Publications by authors named "Trinna L Cuellar"

12 Publications

  • Page 1 of 1

ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages.

Sci Rep 2018 02 28;8(1):3788. Epub 2018 Feb 28.

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

The NLRC4 inflammasome recognizes bacterial flagellin and components of the type III secretion apparatus. NLRC4 stimulation leads to caspase-1 activation followed by a rapid lytic cell death known as pyroptosis. NLRC4 is linked to pathogen-free auto-inflammatory diseases, suggesting a role for NLRC4 in sterile inflammation. Here, we show that NLRC4 activates an alternative cell death program morphologically similar to apoptosis in caspase-1-deficient BMDMs. By performing an unbiased genome-wide CRISPR/Cas9 screen with subsequent validation studies in gene-targeted mice, we highlight a critical role for caspase-8 and ASC adaptor in an alternative apoptotic pathway downstream of NLRC4. Furthermore, caspase-1 catalytically dead knock-in (Casp1 C284A KI) BMDMs genetically segregate pyroptosis and apoptosis, and confirm that caspase-1 does not functionally compete with ASC for NLRC4 interactions. We show that NLRC4/caspase-8-mediated apoptotic cells eventually undergo plasma cell membrane damage in vitro, suggesting that this pathway can lead to secondary necrosis. Unexpectedly, we found that DFNA5/GSDME, a member of the pore-forming gasdermin family, is dispensable for the secondary necrosis that follows NLRC4-mediated apoptosis in macrophages. Together, our data confirm the existence of an alternative caspase-8 activation pathway diverging from the NLRC4 inflammasome in primary macrophages.
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http://dx.doi.org/10.1038/s41598-018-21998-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830643PMC
February 2018

Silencing of retrotransposons by SETDB1 inhibits the interferon response in acute myeloid leukemia.

J Cell Biol 2017 11 8;216(11):3535-3549. Epub 2017 Sep 8.

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

A propensity for rewiring genetic and epigenetic regulatory networks, thus enabling sustained cell proliferation, suppression of apoptosis, and the ability to evade the immune system, is vital to cancer cell propagation. An increased understanding of how this is achieved is critical for identifying or improving therapeutic interventions. In this study, using acute myeloid leukemia (AML) human cell lines and a custom CRISPR/Cas9 screening platform, we identify the H3K9 methyltransferase SETDB1 as a novel, negative regulator of innate immunity. SETDB1 is overexpressed in many cancers, and loss of this gene in AML cells triggers desilencing of retrotransposable elements that leads to the production of double-stranded RNAs (dsRNAs). This is coincident with induction of a type I interferon response and apoptosis through the dsRNA-sensing pathway. Collectively, our findings establish a unique gene regulatory axis that cancer cells can exploit to circumvent the immune system.
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http://dx.doi.org/10.1083/jcb.201612160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5674883PMC
November 2017

Quantitative evaluation of first, second, and third generation hairpin systems reveals the limit of mammalian vector-based RNAi.

RNA Biol 2016 ;13(1):25-33

b Molecular Biology South San Francisco , CA 94080.

Incorporating miRNA-like features into vector-based hairpin scaffolds has been shown to augment small RNA processing and RNAi efficiency. Therefore, defining an optimal, native hairpin context may obviate a need for hairpin-specific targeting design schemes, which confound the movement of functional siRNAs into shRNA/artificial miRNA backbones, or large-scale screens to identify efficacious sequences. Thus, we used quantitative cell-based assays to compare separate third generation artificial miRNA systems, miR-E (based on miR-30a) and miR-3G (based on miR-16-2 and first described in this study) to widely-adopted, first and second generation formats in both Pol-II and Pol-III expression vector contexts. Despite their unique structures and strandedness, and in contrast to first and second-generation RNAi triggers, the third generation formats operated with remarkable similarity to one another, and strong silencing was observed with a significant fraction of the evaluated target sequences within either promoter context. By pairing an established siRNA design algorithm with the third generation vectors we could readily identify targeting sequences that matched or exceeded the potency of those discovered through large-scale sensor-based assays. We find that third generation hairpin systems enable the maximal level of siRNA function, likely through enhanced processing and accumulation of precisely-defined guide RNAs. Therefore, we predict future gains in RNAi potency will come from improved hairpin expression and identification of optimal siRNA-intrinsic silencing properties rather than further modification of these scaffolds. Consequently, third generation systems should be the primary format for vector-based RNAi studies; miR-3G is advantageous due to its small expression cassette and simplified, cost-efficient cloning scheme.
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http://dx.doi.org/10.1080/15476286.2015.1128062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829305PMC
October 2016

Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB-siRNA conjugates.

Nucleic Acids Res 2015 Jan 30;43(2):1189-203. Epub 2014 Dec 30.

Department of Discovery Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-0511, USA

Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody-siRNA complexes provide a possible solution. However, initial reports of antibody-siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody-siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.
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http://dx.doi.org/10.1093/nar/gku1362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333408PMC
January 2015

Real-time quantification of antibody-short interfering RNA conjugate in serum by antigen capture reverse transcription-polymerase chain reaction.

Anal Biochem 2012 Nov 26;430(2):171-8. Epub 2012 Aug 26.

Biochemical and Cellular Pharmacology, Genentech, South San Francisco, CA 94080, USA.

Short interfering RNA (siRNA) has therapeutic potential. However, efficient delivery is a formidable task. To facilitate delivery of siRNA into cells, we covalently conjugated siRNA to antibodies that bind to cell surface proteins and internalize. Understanding how these antibody-siRNA conjugates function in vivo requires pharmacokinetic analysis. Thus, we developed a simple real-time antigen capture reverse transcription-polymerase chain reaction (RT-PCR) assay to detect intact antibody-siRNA conjugates. Biotinylated antigen bound to streptavidin-coated PCR tubes was used to capture antibody-siRNA conjugate. The captured antibody-siRNA conjugate was then reverse-transcribed in the same tube, avoiding a sample transfer step. This reproducible assay had a wide standard curve range of 0.029 to 480ng/ml and could detect as low as 0.58ng/ml antibody-siRNA conjugates in mouse serum. The presence of unconjugated antibody that could be generated from siRNA degradation in vivo did not affect the assay as long as the total antibody concentration in the antigen capture step did not exceed 480ng/ml. Using this assay, we observed a more rapid decrease in serum antibody-siRNA conjugate concentrations than the total antibody concentrations in mice dosed with antibody-siRNA conjugates, suggesting loss of siRNA from the antibody. This assay is useful for optimizing antibody-siRNA and likely aptamer-siRNA conjugates to improve pharmacokinetics and aid siRNA delivery.
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http://dx.doi.org/10.1016/j.ab.2012.08.018DOI Listing
November 2012

Right- and left-loop short shRNAs have distinct and unusual mechanisms of gene silencing.

Nucleic Acids Res 2012 Oct 18;40(18):9255-71. Epub 2012 Jul 18.

SomaGenics, Inc., 2161 Delaware Avenue, Santa Cruz, CA 95060, USA.

Small hairpin RNAs (shRNAs) having duplex lengths of 25-29 bp are normally processed by Dicer into short interfering RNAs (siRNAs) before incorporation into the RNA-induced silencing complex (RISC). However, shRNAs of ≤ 19 bp [short shRNAs (sshRNAs)] are too short for Dicer to excise their loops, raising questions about their mechanism of action. sshRNAs are designated as L-type or R-type according to whether the loop is positioned 3' or 5' to the guide sequence, respectively. Using nucleotide modifications that inhibit RNA cleavage, we show that R- but not L-sshRNAs require loop cleavage for optimum activity. Passenger-arm slicing was found to be important for optimal functioning of L-sshRNAs but much less important for R-sshRNAs that have a cleavable loop. R-sshRNAs could be immunoprecipitated by antibodies to Argonaute-1 (Ago1); complexes with Ago1 contained both intact and loop-cleaved sshRNAs. In contrast, L-sshRNAs were immunoprecipitated with either Ago1 or Ago2 and were predominantly sliced in the passenger arm of the hairpin. However, 'pre-sliced' L-sshRNAs were inactive. We conclude that active L-sshRNAs depend on slicing of the passenger arm to facilitate opening of the duplex, whereas R-sshRNAs primarily act via loop cleavage to generate a 5'-phosphate at the 5'-end of the guide strand.
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http://dx.doi.org/10.1093/nar/gks662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3467060PMC
October 2012

Dicer1 and miR-219 Are required for normal oligodendrocyte differentiation and myelination.

Neuron 2010 Mar;65(5):597-611

Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305-5125, USA.

To investigate the role of microRNAs in regulating oligodendrocyte (OL) differentiation and myelination, we utilized transgenic mice in which microRNA processing was disrupted in OL precursor cells (OPCs) and OLs by targeted deletion of Dicer1. We found that inhibition of OPC-OL miRNA processing disrupts normal CNS myelination and that OPCs lacking mature miRNAs fail to differentiate normally in vitro. We identified three miRNAs (miR-219, miR-138, and miR-338) that are induced 10-100x during OL differentiation; the most strongly induced of these, miR-219, is necessary and sufficient to promote OL differentiation, and partially rescues OL differentiation defects caused by total miRNA loss. miR-219 directly represses the expression of PDGFRalpha, Sox6, FoxJ3, and ZFP238 proteins, all of which normally help to promote OPC proliferation. Together, these findings show that miR-219 plays a critical role in coupling differentiation to proliferation arrest in the OL lineage, enabling the rapid transition from proliferating OPCs to myelinating OLs.
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http://dx.doi.org/10.1016/j.neuron.2010.01.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2843397PMC
March 2010

Expanded RNA-binding activities of mammalian Argonaute 2.

Nucleic Acids Res 2009 Dec;37(22):7533-45

Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

Mammalian Argonaute 2 (Ago2) protein associates with microRNAs (miRNAs) or small interfering RNAs (siRNAs) forming RNA-induced silencing complexes (RISCs/miRNPs). In the present work, we characterize the RNA-binding and nucleolytic activity of recombinant mouse Ago2. Our studies show that recombinant mouse Ago2 binds efficiently to miRNAs forming active RISC. Surprisingly, we find that recombinant mouse Ago2 forms active RISC using pre-miRNAs or long unstructured single stranded RNAs as guides. Furthermore, we demonstrate that, in vivo, endogenous human Ago2 binds directly to pre-miRNAs independently of Dicer, and that Ago2:pre-miRNA complexes are found both in the cytoplasm and in the nucleus of human cells.
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http://dx.doi.org/10.1093/nar/gkp812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794174PMC
December 2009

Conditional loss of Dicer disrupts cellular and tissue morphogenesis in the cortex and hippocampus.

J Neurosci 2008 Apr;28(17):4322-30

Department of Ophthalmology, University of California, San Francisco, San Francisco, California 94143-0730, USA.

To investigate the role of Dicer and microRNAs in the mammalian CNS, we used mice in which the second RNase III domain of Dicer was conditionally floxed. Conditional Dicer mice were bred with mice expressing an alpha-calmodulin kinase II Cre to selectively inactivate Dicer in excitatory forebrain neurons in vivo. Inactivation of Dicer results in an array of phenotypes including microcephaly, reduced dendritic branch elaboration, and large increases in dendritic spine length with no concomitant change in spine density. Microcephaly is likely caused by a 5.5-fold increase in early postnatal apoptosis in these animals as determined by active caspase-3 and TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling) staining in the cortex. Loss of Dicer function had no measurable effect on cortical lamination as determined by in situ hybridization, suggesting that microcephaly is not caused by defects in neuronal migration. Together, these results illustrate the in vivo significance of Dicer and miRNAs in the mammalian CNS and provide additional support for previous in vitro studies indicating that misregulation of this pathway may result in gross abnormalities in cell number and function that may contribute to a variety of neurological disorders.
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http://dx.doi.org/10.1523/JNEUROSCI.4815-07.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3844796PMC
April 2008

Dicer loss in striatal neurons produces behavioral and neuroanatomical phenotypes in the absence of neurodegeneration.

Proc Natl Acad Sci U S A 2008 Apr 2;105(14):5614-9. Epub 2008 Apr 2.

Department of Microbiology and Immunology, Diabetes Center, Biomedical Sciences Graduate Program, and Departments of Ophthalmology and Physiology, University of California, San Francisco, CA 94122-0534, USA.

MicroRNAs (miRNAs) are small noncoding RNAs that can act to repress target mRNAs by suppressing translation and/or reducing mRNA stability. Although it is clear that miRNAs and Dicer, an RNase III enzyme that is central to the production of mature miRNAs, have a role in the early development of neurons, their roles in the postmitotic neuron in vivo are largely unknown. To determine the roles of Dicer in neurons, we ablated Dicer in dopaminoceptive neurons. Mice that have lost Dicer in these cells display a range of phenotypes including ataxia, front and hind limb clasping, reduced brain size, and smaller neurons. Surprisingly, dopaminoceptive neurons without Dicer survive over the life of the animal. The lack of profound cell death contrasts with other mouse models in which Dicer has been ablated. These studies highlight the complicated nature of Dicer ablation in the brain and provide a useful mouse model for studying dopaminoceptive neuron function.
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http://dx.doi.org/10.1073/pnas.0801689105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291142PMC
April 2008

An AICD-based functional screen to identify APP metabolism regulators.

Mol Neurodegener 2007 Aug 24;2:15. Epub 2007 Aug 24.

Department of Bioscience & Biotechnology, Drexel University, Philadelphia, PA, USA.

Background: A central event in Alzheimer's disease (AD) is the regulated intramembraneous proteolysis of the beta-amyloid precursor protein (APP), to generate the beta-amyloid (Abeta) peptide and the APP intracellular domain (AICD). Abeta is the major component of amyloid plaques and AICD displays transcriptional activation properties. We have taken advantage of AICD transactivation properties to develop a genetic screen to identify regulators of APP metabolism. This screen relies on an APP-Gal4 fusion protein, which upon normal proteolysis, produces AICD-Gal4. Production of AICD-Gal4 induces Gal4-UAS driven luciferase expression. Therefore, when regulators of APP metabolism are modulated, luciferase expression is altered.

Results: To validate this experimental approach we modulated alpha-, beta-, and gamma-secretase levels and activities. Changes in AICD-Gal4 levels as measured by Western blot analysis were strongly and significantly correlated to the observed changes in AICD-Gal4 mediated luciferase activity. To determine if a known regulator of APP trafficking/maturation and Presenilin1 endoproteolysis could be detected using the AICD-Gal4 mediated luciferase assay, we knocked-down Ubiquilin 1 and observed decreased luciferase activity. We confirmed that Ubiquilin 1 modulated AICD-Gal4 levels by Western blot analysis and also observed that Ubiquilin 1 modulated total APP levels, the ratio of mature to immature APP, as well as PS1 endoproteolysis.

Conclusion: Taken together, we have shown that this screen can identify known APP metabolism regulators that control proteolysis, intracellular trafficking, maturation and levels of APP and its proteolytic products. We demonstrate for the first time that Ubiquilin 1 regulates APP metabolism in the human neuroblastoma cell line, SH-SY5Y.
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http://dx.doi.org/10.1186/1750-1326-2-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2071909PMC
August 2007

MicroRNAs and endocrine biology.

J Endocrinol 2005 Dec;187(3):327-32

UCSF Diabetes Center, Department of Microbiology and Immunology, University of California, San Francisco, California 94122-0534, USA.

microRNAs (miRNAs) are highly conserved, non-coding RNAs that powerfully regulate gene expression at the post-transcriptional level. These fascinating molecules play essential roles in many biological processes in mammals, including insulin secretion, B-cell development, and adipocyte differentiation. This review provides a general background regarding current knowledge about miRNA biogenesis and the potential contributions of these RNAs to endocrine function.
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http://dx.doi.org/10.1677/joe.1.06426DOI Listing
December 2005