230 results on '"Steven O. Smith"'
Search Results
2. Impact of Aβ40 and Aβ42 Fibrils on the Transcriptome of Primary Astrocytes and Microglia
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Xiaoyue Zhu, Joseph M. Schrader, Brandon A. Irizarry, Steven O. Smith, and William E. Van Nostrand
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amyloid β-protein ,fibrils ,astrocytes ,microglia ,RNA sequencing ,Biology (General) ,QH301-705.5 - Abstract
Fibrillar amyloid β-protein (Aβ) deposits in the brain, which are primarily composed of Aβ40 or Aβ42 peptides, are key pathological features of Alzheimer’s disease (AD) and related disorders. Although the underlying mechanisms are still not clear, the Aβ fibrils can trigger a number of cellular responses, including activation of astrocytes and microglia. In addition, fibril structures of the Aβ40 and Aβ42 peptides are known to be polymorphic, which poses a challenge for attributing the contribution of different Aβ sequences and structures to brain pathology. Here, we systematically treated primary astrocytes and microglia with single, well-characterized polymorphs of Aβ40 or Aβ42 fibrils, and performed bulk RNA sequencing to assess cell-specific changes in gene expression. A greater number of genes were up-regulated by Aβ42 fibril-treated glial cells (251 and 2133 genes in astrocyte and microglia, respectively) compared with the Aβ40 fibril-treated glial cells (191 and 251 genes in astrocytes and microglia, respectively). Immunolabeling studies in an AD rat model with parenchymal fibrillar Aβ42 plaques confirmed the expression of PAI-1, MMP9, MMP12, CCL2, and C1r in plaque-associated microglia, and iNOS, GBP2, and C3D in plaque-associated astrocytes, validating markers from the RNA sequence data. In order to better understand these Aβ fibril-induced gene changes, we analyzed gene expression patterns using the Ingenuity pathway analysis program. These analyses further highlighted that Aβ42 fibril treatment up-regulated cellular activation pathways and immune response pathways in glial cells, including IL1β and TNFα in astrocytes, and microglial activation and TGFβ1 in microglia. Further analysis revealed that a number of disease-associated microglial (DAM) genes were surprisingly suppressed in Aβ40 fibril treated microglia. Together, the present findings indicate that Aβ42 fibrils generally show similar, but stronger, stimulating activity of glial cells compared with Aβ40 fibril treatment.
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- 2022
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3. Dimeric Transmembrane Orientations of APP/C99 Regulate γ-Secretase Processing Line Impacting Signaling and Oligomerization
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Florian Perrin, Nicolas Papadopoulos, Nuria Suelves, Rémi Opsomer, Devkee M. Vadukul, Céline Vrancx, Steven O. Smith, Didier Vertommen, Pascal Kienlen-Campard, and Stefan N. Constantinescu
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Science - Published
- 2021
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4. Dimeric Transmembrane Orientations of APP/C99 Regulate γ-Secretase Processing Line Impacting Signaling and Oligomerization
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Florian Perrin, Nicolas Papadopoulos, Nuria Suelves, Rémi Opsomer, Devkee M. Vadukul, Céline Vrancx, Steven O. Smith, Didier Vertommen, Pascal Kienlen-Campard, and Stefan N. Constantinescu
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Biological Sciences ,Biochemistry ,Molecular Biology ,Neuroscience ,Science - Abstract
Summary: Amyloid precursor protein (APP) cleavage by the β-secretase produces the C99 transmembrane (TM) protein, which contains three dimerization-inducing Gly-x-x-x-Gly motifs. We demonstrate that dimeric C99 TM orientations regulate the precise cleavage lines by γ-secretase. Of all possible dimeric orientations imposed by a coiled-coil to the C99 TM domain, the dimer containing the 33Gly-x-x-x-Gly37 motif in the interface promoted the Aβ42 processing line and APP intracellular domain-dependent gene transcription, including the induction of BACE1 mRNA, enhancing amyloidogenic processing and signaling. Another orientation exhibiting the 25Gly-x-x-x-Gly29 motif in the interface favored processing to Aβ43/40. It induced significantly less gene transcription, while promoting formation of SDS-resistant “Aβ-like” oligomers, reminiscent of Aβ peptide oligomers. These required both Val24 of a pro-β motif and the 25Gly-x-x-x-Gly29 interface. Thus, crossing angles imposed by precise dimeric orientations control γ-secretase initial cleavage at Aβ48 or Aβ49, linking the former to enhanced signaling and Aβ42 production.
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- 2020
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5. Cerebral vascular amyloid seeds drive amyloid β-protein fibril assembly with a distinct anti-parallel structure
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Feng Xu, Ziao Fu, Sharmila Dass, AnnMarie E. Kotarba, Judianne Davis, Steven O. Smith, and William E. Van Nostrand
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Science - Abstract
Abstract Cerebrovascular accumulation of amyloid β-protein (Aβ), a condition known as cerebral amyloid angiopathy (CAA), is a common pathological feature of patients with Alzheimer’s disease. Familial Aβ mutations, such as Dutch-E22Q and Iowa-D23N, can cause severe cerebrovascular accumulation of amyloid that serves as a potent driver of vascular cognitive impairment and dementia. The distinctive features of vascular amyloid that underlie its unique pathological properties remain unknown. Here, we use transgenic mouse models producing CAA mutants (Tg-SwDI) or overproducing human wild-type Aβ (Tg2576) to demonstrate that CAA-mutant vascular amyloid influences wild-type Aβ deposition in brain. We also show isolated microvascular amyloid seeds from Tg-SwDI mice drive assembly of human wild-type Aβ into distinct anti-parallel β-sheet fibrils. These findings indicate that cerebrovascular amyloid can serve as an effective scaffold to promote rapid assembly and strong deposition of Aβ into a unique structure that likely contributes to its distinctive pathology.
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- 2016
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6. Anti-Parallel β-Hairpin Structure in Soluble Aβ Oligomers of Aβ40-Dutch and Aβ40-Iowa
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Ziao Fu, William E. Van Nostrand, and Steven O. Smith
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Alzheimer’s disease ,cerebral amyloid angiopathy ,amyloid-β ,Aβ40-Dutch ,Aβ40-Iowa ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The amyloid-β (Aβ) peptides are associated with two prominent diseases in the brain, Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA). Aβ42 is the dominant component of cored parenchymal plaques associated with AD, while Aβ40 is the predominant component of vascular amyloid associated with CAA. There are familial CAA mutations at positions Glu22 and Asp23 that lead to aggressive Aβ aggregation, drive vascular amyloid deposition and result in degradation of vascular membranes. In this study, we compared the transition of the monomeric Aβ40-WT peptide into soluble oligomers and fibrils with the corresponding transitions of the Aβ40-Dutch (E22Q), Aβ40-Iowa (D23N) and Aβ40-Dutch, Iowa (E22Q, D23N) mutants. FTIR measurements show that in a fashion similar to Aβ40-WT, the familial CAA mutants form transient intermediates with anti-parallel β-structure. This structure appears before the formation of cross-β-sheet fibrils as determined by thioflavin T fluorescence and circular dichroism spectroscopy and occurs when AFM images reveal the presence of soluble oligomers and protofibrils. Although the anti-parallel β-hairpin is a common intermediate on the pathway to Aβ fibrils for the four peptides studied, the rate of conversion to cross-β-sheet fibril structure differs for each.
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- 2021
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7. Retinal orientation and interactions in rhodopsin reveal a two-stage trigger mechanism for activation
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Naoki Kimata, Andreyah Pope, Markus Eilers, Chikwado A. Opefi, Martine Ziliox, Amiram Hirshfeld, Ekaterina Zaitseva, Reiner Vogel, Mordechai Sheves, Philip J. Reeves, and Steven O. Smith
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Science - Abstract
Rhodopsin signalling is triggered by the light-induced isomerization of its 11-cisretinal chromophore. Here, the authors use NMR spectroscopy to define retinal orientation and interactions in the active metarhodopsin II intermediate, proposing a two-stage mechanism for rhodopsin activation.
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- 2016
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8. Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing
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Marie Decock, Laetitia El Haylani, Serena Stanga, Ilse Dewachter, Jean-Noël Octave, Steven O. Smith, Stefan N. Constantinescu, and Pascal Kienlen-Campard
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Alzheimer disease ,APP ,Dimerization ,GXXXG motifs ,Amyloid beta peptide ,Split luciferase ,Biology (General) ,QH301-705.5 - Abstract
Alzheimer's disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the β‐amyloid peptide (Aβ) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aβ production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full‐length APP dimerizes more than its α and β C‐terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ‐cleavage site. We show that both non‐familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C‐terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non‐amyloidogenic processing.
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- 2015
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9. Mechanism of Activation of the Visual Receptor Rhodopsin
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Steven O. Smith
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Structural Biology ,Biophysics ,Bioengineering ,Cell Biology ,Biochemistry - Abstract
Rhodopsin is the photoreceptor in human rod cells responsible for dim-light vision. The visual receptors are part of the large superfamily of G protein–coupled receptors (GPCRs) that mediate signal transduction in response to diverse diffusible ligands. The high level of sequence conservation within the transmembrane helices of the visual receptors and the family A GPCRs has long been considered evidence for a common pathway for signal transduction. I review recent studies that reveal a comprehensive mechanism for how light absorption by the retinylidene chromophore drives rhodopsin activation and highlight those features of the mechanism that are conserved across the ligand-activated GPCRs.
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- 2023
10. Insights into Cerebral Amyloid Angiopathy Type 1 and Type 2 from Comparisons of the Fibrillar Assembly and Stability of the Aβ40-Iowa and Aβ40-Dutch Peptides
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Jitika Rajpoot, Elliot J. Crooks, Brandon A. Irizarry, Ashley Amundson, William E. Van Nostrand, and Steven O. Smith
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Amyloid ,Cerebral Amyloid Angiopathy ,Amyloid beta-Peptides ,Alzheimer Disease ,Humans ,Plaque, Amyloid ,Iowa ,Biochemistry ,Peptide Fragments - Abstract
Two distinct diseases are associated with the deposition of fibrillar amyloid-β (Aβ) peptides in the human brain in an age-dependent fashion. Alzheimer's disease is primarily associated with parenchymal plaque deposition of Aβ42, while cerebral amyloid angiopathy (CAA) is associated with amyloid formation of predominantly Aβ40 in the cerebral vasculature. In addition, familial mutations at positions 22 and 23 of the Aβ sequence can enhance vascular deposition in the two major subtypes of CAA. The E22Q (Dutch) mutation is associated with CAA type 2, while the D23N (Iowa) mutation is associated with CAA type 1. Here we investigate differences in the formation and structure of fibrils of these mutant Aβ peptides
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- 2022
11. Magic angle spinning NMR of G protein-coupled receptors
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Steven O. Smith, Bianca Chandler, and Lauren Todd
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Nuclear and High Energy Physics ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Chemistry ,Molecular Conformation ,Drug design ,Nuclear magnetic resonance spectroscopy ,Ligands ,Biochemistry ,Small molecule ,Receptors, G-Protein-Coupled ,Analytical Chemistry ,Transmembrane domain ,Magic angle spinning ,Biophysics ,Humans ,Receptor ,Spectroscopy ,Function (biology) ,Protein Binding ,G protein-coupled receptor - Abstract
G protein-coupled receptors (GPCRs) have a simple seven transmembrane helix architecture which has evolved to recognize a diverse number of chemical signals. The more than 800 GPCRs encoded in the human genome function as receptors for vision, smell and taste, and mediate key physiological processes. Consequently, these receptors are a major target for pharmaceuticals. Protein crystallography and electron cryo-microscopy have provided high resolution structures of many GPCRs in both active and inactive conformations. However, these structures have not sparked a surge in rational drug design, in part because GPCRs are inherently dynamic and the structural changes induced by ligand or drug binding to stabilize inactive or active conformations are often subtle rearrangements in packing or hydrogen-bonding interactions. NMR spectroscopy provides a sensitive probe of local structure and dynamics at specific sites within these receptors as well as global changes in receptor structure and dynamics. These methods can also capture intermediate states and conformations with low populations that provide insights into the activation pathways. We review the use of solid-state magic angle spinning NMR to address the structure and activation mechanisms of GPCRs. The focus is on the large and diverse class A family of receptors. We highlight three specific class A GPCRs in order to highlight how solid-state, as well as solution-state NMR spectroscopy, can answer questions in the field involving how different GPCR classes and subfamilies are activated by their associated ligands, and how small molecule drugs can modulate GPCR activation.
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- 2022
12. An electrostatic cluster guides Aβ40 fibril formation in cerebral amyloid angiopathy
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Elliot J. Crooks, Ziao Fu, Brandon A. Irizarry, Xiaoyue Zhu, William E. Van Nostrand, Saikat Chowdhury, and Steven O. Smith
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Cerebral amyloid angiopathy (CAA) is associated with the accumulation of fibrillar Aβ peptides upon and within the cerebral vasculature, which leads to loss of vascular integrity and contributes to disease progression in Alzheimer’s disease (AD). We investigate the structure of human-derived Aβ40 fibrils obtained from patients diagnosed with sporadic or familial Dutch-type (E22Q) CAA. Using cryo-EM, two primary structures are identified containing elements that have not been observed in in vitro Aβ40 fibril structures. One population has an ordered N-terminal fold comprised of two β-strands stabilized by electrostatic interactions involving D1, E22, D23 and K28. This charged cluster is disrupted in the second population, which exhibits a disordered N-terminus and is favored in fibrils derived from the familial Dutch-type CAA patient. These results illustrate differences between human-derived CAA and AD fibrils, and how familial CAA mutations guide fibril formation.
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- 2022
13. Constitutive Activation and Oncogenicity Are Mediated by Loss of Helical Structure at the Cytosolic Boundary of the Thrombopoietin Receptor
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Jean-Philippe Defour, Emilie Leroy, Sharmila Dass, Thomas Balligand, Gabriel Levy, Céline Mouton, Lidvine Genet, Christian Pecquet, Steven O. Smith, and Stefan N. Constantinescu
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Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with either proximity of Asn substitutions to the cytosolic boundary or mutation of W515 in the motif. Mutational studies in the TpoR cytosolic JM region show that the helical loss by itself can induce activation, but is localized to a maximum of 6 amino acids downstream of W515, the rest of the region until Box 1 requiring helicity for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together these data allow us to develop a general model for activation and to explain the critical role of the JM W515 residue in the regulation of receptor activity.(Total manuscript without References and Figure Legends 6507 words, main text (without methods and materials):)
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- 2022
14. Glycines from the APP GXXXG/GXXXA transmembrane motifs promote formation of pathogenic Aβ oligomers in cells
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Marie eDecock, Serena eStanga, Jean-Noël eOctave, Ilse eDewachter, Steven O Smith, Stefan N Constantinescu, and Pascal eKienlen-Campard
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oligomers ,Alzheimer’s disease ,amyloid precursor protein ,neuronal differentiation ,Beta-amyloid peptide ,GXXXG motifs ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Alzheimer’s disease (AD) is the most common neurodegenerative disorder characterized by progressive cognitive decline leading to dementia. The amyloid precursor protein (APP) is a ubiquitous type I transmembrane (TM) protein sequentially processed to generate the β-amyloid peptide (Aβ), the major constituent of senile plaques that are typical AD lesions. There is a growing body of evidence that soluble Aβ oligomers correlate with clinical symptoms associated with the disease. The Aβ sequence begins in the extracellular juxtamembrane region of APP and includes roughly half of the TM domain. This region contains GXXXG and GXXXA motifs, which are critical for both TM protein interactions and fibrillogenic properties of peptides derived from TM α-helices. Glycine-to-leucine mutations of these motifs were previously shown to affect APP processing and Aβ production in cells. However, the detailed contribution of these motifs to APP dimerization, their relation to processing, and the conformational changes they can induce within Aβ species remains undefined. Here, we describe highly resistant Aβ42 oligomers that are produced in cellular membrane compartments. They are formed in cells by processing of the APP amyloidogenic C-terminal fragment (C99), or by direct expression of a peptide corresponding to Aβ42, but not to Aβ40. By a point-mutation approach, we demonstrate that glycine-to-leucine mutations in the G29XXXG33 and G38XXXA42 motifs dramatically affect the Aβ oligomerization process. G33 and G38 in these motifs are specifically involved in Aβ oligomerization; the G33L mutation strongly promotes oligomerization, while G38L blocks it with a dominant effect on G33 residue modification. Finally, we report that the secreted Aβ42 oligomers display pathological properties consistent with their suggested role in AD, but do not induce toxicity in survival assays with neuronal cells. Exposure of neurons to these Aβ42 oligomers dramatically affects neuronal differentiation and, consequently, neuronal network maturation.
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- 2016
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15. Human cerebral vascular amyloid contains both antiparallel and parallel in-register Aβ40 fibrils
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Xiaoyue Zhu, Annemieke J.M. Rozemuller, Baayla D.C. Boon, Steven O. Smith, Judianne Davis, William E. Van Nostrand, Brandon A. Irizarry, Pathology, and Amsterdam Neuroscience - Neurodegeneration
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Male ,Aβ, amyloid β ,cryo-EM, electron cryo-microscopy ,Amyloid ,Mutation, Missense ,macromolecular substances ,amyloid-β ,Fibril ,Antiparallel (biochemistry) ,Biochemistry ,polymorphism ,Alzheimer Disease ,APP, amyloid precursor protein ,ATR, attenuated total reflectance ,medicine ,Amyloid precursor protein ,Humans ,CAA, cerebral amyloid angiopathy ,TEM, transmission electron microscopy ,Nuclear Magnetic Resonance, Biomolecular ,cerebral amyloid angiopathy ,Molecular Biology ,Vascular tissue ,Aged ,Laser capture microdissection ,Amyloid beta-Peptides ,biology ,Chemistry ,DARR, dipolar-assisted rotational resonance ,CAA/s, sporadic cerebral amyloid angiopathy ,Brain ,ELISA, enzyme-linked immunosorbent assay ,Cell Biology ,Human brain ,Alzheimer's disease ,MAS, magic angle spinning ,medicine.disease ,Peptide Fragments ,TBS, Tris-HCl buffered saline ,FTIR, Fourier transform infrared ,medicine.anatomical_structure ,Amino Acid Substitution ,Biophysics ,biology.protein ,antiparallel fibrils ,AD, Alzheimer's disease ,Cerebral amyloid angiopathy ,LCM, laser capture microdissection ,Research Article - Abstract
The accumulation of fibrillar amyloid-β (Aβ) peptides alongside or within the cerebral vasculature is the hallmark of cerebral amyloid angiopathy (CAA). This condition commonly co-occurs with Alzheimer's disease (AD) and leads to cerebral microbleeds, intracranial hemorrhages, and stroke. CAA also occurs sporadically in an age-dependent fashion and can be accelerated by the presence of familial Aβ mutant peptides. Recent studies using Fourier transform infrared (FTIR) spectroscopy of vascular Aβ fibrils derived from rodents containing the double E22Q/D23N mutations indicated the presence of a novel antiparallel β-sheet structure. To address whether this structure is associated solely with the familial mutations or is a common feature of CAA, we propagated Aβ fibrils from human brain vascular tissue of patients diagnosed with nonfamilial CAA. Aβ fibrils were isolated from cerebral blood vessels using laser capture microdissection in which specific amyloid deposits were removed from thin slices of the brain tissue. Transmission electron microscopy revealed that these deposits were organized into a tight meshwork of fibrils, which FTIR measurements showed could serve as seeds to propagate the growth of Aβ40 fibrils for structural studies. Solid-state NMR measurements of the fibrils propagated from vascular amyloid showed they contained a mixture of parallel, in-register, and antiparallel β-sheet structures. The presence of fibrils with antiparallel structure derived from vascular amyloid is distinct from the typical parallel, in-register β-sheet structure that appears in fibrils derived from parenchymal amyloid in AD. These observations reveal that different microenvironments influence the structures of Aβ fibrils in the human brain.
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- 2021
16. Deconstructing the transmembrane core of class A G protein-coupled receptors
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Steven O. Smith
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Magnetic Resonance Spectroscopy ,biology ,Chemistry ,Nuclear magnetic resonance spectroscopy ,Ligands ,Biochemistry ,Transmembrane protein ,Article ,Receptors, G-Protein-Coupled ,Cell surface receptor ,Rhodopsin ,Helix ,Core (graph theory) ,biology.protein ,Biophysics ,Receptor ,Molecular Biology ,G protein-coupled receptor - Abstract
Class A G protein-coupled receptors have evolved to recognize ligands ranging from small-molecule odorants to proteins. Although they are among the most diverse membrane receptors in eukaryotic organisms, they possess a highly conserved core within their seven-transmembrane helix framework. The conservation of the transmembrane core has led to the idea of a common mechanism by which ligand binding is coupled to the outward rotation of helix H6, the hallmark of an active receptor. Nevertheless, there is still no consensus on the mechanism of coupling or on the roles of specific residues within the core. Recent insights from crystallography and NMR spectroscopy provide a way to decompose the core into its essential structural and functional elements that shed new light on this important region.
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- 2021
17. The Good, the Bad and the Ugly: Current Feedback - Technical Contributions and Limitations.
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Steven O. Smith
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- 1993
18. Influence of the familial Alzheimer's disease–associated T43I mutation on the transmembrane structure and γ-secretase processing of the C99 peptide
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Tzu-Chun Tang, Stefan N. Constantinescu, Florian Perrin, Jean-Noël Octave, Pascal Kienlen-Campard, Steven O. Smith, Rémi Opsomer, Yi Hu, UCL - SSS/DDUV/SIGN - Cell signalling, UCL - SSS/IONS/CEMO - Pôle Cellulaire et moléculaire, and UCL - (SLuc) Service d'hématologie
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0301 basic medicine ,Amyloid-beta (Aβ) ,Mutant ,Mutation, Missense ,Peptide ,Aβ42/Aβ40 ratio ,Cleavage (embryo) ,Biochemistry ,Fluorescence ,03 medical and health sciences ,Protein Domains ,Alzheimer Disease ,Extracellular ,medicine ,Humans ,Neurodegeneration ,Amyloid precursor protein (APP) ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Fourier transform IR (FTIR) ,chemistry.chemical_classification ,Amyloid beta-Peptides ,030102 biochemistry & molecular biology ,biology ,Molecular Bases of Disease ,Cell Biology ,Secretase ,medicine.disease ,Peptide Fragments ,Transmembrane protein ,Transmembrane domain ,Membrane interaction ,030104 developmental biology ,Amino Acid Substitution ,chemistry ,biology.protein ,Biophysics ,Amyloid Precursor Protein Secretases ,Nuclear magnetic resonance (NMR) ,Peptides ,Amyloid precursor protein secretase - Abstract
Extracellular deposition of β-amyloid (Aβ) peptides in the brain is a hallmark of Alzheimer's disease (AD). Upon β-secretase–mediated cleavage of the β C-terminal fragment (β-CTF) from the Aβ precursor protein, the γ-secretase complex produces the Aβ peptides associated with AD. The familial T43I mutation within the transmembrane domain of the β-CTF (also referred to as C99) increases the ratio between the Aβ42 and Aβ40 peptides largely due to a decrease in Aβ40 formation. Aβ42 is the principal component of amyloid deposits within the brain parenchyma, and an increase in the Aβ42/Aβ40 ratio is correlated with early-onset AD. Using NMR and FTIR spectroscopy, here we addressed how the T43I substitution influences the structure of C55, the minimal sequence containing the entire extracellular and transmembrane (TM) domains of C99 needed for γ-secretase processing. (13)C NMR chemical shifts indicated that the T43I substitution increases helical structure within the TM domain of C55. These structural changes were associated with a shift of the C55 dimer to the monomer and an increase in the tilt of the TM helix relative to the membrane normal in the T43I mutant compared with that of WT C55. The A21G (Flemish) mutation was previously found to increase secreted Aβ40 levels; here, we combined this mutation in the extracellular domain of C99 with T43I and observed that the T43I/A21G double mutant decreases Aβ40 formation. We discuss how the observed structural changes in the T43I mutant may decrease Aβ40 formation and increase the Aβ42/Aβ40 ratio.
- Published
- 2019
19. A Novel Transgenic Rat Model of Robust Cerebral Microvascular Amyloid with Prominent Vasculopathy
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Feng Xu, William E. Van Nostrand, Michael D. Hoos, Helene Benveniste, John K. Robinson, Steven O. Smith, Elliot J. Crooks, Regina Kim, Judianne Davis, Hedok Lee, Dominique L. Popescu, and Joshua Hatfield
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0301 basic medicine ,Genetically modified mouse ,Pathology ,medicine.medical_specialty ,Amyloid ,Transgene ,Plaque, Amyloid ,Article ,Pathology and Forensic Medicine ,Pathogenesis ,Amyloid beta-Protein Precursor ,03 medical and health sciences ,0302 clinical medicine ,mental disorders ,Animals ,Humans ,Medicine ,Dementia ,Neuroinflammation ,Amyloid beta-Peptides ,Behavior, Animal ,business.industry ,Brain ,medicine.disease ,Rats ,Cerebral Amyloid Angiopathy ,030104 developmental biology ,Mutation ,Cerebral amyloid angiopathy ,Rats, Transgenic ,Alzheimer's disease ,business ,030217 neurology & neurosurgery - Abstract
Accumulation of fibrillar amyloid β protein in blood vessels of the brain, a condition known as cerebral amyloid angiopathy (CAA), is a common pathology of elderly individuals, a prominent comorbidity of Alzheimer disease, and a driver of vascular cognitive impairment and dementia. Although several transgenic mouse strains have been generated that develop varying levels of CAA, consistent models of associated cerebral microhemorrhage and vasculopathy observed clinically have been lacking. Reliable preclinical animal models of CAA and microhemorrhage are needed to investigate the molecular pathogenesis of this condition. Herein, we describe the generation and characterization of a novel transgenic rat (rTg-DI) that produces low levels of human familial CAA Dutch/Iowa E22Q/D23N mutant amyloid β protein in brain and faithfully recapitulates many of the pathologic aspects of human small-vessel CAA. rTg-DI rats exhibit early-onset and progressive accumulation of cerebral microvascular fibrillar amyloid accompanied by early-onset and sustained behavioral deficits. Comparable to CAA in humans, the cerebral microvascular amyloid in rTg-DI rats causes capillary structural alterations, promotes prominent perivascular neuroinflammation, and produces consistent, robust microhemorrhages and small-vessel occlusions that are readily detected by magnetic resonance imaging. The rTg-DI rats provide a new model to investigate the pathogenesis of small-vessel CAA and microhemorrhages, to develop effective biomarkers for this condition and to test therapeutic interventions.
- Published
- 2018
20. A General Purpose, Broadband Transimpedance Amplifier.
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Steven O. Smith
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- 1994
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21. High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.
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Bing Xu, Raja Chakraborty, Markus Eilers, Shyamala Dakshinamurti, Joe D O'Neil, Steven O Smith, Rajinder P Bhullar, and Prashen Chelikani
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Medicine ,Science - Abstract
G protein-coupled receptors (GPCRs) exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM) of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP) is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯)-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(-))-TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/10⁶ cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD) spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM.
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- 2013
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22. Copper stabilizes antiparallel β-sheet fibrils of the amyloid β40 (Aβ40)-Iowa variant
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Toru Kawakami, Elliot J. Crooks, Carlos Simmerling, Tiffany W Victor, Steven O. Smith, Lisa M. Miller, Feng Xu, William E. Van Nostrand, Hironobu Hojo, Martine Ziliox, Kelley Chiu, and Brandon A. Irizarry
- Subjects
0301 basic medicine ,Amyloid ,Magnetic Resonance Spectroscopy ,Beta sheet ,Molecular Conformation ,Peptide ,Plaque, Amyloid ,macromolecular substances ,Antiparallel (biochemistry) ,Fibril ,Microscopy, Atomic Force ,Biochemistry ,03 medical and health sciences ,Alzheimer Disease ,mental disorders ,medicine ,Humans ,Molecular Biology ,chemistry.chemical_classification ,Amyloid beta-Peptides ,030102 biochemistry & molecular biology ,Chemistry ,Neurodegeneration ,Brain ,Molecular Bases of Disease ,Cell Biology ,medicine.disease ,Peptide Fragments ,Cerebral Amyloid Angiopathy ,030104 developmental biology ,Biophysics ,Protein Conformation, beta-Strand ,Cerebral amyloid angiopathy ,Alzheimer's disease ,Copper - Abstract
Cerebral amyloid angiopathy (CAA) is a vascular disorder that primarily involves deposition of the 40-residue-long β-amyloid peptide (Aβ40) in and along small blood vessels of the brain. CAA is often associated with Alzheimer's disease (AD), which is characterized by amyloid plaques in the brain parenchyma enriched in the Aβ42 peptide. Several recent studies have suggested a structural origin that underlies the differences between the vascular amyloid deposits in CAA and the parenchymal plaques in AD. We previously have found that amyloid fibrils in vascular amyloid contain antiparallel β-sheet, whereas previous studies by other researchers have reported parallel β-sheet in fibrils from parenchymal amyloid. Using X-ray fluorescence microscopy, here we found that copper strongly co-localizes with vascular amyloid in human sporadic CAA and familial Iowa-type CAA brains compared with control brain blood vessels lacking amyloid deposits. We show that binding of Cu(II) ions to antiparallel fibrils can block the conversion of these fibrils to the more stable parallel, in-register conformation and enhances their ability to serve as templates for seeded growth. These results provide an explanation for how thermodynamically less stable antiparallel fibrils may form amyloid in or on cerebral vessels by using Cu(II) as a structural cofactor.
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- 2020
23. MPL mutations in essential thrombocythemia uncover a common path of activation with eltrombopag dependent on W491
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Jean-Marc Zini, Serge Carillo, Benjamin Papoular, Stefan N. Constantinescu, Emilie Leroy, Bruno Cassinat, Ilyas Chachoua, Leila N. Varghese, Steven O. Smith, Gabriel Levy, Jean-Philippe Defour, De Duve Institute, Université Catholique de Louvain = Catholic University of Louvain (UCL), Ludwig Institute for Cancer Research, Walloon Excellence in Life sciences and BIOtechnology [Liège] (WELBIO), Cliniques Universitaires Saint-Luc [Bruxelles], Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Universitaire de Nîmes (CHU Nîmes), Hopital Saint-Louis [AP-HP] (AP-HP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Hématopoïèse normale et pathologique : émergence, environnement et recherche translationnelle [Paris] ((UMR_S1131 / U1131)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Service d'Hémato-oncologie [CHU Saint-Louis], Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), De Duve Institute, Université Catholique de Louvain, Stony Brook University [SUNY] (SBU), State University of New York (SUNY), UCL - SSS/DDUV/SIGN - Cell signalling, UCL - (SLuc) Service de biologie hématologique, UCL - (SLuc) Service d'hématologie, and UCL - (SLuc) Service d'hématologie et d'oncologie pédiatrique
- Subjects
Immunology ,Mutant ,Eltrombopag ,Mutagenesis (molecular biology technique) ,MESH: Receptors, Thrombopoietin / genetics ,Signal Transduction / drug effects ,Thrombocythemia, Essential / diagnosis ,Thrombocythemia, Essential / genetics ,Thrombocythemia, Essential / metabolism ,[SDV.CAN]Life Sciences [q-bio]/Cancer ,MESH: Alleles ,Amino Acid Substitution ,Benzoates / pharmacology ,Cell Line ,Genetic Association Studies ,Genetic Predisposition to Disease ,medicine.disease_cause ,Biochemistry ,Benzoates ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein-fragment complementation assay ,medicine ,Humans ,Alleles ,030304 developmental biology ,Thrombopoietin receptor ,0303 health sciences ,Mutation ,[SDV.MHEP.HEM]Life Sciences [q-bio]/Human health and pathology/Hematology ,Cell Biology ,Hematology ,[SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences ,Phenotype ,Transmembrane protein ,3. Good health ,Cell biology ,Hydrazines ,chemistry ,030220 oncology & carcinogenesis ,Pyrazoles ,MESH: Humans ,Hydrazines / pharmacology ,Pyrazoles / pharmacology ,Receptors, Thrombopoietin / agonists ,Receptors, Thrombopoietin ,Signal Transduction ,Thrombocythemia, Essential - Abstract
Mutations in the MPL gene encoding the human thrombopoietin receptor (TpoR) drive sporadic and familial essential thrombocythemias (ETs). We identified 2 ET patients harboring double mutations in cis in MPL, namely, L498W-H499C and H499Y-S505N. Using biochemical and signaling assays along with partial saturation mutagenesis, we showed that L498W is an activating mutation potentiated by H499C and that H499C and H499Y enhance the activity of the canonical S505N mutation. L498W and H499C can activate a truncated TpoR mutant, which lacks the extracellular domain, indicating these mutations act on the transmembrane (TM) cytosolic domain. Using a protein complementation assay, we showed that L498W and H499C strongly drive dimerization of TpoR. Activation by tryptophan substitution is exquisitely specific for position 498. Using structure-guided mutagenesis, we identified upstream amino acid W491 as a key residue required for activation by L498W or canonical activating mutations such as S505N and W515K, as well as by eltrombopag. Structural data point to a common dimerization and activation path for TpoR via its TM domain that is shared between the small-molecule agonist eltrombopag and canonical and novel activating TpoR mutations that all depend on W491, a potentially accessible extracellular residue that could become a target for therapeutic intervention.
- Published
- 2020
24. Dimeric Transmembrane Orientations of APP/C99 Regulate γ-Secretase Processing Line Impacting Signaling and Oligomerization
- Author
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Rémi Opsomer, Stefan N. Constantinescu, Pascal Kienlen-Campard, Florian Perrin, Steven O. Smith, Céline Vrancx, Devkee M. Vadukul, Nicolas Papadopoulos, Nuria Suelves, Didier Vertommen, UCL - SSS/DDUV - Institut de Duve, UCL - SSS/DDUV/PHOS - Protein phosphorylation, UCL - SSS/DDUV/SIGN - Cell signalling, UCL - SSS/IONS/CEMO - Pôle Cellulaire et moléculaire, and UCL - (SLuc) Service d'hématologie
- Subjects
0301 basic medicine ,animal structures ,Dimer ,Peptide ,02 engineering and technology ,Cleavage (embryo) ,Biochemistry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Amyloid precursor protein ,γ secretase ,lcsh:Science ,Molecular Biology ,chemistry.chemical_classification ,Coiled coil ,Messenger RNA ,Multidisciplinary ,biology ,integumentary system ,Aβ peptide ,Correction ,Biological Sciences ,021001 nanoscience & nanotechnology ,Transmembrane protein ,030104 developmental biology ,chemistry ,biology.protein ,Biophysics ,lcsh:Q ,0210 nano-technology ,Amyloid precursor protein secretase ,Intracellular ,Neuroscience - Abstract
Summary Amyloid precursor protein (APP) cleavage by the β-secretase produces the C99 transmembrane (TM) protein, which contains three dimerization-inducing Gly-x-x-x-Gly motifs. We demonstrate that dimeric C99 TM orientations regulate the precise cleavage lines by γ-secretase. Of all possible dimeric orientations imposed by a coiled-coil to the C99 TM domain, the dimer containing the 33Gly-x-x-x-Gly37 motif in the interface promoted the Aβ42 processing line and APP intracellular domain-dependent gene transcription, including the induction of BACE1 mRNA, enhancing amyloidogenic processing and signaling. Another orientation exhibiting the 25Gly-x-x-x-Gly29 motif in the interface favored processing to Aβ43/40. It induced significantly less gene transcription, while promoting formation of SDS-resistant “Aβ-like” oligomers, reminiscent of Aβ peptide oligomers. These required both Val24 of a pro-β motif and the 25Gly-x-x-x-Gly29 interface. Thus, crossing angles imposed by precise dimeric orientations control γ-secretase initial cleavage at Aβ48 or Aβ49, linking the former to enhanced signaling and Aβ42 production., Graphical Abstract, Highlights • C99 dimeric transmembrane orientations regulate γ-secretase processing line • Aβ42 production and signaling are linked to the 33Gly-x-x-x-Gly37 interface • SDS-resistant oligomers require the 25Gly-x-x-x-Gly29 interface and a pro-β motif • C99 dimeric orientations impact its localization and processing by PSEN1 or PSEN2, Biological Sciences; Biochemistry; Molecular Biology; Neuroscience
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- 2020
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25. A Conserved Proline Hinge Mediates Helix Dynamics and Activation of Rhodopsin
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Steven O. Smith, Philip J. Reeves, Kieron South, Reiner Vogel, Martine Ziliox, Omar B. Sanchez-Reyes, Andreyah Pope, and Ekaterina Zaitseva
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Models, Molecular ,Rhodopsin ,Magnetic Resonance Spectroscopy ,Proline ,Protein Conformation ,Allosteric regulation ,Article ,03 medical and health sciences ,Structural Biology ,Spectroscopy, Fourier Transform Infrared ,Humans ,Receptor ,Molecular Biology ,030304 developmental biology ,G protein-coupled receptor ,0303 health sciences ,biology ,Chemistry ,030302 biochemistry & molecular biology ,Tryptophan ,Hydrogen Bonding ,Transmembrane domain ,HEK293 Cells ,Helix ,Mutation ,biology.protein ,Biophysics ,Tyrosine ,Intracellular - Abstract
Despite high-resolution crystal structures of both inactive and active G protein-coupled receptors (GPCRs), it is still not known how ligands trigger the large structural change on the intracellular side of the receptor since the conformational changes that occur within the extracellular ligand-binding region upon activation are subtle. Here, we use solid-state NMR and Fourier transform infrared spectroscopy on rhodopsin to show that Trp2656.48 within the CWxP motif on transmembrane helix H6 constrains a proline hinge in the inactive state, suggesting that activation results in unraveling of the H6 backbone within this motif, a local change in dynamics that allows helix H6 to swing outward. Notably, Tyr3017.48 within activation switch 2 appears to mimic the negative allosteric sodium ion found in other family A GPCRs, a finding that is broadly relevant to the mechanism of receptor activation.
- Published
- 2020
26. ASSOCIATION BETWEEN SHORT-TERM VISIT-TO-VISIT BLOOD PRESSURE VARIABILITY AND LONG-TERM MORTALITY AMONG PATIENTS WITH CORONARY ARTERY DISEASE
- Author
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Carl J. Pepine, Steven O. Smith, Eileen M. Handberg, Osama Dasa, and Rhonda M. Cooper-DeHoff
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Coronary artery disease ,medicine.medical_specialty ,Blood pressure ,business.industry ,Internal medicine ,medicine ,Cardiology ,Long term mortality ,Cardiology and Cardiovascular Medicine ,business ,medicine.disease ,Term (time) - Published
- 2021
27. G Protein-Coupled Receptors Contain Two Conserved Packing Clusters
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Markus Eilers, Dale B. Tranter, Philip J. Reeves, Dawood Rashid, Steven O. Smith, Aidan L.G. Cooke, and Omar B. Sanchez-Reyes
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Models, Molecular ,0301 basic medicine ,Protein Folding ,Rhodopsin ,Protein Conformation ,Biophysics ,Receptors, G-Protein-Coupled ,Motion ,03 medical and health sciences ,Protein structure ,Animals ,Channels and Transporters ,Amino Acid Sequence ,Peptide sequence ,G protein-coupled receptor ,Alanine ,biology ,Hydrogen Bonding ,Crystallography ,Transmembrane domain ,030104 developmental biology ,COS Cells ,Mutation ,Helix ,biology.protein ,Protein folding - Abstract
G protein-coupled receptors (GPCRs) have evolved a seven-transmembrane helix framework that is responsive to a wide range of extracellular signals. An analysis of the interior packing of family A GPCR crystal structures reveals two clusters of highly packed residues that facilitate tight transmembrane helix association. These clusters are centered on amino acid positions 2.47 and 4.53, which are highly conserved as alanine and serine, respectively. Ala2.47 mediates the interaction between helices H1 and H2, while Ser4.53 mediates the interaction between helices H3 and H4. The helical interfaces outside of these clusters are lined with residues that are more loosely packed, a structural feature that facilitates motion of helices H5, H6, and H7, which is required for receptor activation. Mutation of the conserved small side chain at position 4.53 within packing cluster 2 is shown to disrupt the structure of the visual receptor rhodopsin, whereas sites in packing cluster 1 (e.g., positions 1.46 and 2.47) are more tolerant to mutation but affect the overall stability of the protein. These findings reveal a common structural scaffold of GPCRs that is important for receptor folding and activation.
- Published
- 2017
28. The S505A thrombopoietin receptor mutation in childhood hereditary thrombocytosis and essential thrombocythemia is S505N: single letter amino acid code matters
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Stefan N. Constantinescu, Jean-Philippe Defour, Gabriel Levy, Emilie Leroy, Steven O. Smith, UCL - SSS/DDUV/SIGN - Cell signalling, UCL - (SLuc) Service de biologie hématologique, UCL - (SLuc) Service d'hématologie, and UCL - (SLuc) Service d'hématologie et d'oncologie pédiatrique
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chemistry.chemical_classification ,Genetics ,Thrombopoietin receptor ,Thrombocytosis ,Cancer Research ,Code (set theory) ,business.industry ,Single letter ,Essential thrombocythemia ,Hematology ,medicine.disease ,Amino acid ,Oncology ,chemistry ,Mutation (genetic algorithm) ,Mutation ,Medicine ,Humans ,Amino Acids ,business ,Receptor ,Child ,Receptors, Thrombopoietin ,Thrombocythemia, Essential - Abstract
Dear Editor, Myeloproliferative neoplasms (MPNs) are rare diseases in children [1] when compared to adults [2]. Targeted sequencing revealed differences in the mutational landscape of pediatric and adult MPNs [1]. Children exhibit a lower frequency of mutations in the MPN driver genes JAK2, MPL, and CALR, and a much higher proportion of children exhibit no mutation among the 104 classically involved genes; these children exhibiting a trend towards essential thrombocythemia (ET) [1]. We were surprised to read about an activating thrombopoietin receptor (TpoR/Mpl) S505A transmembrane domain (TM) mutation in childhood hereditary thrombocytosis (HT) and ET [3–5]. [...]
- Published
- 2018
29. Anti-Parallel β-Hairpin Structure in Soluble Aβ Oligomers of Aβ40-Dutch and Aβ40-Iowa
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Steven O. Smith, Ziao Fu, and William E. Van Nostrand
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0301 basic medicine ,Amyloid ,Circular dichroism ,Mutant ,Plaque, Amyloid ,Peptide ,amyloid-β ,Microscopy, Atomic Force ,Fibril ,Article ,Fluorescence ,Catalysis ,lcsh:Chemistry ,Inorganic Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Aβ40-Iowa ,0302 clinical medicine ,Alzheimer Disease ,Spectroscopy, Fourier Transform Infrared ,mental disorders ,medicine ,Benzothiazoles ,Physical and Theoretical Chemistry ,lcsh:QH301-705.5 ,Molecular Biology ,Spectroscopy ,chemistry.chemical_classification ,Amyloid beta-Peptides ,Circular Dichroism ,Organic Chemistry ,General Medicine ,medicine.disease ,Computer Science Applications ,Cerebral Amyloid Angiopathy ,030104 developmental biology ,Membrane ,Monomer ,lcsh:Biology (General) ,lcsh:QD1-999 ,chemistry ,Mutation ,Biophysics ,Protein Conformation, beta-Strand ,Thioflavin ,Cerebral amyloid angiopathy ,Aβ40-Dutch ,Alzheimer’s disease ,030217 neurology & neurosurgery - Abstract
The amyloid-&beta, (A&beta, ) peptides are associated with two prominent diseases in the brain, Alzheimer&rsquo, s disease (AD) and cerebral amyloid angiopathy (CAA). A&beta, 42 is the dominant component of cored parenchymal plaques associated with AD, while A&beta, 40 is the predominant component of vascular amyloid associated with CAA. There are familial CAA mutations at positions Glu22 and Asp23 that lead to aggressive A&beta, aggregation, drive vascular amyloid deposition and result in degradation of vascular membranes. In this study, we compared the transition of the monomeric A&beta, 40-WT peptide into soluble oligomers and fibrils with the corresponding transitions of the A&beta, 40-Dutch (E22Q), A&beta, 40-Iowa (D23N) and A&beta, 40-Dutch, Iowa (E22Q, D23N) mutants. FTIR measurements show that in a fashion similar to A&beta, 40-WT, the familial CAA mutants form transient intermediates with anti-parallel &beta, structure. This structure appears before the formation of cross-&beta, sheet fibrils as determined by thioflavin T fluorescence and circular dichroism spectroscopy and occurs when AFM images reveal the presence of soluble oligomers and protofibrils. Although the anti-parallel &beta, hairpin is a common intermediate on the pathway to A&beta, fibrils for the four peptides studied, the rate of conversion to cross-&beta, sheet fibril structure differs for each.
- Published
- 2021
30. Disaggregation of Aβ42 for Structural and Biochemical Studies
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Hyewon, Chung, Elliot J, Crooks, Martine, Ziliox, and Steven O, Smith
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Amyloid ,Protein Aggregates ,Amyloid beta-Peptides ,Magnetic Resonance Spectroscopy ,Molecular Structure ,Protein Stability ,Spectroscopy, Fourier Transform Infrared - Abstract
The amyloid-β (Aβ) peptides that form the amyloid fibrils associated with Alzheimer's disease are generated by sequential proteolysis of the amyloid precursor protein by β- and γ-secretase. The two predominant Aβ peptides, Aβ40 and Aβ42, differ by two amino acids, are soluble as monomers at low concentration (and/or low temperature) and are normally cleared from the brain parenchyma. In order to study the structure and assembly of these peptides, they are often synthesized using solid-phase peptide synthesis and purified. Here, we outline the method we use to prepare monomeric Aβ for structural and biochemical studies.
- Published
- 2018
31. Disaggregation of Aβ42 for Structural and Biochemical Studies
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Steven O. Smith, Hye Won Chung, Martine Ziliox, and Elliot J. Crooks
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0301 basic medicine ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,medicine.diagnostic_test ,Proteolysis ,Amino acid ,03 medical and health sciences ,chemistry.chemical_compound ,Monomer ,Biochemistry ,chemistry ,Parenchyma ,Peptide synthesis ,Amyloid precursor protein ,biology.protein ,medicine ,Volume concentration ,Clearance - Abstract
The amyloid-β (Aβ) peptides that form the amyloid fibrils associated with Alzheimer's disease are generated by sequential proteolysis of the amyloid precursor protein by β- and γ-secretase. The two predominant Aβ peptides, Aβ40 and Aβ42, differ by two amino acids, are soluble as monomers at low concentration (and/or low temperature) and are normally cleared from the brain parenchyma. In order to study the structure and assembly of these peptides, they are often synthesized using solid-phase peptide synthesis and purified. Here, we outline the method we use to prepare monomeric Aβ for structural and biochemical studies.
- Published
- 2018
32. β-Sheet Structure within the Extracellular Domain of C99 Regulates Amyloidogenic Processing
- Author
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Steven O. Smith, Rémi Opsomer, Stefan N. Constantinescu, Yi Hu, Tzu-Chun Tang, Xiaoshu Pan, Florian Perrin, Jean-Noël Octave, Marie Decock, Pascal Kienlen-Campard, UCL - SSS/DDUV/SIGN - Cell signalling, and UCL - SSS/IONS/CEMO - Pôle Cellulaire et moléculaire
- Subjects
Models, Molecular ,0301 basic medicine ,Amyloid ,Proteolysis ,Protein domain ,Beta sheet ,lcsh:Medicine ,Sequence (biology) ,Article ,Amyloid beta-Protein Precursor ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,Protein Domains ,Extracellular ,medicine ,Humans ,lcsh:Science ,Multidisciplinary ,medicine.diagnostic_test ,Chemistry ,lcsh:R ,Small molecule ,Peptide Fragments ,030104 developmental biology ,Membrane ,Biophysics ,Protein Conformation, beta-Strand ,lcsh:Q ,Amyloid Precursor Protein Secretases ,030217 neurology & neurosurgery - Abstract
Familial mutations in C99 can increase the total level of the soluble Aβ peptides produced by proteolysis, as well as the Aβ42/Aβ40 ratio, both of which are linked to the progression of Alzheimer’s disease. We show that the extracellular sequence of C99 forms β-sheet structure upon interaction with membrane bilayers. Mutations that disrupt this structure result in a significant increase in Aβ production and, in specific cases, result in an increase in the amount of Aβ42 relative to Aβ40. Fourier transform infrared and solid-state NMR spectroscopic studies reveal a central β-hairpin within the extracellular sequence comprising Y10-E11-V12 and L17-V18-F19 connected by a loop involving H13-H14-Q15. These results suggest how familial mutations in the extracellular sequence influence C99 processing and provide a structural basis for the development of small molecule modulators that would reduce Aβ production.
- Published
- 2017
33. Capping of Aβ42 Oligomers by Small Molecule Inhibitors
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Ziao Fu, William E. Van Nostrand, Darryl Aucoin, Mahiuddin Ahmed, Steven O. Smith, and Martine Ziliox
- Subjects
chemistry.chemical_classification ,Amyloid beta-Peptides ,Curcumin ,Anti-Inflammatory Agents, Non-Steroidal ,Indomethacin ,Peptide ,Sequence (biology) ,Nuclear magnetic resonance spectroscopy ,Protein aggregation ,Microscopy, Atomic Force ,Fibril ,Biochemistry ,Small molecule ,Peptide Fragments ,Article ,Protein Structure, Tertiary ,Protein Aggregates ,chemistry.chemical_compound ,Sulindac ,Monomer ,Protein structure ,chemistry ,Biophysics ,Humans ,Organic chemistry - Abstract
Aβ42 peptides associate into soluble oligomers and protofibrils in the process of forming the amyloid fibrils associated with Alzheimer’s disease. The oligomers have been reported to be more toxic to neurons than fibrils, and have been targeted by a wide range of small molecule and peptide inhibitors. With single touch atomic force microscopy (AFM), we show that monomeric Aβ42 forms two distinct types of oligomers, low molecular weight (MW) oligomers with heights of 1–2 nm and high MW oligomers with heights of 3–5 nm. In both cases, the oligomers are disc-shaped with diameters of ∼10–15 nm. The similar diameters suggest that the low MW species stack to form the high MW oligomers. The ability of Aβ42 inhibitors to interact with these oligomers is probed using atomic force microscopy and NMR spectroscopy. We show that curcumin and resveratrol bind to the N-terminus (residues 5–20) of Aβ42 monomers and cap the height of the oligomers that are formed at 1–2 nm. A second class of inhibitors, which includes sulindac sulfide and indomethacin, exhibit very weak interactions across the Aβ42 sequence and do not block the formation of the high MW oligomers. The correlation between N-terminal interactions and capping of the height of the Aβ oligomers provides insights into the mechanism of inhibition and the pathway of Aβ aggregation.
- Published
- 2014
34. Early-onset Formation of Parenchymal Plaque Amyloid Abrogates Cerebral Microvascular Amyloid Accumulation in Transgenic Mice
- Author
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Steven O. Smith, Judianne Davis, Feng Xu, Ming-Hsuan Ou-Yang, William E. Van Nostrand, AnnMarie E. Kotarba, and Ziao Fu
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Pathology ,medicine.medical_specialty ,Amyloid ,BACE1-AS ,Mice, Transgenic ,Plaque, Amyloid ,Biochemistry ,Mice ,mental disorders ,medicine ,Animals ,Humans ,Senile plaques ,Molecular Biology ,Cerebral Cortex ,Amyloid beta-Peptides ,Microglia ,Chemistry ,P3 peptide ,Molecular Bases of Disease ,Cell Biology ,medicine.disease ,Biochemistry of Alzheimer's disease ,Cerebral Amyloid Angiopathy ,medicine.anatomical_structure ,Cerebrovascular Circulation ,Mutation ,Cerebral amyloid angiopathy ,Alzheimer's disease - Abstract
The fibrillar assembly and deposition of amyloid β (Aβ) protein, a key pathology of Alzheimer disease, can occur in the form of parenchymal amyloid plaques and cerebral amyloid angiopathy (CAA). Familial forms of CAA exist in the absence of appreciable parenchymal amyloid pathology. The molecular interplay between parenchymal amyloid plaques and CAA is unclear. Here we investigated how early-onset parenchymal amyloid plaques impact the development of microvascular amyloid in transgenic mice. Tg-5xFAD mice, which produce non-mutated human Aβ and develop early-onset parenchymal amyloid plaques, were bred to Tg-SwDI mice, which produce familial CAA mutant human Aβ and develop cerebral microvascular amyloid. The bigenic mice presented with an elevated accumulation of Aβ and fibrillar amyloid in the brain compared with either single transgenic line. Tg-SwDI/Tg-5xFAD mice were devoid of microvascular amyloid, the prominent pathology of Tg-SwDI mice, but exhibited larger parenchymal amyloid plaques compared with Tg-5xFAD mice. The larger parenchymal amyloid deposits were associated with a higher loss of cortical neurons and elevated activated microglia in the bigenic Tg-SwDI/Tg-5xFAD mice. The periphery of parenchymal amyloid plaques was largely composed of CAA mutant Aβ. Non-mutated Aβ fibril seeds promoted CAA mutant Aβ fibril formation in vitro. Further, intrahippocampal administration of biotin-labeled CAA mutant Aβ peptide accumulated on and adjacent to pre-existing parenchymal amyloid plaques in Tg-5xFAD mice. These findings indicate that early-onset parenchymal amyloid plaques can serve as a scaffold to capture CAA mutant Aβ peptides and prevent their accumulation in cerebral microvessels.
- Published
- 2014
35. Amino acid conservation and interactions in rhodopsin: Probing receptor activation by NMR spectroscopy
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Steven O. Smith, Andreyah Pope, Markus Eilers, and Philip J. Reeves
- Subjects
Models, Molecular ,Rhodopsin ,Magnetic Resonance Spectroscopy ,Light ,genetic structures ,Stereochemistry ,Molecular Sequence Data ,Biophysics ,Crystallography, X-Ray ,Biochemistry ,Article ,Protein Structure, Secondary ,chemistry.chemical_compound ,GPCR ,Isomerism ,Animals ,Humans ,Inverse agonist ,Amino Acid Sequence ,Solid-state NMR spectroscopy ,Receptor ,Conserved Sequence ,G protein-coupled receptor ,chemistry.chemical_classification ,biology ,Hydrogen Bonding ,Retinal ,Nuclear magnetic resonance spectroscopy ,Cell Biology ,Protein Structure, Tertiary ,Amino acid ,chemistry ,Retinaldehyde ,Visual pigment ,biology.protein ,sense organs - Abstract
Rhodopsin is a classical two-state G protein-coupled receptor (GPCR). In the dark, its 11-cis retinal chromophore serves as an inverse agonist to lock the receptor in an inactive state. Retinal–protein and protein–protein interactions have evolved to reduce the basal activity of the receptor in order to achieve low dark noise in the visual system. In contrast, absorption of light triggers rapid isomerization of the retinal, which drives the conversion of the receptor to a fully active conformation. Several specific protein–protein interactions have evolved that maintain the lifetime of the active state in order to increase the sensitivity of this receptor for dim-light vision in vertebrates. In this article, we review the molecular interactions that stabilize rhodopsin in the dark-state and describe the use of solid-state NMR spectroscopy for probing the structural changes that occur upon light-activation. Amino acid conservation provides a guide for those interactions that are common in the class A GPCRs as well as those that are unique to the visual system. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.
- Published
- 2014
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36. Conformational Changes Induced by the A21G Flemish Mutation in the Amyloid Precursor Protein Lead to Increased Aβ Production
- Author
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Marie Decock, Laetitia El Haylani, Yi Hu, Steven O. Smith, Joanne Van Hees, Tzu-Chun Tang, Jean-Noël Octave, Ziao Fu, Stefan N. Constantinescu, and Pascal Kienlen-Campard
- Subjects
Mutation ,medicine.diagnostic_test ,biology ,Chemistry ,Proteolysis ,Mutant ,Beta sheet ,medicine.disease_cause ,Cell biology ,Cell membrane ,Protein structure ,medicine.anatomical_structure ,Biochemistry ,Structural Biology ,medicine ,Amyloid precursor protein ,biology.protein ,Secretion ,Molecular Biology - Abstract
SummaryProteolysis of the β C-terminal fragment (β-CTF) of the amyloid precursor protein generates the Aβ peptides associated with Alzheimer’s disease. Familial mutations in the β-CTF, such as the A21G Flemish mutation, can increase Aβ secretion. We establish how the Flemish mutation alters the structure of C55, the first 55 residues of the β-CTF, using FTIR and solid-state NMR spectroscopy. We show that the A21G mutation reduces β sheet structure of C55 from Leu17 to Ala21, an inhibitory region near the site of the mutation, and increases α-helical structure from Gly25 to Gly29, in a region near the membrane surface and thought to interact with cholesterol. Cholesterol also increases Aβ peptide secretion, and we show that the incorporation of cholesterol into model membranes enhances the structural changes induced by the Flemish mutant, suggesting a common link between familial mutations and the cellular environment.
- Published
- 2014
37. P100: A needs assessment to guide the development of multidisciplinary simulation-based modules relevant to emergency department nurses in Newfoundland and Labrador
- Author
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Steven O. Smith, K. Bursey, and M. Parsons
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Engineering ,Multidisciplinary approach ,business.industry ,Needs assessment ,Emergency Medicine ,medicine ,Emergency department ,Medical emergency ,medicine.disease ,business ,Simulation based - Abstract
Introduction: Efficient multidisciplinary team dynamics are crucial to the provision of optimal ED care. Physicians and nurses must use a collaborative approach to meet patient needs in this busy setting. This is especially important for high-acuity low-occurrence (HALO) procedures and clinical encounters. Simulation provides a safe environment where learning is enhanced through deliberate practice. Multidisciplinary participation in simulation-based education may augment team cohesiveness and performance. Methods: A web-based needs assessment survey was distributed to ED nurses, collecting information on demographics, opinions about simulation-based instruction and perceptions on the value of the proposed collaborative educational approach of the project. Experience and comfort with nursing roles in specific procedures (TV pacer, surgical airway, chest tube, central line, sedation) and clinical encounters (STEMI, CVA, sepsis, anaphylaxis, GI bleed) seen in the ED were also assessed. There were a number of suggestions for topics in addition to those listed. Responses will guide the collaborative development of simulation modules with nursing colleagues on desired topics. Results: 58/97 potential nurse participants from 2 urban ED's responded to the survey over an 8-week period, giving a response rate of 58.8%. 76% of respondents had less than 10 years of ED nursing experience, and 34.48% less than 5 years. Responses indicate limited familiarity with simulation-based education (SBE) on ED scenarios with 33.93% being not familiar; 55.36% somewhat familiar. Most prior simulation experience was with role-playing (82%) or low-fidelity setups (42%). Perceived benefit of SBE sessions was substantial (43.86%- very significant; 45.61%- significant). Most respondents had limited past exposure (22.81%- none; 64.91%- 1-5 sims). Similarly, there was little ongoing participation in SBE events with none in 43.64% and 40% just annually. For the 5 clinical scenarios, average responses were: Comfort with assisting 87.45%; Interest in further training 91.43%; Willingness to participate 94.13%. For the 5 procedures, averages were 36.35% (21.36% excluding sedation), 91.27%, 89.09%, respectively. Conclusion: Results indicate a low level of familiarity, experience and ongoing exposure with SBE relating to ED training and practice. Participants recognize the potential benefits of using simulation in a multidisciplinary educational setting and indicate a willingness to participate in collaborative teaching sessions.
- Published
- 2019
38. INTERINDIVIDUAL VARIABILITY IN WEIGHT LOSS WITH LORCASERIN: AN ANALYSIS FROM THE CAMELLIA-TIMI 61 STUDY
- Author
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Steven O. Smith, Silvio Inzucchi, Wenfeng Miao, Arman Qamar, Stephen D. Wiviott, Christina Fanola, Tushar Patel, KyungAh Im, Benjamin M. Scirica, Erin A. Bohula, Darren K. McGuire, Marc S. Sabatine, Carlos Perdomo, Anthony Keech, and Sabina A. Murphy
- Subjects
Agonist ,medicine.drug_class ,business.industry ,Pharmacology ,Placebo ,Lorcaserin ,Lifestyle modification ,Weight loss ,medicine ,medicine.symptom ,Cardiology and Cardiovascular Medicine ,business ,TIMI ,medicine.drug - Abstract
Lorcaserin, a 5HT2c agonist, safely facilitates weight (wt.) loss with favorable effects on cardiometabolic profile. The interindividual variability in wt. loss in response to lorcaserin on a background of lifestyle modification is not well-characterized. CAMELLIA-TIMI 61 was a randomized, placebo
- Published
- 2019
39. Retinal orientation and interactions in rhodopsin reveal a two-stage trigger mechanism for activation
- Author
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Reiner Vogel, Chikwado A. Opefi, Naoki Kimata, Steven O. Smith, Martine Ziliox, Philip J. Reeves, Amiram Hirshfeld, Mordechai Sheves, Ekaterina Zaitseva, Markus Eilers, and Andreyah Pope
- Subjects
0301 basic medicine ,Steric effects ,Models, Molecular ,Rhodopsin ,Science ,General Physics and Astronomy ,Protonation ,General Biochemistry, Genetics and Molecular Biology ,Article ,Retina ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Spectroscopy, Fourier Transform Infrared ,Humans ,Nuclear Magnetic Resonance, Biomolecular ,Multidisciplinary ,biology ,Retinal ,General Chemistry ,Protein Structure, Tertiary ,Transmembrane domain ,030104 developmental biology ,HEK293 Cells ,chemistry ,Biochemistry ,Retinaldehyde ,Biophysics ,biology.protein ,Isomerization - Abstract
The 11-cis retinal chromophore is tightly packed within the interior of the visual receptor rhodopsin and isomerizes to the all-trans configuration following absorption of light. The mechanism by which this isomerization event drives the outward rotation of transmembrane helix H6, a hallmark of activated G protein-coupled receptors, is not well established. To address this question, we use solid-state NMR and FTIR spectroscopy to define the orientation and interactions of the retinal chromophore in the active metarhodopsin II intermediate. Here we show that isomerization of the 11-cis retinal chromophore generates strong steric interactions between its β-ionone ring and transmembrane helices H5 and H6, while deprotonation of its protonated Schiff's base triggers the rearrangement of the hydrogen-bonding network involving residues on H6 and within the second extracellular loop. We integrate these observations with previous structural and functional studies to propose a two-stage mechanism for rhodopsin activation., Rhodopsin signalling is triggered by the light-induced isomerization of its 11-cis retinal chromophore. Here, the authors use NMR spectroscopy to define retinal orientation and interactions in the active metarhodopsin II intermediate, proposing a two-stage mechanism for rhodopsin activation.
- Published
- 2016
40. O5‐06‐03: Cerebral Vascular Amyloid Seeds Drive Amyloid Ss‐Protein Fibril Assembly with a Distinct Anti‐Parallel Structure
- Author
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Michael D. Hoos, AnnMarie E. Kotarba, Judianne Davis, Sharmilla Dass, Steven O. Smith, Feng Xu, William E. Van Nostrand, and Ziao Fu
- Subjects
Psychiatry and Mental health ,Cellular and Molecular Neuroscience ,Developmental Neuroscience ,Amyloid ,Epidemiology ,Chemistry ,Health Policy ,P3 peptide ,Biophysics ,Neurology (clinical) ,Geriatrics and Gerontology ,Fibril ,Vascular amyloid - Published
- 2016
41. Glycines from the APP GXXXG/GXXXA transmembrane motifs promote formation of pathogenic Aβ oligomers in cells
- Author
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Steven O. Smith, Stefan N. Constantinescu, Pascal Kienlen-Campard, Ilse Dewachter, Jean-Noël Octave, Serena Stanga, and Marie Decock
- Subjects
0301 basic medicine ,Aging ,Cognitive Neuroscience ,Peptide ,amyloid precursor protein ,Biology ,medicine.disease_cause ,Protein–protein interaction ,lcsh:RC321-571 ,GXXXG motifs ,03 medical and health sciences ,Beta-amyloid peptide ,0302 clinical medicine ,medicine ,Extracellular ,Amyloid precursor protein ,Alzheimer's disease ,Neuronal differentiation ,Oligomers ,Senile plaques ,oligomers ,neuronal differentiation ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Original Research ,chemistry.chemical_classification ,Mutation ,P3 peptide ,Transmembrane protein ,Cell biology ,030104 developmental biology ,Biochemistry ,chemistry ,biology.protein ,Alzheimer’s disease ,030217 neurology & neurosurgery ,Neuroscience - Abstract
Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by progressive cognitive decline leading to dementia. The amyloid precursor protein (APP) is a ubiquitous type I transmembrane (TM) protein sequentially processed to generate the β-amyloid peptide (Aβ), the major constituent of senile plaques that are typical AD lesions. There is a growing body of evidence that soluble Aβ oligomers correlate with clinical symptoms associated with the disease. The Aβ sequence begins in the extracellular juxtamembrane region of APP and includes roughly half of the TM domain. This region contains GXXXG and GXXXA motifs, which are critical for both TM protein interactions and fibrillogenic properties of peptides derived from TM α-helices. Glycine-to-leucine mutations of these motifs were previously shown to affect APP processing and Aβ production in cells. However, the detailed contribution of these motifs to APP dimerization, their relation to processing, and the conformational changes they can induce within Aβ species remains undefined. Here, we describe highly resistant Aβ42 oligomers that are produced in cellular membrane compartments. They are formed in cells by processing of the APP amyloidogenic C-terminal fragment (C99), or by direct expression of a peptide corresponding to Aβ42, but not to Aβ40. By a point-mutation approach, we demonstrate that glycine-to-leucine mutations in the G(29)XXXG(33) and G(38)XXXA(42) motifs dramatically affect the Aβ oligomerization process. G33 and G38 in these motifs are specifically involved in Aβ oligomerization; the G33L mutation strongly promotes oligomerization, while G38L blocks it with a dominant effect on G33 residue modification. Finally, we report that the secreted Aβ42 oligomers display pathological properties consistent with their suggested role in AD, but do not induce toxicity in survival assays with neuronal cells. Exposure of neurons to these Aβ42 oligomers dramatically affects neuronal differentiation and, consequently, neuronal network maturation.
- Published
- 2016
42. Conformational Differences between Two Amyloid β Oligomers of Similar Size and Dissimilar Toxicity
- Author
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Judianne Davis, Darryl Aucoin, Ravi S. Kane, Swarnim Ranjan, Steven O. Smith, Peter M. Tessier, Ali Reza A. Ladiwala, Jeffrey Litt, and William E. Van Nostrand
- Subjects
Amyloid ,Protein Folding ,Cell Survival ,Peptide ,Microscopy, Atomic Force ,Fibril ,PC12 Cells ,Biochemistry ,Oligomer ,Protein Structure, Secondary ,chemistry.chemical_compound ,Protein structure ,Animals ,Humans ,Molecular Biology ,chemistry.chemical_classification ,Amyloid beta-Peptides ,Circular Dichroism ,P3 peptide ,Cell Biology ,Rats ,Monomer ,chemistry ,Protein Structure and Folding ,Chromatography, Gel ,Protein folding ,Hydrophobic and Hydrophilic Interactions - Abstract
Several protein conformational disorders (Parkinson and prion diseases) are linked to aberrant folding of proteins into prefibrillar oligomers and amyloid fibrils. Although prefibrillar oligomers are more toxic than their fibrillar counterparts, it is difficult to decouple the origin of their dissimilar toxicity because oligomers and fibrils differ both in terms of structure and size. Here we report the characterization of two oligomers of the 42-residue amyloid β (Aβ42) peptide associated with Alzheimer disease that possess similar size and dissimilar toxicity. We find that Aβ42 spontaneously forms prefibrillar oligomers at Aβ concentrations below 30 μm in the absence of agitation, whereas higher Aβ concentrations lead to rapid formation of fibrils. Interestingly, Aβ prefibrillar oligomers do not convert into fibrils under quiescent assembly conditions but instead convert into a second type of oligomer with size and morphology similar to those of Aβ prefibrillar oligomers. Strikingly, this alternative Aβ oligomer is non-toxic to mammalian cells relative to Aβ monomer. We find that two hydrophobic peptide segments within Aβ (residues 16-22 and 30-42) are more solvent-exposed in the more toxic Aβ oligomer. The less toxic oligomer is devoid of β-sheet structure, insoluble, and non-immunoreactive with oligomer- and fibril-specific antibodies. Moreover, the less toxic oligomer is incapable of disrupting lipid bilayers, in contrast to its more toxic oligomeric counterpart. Our results suggest that the ability of non-fibrillar Aβ oligomers to interact with and disrupt cellular membranes is linked to the degree of solvent exposure of their central and C-terminal hydrophobic peptide segments.
- Published
- 2012
43. Insights into the activation mechanism of the visual receptor rhodopsin
- Author
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Steven O. Smith
- Subjects
Rhodopsin ,genetic structures ,biology ,Mechanism (biology) ,Biochemistry ,Protein Structure, Secondary ,Cell biology ,Conserved sequence ,Isomerism ,Structural biology ,Retinaldehyde ,Rosaniline Dyes ,biology.protein ,Animals ,Humans ,sense organs ,Receptor ,Neuroscience ,Conserved Sequence ,Function (biology) ,G protein-coupled receptor - Abstract
Recent advances in the structural biology of GPCRs (G-protein-coupled receptors) have provided insights into their structure and function. Comparisons of the visual and ligand-activated receptors highlight the unique elements of rhodopsin that allow it to function as a highly sensitive dim-light photoreceptor in vertebrates, as well as the common elements that it shares with the large class A GPCR family. However, despite progress, a number of questions remain unanswered about how these receptors are activated.
- Published
- 2012
44. Cerebral vascular amyloid seeds drive amyloid β-protein fibril assembly with a distinct anti-parallel structure
- Author
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Steven O. Smith, AnnMarie E. Kotarba, Sharmila Dass, Judianne Davis, Feng Xu, William E. Van Nostrand, and Ziao Fu
- Subjects
0301 basic medicine ,Genetically modified mouse ,Amyloid ,Science ,BACE1-AS ,General Physics and Astronomy ,Mice, Transgenic ,Biology ,Fibril ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Amyloid beta-Protein Precursor ,Mice ,0302 clinical medicine ,mental disorders ,medicine ,Animals ,Senile plaques ,Multidisciplinary ,Amyloid beta-Peptides ,P3 peptide ,General Chemistry ,Amyloidosis ,medicine.disease ,Cell biology ,Biochemistry of Alzheimer's disease ,Cerebral Amyloid Angiopathy ,030104 developmental biology ,Cerebrovascular Circulation ,Cerebral amyloid angiopathy ,030217 neurology & neurosurgery - Abstract
Cerebrovascular accumulation of amyloid β-protein (Aβ), a condition known as cerebral amyloid angiopathy (CAA), is a common pathological feature of patients with Alzheimer's disease. Familial Aβ mutations, such as Dutch-E22Q and Iowa-D23N, can cause severe cerebrovascular accumulation of amyloid that serves as a potent driver of vascular cognitive impairment and dementia. The distinctive features of vascular amyloid that underlie its unique pathological properties remain unknown. Here, we use transgenic mouse models producing CAA mutants (Tg-SwDI) or overproducing human wild-type Aβ (Tg2576) to demonstrate that CAA-mutant vascular amyloid influences wild-type Aβ deposition in brain. We also show isolated microvascular amyloid seeds from Tg-SwDI mice drive assembly of human wild-type Aβ into distinct anti-parallel β-sheet fibrils. These findings indicate that cerebrovascular amyloid can serve as an effective scaffold to promote rapid assembly and strong deposition of Aβ into a unique structure that likely contributes to its distinctive pathology., Cerebrovascular accumulation of Aβ is a common feature of Alzheimer's disease, though it is unclear whether mutant vascular amyloid is capable of Aβ seeding. Here, the authors show microvascular amyloid seeds are capable of driving wild-type Aβ to assemble into distinctive anti-parallel fibrillary structures.
- Published
- 2015
45. Orientation-specific signalling by thrombopoietin receptor dimers
- Author
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William Vainchenker, Ian C. Brett, Emilie Leroy, Judith Staerk, Stefan N. Constantinescu, Hélène Antoine-Poirel, Steven O. Smith, Miki Itaya, Christian Pecquet, and Jean-Philippe Defour
- Subjects
Thrombopoietin receptor ,Coiled coil ,General Immunology and Microbiology ,General Neuroscience ,Megakaryocyte differentiation ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Transmembrane protein ,Cell biology ,Biochemistry ,Signal transduction ,Cytokine receptor ,Receptor ,Molecular Biology ,Thrombopoietin - Abstract
Ligand binding to the thrombopoietin receptor is thought to stabilize an active receptor dimer that regulates megakaryocyte differentiation and platelet formation, as well as haematopoietic stem cell renewal. By fusing a dimeric coiled coil in all seven possible orientations to the thrombopoietin receptor transmembrane (TM)-cytoplasmic domains, we show that specific biological effects and in vivo phenotypes are imparted by distinct dimeric orientations, which can be visualized by cysteine mutagenesis and crosslinking. Using functional assays and computational searches, we identify one orientation that represents the inactive dimeric state and another similar to a physiologically activated receptor. Several other dimeric orientations are identified that induce proliferation and in vivo myeloproliferative and myelodysplastic disorders, indicating the receptor can signal from several dimeric interfaces. The set of dimeric thrombopoietin receptors with different TM orientations may offer new insights into the activation of distinct signalling pathways by a single receptor and suggests that subtle differences in cytokine receptor dimerization provide a new layer of signalling regulation that is relevant for disease.
- Published
- 2011
46. Structural and functional roles of small group-conserved amino acids present on helix-H7 in the β2-adrenergic receptor
- Author
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Steven O. Smith, Jasbir D. Upadhyaya, Raja Chakraborty, Markus Eilers, Prashen Chelikani, Makoto Arakawa, and Philip J. Reeves
- Subjects
Models, Molecular ,Receptor expression ,Mutant ,Biochemistry ,Protein Structure, Secondary ,Serine ,Radioligand Assay ,Protein structure ,Site-directed mutagenesis ,Cricetinae ,Helix packing ,Chlorocebus aethiops ,Cyclic AMP ,Amino Acids ,chemistry.chemical_classification ,0303 health sciences ,030302 biochemistry & molecular biology ,Adrenergic beta-Agonists ,Amino acid ,Transmembrane domain ,COS Cells ,Leucine ,Protein Binding ,Stereochemistry ,Adrenergic beta-Antagonists ,Glycine ,Biophysics ,Article ,03 medical and health sciences ,Structure-Activity Relationship ,G-protein coupled receptors (GPCRs) ,Animals ,Humans ,030304 developmental biology ,G protein-coupled receptor ,Beta2-adrenergic receptor ,Binding Sites ,Isoproterenol ,Hydrogen Bonding ,Cell Biology ,Protein Structure, Tertiary ,HEK293 Cells ,chemistry ,Amino Acid Substitution ,Mutation ,Dihydroalprenolol ,GPCR activation ,Receptors, Adrenergic, beta-2 - Abstract
Sequence analysis of the class A G protein-coupled receptors (GPCRs) reveals that most of the highly conserved sites are located in the transmembrane helices. A second level of conservation exists involving those residues that are conserved as a group characterized by small and/or weakly polar side chains (Ala, Gly, Ser, Cys, Thr). These positions can have group conservation levels of up to 99% across the class A GPCRs and have been implicated in mediating helix-helix interactions in membrane proteins. We have previously shown that mutation of group-conserved residues present on transmembrane helices H2-H4 in the β(2)-adrenergic receptor (β(2)-AR) can influence both receptor expression and function. We now target the group-conserved sites, Gly315(7.42) and Ser319(7.46), on H7 for structure-function analysis. Replacing Ser319(7.46) with smaller amino acids (Ala or Gly) did not influence the ability of the mutant receptors to bind to the antagonist dihydroalprenolol (DHA) but resulted in ~15-20% agonist-independent activity. Replacement of Ser319(7.46) with the larger amino acid leucine lowered the expression of the S319L mutant and its ability to bind DHA. Both the G315A and G315S mutants also exhibited agonist-independent signaling, while the G315L mutant did not show specific binding to DHA. These data indicate that Gly315(7.42) and Ser319(7.46) are stabilizing β(2)-AR in an inactive conformation. We discuss our results in the context of van der Waals interactions of Gly315(7.42) with Trp286(6.48) and hydrogen bonding interactions of Ser319(7.46) with amino acids on H1-H2-H7 and with structural water.
- Published
- 2011
- Full Text
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47. N-terminal Domain of Myelin Basic Protein Inhibits Amyloid β-Protein Fibril Assembly
- Author
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William E. Van Nostrand, Darryl Aucoin, Judianne Davis, Steven O. Smith, Mei-Chen Liao, Michael D. Hoos, and Mahiuddin Ahmed
- Subjects
Amyloid ,Cell Survival ,Molecular Sequence Data ,Amylin ,Peptide ,Microscopy, Atomic Force ,Fibril ,Biochemistry ,Mice ,Microscopy, Electron, Transmission ,Escherichia coli ,Animals ,Humans ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Peptide sequence ,Cells, Cultured ,Neurons ,chemistry.chemical_classification ,Amyloid beta-Peptides ,biology ,Brain ,Myelin Basic Protein ,Fibrillogenesis ,Cell Biology ,Surface Plasmon Resonance ,Peptide Fragments ,Recombinant Proteins ,Rats ,Myelin basic protein ,chemistry ,Protein Structure and Folding ,biology.protein ,Protein Binding - Abstract
Accumulation of amyloid β-protein (Aβ) into brain parenchymal plaques and the cerebral vasculature is a pathological feature of Alzheimer disease and related disorders. Aβ peptides readily form β-sheet-containing oligomers and fibrils. Previously, we reported a strong interaction between myelin basic protein (MBP) and Aβ peptides that resulted in potent inhibition of fibril assembly (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). MBP is recognized as a highly post-translationally modified protein. In the present study, we demonstrate that human MBP purified from either brain or a bacterial recombinant expression system comparably bound to Aβ and inhibited Aβ fibril assembly indicating that post-translational modifications are not required for this activity. We also show that purified mouse brain MBP and recombinantly expressed mouse MBP similarly inhibited Aβ fibril formation. Through a combination of biochemical and ultrastructural techniques, we demonstrate that the binding site for Aβ is located in the N-terminal 64 amino acids of MBP and that a stable peptide (MBP1) comprising these residues was sufficient to inhibit Aβ fibrillogenesis. Under conditions comparable with those used for Aβ, the fibrillar assembly of amylin, another amyloidogenic peptide, was not inhibited by MBP1, although MBP1 still bound to it. This observation suggests that the potent inhibitory effect of MBP on fibril formation is not general to amyloidogenic peptides. Finally, MBP1 could prevent the cytotoxic effects of Aβ in primary cortical neurons. Our findings suggest that inhibition of Aβ fibril assembly by MBP, mediated through its N-terminal domain, could play a role in influencing amyloid formation in Alzheimer disease brain and corresponding mouse models.
- Published
- 2010
48. Structure and function of G protein-coupled receptors using NMR spectroscopy
- Author
-
Shivani Ahuja, Steven O. Smith, Markus Eilers, Sina Erfani, and Joseph A. Goncalves
- Subjects
Models, Molecular ,Rhodopsin ,Nuclear and High Energy Physics ,biology ,Protein Conformation ,Chemistry ,Class C GPCR ,Biochemistry ,Article ,Rhodopsin-like receptors ,Receptors, G-Protein-Coupled ,Analytical Chemistry ,Transmembrane domain ,Metabotropic glutamate receptor ,Heterotrimeric G protein ,Biophysics ,biology.protein ,Animals ,Humans ,Receptor ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,G protein-coupled receptor - Abstract
Nathans and Hogness [1] cloned the gene for the visual pigment rhodopsin in 1983, and found that the receptor contained seven hydrophobic membrane-spanning helices. At roughly the same time, the paradigm in the visual receptor field that rhodopsin functions as a light-activated Ca2+ channel was being overturned in favor of a competing model that rhodopsin is responsible for activating a heterotrimeric G-protein [2]. In 1986, a landmark paper by Strader, Lefkowitz and co-workers [3] reported the sequence of the β2-adrenergic receptor. They recognized that the β2-adrenergic receptor shares significant sequence homology with rhodopsin, including seven transmembrane (TM) segments. They proposed that the amine ligands of the adrenergic receptors intercalate into the TM helices and trigger receptor activation in the same manner as the retinal in rhodopsin. Because the receptors of the adenylate cyclase system were known to activate G-proteins [2,4,5], the similarity between the β2-adrenergic receptor and rhodopsin provided the first hint of the existence of a large G protein-coupled receptor (GPCR) family. Early structural work in the emerging GPCR field was guided by two pioneering studies. In 1993, Schertler, Villa and Henderson [6] reported an electron density projection map based on cryo-EM images of rhodopsin that showed the seven-helix architecture is distinctly different from that of bacteriorhodopsin, which had been a favored template for modeling GPCRs. In 1996, Hubbell and Khorana [7] used site-directed spin labeling in combination with EPR spectroscopy to reveal that activation is mediated by rigid body rotation of the TM helices. A breakthrough in the GPCR field came in 2000 with the determination of the rhodopsin crystal structure [8] (Fig. 1). The structure was solved of the dark, inactive conformation of the protein. The structure of the active state, however, has been more elusive. When illuminated, the crystals of rhodopsin crack and dissolve [9], presumably due to rigid body helix motions that accompany receptor activation. In 2007, the crystal structure of the β2-adrenergic receptor was solved with the inverse agonist carazolol bound in the active site [10]. The structure of this first ligand-activated GPCR confirmed that the overall seven-transmembrane helix architecture is similar to that of rhodopsin. Together the rhodopsin and β2-adrenergic receptor structures provide a high-resolution framework for understanding the structural features that are responsible for locking these receptors in the off conformation, and a starting point for investigating the mechanism of receptor activation. Fig. 1 Crystal structure of rhodopsin. The first crystal structure of a GPCR was obtained in 2000 by Palczewski and Okada [8]. Views of rhodopsin spanning the membrane bilayer (a) and from the extracellular (or intradiscal) surface of the membrane (b) show the ... In this review, we describe the progress that has been made using NMR spectroscopy to characterize the structure and dynamics of GPCRs in membrane environments. NMR methods have been used extensively to establish the structural changes occurring upon the activation of rhodopsin, as well as several ligand-activated receptors. We describe below the importance of these receptors in terms of their cell biology and pharmacology, and then outline the role that NMR can play in answering questions of structure and function. 1.1 Cell biology The 7-transmembrane helix GPCRs have evolved to recognize and transduce signals as diverse as light, Ca2+, small organic molecules and proteins. These receptors are found in both vertebrates and invertebrates, and are typically divided into six classes (Class A–F) based on sequence homology and functional similarity [11–13]. However, the classification is still open to debate. For example, on the basis of phylogeny, the human GPCRs have been divided into five families (Rhodopsin-like, Secretin, Adhesion, Glutamate, and Frizzled/Taste2) [14]. In this scheme, the Class A receptors correspond to the Rhodopsin-like family, but the Class B receptors are divided into the Secretin and Adhesion families. Nevertheless, in all classification schemes proposed to date, the lack of homology between classes or families suggests that nature has converged on the same seven transmembrane helix framework multiple times. The Class A (Rhodopsin-like family) receptors respond to the presence of diverse stimuli ranging from light absorption to the binding of various ligands, which include small molecule amines and hormones. Class B (Secretin and Adhesion families) receptors are activated by peptides of the glucagon hormone family [15,16]. The Class C (Glutamate family) GPCRs are comprised of the metabotropic glutamate receptors. These receptors are characterized by a large N-terminal ligand binding domain [17], which appears to be structurally homologous to the amino terminal domain of the ligand-gated ionotropic glutamate receptors in postsynaptic neuronal membranes [18]. Pheromones (e.g. α-factor) secreted by Saccharomyces cerevisiae bind to Class D GPCRs (e.g. STE2) during the mating process. Similar mechanisms are involved in the mating of several fungi [19]. Class E receptors have been implicated in the chemotactic migration of slime mold and can potentially be exploited as antifungal targets [20,21]. Class F (Frizzled/smoothened/taste2 family) contains receptors in the Wnt signaling pathway [14], which perform indispensable roles in embryonic development [22]. The Class A receptors are by far the most populated class of GPCRs. In the GPCR database there are over 20,000 Class A sequences (http://www.gpcr.org/). In humans, 952 of 1061 GPCRs identified in the human genome are in Class A. Of the 952 human Class A receptors, most (509) are olfactory receptors. The remaining Class A GPCRs are subdivided into 18 subfamilies including the well studied visual and small molecule amine receptors, as well as hormone and peptide receptors. Despite the breadth of this group, there exists a degree of sequence conservation among these receptors. Furthermore, the Class A receptors share similar intracellular proteins (e.g. protein kinases, arrestins) that mediate receptor desensitization.
- Published
- 2010
49. Structural conversion of neurotoxic amyloid-β1–42 oligomers to fibrils
- Author
-
Steven O. Smith, Mahiuddin Ahmed, James I. Elliott, Shivani Ahuja, Takeshi Sato, Darryl Aucoin, William E. Van Nostrand, Judianne Davis, and Saburo Aimoto
- Subjects
Models, Molecular ,Cell Survival ,Amyloid beta ,Molecular Sequence Data ,Peptide ,macromolecular substances ,010402 general chemistry ,Fibril ,01 natural sciences ,Protein Structure, Secondary ,Article ,Turn (biochemistry) ,Mice ,03 medical and health sciences ,Protein structure ,Alzheimer Disease ,Structural Biology ,Animals ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Peptide sequence ,Cells, Cultured ,030304 developmental biology ,Neurons ,chemistry.chemical_classification ,0303 health sciences ,Amyloid beta-Peptides ,biology ,Chemistry ,Nuclear magnetic resonance spectroscopy ,Peptide Fragments ,0104 chemical sciences ,Cold Temperature ,Crystallography ,Polymerization ,biology.protein ,Salts ,Protein Multimerization - Abstract
The amyloid-beta(1-42) (Abeta42) peptide rapidly aggregates to form oligomers, protofibils and fibrils en route to the deposition of amyloid plaques associated with Alzheimer's disease. We show that low-temperature and low-salt conditions can stabilize disc-shaped oligomers (pentamers) that are substantially more toxic to mouse cortical neurons than protofibrils and fibrils. We find that these neurotoxic oligomers do not have the beta-sheet structure characteristic of fibrils. Rather, the oligomers are composed of loosely aggregated strands whose C termini are protected from solvent exchange and which have a turn conformation, placing Phe19 in contact with Leu34. On the basis of NMR spectroscopy, we show that the structural conversion of Abeta42 oligomers to fibrils involves the association of these loosely aggregated strands into beta-sheets whose individual beta-strands polymerize in a parallel, in-register orientation and are staggered at an intermonomer contact between Gln15 and Gly37.
- Published
- 2010
50. What is the role of amyloid precursor protein dimerization?
- Author
-
Jean-Noël Octave, Pascal Kienlen-Campard, Naouel Ben Khalifa, Sandra Huysseune, Stefan N. Constantinescu, Joanne Van Hees, Steven O. Smith, Bernadette Tasiaux, UCL - SSS/DDUV - Institut de Duve, UCL - SSS/IONS - Institute of NeuroScience, and UCL - SSS/IONS/CEMO - Pôle Cellulaire et moléculaire
- Subjects
chemistry.chemical_classification ,biology ,P3 peptide ,Peptide ,Cell Biology ,Special Focus: The Role of Transmembrane Domains in Adhesion ,Bioinformatics ,Cleavage (embryo) ,Models, Biological ,Transmembrane protein ,Cell biology ,Amyloid beta-Protein Precursor ,Cellular and Molecular Neuroscience ,Alpha secretase ,chemistry ,Alzheimer Disease ,mental disorders ,Amyloid precursor protein ,biology.protein ,Humans ,Animals ,Senile plaques ,Receptor ,Dimerization - Abstract
Extensive research efforts have been conducted over the past decades to understand the processing of the Amyloid Precursor Protein (APP). APP cleavage leads to the production of the beta-amyloid peptide (Abeta), which is the major constituent of the amyloid core of senile plaques found in the brains of patients with Alzheimer's disease (AD). Abeta is produced by the sequential cleavage of APP by beta- and gamma-secretases. Cleavage of APP by gamma-secretase also generates the APP Intracellular C-terminal Domain (AICD) peptide, which might be involved in regulation of gene transcription. Up to now, our understanding of the mechanisms controlling APP processing has been elusive. Recently, APP was found to form homo- or hetero-complexes with the APP-like proteins (APLPs), which belong to the same family and share some important structural properties with receptors having a single membrane spanning domain. Homodimerization of APP is driven by motifs present in the extracellular domain and possibly in the juxtamembrane and transmembrane (JM/TM) domains of the protein. These striking observations raise important questions about APP processing and function: How and where is APP dimerizing? What is the role of dimerization in APP processing and function? Can dimerization be targeted by small molecule therapeutics?
- Published
- 2010
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