Publications by authors named "Meredith Sagolla"

20 Publications

  • Page 1 of 1

NINJ1 mediates plasma membrane rupture during lytic cell death.

Nature 2021 Mar 20;591(7848):131-136. Epub 2021 Jan 20.

Department of Physiological Chemistry, Genentech Inc., South San Francisco, CA, USA.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1 macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1 macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1 mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.
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http://dx.doi.org/10.1038/s41586-021-03218-7DOI Listing
March 2021

Unstable Mechanisms of Resistance to Inhibitors of Escherichia coli Lipoprotein Signal Peptidase.

mBio 2020 09 8;11(5). Epub 2020 Sep 8.

Department of Infectious Diseases, Genentech, South San Francisco, California, USA

Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use. Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.
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http://dx.doi.org/10.1128/mBio.02018-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7482066PMC
September 2020

The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins.

Nat Genet 2020 01 6;52(1):106-117. Epub 2020 Jan 6.

Molecular Biology Department, Genentech, Inc., South San Francisco, CA, USA.

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the 'venom-ome' and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 'venom-ome-specific toxins' (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery.
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http://dx.doi.org/10.1038/s41588-019-0559-8DOI Listing
January 2020

Activity of caspase-8 determines plasticity between cell death pathways.

Nature 2019 11 13;575(7784):679-682. Epub 2019 Nov 13.

Department of Physiological Chemistry, Genentech, South San Francisco, CA, USA.

Caspase-8 is a protease with both pro-death and pro-survival functions: it mediates apoptosis induced by death receptors such as TNFR1, and suppresses necroptosis mediated by the kinase RIPK3 and the pseudokinase MLKL. Mice that lack caspase-8 display MLKL-dependent embryonic lethality, as do mice that express catalytically inactive CASP8(C362A). Casp8Mlkl mice die during the perinatal period, whereas Casp8Mlkl mice are viable, which indicates that inactive caspase-8 also has a pro-death scaffolding function. Here we show that mutant CASP8(C362A) induces the formation of ASC (also known as PYCARD) specks, and caspase-1-dependent cleavage of GSDMD and caspases 3 and 7 in MLKL-deficient mouse intestines around embryonic day 18. Caspase-1 and its adaptor ASC contributed to the perinatal lethal phenotype because a number of Casp8MlklCasp1 and Casp8MlklAsc mice survived beyond weaning. Transfection studies suggest that inactive caspase-8 adopts a distinct conformation to active caspase-8, enabling its prodomain to engage ASC. Upregulation of the lipopolysaccharide sensor caspase-11 in the intestines of both Casp8Mlkl and Casp8MlklCasp1 mice also contributed to lethality because Casp8MlklCasp1Casp11 (Casp11 is also known as Casp4) neonates survived more often than Casp8MlklCasp1 neonates. Finally, Casp8Ripk3Casp1Casp11 mice survived longer than Casp8MlklCasp1Casp11 mice, indicating that a necroptosis-independent function of RIPK3 also contributes to lethality. Thus, unanticipated plasticity in death pathways is revealed when caspase-8-dependent apoptosis and MLKL-dependent necroptosis are inhibited.
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http://dx.doi.org/10.1038/s41586-019-1752-8DOI Listing
November 2019

Unobstructed Multiscale Imaging of Tissue Sections for Ultrastructural Pathology Analysis by Backscattered Electron Scanning Microscopy.

J Histochem Cytochem 2020 01 6;68(1):9-23. Epub 2019 Aug 6.

Department of Pathology, Genentech Inc., South San Francisco, California.

Ultrastructural analysis of healthy, diseased, or experimental tissues is essential in diagnostic and investigative pathology. Evaluation of large tissue areas with suborganelle resolution is challenging because biological structures ranging from several millimeters to nanometers in size need to be identified and imaged while maintaining context over multiple scales. Imaging with field emission scanning electron microscopes (FE-SEMs) is uniquely suited for this task. We describe an efficient workflow for the preparation and unobstructed multiscale imaging of tissue sections with backscattered electron scanning electron microscopy (BSE-SEM) for applications in ultrastructural pathology. We demonstrate that a diverse range of tissues, processed by conventional electron microscopy protocols and avoiding the use of mordanting agents, can be imaged on standard glass slides over multiple scales, from the histological to the ultrastructural level, without any visual obstructions. Our workflow takes advantage of the very large scan fields possible with modern FE-SEMs that allow for the acquisition of wide-field overview images which can be explored at the ultrastructural level by digitally zooming into the images. Examples from applications in pulmonary research and neuropathology demonstrate the versatility and efficiency of this method. This BSE-SEM-based multiscale imaging procedure promises to substantially simplify and accelerate ultrastructural tissue analysis in pathology.
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http://dx.doi.org/10.1369/0022155419868992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6931167PMC
January 2020

LACC1 Regulates TNF and IL-17 in Mouse Models of Arthritis and Inflammation.

J Immunol 2019 01 3;202(1):183-193. Epub 2018 Dec 3.

Department of Human Genetics, Genentech, Inc., South San Francisco, CA 94080.

Both common and rare genetic variants of laccase domain-containing 1 (, previously C13orf31) are associated with inflammatory bowel disease, leprosy, Behcet disease, and systemic juvenile idiopathic arthritis. However, the functional relevance of these variants is unclear. In this study, we use LACC1-deficient mice to gain insight into the role of LACC1 in regulating inflammation. Following oral administration of , LACC1 knockout (KO) mice had more severe colon lesions compared with wildtype (WT) controls. Immunization with collagen II, a collagen-induced arthritis (CIA) model, resulted in an accelerated onset of arthritis and significantly worse arthritis and inflammation in LACC1 KO mice. Similar results were obtained in a mannan-induced arthritis model. Serum and local TNF in CIA paws and colons were significantly increased in LACC1 KO mice compared with WT controls. The percentage of IL-17A-producing CD4 T cells was elevated in LACC1 KO mice undergoing CIA as well as aged mice compared with WT controls. Neutralization of IL-17, but not TNF, prevented enhanced mannan-induced arthritis in LACC1 KO mice. These data provide new mechanistic insight into the function of LACC1 in regulating TNF and IL-17 during inflammatory responses. We hypothesize that these effects contribute to immune-driven pathologies observed in individuals carrying variants.
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http://dx.doi.org/10.4049/jimmunol.1800636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305794PMC
January 2019

Non-canonical reader modules of BAZ1A promote recovery from DNA damage.

Nat Commun 2017 10 11;8(1):862. Epub 2017 Oct 11.

Department of Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080, USA.

Members of the ISWI family of chromatin remodelers mobilize nucleosomes to control DNA accessibility and, in some cases, are required for recovery from DNA damage. However, it remains poorly understood how the non-catalytic ISWI subunits BAZ1A and BAZ1B might contact chromatin to direct the ATPase SMARCA5. Here, we find that the plant homeodomain of BAZ1A, but not that of BAZ1B, has the unusual function of binding DNA. Furthermore, the BAZ1A bromodomain has a non-canonical gatekeeper residue and binds relatively weakly to acetylated histone peptides. Using CRISPR-Cas9-mediated genome editing we find that BAZ1A and BAZ1B each recruit SMARCA5 to sites of damaged chromatin and promote survival. Genetic engineering of structure-designed bromodomain and plant homeodomain mutants reveals that reader modules of BAZ1A and BAZ1B, even when non-standard, are critical for DNA damage recovery in part by regulating ISWI factors loading at DNA lesions and supporting transcriptional programs required for survival.ISWI chromatin remodelers regulate DNA accessibility and have been implicated in DNA damage repair. Here, the authors uncover functions, in response to DNA damage, for the bromodomain of the ISWI subunit BAZ1B and for the non-canonical PHD and bromodomain modules of the paralog BAZ1A.
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http://dx.doi.org/10.1038/s41467-017-00866-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5636791PMC
October 2017

Ubiquilin1 promotes antigen-receptor mediated proliferation by eliminating mislocalized mitochondrial proteins.

Elife 2017 09 21;6. Epub 2017 Sep 21.

Department of Infectious Disease, Genentech, South San Francisco, United States.

Ubiquilins (Ubqlns) are a family of ubiquitin receptors that promote the delivery of hydrophobic and aggregated ubiquitinated proteins to the proteasome for degradation. We carried out a proteomic analysis of a B cell lymphoma-derived cell line, BJAB, that requires UBQLN1 for survival to identify UBQLN1 client proteins. When UBQLN1 expression was acutely inhibited, 120 mitochondrial proteins were enriched in the cytoplasm, suggesting that the accumulation of mitochondrial client proteins in the absence of UBQLN1 is cytostatic. Using a mouse strain, we found that B cell receptor (BCR) ligation of B cells led to a defect in cell cycle entry. As in BJAB cells, mitochondrial proteins accumulated in BCR-stimulated cells, leading to protein synthesis inhibition and cell cycle block. Thus, UBQLN1 plays an important role in clearing mislocalized mitochondrial proteins upon cell stimulation, and its absence leads to suppression of protein synthesis and cell cycle arrest.
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http://dx.doi.org/10.7554/eLife.26435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608509PMC
September 2017

Palmitoylation of TEAD Transcription Factors Is Required for Their Stability and Function in Hippo Pathway Signaling.

Structure 2016 Jan 24;24(1):179-186. Epub 2015 Dec 24.

Department of Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address:

The Hippo signaling pathway is responsible for regulating the function of TEAD family transcription factors in metazoans. TEADs, with their co-activators YAP/TAZ, are critical for controlling cell differentiation and organ size through their transcriptional activation of genes involved in cell growth and proliferation. Dysregulation of the Hippo pathway has been implicated in multiple forms of cancer. Here, we identify a novel form of regulation of TEAD family proteins. We show that human TEADs are palmitoylated at a universally conserved cysteine, and report the crystal structures of the human TEAD2 and TEAD3 YAP-binding domains in their palmitoylated forms. These structures show a palmitate bound within a highly conserved hydrophobic cavity at each protein's core. Our findings also demonstrate that this modification is required for proper TEAD folding and stability, indicating a potential new avenue for pharmacologically regulating the Hippo pathway through the modulation of TEAD palmitoylation.
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http://dx.doi.org/10.1016/j.str.2015.11.005DOI Listing
January 2016

β-Cell Insulin Secretion Requires the Ubiquitin Ligase COP1.

Cell 2015 Dec 25;163(6):1457-67. Epub 2015 Nov 25.

Department of Physiological Chemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address:

A variety of signals finely tune insulin secretion by pancreatic β cells to prevent both hyper-and hypoglycemic states. Here, we show that post-translational regulation of the transcription factors ETV1, ETV4, and ETV5 by the ubiquitin ligase COP1 (also called RFWD2) in β cells is critical for insulin secretion. Mice lacking COP1 in β cells developed diabetes due to insulin granule docking defects that were fully rescued by genetic deletion of Etv1, Etv4, and Etv5. Genes regulated by ETV1, ETV4, or ETV5 in the absence of mouse COP1 were enriched in human diabetes-associated genes, suggesting that they also influence human β-cell pathophysiology. In normal β cells, ETV4 was stabilized upon membrane depolarization and limited insulin secretion under hyperglycemic conditions. Collectively, our data reveal that ETVs negatively regulate insulin secretion for the maintenance of normoglycemia.
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http://dx.doi.org/10.1016/j.cell.2015.10.076DOI Listing
December 2015

Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents.

Breast Cancer Res 2015 Apr 23;17:59. Epub 2015 Apr 23.

Department of Translational Oncology, Genentech, Inc., South San Francisco, CA, USA.

Introduction: Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis.

Methods: PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n=980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting.

Results: We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P=1.29×10(-4) and P=0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis.

Conclusions: Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.
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http://dx.doi.org/10.1186/s13058-015-0564-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445529PMC
April 2015

Enhancing the antitumor efficacy of a cell-surface death ligand by covalent membrane display.

Proc Natl Acad Sci U S A 2015 May 20;112(18):5679-84. Epub 2015 Apr 20.

Cancer Immunology,

TNF superfamily death ligands are expressed on the surface of immune cells and can trigger apoptosis in susceptible cancer cells by engaging cognate death receptors. A recombinant soluble protein comprising the ectodomain of Apo2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) has shown remarkable preclinical anticancer activity but lacked broad efficacy in patients, possibly owing to insufficient exposure or potency. We observed that antibody cross-linking substantially enhanced cytotoxicity of soluble Apo2L/TRAIL against diverse cancer cell lines. Presentation of the ligand on glass-supported lipid bilayers enhanced its ability to drive receptor microclustering and apoptotic signaling. Furthermore, covalent surface attachment of Apo2L/TRAIL onto liposomes--synthetic lipid-bilayer nanospheres--similarly augmented activity. In vivo, liposome-displayed Apo2L/TRAIL achieved markedly better exposure and antitumor activity. Thus, covalent synthetic-membrane attachment of a cell-surface ligand enhances efficacy, increasing therapeutic potential. These findings have translational implications for liposomal approaches as well as for Apo2L/TRAIL and other clinically relevant TNF ligands.
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http://dx.doi.org/10.1073/pnas.1418962112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426393PMC
May 2015

Rasip1 regulates vertebrate vascular endothelial junction stability through Epac1-Rap1 signaling.

Blood 2013 Nov 25;122(22):3678-90. Epub 2013 Jul 25.

Department of Molecular Biology, Genentech, Inc., South San Francisco, CA;

Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.
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http://dx.doi.org/10.1182/blood-2013-02-483156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837516PMC
November 2013

Lkb1 regulates organogenesis and early oncogenesis along AMPK-dependent and -independent pathways.

J Cell Biol 2012 Dec;199(7):1117-30

Genentech, South San Francisco, CA 94080, USA.

The tumor suppressor Lkb1/STK11/Par-4 is a key regulator of cellular energy, proliferation, and polarity, yet its mechanisms of action remain poorly defined. We generated mice harboring a mutant Lkb1 knockin allele that allows for rapid inhibition of Lkb1 kinase. Culturing embryonic tissues, we show that acute loss of kinase activity perturbs epithelial morphogenesis without affecting cell polarity. In pancreas, cystic structures developed rapidly after Lkb1 inhibition. In lung, inhibition resulted in cell-autonomous branching defects. Although the lung phenotype was rescued by an activator of the Lkb1 target adenosine monophosphate-activated kinase (AMPK), pancreatic cyst development was independent of AMPK signaling. Remarkably, the pancreatic phenotype evolved to resemble precancerous lesions, demonstrating that loss of Lkb1 was sufficient to drive the initial steps of carcinogenesis ex vivo. A similar phenotype was induced by expression of mutant K-Ras with p16/p19 deletion. Combining culture of embryonic tissues with genetic manipulation and chemical genetics thus provides a powerful approach to unraveling developmental programs and understanding cancer initiation.
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http://dx.doi.org/10.1083/jcb.201208080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3529533PMC
December 2012

Disruption of PH-kinase domain interactions leads to oncogenic activation of AKT in human cancers.

Proc Natl Acad Sci U S A 2012 Nov 7;109(47):19368-73. Epub 2012 Nov 7.

Department of Molecular Biology, Genentech Inc., South San Francisco, CA 94080, USA.

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.
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http://dx.doi.org/10.1073/pnas.1204384109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511101PMC
November 2012

Equilibrative nucleoside transporter 3 deficiency perturbs lysosome function and macrophage homeostasis.

Science 2012 Jan 15;335(6064):89-92. Epub 2011 Dec 15.

Immunology, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Lysosomal storage diseases (LSDs) are a group of heterogeneous disorders caused by defects in lysosomal enzymes or transporters, resulting in accumulation of undegraded macromolecules or metabolites. Macrophage numbers are expanded in several LSDs, leading to histiocytosis of unknown pathophysiology. Here, we found that mice lacking the equilibrative nucleoside transporter 3 (ENT3) developed a spontaneous and progressive macrophage-dominated histiocytosis. In the absence of ENT3, defective apoptotic cell clearance led to lysosomal nucleoside buildup, elevated intralysosomal pH, and altered macrophage function. The macrophage accumulation was partly due to increased macrophage colony-stimulating factor and receptor expression and signaling secondary to the lysosomal defects. These studies suggest a cellular and molecular basis for the development of histiocytosis in several human syndromes associated with ENT3 mutations and potentially other LSDs.
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http://dx.doi.org/10.1126/science.1213682DOI Listing
January 2012

Ubiquitin hydrolase Dub3 promotes oncogenic transformation by stabilizing Cdc25A.

Nat Cell Biol 2010 Apr 14;12(4):400-6. Epub 2010 Mar 14.

Department of Physiological Chemistry, 1 DNA Way, South San Francisco, California, 94080, USA.

The dual specificity (Tyr/Thr) phosphatase Cdc25A activates cyclin-dependent kinases (Cdks) to promote cell-cycle progression and has significant oncogenic potential. Cdc25A protein levels are regulated tightly in normal tissues, but many human cancers overexpress Cdc25A. The underlying mechanism for overexpression has been enigmatic. Here we show that Cdc25A is stabilized by the ubiquitin hydrolase Dub3. Upon binding Cdc25A, Dub3 removes the polyubiquitin modifications that mark Cdc25A for proteasomal degradation. Dub3 knockdown in cells increased Cdc25A ubiquitylation and degradation, resulting in reduced Cdk/Cyclin activity and arrest at G1/S and G2/M phases of the cell cycle. In contrast, acute Dub3 overexpression produced a signature response to oncogene induction: cells accumulated in S and G2 because of replication stress, and activated a DNA damage response. Dub3 also transformed NIH-3T3 cells and cooperated with activated H-Ras to promote growth in soft agar. Importantly, we show that Dub3 overexpression is responsible for an abnormally high level of Cdc25A in a subset of human breast cancers. Moreover, Dub3 knockdown significantly retarded the growth of breast tumour xenografts in nude mice. As a major regulator of Cdc25A, Dub3 is an example of a transforming ubiquitin hydrolase that subverts a key component of the cell cycle machinery.
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http://dx.doi.org/10.1038/ncb2041DOI Listing
April 2010

Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis.

Eukaryot Cell 2007 Dec 31;6(12):2354-64. Epub 2007 Aug 31.

Department of Molecular and Cell Biology, University of California-Berkeley, 341 LSA Bldg., Berkeley, California 94720, USA.

Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.
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http://dx.doi.org/10.1128/EC.00128-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168246PMC
December 2007

Three-dimensional analysis of mitosis and cytokinesis in the binucleate parasite Giardia intestinalis.

J Cell Sci 2006 Dec 14;119(Pt 23):4889-900. Epub 2006 Nov 14.

Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA 94720, USA.

In the binucleate parasite Giardia intestinalis, two diploid nuclei and essential cytoskeletal structures including eight flagella are duplicated and partitioned into two daughter cells during cell division. The mechanisms of mitosis and cytokinesis in the binucleate parasite Giardia are poorly resolved, yet have important implications for the maintenance of genetic heterozygosity. To articulate the mechanism of mitosis and the plane of cell division, we used three-dimensional deconvolution microscopy of each stage of mitosis to monitor the spatial relationships of conserved cytological markers to the mitotic spindles, the centromeres and the spindle poles. Using both light- and transmission electron microscopy, we determined that Giardia has a semi-open mitosis with two extranuclear spindles that access chromatin through polar openings in the nuclear membranes. In prophase, the nuclei migrate to the cell midline, followed by lateral chromosome segregation in anaphase. Taxol treatment results in lagging chromosomes and half-spindles. Our analysis supports a nuclear migration model of mitosis with lateral chromosome segregation in the left-right axis and cytokinesis along the longitudinal plane (perpendicular to the spindles), ensuring that each daughter inherits one copy of each parental nucleus with mirror image symmetry. Fluorescence in situ hybridization (FISH) to an episomal plasmid confirms that the nuclei remain separate and are inherited with mirror image symmetry.
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http://dx.doi.org/10.1242/jcs.03276DOI Listing
December 2006

Individual microtubule dynamics contribute to the function of mitotic and cytoplasmic arrays in fission yeast.

J Cell Sci 2003 Dec;116(Pt 24):4891-903

Department of Molecular and Cell Biology and Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA 94720-3200, USA.

Schizosaccharomyces pombe is an excellent organism for studying microtubule dynamics owing to the presence of well-defined microtubule arrays that undergo dramatic rearrangements during various stages of the cell cycle. Using sensitive time-lapse video microscopy and kymographic analysis, we have determined the polymerization/depolymerization kinetics of individual microtubules within these arrays throughout the fission yeast cell cycle. Interphase bundles are composed of 4-7 microtubules that act autonomously, demonstrating that individual microtubules are responsible for mediating the functions ascribed to these arrays. The nucleation and growth of cytoplasmic microtubules is inhibited upon cellular transition into mitosis, leading to their gradual disappearance. At the onset of mitosis, microtubules form on the nuclear face of the spindle pole body and exhibit dramatically increased dynamics. The presence of these intra-nuclear astral microtubules (INA) is reminiscent of spindle assembly and the search and chromosome capture mechanism observed in metazoan cells. Consistent with other in vivo studies, we do not observe microtubule flux in the anaphase B spindle. Finally, the depolymerization of individual microtubules alternates between each half-spindle, resulting in spindle collapse during telophase. On the basis of these observations, we conclude that microtubules in these diverse cytoskeletal arrays have autonomous behaviors that are an essential component of any model describing cell-cycle-dependent changes in the behavior and function of microtubule arrays.
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http://dx.doi.org/10.1242/jcs.00796DOI Listing
December 2003