Downregulation of myosin II-B by siRNA alters the subcellular localization of the amyloid precursor protein and increases amyloid-beta deposition in N2a cells.

The Alzheimer's disease (AD) brain pathology is characterized by extracellular deposits of amyloid-beta (Abeta) peptides and intraneuronal fibrillar structures. These pathological features may be functionally linked, but the mechanism by which Abeta accumulation relates to neuronal degeneration is still poorly understood. Abeta peptides are fragments cleaved from the amyloid precursor protein (APP), a transmembrane protein ubiquitously expressed in the nervous system. Although the proteolytic processing of APP has been implicated in AD, the physiological function of APP and the subcellular site of APP cleavages remain unknown. The overall structure of the protein and its fast anterograde transport along the axon support the idea that APP functions as a vesicular receptor for cytoskeletal motor proteins. In the current study, we test the hypothesis that myosin II, important contributor to the cytoskeleton of neuronal cells, may influence the trafficking and/or the processing of APP. Our results demonstrate that downregulation of myosin II-B, the major myosin isoform in neurons, is able to increase Abeta deposition, concomitantly altering the subcellular localization of APP. These new insights might be important for the understanding of the function of APP and provide a novel conceptual framework in which to analyze its pathological role.

Identification of a new truncated form and deamidation products of fibrinopeptide B released by thrombin from human fibrinogen.

Quantification of fibrinopeptides release is widely used to investigate fibrinogen activation, and standard chromatographic or capillary electrophoretic procedures are readily available. However, in the analyses of fibrinopeptide mixtures derived from the action of thrombin on human fibrinogen, a few unidentified peaks are usually present. The composition of these peaks was studied by reverse-phase HPLC/MS, revealing a single major anomalous peptide having a molecular mass of 1384.4. A further MS/MS analysis allowed the identification of this form, as a Nterminally truncated fibrinopeptide B (fpB) lacking the first two residues (pyroglutamic acid and glycine). This previously unidentified, relatively low-abundance form ( approximately 7%) has been found consistently in our fibrinopeptides preparations, and analysis of the parent Bbeta-chain suggest that it is likely present in circulating fibrinogen. In addition, deamidated forms of all fpB species (including desArgB), resulting from the conversion of asparagine to aspartic acid, were also identified. Overall, these previously unreported forms constitute a substantial amount of fpB (up to approximately 17% of the total), and should be taken into account for a reliable quantitative analysis of fpB release.

NAADP+ synthesis from cADPRP and nicotinic acid by ADP-ribosyl cyclases.

ADP-ribosyl cyclases (ADPRCs) are present from lower Metazoa to mammals and synthesize the Ca2+-active (di)nucleotides cyclic ADP-ribose (cADPR), NAADP+, and ADP-ribose (ADPR), involved in the regulation of important cellular functions. NAADP+ can be synthesized by ADPRCs from NADP+ through a base-exchange reaction, which substitutes nicotinamide for nicotinic acid (NA). Here we demonstrate that ADPRCs from both lower and higher Metazoa (including human CD38) can also synthesize NAADP+ starting from 2'-phospho-cyclic ADP-ribose (cADPRP) and NA. Comparison, on the two substrates cADPRP and NADP+, of the relative rates of the reactions introducing NA and hydrolyzing/cyclizing the substrate, respectively, indicates that with all ADPRCs tested cADPRP is preferentially transformed into NAADP+, while NADP+ is preferentially cyclized or hydrolyzed to cADPRP/2'-phospho-ADP-ribose. cADPRP was detectable in retinoic acid-differentiated, CD38+ HL-60 cells, but not in undifferentiated, CD38- cells. These results suggest that cADPRP may be a NAADP+ precursor in ADPRC+ cells.