Publications by authors named "Vishva M Dixit"

129 Publications

Shigella ubiquitin ligase IpaH7.8 targets gasdermin D for degradation to prevent pyroptosis and enable infection.

Cell Host Microbe 2021 Sep 1. Epub 2021 Sep 1.

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

The pore-forming protein gasdermin D (GSDMD) executes lytic cell death called pyroptosis to eliminate the replicative niche of intracellular pathogens. Evolution favors pathogens that circumvent this host defense mechanism. Here, we show that the Shigella ubiquitin ligase IpaH7.8 functions as an inhibitor of GSDMD. Shigella is an enteroinvasive bacterium that causes hemorrhagic gastroenteritis in primates, but not rodents. IpaH7.8 contributes to species specificity by ubiquitinating human, but not mouse, GSDMD and targeting it for proteasomal degradation. Accordingly, infection of human epithelial cells with IpaH7.8-deficient Shigella flexneri results in increased GSDMD-dependent cell death compared with wild type. Consistent with pyroptosis contributing to murine disease resistance, eliminating GSDMD from NLRC4-deficient mice, which are already sensitized to oral infection with Shigella flexneri, leads to further enhanced bacterial replication and increased disease severity. This work highlights a species-specific pathogen arms race focused on maintenance of host cell viability.
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http://dx.doi.org/10.1016/j.chom.2021.08.010DOI Listing
September 2021

Selective activation of PFKL suppresses the phagocytic oxidative burst.

Cell 2021 Aug 27;184(17):4480-4494.e15. Epub 2021 Jul 27.

Physiological Chemistry Department, Genentech, South San Francisco, CA 94080, USA. Electronic address:

In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.
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http://dx.doi.org/10.1016/j.cell.2021.07.004DOI Listing
August 2021

NINJ1 mediates plasma membrane rupture during lytic cell death.

Nature 2021 03 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

Integration of innate immune signalling by caspase-8 cleavage of N4BP1.

Nature 2020 11 24;587(7833):275-280. Epub 2020 Sep 24.

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

Mutations in the death receptor FAS or its ligand FASL cause autoimmune lymphoproliferative syndrome, whereas mutations in caspase-8 or its adaptor FADD-which mediate cell death downstream of FAS and FASL-cause severe immunodeficiency in addition to autoimmune lymphoproliferative syndrome. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses, but the mechanisms that underlie immunodeficiency remain undefined. Here we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased the production of select cytokines upon stimulation of the Toll-like receptor (TLR)1-TLR2 heterodimer (referred to herein as TLR1/2), TLR7 or TLR9, but not upon engagement of TLR3 or TLR4. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages, owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, the impaired production of cytokines in response to TLR3 and TLR4 stimulation of caspase-8-deficient macrophages was largely rescued by co-deletion of N4BP1. Thus, the persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumour necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by mutations of FADD and caspase-8.
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http://dx.doi.org/10.1038/s41586-020-2796-5DOI Listing
November 2020

Ubiquitin Ligase COP1 Suppresses Neuroinflammation by Degrading c/EBPβ in Microglia.

Cell 2020 09 13;182(5):1156-1169.e12. Epub 2020 Aug 13.

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

Dysregulated microglia are intimately involved in neurodegeneration, including Alzheimer's disease (AD) pathogenesis, but the mechanisms controlling pathogenic microglial gene expression remain poorly understood. The transcription factor CCAAT/enhancer binding protein beta (c/EBPβ) regulates pro-inflammatory genes in microglia and is upregulated in AD. We show expression of c/EBPβ in microglia is regulated post-translationally by the ubiquitin ligase COP1 (also called RFWD2). In the absence of COP1, c/EBPβ accumulates rapidly and drives a potent pro-inflammatory and neurodegeneration-related gene program, evidenced by increased neurotoxicity in microglia-neuronal co-cultures. Antibody blocking studies reveal that neurotoxicity is almost entirely attributable to complement. Remarkably, loss of a single allele of Cebpb prevented the pro-inflammatory phenotype. COP1-deficient microglia markedly accelerated tau-mediated neurodegeneration in a mouse model where activated microglia play a deleterious role. Thus, COP1 is an important suppressor of pathogenic c/EBPβ-dependent gene expression programs in microglia.
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http://dx.doi.org/10.1016/j.cell.2020.07.011DOI Listing
September 2020

Paradise revealed III: why so many ways to die? Apoptosis, necroptosis, pyroptosis, and beyond.

Cell Death Differ 2020 05 17;27(5):1740-1742. Epub 2020 Mar 17.

Centro de Terapia Celular e Molecular (CTCMol), Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil.

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http://dx.doi.org/10.1038/s41418-020-0526-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206054PMC
May 2020

Fiery Cell Death: Pyroptosis in the Central Nervous System.

Trends Neurosci 2020 01 13;43(1):55-73. Epub 2019 Dec 13.

Department of Medicine, Neuroscience and Mental Health Institute, University of Alberta, Edmonton AB, Canada. Electronic address:

Pyroptosis ('fiery death') is an inflammatory type of regulated cell death (RCD), which occurs downstream of inflammasome activation. Pyroptosis is mediated directly by the recently identified family of pore-forming proteins known as gasdermins, the best characterized of which is gasdermin D (GSDMD). Recent investigations implicate pyroptosis in the pathogenesis of multiple neurological diseases. In this review, we discuss molecular mechanisms that drive pyroptosis, evidence for pyroptosis within the CNS, and emerging therapeutic strategies for its inhibition in the context of neurological disease.
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http://dx.doi.org/10.1016/j.tins.2019.11.005DOI 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

Rescue from a fiery death: A therapeutic endeavor.

Science 2019 11;366(6466):688-689

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

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http://dx.doi.org/10.1126/science.aaw1177DOI Listing
November 2019

The RIPK4-IRF6 signalling axis safeguards epidermal differentiation and barrier function.

Nature 2019 10 2;574(7777):249-253. Epub 2019 Oct 2.

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

The integrity of the mammalian epidermis depends on a balance of proliferation and differentiation in the resident population of stem cells. The kinase RIPK4 and the transcription factor IRF6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft-tissue fusions that result in neonatal lethality. Our understanding of how these genes control epidermal differentiation is incomplete. Here we show that the role of RIPK4 in mouse development requires its kinase activity; that RIPK4 and IRF6 expressed in the epidermis regulate the same biological processes; and that the phosphorylation of IRF6 at Ser413 and Ser424 primes IRF6 for activation. Using RNA sequencing (RNA-seq), histone chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) of skin in wild-type and IRF6-deficient mouse embryos, we define the transcriptional programs that are regulated by IRF6 during epidermal differentiation. IRF6 was enriched at bivalent promoters, and IRF6 deficiency caused defective expression of genes that are involved in the metabolism of lipids and the formation of tight junctions. Accordingly, the lipid composition of the stratum corneum of Irf6 skin was abnormal, culminating in a severe defect in the function of the epidermal barrier. Collectively, our results explain how RIPK4 and IRF6 function to ensure the integrity of the epidermis and provide mechanistic insights into why developmental syndromes that are characterized by orofacial, skin and genital abnormalities result when this axis goes awry.
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http://dx.doi.org/10.1038/s41586-019-1615-3DOI Listing
October 2019

Cleavage of RIPK1 by caspase-8 is crucial for limiting apoptosis and necroptosis.

Nature 2019 10 11;574(7778):428-431. Epub 2019 Sep 11.

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

The aspartate-specific cysteine protease caspase-8 suppresses necroptotic cell death mediated by RIPK3 and MLKL. Indeed, mice that lack caspase-8 die in a RIPK3- and MLKL-dependent manner during embryogenesis. In humans, caspase-8 deficiency is associated with immunodeficiency or very early onset inflammatory bowel disease. The substrates that are cleaved by caspase-8 to prevent necroptosis in vivo have not been defined. Here we show that knock-in mice that express catalytically inactive caspase-8(C362A) die as embryos owing to MLKL-dependent necroptosis, similar to caspase-8-deficient mice. Thus, caspase-8 must cleave itself, other proteins or both to inhibit necroptosis. Mice that express caspase-8(D212A/D218A/D225A/D387A), which cannot cleave itself, were viable, as were mice that express c-FLIP or CYLD proteins that had been mutated to prevent cleavage by caspase-8. By contrast, mice that express RIPK1(D325A), in which the caspase-8 cleavage site Asp325 had been mutated, died mid-gestation. Embryonic lethality was prevented by inactivation of RIPK1, loss of TNFR1, or loss of both MLKL and the caspase-8 adaptor FADD, but not by loss of MLKL alone. Thus, RIPK1(D325A) appears to trigger cell death mediated by TNF, the kinase activity of RIPK1 and FADD-caspase-8. Accordingly, dying endothelial cells that contain cleaved caspase-3 were abnormally abundant in yolk sacs of Ripk1 embryos. Heterozygous Ripk1 cells and mice were viable, but were also more susceptible to TNF-induced cell death than were wild-type cells or mice. Our data show that Asp325 of RIPK1 is essential for limiting aberrant cell death in response to TNF, consistent with the idea that cleavage of RIPK1 by caspase-8 is a mechanism for dismantling death-inducing complexes.
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http://dx.doi.org/10.1038/s41586-019-1548-xDOI Listing
October 2019

Ubiquitin Ligases cIAP1 and cIAP2 Limit Cell Death to Prevent Inflammation.

Cell Rep 2019 05;27(9):2679-2689.e3

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

Cellular inhibitor of apoptosis proteins cIAP1 and cIAP2 ubiquitinate nuclear factor κB (NF-κB)-inducing kinase (NIK) to suppress non-canonical NF-κB signaling and substrates such as receptor interacting protein kinase 1 (RIPK1) to promote cell survival. We investigate how these functions contribute to homeostasis by eliminating cIap2 from adult cIap1-deficient mice. cIAP1 and cIAP2 (cIAP1/2) deficiency causes rapid weight loss and inflammation, with aberrant cell death, indicated by cleaved caspases-3 and -8, prevalent in intestine and liver. Deletion of Casp8 and Ripk3 prevents this aberrant cell death, reduces the inflammation, and prolongs mouse survival, whereas Ripk3 loss alone offers little benefit. Residual inflammation in mice lacking cIap1/2, Casp8, and Ripk3 is reduced by inhibition of NIK. Loss of Casp8 and Mlkl (mixed lineage kinase domain-like), but not Mlkl loss alone, also prevents cIAP1/2-deficient mice from dying around embryonic day 11. Therefore, a major function of cIAP1/2 in vivo is to suppress caspase-8-dependent cell death.
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http://dx.doi.org/10.1016/j.celrep.2019.04.111DOI Listing
May 2019

IRF2 transcriptionally induces expression for pyroptosis.

Sci Signal 2019 05 21;12(582). Epub 2019 May 21.

Department of Physiological Chemistry, Genentech Inc., South San Francisco, CA 94080, USA.

Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1β (IL-1β) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of A forward genetic screen with -ethyl--nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling. expression was substantially attenuated in deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1β secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the promoter to directly drive transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies.
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http://dx.doi.org/10.1126/scisignal.aax4917DOI Listing
May 2019

Intrinsic apoptosis shapes the tumor spectrum linked to inactivation of the deubiquitinase BAP1.

Science 2019 Apr 18;364(6437):283-285. Epub 2019 Apr 18.

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

Malignancies arising from mutation of tumor suppressors have unexplained tissue proclivity. For example, encodes a widely expressed deubiquitinase for histone H2A, but germline mutations are predominantly associated with uveal melanomas and mesotheliomas. We show that BAP1 inactivation causes apoptosis in mouse embryonic stem cells, fibroblasts, liver, and pancreatic tissue but not in melanocytes and mesothelial cells. Ubiquitin ligase RNF2, which silences genes by monoubiquitinating H2A, promoted apoptosis in BAP1-deficient cells by suppressing expression of the prosurvival genes and In contrast, BAP1 loss in melanocytes had little impact on expression of prosurvival genes, instead inducing Thus, BAP1 appears to modulate gene expression by countering H2A ubiquitination, but its loss only promotes tumorigenesis in cells that do not engage an RNF2-dependent apoptotic program.
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http://dx.doi.org/10.1126/science.aav4902DOI Listing
April 2019

The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification.

PLoS One 2019 5;14(4):e0214110. Epub 2019 Apr 5.

Physiological Chemistry Department, Genentech, South San Francisco, California, United States of America.

Peg10 (paternally expressed gene 10) is an imprinted gene that is essential for placental development. It is thought to derive from a Ty3-gyspy LTR (long terminal repeat) retrotransposon and retains Gag and Pol-like domains. Here we show that the Gag domain of PEG10 can promote vesicle budding similar to the HIV p24 Gag protein. Expressed in a subset of mouse endocrine organs in addition to the placenta, PEG10 was identified as a substrate of the deubiquitinating enzyme USP9X. Consistent with PEG10 having a critical role in placental development, PEG10-deficient trophoblast stem cells (TSCs) exhibited impaired differentiation into placental lineages. PEG10 expressed in wild-type, differentiating TSCs was bound to many cellular RNAs including Hbegf (Heparin-binding EGF-like growth factor), which is known to play an important role in placentation. Expression of Hbegf was reduced in PEG10-deficient TSCs suggesting that PEG10 might bind to and stabilize RNAs that are critical for normal placental development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214110PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450627PMC
December 2019

Interview: a conversation with Vishva M Dixit on his journey from remote African village to apoptosis, necroptosis and the inflammasome.

Authors:
Vishva M Dixit

Cell Death Differ 2019 03;26(4):597-604

Genentech, Inc., South San Francisco, CA, United States.

Cv Sketch: Vishva M. Dixit, M.D., Vice President of Physiological Chemistry at Genentech, Inc. has made many contributions to biomedicine, and his early work on apoptosis is prominent in introductory textbooks of biology and medicine.He is a member of the National Academy of Sciences, the National Academy of Medicine, the American Academy of Arts and Sciences, and a Foreign Member, European Molecular Biology Organization.Additionally, he serves on the Boards of the Gates Foundation, Howard Hughes Medical Institute, and Keystone Symposia.
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http://dx.doi.org/10.1038/s41418-019-0294-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460394PMC
March 2019

TBK1 and IKKε restrain cell death.

Nat Cell Biol 2018 12;20(12):1330-1331

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

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http://dx.doi.org/10.1038/s41556-018-0239-4DOI Listing
December 2018

Ubiquitin ligase COP1 coordinates transcriptional programs that control cell type specification in the developing mouse brain.

Proc Natl Acad Sci U S A 2018 10 15;115(44):11244-11249. Epub 2018 Oct 15.

Department of Physiological Chemistry, Genentech, South San Francisco, CA 94080;

The E3 ubiquitin ligase CRL4 is active in the absence of ERK signaling, modifying the transcription factors ETV1, ETV4, ETV5, and c-JUN with polyubiquitin that targets them for proteasomal degradation. Here we show that this posttranslational regulatory mechanism is active in neurons, with ETV5 and c-JUN accumulating within minutes of ERK activation. Mice with () deleted in neural stem cells showed abnormally elevated expression of ETV1, ETV4, ETV5, and c-JUN in the developing brain and spinal cord. Expression of c-JUN target genes and was increased, whereas ETV5 and c-JUN both contributed to an expanded number of cells expressing genes associated with gliogenesis, including , , and The mice had subtle morphological abnormalities in the cerebral cortex, hippocampus, and cerebellum by embryonic day 18 and died soon after birth. Elevated c-JUN, ETV5, and ETV1 contributed to the perinatal lethality, as several -deficient mice also lacking and , or lacking and heterozygous for , were viable.
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http://dx.doi.org/10.1073/pnas.1805033115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217379PMC
October 2018

The tumor suppressor BAP1 cooperates with BRAFV600E to promote tumor formation in cutaneous melanoma.

Pigment Cell Melanoma Res 2019 03 24;32(2):269-279. Epub 2018 Sep 24.

Department of Discovery Oncology, Genentech, Inc., South San Francisco, California.

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk of mesothelioma and melanocytic tumors. Here, we show that Bap1 deletion in melanocytes cooperates with the constitutively active, oncogenic form of BRAF (BRAF ) and UV to cause melanoma in mice, albeit at very low frequency. In addition, Bap1-null melanoma cells derived from mouse tumors are more aggressive and colonize and grow at distant sites more than their wild-type counterparts. Molecularly, Bap1-null melanoma cell lines have increased DNA damage measured by γH2aX and hyperubiquitination of histone H2a. Therapeutically, these Bap1-null tumors are completely responsive to BRAF- and MEK-targeted therapies. Therefore, BAP1 functions as a tumor suppressor and limits tumor progression in melanoma.
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http://dx.doi.org/10.1111/pcmr.12735DOI Listing
March 2019

OTULIN limits cell death and inflammation by deubiquitinating LUBAC.

Nature 2018 07 27;559(7712):120-124. Epub 2018 Jun 27.

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

OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease and embryonic lethality during mouse development. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon.
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http://dx.doi.org/10.1038/s41586-018-0256-2DOI Listing
July 2018

Crystal Structure of Ripk4 Reveals Dimerization-Dependent Kinase Activity.

Structure 2018 05 26;26(5):767-777.e5. Epub 2018 Apr 26.

Department of Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address:

Receptor-interacting protein kinase 4 (RIPK4) is a highly conserved regulator of epidermal differentiation. Members of the RIPK family possess a common kinase domain as well as unique accessory domains that likely dictate subcellular localization and substrate preferences. Mutations in human RIPK4 manifest as Bartsocas-Papas syndrome (BPS), a genetic disorder characterized by severe craniofacial and limb abnormalities. We describe the structure of the murine Ripk4 (MmRipk4) kinase domain, in ATP- and inhibitor-bound forms. The crystallographic dimer of MmRipk4 is similar to those of RIPK2 and BRAF, and we show that the intact dimeric entity is required for MmRipk4 catalytic activity through a series of engineered mutations and cell-based assays. We also assess the impact of BPS mutations on protein structure and activity to elucidate the molecular origins of the disease.
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http://dx.doi.org/10.1016/j.str.2018.04.002DOI Listing
May 2018

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Cell Death Differ 2018 03 23;25(3):486-541. Epub 2018 Jan 23.

Department of Medicine, Division of Pulmonary and Critical Care Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.
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http://dx.doi.org/10.1038/s41418-017-0012-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5864239PMC
March 2018

A new lead to NLRP3 inhibition.

J Exp Med 2017 11 23;214(11):3147-3149. Epub 2017 Oct 23.

VIB-UGent Center for Inflammation Research, VIB; and Department of Internal Medicine, Ghent University, Ghent, Belgium; Department of Physiological Chemistry, Genentech, South San Francisco, CA

The discovery of a small molecule inhibitor that targets the inflammasome sensor NLRP3 offers a new path for the development of selective inflammasome blockers with potential therapeutic benefit in a wide range of human diseases (in this issue, see Jiang et al., https://doi.org/10.1084/jem.20171419).
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http://dx.doi.org/10.1084/jem.20171848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678032PMC
November 2017

Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control.

Cell 2017 Nov 12;171(4):918-933.e20. Epub 2017 Oct 12.

Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA; Howard Hughes Medical Institute, Berkeley, CA, USA. Electronic address:

Posttranslational modification with ubiquitin chains controls cell fate in all eukaryotes. Depending on the connectivity between subunits, different ubiquitin chain types trigger distinct outputs, as seen with K48- and K63-linked conjugates that drive protein degradation or complex assembly, respectively. Recent biochemical analyses also suggested roles for mixed or branched ubiquitin chains, yet without a method to monitor endogenous conjugates, the physiological significance of heterotypic polymers remained poorly understood. Here, we engineered a bispecific antibody to detect K11/K48-linked chains and identified mitotic regulators, misfolded nascent polypeptides, and pathological Huntingtin variants as their endogenous substrates. We show that K11/K48-linked chains are synthesized and processed by essential ubiquitin ligases and effectors that are mutated across neurodegenerative diseases; accordingly, these conjugates promote rapid proteasomal clearance of aggregation-prone proteins. By revealing key roles of K11/K48-linked chains in cell-cycle and quality control, we establish heterotypic ubiquitin conjugates as important carriers of biological information.
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http://dx.doi.org/10.1016/j.cell.2017.09.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669814PMC
November 2017

In Retrospect: The inflammasome turns 15.

Nature 2017 08;548(7669):534-535

Department of Physiological Chemistry, Genentech, South San Francisco, California 94080, USA.

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http://dx.doi.org/10.1038/548534aDOI Listing
August 2017

Transcription factor Etv5 is essential for the maintenance of alveolar type II cells.

Proc Natl Acad Sci U S A 2017 04 28;114(15):3903-3908. Epub 2017 Mar 28.

Department of Physiological Chemistry, Genentech, South San Francisco, CA 94080;

Alveolar type II (AT2) cell dysfunction contributes to a number of significant human pathologies including respiratory distress syndrome, lung adenocarcinoma, and debilitating fibrotic diseases, but the critical transcription factors that maintain AT2 cell identity are unknown. Here we show that the E26 transformation-specific (ETS) family transcription factor Etv5 is essential to maintain AT2 cell identity. Deletion of from AT2 cells produced gene and protein signatures characteristic of differentiated alveolar type I (AT1) cells. Consistent with a defect in the AT2 stem cell population, deficiency markedly reduced recovery following bleomycin-induced lung injury. Lung tumorigenesis driven by mutant KrasG12D was also compromised by Etv5 deficiency. ERK activation downstream of Ras was found to stabilize Etv5 through inactivation of the cullin-RING ubiquitin ligase CRL4 that targets Etv5 for proteasomal degradation. These findings identify Etv5 as a critical output of Ras signaling in AT2 cells, contributing to both lung homeostasis and tumor initiation.
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http://dx.doi.org/10.1073/pnas.1621177114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393225PMC
April 2017

RIPK1 inhibits ZBP1-driven necroptosis during development.

Nature 2016 12 7;540(7631):129-133. Epub 2016 Nov 7.

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

Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival-mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis. However, mice expressing catalytically inactive RIPK1 are viable, and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1 mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis, in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1 mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses but previously had no defined role in development, also prevented perinatal lethality in Ripk1 mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1Mlkl macrophages, but not in wild-type, Mlkl or Ripk1Ripk3 macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.
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http://dx.doi.org/10.1038/nature20559DOI Listing
December 2016

GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes.

Proc Natl Acad Sci U S A 2016 07 23;113(28):7858-63. Epub 2016 Jun 23.

Department of Early Discovery Biochemistry, Genentech, Inc., South San Francisco, CA 94080;

Gasdermin-D (GsdmD) is a critical mediator of innate immune defense because its cleavage by the inflammatory caspases 1, 4, 5, and 11 yields an N-terminal p30 fragment that induces pyroptosis, a death program important for the elimination of intracellular bacteria. Precisely how GsdmD p30 triggers pyroptosis has not been established. Here we show that human GsdmD p30 forms functional pores within membranes. When liberated from the corresponding C-terminal GsdmD p20 fragment in the presence of liposomes, GsdmD p30 localized to the lipid bilayer, whereas p20 remained in the aqueous environment. Within liposomes, p30 existed as higher-order oligomers and formed ring-like structures that were visualized by negative stain electron microscopy. These structures appeared within minutes of GsdmD cleavage and released Ca(2+) from preloaded liposomes. Consistent with GsdmD p30 favoring association with membranes, p30 was only detected in the membrane-containing fraction of immortalized macrophages after caspase-11 activation by lipopolysaccharide. We found that the mouse I105N/human I104N mutation, which has been shown to prevent macrophage pyroptosis, attenuated both cell killing by p30 in a 293T transient overexpression system and membrane permeabilization in vitro, suggesting that the mutants are actually hypomorphs, but must be above certain concentration to exhibit activity. Collectively, our data suggest that GsdmD p30 kills cells by forming pores that compromise the integrity of the cell membrane.
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http://dx.doi.org/10.1073/pnas.1607769113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948338PMC
July 2016

Inflammasomes: mechanism of assembly, regulation and signalling.

Nat Rev Immunol 2016 07 13;16(7):407-20. Epub 2016 Jun 13.

Genentech Inc., South San Francisco, California 94080, USA.

Inflammasomes are multiprotein signalling platforms that control the inflammatory response and coordinate antimicrobial host defences. They are assembled by pattern-recognition receptors following the detection of pathogenic microorganisms and danger signals in the cytosol of host cells, and they activate inflammatory caspases to produce cytokines and to induce pyroptotic cell death. The clinical importance of inflammasomes reaches beyond infectious disease, as dysregulated inflammasome activity is associated with numerous hereditary and acquired inflammatory disorders. In this Review, we discuss the recent developments in inflammasome research with a focus on the molecular mechanisms that govern inflammasome assembly, signalling and regulation.
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http://dx.doi.org/10.1038/nri.2016.58DOI Listing
July 2016
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