Structure-Guided Approach for the Development of MUC1-Glycopeptide-Based Cancer Vaccines with Predictable Responses.

Mucin-1 (MUC1) glycopeptides are exceptional candidates for potential cancer vaccines. However, their autoantigenic nature often results in a weak immune response. To overcome this drawback, we carefully engineered synthetic antigens with precise chemical modifications. To be effective and stimulate an anti-MUC1 response, artificial antigens must mimic the conformational dynamics of natural antigens in solution and have an equivalent or higher binding affinity to anti-MUC1 antibodies than their natural counterparts. As a proof of concept, we have developed a glycopeptide that contains noncanonical amino acid (2S,3R)-3-hydroxynorvaline. The unnatural antigen fulfills these two properties and effectively mimics the threonine-derived antigen. On the one hand, conformational analysis in water shows that this surrogate explores a landscape similar to that of the natural variant. On the other hand, the presence of an additional methylene group in the side chain of this analog compared to the threonine residue enhances a CH/π interaction in the antigen/antibody complex. Despite an enthalpy-entropy balance, this synthetic glycopeptide has a binding affinity slightly higher than that of its natural counterpart. When conjugated with gold nanoparticles, the vaccine candidate stimulates the formation of specific anti-MUC1 IgG antibodies in mice and shows efficacy comparable to that of the natural derivative. The antibodies also exhibit cross-reactivity to selectively target, for example, human breast cancer cells. This investigation relied on numerous analytical (e.g., NMR spectroscopy and X-ray crystallography) and biophysical techniques and molecular dynamics simulations to characterize the antigen-antibody interactions. This workflow streamlines the synthetic process, saves time, and reduces the need for extensive, animal-intensive immunization procedures. These advances underscore the promise of structure-based rational design in the advance of cancer vaccine development.

Stretching the chains: the destabilizing impact of Cu2+ and Zn2+ ions on K48-linked diubiquitin.

Ubiquitin signalling and metal homeostasis play key roles in controlling several physiological cellular activities, including protein trafficking and degradation. While some relationships between these two biochemical pathways have started to surface, our knowledge of their interplay remains limited. Here, we employ a variety of techniques, such as circular dichroism, differential scanning calorimetry, pressure perturbation calorimetry, fluorescence emission, SDS-PAGE, and small-angle X-ray scattering (SAXS) to evaluate the impact of Cu2+ and Zn2+ ions on the structure and stability of K48 linked diubiquitin (K48-Ub2), a simple model for polyubiquitin chains. The SAXS analysis results show that the structure of the metal-free protein is similar to that observed when the protein is bound to the E2 conjugating enzyme, lending support to the idea that the structure of unanchored K48-linked ubiquitin chains is sufficient for identification by conjugating enzymes without the need for an induced fit mechanism. Our results indicate that K48-Ub2 can coordinate up to four metal ions with both copper and zinc ions inducing slight changes to the secondary structure of the protein. However, we noted significant distinctions in their impacts on protein stability and overall architecture. Specifically, Cu2+ ions resulted in a destabilization of the protein structure, which facilitated the formation of dimer aggregates. Next, we observed a shift in the conformational dynamics of K48-Ub2 toward less compact and more flexible states upon metal ion binding, with Zn2+ inducing a more significant effect than Cu2+ ions. Our structural modelling study demonstrates that both metal ions induced perturbations in the K48-Ub2 structure, leading to the separation of the two monomers thus inhibiting interactions with E2 enzymes. In conclusion, the findings from this study enhance our comprehension of the mechanisms underlying Ub chains recognition. Moreover, they strengthen the notion that drug discovery initiatives aimed at targeting metal-mediated disruptions in Ub signaling hold great potential for treating a wide range of diseases that stem from abnormal protein accumulation.

Crystal Structure of the Human Copper Chaperone ATOX1 Bound to Zinc Ion.

The bioavailability of copper (Cu) in human cells may depend on a complex interplay with zinc (Zn) ions. We investigated the ability of the Zn ion to target the human Cu-chaperone Atox1, a small cytosolic protein capable of anchoring Cu(I), by a conserved surface-exposed Cys-X-X-Cys (CXXC) motif, and deliver it to Cu-transporting ATPases in the trans-Golgi network. The crystal structure of Atox1 loaded with Zn displays the metal ion bridging the CXXC motifs of two Atox1 molecules in a homodimer. The identity and location of the Zn ion were confirmed through the anomalous scattering of the metal by collecting X-ray diffraction data near the Zn K-edge. Furthermore, soaking experiments of the Zn-loaded Atox1 crystals with a strong chelating agent, such as EDTA, caused only limited removal of the metal ion from the tetrahedral coordination cage, suggesting a potential role of Atox1 in Zn metabolism and, more generally, that Cu and Zn transport mechanisms could be interlocked in human cells.

Structural Characterization of the Full-Length Anti-CD20 Antibody Rituximab.

Rituximab, a murine-human chimera, is the first monoclonal antibody (mAb) developed as a therapeutic agent to target CD20 protein. Its Fab domain and its interaction with CD20 have been extensively studied and high-resolution atomic models obtained by X-ray diffraction or cryo-electron microscopy are available. However, the structure of the full-length antibody is still missing as the inherent protein flexibility hampers the formation of well-diffracting crystals and the reconstruction of 3D microscope images. The global structure of rituximab from its dilute solution is here elucidated by small-angle X-ray scattering (SAXS). The limited data resolution achievable by this technique has been compensated by intensive computational modelling that led to develop a new and effective procedure to characterize the average mAb conformation as well as that of the single domains. SAXS data indicated that rituximab adopts an asymmetric average conformation in solution, with a radius of gyration and a maximum linear dimension of 52 Å and 197 Å, respectively. The asymmetry is mainly due to an uneven arrangement of the two Fab units with respect to the central stem (the Fc domain) and reflects in a different conformation of the individual units. As a result, the Fab elbow angle, which is a crucial determinant for antigen recognition and binding, was found to be larger (169°) in the more distant Fab unit than that in the less distant one (143°). The whole flexibility of the antibody has been found to strongly depend on the relative inter-domain orientations, with one of the Fab arms playing a major role. The average structure and the amount of flexibility has been studied in the presence of different buffers and additives, and monitored at increasing temperature, up to the complete unfolding of the antibody. Overall, the structural characterization of rituximab can help in designing next-generation anti-CD20 antibodies and finding more efficient routes for rituximab production at industrial level.

Structural characterization of an unprecedented lectin-like antitumoral anti-MUC1 antibody.

The molecular basis of antibody 5E5, which recognizes the entire GalNAc unit as a primary epitope is disclosed. The antibody's contacts with the peptide are mostly limited to two residues, allowing it to show some degree of promiscuity. These findings open the door to the chemical design of peptide-mimetics for developing efficient anti-cancer vaccines and diagnostic tools.

An altered lipid metabolism characterizes Charcot-Marie-Tooth type 2B peripheral neuropathy.

Charcot-Marie Tooth type 2B (CMT2B) is a rare inherited peripheral neuropathy caused by five missense mutations in the RAB7A gene, which encodes a small GTPase of the RAB family. Currently, no cure is available for this disease. In this study, we approached the disease by comparing the lipid metabolism of CMT2B-derived fibroblasts to that of healthy controls. We found that CMT2B cells showed increased monounsaturated fatty acid level and increased expression of key enzymes of monounsaturated and polyunsaturated fatty acid synthesis. Moreover, in CMT2B cells a higher expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), key enzymes of de novo fatty acid synthesis, with a concomitantly increased [1-14C]acetate incorporation into fatty acids, was observed. The expression of diacylglycerol acyltransferase 2, a rate-limiting enzyme in triacylglycerol synthesis, as well as triacylglycerol levels were increased in CMT2B compared to control cells. In addition, as RAB7A controls lipid droplet breakdown and lipid droplet dynamics have been linked to diseases, we analyzed these organelles and showed that in CMT2B cells there is a strong accumulation of lipid droplets compared to control cells, thus reinforcing our data on abnormal lipid metabolism in CMT2B. Furthermore, we demonstrated that ACC and FAS expression levels changed upon RAB7 silencing or overexpression in HeLa cells, thus suggesting that metabolic modifications observed in CMT2B-derived fibroblasts can be, at least in part, related to RAB7 mutations.

Conopeptide-Functionalized Nanoparticles Selectively Antagonize Extrasynaptic N-Methyl-d-aspartate Receptors and Protect Hippocampal Neurons from Excitotoxicity In Vitro.

N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors controlling fundamental physiological processes in the central nervous system, such as learning and memory. Excessive activation of NMDARs causes excitotoxicity and results in neurodegeneration, which is observed in a number of pathological conditions. Because of their dichotomous role, therapeutic targeting of NMDAR is difficult. However, several lines of evidence suggest that excitotoxicity is predominantly linked to extrasynaptically located NMDARs. Here, we report on a nanoparticle-based strategy to inhibit extrasynaptic NMDARs exclusively and subtype selectively, while allowing synaptic NMDARs activity. We designed gold nanoparticles (AuNPs) carrying conopeptide derivatives conjugated on their poly(ethylene glycol) coating as allosteric NMDAR inhibitors and show that these nanoparticles antagonize exclusively extrasynaptic NMDAR-mediated currents in cultured hippocampal neurons. Additionally, we show that conopeptide-functionalized AuNPs are neuroprotective in an in vitro model of excitotoxicity. By using AuNPs carrying different allosteric inhibitors with distinct NMDAR subtype selectivity such as peptide conantokin-G or peptide conantokin-R, we suggest activation of extrasynaptic GluN2B-containing diheteromeric NMDARs as the main cause of excitotoxicity.

Directional Immobilization of Proteins on Gold Nanoparticles Is Essential for Their Biological Activity: Leptin as a Case Study.

Gold nanomaterials hold great potential for biomedical applications. While this field is evolving rapidly, little attention has been paid to precise nanoparticle design and functionalization. Here, we show that when using proteins as targeting moieties, it is fundamental to immobilize them directionally to preserve their biological activity. Using full-length leptin as a case study, we have developed two alternative conjugation strategies for protein immobilization based on either a site-selective or a nonselective derivatization approach. We show that only nanoparticles with leptin immobilized site-selectively fully retain the ability to interact with the cognate leptin receptor. These results demonstrate the importance of a specified molecular design when preparing nanoparticles labeled with proteins.

Structure-Based Design of Potent Tumor-Associated Antigens: Modulation of Peptide Presentation by Single-Atom O/S or O/Se Substitutions at the Glycosidic Linkage.

GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α- O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors.

Concentration-dependent effects of mercury and lead on Aß42: possible implications for Alzheimer's disease.

Mercury (Hg) and lead (Pb) are known to be toxic non-radioactive elements, with well-described neurotoxicology. Much evidence supports the implication of metals as potential risk cofactors in Alzheimer's disease (AD). Although the action mechanism of the two metals remains unclear, Hg and Pb toxicity in AD could depend on their ability to favour misfolding and aggregation of amyloid beta proteins (Aßs) that seem to have toxic properties, particularly in their aggregated state. In our study, we evaluated the effect of Hg and Pb both on the Aß42 ion channel incorporated in a planar lipid membrane made up of phosphatidylcholine containing 30% cholesterol and on the secondary structure of Aß42 in an aqueous environment. The effects of Hg and Pb on the Aß42 peptide were observed for its channel incorporated into a membrane as well as for the peptide in solution. A decreasing Aß42 channel frequency and the formation of large and amorphous aggregates in solution that are prone to precipitate were both dependent on metal concentration. These experimental data suggest that Hg and Pb interact directly with Aßs, strengthening the hypothesis that the two metals may be a risk factor in AD.

Aggregation Pathways of Native-Like Ubiquitin Promoted by Single-Point Mutation, Metal Ion Concentration, and Dielectric Constant of the Medium.

Ubiquitin-positive protein aggregates are biomarkers of neurodegeneration, but the molecular mechanism responsible for their formation and accumulation is still unclear. Possible aggregation pathways of human ubiquitin (hUb) promoted by both intrinsic and extrinsic factors, are here investigated. By a computational analysis, two different hUb dimers are indicated as possible precursors of amyloid-like structures, but their formation is disfavored by an electrostatic repulsion involving Glu16 and other carboxylate residues present at the dimer interface. Experimental data on the E16V mutant of hUb shows that this single-point mutation, although not affecting the overall protein conformation, promotes protein aggregation. It is sufficient to shift the same mutation by only two residues (E18V) to regain the behavior of wild-type hUb. The neutralization of Glu16 negative charge by a metal ion and a decrease of the dielectric constant of the medium by addition of trifluoroethanol (TFE), also promote hUb aggregation. The outcomes of this research have important implications for the prediction of physiological parameters that favor aggregate formation.

Intranasal delivery of dopamine to the striatum using glycol chitosan/sulfobutylether-ß-cyclodextrin based nanoparticles.

The aim of this study was to evaluate chitosan (CS)-, glycol chitosan (GCS)- and corresponding thiomer-based nanoparticles (NPs) for delivering dopamine (DA) to the brain by nasal route. Thus, the polyanions tripolyphosphate and sulfobutylether-ß-cyclodextrin (SBE-ß-CD), respectively, were used as polycation crosslinking agents and SBE-ß-CD also in order to enhance the DA stability. The most interesting formulation, containing GCS and SBE-ß-CD, was denoted as DA GCS/DA-CD NPs. NMR spectroscopy demonstrated an inclusion complex formation between SBE-ß-CD and DA. X-ray photoelectron spectroscopy analysis revealed the presence of DA on the external surface of NPs. DA GCS/DA-CD NPs showed cytotoxic effect toward Olfactory Ensheathing Cells only at higher dosage. Acute administration of DA GCS/DA-CD NPs into the right nostril of rats did not modify the levels of the neurotransmitter in both right and left striatum. Conversely, repeated intranasal administration of DA GCS/DA-CD NPs into the right nostril significantly increased DA in the ipsilateral striatum. Fluorescent microscopy of olfactory bulb after acute administration of DA fluorescent-labeled GCS/DA-CD NPs into the right nostril showed the presence of NPs only in the right olfactory bulb and no morphological tissue damage occurred. Thus, these GCS based NPs could be potentially used as carriers for nose-to-brain DA delivery for the Parkinson's disease treatment.

Amyloid transition of ubiquitin on silver nanoparticles produced by pulsed laser ablation in liquid as a function of stabilizer and single-point mutations.

The interaction of nanoparticles with proteins has emerged as a key issue in addressing the problem of nanotoxicity. We investigated the interaction of silver nanoparticles (AgNPs), produced by laser ablation with human ubiquitin (Ub), a protein essential for degradative processes in cells. The surface plasmon resonance peak of AgNPs indicates that Ub is rapidly adsorbed on the AgNP surface yielding a protein corona; the Ub-coated AgNPs then evolve into clusters held together by an amyloid form of the protein, as revealed by binding of thioflavin T fluorescent dye. Transthyretin, an inhibitor of amyloid-type aggregation, impedes aggregate formation and disrupts preformed AgNP clusters. In the presence of sodium citrate, a common stabilizer that confers an overall negative charge to the NPs, Ub is still adsorbed on the AgNP surface, but no clustering is observed. Ub mutants bearing a single mutation at one edge ß strand (i.e. Glu16Val) or in loop (Glu18Val) behave in a radically different manner.

Conformational selection of ubiquitin quaternary structures driven by zinc ions.

Zinc ions bridging two ubiquitin molecules (with His68 at the interface) contribute to select a subset of conformers from the noncovalent dimer ensemble, thus restricting quaternary structure dynamics, which hampers apo-protein crystallization. The type of selected conformer is shown to determine the crystal packing, which varies from orthorhombic to cubic symmetry.

The light-activated proton pump Bop I of the archaeon Haloquadratum walsbyi.

We have isolated and characterized the light-driven proton pump Bop I from the ultrathin square archaeon Haloquadratum walsbyi, the most abundant component of the dense microbial community inhabiting hypersaline environments. The disruption of cells by hypo-osmotic shock yielded Bop I retinal protein highly enriched membranes, which contain one main 27 kDa protein band together with a high content of the carotenoid bacterioruberin. Light-induced pH changes were observed in suspensions of Bop I retinal protein-enriched membranes under sustained illumination. Solubilization of H. walsbyi cells with Triton X-100, followed by phenyl-Sepharose chromatography, resulted in isolation of two purified Bop I retinal protein bands; mass spectrometry analysis revealed that the Bop I was present as only protein in both the bands. The study of light/dark adaptations, M-decay kinetics, responses to titration with alkali in the dark and endogenous lipid compositions of the two Bop I retinal protein bands showed functional differences that could be attributed to different protein aggregation states. Proton-pumping activity of Bop I during the photocycle was observed in liposomes constituted of archaeal lipids. Similarities and differences of Bop I with other archaeal proton-pumping retinal proteins will be discussed.