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《Molecular cell》2014,53(4):663-671
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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 (46). 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.  相似文献   

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Protein kinase C (PKC) isoenzymes are multi-modular proteins activated at the membrane surface to regulate signal transduction processes. When activated by second messengers, PKC undergoes a drastic conformational and spatial transition from the inactive cytosolic state to the activated membrane-bound state. The complete structure of either state of PKC remains elusive. We demonstrate, using NMR spectroscopy, that the isolated Ca2+-sensing membrane-binding C2 domain of the conventional PKCα interacts with a conserved hydrophobic motif of the kinase C-terminal region, and we report a structural model of the complex. Our data suggest that the C-terminal region plays a dual role in regulating the PKC activity: activating, through sensitization of PKC to intracellular Ca2+ oscillations; and auto-inhibitory, through its interaction with a conserved positively charged region of the C2 domain.  相似文献   

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While conformational flexibility of proteins is widely recognized as one of their functionally crucial features and enjoys proper attention for this reason, their elastic properties are rarely discussed. In ion channel studies, where the voltage-induced or ligand-induced conformational transitions, gating, are the leading topic of research, the elastic structural deformation by the applied electric field has never been addressed at all. Here we examine elasticity using a model channel of known crystal structure—Staphylococcus aureus -hemolysin. Working with single channels reconstituted into planar lipid bilayers, we first show that their ionic conductance is asymmetric with voltage even at the highest salt concentration used where the static charges in the channel interior are maximally shielded. Second, choosing 18-crown-6 as a molecular probe whose size is close to the size of the narrowest part of the -hemolysin pore, we analyze the blockage of the channel by the crown/K+ complex. Analysis of the blockage within the framework of the Woodhull model in its generalized form demonstrates that the model is able to correctly describe the crown effect only if the parameters of the model are considered to be voltage-dependent. Specifically, one has to include either a voltage-dependent barrier for crown release to the cis side of the channel or voltage-dependent interactions between the binding site and the crown. We suggest that the voltage sensitivity of both the ionic conductance of the channel seen at the highest salt concentration and its blockage by the crown reflects a field-induced deformation of the pore.  相似文献   

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Aggregation of aspartate-β-semialdehyde dehydrogenase (ASD) was analyzed by applying modified Lumry–Eyring with nucleated polymerization (LENP) model. Intrinsic nucleation time scales were determined. In absence of glycerol, ASD undergoes concentration and time-dependent polymerization into low-molecular weight soluble aggregates and thereafter condensation into insoluble aggregates. In the presence of increasing solvent glycerol concentration, the aggregation becomes more and more nucleation dominated, with slower polymerization to low-molecular weights soluble aggregates, without any condensation into insoluble aggregates. Effective nucleus size as well as the number of monomers in each irreversible growth event were sensitive to the changes in solvent glycerol concentration. Glycerol-directed diminution of aggregation appears to be largely due to the inhibition of rearrangement (decreased nucleation rearrangement rate coefficient, K r,x ) because of compaction induced due to preferential hydration, thus, preventing the soluble aggregates from locking into irreversible soluble nuclei. Appreciably decreased K r,x (as compared to nucleation dissociation constant, K d,x ), appears to be responsible for increased nucleus size at higher solvent glycerol concentration. This study explains how modified LENP model can be applied to determine the predominant mechanism responsible for the diminution of aggregation by polyhydric alcohols (glycerol).  相似文献   

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The amyloid precursor protein (APP) is a widely expressed type I transmembrane (TM) glycoprotein present at the neuronal synapse. The proteolytic cleavage by γ-secretase of its C-terminal fragment produces amyloid-β (Aβ) peptides of different lengths, the deposition of which is an early indicator of Alzheimer disease. At present, there is no consensus on the conformation of the APP-TM domain at the biological membrane. Although structures have been determined by NMR in detergent micelles, their conformation is markedly different. Here we show by using molecular simulations that the APP-TM region systematically prefers a straight α-helical conformation once embedded in a membrane bilayer. However, APP-TM is highly flexible, and its secondary structure is strongly influenced by the surrounding lipid environment, as when enclosed in detergent micelles. This behavior is confirmed when analyzing in silico the atomistic APP-TM population observed by residual dipolar couplings and double electron-electron resonance spectroscopy. These structural and dynamic features are critical in the proteolytic processing of APP by the γ-secretase enzyme, as suggested by a series of Gly700 mutants. Affecting the hydration and flexibility of APP-TM, these mutants invariantly show an increase in the production of Aβ38 compared with Aβ40 peptides, which is reminiscent of the effect of γ-secretase modulators inhibitors.  相似文献   

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RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4 and an AlF4-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.  相似文献   

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Pirh2 is a p53 inducible gene that encodes a RING-H2 domain and is proposed to be a main regulator of p53 protein, thus fine tuning the DNA damage response. Pirh2 interacts physically with p53 and promotes its MDM2-independent ubiquitination and subsequent degradation as well as participates in an auto-regulatory feedback loop that controls p53 function. Pirh2 also self-ubiquitinates. Interestingly, Pirh2 is overexpressed in a wide range of human tumors. In this study, we investigated the domains and residues essential for Pirh2 self-ubiquitination. Deletions were made in each of the three major domains of Pirh2: the N-terminal domain (NTD), Ring domain (RING), and C-terminal domain (CTD). The effects of these deletions on Pirh2 self-ubiquitination were then assessed using in vitro ubiquitination assays. Our results demonstrate that the RING domain is essential, but not sufficient, for Pirh2 self-ubiquitination and that residues 240–250 of the C-terminal domain are also essential. Our results demonstrate that Pirh2 mediated p53 polyubiquitination occurs mainly through the K48 residue of ubiquitin in vitro. Our data further our understanding of the mechanism of Pirh2 self-ubiquitination and may help identify valuable therapeutic targets that play roles in reducing the effects of the overexpression of Pirh2, thus maximizing p53''s response to DNA damage.  相似文献   

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Disulfide formation in newly synthesized proteins entering the mammalian endoplasmic reticulum is catalyzed by protein disulfide isomerase (PDI), which is itself thought to be directly oxidized by Ero1α. The activity of Ero1α is tightly regulated by the formation of noncatalytic disulfides, which need to be broken to activate the enzyme. Here, we have developed a novel PDI oxidation assay, which is able to simultaneously determine the redox status of the individual active sites of PDI. We have used this assay to confirm that when PDI is incubated with Ero1α, only one of the active sites of PDI becomes directly oxidized with a slow turnover rate. In contrast, a deregulated mutant of Ero1α was able to oxidize both PDI active sites at an equivalent rate to the wild type enzyme. When the active sites of PDI were mutated to decrease their reduction potential, both were now oxidized by wild type Ero1α with a 12-fold increase in activity. These results demonstrate that the specificity of Ero1α toward the active sites of PDI requires the presence of the regulatory disulfides. In addition, the rate of PDI oxidation is limited by the reduction potential of the PDI active site disulfide. The inability of Ero1α to oxidize PDI efficiently likely reflects the requirement for PDI to act as both an oxidase and an isomerase during the formation of native disulfides in proteins entering the secretory pathway.  相似文献   

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Kim DS  Lim HK  Jang S  Oh S 《Neurochemical research》2003,28(12):1771-1778
Butorphanol was infused continuously into cerebral ventricle at a constant rate of 26 nmol/l/h for 3 days, and the withdrawal from opioid was rendered 7 h after the cessation of infusion. The G-protein -subunit has been implicated in opioid tolerance and withdrawal. The effects of continuous infusion of butorphanol on the modulation of G protein -subunit mRNA were investigated by using in situ hybridization techniques. In situ hybridization showed marked changes in the levels of Gs during butorphanol tolerance and withdrawal. Specifically, the level of Gs mRNA was significantly decreased in almost all areas of brain except hippocampus during the butorphanol withdrawal. It was also decreased in the septum and cerebellar granule layer in butorphanol tolerant rats. The level of Gi mRNA was significantly decreased only in the cerebral cortex of butorphanol tolerant rat. However, no such change was noted during the withdrawal from butorphanol. The level of Go mRNA was not changed either in butorphanol tolerant or in the butorphanol withdrawal rats. No alterations were noted in the level of [3H]forskolin binding to adenylyl cyclase in butorphanol tolerant as well as withdrawing rats. The levels of pCREB were significantly elevated in the hippocampus in the butorphanol withdrawal rats. These results suggest that region-specific changes of G protein -subunit mRNA and pCREB without marked changes in the level of adenylyl cyclase may underlie the tolerance to and withdrawal from butorphanol.  相似文献   

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Vascular smooth muscle cell (VSMC) tone is regulated by the state of myosin light chain (MLC) phosphorylation, which is in turn regulated by the balance between MLC kinase and MLC phosphatase (MLCP) activities. RhoA activates Rho kinase, which phosphorylates the regulatory subunit of MLC phosphatase, thereby inhibiting MLC phosphatase activity and increasing contraction and vascular tone. Nitric oxide is an important mediator of VSMC relaxation and vasodilation, which acts by increasing cyclic GMP (cGMP) levels in VSMC, thereby activating cGMP-dependent protein kinase Iα (PKGIα). PKGI is known to phosphorylate Rho kinase, preventing Rho-mediated inhibition of MLC phosphatase, promoting vasorelaxation, although the molecular mechanisms that mediate this are unclear. Here we identify RhoA as a target of activated PKGIα and show further that PKGIα binds directly to RhoA, inhibiting its activation and translocation. In protein pulldown and immunoprecipitation experiments, binding of RhoA and PKGIα was demonstrated via a direct interaction between the amino terminus of RhoA (residues 1–44), containing the switch I domain of RhoA, and the amino terminus of PKGIα (residues 1–59), which includes a leucine zipper heptad repeat motif. Affinity assays using cGMP-immobilized agarose showed that only activated PKGIα binds RhoA, and a leucine zipper mutant PKGIα was unable to bind RhoA even if activated. Furthermore, a catalytically inactive mutant of PKGIα bound RhoA but did not prevent RhoA activation and translocation. Collectively, these results support that RhoA is a PKGIα target and that direct binding of activated PKGIα to RhoA is central to cGMP-mediated inhibition of the VSMC Rho kinase contractile pathway.  相似文献   

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AMP-activated protein kinase (AMPK) is a heterotrimeric complex playing a crucial role in maintaining cellular energy homeostasis. Recently, homodimerization of mammalian AMPK and yeast ortholog SNF1 was shown by us and others. In SNF1, it involved specific hydrophobic residues in the kinase domain αG-helix. Mutation of the corresponding AMPK α-subunit residues (Val-219 and Phe-223) to glutamate reduced the tendency of the kinase to form higher order homo-oligomers, as was determined by the following three independent techniques in vitro: (i) small angle x-ray scattering, (ii) surface plasmon resonance spectroscopy, and (iii) two-dimensional blue native/SDS-PAGE. Recombinant protein as well as AMPK in cell lysates of primary cells revealed distinct complexes of various sizes. In particular, the assembly of very high molecular mass complexes was dependent on both the αG-helix-mediated hydrophobic interactions and kinase activation. In vitro and when overexpressed in double knock-out (α1−/−, α2−/−) mouse embryonic fibroblast cells, activation of mutant AMPK was impaired, indicating a critical role of the αG-helix residues for AMPK activation via its upstream kinases. Also inactivation by protein phosphatase 2Cα was affected in mutant AMPK. Importantly, activation of mutant AMPK by LKB1 was restored by exchanging the corresponding and conserved hydrophobic αG-helix residues of LKB1 (Ile-260 and Phe-264) to positively charged amino acids. These mutations functionally rescued LKB1-dependent activation of mutant AMPK in vitro and in cell culture. Our data suggest a physiological role for the hydrophobic αG-helix residues in homo-oligomerization of heterotrimers and cellular interactions, in particular with upstream kinases, indicating an additional level of AMPK regulation.The maintenance of energy homeostasis is a basic requirement of all living organisms. The AMP-activated protein kinase (AMPK)2 is crucially involved in this essential process by playing a central role in sensing and regulating energy metabolism on the cellular and whole body level (16). AMPK is also participating in several signaling pathways associated with cancer and metabolic diseases, like type 2 diabetes mellitus, obesity, and other metabolic disorders (79).Mammalian AMPK belongs to a highly conserved family of serine/threonine protein kinases with homologs found in all eukaryotic organisms examined (1, 3, 10). Its heterotrimeric structure includes a catalytic α-subunit and regulatory β- and γ-subunits. These subunits exist in different isoforms (α1, α2, β1, β2, γ1, γ2, and γ3) and splice variants (for γ2 and γ3) and can thus assemble to a broad variety of heterotrimeric isoform combinations. The α- and β-subunits possess multiple autophosphorylation sites, which have been implicated in regulation of subcellular localization and kinase activation (1115). The most critical step of AMPK activation, however, is phosphorylation of Thr-172 within the activation segment of the α-subunit kinase domain. At least two AMPK upstream kinases (AMPKKs) have been identified so far, namely the tumor suppressor kinase LKB1 in complex with MO25 and STRAD (16) and Ca2+/calmodulin-dependent protein kinase kinase-2 (CamKK2) (17). Furthermore, the transforming growth factor-β-activated kinase 1 was also shown to activate AMPK using a variety of in vitro approaches (18), but the physiological relevance of these findings remains unclear. Besides direct phosphorylation of Thr-172, AMPK activity is stimulated by the allosteric activator AMP, which can bind to two Bateman domains formed by two pairs of CBS domains within the γ-subunit (1922). Hereby bound AMP not only allosterically stimulates AMPK but also protects Thr-172 from dephosphorylation by protein phosphatase 2Cα (PP2Cα) and thus hinders inactivation of the kinase (19, 22, 23). Consequently, on the cellular level, AMPK is activated upon metabolic stress increasing the AMP/ATP ratio. Furthermore, AMPK activation can also be induced by several chemical compounds, like nucleoside 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (24) and the anti-diabetic drug Metformin (2528). In addition, the small molecule compound A-769662 was recently developed as a direct allosteric activator of AMPK (29, 30).Previous work in our groups proposed a model of AMPK regulation by AMP, which incorporates the major functional features and the latest structural information (31). The latter mainly included truncated core complexes of AMPK from different species (3235). Further valuable structural information is provided by the x-ray structures of the isolated catalytic domains, in particular of the human AMPK α2-subunit (Protein Data Bank code 2H6D) and its yeast ortholog SNF1 (36, 37). The kinase domain of SNF1 is capable of forming homodimers in the protein crystal, as well as in vitro in solution, in a unique way, which has not been observed previously in any other kinase (36). The dimer interface is predominantly formed by hydrophobic interactions of the loop-αG region, also known as subdomain X situated on the large kinase lobe (36, 38, 39), and it mainly involves Ile-257 and Phe-261. Because the T-loop activation segment was buried within the dimer interface, it was suggested that the dimeric state of the SNF1 catalytic domain represents the inactive form of the kinase. Intriguingly, it was shown in our groups by small angle x-ray scattering that AMPK self-organizes in a concentration-dependent manner to form homo-oligomers in solution (31). However, the interface responsible for oligomerization of the AMPK heterotrimer has remained elusive.Here we further investigate the distinct oligomeric states of the AMPK heterotrimer and suggest a possible regulatory function for this process. Most importantly, we provide conclusive evidence for participation of αG-helix residues in the recognition of AMPK by its upstream kinases LKB1 and CamKK2.  相似文献   

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