Publications by authors named "Eigen R Peralta"

7 Publications

  • Page 1 of 1

The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.

PLoS One 2015 7;10(12):e0144409. Epub 2015 Dec 7.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, United States of America.

Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as "central" interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, "peripheral" interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144409PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4671683PMC
June 2016

Sphingolipid-based drugs selectively kill cancer cells by down-regulating nutrient transporter proteins.

Biochem J 2011 Oct;439(2):299-311

Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA.

Cancer cells are hypersensitive to nutrient limitation because oncogenes constitutively drive glycolytic and TCA (tricarboxylic acid) cycle intermediates into biosynthetic pathways. As the anaplerotic reactions that replace these intermediates are fueled by imported nutrients, the cancer cell's ability to generate ATP becomes compromised under nutrient-limiting conditions. In addition, most cancer cells have defects in autophagy, the catabolic process that provides nutrients from internal sources when external nutrients are unavailable. Normal cells, in contrast, can adapt to the nutrient stress that kills cancer cells by becoming quiescent and catabolic. In the present study we show that FTY720, a water-soluble sphingolipid drug that is effective in many animal cancer models, selectively starves cancer cells to death by down-regulating nutrient transporter proteins. Consistent with a bioenergetic mechanism of action, FTY720 induced homoeostatic autophagy. Cells were protected from FTY720 by cell-permeant nutrients or by reducing nutrient demand, but blocking apoptosis was ineffective. Importantly, AAL-149, a FTY720 analogue that lacks FTY720's dose-limiting toxicity, also triggered transporter loss and killed patient-derived leukaemias while sparing cells isolated from normal donors. As they target the metabolic profile of cancer cells rather than specific oncogenic mutations, FTY720 analogues such as AAL-149 should be effective against many different tumour types, particularly in combination with drugs that inhibit autophagy.
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http://dx.doi.org/10.1042/BJ20110853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3454501PMC
October 2011

Differential effects of TBC1D15 and mammalian Vps39 on Rab7 activation state, lysosomal morphology, and growth factor dependence.

J Biol Chem 2010 May 2;285(22):16814-21. Epub 2010 Apr 2.

Department of Developmental and Cell Biology, University of California, Irvine, California 92697-2300, USA.

The small GTPase Rab7 promotes fusion events between late endosomes and lysosomes. Rab7 activity is regulated by extrinsic signals, most likely via effects on its guanine nucleotide exchange factor (GEF) or GTPase-activating protein (GAP). Based on their homology to the yeast proteins that regulate the Ypt7 GTP binding state, TBC1D15, and mammalian Vps39 (mVps39) have been suggested to function as the Rab7 GAP and GEF, respectively. We developed an effector pull-down assay to test this model. TBC1D15 functioned as a Rab7 GAP in cells, reducing Rab7 binding to its effector protein RILP, fragmenting the lysosome, and conferring resistance to growth factor withdrawal-induced cell death. In a cellular context, TBC1D15 GAP activity was selective for Rab7. TBC1D15 overexpression did not inhibit transferrin internalization or recycling, Rab7-independent processes that require Rab4, Rab5, and Rab11 activation. TBC1D15 was thus renamed Rab7-GAP. Contrary to expectations for a Rab7 GEF, mVps39 induced lysosomal clustering without increasing Rab7 GTP binding. Moreover, a dominant-negative mVps39 mutant fragmented the lysosome and promoted growth factor independence without decreasing Rab7-GTP levels. These findings suggest that a protein other than mVps39 serves as the Rab7 GEF. In summary, although only TBC1D15/Rab7-GAP altered Rab7-GTP levels, both Rab7-GAP and mVps39 regulate lysosomal morphology and play a role in maintaining growth factor dependence.
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http://dx.doi.org/10.1074/jbc.M110.111633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2878074PMC
May 2010

Rab7 activation by growth factor withdrawal contributes to the induction of apoptosis.

Mol Biol Cell 2009 Jun 22;20(12):2831-40. Epub 2009 Apr 22.

Department of Developmental and Cell Biology, University of California-Irvine, Irvine, CA 92697-2300, USA.

The Rab7 GTPase promotes membrane fusion reactions between late endosomes and lysosomes. In previous studies, we demonstrated that Rab7 inactivation blocks growth factor withdrawal-induced cell death. These results led us to hypothesize that growth factor withdrawal activates Rab7. Here, we show that growth factor deprivation increased both the fraction of Rab7 that was associated with cellular membranes and the percentage of Rab7 bound to guanosine triphosphate (GTP). Moreover, expressing a constitutively GTP-bound mutant of Rab7, Rab7-Q67L, was sufficient to trigger cell death even in the presence of growth factors. This activated Rab7 mutant was also able to reverse the growth factor-independent cell survival conferred by protein kinase C (PKC) delta inhibition. PKCdelta is one of the most highly induced proteins after growth factor withdrawal and contributes to the induction of apoptosis. To evaluate whether PKCdelta regulates Rab7, we first examined lysosomal morphology in cells with reduced PKCdelta activity. Consistent with a potential role as a Rab7 activator, blocking PKCdelta function caused profound lysosomal fragmentation comparable to that observed when Rab7 was directly inhibited. Interestingly, PKCdelta inhibition fragmented the lysosome without decreasing Rab7-GTP levels. Taken together, these results suggest that Rab7 activation by growth factor withdrawal contributes to the induction of apoptosis and that Rab7-dependent fusion reactions may be targeted by signaling pathways that limit growth factor-independent cell survival.
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http://dx.doi.org/10.1091/mbc.e08-09-0911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2695791PMC
June 2009

Ceramide-induced starvation triggers homeostatic autophagy.

Autophagy 2009 Apr 9;5(3):407-9. Epub 2009 Apr 9.

Department of Developmental and Cell Biology, University of California-Irvine, Irvine, CA 92697-2300, USA.

Autophagy is triggered by ceramide, a sphingolipid that regulates diverse cellular processes including survival, differentiation and senescence. Both ceramide and autophagy play important, but incompletely understood, roles in type 2 diabetes and cancer. We reasoned that defining the connection between ceramide and autophagy might provide an important insight into these highly prevalent diseases. Our recently published work demonstrates that ceramide-induced autophagy is a homeostatic response to starvation caused by nutrient transporter downregulation. Preventing nutrient transporter loss or supplementation with transporter-independent nutrients protects cells from ceramide-induced death and delays the onset of autophagy. Thus, we propose a model where ceramide kills cells by inducing acute and severe intracellular nutrient limitation. Consistent with this idea, AMPK-deficient cells that are less able to deal with bioenergetic stress are also more sensitive to ceramide than wild-type cells. Our observation that gradually adapting cells to tolerate low levels of extracellular nutrients confers striking resistance to ceramide toxicity further supports this model. These results highlight the value of measuring nutrient transporter expression in cells undergoing protective autophagy. In addition, this novel mechanism for ceramide-induced cell death suggests new approaches to studying and treating multiple human diseases.
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http://dx.doi.org/10.4161/auto.5.3.7809DOI Listing
April 2009

Ceramide starves cells to death by downregulating nutrient transporter proteins.

Proc Natl Acad Sci U S A 2008 Nov 3;105(45):17402-7. Epub 2008 Nov 3.

Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA.

Ceramide induces cell death in response to many stimuli. Its mechanism of action, however, is not completely understood. Ceramide induces autophagy in mammalian cells maintained in rich media and nutrient permease downregulation in yeast. These observations suggested to us that ceramide might kill mammalian cells by limiting cellular access to extracellular nutrients. Consistent with this proposal, physiologically relevant concentrations of ceramide produced a profound and specific downregulation of nutrient transporter proteins in mammalian cells. Blocking ceramide-induced nutrient transporter loss or supplementation with the cell-permeable nutrient, methyl pyruvate, reversed ceramide-dependent toxicity. Conversely, cells became more sensitive to ceramide when nutrient stress was increased by acutely limiting extracellular nutrients, inhibiting autophagy, or deleting AMP-activated protein kinase (AMPK). Observations that ceramide can trigger either apoptosis or caspase-independent cell death may be explained by this model. We found that methyl pyruvate (MP) also protected cells from ceramide-induced, nonapoptotic death consistent with the idea that severe bioenergetic stress was responsible. Taken together, these studies suggest that the cellular metabolic state is an important arbiter of the cellular response to ceramide. In fact, increasing nutrient demand by incubating cells in high levels of growth factor sensitized cells to ceramide. On the other hand, gradually adapting cells to tolerate low levels of extracellular nutrients completely blocked ceramide-induced death. In sum, these results support a model where ceramide kills cells by inducing intracellular nutrient limitation subsequent to nutrient transporter downregulation.
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http://dx.doi.org/10.1073/pnas.0802781105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2582319PMC
November 2008

Transfection protocol for antisense oligonucleotides affects uniformity of transfection in cell culture and efficiency of mRNA target reduction.

Oligonucleotides 2005 ;15(1):12-22

Isis Pharmaceuticals, Carlsbad, CA 92008, USA.

In an effort to optimize the transfection of cell lines with antisense oligonucleotides, we examined cellular accumulation of a labeled oligonucleotide by flow cytometry. We were surprised to observe that a routinely used transfection protocol, a fixed lipid/oligonucleotide ratio, resulted in variable transfection efficiency depending on the concentration of oligonucleotide used. A significant population of cells, especially at lower doses of oligonucleotide and cationic lipid, were untransfected. We investigated lipid/oligonucleotide ratios, different lipid preparations, and different cell types and found that these variables did not alter the percentage of cells transfected at these lower doses of oligonucleotide. However, when lipid-oligonucleotide complexes were formed at the high dose and then diluted into a solution of lipid or a complex of lipid and unlabeled, negative control oligonucleotide, a constant percentage of cells was transfected. Under these conditions, mRNA target reduction dose-response curves were also shifted to lower doses. We hypothesize that poor transfection observed at a low concentration of lipid-oligonucleotide complex when diluted in medium is due to loss of active complexes, either by adsorption to the substrate or by changes in physical characteristics of complexes. By maintaining a constant lipid concentration, more consistent transfection was achieved.
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http://dx.doi.org/10.1089/oli.2005.15.12DOI Listing
July 2005
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