Deconstruction of Neurotrypsin Reveals a Multi-factorially Regulated Activity Affecting Myotube Formation and Neuronal Excitability

Neurotrypsin (NT) is a highly specific nervous system multi-domain serine protease best known for its selective processing of the potent synaptic organizer agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. These biological effects are likely to stem from NT-based regulation of agrin signaling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, due to the scarce molecular detail regarding NT activity and NT-agrin interactions. We developed a strategy to reliably produce and purify a catalytically competent engineered variant of NT called "NT-mini" and a library of C-terminal agrin fragments, with which we performed a thorough biochemical and biophysical characterization of NT enzyme functionality. We studied the regulatory effects of calcium ions and heparin, identified NT's heparin-binding domain, and discovered how zinc ions induce modulation of enzymatic activity. Additionally, we investigated myotube differentiation and hippocampal neuron excitability, evidencing a dose-dependent increase in neuronal activity alongside a negative impact on myoblast fusion when using the active NT enzyme. Collectively, our results provide in vitro and cellular foundations to unravel the molecular underpinnings and biological significance of NT-agrin interactions

Signalling strategies for osteogenic differentiation of human umbilical cord mesenchymal stromal cells for 3D bone tissue engineering

Human umbilical cord mesenchymal stromal cells (hUCMSCs) have recently shown the capacity to differentiate into multiple cell lineages in all three embryonic germ layers. The osteogenic differentiation of hUCMSCs in monolayer culture has been reported, while the differentiation in three-dimensional biomaterials has not yet been reported for tissue-engineering applications. Thus, the aim of this study was to evaluate the feasibility of using hUCMSCs for bone tissue engineering. hUCMSCs were cultured in poly( L -lactic acid) (PLLA) scaffolds in osteogenic medium (OM) for 3 weeks, after which the scaffolds were exposed to several different media, including the OM, a mineralization medium (MM) and the MM with either 10 or 100 ng/ml insulin-like growth factor (IGF)-1. The osteogenic differentiation was confirmed by the up-regulation of Runx2 and OCN , calcium quantification and bone histology. Switching from the OM to the MM promoted collagen synthesis and calcium content per cell, while continuing in the OM retained more cells in the constructs and promoted higher osteogenic gene expression. The addition of IGF-1 into the MM had no effect on cell proliferation, differentiation and matrix synthesis. In conclusion, hUCMSCs show significant potential for bone tissue engineering and culturing in the OM throughout the entire period is beneficial for osteogenic differentiation of these cells. Copyright © 2009 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63045/1/176_ftp.pd

Effect of 5-FU and MTX on the Expression of Drug-resistance Related Cancer Stem Cell Markers in Non-small Cell Lung Cancer Cells

Cancer stem cells (CSCs) are often characterized by the elevated expression of drug-resistance related stem-cell surface markers, such as CD133 and ABCG2. Recently, we reported that CSCs have a high level of expression of the IL-6 receptor (IL-6R). The purpose of this study was to investigate the effect of anticancer drugs on the expression of the drug resistance-related cancer stem cell markers, ABCG2, IL-6R, and CD133 in non-small cell lung cancer (NSCLC) cell lines. A549, H460, and H23 NSCLC cell lines were treated with the anticancer drugs 5-fluorouracil (5-FU; 25 µg/ml) and methotrexate (MTX; 50 µg/ml), and the expression of putative CSC markers was analyzed by fluorescent activated cell sorter (FACS) and the gene expression level of abcg2, il-6r and cd133 by reverse transcriptasepolymerase chain reaction (RT-PCR). We found that the fraction of ABCG2-positive(+) cells was significantly increased by treatment with both 5-FU and MTX in NSCLC cells, and the elevation of abcg2, il-6r and cd133 expressions in response to these drugs was also confirmed using RT-PCR. Also, the number of IL-6R(+) cells was increased by MTX in the 3 cell lines mentioned and increased by 5-FU in the H460 cell line. The number of CD133(+) cells was also significantly increased by both 5-FU and MTX treatment in all of the cell lines tested. These results indicate that 5-FU and MTX considerably enhance the expression of drug-resistance related CSC markers in NSCLC cell lines. Thus, we suggest that antimetabolite cancer drugs, such as 5-FU and MTX, can lead to the propagation of CSCs through altering the expression of CSC markers

Fenton chemistry and oxidative stress mediate the toxicity of the β-amyloid peptide in a Drosophila model of Alzheimer’s disease

The mechanism by which aggregates of the β-amyloid peptide (Aβ) mediate their toxicity is uncertain. We show here that the expression of the 42-amino-acid isoform of Aβ (Aβ1–42) changes the expression of genes involved in oxidative stress in a Drosophila model of Alzheimer’s disease. A subsequent genetic screen confirmed the importance of oxidative stress and a molecular dissection of the steps in the cellular metabolism of reactive oxygen species revealed that the iron-binding protein ferritin and the H2O2 scavenger catalase are the most potent suppressors of the toxicity of wild-type and Arctic (E22G) Aβ1–42. Likewise, treatment with the iron-binding compound clioquinol increased the lifespan of flies expressing Arctic Aβ1–42. The effect of iron appears to be mediated by oxidative stress as ferritin heavy chain co-expression reduced carbonyl levels in Aβ1–42 flies by 65% and restored the survival and locomotion function to normal. This was achieved despite the presence of elevated levels of the Aβ1–42. Taken together, our data show that oxidative stress, probably mediated by the hydroxyl radical and generated by the Fenton reaction, is essential for Aβ1–42 toxicity in vivo and provide strong support for Alzheimer’s disease therapies based on metal chelation

Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors

Altering APP Proteolysis: Increasing sAPPalpha Production by Targeting Dimerization of the APP Ectodomain

One of the events associated with Alzheimer's disease is the dysregulation of α- versus β-cleavage of the amyloid precursor protein (APP). The product of α-cleavage (sAPPα) has neuroprotective properties, while Aβ1-42 peptide, a product of β-cleavage, is neurotoxic. Dimerization of APP has been shown to influence the relative rate of α- and β- cleavage of APP. Thus finding compounds that interfere with dimerization of the APP ectodomain and increase the α-cleavage of APP could lead to the development of new therapies for Alzheimer's disease. Examining the intrinsic fluorescence of a fragment of the ectodomain of APP, which dimerizes through the E2 and Aβ-cognate domains, revealed significant changes in the fluorescence of the fragment upon binding of Aβ oligomers—which bind to dimers of the ectodomain— and Aβ fragments—which destabilize dimers of the ectodomain. This technique was extended to show that RERMS-containing peptides (APP695 328–332), disulfiram, and sulfiram also inhibit dimerization of the ectodomain fragment. This activity was confirmed with small angle x-ray scattering. Analysis of the activity of disulfiram and sulfiram in an AlphaLISA assay indicated that both compounds significantly enhance the production of sAPPα by 7W-CHO and B103 neuroblastoma cells. These observations demonstrate that there is a class of compounds that modulates the conformation of the APP ectodomain and influences the ratio of α- to β-cleavage of APP. These compounds provide a rationale for the development of a new class of therapeutics for Alzheimer's disease

Membrane topology of gp41 and amyloid precursor protein: interfering transmembrane interactions as potential targets for HIV and Alzheimer treatment

The amyloid precursor protein (APP), that plays a critical role in the development of senile plaques in Alzheimer disease (AD), and the gp41 envelope protein of the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), are single-spanning type-1 transmembrane (TM) glycoproteins with the ability to form homo-oligomers. In this review we describe similarities, both in structural terms and sequence determinants of their TM and juxtamembrane regions. The TM domains are essential not only for anchoring the proteins in membranes but also have functional roles. Both TM segments contain GxxxG motifs that drive TM associations within the lipid bilayer. They also each possess similar sequence motifs, positioned at the membrane interface preceding their TM domains. These domains are known as cholesterol recognition/interaction amino acid consensus (CRAC) motif in gp41 and CRAC-like motif in APP. Moreover, in the cytoplasmic domain of both proteins other alpha-helical membranotropic regions with functional implications have been identified. Recent drug developments targeting both diseases are reviewed and the potential use of TM interaction modulators as therapeutic targets is discussed