Laura Zaccaro

A New Nerve Growth Factor-Mimetic Peptide Active on Neuropathic Pain in Rats

Analysis of the structure of nerve growth factor (NGF)-tyrosine kinase receptor A (TrkA) complex, site-directed mutagenesis studies and results from chemical modification of amino acid residues have identified loop 1, loop 4, and the N-terminal region of the NGF molecule as the most relevant for its biological activity. We synthesized several peptides mimicking the two loops (1 and 4) linked together with an appropriate spacer, with or without the N-terminal region. Two peptides named NL1L4 and L1L4 demonstrated good NGF agonist activity at a concentration as low as 3 μm. They induced differentiation of chick dorsal root ganglia and stimulated tyrosine phosphorylation of TrkA, but not TrkB, receptor. In addition L1L4 was able to induce differentiation of PC12 cells. More interestingly, the peptide with the highest “in vitro” activity (L1L4) was shown to reduce neuropathic behavior and restore neuronal function in a rat model of peripheral neuropathic pain, thereby suggesting a potential therapeutic role for this NGF-mimetic peptide.

Reactive astrocytosis-induced perturbation of synaptic homeostasis is restored by nerve growth factor

Reactive gliosis has been implicated in both inflammatory and neurodegenerative diseases. However, mechanisms by which astrocytic activation affects synaptic efficacy have been poorly elucidated. We have used the spared nerve injury (SNI) of the sciatic nerve to induce reactive astrocytosis in the lumbar spinal cord and investigate its potential role in disrupting the neuro-glial circuitry. Analysis of spinal cord sections revealed that SNI was associated with an increase of microglial (Iba1) and astrocytic (GFAP) markers. These changes, indicative of reactive gliosis, were paralleled by (i) a decrease of glial amino acid transporters (GLT1 and GlyT1) and increased levels of (ii) neuronal glutamate transporter EAAC1, (iii) neuronal vesicular GABA transporter (vGAT) and (iv) the GABAergic neuron marker GAD65/67. Besides the increase of Glutamate/GABA ratio, indicative of the perturbation of synaptic circuitry homeostasis, the boost of glutamate also compromised glial function in neuroprotection by up-regulating the xCT subunit of the glutamate-cystine antiport system and reducing glutathione (GSH) production. Finally, this study also shows that all these structural changes were linked to an alteration of endogenous NGF metabolism, as demonstrated by the decrease of endogenous NGF expression levels and increased activity of the NGF-degrading metalloproteinases. All the changes displayed by SNI-animals were reversed by a 7-days i.t. administration of NGF or GM6001, a generic metalloproteinase inhibitor, as compared to vehicle (ACSF)-treated animals. All together, these data strongly support the correlation between reactive astrogliosis and mechanisms underlying the perturbation of the synaptic circuitry in the SNI model of peripheral nerve injury, and the essential role of NGF in restoring both synaptic homeostasis and the neuroprotective function of glia.

Zinc to cadmium replacement in the A. thaliana SUPERMAN Cys2His2 zinc finger induces structural rearrangements of typical DNA base determinant positions

Among heavy metals, whose toxicity cause a steadily increasing of environmental pollution, cadmium is of special concern due to its relatively high mobility in soils and potential toxicity at low concentrations. Given their ubiquitous role, zinc fingers domains have been proposed as mediators for the toxic and carcinogenic effects exerted by xenobiotic metals. To verify the structural effects of zinc replacement by cadmium in zinc fingers, we have determined the high resolution structure of the single Cys₂ His₂ zinc finger of the Arabidopsis thaliana SUPERMAN protein (SUP37) complexed to the cadmium ion by means of UV-vis and NMR techniques. SUP37 is able to bind Cd(II), though with a dissociation constant higher than that measured for Zn(II). Cd-SUP37 retains the ββα fold but experiences a global structural rearrangement affecting both the relative orientation of the secondary structure elements and the position of side chains involved in DNA recognition: among them Ser17 side chain, which we show to be essential for DNA binding, experiences the largest displacement.

BB14, a Nerve Growth Factor (NGF)-like peptide shown to be effective in reducing reactive astrogliosis and restoring synaptic homeostasis in a rat model of peripheral nerve injury

Peptidomimetics hold a great promise as therapeutic agents for neurodegenerative disorders. We previously described a Nerve Growth Factor (NGF)-like peptide, now named BB14, which was found to act as a strong TrkA agonist and to be effective in the sciatic nerve injury model of neuropathic pain. In this report we present the effects of BB14 in reducing reactive astrocytosis and reverting neuroplastic changes of the glutamate/GABAergic circuitry in the lumbar spinal cord following spared nerve injury (SNI) of the sciatic nerve. Immunohistochemical analysis of spinal cord sections revealed that SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) responses, indicative of reactive gliosis. These changes were paralleled by (i) decreased glial aminoacid transporters (GLT1 and GlyT1) and increased levels of (ii) neuronal glutamate transporter EAAC1, (iii) neuronal vesicular GABA transporter (vGAT) and (iv) the GABAergic neuron marker GAD65/67. A remarkable increase of the Glutamate/GABA ratio and the reduction of glutathione (GSH) levels were also indicative of modifications of glial function in neuroprotection. All these molecular changes were found to be linked to an alteration of endogenous NGF metabolism, as demonstrated by decreased levels of mature NGF, increase of proNGF and increased activity of NGF-degrading methallo-proteinases (MMPs). Biochemical alterations and SNI-related neuropathic behavior, characterized by allodynia and hyperalgesia, were reversed by 7-days i.t. administration of the NGF-like peptide BB14, as well as by increasing endogenous NGF levels by i.t. infusion of GM6001, a MMPs inhibitor. All together, while confirming the correlation between reactive astrogliosis and perturbation of synaptic circuitry in the SNI model of peripheral nerve injury, these data strongly support the beneficial effect of BB14 in reducing reactive astrogliosis and restoring synaptic homeostasis under pathological conditions linked to alteration of NGF availability and signaling, thereby suggesting a potential role of BB14 as a therapeutic agent.

Deciphering the zinc coordination properties of the prokaryotic zinc finger domain: The solution structure characterization of Ros87 H42A functional mutant

The zinc coordination sphere in prokaryotic zinc finger domain is extremely versatile and influences the stability and the folding property of the domain. Of a particular interest is the fourth zinc coordinating position, which is frequently occupied by two successive histidines, both able to coordinate the metal ion. To clarify their structural and functional role we report the NMR solution structure and the dynamics behavior of Ros87 H42A, which is a functional mutant of Ros87, the DNA binding domain of the Ros protein containing a prokaryotic Cys2His2 zinc finger domain. The structural analysis indicates that reducing the spacer among the two coordinating histidines from 4 (among His37 and His42) amino acids to 3 (among His37 and His41) increases the helicity of the first α-helix. At the same time, the second helix appears more mobile in the μs-ms timescale and the hydrophobic core is reduced. These data explain the high frequency of three-residue His spacers in the eukaryotic zinc finger domain and their absence in the prokaryotic counterpart. Furthermore, the structural comparison shows that the second coordination position is more sensitive to H42A mutation with respect to the first and the third position, providing the rationale of the high variability of the second and the fourth zinc coordinating position in Ros homologs, which adopt different metal coordination but preserve similar tertiary structures and DNA binding activities. Finally, H/D exchange measurements and NMR thermal unfolding analysis indicate that this mutant likely unfolds via a different mechanism with respect to the wild-type.

The (unusual) aspartic acid in the metal coordination sphere of the prokaryotic zinc finger domain.

The possibility of choices of protein ligands and coordination geometries leads to diverse Zn(II) binding sites in zinc-proteins, allowing a range of important biological roles. The prokaryotic Cys2His2 zinc finger domain (originally found in the Ros protein from Agrobacterium tumefaciens) tetrahedrally coordinates zinc through two cysteine and two histidine residues and it does not adopt a correct fold in the absence of the metal ion. Ros is the first structurally characterized member of a family of bacterial proteins that presents several amino acid changes in the positions occupied in Ros by the zinc coordinating residues. In particular, the second position is very often occupied by an aspartic acid although the coordination of structural zinc by an aspartate in eukaryotic zinc fingers is very unusual. Here, by appropriately mutating the protein Ros, we characterize the aspartate role within the coordination sphere of this family of proteins demonstrating how the presence of this residue only slightly perturbs the functional structure of the prokaryotic zinc finger domain while it greatly influences its thermodynamic properties.

A new cryptic host defense peptide identified in human 11-hydroxysteroid dehydrogenase-1 β-like: from in silico identification to experimental evidence

BACKGROUNDnHost defence peptides (HDPs) are evolutionarily conserved components of innate immunity. Human HDPs, produced by a variety of immune cells of hematopoietic and epithelial origin, are generally grouped into two families: beta structured defensins and variably-structured cathelicidins. We report the characterization of a very promising cryptic human HDP, here called GVF27, identified in 11-hydroxysteroid dehydrogenase-1 β-like protein.nnnMETHODSnConformational analysis of GVF27 and its propensity to bind endotoxins were performed by NMR, Circular Dichroism, Fluorescence and Dynamic Light Scattering experiments. Crystal violet and WST-1 assays, ATP leakage measurement and colony counting procedures were used to investigate antimicrobial, anti-biofilm, cytotoxicity and hemolytic activities. Anti-inflammatory properties were evaluated by ELISA.nnnRESULTSnGVF27 possesses significant antibacterial properties on planktonic cells and sessile bacteria forming biofilm, as well as promising dose dependent abilities to inhibit attachment or eradicate existing mature biofilm. It is unstructured in aqueous buffer, whereas it tends to assume a helical conformation in mimic membrane environments as well as it is able to bind lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Notably it is not toxic towards human and murine cell lines and triggers a significant innate immune response by attenuating expression levels of pro-inflammatory interleukins and release of nitric oxide in LPS induced macrophages.nnnCONCLUSIONnHuman GVF27 may offer significant advantages as leads for the design of human-specific therapeutics.nnnGENERAL SIGNIFICANCEnHuman cryptic host defence peptides are naturally no immunogenic and for this they are a real alternative for solving the lack of effective antibiotics to control bacterial infections.

Insights into the anticancer properties of the first antimicrobial peptide from Archaea

BACKGROUNDnThe peptide VLL-28, identified in the sequence of an archaeal protein, the transcription factor Stf76 from Sulfolobus islandicus, was previously identified and characterized as an antimicrobial peptide, possessing a broad-spectrum antibacterial activity.nnnMETHODSnThrough a combined approach of NMR and Circular Dichroism spectroscopy, Dynamic Light Scattering, confocal microscopy and cell viability assays, the interaction of VLL-28 with the membranes of both parental and malignant cell lines has been characterized and peptide mechanism of action has been studied.nnnRESULTSnIt is here demonstrated that VLL-28 selectively exerts cytotoxic activity against murine and human tumor cells. By means of structural methodologies, VLL-28 interaction with the membranes has been proven and the binding residues have been identified. Confocal microscopy data show that VLL-28 is internalized only into tumor cells. Finally, it is shown that cell death is mainly caused by a time-dependent activation of apoptotic pathways.nnnCONCLUSIONSnVLL-28, deriving from the archaeal kingdom, is here found to be endowed with selective cytotoxic activity towards both murine and human cancer cells and consequently can be classified as an ACP.nnnGENERAL SIGNIFICANCEnVLL-28 represents the first ACP identified in an archaeal microorganism, exerting a trans-kingdom activity.

Deciphering RGDechi peptide-α5β1 integrin interaction mode in isolated cell membranes

Integrins are a large family of heterodimeric receptors critically engaged in pathological processes such as tumor progression and metastasis. Although they are validated therapeutic targets, the molecular determinants governing integrin‐ligand interactions are not yet fully understood, leading to a scarcity of integrin sub‐type exclusive antagonists. In the past decade, we have investigated the biological behavior of the RGDechi, a chimeric peptide able to specifically bind αvβ3 integrin without cross reacting with αvβ5 and αIIbβ3 integrins. Here we have investigated the capability of the peptide to bind α5β1 integrin and characterized the molecular determinants governing this interaction through a combined experimental and computational approach. The detailed comparison of RGDechi‐α5β1 structural model with that previously determined of RGDechi in complex with αvβ3 shows how the bifunctional nature of the peptide renders the molecule an important tool to recognize integrins with different recognition modalities, providing novel insight on the structural requirements needed to their specific recognition.

A selective αvβ5 integrin antagonist hidden into the anophelin family protein cE5 from the malaria vector Anopheles gambiae

A RGD motif was identified in the N‐terminal region of cE5, a potent salivary thrombin inhibitor from the African malaria vector Anopheles gambiae. A peptide (APQ30) encompassing the first 30 amino acids residues of the protein and including the RGD tripeptide was tested in cell adhesion assays and found to inhibit αvβ3 and αvβ5 mediated adhesion. A shorter peptide (APQ16), strongly conserved among members of the A. gambiae species complex and including only the first 16 residues, retained adhesion inhibitory properties, however with enhanced specificity toward αvβ5. In addition, migration and invasion assays showed its capacity to inhibit the invasiveness of the malignant cell lines HepG2 and MDA‐MB231. Altogether our data point to APQ16 as a new promising candidate as theranostic agent.