Role of non-coding RNAs in age-related vascular cognitive impairment: An overview on diagnostic/prognostic value in Vascular Dementia and Vascular Parkinsonism.

Age is the pivotal risk factor for different common medical conditions such as cardiovascular diseases, cancer and dementia. Among age-related disorders, cardiovascular and cerebrovascular diseases, represent the leading causes of premature mortality strictly related to vascular ageing, a pathological condition characterized by endothelial dysfunction, atherosclerosis, hypertension, heart disease and stroke. These features negatively impact on the brain, owing to altered cerebral blood flow, neurovascular coupling and impaired endothelial permeability leading to cerebrovascular diseases (CVDs) as Vascular Dementia (VD) and Parkinsonism (VP). It is an increasing opinion that neurodegenerative disorders and cerebrovascular diseases are associated from a pathogenetic point of view, and in this review, we discuss how cerebrovascular dysfunctions, due to epigenetic alterations, are linked with neuronal degeneration/dysfunction that lead to cognitive impairment. The relation between neurodegenerative and cerebrovascular diseases are reviewed with a focus on role of ncRNAs in age-related vascular diseases impairing the endothelium in the blood-brain barrier with consequent dysfunction of cerebral blood flow. In this review we dissert about different regulatory mechanisms of gene expression implemented by ncRNAs in the pathogenesis of age-related neurovascular impairment, aiming to highlight the potential use of ncRNAs as biomarkers for diagnostic/prognostic purposes as well as novel therapeutic targets.

Epidermal growth factor pathway substrate 15, Eps15.

Eps15 was originally identified as a substrate for the kinase activity of the epidermal growth factor receptor (EGFR). Eps15 has a tripartite structure comprising a NH2-terminal portion, which contains three EH domains, a central putative coiled-coil region, and a COOH-terminal domain containing multiple copies of the amino acid triplet Aspartate-Proline-Phenylalanine. A pool of Eps15 is localized at clathrin coated pits where it interacts with the clathrin assembly complex AP-2 and a novel AP-2 binding protein, Epsin. Perturbation of Eps15 and Epsin function inhibits receptor-mediated endocytosis of EGF and transferrin, demonstrating that both proteins are components of the endocytic machinery. Since the family of EH-containing proteins is implicated in various aspects of intracellular sorting, biomolecular strategies aimed at interfering with these processes can now be envisioned. These strategies have potentially far reaching implications extending to the control of cell proliferation. In this regard, it is of note that Eps15 has the potential of transforming NIH-3T3 cells and that the eps15 gene is rearranged with the HRX/ALL/MLL gene in acute myelogeneous leukemias, thus implicating this protein in the subversion of cell proliferation in neoplasia.

Interleukin 10 increases CCR5 expression and HIV infection in human monocytes.

The immunosuppressive and antiinflammatory cytokine interleukin (IL) 10 selectively upregulates the expression of the CC chemokine receptors CCR5, 2, and 1 in human monocytes by prolonging their mRNA half-life. IL-10-stimulated monocytes display an increased number of cell surface receptors for, and better chemotactic responsiveness to, relevant agonists than do control cells. In addition, IL-10-stimulated monocytes are more efficiently infected by HIV BaL. This effect was associated to the enhancement of viral entry through CCR5. These data add support to an emerging paradigm in which pro- and antiinflammatory molecules exert reciprocal and opposing influence on chemokine agonist production and receptor expression.

Construction, exploitation and evolution of a new peptide library displayed at high density by fusion to the major coat protein of filamentous phage.

The amino-terminus of the major coat protein (PVIII) of filamentous phage can be extended, up to 6-7 residues, without interfering with the phage life cycle. We have constructed a library of approximately ten millions different phage each displaying a different octapeptide joined to the amino-terminus of the 2700 copies of PVIII. Most of the resulting clones are able to produce infective particles. This molecular repertoire constituted by the periodic regular decoration of the phage filament surface, can be utilized to search elements that bind proteins or relatively small organic molecules like the textile dye Cibacron blue. By sequential growth cycles we have performed a library evolution experiment to select phage clones that have a growth advantage in the absence of any requirement for binding a specific target. The consensus of the best growers reveals a Pro rich sequence with large hydrophobic residues at position 7 and Asn at position 1 of the random peptide insert. We propose that the assembly secretion process is favoured in phages displaying this family of peptides since they fit the groove between two adjacent PVIII subunits by making advantageous molecular contacts on the phage surface.

Mapping of the molecular determinants involved in the interaction between eps15 and AP-2.

eps15, a substrate for the epidermal growth factor receptor and other receptor tyrosine kinases, possesses a discrete domain structure with protein-binding properties. It interacts with a number of cellular proteins through an evolutionarily conserved protein-binding domain, the eps15 homology domain, located in its NH2-terminal region. In addition, a proline-rich region, located in the COOH-terminal portion of eps15, can bind to the Src homology 3 domain of the crk proto-oncogene product in vitro. Recently, coimmunoprecipitation between eps15 and AP-2, a major component of coated pits, was reported. Here, we characterize the molecular determinants of the eps15/AP-2 interaction. The AP-2 binding region of eps15 is localized in its COOH-terminal region and spans approximately 80 amino acids. At least three molecular determinants, located at residues 650-660, 680-690, and 720-730, are involved in the binding. AP-2 binds to eps15 through its alpha subunit (alpha-adaptin); in particular, the COOH-terminal region of alpha-adaptin, the so-called alpha-ear, contains the eps15 binding region.

eps15 and eps15R are essential components of the endocytic pathway.

eps15 and eps1SR are substrates of the epidermal growth factor (EGF) receptor kinase that are characterized by the presence of a protein:protein interaction domain, the EH domain, and by their ability to bind to the clathrin adaptor protein complex adaptor protein 2. Indirect evidence suggests that eps15 and eps15R are involved in endocytosis. Here we show that microinjection of antibodies against eps15 and eps15R inhibits internalization of EGF and transferrin. In addition, fragments of eps15 (encompassing its EH domains or the COOH-terminal region that binds to adaptor protein 2) inhibit EGF internalization or endocytosis of Sindbis virus. These results demonstrate that eps15 and eps15R are essential components of the endocytic machinery.

Modifying filamentous phage capsid: limits in the size of the major capsid protein.

Ff filamentous phages are long thin cylindrical structures that infect bacteria displaying the F pilus and replicate without lysing the host. These structures are exploited to display peptides by fusing them to the amino terminus of either the bacterial receptor protein (pIII) or the major coat protein (pVIII). We have analysed a vast collection of phage mutants containing substitutions and insertions in the amino terminus of pVIII to ask whether any chemical group of this solvent exposed region of the phage capsid has any key function in the phage life cycle. Any of the five amino-terminal residues can be substituted by most amino acids without affecting phage assembly suggesting that this region does not play any essential role in morphogenesis. However, a deletion of three residues delta (Gly3Asp4Asp5) results in a phage clone with an decreased ability to produce infective particles. By engineering phages designed to display peptides by fusion to the amino terminus of the major coat protein we have found that phage viability is affected by peptide length while peptide sequence plays a minor "tuning" role. Most peptides of six residues are tolerated irrespective of their sequence while only 40% of the phages carrying an amino-terminal extension of eight residues can form infective particles. This fraction drops to 20% and 1% when we attempt to insert peptides 10 and 16 amino acids long. We have used this information to build phage libraries where each phage displays approximately 2700 copies of a different octapeptide all over the phage surface.