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1.  Chlamydia trachomatis-induced alterations in the host cell proteome are required for intracellular growth 
Cell host & microbe  2014;15(1):113-124.
Intracellular pathogens directly alter host cells in order to replicate and survive. While infection-induced changes in host transcription can be readily assessed, post-transcriptional alterations are more difficult to catalog. We applied the global protein stability (GPS) platform, which assesses protein stability based on relative changes in an adjoining fluorescent tag, to identify changes in the host proteome following infection with the obligate intracellular bacteria Chlamydia trachomatis. Our results indicate that C. trachomatis profoundly remodels the host proteome independently of changes in transcription. Additionally, C. trachomatis replication depends on a subset of altered proteins, such as Pin1 and Men1, which regulate the host transcription factor AP-1 controlling host inflammation, stress, and cell survival. Furthermore, AP-1-dependent transcription is activated during infection, and required for efficient Chlamydia growth. In summary, this experimental approach revealed that C. trachomatis broadly alters host proteins and can be applied to examine host-pathogen interactions and develop host-based therapeutics.
PMCID: PMC3932326  PMID: 24439903
2.  A sumoylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis 
Science (New York, N.Y.)  2011;335(6066):348-353.
Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we employed a genome-wide RNAi screen for Myc-synthetic-lethal genes and uncovered a role for the SUMO-activating-enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation of SAE2 leads to mitotic catastrophe and cell death selectively upon Myc hyper-activation. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent-Myc-switchers (SMS genes) is required for mitotic spindle function and to support the Myc oncogenic program. SAE2 is required for Myc-dependent tumor growth, and patient survival significantly correlates with SAE1/SAE2 levels in Myc-high tumors. These studies reveal a mitotic vulnerability of Myc-driven cancers, demonstrate that inhibiting sumoylation impairs Myc-dependent tumorigenesis, and suggest inhibiting SUMOylation may have therapeutic benefits for patients with Myc-driven cancer.
PMCID: PMC4059214  PMID: 22157079
3.  Ontogeny of Recognition Specificity and Functionality for the Broadly Neutralizing Anti-HIV Antibody 4E10 
PLoS Pathogens  2014;10(9):e1004403.
The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies.
Author Summary
4E10 is an antibody that neutralizes a broad variety of HIV strains. However, 4E10 is uncommon in infected patients and has not been successfully elicited by any vaccine approach attempted. Hurdles to re-eliciting 4E10 include the accumulation of many mutations during development, demonstrated reactivity against host proteins and significant structural flexibility. Lacking a confirmed sequence for precursors of 4E10, we studied the recognition and biophysical properties of an ensemble of eight of the likeliest candidates. Surprisingly, 4E10 gained host reactivity and structural flexibility, but lost stability during development when compared to candidate precursors. However, recognition of HIV was remarkably conserved, despite a considerable improvement in binding. Since these results run counter to those expected from conventional vaccination protocols, 4E10 is unlikely to serve as the basis of a useful HIV vaccine.
PMCID: PMC4177983  PMID: 25254371
4.  Application of a synthetic human proteome to autoantigen discovery through PhIP-Seq 
Nature biotechnology  2011;29(6):535-541.
In this study, we improve on current autoantigen discovery approaches by creating a synthetic representation of the complete human proteome, the T7 “peptidome” phage display library (T7-Pep), and use it to profile the autoantibody repertoires of individual patients. We provide methods for 1) designing and cloning large libraries of DNA microarray-derived oligonucleotides encoding peptides for display on bacteriophage, and 2) analysis of the peptide libraries using high throughput DNA sequencing. We applied phage immunoprecipitation sequencing (PhIP-Seq) to identify both known and novel autoantibodies contained in the spinal fluid of three patients with paraneoplastic neurological syndromes. We also show how our approach can be used more generally to identify peptide-protein interactions and point toward ways in which this technology will be further developed in the future. We envision that PhIP-Seq can become an important new tool in autoantibody analysis, as well as proteomic research in general.
PMCID: PMC4169279  PMID: 21602805
Synthetic biology; proteomics; phage display; humoral autoimmunity; paraneoplastic neurological disorder; protein-protein interactions
5.  Protein interaction mapping with ribosome-displayed using PLATO ORF libraries 
Nature protocols  2013;9(1):90-103.
Identifying physical interactions between proteins and other molecules is a critical aspect of biological analysis. Here we describe PLATO, an in vitro method for mapping such interactions by affinity enrichment of a library of full-length open reading frames displayed on ribosomes, followed by massively parallel analysis using DNA sequencing. We demonstrate the broad utility of the method by identifying known and new interacting partners of LYN kinase, patient autoantibodies and the small molecules gefitinib and dasatinib.
PMCID: PMC4129458  PMID: 24336473
6.  PhIP-Seq characterization of autoantibodies from patients with multiple sclerosis, type 1 diabetes and rheumatoid arthritis 
Journal of autoimmunity  2013;43:1-9.
Autoimmune disease results from a loss of tolerance to self-antigens in genetically susceptible individuals. Completely understanding this process requires that targeted antigens be identified, and so a number of techniques have been developed to determine immune receptor specificities. We previously reported the construction of a phage-displayed synthetic human peptidome and a proof-of-principle analysis of antibodies from three patients with neurological autoimmunity. Here we present data from a large-scale screen of 298 independent antibody repertoires, including those from 73 healthy sera, using phage immunoprecipitation sequencing. The resulting database of peptide-antibody interactions characterizes each individual’s unique autoantibody fingerprint, and includes specificities found to occur frequently in the general population as well as those associated with disease. Screening type 1 diabetes (T1D) patients revealed a prematurely polyautoreactive phenotype compared with their matched controls. A collection of cerebrospinal fluids and sera from 63 multiple sclerosis patients uncovered novel, as well as previously reported antibody-peptide interactions. Finally, a screen of synovial fluids and sera from 64 rheumatoid arthritis patients revealed novel disease-associated antibody specificities that were independent of seropositivity status. This work demonstrates the utility of performing PhIP-Seq screens on large numbers of individuals and is another step toward defining the full complement of autoimmunoreactivities in health and disease.
PMCID: PMC3677742  PMID: 23497938
autoantigen discovery; high throughput screening; PhIP-Seq; proteomics
7.  When noise makes music: HIV reactivation with transcriptional noise enhancers 
Genome Medicine  2014;6(7):55.
Reactivating latent HIV is key to depleting the virus reservoir in AIDS patients. A recent paper has described the rationale for and discovery of a new class of drugs - transcriptional noise enhancers - that can synergize with conventional transcription activators to more effectively reactivate latently infected T cells. As well as describing a promising new strategy in the bid to find a cure for AIDS, this study more broadly highlights the utility of exploring drug combinations in treatment of human disease.
PMCID: PMC4179226  PMID: 25276233
8.  Protein interaction discovery using parallel analysis of translated ORFs (PLATO) 
Nature biotechnology  2013;31(4):331-334.
Identifying physical interactions between proteins and other molecules is a critical aspect of biological analysis. Here we describe PLATO, an in vitro method for mapping such interactions by affinity enrichment of a library of full-length open reading frames displayed on ribosomes, followed by massively parallel analysis using DNA sequencing. We demonstrate the broad utility of the method for human proteins by identifying known and previously unidentified interacting partners of LYN kinase, patient autoantibodies, and the small-molecules gefitinib and dasatinib.
PMCID: PMC4110636  PMID: 23503679
9.  Recurrent Hemizygous Deletions in Cancers May Optimize Proliferative Potential 
Science (New York, N.Y.)  2012;337(6090):104-109.
Tumors exhibit numerous recurrent hemizygous focal deletions that contain no known tumor suppressors and are poorly understood. To investigate whether these regions contribute to tumorigenesis, we searched genetically for genes with cancer-relevant properties within these hemizygous deletions. We identified STOP and GO genes, which negatively and positively regulate proliferation, respectively. STOP genes include many known tumor suppressors, whereas GO genes are enriched for essential genes. Analysis of their chromosomal distribution revealed that recurring deletions preferentially over represent STOP genes and under represent GO genes. We propose a hypothesis called the cancer gene island model whereby gene islands encompassing high densities of STOP genes and low densities of GO genes are hemizygously deleted to maximize proliferative fitness through cumulative haploinsufficiencies. Because hundreds to thousands of genes are hemizygously deleted per tumor, this mechanism may help drive tumorigenesis across many cancer types.
PMCID: PMC4027969  PMID: 22628553
10.  Cumulative Haploinsufficiency and Triplosensitivity Drive Aneuploidy Patterns to Shape the Cancer Genome 
Cell  2013;155(4):948-962.
Aneuploidy has been recognized as a hallmark of cancer for over 100 years, yet no general theory to explain the recurring patterns of aneuploidy in cancer has emerged. Here we develop Tumor Suppressor and Oncogene (TUSON) Explorer, a computational method that analyzes the patterns of mutational signatures in tumors and predicts the likelihood that any individual gene functions as a tumor suppressor (TSG) or oncogene (OG). By analyzing >8200 tumor-normal pairs we provide statistical evidence suggesting many more genes possess cancer driver properties than anticipated, forming a continuum of oncogenic potential. Integrating our driver predictions with information on somatic copy number alterations, we find that the distribution and the potency of TSGs (STOP genes), OGs and essential genes (GO genes) on chromosomes can predict the complex patterns of aneuploidy and copy number variation characteristic of cancer genomes. We propose that the cancer genome is shaped through a process of cumulative haploinsufficiency and triplosensitivity.
PMCID: PMC3891052  PMID: 24183448
11.  Abraxas and Rap80 form a novel BRCA1 protein complex required for the DNA damage response 
Science (New York, N.Y.)  2007;316(5828):1194-1198.
The BRCT repeats of BRCA1 are essential for tumor suppression. Phospho-peptide affinity proteomic analysis identified a novel protein, Abraxas, that directly binds the BRCA1 BRCT repeats through a phospho-SXXF motif. Abraxas binds BRCA1 mutually exclusively with BACH1 and CTIP, forming a third Brca1 complex. Abraxas recruits the ubiquitin-interacting motif (UIM) containing protein Rap80 to BRCA1. Both Abraxas and Rap80 are required for DNA damage resistance, G2/M checkpoint control and DNA repair. Rap80 is required for a subset of Brca1-foci formation in response to IR and the UIM domains alone are capable of foci formation. The Rap80/Abraxas complex may help recruit Brca1 to DNA damage sites in part through recognition of ubiquitinated proteins.
PMCID: PMC3573690  PMID: 17525340
12.  A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA damage response 
Nature cell biology  2012;14(3):318-328.
Repair of DNA double strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We performed a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA damage response and pre-mRNA processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions via multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of Rad51 is a common source of RNAi false-positives, sounding a cautionary note for siRNA screens and RNAi-based studies of HR.
PMCID: PMC3290715  PMID: 22344029
13.  The CD225 Domain of IFITM3 Is Required for both IFITM Protein Association and Inhibition of Influenza A Virus and Dengue Virus Replication 
Journal of Virology  2013;87(14):7837-7852.
The interferon-induced transmembrane protein 3 (IFITM3) gene is an interferon-stimulated gene that inhibits the replication of multiple pathogenic viruses in vitro and in vivo. IFITM3 is a member of a large protein superfamily, whose members share a functionally undefined area of high amino acid conservation, the CD225 domain. We performed mutational analyses of IFITM3 and identified multiple residues within the CD225 domain, consisting of the first intramembrane domain (intramembrane domain 1 [IM1]) and a conserved intracellular loop (CIL), that are required for restriction of both influenza A virus (IAV) and dengue virus (DENV) infection in vitro. Two phenylalanines within IM1 (F75 and F78) also mediate a physical association between IFITM proteins, and the loss of this interaction decreases IFITM3-mediated restriction. By extension, similar IM1-mediated associations may contribute to the functions of additional members of the CD225 domain family. IFITM3's distal N-terminal domain is also needed for full antiviral activity, including a tyrosine (Y20), whose alteration results in mislocalization of a portion of IFITM3 to the cell periphery and surface. Comparative analyses demonstrate that similar molecular determinants are needed for IFITM3's restriction of both IAV and DENV. However, a portion of the CIL including Y99 and R87 is preferentially needed for inhibition of the orthomyxovirus. Several IFITM3 proteins engineered with rare single-nucleotide polymorphisms demonstrated reduced expression or mislocalization, and these events were associated with enhanced viral replication in vitro, suggesting that possessing such alleles may impact an individual's risk for viral infection. On the basis of this and other data, we propose a model for IFITM3-mediated restriction.
PMCID: PMC3700195  PMID: 23658454
14.  Amphotericin B Increases Influenza A Virus Infection by Preventing IFITM3-Mediated Restriction 
Cell Reports  2013;5(4):895-908.
The IFITMs inhibit influenza A virus (IAV) replication in vitro and in vivo. Here, we establish that the antimycotic heptaen, amphotericin B (AmphoB), prevents IFITM3-mediated restriction of IAV, thereby increasing viral replication. Consistent with its neutralization of IFITM3, a clinical preparation of AmphoB, AmBisome, reduces the majority of interferon’s protective effect against IAV in vitro. Mechanistic studies reveal that IFITM1 decreases host-membrane fluidity, suggesting both a possible mechanism for IFITM-mediated restriction and its negation by AmphoB. Notably, we reveal that mice treated with AmBisome succumbed to a normally mild IAV infection, similar to animals deficient in Ifitm3. Therefore, patients receiving antifungal therapy with clinical preparations of AmphoB may be functionally immunocompromised and thus more vulnerable to influenza, as well as other IFITM3-restricted viral infections.
Graphical Abstract
•Amphotericin B or AmBisome prevents IFITM3-mediated restriction of IAV•AmBisome overcomes the majority of IFN’s antiviral effects in vitro•IFITM1 decreases membrane fluidity and inhibits membrane fusion•AmBisome increases the morbidity and mortality of influenza
IFITM3 is a ubiquitously expressed antiviral protein that inhibits multiple human pathogenic viruses, including influenza A virus (IAV). Brass and colleagues now show that a widely used antifungal therapy, AmBisome, prevents IFITM3 from blocking IAV replication and that mice given AmBisome succumb to a normally mild influenza virus infection. Therefore, patients receiving antifungal therapy with AmBisome may be functionally immunocompromised and thus more vulnerable to influenza as well as other IFITM3-restricted viral infections.
PMCID: PMC3898084  PMID: 24268777
15.  Global Identification of Modular Cullin-Ring Ligase Substrates 
Cell  2011;147(2):459-474.
Cullin Ring Ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. One substrate, NUSAP1, we demonstrate is an SCFCyclin F substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology, and provides a set of proteins likely to be key indicators of cellular physiology.
PMCID: PMC3226719  PMID: 21963094
16.  Dif1 is a DNA Damage Regulated Facilitator of Nuclear Import for Ribonucleotide Reductase 
Molecular cell  2008;32(1):70-80.
The control of dNTP concentrations is critical to the fidelity of DNA synthesis and repair. One level of regulation is through subcellular localization of ribonucleotide reductase. In S. cerevisiae, the small subunit, Rnr2-Rnr4, is nuclear while the large subunit, Rnr1, is cytoplasmic. In response to S-phase or DNA-damage, Rnr2-Rnr4 enters the cytoplasm to bind Rnr1, forming an active complex. We previously reported that Wtm1 anchors Rnr2-Rnr4 in the nucleus. Here, we identify DIF1 which regulates localization of Rnr2-Rnr4. Dif1 binds directly to the Rnr2-Rnr4 complex through a conserved Hug domain to drive nuclear import. Dif1 is both cell cycle- and DNA-damage regulated, the latter through the Mec1-Dun1 pathway. In response to DNA damage, Dun1 directly phosphorylates Dif1 to both inactivate and degrade Dif1, allowing Rnr2-Rnr4 to become cytoplasmic. We propose that Rnr2-Rnr4 nuclear localization is achieved by a dynamic combination of Wtm1-mediated nuclear retention to limit export, coupled with regulated nuclear import through Dif1.
PMCID: PMC3245869  PMID: 18851834
17.  Characterization of Torin2, an ATP-competitive inhibitor of mTOR, ATM and ATR 
Cancer research  2013;73(8):2574-2586.
mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here we report the characterization of Torin2, a second generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC50 of 250 pM with approximately 800-fold selectivity for cellular mTOR versus PI3K. Torin2 also exhibited potent biochemical and cellular activity against PIKK family kinases including ATM (EC50 28 nM), ATR (EC50 35 nM) and DNA-PK (EC50 118 nM) (PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with MEK inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncological settings where mTOR signaling has a pathogenic role.
PMCID: PMC3760004  PMID: 23436801
mTOR; ATM; ATR; lung cancer; kinase inhibitors
18.  Autoreactivity and Exceptional CDR Plasticity (but Not Unusual Polyspecificity) Hinder Elicitation of the Anti-HIV Antibody 4E10 
PLoS Pathogens  2013;9(9):e1003639.
The broadly-neutralizing anti-HIV antibody 4E10 recognizes an epitope in the membrane-proximal external region of the HIV envelope protein gp41. Previous attempts to elicit 4E10 by vaccination with envelope-derived or reverse-engineered immunogens have failed. It was presumed that the ontogeny of 4E10-equivalent responses was blocked by inherent autoreactivity and exceptional polyreactivity. We generated 4E10 heavy-chain knock-in mice, which displayed significant B cell dysregulation, consistent with recognition of autoantigen/s by 4E10 and the presumption that tolerance mechanisms may hinder the elicitation of 4E10 or 4E10-equivalent responses. Previously proposed candidate 4E10 autoantigens include the mitochondrial lipid cardiolipin and a nuclear splicing factor, 3B3. However, using carefully-controlled assays, 4E10 bound only weakly to cardiolipin-containing liposomes, but also bound negatively-charged, non-cardiolipin-containing liposomes comparably poorly. 4E10/liposome binding was predominantly mediated by electrostatic interactions rather than presumed hydrophobic interactions. The crystal structure of 4E10 free of bound ligands showed a dramatic restructuring of the combining site, occluding the HIV epitope binding site and revealing profound flexibility, but creating an electropositive pocket consistent with non-specific binding of phospholipid headgroups. These results strongly suggested that antigens other than cardiolipin mediate 4E10 autoreactivity. Using a synthetic peptide library spanning the human proteome, we determined that 4E10 displays limited and focused, but unexceptional, polyspecificity. We also identified a novel autoepitope shared by three ER-resident inositol trisphosphate receptors, validated through binding studies and immunohistochemistry. Tissue staining with 4E10 demonstrated reactivity consistent with the type 1 inositol trisphosphate receptor as the most likely candidate autoantigen, but is inconsistent with splicing factor 3B3. These results demonstrate that 4E10 recognition of liposomes competes with MPER recognition and that HIV antigen and autoepitope recognition may be distinct enough to permit eliciting 4E10-like antibodies, evading autoimmunity through directed engineering. However, 4E10 combining site flexibility, exceptional for a highly-matured antibody, may preclude eliciting 4E10 by conventional immunization strategies.
Author Summary
4E10 is an example of an anti-HIV, broadly neutralizing antibody that is uncommon in infected patients and has not been successfully elicited by any vaccine approach attempted. 4E10 has been proposed to neutralize HIV through a mechanism that requires broad recognition of other antigens, including membrane phospholipids. Such a mechanism would also block the generation of 4E10 during B cell development, confounding vaccination strategies. Analysis of B cell development in 4E10 heavy-chain knock-in mice confirmed that 4E10 does recognize self-antigens. However, a previously proposed autoantigen candidate, the mitochondrial lipid cardiolipin, was not consistent with binding studies which showed that while 4E10 does bind liposomes containing cardiolipin, it does so only weakly and nonspecifically, also binding liposomes without cardiolipin. Using a synthetic human peptidome, 4E10 was shown to be polyreactive, binding peptides from various proteins, but only in a limited manner. Three of the top five hits are from types 1, 2 and 3 inositol trisphosphate receptors, with high scoring peptides sharing a conserved sequence motif. Validation of the top hits was performed by binding analyses and staining of tissue sections, which combined to identify the type 1 inositol trisphosphate receptor as the most likely 4E10 physiological autoantigen.
PMCID: PMC3784475  PMID: 24086134
19.  Systematic Identification of Synergistic Drug Pairs Targeting HIV 
Nature biotechnology  2012;30(11):1125-1130.
The systematic identification of effective drug combinations has been hindered by the unavailability of methods that can explore the large combinatorial search space of drug interactions. Here we present a multiplex screening method named MuSIC (Multiplex Screening for Interacting Compounds), which expedites the comprehensive assessment of pair-wise compound interactions. We examined ~500,000 drug pairs from 1000 FDA-approved or clinically tested drugs and identified drugs that synergize to inhibit HIV replication. Our analysis reveals an enrichment of anti-inflammatory drugs in drug combinations that synergize against HIV, indicating HIV benefits from inflammation that accompanies its infection. Multiple drug pairs identified in this study, including glucocorticoid and nitazoxanide, synergize by targeting different steps of the HIV life cycle. As inflammation accompanies HIV infection, our findings indicate that inhibiting inflammation could curb HIV propagation. MuSIC can be applied to a wide variety of disease-relevant screens to facilitate efficient identification of compound combinations.
PMCID: PMC3494743  PMID: 23064238
Combination therapy; FDA-approved drug library; HIV
20.  A role for PVRL4-driven cell–cell interactions in tumorigenesis 
eLife  2013;2:e00358.
During all stages of tumor progression, cancer cells are subjected to inappropriate extracellular matrix environments and must undergo adaptive changes in order to evade growth constraints associated with the loss of matrix attachment. A gain of function screen for genes that enable proliferation independently of matrix anchorage identified a cell adhesion molecule PVRL4 (poliovirus-receptor-like 4), also known as Nectin-4. PVRL4 promotes anchorage-independence by driving cell-to-cell attachment and matrix-independent integrin β4/SHP-2/c-Src activation. Solid tumors frequently have copy number gains of the PVRL4 locus and some have focal amplifications. We demonstrate that the transformation of breast cancer cells is dependent on PVRL4. Furthermore, growth of orthotopically implanted tumors in vivo is inhibited by blocking PVRL4-driven cell-to-cell attachment with monoclonal antibodies, demonstrating a novel strategy for targeted therapy of cancer.
eLife digest
Epithelial tissue is one of the four major types of tissue found in animals, and is the only type of tissue that is able to form and maintain layers of cells that are just one cell thick. These layers provide inner linings to various cavities and hollow organs throughout the body—including the lungs and glandular organs such as mammary glands. A single-cell layer of epithelium is separated from the tissues beneath it by a supporting substance called the extracellular matrix. The individual cells within a single-cell layer are physically attached to the matrix, and when displaced from it, they promptly undergo programmed cell death. This mechanism preserves the single-cell layer pattern throughout the body and prevents epithelial cells from growing in inappropriate locations.
It is estimated that up to 90% of cancers in humans originate in epithelial tissue, and the cells within such tumors are known to survive and divide even when they are no longer attached to the extracellular matrix. Understanding how cancerous cells gain this ability may lead to new approaches to stopping tumor cells from dividing and colonizing tissues around the body.
To address this problem, Pavlova et al. explored which genes enable epithelial cells from the human mammary gland to grow without being attached to the extracellular matrix. They found that the gene that codes for a protein called poliovirus receptor-like 4 (PVRL4) allows attachment-free cell growth and also makes cells cluster together once detached from the matrix.
Normally, the PVRL4 gene is not active in breast epithelial cells, but its activity is detected in many breast, lung, and ovarian tumors. Moreover, cancerous cells tend to cluster together when they are detached from the extracellular matrix. This behavior is particularly evident in the cells that divide aggressively to form tumors that subsequently migrate and colonize other tissues around the body. When Pavlova et al. used genetic techniques to silence PVRL4 in cells from breast tumors, they found that it reduced the formation of clusters by the cancer cells and also reduced their ability to grow in the absence of attachment.
Pavlova et al. also showed that interactions between the PVRL4 in one cell and a related protein called PVRL1 in a neighboring cell were responsible for holding the cells together in clusters. Moreover, PVRL4 triggers a form of signaling between the cells called integrin β4 signaling that allows them to survive without being anchored to the extracellular matrix.
Finally, Pavlova et al. found that injecting anti-PVRL4 antibodies (mouse proteins that attach to PVRL4 and prevent the formation of clusters) slows down the growth of breast tumors in mice. These findings suggest that inhibiting PVRL4 action with antibodies can be used as a new approach to the treatment of breast, lung, and ovarian cancers in humans.
PMCID: PMC3641523  PMID: 23682311
anchorage-independence; transformation; genetic screen; Human; Mouse
21.  The ZRANB3 translocase associates with poly-ubiquitinated PCNA to promote fork restart and limit recombination after replication stress 
Molecular cell  2012;47(3):396-409.
Completion of DNA replication after replication stress depends on PCNA, which undergoes mono-ubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is poly-ubiquitinated to promote recombination dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by poly-ubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.
PMCID: PMC3613862  PMID: 22704558
22.  SCFβTRCPControls Oncogenic Transformation and Neural Differentiation Through REST Degradation 
Nature  2008;452(7185):370-374.
The transcription factor REST/NSRF (RE1-Silencing Transcription Factor) is a master repressor of neuronal gene expression and neuronal programs in non-neuronal lineages1−3. Recently, REST was identified as a human tumor suppressor in epithelial tissues4, suggesting that REST regulation may have important physiologic and pathologic consequences. However, the pathways controlling REST have yet to be elucidated. Here, we demonstrate that REST is regulated by ubiquitin-mediated proteolysis, and use an RNAi screen to identify SCFβTRCP as an E3 ubiquitin ligase responsible for REST degradation. βTRCP binds and ubiquitinates REST and controls its stability through a conserved phosphodegron. During neural differentiation REST is degraded in a βTRCP-dependent manner. βTRCP is required for proper neural differentiation only in the presence of REST, indicating that βTRCP facilitates this process through degradation of REST. Conversely, failure to degrade REST attenuates differentiation. Furthermore, we find that βTRCP overexpression, which is common in human epithelial cancers, causes oncogenic transformation of human mammary epithelial cells and this pathogenic function requires REST degradation. Thus, REST is a key target in βTRCP-driven transformation and the βTRCP-REST axis is a new regulatory pathway controlling neurogenesis.
PMCID: PMC2688689  PMID: 18354483
23.  Reactivation of Latent HIV-1 by Inhibition of BRD4 
Cell reports  2012;2(4):807-816.
HIV-1 depends on many host factors for propagation. Other host factors, however, antagonize HIV-1 and may have profound effects on viral activation. Curing HIV-1 requires the reduction of latent viral reservoirs that remain in the face of antiretroviral therapy (ART). Using orthologous genetic screens, we identified bromodomain containing 4 (BRD4) as a negative regulator of HIV-1 replication. Antagonism of BRD4, via RNA interference or with a small molecule inhibitor, JQ1, both increased proviral transcriptional elongation and alleviated HIV-1 latency in cell line models. In multiple instances, JQ1 when used in combination with the NF-κB activators, Prostratin or PHA, enhanced the in vitro reactivation of latent HIV-1 in primary human T cells. These data are consistent with a model wherein BRD4 competes with the virus for HIV-1 dependency factors (HDFs) and suggests that combinatorial therapies that activate HDFs and antagonize HIV-1 competitive factors may be useful for curing HIV-1 infection.
PMCID: PMC3523124  PMID: 23041316
24.  Identification of the Fanconi anemia (FANC) I protein, a monoubiquitinated FANCD2 paralog required for crosslink repair 
Cell  2007;129(2):289-301.
Fanconi anemia (FA) is a developmental and cancer-predisposition syndrome caused by mutations in genes controlling crosslink repair. Several FA proteins form a ubiquitin ligase that controls monoubiquitination of the FANCD2 protein in an ATR-dependent manner. Here we describe a new FA protein FANCI, identified as a ATM/ATR kinase substrate required for resistance to mitomycin C. FANCI shares sequence similarity with FANCD2, likely evolving from a common ancestral gene. The FANCI protein associates with FANCD2 and together, as the FANCI-FANCD2 (ID) complex, load onto chromatin in response to DNA damage. Like FANCD2, FANCI is monoubiquitinated and unexpectedly, ubiquitination of each protein is important for the maintenance of ubiquitin on the other, indicating the existence of a dual ubiquitin locking mechanism required for the ability of the ID complex to function. Mutation in FANCI is responsible for loss of a functional FA pathway in a patient with Fanconi anemia complementation group I.
PMCID: PMC2175179  PMID: 17412408
Fanconi anemia; FANCI; FANCD2; ubiquitination; phosphorylation; DNA repair; Mitomycin C
25.  BRCA1 as tumor suppressor: lord without its RING? 
BRCA1 is a tumor suppressor with critical roles in the maintenance of genomic stability. It encodes a large protein with an amino-terminal RING domain that possesses ubiquitin-ligase activity. Given the occurrence of numerous cancer-causing mutations within its RING domain, investigators have long suspected that BRCA1's ubiquitin ligase is important for its tumor suppression and DNA repair activities. Using genetically engineered mouse models, two recent studies shed light on this age-old hypothesis.
PMCID: PMC3446363  PMID: 22494569

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