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Selective removal of dysfunctional mitochondria via autophagy is crucial for the maintenance of cellular homeostasis. This event is initiated by the translocation of the E3 ubiquitin ligase Parkin to damaged mitochondria, and it requires the Serine/Threonine-protein kinase PINK1. In a coordinated set of events, PINK1 operates upstream of Parkin in a linear pathway that leads to the phosphorylation of Parkin, Ubiquitin, and Parkin mitochondrial substrates, to promote ubiquitination of outer mitochondrial membrane proteins. Ubiquitin decorated mitochondria are selectively recruiting autophagy receptors,which are required to terminate the organelle via autophagy. In this work we show a previously uncharacterized molecular pathway that correlates the activation of the Ca2+-dependent phosphatase Calcineurin to Parkin-dependent mitophagy. Calcineurin downregulation or genetic inhibition prevents Parkin translocation to CCCP-treated mitochondria, and impairs stress-induced mitophagy, whereas Calcineurin activation promotes Parkin mitochondrial recruitment and basal mitophagy. Calcineurin interacts with Parkin, and promotes Parkin translocation in the absence of PINK1, but requires PINK1 expression to execute mitophagy in MEF cells. Genetic activation of Calcineurinin vivoboosts basal mitophagy in neurons, and corrects locomotor dysfunction and mitochondrial respiratory defects of aDrosophilamodel of impaired mitochondrial functions.Our study identifies Calcineurin as a novel key player in the regulation of Parkin translocation and mitophagy.

The integration of ex vivo and in vitro data, described in this manuscript, together with our previous demonstration that sympathetic neurons (SNs) contact target cardiomyocytes (CMs) at the neuro-cardiac junction (NCJ), which underlies intercellular synaptic communication (Prando et al., 47), demonstrate that: CMs are the cell source of Nerve Growth Factor (NGF), required to sustain innervating cardiac SNs; NCJ is the place of the intimate liaison, between SNs and CMs, allowing on the one hand neurons to peremptorily control CM activity, and on the other, CMs to adequately sustain the contacting, everchanging, neuronal actuators; alterations in NCJ integrity may compromise the efficiency of 'CM-to-SN' signaling, thus representing a potentially novel mechanism of sympathetic denervation in cardiac diseases. ABSTRACT: Background Sympathetic neurons densely innervate the myocardium with non-random topology and establish structured contacts (i.e. neuro-cardiac junctions, NCJ) with cardiomyocytes, allowing synaptic intercellular communication. Establishment of heart innervation is regulated by molecular mediators released by myocardial cells. The mechanisms underlying maintenance of cardiac innervation in the fully developed heart, are, however, less clear. Notably, several cardiac diseases, primarily affecting cardiomyocytes, are associated to sympathetic denervation, supporting that retrograde 'cardiomyocyte-to-sympathetic neuron' communication is essential for heart cellular homeostasis. Objective We aimed to determine whether cardiomyocytes provide Nerve Growth Factor (NGF) to sympathetic neurons, and the role of the NCJ in supporting such retrograde neurotrophic signaling. Methods and Results Immunofluorescence on murine and human heart slices shows that NGF and its receptor, Tropomyosin-receptor-kinase-A, accumulate respectively in the pre- and post-junctional sides of the NCJ. Confocal immunofluorescence, scanning ion conductance microscopy and molecular analyses, in co-cultures, demonstrate that cardiomyocytes feed NGF to sympathetic neurons, and that such mechanism requires a stable intercellular contact at the NCJ. Consistently, cardiac fibroblasts, devoid of NCJ, are unable to sustain SN viability. ELISA assay and competition binding experiments suggest that this depends on the NCJ being an insulated microenvironment, characterized by high [NGF]. In further support, real-time imaging of Tropomyosin-receptor-kinase-A-vesicle movements demonstrate that efficiency of neurotrophic signaling parallels the maturation of such structured intercellular contacts. Conclusions Altogether, our results demonstrate the mechanisms which link sympathetic neuron survival to neurotrophin release by directly innervated cardiomyocytes, conceptualizing sympathetic neurons as cardiomyocyte-driven heart drivers. Abstract figure legend Sympathetic neuron (SN, green) varicosities establish synaptic contacts with target cardiomyocytes (CMs, pink), which we previously called Neuro-Cardiac Junction (NCJ, Prando et al. J Physiol 47). At NCJs, CMs release selectively NGF, which by activating TrkA signaling, is key to sustain neuronal survival. This article is protected by copyright. All rights reserved.

Osteopontin (OPN) is a phosphoglycoprotein secreted into the extracellular matrix upon liver injury, acting as a cytokine stimulates the deposition of fibrillary collagen in liver fibrogenesis. In livers of mice subjected to bile duct ligation (BDL) and in cultured activated hepatic stellate cells (HSCs), we show that OPN, besides being overexpressed, is substantially phosphorylated by family with sequence similarity 20, member C (Fam20C), formerly known as Golgi casein kinase (G-CK), which is exclusively resident in the Golgi apparatus. In both experimental models, Fam20C becomes overactive when associated with a 500-kDa multiprotein complex, as compared with the negligible activity in livers of sham-operated rats and in quiescent HSCs. Fam20C knockdown not only confirmed the role of Fam20C itself in OPN phosphorylation, but also revealed that phosphorylation was essential for OPN secretion. However, OPN acts as a fibrogenic factor independently of its phosphorylation state, as demonstrated by the increased expression of Collagen-I by HSCs incubated with either a phosphorylated or nonphosphorylated form of recombinant OPN. Collectively, our results confirm that OPN promotes liver fibrosis and highlight Fam20C as a novel factor driving this process by favoring OPN secretion from HSCs, opening new avenues for deciphering yet unidentified mechanisms underlying liver fibrogenesis.

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

SummaryParkinson’s disease (PD) is characterized by the progressive degeneration of dopaminergic neurons within the substantia nigra pars compacta and the presence of protein aggregates in surviving neurons. LRRK2 G2019S mutation is one of the major determinants of familial PD cases and leads to late-onset PD with pleomorphic pathology, including alpha-synuclein accumulation and deposition of protein inclusions. We demonstrated that LRRK2 phosphorylates N-ethylmaleimide sensitive factor (NSF). We observed aggregates containing NSF in basal ganglia specimens from G2019S carrier PD patients and in cellular and animal models expressing the LRRK2 G2019S variant. We found that LRRK2 G2019S kinase activity induces the accumulation of NSF in toxic aggregates. Noteworthy, the induction of autophagy cleared NSF aggregation and rescued motor and cognitive impairment observed in aged hG2019S BAC mice. We suggest that LRRK2 G2019S pathological phosphorylation hampers substrate catabolism, thus causing the formation of cytotoxic protein inclusions.HighlightsLRRK2 phosphorylates NSF in vivoNSF aggregates in complementary LRRK2 G2019S modelsLRRK2 G2019S kinase activity induces NSF accumulation in toxic aggregatesAutophagy induction rescues hG2019S BAC mice motor and cognitive impairment

Redox status and inflammation are related to the pathogenesis of the majority of diseases. Therefore, understanding the role of specific food-derived molecules in the regulation of their specific pathways is a relevant issue. Our previous studies indicated that K-8-K and S-10-S, milk and soy-derived bioactive peptides, respectively, exert antioxidant effects through activation of the Keap1/Nrf2 pathway. A crosstalk between Nrf2 and NF-κB, mediated by the action of heme oxygenase (HO-1), is well known. On this basis, we studied if these peptides, in addition to their antioxidant activity, could exert anti-inflammatory effects in human cells. First, we observed an increase of HO-1 expression in Caco-2 cells treated with K-8-K and S-10-S, following the activation of the Keap1/Nrf2 pathway. Moreover, when cells are treated with the two peptides and stimulated by TNF-α, the levels of NF-κB in the nucleus decreased in comparison with TNF-α alone. In the same conditions, we observed the downregulation of the gene expression of proinflammatory cytokines (IL1B, IL6, and TNF), while the anti-inflammatory cytokine gene, IL1RN, was upregulated in Caco-2 cells processed as reported above. Then, when the cells were pretreated with the two peptides and stimulated with LPS, a different proinflammatory factor, (TNF-α) was estimated to have a lower secretion in the supernatant of cells. In conclusion, these observations confirmed that Nrf2-activating bioactive peptides, K-8-K and S-10-S, exerted anti-inflammatory effects by inhibiting the NF-κB pathway.

The fine regulation of intracellular calcium is fundamental for all eukaryotic cells. In neurons, Ca2+ oscillations govern the synaptic development, the release of neurotransmitters and the expression of several genes. Alterations of Ca2+ homeostasis were found to play a pivotal role in neurodegenerative progression. The maintenance of proper Ca2+ signaling in neurons demands the continuous activity of Ca2+ pumps and exchangers to guarantee physiological cytosolic concentration of the cation. The plasma membrane Ca2+ATPases (PMCA pumps) play a key role in the regulation of Ca2+ handling in selected sub-plasma membrane microdomains. Among the four basic PMCA pump isoforms existing in mammals, isoforms 2 and 3 are particularly enriched in the nervous system. In humans, genetic mutations in the PMCA2 gene in association with cadherin 23 mutations have been linked to hearing loss phenotypes, while those occurring in the PMCA3 gene were associated with X-linked congenital cerebellar ataxias. Here we describe a novel missense mutation (V1143F) in the calmodulin binding domain (CaM-BD) of the PMCA2 protein. The mutant pump was present in a patient showing congenital cerebellar ataxia but no overt signs of deafness, in line with the absence of mutations in the cadherin 23 gene. Biochemical and molecular dynamics studies on the mutated PMCA2 have revealed that the V1143F substitution alters the binding of calmodulin to the CaM-BD leading to impaired Ca2+ ejection.

Milk and milk-derived products are a relevant source of bioactive peptides, which are also potential components of functional foods. Bioactive peptides exert multiple actions including an antioxidant role. In the present paper, four synthetic peptides (NPYVPR, AVPYPQR, KVLPVPEK, and ARHPHPHLSFM), corresponding to milk-derived peptides were studied. Although with different features, as revealed by RP-HPLC chromatography and MS analysis, the obtained peptides were shown to be taken up by Caco-2 cells arranged in an epithelial monolayer formation. The four peptides were all able to preserve cell viability against induced oxidative stress indicating that they might have a role in the control of oxidative stress. Therefore, an estimation of total thiols and glutathione content was performed after cell treatment with oxidants like hydrogen peroxide (H2O2) or tert-butyl hydroperoxide (TbOOH). The peptides were able to prevent the decrease of both total thiols and glutathione induced by H2O2 or TbOOH, and, in addition, they showed a protective effect on the thiol-related antioxidant enzymes thioredoxin reductase and glutathione reductase. Finally, they caused a decrease of ROS production induced by TbOOH in Caco-2 cells. The reported results highlight the relevant antioxidant role played by bioactive peptides in cells, which adds to other previously known properties.

Bioactive peptides are a group of molecules with health beneficial properties, deriving from food matrices. They are protein fragments consisting of 2&ndash, 20 amino acids that can be released by microbial fermentation, food processing and gastrointestinal digestion. Once hydrolyzed from their native proteins, they can have different functions including antioxidant activity, which is important for cell protection by oxidant agents. In this work, fermented soy products were digested in vitro in order to improve the release of bioactive peptides. These were extracted, purified and analyzed in vitro and in a cellular model to assess their antioxidant activity. Peptide sequences were identified by LC-MS/MS analysis and a molecular docking approach was used to predict their ability to interact with Keap1, one of the key proteins of the Keap1/Nrf2 pathway, the major system involved in redox regulation. Peptides showing a high score of interaction were selected and tested for their antioxidant properties in a cellular environment using the Caco-2 cell line and examined for their capability to defend cells against oxidative stress. Our results indicate that several of the selected peptides were indeed able to activate the Keap1/Nrf2 pathway with the consequent overexpression of antioxidant and phase II enzymes.

Bioactive peptides are relevant nutritional factors that exhibit many functions including antioxidant, antihypertensive, anticancer and antimicrobial properties. In this paper, four synthetic peptides ARHPHPHLSFM (A-11-M), AVPYPQR (A-7-R), NPYVPR (N-6-R) and KVLPVPEK (K-8-K) with sequences present in milk proteins were examined for their antioxidant properties. The compounds show moderate free radical scavenging activity in the ABTS and crocin assays (A-7-R and N-6-R) and lipid peroxidation inhibition in Caco-2 cells (N-6-R and K-8-K). All peptides, in particular K-8-K, activate the Keap1-Nrf2 system by allowing the translocation of the transcription factor Nrf2 from the cytosol to nucleus. This activation triggers the overexpression of the antioxidant enzymes Trx1, TrxR1, GR, NQO1 and SOD1. Furthermore, molecular modeling shows that K-8-K is able to hinder the interaction of Nrf2 with Keap1. The reported results show that the antioxidant action in cells of these bioactive peptides is mostly due to the activation of Keap1-Nrf2 signaling pathway.

Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+ -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.

The PI3K/Akt pathway is interconnected to protein kinase CK2, which directly phosphorylates Akt1 at S129. We have previously found that, in HK-2 renal cells, downregulation of the CK2 regulatory subunit β (shCK2β cells) reduces S129 Akt phosphorylation. Here, we investigated in more details how the different CK2 isoforms impact on Akt and other signaling pathways. We found that all CK2 isoforms phosphorylate S129 in vitro, independently of CK2β. However, in HK-2 cells the dependence on CK2β was confirmed by rescue experiments (CK2β re-expression in shCK2β HK-2 cells), suggesting the presence of additional components that drive Akt recognition by CK2 in cells. We also found that CK2β downregulation altered the phosphorylation ratio between the two canonical Akt activation sites (pT308 strongly reduced, pS473 slightly increased) in HK-2 cells. Similar results were found in other cell lines where CK2β was stably knocked out by CRISPR-Cas9 technology. The phosphorylation of rpS6 S235/S236, a downstream effector of Akt, was strongly reduced in shCK2β HK-2 cells, while the phosphorylation of two Akt direct targets, PRAS40 T246 and GSK3β S9, was increased. Differently to what observed in response to CK2β down-regulation, the chemical inhibition of CK2 activity by cell treatment with the specific inhibitor CX-4945 reduced both the Akt canonical sites, pT308 and pS473. In CX-4945-treated cells, the changes in rpS6 pS235/S236 and GSK3β pS9 mirrored those induced by CK2β knock-down (reduction and slight increase, respectively); on the contrary, the effect on PRAS40 pT246 phosphorylation was sharply different, being strongly reduced by CK2 inhibition; this suggests that this Akt target might be dependent on Akt pS473 status in HK-2 cells. Since PI3K/Akt and ERK1/2/p90rsk pathways are known to be interconnected and both modulated by CK2, with GSK3β pS9 representing a convergent point, we investigated if ERK1/2/p90rsk signaling was affected by CK2β knock-down and CX-4945 treatment in HK-2 cells. We found that p90rsk was insensitive to any kind of CK2 targeting; therefore, the observation that, similarly, GSK3β pS9 was not reduced by CK2 blockade suggests that GSK3β phosphorylation is mainly under the control of p90rsk in these cells. However, we found that the PI3K inhibitor LY294002 reduced GSK3β pS9, and concomitantly decreased Snail1 levels (a GSK3β target and Epithelial-to-Mesenchymal transition marker). The effects of LY294002 were observed also in CK2β-downregulated cells, suggesting that reducing GSK3β pS9 could be a strategy to control Snail1 levels in any situation where CK2β is defective, as possibly occurring in cancer cells.

Due to their beneficial properties, fermented foods are considered important constituents of the human diet. They also contain bioactive peptides, health-promoting compounds studied for a wide range of effects. In this work, several antioxidant peptides extracted from fermented milk proteins were investigated. First, enriched peptide fractions were purified and analysed for their antioxidant capacity in vitro and in a cellular model. Subsequently, from the most active fractions, 23 peptides were identified by mass spectrometry MS/MS), synthesized and tested. Peptides N-15-M, E-11-F, Q-14-R and A-17-E were selected for their antioxidant effects on Caco-2 cells both in the protection against oxidative stress and inhibition of ROS production. To define their action mechanism, the activation of the Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2(Keap1/Nrf2) pathway was studied evaluating the translocation of Nrf2 from cytosol to nucleus. In cells treated with N-15-M, Q-14-R and A-17-E, a higher amount of Nrf2 was found in the nucleus with respect to the control. In addition, the three active peptides, through the activation of Keap1/Nrf2 pathway, led to overexpression and increased activity of antioxidant enzymes. Molecular docking analysis confirmed the potential ability of N-15-M, Q-14-R and A-17-E to bind Keap1, showing their destabilizing effect on Keap1/Nrf2 interaction.

Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria‐endoplasmic reticulum (ER) contact sites called MAMs (mitochondria‐associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol‐3‐phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+‐dependent calpain activation, mitochondrial depolarization and cell death. The HK2‐targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti‐neoplastic strategy., HK2 localizes at mitochondria‐associated membranes of tumor cells. Its displacement with a cell‐penetrating peptide prompts IP3R opening, mitochondria Ca2+ overload, calpain activation and tumor cell death.

Mucopolysaccharidosis type II (MPSII) is a lysosomal storage disorder due to the deficit of the enzyme iduronate 2-sulfatase (IDS), which leads to the accumulation of glycosaminoglycans in most organ-systems, including the brain, and resulting in neurological involvement in about two-thirds of the patients. The main treatment is represented by a weekly infusion of the functional enzyme, which cannot cross the blood-brain barrier and reach the central nervous system. In this study, a tailored nanomedicine approach based on brain-targeted polymeric nanoparticles (g7-NPs), loaded with the therapeutic enzyme, was exploited. Fibroblasts from MPSII patients were treated for 7 days with NPs loaded with the IDS enzyme, an induced IDS activity like the one detected in healthy cells was measured, together with a reduction of GAG content to non-pathological levels. An in vivo short-term study in MPSII mice was performed by weekly administration of g7-NPs-IDS. Biochemical, histological, and immunohistochemical evaluations of liver and brain were performed. The 6-weeks treatment produced a significant reduction of GAG deposits in liver and brain tissues, as well as a reduction of some neurological and inflammatory markers (i.e., LAMP2, CD68, GFAP), highlighting a general improvement of the brain pathology. The g7-NPs-IDS approach allowed a brain-targeted enzyme replacement therapy. Based on these positive results, the future aim will be to optimize NP formulation further to gain a higher efficacy of the proposed approach.

Milk is a nutritionally important source of bioactive peptides with anti-inflammatory, immunomodulatory, anticancer, and antioxidant properties. These compounds can be useful as ingredients of functional food. For this reason, in the last decades, bioactive peptides attracted the interest of researchers and food companies. In this work, the results obtained with six milk-derived bioactive peptides (Y-4-R, V-6-R, V-7-K, A-10-F, R-10-M, and H-9-M) synthesized and studied for their antioxidant properties in vitro and in a cellular model, are reported. These molecules correspond to peptide fragments derived from parent compounds able to cross the apical membrane of Caco-2 cell layer and released in the basolateral compartment. In vitro, antioxidant tests such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and crocin bleaching showed antioxidant activity mainly for peptides Y-4-R and V-6-R, respectively. In Caco-2 cells, peptides V-6-R, H-9-R, Y-4-R, and particularly R-10-M and V-7-K are able to prevent the decrease of viability due to oxidative stress. The latter peptide is also the most effective in protecting cells from lipid peroxidation. In conclusion, the reported hydrolyzed peptides are shown to exert the antioxidant properties both in vitro and in a cellular model.

Egg yolk phosvitins, generated through the fragmentation of vitellogenins (VTGs), are among the most heavily phosphorylated proteins ever described. Despite the early discovery in 1900 that chicken phosvitin is a phosphoprotein and its subsequent employment as an artificial substrate for a number of protein kinases, the identity of the enzyme(s) responsible for its phosphorylation remained a matter of conjecture until present. Here, we provide evidence that phosvitin phosphorylation is catalyzed by a family with sequence similarity 20, member C (Fam20C), an atypical protein kinase recently identified as the genuine casein kinase and responsible for the phosphorylation of many other secreted proteins at residues specified by the S-x-E/pS consensus. Such a conclusion is grounded on the following observations: (a) the levels of Fam20C and phosphorylated VTG rise in parallel upon treatment of zebrafish with oestrogens; (b) zebrafish phosvitin is readily phosphorylated upon coexpression in U2OS cells with Fam20C, but not with its catalytically inactive mutant; (c) a peptide reproducing a stretch of 12 serines, which are phosphorylated in chicken phosvitin despite lacking the C-terminal priming motif S-x-E, is efficiently phosphorylated by both recombinant and native Fam20C. The last finding expands the repertoire of potential targets of Fam20C to include several proteins known to harbor (p-Ser)n clusters not specified by any known kinase consensus.

Phospholipases A2 are a major component of snake venoms. Some of them cause severe muscle necrosis through an unknown mechanism. Phospholipid hydrolysis is a possible explanation of their toxic action, but catalytic and toxic properties of PLA2s are not directly connected. In addition, viperid venoms contain PLA2-like proteins, which are very toxic even if they lack catalytic activity due to a critical mutation in position 49. In this work, the PLA2-like Bothrops asper myotoxin-II, conjugated with the fluorophore TAMRA, was found to be internalized in mouse myotubes, and in RAW264.7 cells. Through experiments of protein fishing and mass spectrometry analysis, using biotinylated Mt-II as bait, we found fifteen proteins interacting with the toxin and among them nucleolin, a nucleolar protein present also on cell surface. By means of confocal microscopy, Mt-II and nucleolin were shown to colocalise, at 4 °C, on cell membrane where they form Congo-red sensitive assemblies, while at 37 °C, 20 minutes after the intoxication, they colocalise in intracellular spots going from plasmatic membrane to paranuclear and nuclear area. Finally, nucleolin antagonists were found to inhibit the Mt-II internalization and toxic activity and were used to identify the nucleolin regions involved in the interaction with the toxin Universidad de Costa Rica/[741-B4-100]/UCR/Costa Rica Universidad de Costa Rica/[741-B5-602]/UCR/Costa Rica International Center for Genetic Engineering and Biotechnology/[CRP/13/006]/ICGEB/India UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP) UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología

CK2 is a pleiotropic S/T protein kinase (formerly known as casein kinase 2) which is attracting increasing interest as therapeutic target, and the identification of its substrates is a crucial step in determining its involvement in different pathological conditions. We recently found that S131 of Akt2 (homologous to the well established CK2 target S129 of Akt1) is not phosphorylated by CK2 either in vitro or in vivo, although the consensus sequence recognized by CK2 (S/T-x-x-E/D/pS/pT) is conserved in it. Here, by exploiting synthetic peptides, in cell transfection experiments, and computational analysis, we show that a single sequence element, a T at position n+1, hampers phosphorylation, causing an α-helix structure organization which prevents the recognition of its own consensus by CK2. Our results highlight the role of negative determinants as crucial modulators of CK2 targeting and corroborate the concept that Akt1 and Akt2 display isoform specific features. Experiments with synthetic peptides suggest that Akt2 S131 could be phosphorylated by kinases of the Plk (Polo-like kinase) family, which are insensitive to the presence of the n+1 T. The low phylogenetic conservation of the Akt2 sequence around S131, as opposed to the extremely well-conserved Akt1 homologous sequence, would indicate a dominant positive role in the selective pressure only for the Akt1 phosphoacceptor site committed to undergo phosphorylation by CK2. By contrast, Akt2 S131 may mediate the response to specific physio/pathological conditions, being consequently shielded against basal CK2 targeting.

Phospholipases A2 (PLA2s) are a major component of snake venoms. Some of them cause severe muscle necrosis through a still unknown mechanism. Phospholipid hydrolysis is a possible explanation of their toxic action, but catalytic and toxic properties of PLA2s are not directly connected. In addition, viperid venoms contain PLA2-like proteins, which are very toxic even if they lack catalytic activity due to a critical mutation in position 49. Nucleolin, a main component of the nucleolus, is a disordered protein involved in many protein assembly and phase separation phenomena. In some circumstances nucleolin is exposed on the cell surface from where it is involved in the internalization of many ligands.In this work we demonstrate that Bothrops asper myotoxin II (Mt-II), a Lys49 PLA2-like toxin, interacts with, and is internalized in cells by nucleolin. The internalization process is functional to the toxicity of the protein, as both an antibody and an aptamer specific for nucleolin protect cells from intoxication. We identified central RRM and the C-terminal R/F-GG domain of nucleolin as the regions involved in the interaction with Mt-II. Finally we observed that Mt-II forms, on the cell surface, amyloid-like assemblies that colocalize with nucleolin and that can be involved in the activation of the internalization process. The presence, in the three dimensional structure of Mt-II and related PLA2 homologues, of four exposed loops enriched in prion-like amino acid sequences reinforces this hypothesis.Phospholipases A2 | Lys49 myotoxins | nucleolin | amyloid-like | molecular assembliesSIGNIFICANCEThe main finding of this work, the role of nucleolin as Bothrops asper Mt-II receptor, is a remarkable step forward in understanding the mechanism of action of cytotoxic PLA2s. It may suggest new strategies for anti-venom therapies and explain the anti-tumoral and anti-viral pharmacological action of snake PLA2s, since nucleolin is a receptor for many growth factors and virus.The proposed internalization mechanism, via formation of molecular assemblies among Mt-II amyloid-like structures and other proteins, including nucleolin, can be of general validity. Cell surface molecular assemblies couldbepointsofselectionandconcentrationnotonlyofsnake,butalsoofmammaliansecretedPLA2s, proteins involved in different pathologies, and trigger the internalization pathway only when their molarity exceeds a threshold dose.

The motif "SYDE", incorporating the protein kinase CK2 consensus sequence (S-x-x-E) has been found to be phosphorylated at both its serine and tyrosine residues in several proteins. Of special interest is the case of cystic fibrosis Transmembrane-conductance Regulator (CFTR), where this motif is close to the residue (F508), whose deletion is the by far commonest cause of cystic fibrosis. Intriguingly, however, CFTR S511 cannot be phosphorylated by CK2 to any appreciable extent. Using a number of peptide substrates encompassing the CFTR "SYDE" site we have recently shown that: (1) failure of CK2 to phosphorylate the S(511)YDE motif is due to the presence of Y512; (2) CK2 readily phosphorylates S511 if Y512 is replaced by a phospho-tyrosine; (3) the Src family protein tyrosine kinase Lyn phosphorylates Y512 in a manner that is enhanced by the deletion of F508. These data, in conjunction with the recent observation that by inhibiting CK2 the degradation of F508delCFTR is reduced, lead us to hypothesize that the hierarchical phosphorylation of the motif SYDE by the concerted action of protein tyrosine kinases and CK2 is one of the mechanisms that cooperate to the premature degradation of F508delCFTR.

International audience; The plasma membrane Ca(2+) ATPases (PMCA pumps) have a long, cytosolic C-terminal regulatory region where a calmodulin-binding domain (CaM-BD) is located. Under basal conditions (low Ca(2+)), the C-terminal tail of the pump interacts with autoinhibitory sites proximal to the active center of the enzyme. In activating conditions (i.e., high Ca(2+)), Ca(2+)-bound CaM displaces the C-terminal tail from the autoinhibitory sites, restoring activity. We have recently identified a G1107D replacement within the CaM-BD of isoform 3 of the PMCA pump in a family affected by X-linked congenital cerebellar ataxia. Here, we investigate the effects of the G1107D replacement on the interplay of the mutated CaM-BD with both CaM and the pump core, by combining computational, biochemical and functional approaches. We provide evidence that the affinity of the isolated mutated CaM-BD for CaM is significantly reduced with respect to the wild type (wt) counterpart, and that the ability of CaM to activate the pump in vitro is thus decreased. Multiscale simulations support the conclusions on the detrimental effect of the mutation, indicating reduced stability of the CaM binding. We further show that the G1107D replacement impairs the autoinhibition mechanism of the PMCA3 pump as well, as the introduction of a negative charge perturbs the contacts between the CaM-BD and the pump core. Thus, the mutation affects both the ability of the pump to optimally transport Ca(2+) in the activated state, and the autoinhibition mechanism in its resting state.

Mutations of the GJB1 gene encoding connexin 32 (Cx32) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX1), a demyelinating peripheral neuropathy for which there is no cure. A growing body of evidence indicates that ATP release through Cx32 hemichannels in Schwann cells could be critical for nerve myelination, but it is unknown if CMTX1 mutations alter the cytosolic Ca2+-dependent gating mechanism that controls Cx32 hemichannel opening and ATP release. The current study uncovered that loss of the C-terminus in Cx32 (R220X mutation), which causes a severe CMTX1 phenotype, inhibits hemichannel opening during a canonical IP3-mediated increase in cytosolic Ca2+ in HeLa cells. Interestingly, the gating function of R220X hemichannels was completely restored by both the intracellular and extracellular application of a peptide that mimics the Cx32 cytoplasmic loop. All-atom molecular dynamics simulations suggest that loss of the C-terminus in the mutant hemichannel triggers abnormal fluctuations of the cytoplasmic loop which are prevented by binding to the mimetic peptide. Experiments that stimulated R220X hemichannel opening by cell depolarization displayed reduced voltage sensitivity with respect to wild-type hemichannels which was explained by loss of subconductance states at the single channel level. Finally, experiments of intercellular diffusion mediated by wild-type or R220X gap junction channels revealed similar unitary permeabilities to ions, signalling molecules (cAMP) or larger solutes (Lucifer yellow). Taken together, our findings support the hypothesis that paracrine signalling alteration due to Cx32 hemichannel dysfunction underlies CMTX1 pathogenesis and suggest a candidate molecule for novel studies investigating a therapeutic approach.

A phosphorylated peptide, named K40H, derived from the constant region of IgMs was detected in human serum by liquid chromatography coupled to high-resolution mass spectrometry. Synthetic K40H proved to exert a potent in vitro activity against fungal pathogens, and to inhibit HIV-1 replication in vitro and ex vivo. It also showed a therapeutic effect against an experimental infection by Candida albicans in the invertebrate model Galleria mellonella. K40H represents the proof of concept of the innate role that naturally occurring antibody fragments may exert against infectious agents, shedding a new light upon the posthumous role of antibodies and opening a new scenario on the multifaceted functionality of humoral immunity.

The plasma membrane Ca

Lysosomal Storage Disorders (LSDs) are a group of metabolic syndromes, each one due to the deficit of one lysosomal enzyme. Many LSDs affect most of the organ systems and overall about 75% of the patients present neurological impairment. Enzyme Replacement Therapy, although determining some systemic clinical improvements, is ineffective on the CNS disease, due to enzymes' inability to cross the blood-brain barrier (BBB). With the aim to deliver the therapeutic enzymes across the BBB, we here assayed biodegradable and biocompatible PLGA-nanoparticles (NPs) in two murine models for LSDs, Mucopolysaccharidosis type I and II (MPS I and MPS II). PLGA-NPs were modified with a 7-aminoacid glycopeptide (g7), yet demonstrated to be able to deliver low molecular weight (MW) molecules across the BBB in rodents. We specifically investigated, for the first time, the g7-NPs ability to transfer a model drug (FITC-albumin) with a high MW, comparable to the enzymes to be delivered for LSDs brain therapy. In vivo experiments, conducted on wild-type mice and knockout mouse models for MPS I and II, also included a whole series of control injections to obtain a broad preliminary view of the procedure efficiency. Results clearly showed efficient BBB crossing of albumin in all injected mice, underlying the ability of NPs to deliver high MW molecules to the brain. These results encourage successful experiments with enzyme-loaded g7-NPs to deliver sufficient amounts of the drug to the brain district on LSDs, where exerting a corrective effect on the pathological phenotype.

NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH 2 -terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH 2 -terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH 2 globular head domain and the NH 2 dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen.