Role of aromatic interactions in amyloid formation by peptides derived from human Amylin

Numerous polypeptides and proteins form amyloid deposits in vivo or in vitro. The mechanism of amyloid formation is not well-understood particularly in the case where unstructured polypeptides assemble to form amyloid. Aromatic-aromatic interactions are known to be important in globular proteins, and the possibility that they might play a key role in amyloid formation has been raised. The results of Ala-scanning experiments on short polypeptides derived from Amylin have suggested that aromatic interactions could be particularly important for this system. Here, we examine a set of Amylin-derived polypeptides in which the single aromatic residue has been substituted with a Leu and Ala. A peptide corresponding to residues 21-29 with a Phe-23 to Leu substitution, a free N terminus, and amidated C terminus readily forms amyloid. Shorter peptides derived from the putative minimal amyloid-forming segment of Amylin, residues 22-27, also form amyloid when Phe-23 is replaced by Leu. Amyloid formation is more facile when the N terminus is deprotonated and the peptide is uncharged. Substitution of the Phe with Ala results in a peptide that is noticeably less prone to form amyloid. A peptide corresponding to residues 10-19 of human Amylin with blocked termini and the sole aromatic residue, Phe-15, substituted by Leu readily forms amyloid. A Phe-15 to Ala substitution reduces significantly the ability to form amyloid. These results indicate that an aromatic residue is not required for amyloid formation in these systems and indicates that other factors such as size, beta-sheet propensity, and hydrophobicity of the side chain in question are also important.     

Amyloid formation by pro-islet amyloid polypeptide processing intermediates: examination of the role of protein heparan sulfate interactions and implications for islet amyloid formation in type 2 diabetes

Amyloid formation has been implicated in a wide range of human diseases including Alzheimer's disease, Parkinson's disease, and type 2 diabetes. In type 2 diabetes, islet amyloid polypeptide (IAPP, also known as amylin) forms cytotoxic amyloid deposits in the pancreas, and these are believed to contribute to the pathology of the disease. The mechanism of islet amyloid formation is not understood; however, recent proposals have invoked a role for incompletely processed proIAPP. In this model, incompletely processed proIAPP containing the N-terminal pro region is excreted and binds to heparan sulfate proteoglycans (HSPGs) of the basement membrane thereby establishing a high local concentration which can act as a seed for amyloid formation. Here we report biophysical proof-of-principle experiments designed to test the viability of this model. The model predicts that interactions with HSPGs should accelerate amyloid formation by the proIAPP processing intermediate, and this is indeed what is observed. Interaction with heparan sulfate leads to the rapid formation of an intermediate state with partial helical content which then converts, on a slower time scale, to amyloid fibrils. TEM shows that fibrils formed by the proIAPP processing intermediate in the presence and in the absence of heparan sulfate have the classic features of amyloid. Fibrils formed by the proIAPP processing intermediate are competent to seed amyloid formation by mature IAPP. The seeding experiments support a second major premise of the model, namely, that fibrils formed by the processing intermediate are capable of seeding amyloid formation by the mature peptide.     

Aromatic interactions are not required for amyloid fibril formation by islet amyloid polypeptide but do influence the rate of fibril formation and fibril morphology

Amyloid formation has been implicated in a wide range of human diseases, and a diverse set of proteins is involved. There is considerable interest in elucidating the interactions which lead to amyloid formation and which contribute to amyloid fibril stability. Recent attention has been focused upon the potential role of aromatic-aromatic and aromatic-hydrophobic interactions in amyloid formation by short to midsized polypeptides. Here we examine whether aromatic residues are necessary for amyloid formation by islet amyloid polypeptide (IAPP). IAPP is responsible for the formation of islet amyloid in type II diabetes which is thought to play a role in the pathology of the disease. IAPP is 37 residues in length and contains three aromatic residues, Phe-15, Phe-23, and Tyr-37. Structural models of IAPP amyloid fibrils postulate that Tyr-37 is near one of the phenylalanine residues, and it is known that Tyr-37 interacts with one of the phenylalanines during fibrillization; however, it is not known if aromatic-aromatic or aromatic-hydrophobic interactions are absolutely required for amyloid formation. An F15L/F23L/Y37L triple mutant (IAPP-3XL) was prepared, and its ability to form amyloid was tested. CD, thioflavin binding assays, AFM, and TEM measurements all show that the triple leucine mutant readily forms amyloid fibrils. The substitutions do, however, decrease the rate of fibril formation and alter the tendency of fibrils to aggregate. Thus, while aromatic residues are not an absolute requirement for amyloid formation by IAPP, they do play a role in the fibril assembly process.     

Detection of different adenosine triphosphatases in human placental brush border membranes

The microvillous membrane of human placental syncytiotrophoblast cells contains a high ATPase activity. The purpose of this study was to characterize this activity and to investigate the presence of vacuolar type H⁺ ATPase in this membrane. Intact brush border membrane vesicles strongly hydrolyzed ATP, reflecting the presence of ATPase on the external side of the membrane. The ATPase activity was entirely Mg²⁺ dependent and increased with pH. At pH 7.5, Vmax was 31.0 ± 1.7 mol/mg/20 min and Km 0.18 ± 0.03 mm ATP. Hydrolysis of ATP was not influenced by the presence of bicarbonate or alkaline phosphatase inhibitors, but at pH 8 it decreased by half following addition of 100 m dicyclohexylcarbodiimide (DCCD). At pH 7.5, 1 mm N-ethylmaleimide (NEM) depressed this activity by less than 5%. Opening the membrane vesicles with 0.1% desoxycholate (DOC) or Triton-X neither revealed any additional ATPase activity nor altered the low sensitivity to NEM. Treatment of these membranes with 1% cholate decreased the ATPase activity by more than 70% and did not enhance the sensitivity of ATP hydrolysis to NEM. 10^–7 m Bafilomycin, which reduced by 56 ± 9% the ATPase activity in dog kidney brash border membranes treated with 0.1% DOC, had no effect on placental brush border membranes subjected to the same procedure. Finally, neither immunocytochemical staining using monoclonal antibody to the Mr 31000 subunit of V-type H⁺ ATPase, nor electron microscopic examination detected the presence of H⁺ ATPase in placental membranes.In conclusion, the placental brush border membrane is the site of a strong ecto ATPase activity which is partially DCCD sensitive. However, our results did not detect the presence of any vacuolar type H⁺ ATPase activity in these membranes.This study was supported by the MRC grant N MA 9565 and by an extramural grant from Baxter Health Care Corporation. The authors are indepted to Dr. Patrick Vinay for this help in providing Bafilomycin and dog kidney membranes, as well as for his insightful discussion.     

The role of His-18 in amyloid formation by human islet amyloid polypeptide

The 37-residue islet amyloid polypeptide (IAPP) is the major protein component of the amyloid deposits found in type-II diabetes. IAPP is stored in a relatively low pH environment in the pancreatic secretory granules prior to its release to the extracellular environment. Human IAPP contains a single histidine at position 18. Aggregation of IAPP is considerably faster at a lower pH (4.0 +/- 0.3) than at high pH (8.8 +/- 0.3), as judged by turbidity and thioflavine-T fluorescence studies. The rate of aggregation at low pH increases drastically in the presence of salt. CD experiments show that the conversion of largely unstructured monomers to beta-sheet-rich structures is faster at high pH. TEM studies show that fibrils are formed at both pH values but are more prevalent at pH 8.8 (+/-0.3). Both the free N terminus of IAPP and His-18 will titrate over the pH range studied. An N-terminal acetylated fragment consisting of residues 8-37 of human IAPP was also studied to isolate contributions from the protonation of His-18. Previous studies have shown that this fragment forms protofibrils that are very similar to those formed by intact IAPP. The effects of varying the protonation state of His-18 in the 8-37 analogue indicate that the rate of aggregation and fibril formation is noticeably faster when His-18 is deprotonated, similar to the wild type. However, the pH-dependent effects are larger for full-length IAPP than for the disulfide-truncated, acetylated analogue. TEM studies indicate differences in the morphology of the deposits formed at high and low pH. These results are discussed in light of recent structural models of IAPP fibrils.     

A dynamically growing self-organizing tree (DGSOT) for hierarchical clustering gene expression profiles

MOTIVATION: The increasing use of microarray technologies is generating large amounts of data that must be processed in order to extract useful and rational fundamental patterns of gene expression. Hierarchical clustering technology is one method used to analyze gene expression data, but traditional hierarchical clustering algorithms suffer from several drawbacks (e.g. fixed topology structure; mis-clustered data which cannot be reevaluated). In this paper, we introduce a new hierarchical clustering algorithm that overcomes some of these drawbacks. RESULT: We propose a new tree-structure self-organizing neural network, called dynamically growing self-organizing tree (DGSOT) algorithm for hierarchical clustering. The DGSOT constructs a hierarchy from top to bottom by division. At each hierarchical level, the DGSOT optimizes the number of clusters, from which the proper hierarchical structure of the underlying dataset can be found. In addition, we propose a new cluster validation criterion based on the geometric property of the Voronoi partition of the dataset in order to find the proper number of clusters at each hierarchical level. This criterion uses the Minimum Spanning Tree (MST) concept of graph theory and is computationally inexpensive for large datasets. A K-level up distribution (KLD) mechanism, which increases the scope of data distribution in the hierarchy construction, was used to improve the clustering accuracy. The KLD mechanism allows the data misclustered in the early stages to be reevaluated at a later stage and increases the accuracy of the final clustering result. The clustering result of the DGSOT is easily displayed as a dendrogram for visualization. Based on a yeast cell cycle microarray expression dataset, we found that our algorithm extracts gene expression patterns at different levels. Furthermore, the biological functionality enrichment in the clusters is considerably high and the hierarchical structure of the clusters is more reasonable. AVAILABILITY: DGSOT is available upon request from the authors.     

A Machine Learned Classifier That Uses Gene Expression Data to Accurately Predict Estrogen Receptor Status

Background

Selecting the appropriate treatment for breast cancer requires accurately determining the estrogen receptor (ER) status of the tumor. However, the standard for determining this status, immunohistochemical analysis of formalin-fixed paraffin embedded samples, suffers from numerous technical and reproducibility issues. Assessment of ER-status based on RNA expression can provide more objective, quantitative and reproducible test results.

Methods

To learn a parsimonious RNA-based classifier of hormone receptor status, we applied a machine learning tool to a training dataset of gene expression microarray data obtained from 176 frozen breast tumors, whose ER-status was determined by applying ASCO-CAP guidelines to standardized immunohistochemical testing of formalin fixed tumor.

Results

This produced a three-gene classifier that can predict the ER-status of a novel tumor, with a cross-validation accuracy of 93.17±2.44%. When applied to an independent validation set and to four other public databases, some on different platforms, this classifier obtained over 90% accuracy in each. In addition, we found that this prediction rule separated the patients'' recurrence-free survival curves with a hazard ratio lower than the one based on the IHC analysis of ER-status.

Conclusions

Our efficient and parsimonious classifier lends itself to high throughput, highly accurate and low-cost RNA-based assessments of ER-status, suitable for routine high-throughput clinical use. This analytic method provides a proof-of-principle that may be applicable to developing effective RNA-based tests for other biomarkers and conditions.     

Current approaches in identification and isolation of cancer stem cells

Cancer stem cells (CSCs) are tumor cells with initiating ability, self-renewal potential, and intrinsic resistance to conventional therapeutics. Efficient isolation and characterization of CSCs pave the way for more comprehensive knowledge about tumorigenesis, heterogeneity, and chemoresistance. Also a better understanding of CSCs will lead to novel era of both basic and clinical cancer research, reclassification of human tumors, and development of innovative therapeutic strategies. Finding novel diagnostic and effective therapeutic strategies also enhance the success of treatment in cancer patients. There are various methods based on the characteristics of the CSCs to detect and isolate these cells, some of which have recently developed. This review summarized current techniques for effective isolation and characterization of CSCs with a focus on advantages and limitations of each method with clinical applications.     

Aluminum alleviates boron-deficiency induced growth impairment in tea plants

Interaction between aluminum (Al) and boron (B) in Al accumulator species has not been characterized so far. In this work, tea [Camellia sinensis (L.) O. Kuntze] plants were cultivated hydroponically and treated with adequate (control) or low B supply (-B) without or with 300 μM Al (-B+Al) for 14 weeks. Growth of B-deficient plants was completely resumed by Al supplementation or even surpassed control plants regarding shoot biomass. Net photosynthetic rate was negatively influenced by the low B supply, and the Al treatment increased it up to the level of the control plants that was reflected in the higher content of saccharides. The activity of ascorbate peroxidase (APX) in the younger leaves decreased at the low B supply accompanied with an increased H₂O₂ content. The Al treatment increased the APX activity up to the level of the control plants simultaneously with the reduction of H₂O₂. Activities of superoxide dismutase (SOD) and peroxidase (POD) increased in the low B plants and the Al treatment augmented this effect. The content of malondialdehyde (MDA) in the leaves increased by low B but declined upon the Al treatment. In the Al-treated plants, the activity of nitrate reductase (NR) and the content of free α-amino acids exceeded those of the control plants, and nitrite concentration diminished. The shoot and root B content of the B-deficient plants supplemented with Al was similar with the B-sufficient ones. The results demonstrate that the up-regulation of C and N metabolism, the activation of antioxidative defense, and the enhancement of B uptake and transport were mechanisms for growth amelioration of the B-deficient plants by Al supplementation in tea.     

Characterization of the heparin binding site in the N-terminus of human pro-islet amyloid polypeptide: implications for amyloid formation

Islet amyloid deposits are a characteristic pathological hallmark of type 2 diabetes mellitus. Islet amyloid polypeptide (IAPP), also referred to as amylin, aggregates in the islet extracellular space to form amyloid deposits in up to 95% of patients with the disease. IAPP is stored with insulin in beta-islet cells and is processed in parallel by subtilisin-like prohormone convertases prior to secretion. There is indirect evidence that normal processing of the prohormone precursor, proIAPP, at the N-terminal cleavage site is defective in type 2 diabetes and results in secretion of an N-terminal extended proIAPP intermediate. The N-terminal flanking region of proIAPP is detected in amyloid deposits; however, the C-terminal flanking region is not. Immunohistochemical studies implicate the presence of the heparan sulfate proteoglycan (HSPG) perlecan in islet amyloid deposits, suggesting a role for HSPGs in mediating amyloid deposition in type 2 diabetes and implicating a binding domain in the N-terminus of proIAPP. Initial studies of proIAPP indicated that the HSPG binding region is contained within the first 30 residues. Here, we characterize the potential HSPG binding site of proIAPP in detail by analyzing a set of peptide fragments. Binding is tighter at low pH due to protonation of histidine residues. Deletion studies show that Arg-22 and His-29 play a role in binding. Reduction of the Cys-13 to Cys-18 disulfide leads to a noticeable decrease in binding. We demonstrate the ability of heparan sulfate to induce amyloid formation in N-terminal fragments of proIAPP. The oxidized peptide forms amyloid more rapidly than the reduced variant in the presence of heparan sulfate, but the reduced peptide ultimately forms more extensive amyloid deposits. The potential implications for islet amyloid formation in vivo are discussed.