RESUMO
Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essential to the pathogen's long term intracellular survival in the amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1' recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells.
Assuntos
Proteínas de Bactérias/química , Carboxipeptidases/química , Francisella tularensis/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Carboxipeptidases/fisiologia , Domínio Catalítico , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Monócitos/microbiologia , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína , Difração de Raios XRESUMO
Subunit antigen-based vaccines can provide a number of important benefits over traditional vaccine candidates, such as overall safety. However, because of the inherently low immunogenicity of these antigens, methods for colocalized delivery of antigen and immunostimulatory molecules (i.e., adjuvants) are needed. Here we report a robust nanolipoprotein particle (NLP)-based vaccine delivery platform that facilitates the codelivery of both subunit antigens and adjuvants. Ni-chelating NLPs (NiNLPs) were assembled to incorporate the amphipathic adjuvants monophosphoryl lipid A and cholesterol-modified CpG oligodeoxynucleotides, which can bind His-tagged protein antigens. Colocalization of antigen and adjuvant delivery using the NiNLP platform resulted in elevated antibody production against His-tagged influenza hemagglutinin 5 and Yersinia pestis LcrV antigens. Antibody titers in mice immunized with the adjuvanted NLPs were 5-10 times higher than those observed with coadministration formulations and nonadjuvanted NiNLPs. Colocalized delivery of adjuvant and antigen provides significantly greater immune stimulation in mice than coadministered formulations.
Assuntos
Adjuvantes Imunológicos/química , Antígenos de Bactérias/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Lipoproteínas/química , Nanopartículas/química , Proteínas Citotóxicas Formadoras de Poros/imunologia , Vacinas/química , Animais , Antígenos de Bactérias/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Lipoproteínas/imunologia , Camundongos , Níquel/química , Níquel/imunologia , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Vacinas/imunologiaRESUMO
Nanolipoprotein particles (NLPs) are discoidal self-assembling membrane mimetics that have been primarily used as a platform for the solubilization and stabilization of membrane proteins. Nickel-chelating nanolipoprotein particles (NiNLPs) containing nickel-chelating lipids (Ni-lipid) for the targeted immobilization of His-tagged proteins hold promise as carriers of hydrophilic biological molecules for a range of applications. The effect of protein loading (i.e., the number of proteins bound per NiNLP) and Ni-lipid content on the time scales and kinetics of binding are important to various applications such as vaccine development, diagnostic imaging, and drug delivery. We have immobilized hexa-His-tagged LsrB, a Yersinia pestis transport protein, onto NiNLPs to examine the effect of protein binding stoichiometry and Ni-lipid content on the time scales and kinetics of protein binding by surface plasmon resonance (SPR). Data indicate that the dissociation half-time increases with Ni-lipid content up to a molar concentration of 35% and decreases as the number of bound protein per NiNLP increases. These findings indicate that the kinetics of protein binding are highly dependent on both the number of bound protein per NiNLP and Ni-lipid content.
Assuntos
Proteínas de Bactérias/química , Quelantes/química , Histidina/química , Lipoproteínas/química , Nanopartículas/química , Níquel/química , Proteínas de Bactérias/metabolismo , Quelantes/metabolismo , Histidina/metabolismo , Cinética , Lipídeos/química , Lipoproteínas/metabolismo , Níquel/metabolismo , Tamanho da Partícula , Ligação Proteica , Proteínas Recombinantes/química , Ressonância de Plasmônio de Superfície , Yersinia pestis/químicaRESUMO
BACKGROUND: A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL) (DvLPBaPPP)2LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma. RESULTS: An analog of the SHAL (DvLPBaPPP)2LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more 111In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the 111In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus. CONCLUSION: The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP)2LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP)2LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a short polypeptide.
Assuntos
Arginina/metabolismo , Compostos Heterocíclicos com 1 Anel/metabolismo , Linfoma de Células B/metabolismo , Oligopeptídeos/metabolismo , Peptídeos/metabolismo , Arginina/química , Sítios de Ligação , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Antígenos HLA-DR/metabolismo , Subtipos Sorológicos de HLA-DR , Compostos Heterocíclicos com 1 Anel/química , Humanos , Ligantes , Microscopia Confocal , Oligopeptídeos/química , Peptídeos/químicaRESUMO
Nanolipoprotein particles (NLPs) are nanometer-sized, discoidal particles that self-assemble from purified apolipoprotein and phospholipid. Their size and facile functionalization suggest potential application of NLPs as platforms for the presentation and delivery of recombinant proteins. To this end, we investigated incorporation of nickel-chelating lipids into NLPs (NiNLPs) and subsequent sequestration of polyhistidine (His)-tagged proteins. From initial lipid screens for NLP formation, the two phospholipids DMPC and DOPC were identified as suitable bulk lipids for incorporation of the nickel-chelating lipid DOGS-NTA-Ni into NLPs, and NiNLPs were successfully formed with varying amounts of DOGS-NTA-Ni. NiNLPs consisting of 10% DOGS-NTA-Ni with 90% bulk lipid (either DMPC or DOPC) were thoroughly characterized by size exclusion chromatography (SEC), non-denaturing gradient gel electrophoresis (NDGGE), and atomic force microscopy (AFM). Three different His-tagged proteins were sequestered on NiNLPs in a nickel-dependent manner, and the amount of immobilized protein was contingent on the size and composition of the NiNLP.
Assuntos
Proteínas de Bactérias/metabolismo , Quelantes/química , Lipídeos/química , Lipoproteínas/química , Nanopartículas/química , Níquel/química , Proteínas de Bactérias/química , Quelantes/metabolismo , Histidina/química , Histidina/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Níquel/metabolismo , Tamanho da Partícula , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Yersinia pestis/químicaRESUMO
Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell-cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring of amphipathic protein of defined diameter. The NLPs act as a platform for inserting, solubilizing and characterizing functional membrane proteins. NLP component proteins (apolipoproteins), as well as membrane proteins can be produced by either traditional cell-based or as discussed here, cell-free expression methodologies.
Assuntos
Lipoproteínas/metabolismo , Proteínas de Membrana/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Animais , Biotinilação , Fracionamento Celular/métodos , Escherichia coli/genética , Lipoproteínas/química , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Nanopartículas/química , Análise Serial de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Heterogeneity is a fact that plagues the characterization and application of many self-assembled biological constructs. The importance of obtaining particle homogeneity in biological assemblies is a critical goal, as bulk analysis tools often require identical species for reliable interpretation of the results-indeed, important tools of analysis such as x-ray diffraction typically require over 90% purity for effectiveness. This issue bears particular importance in the case of lipoproteins. Lipid-binding proteins known as apolipoproteins can self assemble with liposomes to form reconstituted high density lipoproteins (rHDLs) or nanolipoprotein particles (NLPs) when used for biotechnology applications such as the solubilization of membrane proteins. Typically, the apolipoprotein and phospholipids reactants are self assembled and even with careful assembly protocols the product often contains heterogeneous particles. In fact, size polydispersity in rHDLs and NLPs published in the literature are frequently observed, which may confound the accurate use of analytical methods. In this article, we demonstrate a procedure for producing a pure, monodisperse NLP subpopulation from a polydisperse self-assembly using size exclusion chromatography (SEC) coupled with high resolution particle imaging by atomic force microscopy (AFM). In addition, NLPs have been shown to self assemble both in the presence and absence of detergents such as cholate, yet the effects of cholate on NLP polydispersity and separation has not been systematically examined. Therefore, we examined the separation properties of NLPs assembled in both the absence and presence of cholate using SEC and native gel electrophoresis. From this analysis, NLPs prepared with and without cholate showed particles with well defined diameters spanning a similar size range. However, cholate was shown to have a dramatic affect on NLP separation by SEC and native gel electrophoresis. Furthermore, under conditions where different sized NLPs were not sufficiently separated or purified by SEC, AFM was used to deconvolute the elution pattern of different sized NLPs. From this analysis we were able to purify an NLP subpopulation to 90% size homogeneity by taking extremely fine elutions from the SEC. With this purity, we generate high quality NLP crystals that were over 100 microm in size with little precipitate, which could not be obtained utilizing the traditional size exclusion techniques. This purification procedure and the methods for validation are broadly applicable to other lipoprotein particles.
Assuntos
Lipoproteínas HDL/química , Nanopartículas/química , Colatos/química , Cromatografia em Gel , Bicamadas Lipídicas/químicaRESUMO
PURPOSE: More than two decades of research and clinical trials have shown radioimmunotherapy to be a promising approach for treating various forms of cancer. Lym-1 antibody, which binds selectively to HLA-DR10 on malignant B-cell lymphocytes, has proved to be effective in delivering radionuclides to non-Hodgkin's lymphoma and leukemia. Using a new approach to create small synthetic molecules that mimic the targeting properties of the Lym-1 antibody, a prototype, selective high-affinity ligand (SHAL), has been developed to bind to a unique region located within the Lym-1 epitope on HLA-DR10. EXPERIMENTAL DESIGN: Computer docking methods were used to predict two sets of small molecules that bind to neighboring cavities on the beta subunit of HLA-DR10 surrounding a critical amino acid in the epitope, and the ligands were confirmed to bind to the protein by nuclear magnetic resonance spectroscopy. Pairs of these molecules were then chemically linked together to produce a series of bidentate and bisbidentate SHALs. RESULTS: These SHALs bind with nanomolar to picomolar K(d)'s only to cell lines expressing HLA-DR10. Analyses of biopsy sections obtained from patients also confirmed that SHAL bound to both small and large cell non-Hodgkin's lymphomas mimicking the selectivity of Lym-1. CONCLUSIONS: These results show that synthetic molecules less than 1/50th the mass of an antibody can be designed to exhibit strong binding to subtle structural features on cell surface proteins similar to those recognized by antibodies. This approach offers great potential for developing small molecule therapeutics that target other types of cancer and disease.
Assuntos
Anticorpos Monoclonais/química , Antineoplásicos/farmacologia , Materiais Biomiméticos/farmacologia , Antígenos HLA-DR/metabolismo , Linfoma não Hodgkin/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Murinos , Antineoplásicos/síntese química , Antineoplásicos/química , Sítios de Ligação , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Antígenos HLA-DR/química , Antígenos HLA-DR/imunologia , Subtipos Sorológicos de HLA-DR , Humanos , Ligantes , Linfoma não Hodgkin/patologia , Linfoma não Hodgkin/terapia , Modelos Moleculares , Estreptavidina/metabolismoRESUMO
Selective high-affinity ligands (SHALs) were selected as substitutes for monoclonal antibodies (mAbs) to deliver radioisotopes to malignant tumors. Because a SHAL (5 KD) is considerably smaller in comparison to an antibody (150 KD), a significant therapeutic index (TI) enhancement for radioimmunotherapy (RIT) is anticipated. The antibody-antigen (Ab-Ag) model system chosen for the development of SHALs consists of Lym-1, a MAb with proven selectivity in non-Hodgkin's lymphoma (NHL) patients and its well-characterized Ag, the beta subunit of HLA DR10. Whereas Lym-1 is readily available, the subunit of HLA-DR10 is not. Native, heterodimeric (alpha and beta subunits) HLA-DR10 can be purified from Raji cells, which are known to overexpress this Ag. Inconsistent homogeneity between preparations of HLA-DR10 solubilized in the presence of detergents prompted us to express a recombinant form of the beta subunit of HLA-DR10 in Escherichia coli. Negligible production yields (Assuntos
Antígenos HLA-DR/genética
, Antígenos HLA-DR/metabolismo
, Linfoma/imunologia
, Linfoma/metabolismo
, Radioimunoterapia/métodos
, Sequência de Aminoácidos
, Anticorpos Monoclonais/imunologia
, Anticorpos Monoclonais Murinos
, Linhagem Celular Tumoral
, Dicroísmo Circular
, Clonagem Molecular
, Epitopos/imunologia
, Escherichia coli
, Expressão Gênica
, Antígenos HLA-DR/química
, Antígenos HLA-DR/imunologia
, Subtipos Sorológicos de HLA-DR
, Humanos
, Ligantes
, Modelos Moleculares
, Dados de Sequência Molecular
, Conformação Proteica
, Subunidades Proteicas/química
, Subunidades Proteicas/genética
, Subunidades Proteicas/imunologia
, Subunidades Proteicas/metabolismo
, Proteínas Recombinantes/química
, Proteínas Recombinantes/genética
, Proteínas Recombinantes/imunologia
, Proteínas Recombinantes/metabolismo
, Sensibilidade e Especificidade
, Homologia Estrutural de Proteína
, Especificidade por Substrato
, Ressonância de Plasmônio de Superfície
RESUMO
To address the need for vaccine platforms that induce robust cell-mediated immunity, we investigated the potential of utilizing self-assembling biologic nanolipoprotein particles (NLPs) as an antigen and adjuvant delivery system to induce antigen-specific murine T cell responses. We utilized OT-I and OT-II TCR-transgenic mice to investigate the effects of NLP-mediated delivery of the model antigen ovalbumin (OVA) on T cell activation. Delivery of OVA with the TLR4 agonist monophosphoryl lipid A (MPLA) in the context of NLPs significantly enhanced the activation of both CD4+ and CD8+ T cells in vitro compared to co-administration of free OVA and MPLA. Upon intranasal immunization of mice harboring TCR-transgenic cells, NLPs enhanced the adjuvant effects of MPLA and the in vivo delivery of OVA, leading to significantly increased expansion of CD4+ and CD8+ T cells in lung-draining lymph nodes. Therefore, NLPs are a promising vaccine platform for inducing T cell responses following intranasal administration.
Assuntos
Produtos Biológicos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Nanopartículas/administração & dosagem , Vacinas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Animais , Produtos Biológicos/administração & dosagem , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Pulmão/imunologia , Linfonodos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Vacinas/administração & dosagemRESUMO
Nanolipoprotein particles (NLPs) consist of a discoidal phospholipid lipid bilayer confined by an apolipoprotein belt. NLPs are a promising platform for a variety of biomedical applications due to their biocompatibility, size, definable composition, and amphipathic characteristics. However, poor serum stability hampers the use of NLPs for in vivo applications such as drug formulation. In this study, NLP stability was enhanced upon the incorporation and subsequent UV-mediated intermolecular cross-linking of photoactive DiynePC phospholipids in the lipid bilayer, forming cross-linked nanoparticles (X-NLPs). Both the concentration of DiynePC in the bilayer and UV exposure time significantly affected the resulting X-NLP stability in 100% serum, as assessed by size exclusion chromatography (SEC) of fluorescently labeled particles. Cross-linking did not significantly impact the size of X-NLPs as determined by dynamic light scattering and SEC. X-NLPs had essentially no degradation over 48 h in 100% serum, which is a drastic improvement compared to non-cross-linked NLPs (50% degradation by â¼10 min). X-NLPs had greater uptake into the human ATCC 5637 bladder cancer cell line compared to non-cross-linked particles, indicating their potential utility for targeted drug delivery. X-NLPs also exhibited enhanced stability following intravenous administration in mice. These results collectively support the potential utility of X-NLPs for a variety of in vivo applications.
Assuntos
Nanopartículas/química , Animais , Linhagem Celular Tumoral , Cromatografia em Gel , Humanos , Bicamadas Lipídicas , Camundongos , FosfolipídeosRESUMO
The conformation of the non-glycosylated recombinant form of the extracellar domain of rat MOG (rMOG(1-125)) dissolved in different solvent conditions was studied by CD spectroscopy. The results show that rMOG(1-125) exhibits a predominantly beta sheet conformation in aqueous buffer solution at pH 7.5 and that this 'beta-form' is stabilized by zwitterionic phospholipids, DPC and LPCP. The alpha helical content of the protein can increase from 9% to up to 20% when TFE or anionic detergent LPAP and SDS are added.
Assuntos
Glicoproteína Associada a Mielina/química , Oligodendroglia/química , Fosforilcolina/análogos & derivados , Conformação Proteica , Solventes/química , Animais , Dicroísmo Circular , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Concentração de Íons de Hidrogênio , Lisofosfolipídeos/química , Proteínas da Mielina , Glicoproteína Associada a Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Fosforilcolina/química , Estrutura Terciária de Proteína , Ratos , Dodecilsulfato de Sódio/química , Temperatura , Trifluoretanol/químicaRESUMO
Nanoparticles hold great promise for the delivery of therapeutics, yet limitations remain with regards to the use of these nanosystems for efficient long-lasting targeted delivery of therapeutics, including imparting functionality to the platform, in vivo stability, drug entrapment efficiency and toxicity. To begin to address these limitations, we evaluated the functionality, stability, cytotoxicity, toxicity, immunogenicity and in vivo biodistribution of nanolipoprotein particles (NLPs), which are mimetics of naturally occurring high-density lipoproteins (HDLs). We found that a wide range of molecules could be reliably conjugated to the NLP, including proteins, single-stranded DNA, and small molecules. The NLP was also found to be relatively stable in complex biological fluids and displayed no cytotoxicity in vitro at doses as high as 320 µg/ml. In addition, we observed that in vivo administration of the NLP daily for 14 consecutive days did not induce significant weight loss or result in lesions on excised organs. Furthermore, the NLPs did not display overt immunogenicity with respect to antibody generation. Finally, the biodistribution of the NLP in vivo was found to be highly dependent on the route of administration, where intranasal administration resulted in prolonged retention in the lung tissue. Although only a select number of NLP compositions were evaluated, the findings of this study suggest that the NLP platform holds promise for use as both a targeted and non-targeted in vivo delivery vehicle for a range of therapeutics.
Assuntos
Materiais Biomiméticos/farmacocinética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Portadores de Fármacos , Lipoproteínas HDL/farmacocinética , Nanopartículas/química , Administração Intranasal , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Apolipoproteína E4/química , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Materiais Biomiméticos/síntese química , DNA Bacteriano/química , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Estabilidade de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Corantes Fluorescentes , Lipoproteínas HDL/síntese química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/toxicidade , Tamanho da Partícula , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Distribuição TecidualRESUMO
Recent studies have demonstrated that therapies targeting the innate immune system have the potential to provide transient, non-specific protection from a variety of infectious organisms; however, the potential of enhancing the efficacy of such treatments using nano-scale delivery platforms requires more in depth evaluation. As such, we employed a nanolipoprotein (NLP) platform to enhance the efficacy of innate immune agonists. Here, we demonstrate that the synthetic Toll-like receptor (TLR) agonists monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG) can be readily incorporated into NLPs. Conjugation of MPLA and CpG to NLPs (MPLA:NLP and CpG:NLP, respectively) significantly enhanced their immunostimulatory profiles both in vitro and in vivo compared to administration of agonists alone, as evidenced by significant increases in cytokine production, cell surface expression of activation markers, and upregulation of immunoregulatory genes. Importantly, enhancement of cytokine production by agonist conjugation to NLPs was also observed in primary human dendritic cells. Furthermore, BALB/c mice pretreated with CpG:NLP constructs survived a lethal influenza challenge whereas pretreatment with CpG alone had no effect on survival.
Assuntos
Fatores Imunológicos/imunologia , Influenza Humana/imunologia , Nanopartículas/química , Animais , Linhagem Celular , Citocinas/metabolismo , Células Dendríticas/metabolismo , Humanos , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/químicaRESUMO
BACKGROUND: Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs. METHODOLOGY: Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to >25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4 degrees C was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes. CONCLUSIONS: ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utility for biotechnological applications.
Assuntos
Apolipoproteínas/química , Proteínas de Insetos/química , Lipoproteínas/química , Nanopartículas/química , Animais , Bombyx/química , Humanos , Manduca/química , Microscopia de Força AtômicaRESUMO
Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.
Assuntos
DNA/química , DNA/metabolismo , Espermatozoides/citologia , Espermatozoides/patologia , Cromatina/metabolismo , Humanos , Masculino , Microscopia de Fluorescência , Conformação de Ácido Nucleico , Proteínas/química , Proteínas/metabolismo , Análise Espectral Raman , Espermatozoides/metabolismo , Fatores de TempoRESUMO
To mimic the molecular specificity and cell selectivity of monoclonal antibody (mAb) binding while decreasing size, nanomolecules (selective high-affinity ligands; SHALs), based on in silico modeling, have been created to bind to human leukocyte antigen-DR (HLA-DR10), a signaling receptor protein upregulated on the malignant B-lymphocytes of non-Hodgkin's lymphoma and chronic lymphocytic leukemia. SHALs were synthesized with a biotin or DOTA chelate (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), using a solid-phase lysine-polyethyleneglycol backbone to link sets of ligands shown previously to bind to HLA-DR10. Using cell-binding and death assays and confocal microscopy, SHAL uptake, residualization, and cytocidal activity were evaluated in HLA-DR10 expressing and nonexpressing live, human lymphoma cell lines. All of the SHALs tested were selective for, and accumulated in, expressing cells. Reflecting binding to HLA-DR10 inside the cells, SHALs having the Ct ligand (3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid) residualized in expressing cells greater than 179 times more than accountable by cell-surface membrane HLA-DR10. Confocal microscopy confirmed the intracellular residualization of these SHALs. Importantly, SHALs with a Ct ligand had direct cytocidal activity, similar in potency to that of Lym-1 mAb and rituximab, selectively for HLA-DR10 expressing lymphoma cells and xenografts. The results show that SHALs containing the Ct ligand residualize intracellularly and have cytocidal effects mediated by HLA-DR10. These SHALs have extraordinary potential as novel molecules for the selective targeting of lymphoma and leukemia for molecular therapy and imaging. Further, these SHALs can be used to transport and residualize cytotoxic agents near critical sites inside these malignant cells.
Assuntos
Anticorpos Monoclonais/imunologia , Materiais Biomiméticos/farmacologia , Antígenos HLA-DR/imunologia , Leucemia/terapia , Linfoma/terapia , Mimetismo Molecular/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos , Materiais Biomiméticos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Subtipos Sorológicos de HLA-DR , Humanos , Radioisótopos de Índio , Leucemia/imunologia , Linfoma/imunologia , Nanopartículas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Compostos Radiofarmacêuticos , Rituximab , Espectrometria de Massas por Ionização por Electrospray , Distribuição TecidualRESUMO
Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at approximately 14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.
Assuntos
Lipoproteínas/química , Lipoproteínas/ultraestrutura , Nanopartículas/química , Nanopartículas/ultraestrutura , Biologia Computacional , Dimiristoilfosfatidilcolina/isolamento & purificação , Dimiristoilfosfatidilcolina/metabolismo , Eletroforese em Gel de Poliacrilamida , Lipoproteínas/isolamento & purificação , Lipoproteínas/metabolismo , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein [MOG(1-120)]. METHODS: Raman spectra were acquired from individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of 3 h. Data were also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. RESULTS: Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1,257, 1,340, 1,453, and 1,660 cm(-1), are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. CONCLUSION: It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in flow cytometry, open up new possibilities for analyzing cellular processes occurring in single microbial and eukaryotic cells.
Assuntos
Escherichia coli/genética , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , Pinças Ópticas , Análise Espectral Raman/métodos , Animais , Perfilação da Expressão Gênica/instrumentação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Isopropiltiogalactosídeo/farmacologia , Modelos Biológicos , Proteínas da Mielina , Glicoproteína Associada a Mielina/genética , Glicoproteína Mielina-Oligodendrócito , Ratos , Proteínas Recombinantes/genética , Análise Espectral Raman/instrumentaçãoRESUMO
We have compared the relative proportion of protamine 1 (P1) and protamine 2 (P2) bound to DNA in the sperm of a variety of eutherian mammals to obtain insight into how these two proteins interact in sperm chromatin. Gel electrophoresis (combined with microdensitometry) and high performance liquid chromatography (HPLC) were used to determine the content of the two protamines, and the identity of each protein was confirmed by amino-terminal sequencing or amino acid analysis. The sperm of all species examined contained P1, but P2 was found to be present only in certain species. Unlike the fixed ratio of core histones that package DNA into nucleosomes in all somatic cells, the proportion of P2 present in mature sperm was found to be continuously variable from 0 to nearly 80%. These results show that P1 and P2 do not interact with each other or DNA to form a discrete complex or subunit structure that is dependent upon particular P1/P2 stoichiometries. Data obtained from a number of closely and distantly related species also indicate that while the P2 content of sperm chromatin is allowed to vary over a wide range during the course of evolution, the relative proportion of P1 and P2 are tightly regulated within a genus.