Spectrofluorimetric analysis of the interaction of amyloid peptides with neuronal nitric oxide synthase: Implications in Alzheimer's disease

Background

The deposition of aggregated β-amyloid peptide senile plaques and the accumulation of arginine within the astrocytes in the brain of an Alzheimer's patient are classic observations in the neuropathology of the disease. It would be logical, in the aetiology and pathogenesis, to investigate arginine-metabolising enzymes and their intimate association with amyloid peptides.

Methods

Neuronal nitric oxide synthase (nNOS) was isolated, purified and shown, through fluorescence quenching spectroscopy and fluorescence resonance energy transfer (FRET), to interact with structural fragments of Aβ_1–40 and be catalytic towards amyloid fibril formation.

Results

Only one binding site on the enzyme was available for binding. Two amyloid peptide fragments of Aβ_1–40 (Aβ_17–28 and Aβ_25–35) had Stern–Volmer values (K_SV) of 0.111 μM⁻¹ and 0.135 μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. The polarity of this active site precludes binding of the predominantly hydrophobic amyloid peptide fragments contained within Aβ_17–28 and within two glycine zipper motifs [G-X-X-X-G-X-X-X-G] [Aβ_29–37] and bind to the enzyme at a site remote to the active region.

Conclusions

The interaction and binding of Aβ_17–28 and Aβ_25–35 to nNOS causes the movement of two critical tryptophan residues of 0.77 nm and 0.57 nm respectively towards the surface of the enzyme.

General significance

The binding of Aβ-peptide fragments with nNOS has been studied by spectrofluorimetry. The information and data presented should contribute towards understanding the mechanism for deposition of aggregated Aβ-peptides and fibrillogenesis in senile plaques in an AD brain.     

Carbamoyl-PROXYL-enhanced MRI detects very small disruptions in brain vascular permeability induced by dietary cholesterol

Background

Gd-DTPA-enhanced magnetic resonance imaging (MRI) is a conventional method for non-invasive investigation of blood-brain-barrier (BBB) permeability in animal models. It allows the visualization of serious injury to the BBB. We developed a novel approach for detecting very small disruptions in BBB permeability induced by dietary cholesterol by using carbamoyl-PROXYL (CMP) as an MRI contrast probe.

Methods

Mice were separated into two groups: normal diet (ND-mice) and high cholesterol diet (CD-mice). MRI-signal dynamics, plasma cholesterol, matrix metalloproteinase (MMP-9, MMP-2), and the white blood cell profile were analyzed. For the MRI analysis, two regions-of-interest (ROI) were selected: brain (ROI-1) and surrounding area (ROI-2).

Results

In the ROI-2 of ND-mice, CMP- or Gd-enhanced MRI-signal followed typical kinetics with a half-life of signal decay (τ_1/2) ~ 8 or ~ 15 min, respectively. In CD-mice, the MRI-signal increased continuously without decay.In the ROI-1 of ND- and CD-mice, MRI-signal enhancement was not detected by Gd-DTPA. In the ROI-1 of ND-mice, CMP-induced MRI-signal enhancement was negligible, while in CD-mice, it was significant (τ_1/2 > 15 min).Hypercholesterolemia increased the plasma levels of MMP-9 and neutrophils.

Conclusions

Hypercholesterolemia increases vascular permeability, which is mediated by MMP-9 and neutrophils.

General significance

Even very small disruptions in brain vascular permeability could be detected by CMP-enhanced MRI but not by Gd-DTPA-enhanced MRI.     

正常人长时数字记忆信息提取的神经基础的功能磁共振研究

本文旨在通过功能磁共振成像(functional magnetic resonance imaging,fMRI)技术研究正常人进行长时数字记忆信息提取的神经基础。选取22名右利手志愿者进行长时数字记忆任务实验,采用组块设计,记忆任务与对照任务交替进行,同时利用Siemens 1.5T超导型磁共振仪进行fMRI成像,采用SPM99软件进行数据分析,脑功能区定位在Talairach坐标中显示。结果显示被试者在进行长时数字记忆提取任务时,激活最显著的皮层是左侧额中回(Brodmann分区9区,BA9区),另外左额叶内侧回、左额下回、右额下回、扣带回、左顶下小叶、左顶上小叶、右顶上小叶、右颞中回、左枕舌回、左枕中回、右中脑、小脑、右尾状核尾部等结构也有激活,各大脑皮层的激活均呈现明显的左侧半球优势。根据上述结果推论,长时数字记忆由以左侧大脑半球为优势的各脑区共同参与完成,其中左侧额叶外侧面可能是信息提取的重要结构,而其它脑叶及其之间的广泛联系可能在数字信息的加工、处理和存储中起重要作用。     

Intramolecular hydrogen bonding in articaine can be related to superior bone tissue penetration: a molecular dynamics study

Local anesthetics (LAs) are drugs that cause reversible loss of nociception during surgical procedures. Articaine is a commonly used LA in dentistry that has proven to be exceptionally effective in penetrating bone tissue and induce anesthesia on posterior teeth in maxilla and mandibula. In the present study, our aim was to gain a deeper understanding of the penetration of articaine through biological membranes by studying the interactions of articaine with a phospholipid membrane. Our approach involves Langmuir monolayer experiments combined with molecular dynamics simulations. Membrane permeability of LAs can be modulated by pH due to a titratable amine group with a pKa value close to physiological pH. A change in protonation state is thus known to act as a lipophilicity switch in LAs. Our study shows that articaine has an additional unique lipophilicity switch in its ability to form an intramolecular hydrogen bond. We suggest this intramolecular hydrogen bond as a novel and additional solvent-dependent mechanism for modulation of lipophilicity of articaine which may enhance its diffusion through membranes and connective tissue.     

Interactions of the DNA polymerase X from African Swine Fever Virus with the ssDNA. Properties of the total DNA-binding site and the strong DNA-binding subsite

Interactions of the polymerase X from the African Swine Fever Virus with the ssDNA have been studied, using quantitative fluorescence titration and fluorescence resonance energy transfer techniques. The primary DNA-binding subsite of the enzyme, independent of the DNA conformation, is located on the C-terminal domain. Association of the bound DNA with the catalytic N-terminal domain finalizes the engagement of the total DNA-binding site of the enzyme and induces a large topological change in the structure of the bound ssDNA. The free energy of binding includes a conformational transition of the protein. Large positive enthalpy changes accompanying the ASFV pol X-ssDNA association indicate that conformational changes of the complex are induced by the engagement of the N-terminal domain. The enthalpy changes are offset by large entropy changes accompanying the DNA binding to the C-terminal domain and the total DNA-binding site, predominantly resulting from the release of water molecules.     

Binding kinetics of calmodulin with target peptides of three nitric oxide synthase isozymes

Efficient electron transfer from reductase domain to oxygenase domain in nitric oxide synthase (NOS) is dependent on the binding of calmodulin (CaM). Rate constants for the binding of CaM to NOS target peptides was only determined previously by surface plasmon resonance (SPR) (Biochemistry 35, 8742-8747, 1996) suggesting that the binding of CaM to NOSs is slow and does not support the fast electron transfer in NOSs measured in previous and this studies. To resolve this contradiction, the binding rates of holo Alexa 350 labeled T34C/T110W CaM (Alexa-CaM) to target peptides from three NOS isozymes were determined using fluorescence stopped-flow. All three target peptides exhibited fast k_on constants at 4.5 °C: 6.6 × 10⁸ M^− 1 s^− 1 for nNOS_726-749, 2.9 × 10⁸ M^− 1 s^− 1 for eNOS_492-511 and 6.1 × 10⁸ M^− 1 s^− 1 for iNOS_507-531, 3-4 orders of magnitude faster than those determined previously by SPR. Dissociation rates of NOS target peptides from Alexa-CaM/peptide complexes were measured by Ca²⁺ chelation with ETDA: 3.7 s^− 1 for nNOS_726-749, 4.5 s^− 1 for eNOS_492-511, and 0.063 s^− 1 for iNOS_507-531. Our data suggest that the binding of CaM to NOS is fast and kinetically competent for efficient electron transfer and is unlikely rate-limiting in NOS catalysis. Only iNOS_507-531 was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM.     

EPR investigation of the role of B10 phenylalanine in neuroglobin - evidence that B10Phe mediates structural changes in the heme region upon disulfide-bridge formation

The function of neuroglobin, a member of the vertebrate globin family, is still unknown. In human neuroglobin (NGB), the formation of a disulfide bridge between the CysCD7 and CysD5 is known to affect the heme environment and its ligand-binding kinetics. Here, we show by means of EPR that the PheB10 residue plays a key role in transmitting the structural information from the disulfide bridge to the heme-pocket region. While formation of a disulfide bridge in ferric wild-type NGB leads to a considerable change of its EPR parameters, only minor changes are observed in the case of ferric PheB10Leu NGB. Furthermore, wild-type NGB is found to be much more stable in the presence of H₂O₂ than its PheB10Leu or its HisE7Leu mutants. While tyrosyl radicals are induced in HisE7Leu NGB by the addition of H₂O₂, this is not the case for wild-type and PheB10Leu NGB. The results will be discussed in terms of the protein's putative functions.     

Gastrins, iron homeostasis and colorectal cancer

The peptide hormone gastrin has been identified as a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. The importance of gastric acid in the absorption of dietary iron first became evident 50 years ago when iron deficiency anemia was recognized as a long-term consequence of partial gastrectomy. This review summarizes the connections between circulating gastrins, iron status and colorectal cancer. Gastrins bind two ferric ions with micromolar affinity and, in the case of non-amidated forms of the hormone, iron binding is essential for biological activity in vitro and in vivo. The demonstration of an interaction between gastrin and transferrin by biochemical techniques led to the proposal that gastrins catalyze the loading of transferrin with iron. Several lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron overload disease hemochromatosis and that transferrin saturation positively correlates with circulating gastrin concentration, suggest the potential involvement of gastrins in iron homeostasis. Conversely, recognition that ferric ions play an unexpected role in the biological activity of gastrins may assist in the development of useful therapies for colorectal carcinoma and other disorders of mucosal proliferation in the gastrointestinal tract.     

Role of somatostatin receptor 1 and 5 on epidermal growth factor receptor mediated signaling

Epidermal growth factor (EGF) regulates normal and tumor cell proliferation via epidermal growth factor receptor (EGFR) phosphorylation, homo- or heterodimerization and activation of mitogen-activated protein kinases (MAPKs) and PI3K/AKT cell survival pathways. In contrast, SST via activation of five different receptor subtypes inhibits cell proliferation and has been potential target in tumor treatment. To gain further insight for the effect of SSTRs on EGFR activated signaling, we determine the role of SSTR1 and SSTR1/5 in human embryonic kidney (HEK) 293 cells. We here demonstrate that cells transfected with SSTR1 or SSTR1/5 negatively regulates EGF mediated effects attributed to the inhibition of EGFR phosphorylation, MAPKs as well as the cell survival signaling. Furthermore, SSTR effects were significantly enhanced in cells when EGFR was knock down using siRNA or treated with selective antagonist (AG1478). Most importantly, the presence of SSTR in addition to modulating signaling pathways leads to the dissociation of the constitutive and EGF induced heteromeric complex of EGFR/ErbB2. Furthermore, cells cotransfected with SSTR1/5 display pronounced effect of SST on the signaling and dissociation of the EGFR/ErbB2 heteromeric complex than the cells expressing SSTR1 alone. Taken together this study provides the first evidence that the presence of SSTR controls EGF mediated cell survival pathway via dissociation of ErbB heteromeric complex. We propose that the activation of SSTR and blockade of EGFR might serve novel therapeutic approach in inhibition of tumor proliferation.     

Integrin binding human antibody constant domains--probing the C-terminal structural loops for grafting the RGD motif

Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL - a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.