IGF2 prevents dopaminergic neuronal loss and decreases intracellular alpha-synuclein accumulation in Parkinson's disease models.

Parkinson's disease (PD) is the second most common late-onset neurodegenerative disease and the predominant cause of movement problems. PD is characterized by motor control impairment by extensive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). This selective dopaminergic neuronal loss is in part triggered by intracellular protein inclusions called Lewy bodies, which are composed mainly of misfolded alpha-synuclein (α-syn) protein. We previously reported insulin-like growth factor 2 (IGF2) as a key protein downregulated in PD patients. Here we demonstrated that IGF2 treatment or IGF2 overexpression reduced the α-syn aggregates and their toxicity by IGF2 receptor (IGF2R) activation in cellular PD models. Also, we observed IGF2 and its interaction with IGF2R enhance the α-syn secretion. To determine the possible IGF2 neuroprotective effect in vivo we used a gene therapy approach in an idiopathic PD model based on α-syn preformed fibrils intracerebral injection. IGF2 gene therapy revealed a significantly preventing of motor impairment in idiopathic PD model. Moreover, IGF2 expression prevents dopaminergic neuronal loss in the SN together with a decrease in α-syn accumulation (phospho-α-syn levels) in the striatum and SN brain region. Furthermore, the IGF2 neuroprotective effect was associated with the prevention of synaptic spines loss in dopaminergic neurons in vivo. The possible mechanism of IGF2 in cell survival effect could be associated with the decrease of the intracellular accumulation of α-syn and the improvement of dopaminergic synaptic function. Our results identify to IGF2 as a relevant factor for the prevention of α-syn toxicity in both in vitro and preclinical PD models.

Functional characterization of the ATPase-like activity displayed by a catalytic amyloid.

BACKGROUND: Amyloids are highly ordered polypeptide aggregates stabilized by a beta-sheet structural core. Though classically associated to pathology, reports on novel functional roles of these proteins have increasingly emerged in the past decade. Moreover, the recent discovery that amyloids formed with rationally designed small peptides can exhibit catalytic reactivity has opened up new opportunities in both biology and biotechnology. The observed activities typically require the binding of divalent metals, giving rise to active metal-amyloid complexes. METHODS: Peptide (SDIDVFI) was aggregated in vitro. The structure of the self-assembled species was analyzed using fluorescence, transmission electron microscopy, circular dichroism and computational modeling. A kinetic characterization of the emerging catalytic activity was performed. RESULTS: The peptide self-assembled into canonical amyloids that exhibited catalytic activity towards hydrolysis of the phosphoanhydride bonds of adenosine triphosphate (ATP), partially mimicking an ATPase-like enzyme. Both amyloid formation and activity are shown to depend on manganese (Mn2+) binding. The activity was not restricted to ATP but also affected all other ribonucleotides (GTP, CTP and UTP). Peptides carrying a single aspartate exhibited a similar activity. CONCLUSIONS: The phosphoanhydride bonds appear as the main specificity target of the Mn2+-amyloid complex. A single aspartate per peptide is sufficient to enable the hydrolytic activity. GENERAL SIGNIFICANCE: Catalytic amyloids are shown for the first time to catalyze the hydrolysis of all four ribonucleotides. Our results should contribute towards understanding the biological implications of amyloid-mediated reactivity as well as in the design of future catalytic amyloids for biotechnological applications.

The oxygen-binding properties of hemocyanin from the mollusk Concholepas concholepas.

Hemocyanins have highly conserved copper-containing active sites that bind oxygen. However, structural differences among the hemocyanins of various mollusks may affect their physicochemical properties. Here, we studied the oxygen-binding cooperativity and affinity of Concholepas concholepas hemocyanin (CCH) and its two isolated subunits over a wide range of temperatures and pH values. Considering the differences in the quaternary structures of CCH and keyhole limpet hemocyanin (KLH), we hypothesized that the heterodidecameric CCH has different oxygen-binding parameters than the homodidecameric KLH. A novel modification of the polarographic method was applied in which rat liver submitochondrial particles containing cytochrome c oxidase were introduced to totally deplete oxygen of the test solution using ascorbate as the electron donor. This method was both sensitive and reproducible. The results showed that CCH, like other hemocyanins, exhibits cooperativity, showing an inverse relationship between the oxygen-binding parameters and temperature. According to their Hill coefficients, KLH has greater cooperativity than CCH at physiological pH; however, CCH is less sensitive to pH changes than KLH. Appreciable differences in binding behavior were found between the CCH subunits: the cooperativity of CCH-A was not only almost double that of CCH-B, but it was also slightly superior to that of CCH, thus suggesting that the oxygen-binding domains of the CCH subunits are different in their primary structure. Collectively, these data suggest that CCH-A is the main oxygen-binding domain in CCH; CCH-B may play a more structural role, perhaps utilizing its surprising predisposition to form tubular polymers, unlike CCH-A, as demonstrated here using electron microscopy.

Overcoming electrostatic repulsions during amyloid assembly: Effect of pH and interaction with divalent metals using model peptides.

Amyloids are polypeptide aggregates involved in many pathologies including Alzheimer's disease. Amyloid assembly is a complex process affected by different interactions including hydrogen bonding, van der Waals forces and electrostatic interactions. The highly regular amyloid structure allows for an arrangement of residues that forces side chains to be closely positioned, giving rise to potentially unfavorable interactions such as electrostatic repulsions. In these cases, amyloid assembly will depend on a balance between stabilizing versus unfavorable interactions. In this study, we rationally designed several amyloid-prone model peptides that had two acidic groups and tested their assembly into amyloids under different conditions. We found that at low pH (pH 4.0), most peptides spontaneously formed amyloids whereas no or little aggregation was observed at higher pHs (pH 8.0). When divalent metals with affinity for carboxylate groups were added at millimolar concentrations, most peptides exhibited a metal-dependent switch to the amyloid state at pH 8.0. Our results show that electrostatic repulsion between amyloid-prone sequences can be overcome in conditions that affect protonation of residue side chains. Moreover, the presence of divalent metals can contribute to electrostatic shielding through specific coordination with acidic groups and thus promote amyloid assembly.

Development of a novel catalytic amyloid displaying a metal-dependent ATPase-like activity.

Amyloids are protein aggregates of highly regular structure that are involved in diverse pathologies such as Alzheimer's and Parkinson's disease. Recent evidence has shown that under certain conditions, small peptides can self-assemble into amyloids that exhibit catalytic reactivity towards certain compounds. Here we report a novel peptide with a sequence derived from the active site of RNA polymerase that displays hydrolytic activity towards ATP. The catalytic reaction proceeds in the presence of the divalent metal manganese and the products are ADP and AMP. The kinetic data shows a substrate-dependent saturation of the activity with a maximum rate achieved at around 1 mM ATP. At higher ATP concentrations, we also observed substrate inhibition of the activity. The self-assembly of the peptide into amyloids is strictly metal-dependent and required for the catalysis. Our results show that aspartate-containing amyloids can also be catalysts under conditions that include interactions with metals. Moreover, we show for the first time an amyloid that exerts reactivity towards a biologically essential molecule.

Binding of dihydroxynaphthyl aryl ketones to tubulin colchicine site inhibits microtubule assembly.

Dihydroxynaphthyl aryl ketones 1-5 have been evaluated for their abilities to inhibit microtubule assembly and the binding to tubulin. Compounds 3, 4 and 5 displayed competitive inhibition against colchicine binding, and docking analysis showed that they bind to the tubulin colchicine-binding pocket inducing sheets instead of microtubules. Remarkable differences in biological activity observed among the assayed compounds seem to be related to the structure and position of the aryl substituent bonded to the carbonyl group. Compounds 2, 3 and 4, which contain a heterocyclic ring, presented higher affinity for tubulin compared to the carbocyclic analogue 5. Compound 4 showed the best affinity of the series, with an IC50 value of 2.1 µM for microtubule polymerization inhibition and a tubulin dissociation constant of 1.0 ± 0.2 µM, as determined by thermophoresis. Compound 4 was more efficacious in disrupting microtubule assembly in vitro than compound 5 although it contains the trimethoxyphenyl ring present in colchicine. Hydrogen bonds with Asn101 of α-tubulin seem to be responsible for the higher affinity of compound 4 respects to the others.

Immunocompetence of breeding females is sensitive to cortisol levels but not to communal rearing in the degu (Octodon degus).

One hypothesis largely examined in social insects is that cooperation in the context of breeding benefits individuals through decreasing the burden of immunocompetence and provide passive immunity through social contact. Similarly, communal rearing in social mammals may benefit adult female members of social groups by reducing the cost of immunocompetence, and through the transfer of immunological compounds during allonursing. Yet, these benefits may come at a cost to breeders in terms of a need to increase investment in individual immunocompetence. We examined how these potential immunocompetence costs and benefits relate to reproductive success and survival in a natural population of the communally rearing rodent, Octodon degus. We related immunocompetence (based on ratios of white blood cell counts, total and specific immunoglobulins of G isotype titers) and fecal glucocorticoid metabolite (FGC) levels of adults immunized with hemocyanin from the mollusk Concholepas concholepas to measures of sociality (group size) and communal rearing (number of breeding females). Offspring immunocompetence was quantified based on circulating levels of the same immune parameters. Neither female nor offspring immunocompetence was influenced by communal rearing or sociality. These findings did not support that communal rearing and sociality enhance the ability of females to respond to immunological challenges during lactation, or contribute to enhance offspring condition (based on immunocompetence) or early survival (i.e., to 3months of age). Instead, levels of humoral and cellular components of immunocompetence were associated with variation in glucorcorticoid levels of females. We hypothesize that this covariation is driven by physiological (life-history) adjustments needed to sustain breeding.

Enhanced structural stability of Concholepas hemocyanin increases its immunogenicity and maintains its non-specific immunostimulatory effects.

Hemocyanins, which boost the immune system of mammals, have been used as carrier-adjuvants to promote Ab production against haptens and peptides, as immunostimulants during therapy for bladder carcinoma and as a component in therapeutic vaccines for cancer. These biomedical applications have led to growing interest in obtaining hemocyanins with high immunogenicity. Here, we study the immunological properties of a modified oxidized Concholepas concholepas hemocyanin (Ox-CCH) obtained by the oxidation of its carbohydrates using sodium periodate. We assessed the internalization of Ox-CCH into DCs and its immunogenicity and antitumor effects. Transmission electron microscopy showed no changes in Ox-CCH quaternary structure with respect to native CCH, although proteolytic treatment followed by SDS-PAGE analysis demonstrated that Schiff bases were formed. Interestingly, DCs internalized Ox-CCH faster than CCH, mainly through macropinocytosis. During this process, Ox-CCH remained inside endosome-like structures for a longer period. Mouse immunization experiments demonstrated that Ox-CCH is more immunogenic and a better carrier than CCH. Moreover, Ox-CCH showed a significant antitumor effect in the B16F10 melanoma model similar to that produced by CCH, inducing IFN-γ secretion. Together, these data demonstrate that the aldehydes formed by the periodate oxidation of sugar moieties stabilizes the CCH structure, increasing its adjuvant/immunostimulatory carrier effects.

[Hemocyanins as immunostimulants]. / Hemocianinas, una herramienta inmunológica de la biomedicina actual.

Hemocyanins, the giant oxygen transporter glycoproteins of diverse mollusks, are xenogenic to the mammalian immune system and they display a remarkable immuno-genicity. Therefore they are ideal non-specific immunostimulants to treat some types of cancer. They are used as an alternative therapy for superficial urinary bladder cancer (SBC), that has been traditionally treated with Bacillus Calmette-Guerin (BCG). In contrast to BCG, hemocyanins do not cause side-effects, making them ideal for long-term repetitive treatments. Hemocyanins have also been exploited as carriers to develop antibodies against hapten molecules and peptides, as carrier-adjuvants for cutting-edge vaccines against cancer, drug addiction, and infectious diseases and in the diagnosis of parasitic diseases, such as Schistosomiasis. The hemocyanin from Megathura crenulata, also known as keyhole limpet hemocyanin (KLH), has been used for over thirty years for the purposes described above. More recently, hemoc yanin from the Chilean mollusk Concholepas concholepas (CCH) has proved to be a reliable alternative to KLH, either as carrier protein, and as a likely alternative for the immunotherapy of SBC. Despite KLH and CCH differ significantly in their origin and structure, we have demonstrated that both hemocyanins stimulate the immune system of mammals in a similar way by inducing a potent Thl-polarized cellular and humoral response.

Hemocianinas, una herramienta inmunológica de la biomedicina actual / Hemocyanins as immunostimulants

Hemocyanins, the giant oxygen transporter glycoproteins of diverse mollusks, are xenogenic to the mammalian immune system and they display a remarkable immuno-genicity. Therefore they are ideal non-specific immunostimulants to treat some types of cancer. They are used as an alternative therapy for superficial urinary bladder cancer (SBC), that has been traditionally treated with Bacillus Calmette-Guerin (BCG). In contrast to BCG, hemocyanins do not cause side-effects, making them ideal for long-term repetitive treatments. Hemocyanins have also been exploited as carriers to develop antibodies against hapten molecules and peptides, as carrier-adjuvants for cutting-edge vaccines against cancer, drug addiction, and infectious diseases and in the diagnosis of parasitic diseases, such as Schistosomiasis. The hemocyanin from Megathura crenulata, also known as keyhole limpet hemocyanin (KLH), has been used for over thirty years for the purposes described above. More recently, hemoc yanin from the Chilean mollusk Concholepas concholepas (CCH) has proved to be a reliable alternative to KLH, either as carrier protein, and as a likely alternative for the immunotherapy of SBC. Despite KLH and CCH differ significantly in their origin and structure, we have demonstrated that both hemocyanins stimulate the immune system of mammals in a similar way by inducing a potent Thl-polarized cellular and humoral response.

Immunodominant role of CCHA subunit of Concholepas hemocyanin is associated with unique biochemical properties.

Hemocyanin, the oxygen transporter metallo-glycoprotein from mollusks, shows strong relationship between its notable structural features and intrinsic immunomodulatory effects. Here we investigated the individual contribution of CCHA and CCHB subunits from Concholepas hemocyanin (CCH) to in vivo humoral immune response and their pre-clinical evaluation as immunotherapeutic agent in a mice bladder cancer model, in relation to their biochemical properties. To this end, subunits were purified and well characterized. Homogeneous subunits were obtained by anionic exchange chromatography, and its purity assessed by electrophoretic and immunochemical methods. While each CCH subunit contains eight functional units showing partial cross reaction, the vibrational spectral analysis showed several spectral differences, suggesting structural differences between them. In addition, we demonstrated differences in the carbohydrate content: CCHA had a 3.6% w/w sugar with both N- and O-linked moieties. In turn, CCHB had a 2.5% w/w sugar with N-linked, while O-linked moieties were nearly absent. Considering these differences, it was not possible to predict a priori whether the immunogenic and immunotherapeutic properties of subunits might be similar. Surprisingly, both subunits by itself induced a humoral response, and showed an antitumor effect in the bladder carcinoma cell line MBT-2. However, when immunologic parameters were analyzed, CCHA showed better efficiency than CCHB. No allergic reactions or any toxic effects were observed in mice treated with CCHA, sustaining its potential therapeutic use. Our study supports that CCHA subunit accounts for the most important features involved in the immunogenicity of CCH, such as better hydrophilicity and higher content of carbohydrates.

4',6-Diamidino-2-phenylindole (DAPI) induces bundling of Escherichia coli FtsZ polymers inhibiting the GTPase activity.

FtsZ (Filamentous temperature sensitivity Z) cell division protein from Escherichia coli binds the fluorescence probe DAPI. Bundling of FtsZ was facilitated in the presence of DAPI, and the polymers in solution remained polymerized longer time than the protofilaments formed in the absence of DAPI. DAPI decreased both the maximal velocity of the GTPase activity and the Michaelis-Menten constant for GTP, indicating that behaves like an uncompetitive inhibitor of the GTPase activity favoring the GTP form of FtsZ in the polymers. The results presented in this work support a cooperative polymerization mechanism in which the binding of DAPI favors protofilament lateral interactions and the stability of the resulting polymers.

Fluorescence resonance energy transfer and molecular modeling studies on 4',6-diamidino-2-phenylindole (DAPI) complexes with tubulin.

The goal of this work was to determine the binding properties and location of 4',6-diamidino-2-phenylindole (DAPI) complexed with tubulin. Using fluorescence anisotropy, a dissociation constant of 5.2+/-0.4 microM for the DAPI-tubulin complex was determined, slightly lower than that for the tubulin S complex. The influence of the C-terminal region on the binding of DAPI to tubulin was also characterized. Using FRET experiments, and assuming a kappa2 value of 2/3, distances between Co2+ bound to its high-affinity binding site and the DAPI-binding site and 2',3'-O-(trinitrophenyl)guanosine 5'-triphosphate bound to the exchangeable nucleotide and the DAPI-binding site were found to be 20+/-2 A and 43+/-2 A, respectively. To locate potential DAPI-binding sites on tubulin, a molecular modeling study was carried out using the tubulin crystal structure and energy minimization calculations. The results from the FRET measurements were used to limit the possible location of DAPI in the tubulin structure. Several candidate binding sites were found and these are discussed in the context of the various properties of bound DAPI.