PubMed Health⌕ Search

Biomedical subjects

Judy Lieberman

Publications and source records attributed to Judy Lieberman.

At least 19 recordsLinked to original sources
PubMed2026

Functional chimeric mRNAs encode proteins in mammalian immunity.

Individual mammalian mRNAs and proteins are typically believed to originate from single genomic loci, with isoform diversity arising through cis-splicing of pre-mRNA. Whether mRNA from distant genes can undergo trans-splicing to generate functionally relevant chimeric transcripts has remained unclear. Here we develop a pipeline combining long-read direct RNA sequencing with non-targeted and targeted validation to identify chimeric transcripts in macrophages. Chromatin conformation capture studies reveal that inflammation induces interchromosomal DNA interactions, positioning parent genes proximally to facilitate the formation of chimeric mRNA. Notably, we identify a protein-coding chimeric mRNA representing a fusion between the pore-forming protein gasdermin D (GSDMD)1,2 and a C-terminal domain translated out of frame from Tmem106a (Gsdmd-Tmem106a) in mice. We show that inflammasome priming upregulates Gsdmd-Tmem106a, with the protein localizing to the plasma membrane. After activation of the inflammasome, GSDMD-TMEM106A directly interacts with canonical GSDMD N termini to accelerate and enhance pore formation and IL-1β release. Finally, we show that GSDMD-TMEM106A balances host defence and immunopathology in vivo: its loss protects against lethal sepsis but compromises antibacterial defence, whereas overexpression enhances host protection while increasing sepsis lethality. We establish that protein-coding chimeric mRNAs formed by regulated transcript fusion events are operative during inflammation and immunity.

Journal Article↗
PubMed2006

Knocking down disease with siRNAs.

Knocking down expression of disease-related genes using small interfering RNAs (siRNAs) has potential for treating a variety of illnesses. This Essay will examine the opportunities for harnessing RNA interference (RNAi) for therapy, as well as the obstacles and possible ways to circumvent them.

Animals↗
PubMed2006

The exonuclease TREX1 is in the SET complex and acts in concert with NM23-H1 to degrade DNA during granzyme A-mediated cell death.

Granzyme A (GzmA) activates a caspase-independent cell death pathway with morphological features of apoptosis. Single-stranded DNA damage is initiated when the endonuclease NM23-H1 becomes activated to nick DNA after granzyme A cleaves its inhibitor, SET. SET and NM23-H1 reside in an endoplasmic reticulum-associated complex (the SET complex) that translocates to the nucleus in response to superoxide generation by granzyme A. We now find the 3'-to-5' exonuclease TREX1, but not its close homolog TREX2, in the SET complex. TREX1 binds to SET and colocalizes and translocates with the SET complex. NM23-H1 and TREX1 work in concert to degrade DNA. Silencing NM23-H1 or TREX1 inhibits DNA damage and death of cells treated with perforin (PFN) and granzyme A, but not of cells treated with perforin and granzyme B (GzmB). After granzyme A activates NM23-H1 to make single-stranded nicks, TREX1 removes nucleotides from the nicked 3' end to reduce the possibility of repair by rejoining the nicked ends.

Cell Death↗
PubMed2006

Determinants of specific RNA interference-mediated silencing of human beta-globin alleles differing by a single nucleotide polymorphism.

A single nucleotide polymorphism (SNP) in the sickle beta-globin gene (beta(S)) leads to sickle cell anemia. Sickling increases sharply with deoxy sickle Hb concentration and decreases with increasing fetal gamma-globin concentration. Measures that decrease sickle Hb concentration should have an antisickling effect. RNA interference (RNAi) uses small interfering (si)RNAs for sequence-specific gene silencing. A beta(S) siRNA with position 10 of the guide strand designed to align with the targeted beta(S) SNP specifically silences beta(S) gene expression without affecting the expression of the gamma-globin or normal beta-globin (beta(A)) genes. Silencing is increased by altering the 5' end of the siRNA antisense (guide) strand to enhance its binding to the RNA-induced silencing complex (RISC). Specific beta(S) silencing was demonstrated by using a luciferase reporter and full-length beta(S) cDNA transfected into HeLa cells and mouse erythroleukemia cells, where it was expressed in the context of the endogenous beta-globin gene promoter and the locus control region enhancers. When this strategy was used to target beta(E), silencing was not limited to the mutant gene but also targeted the normal beta(A) gene. siRNAs, mismatched with their target at position 10, guided mRNA cleavage in all cases except when two bulky purines were aligned. The specific silencing of the beta(S)-globin gene, as compared with beta(E), as well as studies of silencing SNP mutants in other diseases, indicates that siRNAs developed to target a disease-causing SNP will be specific if the mutant residue is a pyrimidine and the normal residue is a purine.

Base Sequence↗
PubMed2006

Granzyme A, which causes single-stranded DNA damage, targets the double-strand break repair protein Ku70.

Granzyme A (GzmA) induces caspase-independent cell death with morphological features of apoptosis. Here, we show that GzmA at nanomolar concentrations cleaves Ku70, a key double-strand break repair (DSBR) protein, in target cells. Ku70 is cut after Arg(301), disrupting Ku complex binding to DNA. Cleaving Ku70 facilitates GzmA-mediated cell death, as silencing Ku70 by RNA interference increases DNA damage and cell death by GzmB cluster-deficient cytotoxic T lymphocytes or by GzmA and perforin, whereas Ku70 overexpression has the opposite effect. Ku70 has two known antiapoptotic effects-facilitating DSBR and sequestering bax to prevent its translocation to mitochondria. However, GzmA triggers single-stranded, not double-stranded, DNA damage, and GzmA-induced cell death does not involve bax. Therefore, Ku70 has other antiapoptotic functions in GzmA-induced cell death, which are blocked when GzmA proteolyses Ku70.

Animals↗
PubMed2006

Running interference: prospects and obstacles to using small interfering RNAs as small molecule drugs.

RNA interference (RNAi) is a well-conserved, ubiquitous, endogenous mechanism that uses small noncoding RNAs to silence gene expression. The endogenous small RNAs, called microRNAs, are processed from hairpin precursors and regulate important genes involved in cell death, differentiation, and development. RNAi also protects the genome from invading genetic elements, encoded by transposons and viruses. When small double-stranded RNAs, called small interfering (si)RNAs, are introduced into cells, they bind to the endogenous RNAi machinery to disrupt the expression of mRNAs containing complementary sequences with high specificity. Any disease-causing gene and any cell type or tissue can potentially be targeted. This technique has been rapidly utilized for gene-function analysis and drug-target discovery and validation. Harnessing RNAi also holds great promise for therapy, although introducing siRNAs into cells in vivo remains an important obstacle. Pilot siRNA clinical studies began just three years after the discovery that RNAi works in mammalian cells. This review discusses recent progress and obstacles to using siRNAs as small molecule drugs.

Animals↗
PubMed2006

NFAT5 regulates HIV-1 in primary monocytes via a highly conserved long terminal repeat site.

To replicate, HIV-1 capitalizes on endogenous cellular activation pathways resulting in recruitment of key host transcription factors to its viral enhancer. RNA interference has been a powerful tool for blocking key checkpoints in HIV-1 entry into cells. Here we apply RNA interference to HIV-1 transcription in primary macrophages, a major reservoir of the virus, and specifically target the transcription factor NFAT5 (nuclear factor of activated T cells 5), which is the most evolutionarily divergent NFAT protein. By molecularly cloning and sequencing isolates from multiple viral subtypes, and performing DNase I footprinting, electrophoretic mobility shift, and promoter mutagenesis transfection assays, we demonstrate that NFAT5 functionally interacts with a specific enhancer binding site conserved in HIV-1, HIV-2, and multiple simian immunodeficiency viruses. Using small interfering RNA to ablate expression of endogenous NFAT5 protein, we show that the replication of three major HIV-1 viral subtypes (B, C, and E) is dependent upon NFAT5 in human primary differentiated macrophages. Our results define a novel host factor-viral enhancer interaction that reveals a new regulatory role for NFAT5 and defines a functional DNA motif conserved across HIV-1 subtypes and representative simian immunodeficiency viruses. Inhibition of the NFAT5-LTR interaction may thus present a novel therapeutic target to suppress HIV-1 replication and progression of AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed2005

gamma-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair.

Phosphorylated histone H2AX (gamma-H2AX) forms foci over large chromatin domains surrounding double-stranded DNA breaks (DSB). These foci recruit DSB repair proteins and dissolve during or after repair is completed. How gamma-H2AX is removed from chromatin remains unknown. Here, we show that protein phosphatase 2A (PP2A) is involved in removing gamma-H2AX foci. The PP2A catalytic subunit [PP2A(C)] and gamma-H2AX coimmunoprecipitate and colocalize in DNA damage foci and PP2A dephosphorylates gamma-H2AX in vitro. The recruitment of PP2A(C) to DNA damage foci is H2AX dependent. When PP2A(C) is inhibited or silenced by RNA interference, gamma-H2AX foci persist, DNA repair is inefficient, and cells are hypersensitive to DNA damage. The effect of PP2A on gamma-H2AX levels is independent of ATM, ATR, or DNA-PK activity.

Animals↗
PubMed2005

An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection.

Herpes simplex virus 2 (HSV-2) infection causes significant morbidity and is an important cofactor for the transmission of HIV infection. A microbicide to prevent sexual transmission of HSV-2 would contribute substantially to controlling the spread of HIV and other infections. Because RNA interference (RNAi) provides effective antiviral defence in plants and other organisms, several studies have focused on harnessing RNAi to inhibit viral infection. Here we show that vaginal instillation of small interfering RNAs (siRNAs) targeting HSV-2 protects mice from lethal infection. siRNAs mixed with lipid are efficiently taken up by epithelial and lamina propria cells and silence gene expression in the mouse vagina and ectocervix for at least nine days. Intravaginal application of siRNAs targeting the HSV-2 UL27 and UL29 genes (which encode an envelope glycoprotein and a DNA binding protein, respectively) was well tolerated, did not induce interferon-responsive genes or cause inflammation, and protected mice when administered before and/or after lethal HSV-2 challenge. These results suggest that siRNAs are attractive candidates for the active component of a microbicide designed to prevent viral infection or transmission.

Administration, Intravaginal↗
PubMed2005

A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery.

One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation of the histone variant H2AX at the carboxy-terminal SQE motif to create gammaH2AX-containing nucleosomes. Budding-yeast histone H2A is phosphorylated in a similar manner by the checkpoint kinases Tel1 and Mec1 (ref. 2; orthologous to mammalian ATM and ATR, respectively) over a 50-kilobase region surrounding the DSB. This modification is important for recruiting numerous DSB-recognition and repair factors to the break site, including DNA damage checkpoint proteins, chromatin remodellers and cohesins. Multiple mechanisms for eliminating gammaH2AX as DNA repair completes are possible, including removal by histone exchange followed potentially by degradation, or, alternatively, dephosphorylation. Here we describe a three-protein complex (HTP-C, for histone H2A phosphatase complex) containing the phosphatase Pph3 that regulates the phosphorylation status of gammaH2AX in vivo and efficiently dephosphorylates gammaH2AX in vitro. gammaH2AX is lost from chromatin surrounding a DSB independently of the HTP-C, indicating that the phosphatase targets gammaH2AX after its displacement from DNA. The dephosphorylation of gammaH2AX by the HTP-C is necessary for efficient recovery from the DNA damage checkpoint.

Cell Cycle↗
PubMed2005

Listeria-infected myeloid dendritic cells produce IFN-beta, priming T cell activation.

The intracellular bacterium Listeria monocytogenes infects dendritic cells (DC) and other APCs and induces potent cell-mediated protective immunity. However, heat-killed bacteria fail to do so. This study explored whether DC differentially respond to live and killed Listeria and how this affects T cell activation. To control for bacterial number, a replication-deficient strain, Lmdd, defective in D-alanine biosynthesis, was used. We found that DC internalize both live and heat-killed Lmdd and similarly up-regulate the expression of costimulatory molecules, a necessary step for T cell activation. However, only live Lmdd-infected DC stimulate T cells to express the early activation marker CD69 and enhance T cell activation upon TCR engagement. Infection with live, but not heat-killed, Lmdd induces myeloid DC to secrete copious amounts of IFN-beta, which requires bacterial cytosolic invasion. Exposure to high concentrations of IFN-beta sensitizes naive T cells for Ag-dependent activation.

Animals↗
PubMed2005

Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.

Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.

Animals↗
PubMed2005

Granzyme B binds to target cells mostly by charge and must be added at the same time as perforin to trigger apoptosis.

Perforin (PFN) delivery of granzymes (Gzm) into the target cell at the immunological synapse is the major pathway for inducing apoptosis of virus-infected cells and tumors. A validated model for how PFN delivers Gzm into the cytosol is still lacking. PFN was originally thought to work by forming pores in the target cell plasma membrane that allow Gzm entry. This model was questioned when it was shown that GzmB is endocytosed without PFN. Moreover, apoptosis could be triggered by adding PFN to washed cells that have previously endocytosed GzmB. In this study, we show that GzmB binds to the plasma membrane mostly via nonspecific charge interactions. Washing in saline does not remove bound Gzm. However, if externally bound GzmB is completely removed, subsequent addition of PFN does not release previously endocytosed GzmB and does not trigger apoptosis. Therefore, PFN must be coendocytosed with GzmB to deliver it into the cytosol.

Animals↗
PubMed2005

Lentiviral delivery of short hairpin RNAs protects CD4 T cells from multiple clades and primary isolates of HIV.

Viral heterogeneity is a major hurdle for potential therapeutic use of RNA interference (RNAi) against HIV-1. To determine the extent of RNAi tolerance to mutations, we tested 3 viral target sites with differing propensity for mutations: a highly variable rev sequence, a gag sequence conserved only among clade B isolates, and a vif sequence highly conserved across clades. Lentiviral expression of all 3 shRNAs inhibited replication of the homologous HIV(IIIB) strain. However, they differed in their ability to protect primary CD4 T cells against multiple isolates within and across HIV clades. The least conserved rev sequence inhibited only 2 of 5 clade B isolates. The gag sequence (conserved within clade B) protected 5 of 5 clade B isolates but not other clade viruses with 2 or 3 mutations in the central region. In contrast, the vif sequence, which was conserved across clades except for single mutations at positions 14 and 17, inhibited viruses from 5 different clades. Moreover, siRNAs with introduced mutations at sites of gag sequence polymorphisms showed reduced antiviral activity, whereas mutations in vif siRNA only modestly decreased silencing. Thus, although 1 or 2 mutations at peripheral sites are tolerated, mutations in the central target cleavage region abolish RNAi activity.

Antiviral Agents↗
PubMed2005

The prospect of silencing disease using RNA interference.

The discovery of RNA interference (RNAi), an endogenous cellular gene-silencing mechanism, has already provided a powerful tool for basic science researchers to study gene function. The subsequent finding that RNAi also operates in mammalian cells has generated excitement regarding potential therapeutic applications. In this article we discuss the basic mechanism of RNAi and the therapeutic opportunities and obstacles for harnessing RNAi for therapy of human disease.

Animals↗
PubMed2005

Granzyme A induces caspase-independent mitochondrial damage, a required first step for apoptosis.

Granzyme A (GzmA) triggers cell death with apoptotic features by targeting the endoplasmic reticulum-associated SET complex, which contains the GzmA-activated DNase NM23-H1, its inhibitor SET, and Ape1. The SET complex was postulated to translocate to the nucleus in response to oxidative stress and participate in its repair. Because mitochondrial damage is important in apoptosis, we investigated whether GzmA damages mitochondria. GzmA induces a rapid increase in reactive oxygen species and mitochondrial transmembrane potential loss, but does not cleave bid or cause apoptogenic factor release. The mitochondrial effect is direct, does not require cytosol, and is insensitive to bcl-2 and caspase inhibition. SET complex nuclear translocation, which occurs within minutes of peroxide or GzmA treatment, is dependent on superoxide generation since superoxide scavengers block it. Superoxide scavengers also block apoptosis by CTLs expressing GzmA and/or GzmB. Therefore, mitochondrial damage is an essential first step in killer cell granule-mediated pathways of apoptosis.

Animals↗
PubMed2005

Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis.

Perforin delivers granzymes to induce target-cell apoptosis. At high concentrations, perforin multimerizes in the plasma membrane to form pores. However, whether granzymes enter target cells via membrane pores is uncertain. Here we find that perforin at physiologically relevant concentrations and during cell-mediated lysis creates pores in the target-cell membrane, transiently allowing Ca(2+) and small dyes into the cell. The Ca(2+) flux triggers a wounded membrane-repair response in which internal vesicles, including lysosomes and endosomes, donate their membranes to reseal the damaged membrane. Perforin also triggers the rapid endocytosis of granzymes into large EEA-1-staining vesicles. The restoration of target-cell membrane integrity by triggering the repair response is necessary for target cells subjected to cytotoxic T lymphocyte attack to avoid necrosis and undergo the slower process of programmed cell death. Thus, the target cell actively participates in determining its own fate during cell-mediated death.

Apoptosis↗