Rina Yamin

Metalloendopeptidase EC 3.4.24.15 is necessary for Alzheimer's amyloid-beta peptide degradation.

We have investigated the functional relationship between metalloendopeptidase EC 3.4.24.15 (MP24.15) and the amyloid precursor protein involved in Alzheimer’s disease (AD) and discovered that the enzyme promotes Aβ degradation. We show here that conditioned medium (CM) of MP24.15 antisense-transfected SKNMC neuroblastoma has significantly higher levels of Aβ. Furthermore, synthetic-Aβ degradation was increased or decreased following incubation with CM of sense or antisense-transfected cells, respectively. Soluble Aβ1–42 was degraded more slowly than soluble Aβ1–40, while aggregated Aβ1–42 showed almost no degradation. Pretreatment of CM with serine proteinase inhibitors 4-(2-aminoethyl)benzenesulfonyl fluoride and diisopropyl fluorophosphate completely inhibited Aβ degradation. Additionally, α1-antichymotrypsin (ACT), a serpin family inhibitor tightly associated with plaques and elevated in AD brain, blocked up to 60% of Aβ degradation. Interestingly, incubation of CM of MP24.15-overexpressing cells with ACT formed an SDS-resistant ACT complex, suggesting an ACT-serine proteinase interaction. Recombinant MP24.15 alone did not degrade Aβ. 14C-Diisopropyl fluorophosphate-radiolabeled CM from MP24.15-overexpressing cells contained increased levels of several active serine proteinases, suggesting that MP24.15 activates one or more Aβ-degrading serine proteases. Thus, ACT may cause Aβ accumulation by inhibiting an Aβ-degrading enzyme or by direct binding to Aβ, rendering it degradation-resistant. Identification of the Aβ-degrading enzyme and MP24.15’s role in its activation is underway. Pharmacological modulation of either enzyme may provide a means of regulating Aβ in the brain.

Deciphering actin cytoskeletal function in the contractile vascular smooth muscle cell

Abstract  This review focuses on the vascular smooth muscle cells present in the medial layer of the blood vessels wall in the fully differentiated state (dVSMCs). The dVSMC contractile phenotype enables these cells to respond in a highly regulated manner to changes in extracellular stimuli. Through modulation of vascular contractile force and vascular compliance dVSMCs regulate blood pressure and blood flow. The cellular and molecular mechanisms by which vascular smooth muscle contractile functions are regulated are not completely elucidated. Recent studies have documented a critical role for actin polymerization and cytoskeletal dynamics in the regulation of contractile function. Here we will review the current understanding of actin cytoskeletal dynamics and focal adhesion function in dVSMCs in order to better understand actin cytoskeleton connections to the extracellular matrix and the effects of cytoskeletal remodelling on vascular contractility and vascular stiffness in health and disease.

Amyloid precursor protein interacts with notch receptors.

The amyloid precursor protein (APP) must fulfill important roles based on its sequence conservation from fly to human. Although multiple functions for APP have been proposed, the best‐known role for this protein is as the precursor of Aβ peptide, a neurotoxic 39–43‐amino acid peptide crucial to the pathogenesis of Alzheimers disease. To investigate additional roles for APP with an eye toward understanding the molecular basis of the pleiotropic effects ascribed to APP, we isolated proteins that interacted with the plasma membrane isoform of APP. We employed a membrane‐impermeable crosslinker to immobilize proteins binding to transmembrane APP in human embryonic kidney (HEK)293 cells expressing APP751 (HEK275) or rat embryonic day 18 primary neurons infected with a virus expressing APP. Notch2 was identified as a potential APP binding partner based on mass spectrometry analysis of APP complexes immunopurified from neurons. To confirm the interaction between Notch2 and APP, we carried out immunoprecipitation studies in HEK275 cells transiently expressing full‐length Notch2 using Notch2 antibodies. The results indicated that APP and Notch2 interact in mammalian cells, and confirmed our initial findings. Interestingly, Notch1 also coimmunoprecipitated with APP, suggesting that APP and Notch family members may engage in intermolecular cross talk to modulate cell function. Finally, cotransfection of APP/CFP and Notch2/YFP into COS cells revealed that these two proteins colocalize on the plasma membrane. Intracellularly, however, although some APP and Notch molecules colocalize, others reside in distinct locations. The discovery of proteins that interact with APP may aid in the identification of new functions for APP.

Acyl Peptide Hydrolase Degrades Monomeric and Oligomeric Amyloid-Beta Peptide

BackgroundThe abnormal accumulation of amyloid-beta peptide is believed to cause malfunctioning of neurons in the Alzheimers disease brain. Amyloid-beta exists in different assembly forms in the aging mammalian brain including monomers, oligomers, and aggregates, and in senile plaques, fibrils. Recent findings suggest that soluble amyloid-beta oligomers may represent the primary pathological species in Alzheimers disease and the most toxic form that impairs synaptic and thus neuronal function. We previously reported the isolation of a novel amyloid-beta-degrading enzyme, acyl peptide hydrolase, a serine protease that degrades amyloid-beta, and is different in structure and activity from other amyloid-beta-degrading enzymes.ResultsHere we report the further characterization of acyl peptide hydrolase activity using mass spectrometry. Acyl peptide hydrolase cleaves the amyloid-beta peptide at amino acids 13, 14 and 19. In addition, by real-time PCR we found elevated acyl peptide hydrolase expression in brain areas rich in amyloid plaques suggesting that this enzymes levels are responsive to increases in amyloid-beta levels. Lastly, tissue culture experiments using transfected CHO cells expressing APP751 bearing the V717F mutation indicate that acyl peptide hydrolase preferentially degrades dimeric and trimeric forms of amyloid-beta.ConclusionThese data suggest that acyl peptide hydrolase is involved in the degradation of oligomeric amyloid-beta, an activity that, if induced, might present a new tool for therapy aimed at reducing neurodegeneration in the Alzheimers brain.

Acyl peptide hydrolase, a serine proteinase isolated from conditioned medium of neuroblastoma cells, degrades the amyloid‐β peptide

Considerable evidence indicates that the amyloid‐β (Aβ) peptide, a proteolytic fragment of the amyloid precursor protein, is the pathogenic agent in Alzheimers disease (AD). A number of proteases have been reported as capable of degrading Aβ, among them: neprilysin, insulin‐degrading enzyme, endothelin‐converting enzyme‐1 and ‐2, angiotensin‐converting enzyme and plasmin. These proteases, originating from a variety of cell types, degrade Aβ of various conformational states and in different cellular locations. We report here the isolation of a serine protease from serum‐free conditioned medium of human neuroblastoma cells. Tandem mass spectrometry (MS/MS)‐based sequencing of the isolated protein identified acyl peptide hydrolase (APH; EC3.4.19.1) as the active peptidase. APH is one of four members of the prolyl oligopeptidase family of serine proteases expressed in a variety of cells and tissues, including erythrocytes, liver and brain, but its precise biological activity is unknown. Here, we describe the identification of APH as an Aβ‐degrading enzyme, and we show that the degradation of Aβ by APH isolated from transfected cells is inhibited by APH‐specific inhibitors, as well as by synthetic Aβ peptide. In addition, we cloned APH from human brain and from neuroblastoma cells. Most importantly, our results indicate that APH expression in AD brain is lower than in age‐matched controls.

α1-Antichymotrypsin Inhibits Aβ Degradation in Vitro and in Vivo

Abstract: The neuropathology of Alzheimers disease (AD) is characterized by extensive deposition of the toxic amyloid β peptide (Aβ) in selected regions of the brain and brain vasculature (Selkoe, 1999). Thus, lowering the levels of Aβ may be beneficial for AD patients. Aβ is a proteolytic fragment derived from the amyloid precursor protein (APP). The mechanisms of Aβ formation from its precursor have been studied extensively; however, considerably less effort has been invested into studying Aβ clearance. We find that the degradation of Aβ in our system is dependent upon the presence of a metallopeptidase E.C.3.4.24.15 (MP24.15) (Yamin et al., 1999). We have previously purified MP24.15 to homogeneity from AD brain and identified it as an APP‐processing protease in vitro (Papastoitsis, 1994). To confirm its role in cell culture, we transfected SKNMC neuroblastoma cells with sense and antisense cDNAs of MP24.15 and with a mock construct. Compared to mock conditioned media (CM), CM of MP24.15‐overexpressing cells had very high Aβ‐degrading activity. Conversely, CM of antisense‐expressing cells lacked Aβ‐degrading activity. These results suggested that MP24.15 is involved in Aβ degradation. Characterization of the proteolytic activity directly responsible for Aβ degradation using a spectrum of protease inhibitors revealed that only serine protease inhibitors completely blocked Aβ degradation. Therefore, MP24.15 appears to activate a serine protease, which then cleaves Aβ. Interestingly, α1‐antichymotrypsin (ACT) which we discovered to be highly elevated in AD brain (Abraham, et al., 1988) also inhibited Aβ degradation. To our delight, ACT proved to be an inhibitor of Aβ degradation in vivo as well. When we crossed transgenic mice expressing human ACT with plaque‐producing mice expressing human APP, the doubly transgenic mice had twice as many plaques at 20 months of age as the APP mice (Mucke et al., 2000). Successful completion of this study could lead to the design of reagents that would reduce the amyloid load in AD patients.

Aging impairs smooth muscle-mediated regulation of aortic stiffness: a defect in shock absorption function?

Increased aortic stiffness is an early and independent biomarker of cardiovascular disease. Here we tested the hypothesis that vascular smooth muscle cells (VSMCs) contribute significantly to aortic stiffness and investigated the mechanisms involved. The relative contributions of VSMCs, focal adhesions (FAs), and matrix to stiffness in mouse aorta preparations at optimal length and with confirmed VSMC viability were separated by the use of small-molecule inhibitors and activators. Using biomechanical methods designed for minimal perturbation of cellular function, we directly quantified changes with aging in aortic material stiffness. An alpha adrenoceptor agonist, in the presence of N(G)-nitro-l-arginine methyl ester (l-NAME) to remove interference of endothelial nitric oxide, increases stiffness by 90-200% from baseline in both young and old mice. Interestingly, increases are robustly suppressed by the Src kinase inhibitor PP2 in young but not old mice. Phosphotyrosine screening revealed, with aging, a biochemical signature of markedly impaired agonist-induced FA remodeling previously associated with Src signaling. Protein expression measurement confirmed a decrease in Src expression with aging. Thus we report here an additive model for the in vitro biomechanical components of the mouse aortic wall in which 1) VSMCs are a surprisingly large component of aortic stiffness at physiological lengths and 2) regulation of the VSMC component through FA signaling and hence plasticity is impaired with aging, diminishing the aortas normal shock absorption function in response to stressors.