Alan C. Rigby

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.

Pilot Study of Extracorporeal Removal of Soluble Fms-Like Tyrosine Kinase 1 in Preeclampsia

Background— Targeted therapies to stabilize the clinical manifestations and prolong pregnancy in preeclampsia do not exist. Soluble fms-like tyrosine kinase 1 (sFlt-1), an alternatively spliced variant of the vascular endothelial growth factor receptor 1, induces a preeclampsia-like phenotype in experimental models and circulates at elevated levels in human preeclampsia. Removing sFlt-1 may benefit women with very preterm (<32 weeks) preeclampsia. Methods and Results— We first show that negatively charged dextran sulfate cellulose columns adsorb sFlt-1 in vitro. In 5 women with very preterm preeclampsia and elevated circulating sFlt-1 levels, we next demonstrate that a single dextran sulfate cellulose apheresis treatment reduces circulating sFlt-1 levels in a dose-dependent fashion. Finally, we performed multiple apheresis treatments in 3 additional women with very preterm (gestational age at admission 28, 30, and 27+4 weeks) preeclampsia and elevated circulating sFlt-1 levels. Dextran sulfate apheresis lowered circulating sFlt-1, reduced proteinuria, and stabilized blood pressure without apparent adverse events to mother and fetus. Pregnancy lasted for 15 and 19 days in women treated twice and 23 days in a woman treated 4 times. In each, there was evidence of fetal growth. Conclusions— This pilot study supports the hypothesis that extracorporeal apheresis can lower circulating sFlt-1 in very preterm preeclampsia. Further studies are warranted to determine whether this intervention safely and effectively prolongs pregnancy and improves maternal and fetal outcomes in this setting.

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.

Successful Applications of Computer Aided Drug Discovery: Moving Drugs from Concept to the Clinic

Drug discovery and development is an interdisciplinary, expensive and time-consuming process. Scientific advancements during the past two decades have changed the way pharmaceutical research generate novel bioactive molecules. Advances in computational techniques and in parallel hardware support have enabled in silico methods, and in particular structure-based drug design method, to speed up new target selection through the identification of hits to the optimization of lead compounds in the drug discovery process. This review is focused on the clinical status of experimental drugs that were discovered and/or optimized using computer-aided drug design. We have provided a historical account detailing the development of 12 small molecules (Captopril, Dorzolamide, Saquinavir, Zanamivir, Oseltamivir, Aliskiren, Boceprevir, Nolatrexed, TMI-005, LY-517717, Rupintrivir and NVP-AUY922) that are in clinical trial or have become approved for therapeutic use.

Small molecule-induced cytosolic activation of protein kinase Akt rescues ischemia-elicited neuronal death

Elevating Akt activation is an obvious clinical strategy to prevent progressive neuronal death in neurological diseases. However, this endeavor has been hindered because of the lack of specific Akt activators. Here, from a cell-based high-throughput chemical genetic screening, we identified a small molecule SC79 that inhibits Akt membrane translocation, but paradoxically activates Akt in the cytosol. SC79 specifically binds to the PH domain of Akt. SC79-bound Akt adopts a conformation favorable for phosphorylation by upstream protein kinases. In a hippocampal neuronal culture system and a mouse model for ischemic stroke, the cytosolic activation of Akt by SC79 is sufficient to recapitulate the primary cellular function of Akt signaling, resulting in augmented neuronal survival. Thus, SC79 is a unique specific Akt activator that may be used to enhance Akt activity in various physiological and pathological conditions.

STAS Domain Structure and Function

Pendrin shares with nearly all SLC26/SulP anion transporters a carboxy-terminal cytoplasmic segment organized around a Sulfate Transporter and Anti-Sigma factor antagonist (STAS) domain. STAS domains of divergent amino acid sequence exhibit a conserved fold of 4 β strands interspersed among 5 α helices. The first STAS domain proteins studied were single-domain anti-sigma factor antagonists (anti-anti-σ). These anti-anti-σ indirectly stimulate bacterial RNA polymerase by inactivating inhibitory anti-σ kinases, liberating σ factors to direct specific transcription of target genes or operons. Some STAS domains are nucleotide-binding phosphoproteins or nucleotidases. Others are interaction/transduction modules within multidomain sensors of light, oxygen and other gasotransmitters, cyclic nucleotides, inositol phosphates, and G proteins. Additional multidomain STAS protein sequences suggest functions in sensing, metabolism, or transport of nutrients such as sugars, amino acids, lipids, anions, vitamins, or hydrocarbons. Still other multidomain STAS polypeptides include histidine and serine/threonine kinase domains and ligand-activated transcription factor domains. SulP/SLC26 STAS domains and adjacent sequences interact with other transporters, cytoskeletal scaffolds, and with enzymes metabolizing transported anion substrates, forming putative metabolons. STAS domains are central to membrane targeting of many SulP/SLC26 anion transporters, and STAS domain mutations are associated with at least three human recessive diseases. This review summarizes STAS domain structure and function.

A novel approach for assesing macromolecular complexes combining soft-docking calculations with NMR data

We present a novel and efficient approach for assessing protein–protein complex formation, which combines ab initio docking calculations performed with the protein docking algorithm BiGGER and chemical shift perturbation data collected with heteronuclear single quantum coherence (HSQC) or TROSY nuclear magnetic resonance (NMR) spectroscopy. This method, termed “restrained soft‐docking,” is validated for several known protein complexes. These data demonstrate that restrained soft‐docking extends the size limitations of NMR spectroscopy and provides an alternative method for investigating macromolecular protein complexes that requires less experimental time, effort, and resources. The potential utility of this novel NMR and simulated docking approach in current structural genomic initiatives is discussed.

The ω-Loop Region of the Human Prothrombin γ-Carboxyglutamic Acid Domain Penetrates Anionic Phospholipid Membranes

The hydrophobic ω-loop within the prothrombin γ-carboxyglutamic acid-rich (Gla) domain is important in membrane binding. The role of this region in membrane binding was investigated using a synthetic peptide, PT-(1–46)F4W, which includes the N-terminal 46 residues of human prothrombin with Phe-4 replaced by Trp providing a fluorescent probe. PT-(1–46)F4W and PT-(1–46) bind calcium ions and phospholipid membranes, and inhibit the prothrombinase complex. PT-(1–46)F4W, but not PT-(1–46), exhibits a blue shift (5 nm) and red-edge excitation shift (28 nm) in the presence of phosphatidylserine (PS)-containing vesicles, suggesting Trp-4 is located within the motionally restricted membrane interfacial region. PS-containing vesicles protect PT-(1–46)F4W, but not PT-(1–46), fluorescence from potassium iodide-induced quenching. Stern-Volmer analysis of the quenching of PT-(1–46)F4W in the presence and absence of 80% phosphatidylcholine/20% PS vesicles suggested that Trp-4 is positioned within the membrane and protected from aqueous quenching agents whereas Trp-41 remains solvent-accessible in the presence of PS-containing vesicles. Fluorescence quenching of membrane-bound PT-(1–46)F4W is optimal with 7- and 10-doxyl-labeled lipids, indicating that Trp-4 is inserted 5 to 7 Å into the bilayer. This report demonstrates that the ω-loop region of prothrombin specifically interacts with PS-containing membranes within the interfacial membrane region.

Probing the Interaction between the Coiled Coil Leucine Zipper of cGMP-dependent Protein Kinase Iα and the C Terminus of the Myosin Binding Subunit of the Myosin Light Chain Phosphatase

Nitric oxide and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase I (PKG-Iα)-mediated activation of myosin phosphatase (MLCP). Mechanistically it has been proposed that protein-protein interactions between the N-terminal leucine zipper (LZ) domain of PKG-Iα ((PKG-Iα1-59) and the LZ and/or coiled coil (CC) domain of the myosin binding subunit (MBS) of MLCP are localized in the C terminus of MBS. Although recent studies have supported these interactions, the critical amino acids responsible for these interactions have not been identified. Here we present structural and biophysical data identifying that the LZ domain of PKG-Iα1-59 interacts with a well defined 42-residue CC motif (MBSct42) within the C terminus of MBS. Using glutathione S-transferase pulldown experiments, chemical cross-linking, size exclusion chromatography, circular dichroism, and isothermal titration calorimetry we identified a weak dimer-dimer interaction between PKG-Iα1-59 and this C-terminal CC domain of MBS. The Kd of this non-covalent complex is 178.0 ± 1.5 μm. Furthermore our 1H-15N heteronuclear single quantum correlation NMR data illustrate that this interaction is mediated by several PKG-Iα residues that are on the a, d, e, and g hydrophobic and electrostatic interface of the C-terminal heptad layers 2, 4, and 5 of PKG-Iα. Taken together these data support a role for the LZ domain of PKG-Iα and the CC domain of MBS in this requisite contractile complex.