Marianne A. Grant

On the nucleation of amyloid β‐protein monomer folding

Neurotoxic assemblies of the amyloid β‐protein (Aβ) have been linked strongly to the pathogenesis of Alzheimers disease (AD). Here, we sought to monitor the earliest step in Aβ assembly, the creation of a folding nucleus, from which oligomeric and fibrillar assemblies emanate. To do so, limited proteolysis/mass spectrometry was used to identify protease‐resistant segments within monomeric Aβ(1–40) and Aβ(1–42). The results revealed a 10‐residue, protease‐resistant segment, Ala21–Ala30, in both peptides. Remarkably, the homologous decapeptide, Aβ(21–30), displayed identical protease resistance, making it amenable to detailed structural study using solution‐state NMR. Structure calculations revealed a turn formed by residues Val24–Lys28. Three factors contribute to the stability of the turn, the intrinsic propensities of the Val‐Gly‐Ser‐Asn and Gly‐Ser‐Asn‐Lys sequences to form a β‐turn, long‐range Coulombic interactions between Lys28 and either Glu22 or Asp23, and hydrophobic interaction between the isopropyl and butyl side chains of Val24 and Lys28, respectively. We postulate that turn formation within the Val24–Lys28 region of Aβ nucleates the intramolecular folding of Aβ monomer, and from this step, subsequent assembly proceeds. This model provides a mechanistic basis for the pathologic effects of amino acid substitutions at Glu22 and Asp23 that are linked to familial forms of AD or cerebral amyloid angiopathy. Our studies also revealed that common C‐terminal peptide segments within Aβ(1–40) and Aβ(1–42) have distinct structures, an observation of relevance for understanding the strong disease association of increased Aβ(1–42) production. Our results suggest that therapeutic approaches targeting the Val24–Lys28 turn or the Aβ(1–42)‐specific C‐terminal fold may hold promise.

Structural basis of membrane binding by Gla domains of vitamin K-dependent proteins.

In a calcium-dependent interaction critical for blood coagulation, vitamin K–dependent blood coagulation proteins bind cell membranes containing phosphatidylserine via γ-carboxyglutamic acid–rich (Gla) domains. Gla domain–mediated protein-membrane interaction is required for generation of thrombin, the terminal enzyme in the coagulation cascade, on a physiologic time scale. We determined by X-ray crystallography and NMR spectroscopy the lysophosphatidylserine-binding site in the bovine prothrombin Gla domain. The serine head group binds Gla domain–bound calcium ions and Gla residues 17 and 21, fixed elements of the Gla domain fold, predicting the structural basis for phosphatidylserine specificity among Gla domains. Gla domains provide a unique mechanism for protein-phospholipid membrane interaction. Increasingly Gla domains are being identified in proteins unrelated to blood coagulation. Thus, this membrane-binding mechanism may be important in other physiologic processes.

Familial Alzheimer's disease mutations alter the stability of the amyloid β-protein monomer folding nucleus

Amyloid β-protein (Aβ) oligomers may be the proximate neurotoxins in Alzheimers disease (AD). Recently, to elucidate the oligomerization pathway, we studied Aβ monomer folding and identified a decapeptide segment of Aβ, 21Ala–22Glu–23Asp–24Val–25Gly–26Ser–27Asn–28Lys–29Gly–30Ala, within which turn formation appears to nucleate monomer folding. The turn is stabilized by hydrophobic interactions between Val-24 and Lys-28 and by long-range electrostatic interactions between Lys-28 and either Glu-22 or Asp-23. We hypothesized that turn destabilization might explain the effects of amino acid substitutions at Glu-22 and Asp-23 that cause familial forms of AD and cerebral amyloid angiopathy. To test this hypothesis, limited proteolysis, mass spectrometry, and solution-state NMR spectroscopy were used here to determine and compare the structure and stability of the Aβ(21–30) turn within wild-type Aβ and seven clinically relevant homologues. In addition, we determined the relative differences in folding free energies (ΔΔGf) among the mutant peptides. We observed that all of the disease-associated amino acid substitutions at Glu-22 or Asp-23 destabilized the turn and that the magnitude of the destabilization correlated with oligomerization propensity. The Ala21Gly (Flemish) substitution, outside the turn proper (Glu-22–Lys-28), displayed a stability similar to that of the wild-type peptide. The implications of these findings for understanding Aβ monomer folding and disease causation are discussed.

Protein S: a conduit between anticoagulation and inflammation.

Objective:To review the multifaceted roles of the anticoagulant protein S, facilitating a better comprehension of this protein’s role in anticoagulation and inflammation pathways and the crosstalk between these pathologic states. Data Sources and Study Selection:Original research and review articles published in English pertaining to protein S, sourced from PubMed, during the last 30 yrs. Data Extraction and Synthesis:The protein C anticoagulant pathway is an essential mechanism for attenuating thrombin generation by the membrane-bound procoagulant complexes, tenase and prothrombinase. Protein S is a nonenzymatic protein. In the absence of activated protein C, it demonstrates anticoagulant activity; in the presence of activated protein C, it functions as a cofactor for activated protein C– dependent proteolytic inactivation of the coagulation cofactors factor Va and factor VIIIa. However, in plasma, these anticoagulant activities are limited by the concentration of free protein S (~40% of the total protein S plasma concentration). The remaining protein S (~60%) is found in a high-affinity, calcium-stabilized complex with C4b-binding protein, which renders this fraction devoid of anticoagulant function. Several recent investigations have attributed novel activated protein C–independent functions of protein S to the association of protein S with C4b-binding protein, thus establishing the importance of this fraction of plasma protein S. Conclusions:Together, these data support a role for protein S in both anticoagulation and inflammation, facilitating a better understanding of the need for both free and C4b-binding protein–bound protein S. Although these physiologic roles are truly dichotomous in terms of functional end point, mechanistically, both involve high-affinity membrane binding to phosphatidylserine-bearing surfaces. This binding is mediated by the n-terminal γ-carboxyglutamic acid-rich domain of this protein.

FoxO1-Mediated Activation of Akt Plays a Critical Role in Vascular Homeostasis

Rationale: Forkhead box-O transcription factors (FOXOs) transduce a wide range of extracellular signals, resulting in changes in cell survival, cell cycle progression, and several cell type-specific responses. FOXO1 is expressed in many cell types, including endothelial cells (ECs). Previous studies have shown that Foxo1 knockout in mice results in embryonic lethality at E11 because of impaired vascular development. In contrast, somatic deletion of Foxo1 is associated with hyperproliferation of ECs. Thus, the precise role of FOXO1 in the endothelium remains enigmatic. Objective: To determine the effect of endothelial-specific knockout and overexpression of FOXO1 on vascular homeostasis. Methods and Results: We show that EC-specific disruption of Foxo1 in mice phenocopies the full knockout. Although endothelial expression of FOXO1 rescued otherwise Foxo1-null animals, overexpression of constitutively active FOXO1 resulted in increased EC size, occlusion of capillaries, elevated peripheral resistance, heart failure, and death. Knockdown of FOXO1 in ECs resulted in marked inhibition of basal and vascular endothelial growth factor–induced Akt-mammalian target of rapamycin complex 1 (mTORC1) signaling. Conclusions: Our findings suggest that in mice, endothelial expression of FOXO1 is both necessary and sufficient for embryonic development. Moreover, FOXO1-mediated feedback activation of Akt maintains growth factor responsive Akt/mTORC1 activity within a homeostatic range.

Netropsin improves survival from endotoxaemia by disrupting HMGA1 binding to the NOS2 promoter.

The inducible form of nitric oxide synthase (NOS2) plays an important role in sepsis incurred as a result of infection with Gram-negative bacteria that elaborate endotoxin. The HMGA1 (high-mobility group A1) architectural transcription factor facilitates NOS2 induction by binding a specific AT-rich Oct (octamer) sequence in the core NOS2 promoter via AT-hook motifs. The small-molecule MGB (minor-groove binder) netropsin selectively targets AT-rich DNA sequences and can interfere with transcription factor binding. We therefore hypothesized that netropsin would improve survival from murine endotoxaemia by attenuating NOS2 induction through interference with HMGA1 DNA binding to the core NOS2 promoter. Netropsin improved survival from endotoxaemia in wild-type mice, yet not in NOS2-deficient mice, supporting an important role for NOS2 in the beneficial effects of MGB administration. Netropsin significantly attenuated NOS2 promoter activity in macrophage transient transfection studies and the AT-rich HMGA1 DNA-binding site was critical for this effect. EMSAs (electrophoretic mobility-shift assays) demonstrated that netropsin interferes with HMGA1 NOS2 promoter binding and NMR spectroscopy was undertaken to characterize this disruption. Chemical shift perturbation analysis identified that netropsin effectively competes both HMGA1 DNA-binding AT-hooks from the AT-rich NOS2 promoter sequence. Furthermore, NOESY data identified direct molecular interactions between netropsin and A/T base pairs within the NOS2 promoter HMGA1-binding site. Finally, we determined a structure of the netropsin/NOS2 promoter Oct site complex from molecular modelling and dynamics calculations. These findings represent important steps toward refined structure-based ligand design of novel compounds for therapeutic benefit that can selectively target key regulatory regions within genes that are important for the development of critical illness.

Integrating computational protein function prediction into drug discovery initiatives

Pharmaceutical researchers must evaluate vast numbers of protein sequences and formulate innovative strategies to identify valid targets and discover leads against them in order to accelerate drug discovery. The ever‐increasing number and diversity of novel protein sequences identified by genomic sequencing projects and the success of worldwide structural genomics initiatives have spurred great interest and impetus in the development of methods for accurate, computationally empowered protein function prediction and active site identification. Previously, in the absence of direct experimental evidence, homology‐based protein function annotation remained the gold standard for in silico analysis and prediction of protein function. However, with the continued exponential expansion of sequence databases, this approach is not always applicable, as fewer query protein sequences demonstrate significant homology to protein gene products of known function. As a result, several non‐homology‐based methods for protein function prediction that are based on sequence features, structure, evolution, biochemical, and genetic knowledge have emerged. This works reviews current bioinformatic programs and approaches for protein function prediction/annotation and discusses their integration into drug discovery initiatives. The development of such methods to annotate protein functional sites and their application to large protein functional families is crucial to successfully using the vast amounts of genomic sequence information available to drug discovery and development processes. Drug Dev Res 72: 4–16, 2011.

Conotoxin therapeutics: a pipeline for success?

The pharmacopoeia of conotoxins from the marine snail Conus has evolved with time, providing a myriad of molecular scaffolds on which critical, molecular pharmacophoric descriptors, responsible for mediating conotoxin receptor–target specificity and selectivity have been grafted. Several reports have defined how these critical determinants contribute to refined, subtype-selective receptor recognition. However, the clinical utility of conotoxins is debatable with a single conotoxin, ω-MVIIA (ziconotide), approved by the US FDA. The authors review the present status of conotoxin-based drug discovery efforts, highlighting ongoing preclinical and clinical studies, while discussing strategies that may be necessary to overcome the barriers inherent to peptide therapeutics. Through the beauty of nature and the art of design it should be possible to expand the Conus pipeline.

Crystallization and preliminary X-ray analysis of a complex of the FOXO1 and Ets1 DNA-binding domains and DNA

The Ets1 transcription factor is a member of the Ets protein family, a group of evolutionarily related DNA-binding transcriptional factors. Ets proteins activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. FOXO1 is a member of the forkhead-box proteins (FOX proteins), which comprise a large family of functionally diverse transcription factors involved in cellular proliferation, transformation and differentiation. The FOXO subgroup of FOX proteins regulates the transcription of genes that control metabolism, cell survival, cellular proliferation, DNA damage responses, stress resistance and longevity. The DNA-binding domains (DBDs) of Ets1 and FOXO1 were crystallized in complex with DNA containing a composite sequence for a noncanonical forkhead binding site (AATAACA) and an ETS site (GGAA), FOX:ETS, by the sitting-drop vapor-diffusion method. The FOX:ETS motif has been shown to be a conserved cis-acting element in several endothelial cell-specific genes, including Vegfr2, Tie2, Mef2c and ve-cadherin. Crystals were grown at 291 K using 30% polyethylene glycol 400, 50 mM Tris pH 8.5, 100 mM KCl, 10 mM MgCl2 as the reservoir solution. The crystals belonged to space group C222(1), with unit-cell parameters a = 68.7, b = 104.9, c = 136.3 Å. Diffraction data were collected to a resolution of 2.2 Å.

Evolutionary conservation of the allosteric activation of factor VIIa by tissue factor in lamprey

Essentials Tissue factor (TF) enhances factor VIIa (FVIIa) activity through structural and dynamic changes. We analyzed conservation of TF‐activated FVIIa allosteric networks in extant vertebrate lamprey. Lamprey Tf/FVIIa molecular dynamics show conserved Tf‐induced structural/dynamic FVIIa changes. Lamprey Tf activation of FVIIa allosteric networks follows molecular pathways similar to human.