Jennifer K. Sabo

Binding hot spot for invasion inhibitory molecules on Plasmodium falciparum apical membrane antigen 1

ABSTRACT Apical membrane antigen 1 (AMA1) is expressed in schizont-stage malaria parasites and sporozoites and is thought to be involved in the invasion of host red blood cells. AMA1 is an important vaccine candidate, as immunization with this antigen induces a protective immune response in rodent and monkey models of human malaria. Additionally, anti-AMA1 polyclonal and monoclonal antibodies inhibit parasite invasion in vitro. We have isolated a 20-residue peptide (R1) from a random peptide library that binds to native AMA1 as expressed by Plasmodium falciparum parasites. Binding of R1 peptide is dependent on AMA1 having the proper conformation, is strain specific, and results in the inhibition of merozoite invasion of host erythrocytes. The solution structure of R1, as determined by nuclear magnetic resonance spectroscopy, contains two structured regions, both involving turns, but the first region, encompassing residues 5 to 10, is hydrophobic and the second, at residues 13 to 17, is more polar. Several lines of evidence reveal that R1 targets a “hot spot” on the AMA1 surface that is also recognized by other peptides and monoclonal antibodies that have previously been shown to inhibit merozoite invasion. The functional consequence of binding to this region by a variety of molecules is the inhibition of merozoite invasion into host erythrocytes. The interaction between these peptides and AMA1 may further our understanding of the molecular mechanisms of invasion by identifying critical functional regions of AMA1 and aid in the development of novel antimalarial strategies.

The SOCS box domain of SOCS3: structure and interaction with the elonginBC-cullin5 ubiquitin ligase.

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.

Differential Modulation of the Oligodendrocyte Transcriptome by Sonic Hedgehog and Bone Morphogenetic Protein 4 via Opposing Effects on Histone Acetylation

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.

Remyelination Is Altered by Bone Morphogenic Protein Signaling in Demyelinated Lesions

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.

Modulation of bone morphogenic protein signalling alters numbers of astrocytes and oligodendroglia in the subventricular zone during cuprizone‐induced demyelination

J. Neurochem. (2010) 115, 11–22.

The D-Diastereomer of ShK Toxin Selectively Blocks Voltage-gated K+ Channels and Inhibits T Lymphocyte Proliferation

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 (TEM); selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by TEM 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 Kd 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 TEM 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.

Rapid Optimization of a Peptide Inhibitor of Malaria Parasite Invasion by Comprehensive N-Methyl Scanning

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.

Effects of Bone Morphogenic Proteins on Neural Precursor Cells and Regulation during Central Nervous System Injury

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.

Solution structure and alanine scan of a spider toxin that affects the activation of mammalian voltage-gated sodium channels

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 β-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 β-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 β-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.

Peptide inhibitors of the malaria surface protein, apical membrane antigen 1: identification of key binding residues.

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 CαH 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.