Head-to-Head Comparison of Soluble vs. Qβ VLP Circumsporozoite Protein Vaccines Reveals Selective Enhancement of NANP Repeat Responses

Circumsporozoite protein (CSP) of Plasmodium falciparum is a promising malaria vaccine target. RTS,S, the most advanced malaria vaccine candidate consists of the central NANP repeat and carboxy-terminal region of CSP displayed on a hepatitis B virus-like particle (VLP). To build upon the success of RTS,S, we produced a near full-length Plasmodium falciparum CSP that also includes the conserved amino-terminal region of CSP. We recently showed that this soluble CSP, combined with a synthetic Toll-like-receptor-4 (TLR4) agonist in stable oil-in-water emulsion (GLA/SE), induces a potent and protective immune response in mice against transgenic parasite challenge. Here we have investigated whether the immunogenicity of soluble CSP could be further augmented by presentation on a VLP. Bacteriophage Qβ VLPs can be readily produced in E.coli, they have a diameter of 25 nm and contain packaged E. coli RNA which serves as a built in adjuvant through the activation of TLR7/8. CSP was chemically conjugated to Qβ and the CSP-Qβ vaccine immunogenicity and efficacy were compared to adjuvanted soluble CSP in the C57Bl/6 mouse model. When formulated with adjuvants lacking a TLR4 agonist (Alum, SE and Montanide) the Qβ-CSP induced higher anti-NANP repeat titers, higher levels of cytophilic IgG2b/c antibodies and a trend towards higher protection against transgenic parasite challenge as compared to soluble CSP formulated in the same adjuvant. The VLP and soluble CSP immunogenicity difference was most pronounced at low antigen dose, and within the CSP molecule, the titers against the NANP repeats were preferentially enhanced by Qβ presentation. While a TLR4 agonist enhanced the immunogenicity of soluble CSP to levels comparable to the VLP vaccine, the TLR4 agonist did not further improve the immunogenicity of the Qβ-CSP vaccine. The data presented here pave the way for further improvement in the Qβ conjugation chemistry and evaluation of both the Qβ-CSP and soluble CSP vaccines in the non-human primate model.     

Colorimetric estimation of human glucose level using γ-Fe2O3 nanoparticles: An easily recoverable effective mimic peroxidase

This article reports simple, green and efficient synthesis of γ-Fe₂O₃ nanoparticles (NPs) (maghemite) through single-source precursor approach for colorimetric estimation of human glucose level. The γ-Fe₂O₃ NPs, having cubic morphology with an average particle size of 30 nm, exhibited effective peroxidase-like activity through the catalytic oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂ producing a blue-colored solution. On the basis of this colored-reaction, we have developed a simple, cheap, highly sensitive and selective colorimetric method for estimation of glucose using γ-Fe₂O₃/TMB/glucose–glucose oxidase (GOx) system in the linear range from 1 to 80 μM with detection limit of 0.21 μM. The proposed glucose sensor displays faster response, good stability, reproducibility and anti-interference ability. Based on this simple reaction process, human blood and urine glucose level can be monitored conveniently.     

Blocking Dynamics of the SMR Transporter EmrE Impairs Efflux Activity

EmrE is a small multidrug resistance transporter that has been well studied as a model for secondary active transport. Because transport requires the protein to convert between at least two states open to opposite sides of the membrane, it is expected that blocking these conformational transitions will prevent transport activity. We have previously shown that NMR can quantitatively measure the transition between the open-in and open-out states of EmrE in bicelles. Now, we have used the antiparallel EmrE crystal structure to design a cross-link to inhibit this conformational exchange process. We probed the structural, dynamic, and functional effects of this cross-link with NMR and in vivo efflux assays. Our NMR results show that our antiparallel cross-link performs as predicted: dramatically reducing conformational exchange while minimally perturbing the overall structure of EmrE and essentially trapping EmrE in a single state. The same cross-link also impairs ethidium efflux activity by EmrE in Escherichia coli. This confirms the hypothesis that transport can be inhibited simply by blocking conformational transitions in a properly folded transporter. The success of our cross-linker design also provides further evidence that the antiparallel crystal structure provides a good model for functional EmrE.     

Blocking Dynamics of the SMR Transporter EmrE Impairs Efflux Activity

EmrE is a small multidrug resistance transporter that has been well studied as a model for secondary active transport. Because transport requires the protein to convert between at least two states open to opposite sides of the membrane, it is expected that blocking these conformational transitions will prevent transport activity. We have previously shown that NMR can quantitatively measure the transition between the open-in and open-out states of EmrE in bicelles. Now, we have used the antiparallel EmrE crystal structure to design a cross-link to inhibit this conformational exchange process. We probed the structural, dynamic, and functional effects of this cross-link with NMR and in vivo efflux assays. Our NMR results show that our antiparallel cross-link performs as predicted: dramatically reducing conformational exchange while minimally perturbing the overall structure of EmrE and essentially trapping EmrE in a single state. The same cross-link also impairs ethidium efflux activity by EmrE in Escherichia coli. This confirms the hypothesis that transport can be inhibited simply by blocking conformational transitions in a properly folded transporter. The success of our cross-linker design also provides further evidence that the antiparallel crystal structure provides a good model for functional EmrE.     

Structural adaptation of tooth enamel protein amelogenin in the presence of SDS micelles

Amelogenin, the major extracellular matrix protein of developing tooth enamel is intrinsically disordered. Through its interaction with other proteins and mineral, amelogenin assists enamel biomineralization by controlling the formation of highly organized enamel crystal arrays. We used circular dichroism (CD), dynamic light scattering (DLS), fluorescence, and NMR spectroscopy to investigate the folding propensity of recombinant porcine amelogenin rP172 following its interaction with SDS, at levels above critical micelle concentration. The rP172‐SDS complex formation was confirmed by DLS, while an increase in the structure moiety of rP172 was noted through CD and fluorescence experiments. Fluorescence quenching analyses performed on several rP172 mutants where all but one Trp was replaced by Tyr at different sequence regions confirmed that the interaction of amelogenin with SDS micelles occurs via the N‐terminal region close to Trp25 where helical segments can be detected by NMR. NMR spectroscopy and structural refinement calculations using CS‐Rosetta modeling confirm that the highly conserved N‐terminal domain is prone to form helical structure when bound to SDS micelles. Our findings reported here reveal interactions leading to significant changes in the secondary structure of rP172 upon treatment with SDS. These interactions may reflect the physiological relevance of the flexible nature of amelogenin and its sequence specific helical propensity that might enable it to structurally adapt with charged and potential targets such as cell surface, mineral, and other proteins during enamel biomineralization. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 525–535, 2014.     

Neurons under viral attack: Victims or warriors?

When the central nervous system (CNS) is under viral attack, defensive antiviral responses must necessarily arise from the CNS itself to rapidly and efficiently curb infections with minimal collateral damage to the sensitive, specialized and non-regenerating neural tissue. This presents a unique challenge because an intact blood–brain barrier (BBB) and lack of proper lymphatic drainage keeps the CNS virtually outside the radar of circulating immune cells that are at constant vigilance for antigens in peripheral tissues. Limited antigen presentation skills of CNS cells in comparison to peripheral tissues is because of a total lack of dendritic cells and feeble expression of major histocompatibility complex (MHC) proteins in neurons and glia. However, research over the past two decades has identified immune effector mechanisms intrinsic to the CNS for immediate tackling, attenuating and clearing of viral infections, with assistance pouring in from peripheral circulation in the form of neutralizing antibodies and cytotoxic T cells at a later stage. Specialized CNS cells, microglia and astrocytes, were regarded as sole sentinels of the brain for containing a viral onslaught but neurons held little recognition as a potential candidate for protecting itself from the proliferation and pathogenesis of neurotropic viruses. Accumulating evidence however indicates that extracellular insult causes neurons to express immune factors characteristic of lymphoid tissues. This article aims to comprehensively analyze current research on this conditional alteration in the protein expression repertoire of neurons and the role it plays in CNS innate immune response to counter viral infections.     

SHP2 Mediates the Localized Activation of Fyn Downstream of the α6β4 Integrin To Promote Carcinoma Invasion

Src family kinase (SFK) activity is elevated in many cancers, and this activity correlates with aggressive tumor behavior. The α6β4 integrin, which is also associated with a poor prognosis in many tumor types, can stimulate SFK activation; however, the mechanism by which it does so is not known. In the current study, we provide novel mechanistic insight into how the α6β4 integrin selectively activates the Src family member Fyn in response to receptor engagement. Both catalytic and noncatalytic functions of SHP2 are required for Fyn activation by α6β4. Specifically, the tyrosine phosphatase SHP2 is recruited to α6β4 and its catalytic activity is stimulated through a specific interaction of its N-terminal SH2 domain with pY1494 in the β4 subunit. Fyn is recruited to the α6β4/SHP2 complex through an interaction with phospho-Y580 in the C terminus of SHP2. In addition to activating Fyn, this interaction with Y580-SHP2 localizes Fyn to sites of receptor engagement, which is required for α6β4-dependent invasion. Of significance for tumor progression, phosphorylation of Y580-SHP2 and SFK activation are increased in orthotopic human breast tumors that express α6β4 and activation of this pathway is dependent upon Y1494.Expression of the α6β4 integrin, a laminin receptor, is associated with poor patient prognosis and reduced survival in many human cancers (32). For this reason, there is considerable interest in understanding how this integrin is regulated and how it functions to promote tumor progression. In normal tissues, the α6β4 integrin plays a major role in maintaining the integrity of epithelia by binding to laminins in the basement membrane and regulating the assembly of hemidesmosomes on the basal epithelial cell surface (7, 17). In pathophysiological conditions such as wound healing and cancer, the stable adhesive interactions of the α6β4 receptor are disrupted by phosphorylation of the β4 cytoplasmic domain, converting α6β4 to a signaling-competent receptor that promotes dynamic adhesion and invasion (18). Phosphorylation of the β4 subunit cytoplasmic domain on serine residues contributes to the dynamic adhesive functions of the receptor by disrupting interactions with hemidesmosomal proteins that regulate stable adhesion (33, 37). Phosphorylation of the β4 cytoplasmic domain on tyrosine residues may also contribute to the regulation of hemidesmosomes, but it is likely that the major contribution of tyrosyl phosphorylation is to mediate interactions that stimulate downstream signaling from the receptor (22).In transformed cells, engagement of the α6β4 integrin stimulates the activation of several signaling molecules, including phosphatidylinositol-3 kinase (PI3K), mitogen-activated protein kinases (MAPK), NFκB, and Src family kinases (SFKs) (10, 12, 21, 40). In previous studies, we identified Y1494 in the β4 subunit cytoplasmic domain as an important mediator of α6β4-dependent signaling by demonstrating that mutation of Y1494 inhibits the ability of α6β4 to stimulate PI3K, MAPK, and SFK activation (10, 39). Restoration of both PI3K and SFK signaling, but not MAPK signaling, rescues invasion in tumor cells expressing Y1494F-β4, indicating that PI3K and SFK signaling pathways cooperate downstream of Y1494 to promote α6β4-dependent invasion (10). Y1494 is localized within an immunoreceptor tyrosine-based inhibition motif (ITIM), a canonical binding site for Src-homology-2 (SH2) domain-containing protein-tyrosine phosphatase 1 (SHP1) and SHP2 (44). Examination of a chimeric receptor containing the extracellular domain of TrkB and the transmembrane and cytoplasmic domains of the β4 subunit demonstrated that SHP2 binds to and is activated by sequences in the β4 cytoplasmic domain in response to dimerization (23). Moreover, Y1494 is one of three tyrosine residues, along with Y1257 and Y1440, that mediate the interaction of SHP2 with the β4 subunit cytoplasmic domain in response to c-Met signaling (6). Importantly, SHP2 is essential for the activation of SFKs both by the chimeric TrkB/β4 receptor and when the β4 subunit functions as a signaling adaptor for c-Met (6, 23). However, the mechanism by which SHP2 activates SFKs in response to α6β4 engagement has not been established.Elevated SFK activity correlates strongly with breast cancer invasion and metastasis, and these kinases are frequently activated in human cancers (15). Given the parallels between α6β4 expression and SFK activation in cancer, investigation of how α6β4 contributes to the activation of this invasion-promoting pathway is warranted. In the current study, we sought to elucidate the mechanism by which engagement of α6β4 activates SFKs and the significance of the β4/SHP2/SFK signaling axis for tumor progression. Our results reveal a novel mechanism for SHP2-dependent activation of the SFK family member Fyn which involves Y580 in the C terminus of SHP2.     

Two‐state conformational equilibrium in the Par‐4 leucine zipper domain

Prostate apoptosis response factor‐4 (Par‐4) is a pro‐apoptotic and tumor‐suppressive protein. A highly conserved heptad repeat sequence at the Par‐4 C‐terminus suggests the presence of a leucine zipper (LZ). This C‐terminal region is essential for Par‐4 self‐association and interaction with various effector proteins. We have used nuclear magnetic resonance (NMR) spectroscopy to fully assign the chemical shift resonances of a peptide comprising the LZ domain of Par‐4 at neutral pH. Further, we have investigated the properties of the Par‐4 LZ domain and two point mutants under a variety of conditions using NMR, circular dichroism (CD), light scattering, and bioinformatics. Results indicate an environment‐dependent conformational equilibrium between a partially ordered monomer (POM) and a predominantly coiled coil dimer (CCD). The combination of techniques used allows the time scales of the equilibrium to be probed and also helps to identify features of the amino acid sequence that may influence the equilibrium. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Effect of dietary fish oil on platelet aggregability: comparison between two oils with different eicosapentaenoic to arachidonic acid ratio.

Rats, acclimatized on a control diet, were fed for 60 days with diets, supplemented with 10% fat of either marine Hilsa fish (Hilsa ilisa) or fresh-water Chital fish (Notopterus chitala). The percentage of eicosapentaenoic acid in chital oil diet was 0.57 times that of the hilsa oil diet, but the eicosapentaenoic to arachidonic acid ratio in the latter (4.08) was 3.2 times that of the former (1.27). Otherwise these two diets were comparable in respect to total saturated, monounsaturated and n-3 polyunsaturated fatty acid contents. Results showed that of the two only hilsa oil diet could significantly lower platelet aggregability and in vitro thromboxane production, through replacement of arachidonic acid in platelet phospholipid by eicosapentaenoic acid. The antithrombic criteria of the oil seems to be a combination of low arachidonic acid content and high eicosapentaenoic to arachidonic acid ratio.