ABSTRACT
Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Animals , Mice , Ribonucleoproteins/metabolism , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , Brain/metabolismABSTRACT
OBJECTIVE: Neonatal white matter injury (NWMI) is a lesion found in preterm infants that can lead to cerebral palsy. Although antagonists of bone morphogenetic protein (BMP) signaling, such as Noggin, promote oligodendrocyte precursor cell (OPC) production after hypoxic-ischemic (HI) injury, the downstream functional targets are poorly understood. The basic helix-loop-helix protein, oligodendrocyte transcription factor 1 (Olig1), promotes oligodendrocyte (OL) development and is essential during remyelination in adult mice. Here, we investigated whether Olig1 function is required downstream of BMP antagonism for response to injury in the neonatal brain. METHODS: We used wild-type and Olig1-null mice subjected to neonatal stroke and postnatal neural progenitor cultures, and we analyzed Olig1 expression in human postmortem samples from neonates that suffered HI encephalopathy (HIE). RESULTS: Olig1-null neonatal mice showed significant hypomyelination after moderate neonatal stroke. Surprisingly, damaged white matter tracts in Olig1-null mice lacked Olig2+ OPCs, and instead proliferating neuronal precursors and GABAergic interneurons were present. We demonstrate that Noggin-induced OPC production requires Olig1 function. In postnatal neural progenitors, Noggin governs production of OLs versus interneurons through Olig1-mediated repression of Dlx1/2 transcription factors. Additionally, we observed that Olig1 and the BMP signaling effector, phosphorylated SMADs (Sma- and Mad-related proteins) 1, 5, and 8, were elevated in the subventricular zone of human infants with HIE compared to controls. INTERPRETATION: These findings indicate that Olig1 has a critical function in regulation of postnatal neural progenitor cell production in response to Noggin. Ann Neurol 2017;81:560-571.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Bone Morphogenetic Proteins/metabolism , Carrier Proteins/metabolism , Hypoxia-Ischemia, Brain/metabolism , Nerve Tissue Proteins/metabolism , Oligodendroglia/metabolism , Stroke/metabolism , Animals , Cell Culture Techniques , Disease Models, Animal , Humans , Infant , Infant, Newborn , Infant, Newborn, Diseases , Mice , Mice, Inbred C57BL , Mice, Knockout , Neural Stem CellsABSTRACT
Multiple sclerosis is an autoimmune disease of the CNS resulting in degeneration of myelin sheaths and loss of oligodendrocytes, which means that protection and electrical insulation of axons and rapid signal propagation are impaired, leading to axonal damage and permanent disabilities. Partial replacement of lost oligodendrocytes and remyelination can occur as a result of activation and recruitment of resident oligodendroglial precursor cells. However, the overall remyelination capacity remains inefficient because precursor cells often fail to generate new oligodendrocytes. Increasing evidence points to the existence of several molecular inhibitors that act on these cells and interfere with their cellular maturation. The p57kip2 gene encodes one such potent inhibitor of oligodendroglial differentiation and this study sheds light on the underlying mode of action. We found that subcellular distribution of the p57kip2 protein changed during differentiation of rat, mouse, and human oligodendroglial cells both in vivo and in vitro. Nuclear export of p57kip2 was correlated with promoted myelin expression, higher morphological phenotypes, and enhanced myelination in vitro. In contrast, nuclear accumulation of p57kip2 resulted in blocked oligodendroglial differentiation. Experimental evidence suggests that the inhibitory role of p57kip2 depends on specific interactions with binding proteins such as LIMK-1, CDK2, Mash1, and Hes5 either by controlling their site of action or their activity. Because functional restoration in demyelinating diseases critically depends on the successful generation of oligodendroglial cells, a therapeutic need that is currently unmet, the regulatory mechanism described here might be of particular interest for identifying suitable drug targets and devising novel therapeutic approaches.
Subject(s)
Cell Differentiation/physiology , Cell Nucleus/metabolism , Cyclin-Dependent Kinase Inhibitor p57/metabolism , Oligodendroglia/metabolism , Active Transport, Cell Nucleus , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cells, Cultured , Cerebellar Cortex/metabolism , Cyclin-Dependent Kinase 2/metabolism , Humans , Lim Kinases/metabolism , Mice , Multiple Sclerosis/metabolism , Myelin Sheath/metabolism , Oligodendroglia/cytology , Protein Transport/physiology , Rats , Rats, Wistar , Repressor Proteins/metabolismABSTRACT
Differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes is regulated by the interplay between extrinsic signals and intrinsic epigenetic determinants. In this study, we analyze the effect that the extracellular ligands sonic hedgehog (Shh) and bone morphogenetic protein 4 (BMP4), have on histone acetylation and gene expression in cultured OPCs. Shh treatment favored the progression toward oligodendrocytes by decreasing histone acetylation and inducing peripheral chromatin condensation. BMP4 treatment, in contrast, inhibited the progression toward oligodendrocytes and favored astrogliogenesis by favoring global histone acetylation and retaining euchromatin. Pharmacological treatment or silencing of histone deacetylase 1 (Hdac1) or histone deacetylase 2 (Hdac2) in OPCs did not affect BMP4-dependent astrogliogenesis, while it prevented Shh-induced oligodendrocyte differentiation and favored the expression of astrocytic genes. Transcriptional profiling of treated OPCs, revealed that BMP4-inhibition of oligodendrocyte differentiation was accompanied by increased levels of Wnt (Tbx3) and Notch-target genes (Jag1, Hes1, Hes5, Hey1, and Hey2), decreased recruitment of Hdac and increased histone acetylation at these loci. Similar upregulation of Notch-target genes and increased histone acetylation were observed in the corpus callosum of mice infused with BMP4 during cuprizone-induced demyelination. We conclude that Shh and Bmp4 differentially regulate histone acetylation and chromatin structure in OPCs and that BMP4 acts as a potent inducer of gene expression, including Notch and Wnt target genes, thereby enhancing the crosstalk among signaling pathways that are known to inhibit myelination and repair.
Subject(s)
Bone Morphogenetic Protein 4/physiology , Hedgehog Proteins/physiology , Histone Deacetylase 1/metabolism , Histone Deacetylase 2/metabolism , Histones/metabolism , Oligodendroglia/physiology , Transcriptome/genetics , Acetylation , Animals , Animals, Newborn , Cells, Cultured , Female , Gene Silencing , Histone Deacetylase 1/antagonists & inhibitors , Histone Deacetylase 1/genetics , Histone Deacetylase 2/antagonists & inhibitors , Histone Deacetylase 2/genetics , Histones/antagonists & inhibitors , Histones/genetics , Mice , Mice, Inbred C57BL , Oligodendroglia/metabolism , RatsABSTRACT
In demyelinating disorders such as Multiple Sclerosis (MS), targets of injury are myelin and oligodendrocytes, leading to severe neurological dysfunction. Regenerative therapies aimed at promoting oligodendrocyte maturation and remyelination are promising strategies for treatment in demyelinating disorders. Endogenous precursor cells or exogenous transplanted cells are potential sources for remyelinating oligodendrocytes in the central nervous system (CNS). Several signalling pathways have been implicated in regulating the capacity of these cell populations for myelin repair. Here, we review neural precursor cells and oligodendrocyte progenitor cells as potential sources for remyelinating oligodendrocytes and evidence for the functional role of key signalling pathways in inhibiting regeneration from these precursor cell populations.
Subject(s)
Demyelinating Diseases/physiopathology , Myelin Sheath/physiology , Neural Stem Cells/physiology , Signal Transduction , Animals , Cell Transplantation/methods , Demyelinating Diseases/therapy , Humans , Multiple Sclerosis/physiopathology , Multiple Sclerosis/therapy , Nerve Regeneration/physiology , Neural Stem Cells/transplantation , Oligodendroglia/physiology , Oligodendroglia/transplantationABSTRACT
Remyelination of the CNS involves the regeneration of mature oligodendrocytes by endogenous oligodendrocyte progenitor cells (OPCs). Previous studies have shown that bone morphogenic proteins (BMPs) inhibit the production of oligodendrocytes in the healthy CNS. However, there is currently no information on the influence of BMP signaling in vivo within demyelinated lesions of the brain or on subsequent remyelination. Here, we determine a role for BMP signaling in modulating oligodendrogliogenesis and remyelination in the brain following cuprizone-induced demyelination. We identified that BMP signaling is active in oligodendroglia and astrocytes within the demyelinated corpus callosum. Intraventricular infusion of BMP4 into the brains of mice during demyelination increased the proliferation of OPCs and, to a lesser extent, microglia and astrocytes in the corpus callosum. In contrast, infusion of Noggin, an extracellular antagonist of BMP4, increased the density of mature oligodendrocytes in the remyelinating corpus callosum. Additional evidence from myelin staining and electron microscopy indicates there is an increase in remyelinated axons in the corpus callosum of Noggin-infused mice. Thus, inhibition of endogenous BMP signaling during demyelination promotes mature oligodendrocyte regeneration and remyelination.
Subject(s)
Bone Morphogenetic Proteins/physiology , Demyelinating Diseases/pathology , Myelin Sheath/physiology , Signal Transduction/physiology , Animals , Antimetabolites , Astrocytes/physiology , Bone Morphogenetic Protein 4/pharmacology , Bromodeoxyuridine , Carrier Proteins/genetics , Carrier Proteins/physiology , Cell Count , Chelating Agents , Corpus Callosum/pathology , Cuprizone , Demyelinating Diseases/chemically induced , Female , Mice , Mice, Inbred C57BL , Microscopy, Electron , Neuroglia/physiology , Oligodendroglia/physiology , Stem Cells/physiologyABSTRACT
The adult subventricular zone (SVZ) is a potential source of precursor cells to replace neural cells lost during demyelination. To better understand the molecular events that regulate neural precursor cell responsiveness in this context we undertook a microarray and quantitative PCR based analysis of genes expressed within the SVZ during cuprizone-induced demyelination. We identified an up-regulation of the genes encoding bone morphogenic protein 4 (BMP4) and its receptors. Immunohistochemistry confirmed an increase in BMP4 protein levels and also showed an increase in phosphorylated SMAD 1/5/8, a key component of BMP4 signalling, during demyelination. In vitro analysis revealed that neural precursor cells isolated from demyelinated animals, as well as those treated with BMP4, produce more astrocytes. Similarly, there were increased numbers of astrocytes in vivo within the SVZ during demyelination. Intraventricular infusion of Noggin, an endogenous antagonist of BMP4, during cuprizone-induced demyelination reduced pSMAD1/5/8, decreased astrocyte numbers and increased oligodendrocyte numbers in the SVZ. Our results suggest that lineage commitment of SVZ neural precursor cells is altered during demyelination and that BMP signalling plays a role in this process.
Subject(s)
Astrocytes/drug effects , Bone Morphogenetic Proteins/physiology , Cerebral Ventricles/pathology , Demyelinating Diseases/pathology , Oligodendroglia/drug effects , Signal Transduction/physiology , Animals , Antimetabolites , Bone Morphogenetic Protein 4/antagonists & inhibitors , Bone Morphogenetic Protein 4/genetics , Bone Morphogenetic Protein 4/physiology , Bone Morphogenetic Protein Receptors/antagonists & inhibitors , Bone Morphogenetic Proteins/genetics , Brain/cytology , Brain/immunology , Bromodeoxyuridine , Carrier Proteins/pharmacology , Cell Count , Cell Differentiation/drug effects , Cell Lineage , Cell Proliferation/drug effects , Cerebral Ventricles/drug effects , Cuprizone , Demyelinating Diseases/chemically induced , Injections, Intraventricular , Mice , Mice, Inbred C57BL , Microdissection , Monoamine Oxidase Inhibitors , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effectsABSTRACT
CRISPR gene editing technology is strategically foreseen to control diseases by correcting underlying aberrant genetic sequences. In order to overcome drawbacks associated with viral vectors, the establishment of an effective non-viral CRISPR delivery vehicle has become an important goal for nanomaterial scientists. Herein, we introduce a monosized lipid-coated mesoporous silica nanoparticle (LC-MSN) delivery vehicle that enables both loading of CRISPR components [145 µg ribonucleoprotein (RNP) or 40 µg plasmid/mg nanoparticles] and efficient release within cancer cells (70%). The RNP-loaded LC-MSN exhibited 10% gene editing in both in vitro reporter cancer cell lines and in an in vivo Ai9-tdTomato reporter mouse model. The structural and chemical versatility of the mesoporous silica core and lipid coating along with framework dissolution-assisted cargo delivery open new prospects towards safe CRISPR component delivery and enhanced gene editing. STATEMENT OF SIGNIFICANCE: After the discovery of CRISPR gene-correcting technology in bacteria. The translation of this technology to mammalian cells may change the face of cancer therapy within the next years. This was first made possible through the use of viral vectors; however, such systems limit the safe translation of CRISPR into clinics because its difficult preparation and immunogenicity. Therefore, biocompatible non-viral nanoparticulate systems are required to successfully deliver CRISPR into cancer cells. The present study presents the use of biomimetic lipid-coated mesoporous silica nanoparticles showing successful delivery of CRISPR ribonucleoprotein and plasmid into HeLa cervical and A549 lung cancer cells as well as successful gene editing in mice brain.
Subject(s)
Nanoparticles , Silicon Dioxide , Animals , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Editing , Humans , Lipid Bilayers , MiceABSTRACT
Bone morphogenic proteins (BMPs) are well known for their influence on cell fate determination, proliferation and differentiation during early embryogenesis. Here, we review evidence for BMPs playing an additional, ongoing role in the proliferation and differentiation of neural precursor and progenitor cells in postnatal and adult central nervous system (CNS) and in CNS injury. The effects of BMPs on CNS cells have been studied using primary cultures of neural precursor and oligodendrocyte lineage cells. In addition, transgenic mice have been used to investigate in vivo effects of altering BMP pathway activation, and rodent models of CNS injury have been used to examine endogenous regulation of BMPs. These results have shown that BMPs promote production of astrocytes and inhibit production and maturation of oligodendroglia. The effects of BMPs on neurogenesis could be dependent on the origin of precursor cells or on the specifics of the microenvironment of the cell niche, as there are reports of inhibition and promotion of neurogenesis by BMPs. There is emerging evidence that BMPs are upregulated in several models of CNS injury; however, the effects of this regulation have not been well characterised. Understanding of the function of endogenous BMP regulation is important for determining how modulation of BMP signalling could improve repair following CNS injury.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Central Nervous System/injuries , Central Nervous System/physiopathology , Neurogenesis/physiology , Neurons/physiology , Stem Cells/physiology , Animals , Central Nervous System/embryology , Humans , Models, Neurological , Signal TransductionABSTRACT
Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.
Subject(s)
Brain Neoplasms/blood supply , Glioma/blood supply , Oligodendrocyte Transcription Factor 2/metabolism , Oligodendroglia/microbiology , Wnt Proteins/metabolism , Animals , Bevacizumab/pharmacology , Blood-Brain Barrier/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/drug effects , Glioma/drug therapy , Glioma/metabolism , Humans , Mice , Neoplasm Transplantation , Oligodendrocyte Transcription Factor 2/genetics , Temozolomide/pharmacology , Tumor Cells, Cultured , Tumor Microenvironment , Wnt Proteins/genetics , Wnt Signaling Pathway/drug effectsABSTRACT
We demonstrate editing of post-mitotic neurons in the adult mouse brain following injection of Cas9 ribonucleoprotein (RNP) complexes in the hippocampus, striatum and cortex. Engineered variants of Cas9 with multiple SV40 nuclear localization sequences enabled a tenfold increase in the efficiency of neuronal editing in vivo. These advances indicate the potential of genome editing in the brain to correct or inactivate the underlying genetic causes of neurological diseases.
Subject(s)
Bacterial Proteins/genetics , Brain/physiology , CRISPR-Associated Proteins/genetics , Endonucleases/genetics , Gene Editing/methods , Nerve Tissue Proteins/physiology , Ribonucleoproteins/genetics , Animals , CRISPR-Associated Protein 9 , CRISPR-Associated Proteins/administration & dosage , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Targeting/methods , Male , Mice , Protein Engineering/methodsABSTRACT
Repair in multiple sclerosis involves remyelination, a process in which axons are provided with a new myelin sheath by new oligodendrocytes. Bone morphogenic proteins (BMPs) are a family of growth factors that have been shown to influence the response of oligodendrocyte progenitor cells (OPCs) in vivo during demyelination and remyelination in the adult brain. We have previously shown that BMP4 infusion increases numbers of OPCs during cuprizone-induced demyelination, while infusion of Noggin, an endogenous antagonist of BMP4 increases numbers of mature oligodendrocytes and remyelinated axons following recovery. Additional studies have shown that insulin-like growth factor-1 (IGF-1) promotes the survival of OPCs during cuprizone-induced demyelination. Based on these data, we investigated whether myelin repair could be further enhanced by sequential infusion of these agents firstly, BMP4 to increase OPC numbers, followed by either Noggin or IGF-1 to increase the differentiation and survival of the newly generated OPCs. We identified that sequential delivery of BMP4 and IGF-1 during cuprizone challenge increased the number of mature oligodendrocytes and decreased astrocyte numbers following recovery compared with vehicle infused mice, but did not alter remyelination. However, sequential delivery of BMP4 and Noggin during cuprizone challenge did not alter numbers of oligodendrocytes or astrocytes in the corpus callosum compared with vehicle infused mice. Furthermore, electron microscopy analysis revealed no change in average myelin thickness in the corpus callosum between vehicle infused and BMP4-Noggin infused mice. Our results suggest that while single delivery of Noggin or IGF-1 increased the production of mature oligodendrocytes in vivo in the context of demyelination, only Noggin infusion promoted remyelination. Thus, sequential delivery of BMP4 and Noggin or IGF-1 does not further enhance myelin repair above what occurs with delivery of Noggin alone.
Subject(s)
Bone Morphogenetic Protein 4/administration & dosage , Demyelinating Diseases/drug therapy , Myelin Sheath/physiology , Animals , Astrocytes/drug effects , Bone Morphogenetic Protein 4/pharmacology , Carrier Proteins/administration & dosage , Cell Differentiation , Corpus Callosum/drug effects , Corpus Callosum/pathology , Cuprizone , Demyelinating Diseases/chemically induced , Demyelinating Diseases/pathology , Drug Evaluation, Preclinical , Drug Therapy, Combination , Insulin-Like Growth Factor I/administration & dosage , Mice , Mice, Inbred C57BL , Neural Stem Cells/physiology , Oligodendroglia/drug effects , Oligodendroglia/physiologyABSTRACT
Apical membrane antigen 1 (AMA1) is essential for malaria parasite invasion of erythrocytes and is therefore an attractive target for drug development. Peptides that bind AMA1 have been identified from random peptide libraries expressed on the surface of phage. Of these, R1, which binds to a hydrophobic ligand binding site on AMA1, was a particularly potent inhibitor of parasite invasion of erythrocytes in vitro. The solution structure of R1 contains a turn-like conformation between residues 5-10. Here the importance of residues in this turn-like structure for binding to AMA1 was examined by site-directed mutagenesis and NMR spectroscopy. The peptide was expressed as a fusion protein following replacement of Met16 by Leu in order to accommodate cyanogen bromide cleavage. This modified peptide (R2) displayed the same affinity for AMA1 as R1, showing that the identity of the side chain at position 16 was not critical for binding. Substitution of Phe5, Pro7, Leu8, and Phe9 with alanine led to significant (7.5- to >350-fold) decreases in affinity for AMA1. Comparison of backbone amide and C(α) H chemical shifts for these R2 analogues with corresponding values for R2 showed no significant changes, with the exception of R2(P7A), where slightly larger differences were observed, particularly for residues flanking position 7. The absence of significant changes in the secondary chemical shifts suggests that these mutations had little effect on the solution conformation of R2. The identification of a nonpolar region of these peptides containing residues essential for AMA1 binding establishes a basis for the design of anti-malarial drugs based on R1 mimetics.
Subject(s)
Antimalarials/pharmacology , Membrane Proteins/antagonists & inhibitors , Peptides/pharmacology , Protozoan Proteins/antagonists & inhibitors , Amino Acid Sequence , Amino Acid Substitution , Animals , Antigens, Protozoan/chemistry , Antigens, Protozoan/genetics , Antimalarials/chemistry , Binding Sites/genetics , Drug Design , Erythrocytes/drug effects , Erythrocytes/parasitology , Humans , In Vitro Techniques , Membrane Proteins/chemistry , Membrane Proteins/genetics , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptide Library , Peptides/chemistry , Peptides/genetics , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Plasmodium falciparum/pathogenicity , Protein Conformation , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Virulence/drug effectsABSTRACT
The SOCS (suppressors of cytokine signalling) family of proteins inhibits the cytokine-induced signalling cascade in part by promoting the ubiquitination of signalling intermediates that are then targeted for proteasomal degradation. This activity relies upon an interaction between the SOCS box domain, the adapter complex elonginBC and a member of the Cullin family, the scaffold protein of an E3 ubiquitin ligase. In this study, we dissected this interaction in vitro using purified components.We found that all eight SOCS proteins bound Cullin5 but required prior recruitment of elonginBC. Neither SOCS nor elonginBC bound Cullin5 when in isolation. Interestingly, the affinity of each SOCS-elonginBC complex for Cullin5 varied by 2 orders of magnitude across the SOCS family. Unexpectedly, the most potent suppressors of signalling, SOCS-1 and SOCS-3, bound most weakly to the E3 ligase scaffold, with affinities 100- and 10-fold lower, respectively, than the rest of the family. The remaining six SOCS proteins all bound Cullin5 with high affinity (K(d) of ~10 nM) due to a slower off-rate and hence a longer halflife of the complex. This difference in affinity may reflect a difference in mode of action as only SOCS-1 and SOCS-3 have been shown to suppress signalling using both SOCS box-dependent and SOCS box-independent mechanisms. This is not the case with the other six SOCS proteins, and our data imply the existence of two distinct subclasses of SOCS proteins with a high affinity for Cullin5, the E3 ligase scaffold, possibly reflecting complete dependence upon ubiquitination for suppression of cytokine signalling.
Subject(s)
Cullin Proteins/metabolism , Cytokines/metabolism , Signal Transduction , Suppressor of Cytokine Signaling Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Animals , Calorimetry , Cullin Proteins/genetics , Mice , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Sequence Homology, Amino Acid , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/chemistry , Suppressor of Cytokine Signaling Proteins/genetics , Surface Plasmon ResonanceABSTRACT
Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum has been implicated in the invasion of host erythrocytes and is an important vaccine candidate. We have previously described a 20-residue peptide, R1, that binds to AMA1 and subsequently blocks parasite invasion. Because this peptide appears to target a site critical for AMA1 function, it represents an important lead compound for anti-malarial drug development. However, the effectiveness of this peptide inhibitor was limited to a subset of parasite isolates, indicating a requirement for broader strain specificity. Furthermore, a barrier to the utility of any peptide as a potential therapeutic is its susceptibility to rapid proteolytic degradation. In this study, we sought to improve the proteolytic stability and AMA1 binding properties of the R1 peptide by systematic methylation of backbone amides (N-methylation). The inclusion of a single N-methyl group in the R1 peptide backbone dramatically increased AMA1 affinity, bioactivity, and proteolytic stability without introducing global structural alterations. In addition, N-methylation of multiple R1 residues further improved these properties. Therefore, we have shown that modifications to a biologically active peptide can dramatically enhance activity. This approach could be applied to many lead peptides or peptide therapeutics to simultaneously optimize a number of parameters.
Subject(s)
Antimalarials/pharmacology , Peptides/pharmacology , Plasmodium falciparum/drug effects , Amino Acid Sequence , Animals , Antimalarials/chemistry , Methylation , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptides/chemistry , Substrate Specificity , Surface Plasmon Resonance , Time FactorsABSTRACT
Suppressor of cytokine signalling 3 (SOCS3) is responsible for regulating the cellular response to a variety of cytokines, including interleukin 6 and leukaemia inhibitory factor. Identification of the SOCS box domain led to the hypothesis that SOCS3 can associate with functional E3 ubiquitin ligases and thereby induce the degradation of bound signalling proteins. This model relies upon an interaction between the SOCS box, elonginBC and a cullin protein that forms the E3 ligase scaffold. We have investigated this interaction in vitro using purified components and show that SOCS3 binds to elonginBC and cullin5 with high affinity. The SOCS3-elonginBC interaction was further characterised by determining the solution structure of the SOCS box-elonginBC ternary complex and by deletion and alanine scanning mutagenesis of the SOCS box. These studies revealed that conformational flexibility is a key feature of the SOCS-elonginBC interaction. In particular, the SOCS box is disordered in isolation and only becomes structured upon elonginBC association. The interaction depends upon the first 12 residues of the SOCS box domain and particularly on a deeply buried, conserved leucine. The SOCS box, when bound to elonginBC, binds tightly to cullin5 with 100 nM affinity. Domains upstream of the SOCS box are not required for elonginBC or cullin5 association, indicating that the SOCS box acts as an independent binding domain capable of recruiting elonginBC and cullin5 to promote E3 ligase formation.
Subject(s)
Cullin Proteins/metabolism , Suppressor of Cytokine Signaling Proteins/chemistry , Suppressor of Cytokine Signaling Proteins/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Alanine/metabolism , Amino Acid Sequence , Animals , Binding Sites , Calorimetry , Cullin Proteins/chemistry , Elongin , Epitopes , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis , Mutant Proteins/metabolism , Protein Binding , Suppressor of Cytokine Signaling 3 Protein , Transcription Factors/chemistry , Transcription Factors/isolation & purification , Ubiquitin-Protein Ligases/chemistry , Ultracentrifugation , src Homology DomainsABSTRACT
The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which are crucial in the activation of human effector memory T cells (T(EM)); selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T(EM) cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. The critical motif on the toxin for potassium channel blockade consists of neighboring lysine and tyrosine residues. Because this motif is sufficient for activity, an ShK analogue was designed based on D-amino acids. D-allo-ShK has a structure essentially identical with that of ShK and is resistant to proteolysis. It blocked Kv1.3 with K(d) 36 nm (2,800-fold lower affinity than ShK), was 2-fold selective for Kv1.3 over Kv1.1, and was inactive against other K(+) channels tested. D-allo-ShK inhibited human T(EM) cell proliferation at 100-fold higher concentration than ShK. Its circulating half-life was only slightly longer than that of ShK, implying that renal clearance is the major determinant of its plasma levels. D-allo-ShK did not bind to the closed state of the channel, unlike ShK. Models of D-allo-ShK bound to Kv1.3 show that it can block the pore as effectively as ShK but makes different interactions with the vestibule, some of which are less favorable than for native ShK. The finding that an all-D analogue of a polypeptide toxin retains biological activity and selectivity is highly unusual. Being resistant to proteolysis and nonantigenic, this analogue should be useful in K(+) channel studies; all-d analogues with improved Kv1.3 potency and specificity may have therapeutic advantages.
Subject(s)
Cell Division/drug effects , Cnidarian Venoms/toxicity , Kv1.3 Potassium Channel/antagonists & inhibitors , Potassium Channels, Voltage-Gated/antagonists & inhibitors , T-Lymphocytes/cytology , Amino Acid Sequence , Cnidarian Venoms/chemistry , Humans , Immunologic Memory , Kinetics , Kv1.3 Potassium Channel/drug effects , Models, Molecular , Molecular Sequence Data , Potassium Channels, Voltage-Gated/drug effects , Potassium Channels, Voltage-Gated/physiology , Protein Conformation , T-Lymphocytes/drug effectsABSTRACT
Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide containing three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltage-gated sodium channels and competes with scorpion beta-toxins, such as Css IV from Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of the mammalian rNav1.2a channel to more hyperpolarized voltages, whereas the insect channel, DmNav1, is not affected. To gain insight into toxin-channel interactions, Magi 5 and 23 analogues were synthesized. The three-dimensional structure of Magi 5 in aqueous solution was determined, and its voltage-gated sodium channel-binding surfaces were mapped onto this structure using data from electrophysiological measurements on a series of Ala-substituted analogues. The structure clearly resembles the inhibitor cystine knot structural motif, although the triple-stranded beta-sheet typically found in that motif is partially distorted in Magi 5. The interactive surface of Magi 5 toward voltage-gated sodium channels resembles in some respects the Janus-faced atracotoxins, with functionally important charged residues on one face of the toxin and hydrophobic residues on the other. Magi 5 also resembles the scorpion beta-toxin Css IV, which has distinct nonpolar and charged surfaces that are critical for channel binding and has a key Glu involved in voltage sensor trapping. These two distinct classes of toxin, with different amino acid sequences and different structures, may utilize similar groups of residues on their surface to achieve the common end of modifying voltage-gated sodium channel function.
Subject(s)
Models, Molecular , Sodium Channels/chemistry , Spider Venoms/chemistry , Animals , Humans , Membrane Potentials/drug effects , Mutation, Missense , Protein Structure, Tertiary/genetics , Scorpion Venoms/chemistry , Scorpion Venoms/pharmacology , Sequence Homology, Amino Acid , Sodium Channels/genetics , Sodium Channels/metabolism , Spider Venoms/pharmacology , Structure-Activity Relationship , Xenopus laevisABSTRACT
Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum is an integral membrane protein that plays a key role in merozoite invasion of host erythrocytes. A monoclonal antibody, 4G2dc1, recognizes correctly folded AMA1 and blocks merozoite invasion. Phage display was used to identify peptides that bind to 4G2dc1 and mimic an important epitope of AMA1. Three of the highest-affinity binders--J1, J3, and J7--were chosen for antigenicity and immunogenicity studies. J1 and J7 were found to be true antigen mimics since both peptides generated inhibitory antibodies in rabbits (J. L. Casey et al., Infect. Immun. 72:1126-1134, 2004). In the present study, the solution structures of all three mimotopes were investigated by nuclear magnetic resonance spectroscopy. J1 adopted a well-defined region of structure, which can be attributed in part to the interactions of Trp11 with surrounding residues. In contrast, J3 and J7 did not adopt an ordered conformation over the majority of residues, although they share a region of local structure across their consensus sequence. Since J1 was the most structured of the peptides, it provided a template for the design of a constrained analogue, J1cc, which shares a structure similar to that of J1 and has a disulfide-stabilized conformation around the Trp11 region. J1cc binds with greater affinity to 4G2dc1 than does J1. These peptide structures provide the foundation for a better understanding of the complex conformational nature of inhibitory epitopes on AMA1. With its greater conformational stability and higher affinity for AMA1, J1cc may be a better in vitro correlate of immunity than the peptides identified by phage display.