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1.
ACS Nano ; 16(8): 11676-11691, 2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-35830573

RESUMO

M13 bacteriophage (phage) are versatile, genetically tunable nanocarriers that have been recently adapted for use as diagnostic and therapeutic platforms. Applying p3 capsid chlorotoxin fusion with the "inho" circular single-stranded DNA (cssDNA) gene packaging system, we produced miniature chlorotoxin inho (CTX-inho) phage particles with a minimum length of 50 nm that can target intracranial orthotopic patient-derived GBM22 glioblastoma tumors in the brains of mice. Systemically administered indocyanine green conjugated CTX-inho phage accumulated in brain tumors, facilitating shortwave infrared detection. Furthermore, we show that our inho phage can carry cssDNA that are transcriptionally active when delivered to GBM22 glioma cells in vitro. The ability to modulate the capsid display, surface loading, phage length, and cssDNA gene content makes the recombinant M13 phage particle an ideal delivery platform.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Camundongos , Animais , Glioblastoma/diagnóstico por imagem , Glioblastoma/genética , Glioblastoma/terapia , Bacteriófago M13 , Capsídeo , Proteínas do Capsídeo , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia
2.
Proc Natl Acad Sci U S A ; 118(5)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33495350

RESUMO

Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan-receptor interactions in living cells.


Assuntos
Antígenos de Diferenciação Mielomonocítica/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma Humano , Lectinas/metabolismo , Polissacarídeos/metabolismo , Motivos de Aminoácidos , Antígenos de Diferenciação Mielomonocítica/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Glicopeptídeos/metabolismo , Humanos , Sinapses Imunológicas/metabolismo , Células Matadoras Naturais/metabolismo , Lectinas/química , Leucossialina/química , Leucossialina/metabolismo , Ligantes , Ligação Proteica
3.
Mol Cell ; 80(4): 578-591.e5, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33171122

RESUMO

Extracellular 2'3'-cyclic-GMP-AMP (cGAMP) is an immunotransmitter exported by diseased cells and imported into host cells to activate the innate immune STING pathway. We previously identified SLC19A1 as a cGAMP importer, but its use across human cell lines is limited. Here, we identify LRRC8A heteromeric channels, better known as volume-regulated anion channels (VRAC), as widely expressed cGAMP transporters. LRRC8A forms complexes with LRRC8C and/or LRRC8E, depending on their expression levels, to transport cGAMP and other 2'3'-cyclic dinucleotides. In contrast, LRRC8D inhibits cGAMP transport. We demonstrate that cGAMP is effluxed or influxed via LRRC8 channels, as dictated by the cGAMP electrochemical gradient. Activation of LRRC8A channels, which can occur under diverse stresses, strongly potentiates cGAMP transport. We identify activator sphingosine 1-phosphate and inhibitor DCPIB as chemical tools to manipulate channel-mediated cGAMP transport. Finally, LRRC8A channels are key cGAMP transporters in resting primary human vasculature cells and universal human cGAMP transporters when activated.


Assuntos
Sistemas CRISPR-Cas , Proteínas de Membrana/metabolismo , Nucleotídeos Cíclicos/metabolismo , Transporte Biológico , Ciclopentanos/farmacologia , Humanos , Indanos/farmacologia , Lisofosfolipídeos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Células U937
4.
Proc Natl Acad Sci U S A ; 117(41): 25293-25301, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32989128

RESUMO

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.


Assuntos
Acetilgalactosamina/metabolismo , Glicoproteínas/metabolismo , Acetilgalactosamina/química , Regulação Enzimológica da Expressão Gênica , Glicosilação , Humanos , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Especificidade por Substrato , Uridina Difosfato N-Acetilgalactosamina/química
5.
Nat Commun ; 10(1): 4063, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492858

RESUMO

Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.


Assuntos
Sistemas CRISPR-Cas , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , RNA Guia de Cinetoplastídeos/genética , Elementos Reguladores de Transcrição/genética , Biologia Computacional/métodos , Epigênese Genética/genética , Epigenômica/métodos , Edição de Genes/métodos , Células HEK293 , Humanos , Células K562
6.
Mol Cell ; 75(2): 372-381.e5, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31126740

RESUMO

2'3'-cyclic-GMP-AMP (cGAMP) is a second messenger that activates the antiviral stimulator of interferon genes (STING) pathway. We recently identified a novel role for cGAMP as a soluble, extracellular immunotransmitter that is produced and secreted by cancer cells. Secreted cGAMP is then sensed by host cells, eliciting an antitumoral immune response. Due to the antitumoral effects of cGAMP, other CDN-based STING agonists are currently under investigation in clinical trials for metastatic solid tumors. However, it is unknown how cGAMP and other CDNs cross the cell membrane to activate intracellular STING. Using a genome-wide CRISPR screen, we identified SLC19A1 as the first known importer of cGAMP and other CDNs, including the investigational new drug 2'3'-bisphosphosphothioate-cyclic-di-AMP (2'3'-CDAS). These discoveries will provide insight into cGAMP's role as an immunotransmitter and aid in the development of more targeted CDN-based cancer therapeutics.


Assuntos
Imunidade Inata/genética , Neoplasias/genética , Nucleotídeos Cíclicos/genética , Proteína Carregadora de Folato Reduzido/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Membrana Celular/genética , Genoma Humano/genética , Humanos , Proteínas de Membrana/genética , Neoplasias/imunologia , Nucleotídeos Cíclicos/imunologia , Transdução de Sinais/genética
7.
Mol Cell ; 67(6): 1037-1048.e6, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28890333

RESUMO

The three-dimensional arrangement of the human genome comprises a complex network of structural and regulatory chromatin loops important for coordinating changes in transcription during human development. To better understand the mechanisms underlying context-specific 3D chromatin structure and transcription during cellular differentiation, we generated comprehensive in situ Hi-C maps of DNA loops in human monocytes and differentiated macrophages. We demonstrate that dynamic looping events are regulatory rather than structural in nature and uncover widespread coordination of dynamic enhancer activity at preformed and acquired DNA loops. Enhancer-bound loop formation and enhancer activation of preformed loops together form multi-loop activation hubs at key macrophage genes. Activation hubs connect 3.4 enhancers per promoter and exhibit a strong enrichment for activator protein 1 (AP-1)-binding events, suggesting that multi-loop activation hubs involving cell-type-specific transcription factors represent an important class of regulatory chromatin structures for the spatiotemporal control of transcription.


Assuntos
Diferenciação Celular , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , DNA/metabolismo , Macrófagos/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/química , Cromatina/genética , DNA/química , DNA/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Conformação de Ácido Nucleico , Fenótipo , Ligação Proteica , Fatores de Tempo , Fator de Transcrição AP-1/genética
8.
Nat Biotechnol ; 35(5): 463-474, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28319085

RESUMO

Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but direct screening of all possible drug combinations is infeasible. Here we introduce a CRISPR-based double knockout (CDKO) system that improves the efficiency of combinatorial genetic screening using an effective strategy for cloning and sequencing paired single guide RNA (sgRNA) libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs. We applied CDKO to generate a large-scale human GI map, comprising 490,000 double-sgRNAs directed against 21,321 pairs of drug targets in K562 leukemia cells and identified synthetic lethal drug target pairs for which corresponding drugs exhibit synergistic killing. These included the BCL2L1 and MCL1 combination, which was also effective in imatinib-resistant cells. We further validated this system by identifying known and previously unidentified GIs between modifiers of ricin toxicity. This work provides an effective strategy to screen synergistic drug combinations in high-throughput and a CRISPR-based tool to dissect functional GI networks.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Proteínas de Neoplasias/genética , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Sinergismo Farmacológico , Humanos , Células K562
9.
Nat Methods ; 13(12): 1036-1042, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27798611

RESUMO

Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.


Assuntos
Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Evolução Molecular Direcionada/métodos , Mutação Puntual , Engenharia de Proteínas/métodos , RNA Guia de Cinetoplastídeos/genética , Bortezomib/farmacologia , Citidina Desaminase/genética , Resistência a Medicamentos/genética , Proteínas de Fluorescência Verde/genética , Humanos , Células K562 , Levivirus/genética , Complexo de Endopeptidases do Proteassoma/genética
10.
Nature ; 534(7609): 719-23, 2016 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-27281202

RESUMO

A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance are insufficient to protect cells from potential dominant consequences. Here, through a combination of transgenics and CRISPR­Cas9 gene editing in Caenorhabditis elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal-extended proteins that result from failure to terminate at stop codons. Sequences encoded by the 3' untranslated region (UTR) were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, in which we observed a comparable tendency for translated human 3' UTR sequences to reduce mature protein expression in tissue culture assays, including 3' UTR sequences from the hypomorphic 'Constant Spring' haemoglobin stop codon variant. We suggest that 3' UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.


Assuntos
Regiões 3' não Traduzidas/genética , Códon de Terminação/genética , Peptídeos/metabolismo , Biossíntese de Proteínas , Proteínas/química , Proteínas/metabolismo , Ribossomos/metabolismo , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Caenorhabditis elegans/genética , Genes/genética , Hemoglobinas Anormais/genética , Humanos , Peptídeos/genética , Biossíntese de Proteínas/genética , Proteínas/análise , Proteínas/genética
11.
ACS Synth Biol ; 2(9): 490-6, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-23713956

RESUMO

M13 bacteriophage has been used as a scaffold to organize materials for various applications. Building more complex multiphage devices requires precise control of interactions between the M13 capsid proteins. Toward this end, we engineered a loop structure onto the pIII capsid protein of M13 bacteriophage to enable sortase-mediated labeling reactions for C-terminal display. Combining this with N-terminal sortase-mediated labeling, we thus created a phage scaffold that can be labeled orthogonally on three capsid proteins: the body and both ends. We show that covalent attachment of different DNA oligonucleotides at the ends of the new phage structure enables formation of multiphage particles oriented in a specific order. These have potential as nanoscale scaffolds for multi-material devices.


Assuntos
Bacteriófago M13/genética , Bacteriófago M13/metabolismo , Proteínas do Capsídeo/química , DNA Viral/metabolismo , Motivos de Aminoácidos , Bacteriófago M13/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Hibridização de Ácido Nucleico/métodos , Imagem Óptica , Engenharia de Proteínas
12.
Bioconjug Chem ; 23(7): 1478-87, 2012 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-22759232

RESUMO

We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriófago M13/metabolismo , Proteínas do Capsídeo/metabolismo , Técnicas de Visualização da Superfície Celular/métodos , Cisteína Endopeptidases/metabolismo , Animais , Bacteriófago M13/química , Bacteriófago M13/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Camundongos , Camundongos Endogâmicos C57BL , Propriedades de Superfície
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