RESUMO
Neurotoxic organophosphorus compounds (OPs) pose a severe threat if misused in military conflicts or by terrorists. Administration of a hydrolytic enzyme that can decompose the circulating nerve agent into non-toxic metabolites in vivo offers a potential treatment. A promising candidate is the homo-dimeric phosphotriesterase originating from the bacterium Brevundimonas diminuta (BdPTE), which has been subject to several rational and combinatorial protein design studies. A series of engineered versions with much improved catalytic efficiencies toward medically relevant nerve agents was described, carrying up to 22 mutations per enzyme subunit. To provide a basis for further rational design, we have determined the crystal structure of the highly active variant 10-2-C3(C59V/C227V)âstabilized against oxidation by substitution of two unpaired Cys residuesâin complex with a substrate analogue at 1.5 Å resolution. Unexpectedly, the long loop segment (residues 253-276) that covers the active site shows a totally new conformation, with drastic structural deviations up to 19 Å, which was neither predicted in any of the preceding protein design studies nor seen in previous crystallographic analyses of less far evolved enzyme versions. Inspired by this structural insight, additional amino acid exchanges were introduced and their effects on protein stability as well as on the catalytic efficiency toward several neurotoxic OPs were investigated. Somewhat surprisingly, our results suggest that the presently available engineered version of BdPTE, in spite of its design on the basis of partly false structural assumptions, constitutes a fairly optimized enzyme for the detoxification of relevant OP nerve agents.
Assuntos
Agentes Neurotóxicos , Hidrolases de Triester Fosfórico , Hidrolases de Triester Fosfórico/metabolismo , Organofosfatos , Domínio Catalítico , Compostos Organofosforados/metabolismoRESUMO
The biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (kcat/KM = 21 to 38 × 106 M-1 min-1) towards VX and a preferential hydrolysis of the more toxic P(-) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD50) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg-1 i.v.: PTE-2 dosed at 1.3 mg kg-1 i.v. (PTE-2.1) and 2.6 mg kg-1 i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg-1 i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.
Assuntos
Substâncias para a Guerra Química/toxicidade , Compostos Organotiofosforados/toxicidade , Hidrolases de Triester Fosfórico/farmacologia , Animais , Caulobacteraceae/enzimologia , Inibidores da Colinesterase/toxicidade , Masculino , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/genética , Engenharia de Proteínas , Ratos , Ratos Wistar , EstereoisomerismoRESUMO
Highly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured kcat/KM values were as high as 6.5 and 1.5 × 108 M-1 min-1 with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC-MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(-) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(-) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo.
Assuntos
Caulobacteraceae/enzimologia , Agentes Neurotóxicos/metabolismo , Hidrolases de Triester Fosfórico/metabolismo , Catálise , Cromatografia Líquida , Hidrólise , Mutação , Agentes Neurotóxicos/química , Agentes Neurotóxicos/toxicidade , Hidrolases de Triester Fosfórico/genética , Estereoisomerismo , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
We have adopted the concept of bispecific antibodies, which can simultaneously block or cross-link two different biomolecular targets, to create bispecific enzymes by exploiting the homodimeric quaternary structure of bacterial phosphotriesterases (PTEs). The PTEs from Brevundimonas diminuta and Agrobacterium radiobacter, whose engineered variants can efficiently hydrolyze organophosphorus (OP) nerve agents and pesticides, respectively, have attracted considerable interest for the treatment of the corresponding intoxications. OP nerve agents and pesticides still pose a severe toxicological threat in military conflicts, including acts of terrorism, as well as in agriculture, leading to >100000 deaths per year. In principle, engineered conventional homodimeric PTEs may provoke hydrolytic inactivation of individual OPs in vivo, and their application as catalytic bioscavengers via administration into the bloodstream has been proposed. However, their narrow substrate specificity would necessitate therapeutic application of a set or mixture of different enzymes, which complicates biopharmaceutical development. We succeeded in combining subunits from both enzymes and to stabilize their heterodimerization by rationally designing electrostatic steering mutations, thus breaking the natural C2 symmetry. The resulting bispecific enzyme from two PTEs with different bacterial origin exhibits an ultrabroad OP substrate profile and allows the efficient detoxification of both nerve agents and pesticides. Our approach of combining two active sites with distinct substrate specificities within one artificial dimeric biocatalyst-retaining the size and general properties of the original enzyme without utilizing protein mixtures or much larger fusion proteins-not only should facilitate biological drug development but also may be applicable to oligomeric enzymes with other catalytic activities.
Assuntos
Anticorpos Biespecíficos/imunologia , Organofosfatos/metabolismo , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/genética , Engenharia de Proteínas , Multimerização Proteica , Domínio Catalítico , Hidrolases de Triester Fosfórico/imunologia , Hidrolases de Triester Fosfórico/metabolismo , Estrutura Quaternária de Proteína , Eletricidade EstáticaRESUMO
A prominent feature of sensitizing environmental compounds that cause allergic contact dermatitis is the rapid induction of an innate inflammatory response that seems to provide danger signals for efficient T cell priming. We generated mouse models of mast cell deficiency, mast cell-specific gene inactivation, and mast cell reporter mice for intravital imaging and showed that these adjuvant effects of contact allergens are mediated by mast cells and histamine. Mast cell deficiency resulted in impaired emigration of skin DCs to the lymph node and contact hypersensitivity was dramatically reduced in the absence of mast cells. In addition, mast cell-specific inactivation of the Il10 gene did not reveal any role for mast cell-derived IL-10 in the regulation of contact allergy. Collectively, we demonstrate that mast cells are essential promoters of contact hypersensitivity, thereby highlighting their potential to promote immune responses to antigens entering via the skin.
Assuntos
Adjuvantes Imunológicos/farmacologia , Dermatite Alérgica de Contato/imunologia , Haptenos/imunologia , Mastócitos/imunologia , Animais , Movimento Celular , Células Dendríticas/imunologia , Histamina/imunologia , Hipertrofia/imunologia , Imunidade Inata , Linfonodos/imunologia , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Neovascularização Patológica/induzido quimicamente , Neovascularização Patológica/imunologiaRESUMO
Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals.
Assuntos
Movimento Celular/fisiologia , Conexina 43/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Osteoblastos/metabolismo , Nicho de Células-Tronco/fisiologia , Animais , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Conexina 43/genética , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Mutantes , Osteoblastos/citologiaRESUMO
Phosphotriesterases (PTE) are a new and promising approach for the treatment of organophosphate poisoning, since the current therapy of such intoxications shows some limitations. A previous rat in vivo study confirmed the therapeutic effect of PTE, which were specifically designed for VX breakdown, and demonstrated rapid degradation of VX in whole blood samples. The present study now focuses on the degradation of VX and its distribution in organ tissues of the animals used in the aforementioned study. In order to gain a broader overview, we have extended the investigations to the VX metabolites EA-2192 and EMPA by using methods developed for an LC-ESI-MS/MS system. Applying these methods, we were able to verify the effectiveness of the PTE treatment and gained an overview of VX tissue distribution in poisoned but untreated rats.
Assuntos
Compostos Organotiofosforados , Hidrolases de Triester Fosfórico , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Animais , Compostos Organotiofosforados/toxicidade , Masculino , Distribuição Tecidual , Hidrolases de Triester Fosfórico/metabolismo , Ratos Wistar , Cromatografia Líquida , RatosRESUMO
Dynamic changes in the epigenome at defined genomic loci play crucial roles during cellular differentiation and disease development. Here, we developed dual-color bimolecular anchor detector (BiAD) sensors for high-sensitivity readout of locus-specific epigenome modifications by fluorescence microscopy. Our BiAD sensors comprise an sgRNA/dCas9 complex as anchor and double chromatin reader domains as detector modules, both fused to complementary parts of a split IFP2.0 fluorophore, enabling its reconstitution upon binding of both parts in close proximity. In addition, a YPet fluorophore is recruited to the sgRNA to mark the genomic locus of interest. With these dual-color BiAD sensors, we detected H3K9me2/3 and DNA methylation and their dynamic changes upon RNAi or inhibitor treatment with high sensitivity at endogenous genomic regions. Furthermore, we showcased locus-specific H3K36me2/3 readout as well as H3K27me3 and H3K9me2/3 enrichment on the inactive X chromosome, highlighting the broad applicability of our dual-color BiAD sensors for single-cell epigenome studies.
Assuntos
Metilação de DNA , Epigenoma , Histonas , Análise de Célula Única , Humanos , Análise de Célula Única/métodos , Histonas/metabolismo , Histonas/genética , Epigênese Genética , Loci Gênicos , Cromatina/metabolismo , Cromatina/genética , Técnicas Biossensoriais/métodos , Cor , Células HEK293 , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismoRESUMO
Through its involvement in gene transcription and heterochromatin formation, DNA methylation regulates how cells interact with their environment. Nevertheless, the extracellular signaling cues that modulate the distribution of this central chromatin modification are largely unclear. DNA methylation is highly abundant at repetitive elements, but its investigation in live cells has been complicated by methodological challenges. Utilizing a CRISPR/dCas9 biosensor that reads DNA methylation of human α-satellite repeats in live cells, we here uncover a signaling pathway linking the chromatin and transcriptional state of repetitive elements to epithelial adherens junction integrity. Specifically, we find that in confluent breast epithelial cell monolayers, α-satellite repeat methylation is reduced by comparison to low density cultures. This is coupled with increased transcriptional activity at repeats. Through comprehensive perturbation experiments, we identify the junctional protein E-cadherin, which links to the actin cytoskeleton, as a central molecular player for signal relay into the nucleus. Furthermore, we find that this pathway is impaired in cancer cells that lack E-cadherin and are not contact-inhibited. This suggests that the molecular connection between cell density and repetitive element methylation could play a role in the maintenance of epithelial tissue homeostasis.
Assuntos
Junções Aderentes , Metilação de DNA , Humanos , Junções Aderentes/genética , Junções Aderentes/metabolismo , Caderinas/genética , Caderinas/metabolismo , Transdução de Sinais , Cromatina/metabolismoRESUMO
Emergency mobilization of neutrophil granulocytes (neutrophils) from the bone marrow (BM) is a key event of early cellular immunity. The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) stimulates this process, but it is unknown how individual neutrophils respond in situ. We show by intravital 2-photon microscopy that a systemic dose of human clinical-grade G-CSF rapidly induces the motility and entry of neutrophils into blood vessels within the tibial BM of mice. Simultaneously, the neutrophil-attracting chemokine KC (Cxcl1) spikes in the blood. In mice lacking the KC receptor Cxcr2, G-CSF fails to mobilize neutrophils and antibody blockade of Cxcr2 inhibits the mobilization and induction of neutrophil motility in the BM. KC is expressed by megakaryocytes and endothelial cells in situ and is released in vitro by megakaryocytes isolated directly from BM. This production of KC is strongly increased by thrombopoietin (TPO). Systemic G-CSF rapidly induces the increased production of TPO in BM. Accordingly, a single injection of TPO mobilizes neutrophils with kinetics similar to G-CSF, and mice lacking the TPO receptor show impaired neutrophil mobilization after short-term G-CSF administration. Thus, a network of signaling molecules, chemokines, and cells controls neutrophil release from the BM, and their mobilization involves rapidly induced Cxcr2-mediated motility controlled by TPO as a pacemaker.
Assuntos
Células da Medula Óssea/citologia , Fator Estimulador de Colônias de Granulócitos/imunologia , Neutrófilos/citologia , Receptores de Interleucina-8B/imunologia , Trombopoetina/imunologia , Animais , Medula Óssea/imunologia , Células da Medula Óssea/imunologia , Osso e Ossos/citologia , Linhagem Celular , Movimento Celular , Células Cultivadas , Humanos , Megacariócitos/citologia , Megacariócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologiaRESUMO
Organophosphorus (OP) pesticides are still widely applied but pose a severe toxicological threat if misused. For in vivo detoxification, the application of hydrolytic enzymes potentially offers a promising treatment. A well-studied example is the phosphotriesterase of Brevundimonas diminuta (BdPTE). Whereas wild-type BdPTE can hydrolyse pesticides like paraoxon, chlorpyrifos-oxon and mevinphos with high catalytic efficiencies, kcat/KM >2 × 107 M-1 min-1, degradation of malaoxon is unsatisfactory (kcat/KM ≈ 1 × 104 M-1 min-1). Here, we report the rational engineering of BdPTE mutants with improved properties and their efficient production in Escherichia coli. As result, the mutant BdPTE(VRNVVLARY) exhibits 37-fold faster malaoxon hydrolysis (kcat/KM = 4.6 × 105 M-1 min-1), together with enhanced expression yield, improved thermal stability and reduced susceptibility to oxidation. Therefore, this BdPTE mutant constitutes a powerful candidate to develop a biocatalytic antidote for the detoxification of this common pesticide metabolite as well as related OP compounds.
Assuntos
Praguicidas , Hidrolases de Triester Fosfórico , Praguicidas/metabolismo , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Malation , Compostos Organofosforados/metabolismoRESUMO
While cytosine-C5 methylation of DNA is an essential regulatory system in higher eukaryotes, the presence and relevance of 6-methyladenine (m6dA) in human cells is controversial. To study the role of m6dA in human DNA, we introduced it in human cells at a genome-wide scale at GANTC and GATC sites by expression of bacterial DNA methyltransferases and observed concomitant reductions in cell viability, in particular after global GANTC methylation. We identified several genes that are directly regulated by m6dA in a GANTC context. Upregulated genes showed m6dA-dependent reduction of H3K27me3 suggesting that the PRC2 complex is inhibited by m6dA. Genes downregulated by m6dA showed enrichment of JUN family transcription factor binding sites. JUN binds m6dA containing DNA with reduced affinity suggesting that m6dA can reduce the recruitment of JUN transcription factors to target genes. Our study documents that global introduction of m6dA in human DNA has physiological effects. Furthermore, we identified a set of target genes which are directly regulated by m6dA in human cells, and we defined two molecular pathways with opposing effects by which artificially introduced m6dA in GANTC motifs can directly control gene expression and phenotypes of human cells.
Assuntos
Metilação de DNA , DNA , Humanos , Células HEK293 , DNA/genética , Expressão GênicaRESUMO
The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A-ADD and MECP2-TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull-down, equilibrium peptide binding assays, and structural analyses. The region D529-D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N-terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214-228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2-TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.
Assuntos
DNA (Citosina-5-)-Metiltransferases , DNA Metiltransferase 3A , Proteína 2 de Ligação a Metil-CpG , Sítios de Ligação , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Proteína 2 de Ligação a Metil-CpG/química , Proteína 2 de Ligação a Metil-CpG/metabolismo , Células NIH 3T3 , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Histonas/química , Histonas/metabolismo , HumanosRESUMO
We report the finding of two copy number variants (CNVs) in a 12-year-old boy presenting both with autism spectrum disorder (ASD) and attention deficit/hyperactivity disorder (ADHD). Clinical features included aggressive behavior, mood instability, suicidal statements, repetitive and restrictive behavior, sensitivity to noise, learning problems and dyslexia, though no intellectual disability was present. Using array-based comparative genomic hybridization (array-CGH), we identified two CNVs, both triplex duplications of 324 kb on 3p26.3, and 284 kb on 4q13.1, respectively. One of the CNVs is located on chromosome 4q13.1 in the region of the gene encoding for adhesion G protein-coupled receptor L3 (ADGRL3, former name: latrophilin-3, LPHN3), the other on chromosome 3p26.3 in the region of the two pseudogenes AC090043.1 and RPL23AP39. The patient described in the present study showed increased symptoms under methylphenidate treatment but responded positively to 3 mg per day of the atypical neuroleptic drug aripiprazole. To our knowledge, this is the first report of a CNV in the ADGRL3 gene and its first association with ASD in humans.
RESUMO
Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.
Assuntos
Phormidium , Synechocystis , Sequência de Bases , Proteínas de Fluorescência Verde/metabolismo , Regiões Promotoras Genéticas , Synechocystis/genéticaRESUMO
Viral warts from immunosuppressed organ transplant recipients (OTR) persist over years and may progress into non-melanoma skin cancer. The types of human papillomaviruses (HPV) in such lesions are different from that seen in the general population. A subset of these lesions is not infected with the classical wart-associated HPV types. In order to gain a better understanding of the HPV types in those lesions, we isolated ten novel HPVs from persisting keratotic lesions of immunosuppressed OTRs by rolling circle amplification and subsequent long-template PCR. Additionally, we sequenced and characterized the whole genome of the ten novel HPV types. Phylogenetic analyses revealed that nine HPV types belonged to the genus Gammapapillomavirus (γ-PV) and one to the genus Betapapillomavirus. In a phylogenetic analysis using L1 fragments of human and non-human PV types, primate papillomaviruses and our novel HPV types nested within the genus γ-PV in a highly polyphyletic pattern. This study significantly broadens the knowledge concerning the diversity and evolution of the poorly known γ-PV types.
Assuntos
Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Dermatopatias/virologia , Idoso , Feminino , Genoma Viral , Genômica , Humanos , Hospedeiro Imunocomprometido , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Transplante de Órgãos/efeitos adversos , Papillomaviridae/classificação , Infecções por Papillomavirus/etiologia , Filogenia , Dermatopatias/imunologiaRESUMO
Aged hematopoietic stem cells (HSCs) are impaired in supporting hematopoiesis. The molecular and cellular mechanisms of stem cell aging are not well defined. HSCs interact with nonhematopoietic stroma cells in the bone marrow forming the niche. Interactions of hematopoietic cells with the stroma/microenvironment inside bone cavities are central to hematopoiesis as they regulate cell proliferation, self-renewal, and differentiation. We recently hypothesized that one underlying cause of altered hematopoiesis in aging might be due to altered interactions of aged stem cells with the microenvironment/niche. We developed time-lapse 2-photon microscopy and novel image analysis algorithms to quantify the dynamics of young and aged hematopoietic cells inside the marrow of long bones of mice in vivo. We report in this study that aged early hematopoietic progenitor cells (eHPCs) present with increased cell protrusion movement in vivo and localize more distantly to the endosteum compared with young eHPCs. This correlated with reduced adhesion to stroma cells as well as reduced cell polarity upon adhesion of aged eHPCs. These data support a role of altered eHPC dynamics and altered cell polarity, and thus altered niche biology in mechanisms of mammalian aging.
Assuntos
Envelhecimento/fisiologia , Osso e Ossos/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Movimento Celular , Macrófagos/citologia , Camundongos , Fatores de TempoRESUMO
The phosphotriesterase of the bacterium Brevundimonas diminuta (BdPTE) is a naturally occurring enzyme that catalyzes the hydrolysis of organophosphate (OP) nerve agents as well as pesticides and offers a potential treatment of corresponding intoxications. While BdPTE mutants with improved catalytic efficiencies against several OPs have been described, unexpectedly, less efficient breakdown of an OP was observed upon application in an animal model compared with in vitro measurements. Here, we describe detailed inhibition studies with the high-activity BdPTE mutant 10-2C3(C59M/C227A) by human plasma components, indicating that this enzyme is inhibited by serum albumin. The inhibitory activity is mediated by depletion of crucial zinc ions from the BdPTE active site, either via the known high-affinity zinc binding site of albumin or via chemical complex formation with its free thiol side chain at position Cys34. Albumin pre-charged with zinc ions or carrying a chemically blocked Cys34 side chain showed significantly reduced inhibitory activity; in fact, the combination of both measures completely abolished BdPTE inhibition. Consequently, the available zinc ion concentration in blood plays an important role for BdPTE activity in vivo and should be taken into account for therapeutic development and application of a catalytic OP scavenger.
Assuntos
Albuminas/farmacologia , Proteínas de Bactérias/farmacologia , Inibidores Enzimáticos/farmacologia , Intoxicação por Organofosfatos/tratamento farmacológico , Hidrolases de Triester Fosfórico/metabolismo , Hidrolases de Triester Fosfórico/uso terapêutico , Compostos de Sulfidrila/metabolismo , Albuminas/metabolismo , Proteínas de Bactérias/metabolismo , Caulobacteraceae/química , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Inibidores Enzimáticos/metabolismo , Modelos Animais , Compostos Organofosforados/metabolismo , Compostos de Sulfidrila/sangueRESUMO
Organophosphorus (OP)1 nerve agents pose a severe toxicological threat, both after dissemination in military conflicts and by terrorists. Hydrolytic enzymes, which may be administered into the blood stream of victims by injection and can decompose the circulating nerve agent into non-toxic metabolites in vivo, could offer a treatment. Indeed, for the phosphotriesterase found in the bacterium Brevundimonas diminuta (BdPTE),2 engineered versions with improved catalytic efficiencies have been described; yet, their biochemical stabilities are insufficient for therapeutic use. Here, we describe the application of rational protein design to develop novel mutants of BdPTE that are less susceptible to oxidative damage. In particular, the replacement of two unpaired cysteine residues by more inert amino acids led to higher stability while maintaining high catalytic activity towards a broad spectrum of substrates, including OP pesticides and V-type nerve agents. The mutant BdPTE enzymes were produced in Escherichia coli, purified to homogeneity, and their biochemical and enzymological properties were assessed. Several candidates both revealed enhanced thermal stability and were less susceptible to oxidative stress, as demonstrated by mass spectrometry. These mutants of BdPTE may show promise for the treatment of acute intoxications by nerve agents as well as OP pesticides.
Assuntos
Antídotos/farmacologia , Proteínas de Bactérias/farmacologia , Caulobacteraceae/enzimologia , Agentes Neurotóxicos/intoxicação , Intoxicação por Organofosfatos/tratamento farmacológico , Compostos Organofosforados/toxicidade , Hidrolases de Triester Fosfórico/farmacologia , Antídotos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacteraceae/genética , Estabilidade de Medicamentos , Estabilidade Enzimática , Temperatura Alta , Mutação , Intoxicação por Organofosfatos/enzimologia , Compostos Organotiofosforados/intoxicação , Oxirredução , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Desnaturação Proteica , Proteínas Recombinantes/farmacologia , Sarina/intoxicação , Soman/intoxicaçãoRESUMO
During bacterial conjugation, plasmid DNA is transferred from cell to cell. In Agrobacterium fabrum, conjugation is regulated by the phytochrome photoreceptors Agp1 and Agp2. Both contribute equally to this regulation. Agp1 and Agp2 are histidine kinases, but, for Agp2, we found no autophosphorylation activity. A clear autophosphorylation signal, however, was obtained with mutants in which the phosphoaccepting Asp of the C-terminal response regulator domain is replaced. Thus, the Agp2 histidine kinase differs from the classical transphosphorylation pattern. We performed size exclusion, photoconversion, dark reversion, autophosphorylation, chromophore assembly kinetics and fluorescence resonance energy transfer measurements on mixed Agp1/Agp2 samples. These assays pointed to an interaction between both proteins. This could partially explain the coaction of both phytochromes in the cell.