Degradation of Amyloid �� Protein by Purified Myelin Basic Protein

The progressive accumulation of β-amyloid (Aβ) in senile plaques and in the cerebral vasculature is the hallmark of Alzheimer disease and related disorders. Impaired clearance of Aβ from the brain likely contributes to the prevalent sporadic form of Alzheimer disease. Several major pathways for Aβ clearance include receptor-mediated cellular uptake, blood-brain barrier transport, and direct proteolytic degradation. Myelin basic protein (MBP) is the major structural protein component of myelin and plays a functional role in the formation and maintenance of the myelin sheath. MBP possesses endogenous serine proteinase activity and can undergo autocatalytic cleavage liberating distinct fragments. Recently, we showed that MBP binds Aβ and inhibits Aβ fibril formation (Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2007) J. Biol. Chem. 282, 9952–9961; Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2009) Biochemistry 48, 4720–4727). Here we show that Aβ40 and Aβ42 peptides are degraded by purified human brain MBP and recombinant human MBP, but not an MBP fragment that lacks autolytic activity. MBP-mediated Aβ degradation is inhibited by serine proteinase inhibitors. Similarly, Cos-1 cells expressing MBP degrade exogenous Aβ40 and Aβ42. In addition, we demonstrate that purified MBP also degrades assembled fibrillar Aβ in vitro. Mass spectrometry analysis identified distinct degradation products generated from Aβ digestion by MBP. Lastly, we demonstrate in situ that purified MBP can degrade parenchymal amyloid plaques as well as cerebral vascular amyloid that form in brain tissue of Aβ precursor protein transgenic mice. Together, these findings indicate that purified MBP possesses Aβ degrading activity in vitro.The progressive accumulation of β-amyloid (Aβ)³ in senile/neuritic plaques and the cerebral vasculature is the hallmark of Alzheimer disease (AD) and is widely used in the pathological diagnosis of the disease. Aβ is generated by proteolytic cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase (1, 2). The main species of Aβ are 40 and 42 amino acids in length. Aβ42 is much more amyloidogenic than Aβ40 because of its two additional hydrophobic amino acids at the carboxyl-terminal end of the peptide (3). The Aβ42 peptide is the predominant form in senile plaques, forming a β-sheet structure, which is insoluble and resistant to proteolysis.Although increased production of Aβ has been implicated in the onset of familial forms of AD, it has been hypothesized that the more common sporadic forms of AD may be caused by the impaired clearance of Aβ peptides from the CNS. Several major pathways for Aβ clearance have been proposed including receptor-mediated cellular uptake, blood-brain barrier transport into the circulation, and direct proteolytic degradation (4–6). In the latter case, several proteinases or peptidases have been identified that are capable of degrading Aβ, including neprilysin (7, 8), insulin-degrading enzyme (9), the urokinase/tissue plasminogen activator-plasmin system (10), endothelin-converting enzyme (11), angiotensin-converting enzyme (12), gelatinase A (matrix metalloproteinase-2) (13, 14), gelatinase B (matrix metalloproteinase-9) (15), and acylpeptide hydrolase (16). Each of these enzymes has been shown to cleave Aβ peptides at multiple sites (5). However, only neprilysin, insulin-degrading enzyme, endothelin-converting enzyme, and matrix metalloproteinase-9 have been shown to have a significant role in regulating Aβ levels in the brains of experimental animal models (8, 17, 18).The “classic” myelin basic proteins (MBP) are major structural components of myelin sheaths accounting for 30% of total myelin protein. There are four different major isoforms generated from alternative splicing with molecular masses of 17.3, 18.5, 20.2, and 21.5 kDa. The 18.5-kDa variant, composed of 180 amino acids including 19 Arg and 12 Lys basic residues, is most abundant in mature myelin (19). One of the major functions of MBP is to hold together the cytoplasmic leaflets of myelin membranes to maintain proper compaction of the myelin sheath through the electrostatic interaction between the positive Arg and Lys residues of MBP and the negatively charged phosphate groups of the membrane lipid (20). MBP plays an important role in the pathology of multiple sclerosis, which is an autoimmune disease characterized by demyelination within white matter (21). Recently, it was reported that purified MBP exhibits autocleavage activity, generating distinct peptide fragments (22). In this study, serine 151 was reported as the active site serine residue involved in autocatalysis.In the early stages of AD, appreciable and diffuse myelin breakdown in the white matter is observed (23). Also, in white matter regions there are much fewer fibrillar amyloid deposits than are commonly found in gray matter regions. Recently, our laboratory has shown that MBP strongly interacts with Aβ peptides and prevents their assembly into mature amyloid fibrils (24, 25). Through the course of these studies we observed that upon longer incubations the levels of Aβ peptides were reduced upon treatment with MBP. In light of this observation, coupled with the report that MBP possesses proteolytic activity, we hypothesized that MBP may degrade Aβ peptides. In the present study, we show that purified human brain MBP and recombinantly expressed human MBP can degrade soluble Aβ40 and Aβ42 peptides in vitro. Purified MBP also degraded fibrillar Aβ in vitro. Mass spectrometry analysis identified distinct degradation products generated from soluble and fibrillar Aβ digestion by MBP. Furthermore, purified MBP degraded parenchymal and vascular fibrillar amyloid deposits in situ in the brain tissue of APP transgenic mice. Together, these findings indicate that purified MBP possesses Aβ degrading activity in vitro.     

Degradation of Cellular Phospholipids and Softening of Pressed Baker’s Yeast

Variations in lipid components of washings and homogenate of pressed baker’s yeast were investigated during the storage of pressed baker’s yeast at 30°C. Washings represents the substances which had leaked out from cells. Homogenate represents those contained in whole cells. Lipids in yeast washings increased toward softening, the phospholipids in yeast homogenate decreased continuously during storage. Two stages, an earlier period of storage (Stage I) and a later period of storage (Stage II) were observed in the degradation of phospholipids. Free fatty acid which was the main degradation product of phospholipid accumulated in Stage II, particularly at softening. The order in phospholipid degradation was PC>PE>PI + PS (PI>PS). Moreover, when washings of stored yeast at softening were assayed using ¹⁴C-acyl PC, the release of ¹⁴C-acyl fatty acid was observed.

These results suggest that phospholipids were degraded by some phospholipid-deacylating enzymes toward softening. From the results of lipid analysis, we inferred that the responsible enzymes were phospholipases.     

Targeting of Integrin ��1 and Kinesin 2�� by MicroRNA 183

MicroRNA 183 (miR-183) has been reported to inhibit tumor invasiveness and is believed to be involved in the development and function of ciliated neurosensory organs. We have recently found that expression of miR-183 increased after the induction of cellular senescence by exposure to H₂O₂. To gain insight into the biological roles of miR-183 we investigated two potential novel targets: integrin β1 (ITGB1) and kinesin 2α (KIF2A). miR-183 significantly decreased the expression of ITGB1 and KIF2A measured by Western blot. Targeting of the 3′-untranslated region (3′-UTR) of ITGB1 and KIF2A by miR-183 was confirmed by luciferase assay. Transfection with miR-183 led to a significant decrease in cell invasion and migration capacities of HeLa cells that could be rescued by expression of ITGB1 lacking the 3′-UTR. Although miR-183 had no effects on cell adhesion in HeLa cells, it significantly decreased adhesion to laminin, gelatin, and collagen type I in normal human diploid fibroblasts and human trabecular meshwork cells. These effects were also rescued by expression of ITGB1 lacking the 3′-UTR. Targeting of KIF2A by miR-183 resulted in some increase in the formation of cells with monopolar spindles in HeLa cells but not in human diploid fibroblast or human trabecular meshwork cells. The regulation of ITGB1 expression by miR-183 provides a new mechanism for the anti-metastatic role of miR-183 and suggests that this miRNA could influence the development and function in neurosensory organs, and contribute to functional alterations associated with cellular senescence in human diploid fibroblasts and human trabecular meshwork cells.     

Phosphoinositide 3-Kinase p110�� Regulates Integrin ��IIb��3 Avidity and the Cellular Transmission of Contractile Forces

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin α_IIbβ₃, necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin α_IIbβ₃ activation (affinity modulation) primarily occurs downstream of G_i-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin α_IIbβ₃ bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110β isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin α_IIbβ₃ activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin α_IIbβ₃ association with the contractile cytoskeleton. Analysis of integrin α_IIbβ₃ adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin α_IIbβ₃ bonds. These studies demonstrate an important role for PI3K p110β in regulating the avidity of high affinity integrin α_IIbβ₃ receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110β inhibitors.     

Smad3 Prevents ��-Catenin Degradation and Facilitates ��-Catenin Nuclear Translocation in Chondrocytes

Our previous study demonstrated that transforming growth factor (TGF)-β activates β-catenin signaling through Smad3 interaction with β-catenin in chondrocytes. In the present studies, we further investigated the detailed molecular mechanism of the cross-talk between TGF-β/Smad3 and Wnt/β-catenin signaling pathways. We found that C-terminal Smad3 interacted with both the N-terminal region and the middle region of β-catenin protein in a TGF-β-dependent manner. Both Smad3 and Smad4 were required for the interaction with β-catenin and protected β-catenin from an ubiquitin-proteasome-dependent degradation. In addition, the formation of the Smad3-Smad4-β-catenin protein complex also mediated β-catenin nuclear translocation. This Smad3-mediated regulatory mechanism of β-catenin protein stability enhanced the activity of β-catenin to activate downstream target genes during chondrogenesis. Our findings demonstrate a novel mechanism between TGF-β and Wnt/β-catenin signaling pathways during chondrocyte development.     

A comparison of the different helices adopted by ��- and ��-peptides suggests different reasons for their stability

The right‐handed α‐helix is the dominant helical fold of α‐peptides, whereas the left‐handed 3₁₄‐helix is the dominant helical fold of β‐peptides. Using molecular dynamics simulations, the properties of α‐helical α‐peptides and 3₁₄‐helical β‐peptides with different C‐terminal protonation states and in the solvents water and methanol are compared. The observed energetic and entropic differences can be traced to differences in the polarity of the solvent‐accessible surface area and, in particular, the solute dipole moments, suggesting different reasons for their stability.     

Characterization of Erg K+ Channels in ��- and ��-Cells of Mouse and Human Islets

Voltage-gated eag-related gene (Erg) K⁺ channels regulate the electrical activity of many cell types. Data regarding Erg channel expression and function in electrically excitable glucagon and insulin producing cells of the pancreas is limited. In the present study Erg1 mRNA and protein were shown to be highly expressed in human and mouse islets and in α-TC6 and Min6 cells α- and β-cell lines, respectively. Whole cell patch clamp recordings demonstrated the functional expression of Erg1 in α- and β-cells, with rBeKm1, an Erg1 antagonist, blocking inward tail currents elicited by a double pulse protocol. Additionally, a small interference RNA approach targeting the kcnh2 gene (Erg1) induced a significant decrease of Erg1 inward tail current in Min6 cells. To investigate further the role of Erg channels in mouse and human islets, ratiometric Fura-2 AM Ca²⁺-imaging experiments were performed on isolated α- and β-cells. Blocking Erg channels with rBeKm1 induced a transient cytoplasmic Ca²⁺ increase in both α- and β-cells. This resulted in an increased glucose-dependent insulin secretion, but conversely impaired glucagon secretion under low glucose conditions. Together, these data present Erg1 channels as new mediators of α- and β-cell repolarization. However, antagonism of Erg1 has divergent effects in these cells; to augment glucose-dependent insulin secretion and inhibit low glucose stimulated glucagon secretion.Voltage-gated eag-related gene (Erg)² potassium (K⁺) channels are part of the larger family of voltage dependent K⁺ (Kv) channels (1). Three channel isoforms Erg1, Erg2, and Erg3 have been discovered (2, 3), and they differ by their activation and inactivation voltage dependence, gating properties, and pharmacological profile (4–7). Erg channels control cellular activity by controlling the repolarization of the action potential (AP). In atrial cells and ventricular myocytes, Erg regulates plateau formation and AP repolarization, as blocking Erg channels increases AP length (8, 9). These channels are also strongly involved in the pacemaking activity of cardiac cells (10, 11). Interestingly, a rare congenital heart condition, the inherited form of long QT syndrome is caused by mutations of Erg channel genes (9, 12). Erg channels also control the resting membrane potential in various cell types. For example, in neurons of the medial vestibular nucleus, blocking Erg channels produce an increase in AP discharge or in smooth muscle cells, blocking Erg channels mediates depolarization up to 20 mV (13–15). Hormone secretion studies also demonstrated the involvement of Erg channels in the secretion of prolactin from neurons of the anterior pituitary. Thyrotropin-releasing factor decreases Erg current, which depolarizes neurons and thereby stimulates prolactin secretion (16, 17).In the pancreas, Kv channels and more specifically Kv2.1, regulate insulin secretion by controlling the repolarization of β-cell membrane potential (18–20), although the contribution of this isoform in humans has recently been questioned (21). In α-cells, Kv2.1 and Kv1.4 channels repolarize the membrane potential (22, 23); however, the involvement of Kv channels in the secretion of glucagon is yet to be investigated. One study showed that Erg1, -2, and -3 are expressed in rat α- and β-cells and the rat insulinoma cell line, INS-1, and that they are involved in decreasing membrane potential. Blocking Erg channels with the channel antagonist E4031 increases insulin secretion from INS1 cells (24); however, definitive data regarding the role of Erg channels in insulin and glucagon secretion is limited.Therefore this study aimed to define the functions of Erg channels in α- and β-cells. We found that Erg1 channels are strongly expressed in pancreatic α- and β-cells. Pharmacological and genetic manipulation combined with whole cell recordings in pancreatic cell lines and primary islet cells determined that Erg1 produces a functional current in α- and β-cells. Blocking Erg1 increased intracellular calcium ([Ca²⁺]_i) in mouse β-cells, but only in a minority of mouse and human α-cells. Secretion studies using isolated mouse islets demonstrated that Erg1 are negative regulators of insulin secretion, but positive regulators of glucagon secretion, suggesting distinct roles for Erg1 in β- and α-cells.     

Degradation of Phospholipids and Liquefaction of Pressed Baker’s Yeast by Acetic Acid Vapour

When pressed baker’s yeast (Saccharomyces cerevisiae) was exposed to the vapour of acetic acid, autolysis of yeast cells was induced in 3 or 4 hr. In order to elucidate the mechanism of the autolysis caused by the AcOH-treatment, we investigated variations in the lipid content of yeast cells during the treatment. The degradation of phospholipids and the accumulation of free fatty acids occurred within 3 hr. Formic acid exerted a similar effect on the pressed yeast. The effect of propionic acid was not seen in 3hr but was after 18 hr. When the homogenate of fresh yeast cells was incubated in the acidic region below pH 4.5 for 1 hr, phospholipids were hydrolyzed and free fatty acids were accumulated. Such deacylation of phospholipids was observed even at pH 6 on incubation for 12hr, but not observed at pH 7 or above pH 9. At pH 8, although phospholipids were somewhat degraded, free fatty acids almost never accumulated but diacylglycerol did accumulate.

Therefore, yeast cells have inherently phospholipid-acylhydrolases and, on AcOH-treatment, such enzymes may degrade membrane phospholipids to induce the autolysis of pressed yeast.     

β-TrCP-Mediated Ubiquitination and Degradation of PHLPP1 Are Negatively Regulated by Akt

PHLPP1 belongs to a novel family of Ser/Thr protein phosphatases that serve as tumor suppressors by negatively regulating Akt signaling. Our recent studies have demonstrated that loss of PHLPP expression occurs at high frequency in colorectal cancer. In this study, we identified PHLPP1 as a proteolytic target of a β-TrCP-containing Skp-Cullin 1-F-box protein (SCF) complex (SCF^β-TrCP) E3 ubiquitin ligase in a phosphorylation-dependent manner. Overexpression of wild-type but not ΔF-box mutant β-TrCP leads to decreased expression and increased ubiquitination of PHLPP1, whereas knockdown of endogenous β-TrCP has the opposite effect. In addition, we show that the β-TrCP-mediated degradation requires phosphorylation of PHLPP1 by casein kinase I and glycogen synthase kinase 3β (GSK-3β), and activation of the phosphatidylinositol 3-kinase/Akt pathway suppresses the degradation of PHLPP1 by inhibiting the GSK-3β activity. Furthermore, expression of a degradation-deficient PHLPP1 mutant in colon cancer cells results in a more effective dephosphorylation of Akt and inhibition of cell growth. Taken together, our findings demonstrate a key role for β-TrCP in controlling the level of PHLPP1, and activation of Akt negatively regulates this degradation process.Hyperactivation of phosphatidylinositol 3-kinase/Akt signaling is commonly associated with human cancers (1, 5, 27). Inability to terminate the growth and survival signals mediated by Akt is one of the major mechanisms contributing to the development of cancer (1, 22, 32). The activation of Akt involves two phosphorylation steps: it is first phosphorylated at the activation loop (Thr308) within the kinase core by PDK-1 and subsequently at the hydrophobic motif (Ser473) in the C terminus by the TORC2 complex (22). Since the activity of Akt is tightly controlled by phosphorylation, dephosphorylation of Akt leads to effective signaling termination by inactivating the kinase. Recently, a novel family of Ser/Thr protein phosphatases, PHLPP, has been identified to fulfill the role of a negative regulator for Akt via direct dephosphorylation (3, 14). Two isoforms of PHLPP, namely PHLPP1 and PHLPP2, are found in this phosphatase family. Although the two isoforms of PHLPP share their ability to dephosphorylate Akt, each PHLPP preferentially regulates a subset of Akt isoforms in human lung cancer cells (3). Several lines of evidence suggest that PHLPP functions as a tumor suppressor. For example, overexpression of PHLPP in glioblastoma and colon cancer cells inhibits tumorigenesis in xenografted nude mice (14, 20), while decreased PHLPP expression correlates with increased metastastic potential in breast cancer cells (26). Furthermore, our recent studies have shown that downregulation of both PHLPP isoforms occurs at high frequency in colorectal cancer clinical samples (20). Loss of tumor suppressor expression can be caused by alterations at the gene level such as loss of heterozygosity or gene methylation. However, dysregulation of protein degradation pathways has also been implicated as a reason for downregulation of tumor suppressors (2, 6, 16).The ubiquitin (Ub) proteasome pathway controls degradation of the majority of eukaryotic proteins (12). β-TrCP belongs to a large family of F-box-containing proteins, and it serves as the substrate recognition subunit in the SCF (Skp1-Cullin 1-F-box protein) Ub-E3 ligase protein complex (4). By regulating the proteolytic process of its substrates, β-TrCP plays an important role in controlling cell cycle and cancer biogenesis (10). It is believed that β-TrCP-mediated ubiquitination requires phosphorylation of its substrates (35). A consensus binding motif with the sequence of DSG(X)_2-nS (so-called “phospho-degron”) has been proposed, in which the two serine residues are phosphorylated prior to binding to β-TrCP (4). However, variations of this motif, including replacement of the serine residues with phosphomimetic residues (e.g., Glu or Asp) in the substrate sequence, have been shown to be equally effective in mediating association with β-TrCP (31, 34).In this study, we report the identification of PHLPP1 as a proteolytic target of β-TrCP. We show that the degradation process of PHLPP1 depends on casein kinase I (CK1)- and glycogen synthase kinase 3 (GSK-3)-mediated phosphorylation, and activation of Akt negatively regulates PHLPP1 turnover. In addition, a PHLPP1 phosphorylation/degradation mutant antagonizes Akt more effectively in colon cancer cells.