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1.
Nucleic Acids Res ; 52(10): 5423-5437, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38742636

RESUMO

Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.


Assuntos
Acetilgalactosamina , RNA Interferente Pequeno , Animais , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Administração Oral , Camundongos , Ratos , Interferência de RNA , Masculino , Disponibilidade Biológica , Humanos , Ratos Sprague-Dawley , Macaca fascicularis , Fígado/metabolismo , Macaca mulatta
2.
J Am Chem Soc ; 145(36): 19691-19706, 2023 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-37638886

RESUMO

Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2'-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4'-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.


Assuntos
Ácidos Nucleicos , Animais , Camundongos , Ratos , RNA Interferente Pequeno , Nucleotídeos , Interferência de RNA , Acetilgalactosamina
3.
Chem Commun (Camb) ; 59(42): 6347-6350, 2023 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-37144553

RESUMO

To ensure specificity of small interfering RNAs (siRNAs), the antisense strand must be selected by the RNA-induced silencing complex (RISC). We have previously demonstrated that a 5'-morpholino-modified nucleotide at the 5'-end of the sense strand inhibits its interaction with RISC ensuring selection of the desired antisense strand. To improve this antagonizing binding property even further, a new set of morpholino-based analogues, Mo2 and Mo3, and a piperidine analogue, Pip, were designed based on the known structure of Argonaute2, the slicer enzyme component of RISC. Sense strands of siRNAs were modified with these new analogues, and the siRNAs were evaluated in vitro and in mice for RNAi activity. Our data demonstrated that Mo2 is the best RISC inhibitor among the modifications tested and that it effectively mitigates sense strand-based off-target activity of siRNA.


Assuntos
RNA Interferente Pequeno , Complexo de Inativação Induzido por RNA , Animais , Camundongos , RNA Interferente Pequeno/química , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Morfolinos/química
4.
Nat Biotechnol ; 40(10): 1500-1508, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35654979

RESUMO

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.


Assuntos
Precursor de Proteína beta-Amiloide , Terapêutica com RNAi , Animais , Camundongos , Primatas/genética , Primatas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico
5.
Drug Metab Dispos ; 50(6): 781-797, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-34154993

RESUMO

Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to N-acetylgalactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterolemia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor-mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conjugated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. SIGNIFICANCE STATEMENT: Several nonclinical ADME studies have been conducted in order to provide a comprehensive overview of the disposition and elimination of GalNAc-conjugated siRNAs and the pharmacokinetic/pharmacodynamic translation between species. These studies demonstrate that the ADME properties of GalNAc-conjugated siRNAs are well correlated and predictable across species, building confidence in the ability to extrapolate to human.


Assuntos
Acetilgalactosamina , Porfirias Hepáticas , Acetilgalactosamina/farmacocinética , Receptor de Asialoglicoproteína/metabolismo , Hepatócitos/metabolismo , Humanos , Porfirias Hepáticas/metabolismo , RNA Interferente Pequeno/genética
6.
Mol Ther ; 29(6): 2053-2066, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-33601052

RESUMO

RNA interference (RNAi) offers the potential to treat disease at the earliest onset by selectively turning off the expression of target genes, such as intracellular oncogenes that drive cancer growth. However, the development of RNAi therapeutics as anti-cancer drugs has been limited by both a lack of efficient and target cell-specific delivery systems and the necessity to overcome numerous intracellular barriers, including serum/lysosomal instability, cell membrane impermeability, and limited endosomal escape. Here, we combine two technologies to achieve posttranscriptional gene silencing in tumor cells: Centyrins, alternative scaffold proteins binding plasma membrane receptors for targeted delivery, and small interfering RNAs (siRNAs), chemically modified for high metabolic stability and potency. An EGFR Centyrin known to internalize in EGFR-positive tumor cells was site-specifically conjugated to a beta-catenin (CTNNb1) siRNA and found to drive potent and specific target knockdown by free uptake in cell culture and in mice inoculated with A431 tumor xenografts (EGFR amplified). The generalizability of this approach was further demonstrated with Centyrins targeting multiple receptors (e.g., BCMA, PSMA, and EpCAM) and siRNAs targeting multiple genes (e.g., CD68, KLKb1, and SSB1). Moreover, by installing multiple conjugation handles, two different siRNAs were fused to a single Centyrin, and the conjugate was shown to simultaneously silence two different targets. Finally, by specifically pairing EpCAM-binding Centyrins that exhibited optimized internalization profiles, we present data showing that an EpCAM Centyrin CTNNb1 siRNA conjugate suppressed tumor cell growth of a colorectal cancer cell line containing an APC mutation but not cells with normal CTNNb1 signaling. Overall, these data demonstrate the potential of Centyrin-siRNA conjugates to target cancer cells and silence oncogenes, paving the way to a new class of anticancer drugs.


Assuntos
Técnicas de Transferência de Genes , Interferência de RNA , RNA Interferente Pequeno/genética , Animais , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Inativação Gênica , Genes erbB-1 , Terapia Genética , Humanos , Ligantes , Camundongos , RNA Mensageiro , RNA Interferente Pequeno/administração & dosagem , Tenascina/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
7.
Nucleic Acids Res ; 48(21): 11827-11844, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-32808038

RESUMO

One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc-siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc-siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.


Assuntos
Acetilgalactosamina/metabolismo , Receptor de Asialoglicoproteína/metabolismo , Portadores de Fármacos , Inativação Gênica , Pré-Albumina/genética , RNA Interferente Pequeno/metabolismo , Acetilgalactosamina/química , Animais , Proteínas Argonautas/genética , Receptor de Asialoglicoproteína/genética , Transporte Biológico , Estabilidade de Medicamentos , Feminino , Glicoconjugados/química , Glicoconjugados/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Fígado/citologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Nanopartículas/metabolismo , Pré-Albumina/antagonistas & inibidores , Pré-Albumina/metabolismo , RNA Interferente Pequeno/genética , Fatores de Tempo
8.
J Biol Chem ; 295(40): 13769-13783, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-32732284

RESUMO

Single-stranded, positive-sense RNA viruses assemble their replication complexes in infected cells from a multidomain replication polyprotein. This polyprotein usually contains at least one protease, the primary function of which is to process the polyprotein into mature proteins. Such proteases also may have other functions in the replication cycle. For instance, cysteine proteases (PRO) frequently double up as ubiquitin hydrolases (DUB), thus interfering with cellular processes critical for virus replication. We previously reported the crystal structures of such a PRO/DUB from Turnip yellow mosaic virus (TYMV) and of its complex with one of its PRO substrates. Here we report the crystal structure of TYMV PRO/DUB in complex with ubiquitin. We find that PRO/DUB recognizes ubiquitin in an unorthodox way: It interacts with the body of ubiquitin through a split recognition motif engaging both the major and the secondary recognition patches of ubiquitin (Ile44 patch and Ile36 patch, respectively, including Leu8, which is part of the two patches). However, the contacts are suboptimal on both sides. Introducing a single-point mutation in TYMV PRO/DUB aimed at improving ubiquitin-binding led to a much more active DUB. Comparison with other PRO/DUBs from other viral families, particularly coronaviruses, suggests that low DUB activities of viral PRO/DUBs may generally be fine-tuned features of interaction with host factors.


Assuntos
Enzimas Desubiquitinantes/química , Peptídeo Hidrolases/química , Tymovirus/enzimologia , Ubiquitina/química , Proteínas Virais/química , Cristalografia por Raios X , Enzimas Desubiquitinantes/genética , Peptídeo Hidrolases/genética , Tymovirus/genética , Ubiquitina/genética , Proteínas Virais/genética
9.
Nucleic Acids Res ; 48(10): 5281-5293, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32347936

RESUMO

Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody-siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to ß-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting ß-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown.


Assuntos
Região Variável de Imunoglobulina/química , Interferência de RNA , RNA Interferente Pequeno/química , Anticorpos/química , Linhagem Celular Tumoral , Humanos , RNA Interferente Pequeno/farmacocinética , beta Catenina/genética
10.
J Med Chem ; 61(3): 734-744, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29376650

RESUMO

(E)-Vinylphosphonate ((E)-VP), a metabolically stable phosphate mimic at the 5'-end of the antisense strand, enhances the in vivo potency of siRNA. Here we describe a straightforward synthetic approach to incorporate a nucleotide carrying a vinylphosphonate (VP) moiety at the 5'-end of oligonucleotides under standard solid-phase synthesis and deprotection conditions by utilizing pivaloyloxymethyl (POM) protected VP-nucleoside phosphoramidites. The POM protection enhances scope and scalability of 5'-VP-modified oligonucleotides and, in a broader sense, the synthesis of oligonucleotides modified with phosphonate moieties. Trivalent N-acetylgalactosamine-conjugated small interfering RNA (GalNAc-siRNA) comprising (E)-geometrical isomer of VP showed improved RISC loading with robust RNAi-mediated gene silencing in mice compared to the corresponding (Z)-isomer despite similar tissue accumulation. We also obtained structural insights into why bulkier 2'-ribosugar substitutions such as 2'-O-[2-(methylamino)-2-oxoethyl] are well tolerated only when combined with 5'-(E)-VP.


Assuntos
Organofosfonatos/química , Organofosfonatos/síntese química , RNA Interferente Pequeno/química , Animais , Proteínas Argonautas/química , Proteínas Argonautas/deficiência , Proteínas Argonautas/genética , Sequência de Bases , Técnicas de Química Sintética , Inativação Gênica , Camundongos , Modelos Moleculares , Domínios Proteicos , RNA Interferente Pequeno/genética , Estereoisomerismo
12.
Biosens Bioelectron ; 89(Pt 2): 789-794, 2017 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-27816596

RESUMO

Current techniques to characterize leukocyte subgroups in blood require long sample preparation times and sizable sample volumes. A simplified method for leukocyte characterization using smaller blood volumes would thus be useful in diagnostic settings. Here we describe a flow system comprised of two functionalized graphene oxide (GO) surfaces that allow the capture of distinct leukocyte populations from small volumes blood using camelid single-domain antibodyfragments (VHHs) as capture agents. We used site-specifically labeled leukocytes to detect and identify cells exposed to fungal challenge. Combining the chemical and optical properties of GO with the versatility of the VHH scaffold in the context of a flow system provides a quick and efficient method for the capture and characterization of functional leukocytes.


Assuntos
Anticorpos Imobilizados/química , Candida albicans/isolamento & purificação , Separação Celular/métodos , Grafite/química , Leucócitos/citologia , Leucócitos/microbiologia , Anticorpos de Domínio Único/química , Aminoaciltransferases/análise , Animais , Proteínas de Bactérias/análise , Candidíase/sangue , Cisteína Endopeptidases/análise , Feminino , Citometria de Fluxo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nanoestruturas/química
13.
Nucleic Acids Res ; 45(6): 3528-3536, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-27903888

RESUMO

Efficient gene silencing by RNA interference (RNAi) in vivo requires the recognition and binding of the 5΄- phosphate of the guide strand of an siRNA by the Argonaute protein. However, for exogenous siRNAs it is limited by the rapid removal of the 5΄- phosphate of the guide strand by metabolic enzymes. Here, we have determined the crystal structure of human Argonaute-2 in complex with the metabolically stable 5΄-(E)-vinylphosphonate (5΄-E-VP) guide RNA at 2.5-Šresolution. The structure demonstrates how the 5΄ binding site in the Mid domain of human Argonaute-2 is able to adjust the key residues in the 5΄-nucleotide binding pocket to compensate for the change introduced by the modified nucleotide. This observation also explains improved binding affinity of the 5΄-E-VP -modified siRNA to human Argonaute-2 in-vitro, as well as the enhanced silencing in the context of the trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA in mice relative to the un-modified siRNA.


Assuntos
Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Organofosfonatos/química , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Compostos de Vinila/química , Animais , Sítios de Ligação , Humanos , Camundongos , Modelos Moleculares , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/metabolismo , Receptores de Albumina/genética , Receptores de Albumina/metabolismo
14.
Chembiochem ; 17(11): 985-9, 2016 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-27121751

RESUMO

Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.


Assuntos
Acetilgalactosamina/química , Organofosfonatos/química , Interferência de RNA , RNA Interferente Pequeno/química , Compostos de Vinila/química , Animais , Apolipoproteínas B/antagonistas & inibidores , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Proteínas Argonautas/antagonistas & inibidores , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Células Cultivadas , Fator IX/antagonistas & inibidores , Fator IX/genética , Fator IX/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Lipoproteínas LDL/sangue , Camundongos , Camundongos Endogâmicos C57BL , Organofosfonatos/farmacologia , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Complexo de Inativação Induzido por RNA/química , Complexo de Inativação Induzido por RNA/metabolismo , Fatores de Transcrição/metabolismo , Compostos de Vinila/farmacologia
15.
Proc Natl Acad Sci U S A ; 113(6): E705-14, 2016 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-26811477

RESUMO

Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12(-/-) Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12(-/-) cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades.


Assuntos
Membrana Celular/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Ubiquitina Tiolesterase/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Núcleo Celular/metabolismo , Separação Celular , Citosol/metabolismo , Endopeptidases/metabolismo , Ácidos Graxos Insaturados/farmacologia , Humanos , Células Jurkat , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Transporte Proteico , Reprodutibilidade dos Testes , Especificidade por Substrato/efeitos dos fármacos , Linfócitos T/metabolismo , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/deficiência , Proteases Específicas de Ubiquitina/metabolismo
16.
Chemistry ; 21(48): 17178-83, 2015 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-26472062

RESUMO

Peripheral blood can provide valuable information on an individual's immune status. Cell-based assays typically target leukocytes and their products. Characterization of leukocytes from whole blood requires their separation from the far more numerous red blood cells.1 Current methods to classify leukocytes, such as recovery on antibody-coated beads or fluorescence-activated cell sorting require long sample preparation times and relatively large sample volumes.2 A simple method that enables the characterization of cells from a small peripheral whole blood sample could overcome limitations of current analytical techniques. We describe the development of a simple graphene oxide surface coated with single-domain antibody fragments. This format allows quick and efficient capture of distinct WBC subpopulations from small samples (∼30 µL) of whole blood in a geometry that does not require any specialized equipment such as cell sorters or microfluidic devices.


Assuntos
Grafite/química , Nanoestruturas/química , Anticorpos de Domínio Único/imunologia , Grafite/sangue , Humanos , Anticorpos de Domínio Único/sangue
17.
Angew Chem Int Ed Engl ; 54(40): 11706-10, 2015 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-26252716

RESUMO

The site-specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self-quenching from multiple fluorophores installed in close proximity, we used Holliday junctions to label proteins site-specifically. Holliday junctions enable modification with multiple fluorophores at reasonably precise spacing. We designed a Holliday junction with three of its four arms modified with a fluorophore of choice and the remaining arm equipped with a dibenzocyclooctyne substituent to render it reactive with an azide-modified fluorescent single-domain antibody fragment or an intact immunoglobulin produced in a sortase-catalyzed reaction. These fluorescent Holliday junctions improve fluorescence yields for both single-domain and full-sized antibodies without deleterious effects on antigen binding.


Assuntos
Alcinos/química , Anticorpos/análise , Anticorpos/química , Azidas/química , DNA Cruciforme/química , Corantes Fluorescentes/química
18.
Nat Protoc ; 10(3): 508-16, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25719269

RESUMO

Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of sortase A on a solid support (Sepharose beads). Immobilization of sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca(2+)-independent variant of sortase A with increased catalytic activity. This heptamutant variant of sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized sortase A takes 1-2 d. Batch reactions take 3-12 h and flow reactions proceed at 0.5 ml h(-1), depending on the geometry of the reactor used.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Enzimas Imobilizadas/metabolismo , Peptidil Transferases/metabolismo , Engenharia de Proteínas/métodos , Proteínas/metabolismo , Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Catálise , Cisteína Endopeptidases/genética , Mutação/genética , Sefarose
19.
PLoS One ; 9(10): e109883, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25275512

RESUMO

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.


Assuntos
Aeromonas hydrophila/metabolismo , Toxinas Bacterianas/metabolismo , Antígenos CD59/metabolismo , Glipicanas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Aeromonas hydrophila/química , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/análise , Biotina/química , Antígenos CD59/análise , Linhagem Celular , Corantes Fluorescentes/química , Glipicanas/análise , Humanos , Dados de Sequência Molecular , Imagem Óptica , Proteínas Citotóxicas Formadoras de Poros/análise , Receptores de Ativador de Plasminogênio Tipo Uroquinase/análise
20.
J Exp Med ; 211(2): 365-79, 2014 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-24493799

RESUMO

Valency requirements for B cell activation upon antigen encounter are poorly understood. OB1 transnuclear B cells express an IgG1 B cell receptor (BCR) specific for ovalbumin (OVA), the epitope of which can be mimicked using short synthetic peptides to allow antigen-specific engagement of the BCR. By altering length and valency of epitope-bearing synthetic peptides, we examined the properties of ligands required for optimal OB1 B cell activation. Monovalent engagement of the BCR with an epitope-bearing 17-mer synthetic peptide readily activated OB1 B cells. Dimers of the minimal peptide epitope oriented in an N to N configuration were more stimulatory than their C to C counterparts. Although shorter length correlated with less activation, a monomeric 8-mer peptide epitope behaved as a weak agonist that blocked responses to cell-bound peptide antigen, a blockade which could not be reversed by CD40 ligation. The 8-mer not only delivered a suboptimal signal, which blocked subsequent responses to OVA, anti-IgG, and anti-kappa, but also competed for binding with OVA. Our results show that fine-tuning of BCR-ligand recognition can lead to B cell nonresponsiveness, activation, or inhibition.


Assuntos
Linfócitos B/imunologia , Ovalbumina/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Linfócitos B/metabolismo , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Epitopos/química , Epitopos/metabolismo , Tolerância Imunológica , Ligantes , Ativação Linfocitária , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Imunológicos , Ovalbumina/metabolismo , Multimerização Proteica/imunologia
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