mutants unable to self-associate are transformation defective, however their oncogenic activity is rescued by the synthetic oligomerization domain of FKBP, which confers conditional transformation properties on E2A-PBX1. In contrast to self-association, PBX1 protein domains that mediate interactions with HOX DNA-binding partners are dispensable. These studies suggest that oligomeric self-association may compensate for the inability of monomeric E2A-PBX1 to stably bind DNA and circumvents protein interactions that otherwise modulate PBX1 stability, nuclear localization, DNA binding, and transcriptional activity. The unique dependence on self-association for E2A-PBX1 oncogenic activity suggests potential approaches for mechanism-based targeted therapies.
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http://dx.doi.org/10.1038/s41598-019-41393-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426973PMC
March 2019

EZH2 Inactivates Primary Cilia to Activate Wnt and Drive Melanoma.

Authors:
Peter K Jackson

Cancer Cell 2018 07;34(1):3-5

Baxter Laboratory, Department of Microbiology and Immunology and Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

EZH2 is frequently amplified in human melanomas. In this issue of Cancer Cell, Zingg et al. find that EZH2 overexpression silences genes for the primary cilium, causing deciliation, Wnt pathway activation, and progression of Braf- or Nras-driven melanomas, thus defining a tumor-suppressor role for cilia in cancer.
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http://dx.doi.org/10.1016/j.ccell.2018.06.011DOI Listing
July 2018

Guanine Nucleotide Exchange Assay Using Fluorescent MANT-GDP.

Bio Protoc 2018 Apr;8(7)

Baxter Laboratory, Department of Microbiology & Immunology and Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

GTPases are molecular switches that cycle between the inactive GDP-bound state and the active GTP-bound state. GTPases exchange nucleotides either by its intrinsic nucleotide exchange or by interaction with guanine nucleotide exchange factors (GEFs). Monitoring the nucleotide exchange , together with reconstitution of direct interactions with regulatory proteins, provides key insights into how a GTPase is activated. In this protocol, we describe core methods to monitor nucleotide exchange using fluorescent N-Methylanthraniloyl (MANT)-guanine nucleotide.
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http://dx.doi.org/10.21769/BioProtoc.2795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6017992PMC
April 2018

Neural Precursor-Derived Pleiotrophin Mediates Subventricular Zone Invasion by Glioma.

Cell 2017 Aug 17;170(5):845-859.e19. Epub 2017 Aug 17.

Department of Neurology, Stanford University, Palo Alto, CA 94305, USA; Department of Pathology, Stanford University, Palo Alto, CA 94305, USA; Department of Neurosurgery, Stanford University, Palo Alto, CA 94305, USA; Department of Pediatrics, Stanford University, Palo Alto, CA 94305, USA. Electronic address:

The lateral ventricle subventricular zone (SVZ) is a frequent and consequential site of pediatric and adult glioma spread, but the cellular and molecular mechanisms mediating this are poorly understood. We demonstrate that neural precursor cell (NPC):glioma cell communication underpins this propensity of glioma to colonize the SVZ through secretion of chemoattractant signals toward which glioma cells home. Biochemical, proteomic, and functional analyses of SVZ NPC-secreted factors revealed the neurite outgrowth-promoting factor pleiotrophin, along with required binding partners SPARC/SPARCL1 and HSP90B, as key mediators of this chemoattractant effect. Pleiotrophin expression is strongly enriched in the SVZ, and pleiotrophin knock down starkly reduced glioma invasion of the SVZ in the murine brain. Pleiotrophin, in complex with the binding partners, activated glioma Rho/ROCK signaling, and ROCK inhibition decreased invasion toward SVZ NPC-secreted factors. These findings demonstrate a pathogenic role for NPC:glioma interactions and potential therapeutic targets to limit glioma invasion. PAPERCLIP.
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http://dx.doi.org/10.1016/j.cell.2017.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587159PMC
August 2017

Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis.

Proc Natl Acad Sci U S A 2017 05 17;114(18):E3642-E3651. Epub 2017 Apr 17.

Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305;

Despite the wide administration of several effective vaccines, rotavirus (RV) remains the single most important etiological agent of severe diarrhea in infants and young children worldwide, with an annual mortality of over 200,000 people. RV attachment and internalization into target cells is mediated by its outer capsid protein VP4. To better understand the molecular details of RV entry, we performed tandem affinity purification coupled with high-resolution mass spectrometry to map the host proteins that interact with VP4. We identified an actin-binding protein, drebrin (DBN1), that coprecipitates and colocalizes with VP4 during RV infection. Importantly, blocking DBN1 function by siRNA silencing, CRISPR knockout (KO), or chemical inhibition significantly increased host cell susceptibility to RV infection. KO mice exhibited higher incidence of diarrhea and more viral antigen shedding in their stool samples compared with the wild-type littermates. In addition, we found that uptake of other dynamin-dependent cargos, including transferrin, cholera toxin, and multiple viruses, was also enhanced in DBN1-deficient cells. Inhibition of cortactin or dynamin-2 abrogated the increased virus entry observed in DBN1-deficient cells, suggesting that DBN1 suppresses dynamin-mediated endocytosis via interaction with cortactin. Our study unveiled an unexpected role of DBN1 in restricting the entry of RV and other viruses into host cells and more broadly to function as a crucial negative regulator of diverse dynamin-dependent endocytic pathways.
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http://dx.doi.org/10.1073/pnas.1619266114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5422808PMC
May 2017

Comparative Proteomics Reveals Strain-Specific β-TrCP Degradation via Rotavirus NSP1 Hijacking a Host Cullin-3-Rbx1 Complex.

PLoS Pathog 2016 Oct 5;12(10):e1005929. Epub 2016 Oct 5.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

Rotaviruses (RVs) are the leading cause of severe gastroenteritis in young children, accounting for half a million deaths annually worldwide. RV encodes non-structural protein 1 (NSP1), a well-characterized interferon (IFN) antagonist, which facilitates virus replication by mediating the degradation of host antiviral factors including IRF3 and β-TrCP. Here, we utilized six human and animal RV NSP1s as baits and performed tandem-affinity purification coupled with high-resolution mass spectrometry to comprehensively characterize NSP1-host protein interaction network. Multiple Cullin-RING ubiquitin ligase (CRL) complexes were identified. Importantly, inhibition of cullin-3 (Cul3) or RING-box protein 1 (Rbx1), by siRNA silencing or chemical perturbation, significantly impairs strain-specific NSP1-mediated β-TrCP degradation. Mechanistically, we demonstrate that NSP1 localizes to the Golgi with the host Cul3-Rbx1 CRL complex, which targets β-TrCP and NSP1 for co-destruction at the proteasome. Our study uncovers a novel mechanism that RV employs to promote β-TrCP turnover and provides molecular insights into virus-mediated innate immunity inhibition.
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http://dx.doi.org/10.1371/journal.ppat.1005929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5051689PMC
October 2016

Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition.

Nat Chem Biol 2016 10 1;12(10):779-86. Epub 2016 Aug 1.

Translational Oncology, Genentech, South San Francisco, California, USA.

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.
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http://dx.doi.org/10.1038/nchembio.2143DOI Listing
October 2016

The ciliopathy-associated CPLANE proteins direct basal body recruitment of intraflagellar transport machinery.

Nat Genet 2016 06 9;48(6):648-56. Epub 2016 May 9.

EA4271GAD Genetics of Developmental Anomalies, FHU-TRANSLAD, Medecine Faculty, Burgundy University, Dijon, France.

Cilia use microtubule-based intraflagellar transport (IFT) to organize intercellular signaling. Ciliopathies are a spectrum of human diseases resulting from defects in cilia structure or function. The mechanisms regulating the assembly of ciliary multiprotein complexes and the transport of these complexes to the base of cilia remain largely unknown. Combining proteomics, in vivo imaging and genetic analysis of proteins linked to planar cell polarity (Inturned, Fuzzy and Wdpcp), we identified and characterized a new genetic module, which we term CPLANE (ciliogenesis and planar polarity effector), and an extensive associated protein network. CPLANE proteins physically and functionally interact with the poorly understood ciliopathy-associated protein Jbts17 at basal bodies, where they act to recruit a specific subset of IFT-A proteins. In the absence of CPLANE, defective IFT-A particles enter the axoneme and IFT-B trafficking is severely perturbed. Accordingly, mutation of CPLANE genes elicits specific ciliopathy phenotypes in mouse models and is associated with ciliopathies in human patients.
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http://dx.doi.org/10.1038/ng.3558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978421PMC
June 2016

Cell biology: Calcium contradictions in cilia.

Nature 2016 Mar 23;531(7596):582-3. Epub 2016 Mar 23.

Departments of Microbiology &Immunology and Pathology, and in the Baxter Laboratory, Stanford University School of Medicine, Stanford, California 94305, USA.

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http://dx.doi.org/10.1038/nature17313DOI Listing
March 2016

Smoothened determines β-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium.

J Cell Biol 2016 Mar 21;212(7):861-75. Epub 2016 Mar 21.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390

Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein-coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia. The first step involves β-arrestin recruitment by the signaling competent receptor, which is facilitated by the GPCR kinase Grk2. An essential factor here is the ciliary trafficking and activation of Smoothened, which by increasing Gpr161-β-arrestin binding promotes Gpr161 removal, both during resting conditions and upon Shh pathway activation. The second step involves clathrin-mediated endocytosis, which functions outside of the ciliary compartment in coordinating Gpr161 removal. Mechanisms determining dynamic compartmentalization of Gpr161 in cilia define a new paradigm for down-regulation of GPCRs during developmental signaling from a specialized subcellular compartment.
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http://dx.doi.org/10.1083/jcb.201506132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810300PMC
March 2016

p73 and FoxJ1: Programming Multiciliated Epithelia.

Trends Cell Biol 2016 Apr 14;26(4):239-240. Epub 2016 Mar 14.

Stanford University School of Medicine, Department of Radiation Oncology and Department of Genetics, Stanford, CA 94305, USA.

The mysteriously diverse phenotypes in mice lacking the p53 homolog p73 are recently unified by new analysis showing p73 is required for formation of multiciliated epithelia. p73 directly activates FoxJ1, the central transcriptional driver for multiciliation, and induces a host of genes critical for ciliogenesis.
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http://dx.doi.org/10.1016/j.tcb.2016.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555749PMC
April 2016

The primary cilium as a cellular receiver: organizing ciliary GPCR signaling.

Curr Opin Cell Biol 2016 Apr 27;39:84-92. Epub 2016 Feb 27.

Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

The primary cilium is an antenna-like cellular protrusion mediating sensory and neuroendocrine signaling. Its localization within tissue architecture and a growing list of cilia-localized receptors, in particular G-protein-coupled receptors, determine a host of crucial physiologies, which are disrupted in human ciliopathies. Here, we discuss recent advances in the identification and characterization of ciliary signaling components and pathways. Recent studies have highlighted the unique signaling environment of the primary cilium and we are just beginning to understand how this design allows for highly amplified and regulated signaling.
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http://dx.doi.org/10.1016/j.ceb.2016.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4828300PMC
April 2016

Tctex1d2 associates with short-rib polydactyly syndrome proteins and is required for ciliogenesis.

Cell Cycle 2015 ;14(7):1116-25

a Department of Chemistry and Biochemistry ; University of California ; Los Angeles , CA USA.

Short-rib polydactyly syndromes (SRPS) arise from mutations in genes involved in retrograde intraflagellar transport (IFT) and basal body homeostasis, which are critical for cilia assembly and function. Recently, mutations in WDR34 or WDR60 (candidate dynein intermediate chains) were identified in SRPS. We have identified and characterized Tctex1d2, which associates with Wdr34, Wdr60 and other dynein complex 1 and 2 subunits. Tctex1d2 and Wdr60 localize to the base of the cilium and their depletion causes defects in ciliogenesis. We propose that Tctex1d2 is a novel dynein light chain important for trafficking to the cilium and potentially retrograde IFT and is a new molecular link to understanding SRPS pathology.
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http://dx.doi.org/10.4161/15384101.2014.985066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614626PMC
December 2015

Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation.

Nat Cell Biol 2015 Mar 16;17(3):228-240. Epub 2015 Feb 16.

NCI-Frederick National Laboratory, Laboratory of Cellular and Developmental Signaling, Frederick, MD 21702, USA.

Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11-Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11-Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby EHD1 and EHD3 reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.
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http://dx.doi.org/10.1038/ncb3109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4344897PMC
March 2015

3D spheroid model of mIMCD3 cells for studying ciliopathies and renal epithelial disorders.

Nat Protoc 2014 Dec 30;9(12):2725-31. Epub 2014 Oct 30.

1] Research Oncology, Genentech, South San Francisco, California, USA. [2] Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.

We have developed a novel 3D cell culture model that uses mouse inner-medullary collecting duct (mIMCD3) cells to generate epithelial spheroids. This model is amenable to efficient siRNA knockdown and subsequent rescue with human patient-derived alleles. Spheroids develop apicobasal polarity and complete lumens, and they are consequently an ideal model for polarity defects seen in renal ciliopathies such as nephronophthisis. Briefly, mIMCD3 cells are transfected and subsequently passaged to a Matrigel mixture, which is seeded in chamber slides and covered in growth medium. Once the spheroids are formed, Matrigel is dissolved and immunocytochemistry is performed in the chamber slides. The technique is amenable to semiautomatic imaging analysis, and it can test multiple genes simultaneously, gene-dosing effects and a variety of therapeutic interventions. The spheroid technique is a unique and simple 6-d in vitro method of interrogating ex vivo tissue organization.
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http://dx.doi.org/10.1038/nprot.2014.181DOI Listing
December 2014

Regulating microtubules and genome stability via the CUL7/3M syndrome complex and CUL9.

Authors:
Peter K Jackson

Mol Cell 2014 Jun;54(5):713-5

Baxter Laboratory for Stem Cell Biology, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

In this issue, two studies from Yue Xiong and colleagues (Li et al., 2014; Yan et al., 2014) show that CUL7, OBSL1, and CCDC8, all mutated in 3M short stature syndrome, form a centrosomal complex that regulates CUL9 and its substrate survivin to link mitosis to cell survival.
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http://dx.doi.org/10.1016/j.molcel.2014.05.024DOI Listing
June 2014