26 research outputs found

    Automated High-Content Live Animal Drug Screening Using C. elegans Expressing the Aggregation Prone Serpin α1-antitrypsin Z

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    The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms

    The biology of non-native proteins

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    Protein misfolding diseases are linked by common principles of protein aggregation, plaque development and tissue damage. There is no adequate therapy for these highly debilitating diseases. This thesis aims to increase the understanding of protein misfolding diseases, which will hopefully lead to development of safe therapeutics. In protein misfolding disorders, increased levels of platelet reactivity was observed, although the underlying mechanisms are not entirely understood. Therefore, we tested platelet activating capacity of misfolded proteins. Our findings illustrate that unrelated misfolded proteins induce platelet aggregation, which is mediated by CD36 and GPIbα. These results reveal novel platelet stimulatory mechanisms. Since misfolding of cellular proteins occurs during apoptosis and infection, one intriguing option is that platelets aid in the clearance of misfolded proteins and maintain homeostasis. There are other blood-born substances that recognize misfolded proteins. Pooled healthy human intravenous immunoglobulins (IVIg) are applied in the therapy of numerous diseases. We found that IVIg binds to unrelated misfolded proteins and inhibits platelet aggregation induced by misfolded proteins. We studied whether misfolded proteins share epitopes for conformational antibodies. Antibodies raised in mice recognize misfolded proteins, although in contrast to human IgG, these antibodies are IgM, which implies a role of human antibodies in clearance and that tolerance against misfolded proteins was not broken in mice. Aggregation aspects of amyloid-β (Aβ) are crucial for the pathology of the peptide. Soluble, rather than fibrillar species are supposed to participate in the progression of Alzheimer's disease, although, the principal mechanisms are not completely understood. To investigate the relationship between amyloidogenic characteristics and platelet stimulatory capacity of Aβ, we used different Aβ fragments. We found that non-fibrillar Aβ1-42 induced platelet aggregation, though Aβ1-40 and small fragments did not stimulate platelets, which suggests that platelet stimulatory capacity resides in non-fibrillar fractions. Proteins acquire dissimilar properties after conformational changes. Previous studies suggest that native CRP does not activate endothelial cells, however its conformational variant mCRP up-regulates adhesion molecule expression, whereas others found the opposite. We illustrate that neither nCRP nor mCRP evoked pro-inflammatory changes in HUVEC and that CRP preparations have toxic properties at high doses. Modified CRP exhibited membrane binding to HUVECs, whereas no detectable binding of nCRP was observed. These results show differences between native and modified CRP in their binding capacity to diverse endothelial compartments. Endothelial disfunction is the early event of protein misfolding diseases and cancer, where levels of endothelial growth factors are increased, which accelerate angiogenesis. Therefore, suppression of tumor capillarization is beneficial. Receptor tyrosine kinase inhibitors are potent tools in tumor therapy; however, their cytotoxic effect has not been studied. We show that protein kinase inhibitors have cytotoxic effects. There is strong positive correlation between EGF receptor inhibition and cytotoxicity and negative correlation between PDGF inhibition and cytotoxicity. These data provide possible explanations for side-effects of medicines and highlight the importance of endothelial cell cytotoxicity measurements of drug-candidate compounds

    Tracking down contact activation - from coagulation in vitro to inflammation in vivo

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    The contact system is a volatile and versatile enzyme system in blood plasma that responds to the presence of nonphysiological surface materials by spontaneous generation of enzymatic activity. In subsequent steps, it can trigger blood coagulation and is responsible for the generation of the proinflammatory peptide bradykinin. The physiological role of the contact system is presently unknown, but it is commonly used to trigger coagulation in a diagnostic setting. In this three-part review, we will first describe the molecular mechanisms that drive contact activation on nonphysiological materials. Next, we will summarize and compare a number of bioassays, which are commonly used to investigate the contact system in health and disease. Finally, we will discuss recent findings from both fundamental and clinical studies on the contributions of contact system to cardiovascular, infectious, and inflammatory disease.published_online: 2014-04-18status: publishe

    Activation of human platelets by misfolded proteins

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    Objective: Protein misfolding diseases result from the deposition of insoluble protein aggregates that often contain fibrils called amyloid. Amyloids are found in Alzheimer disease, atherosclerosis, diabetes mellitus, and systemic amyloidosis,which are diseases where platelet activation might be implicated. Methods and Results:We induced amyloid properties in 6 unrelated proteins and found that all induced platelet aggregation in contrast to fresh controls. Amyloid-induced platelet aggregation was independent of thromboxane A2 formation and ADP secretion but enhanced by feedback stimulation through these pathways. Treatments that raised cAMP (iloprost), sequestered Ca2+ (BAPTA-AM) or prevented amyloid-platelet interaction (sRAGE, tissue-typeplasminogen activator [tPA]) induced almost complete inhibition. Modulation of the function of CD36 (CD36-/- mice), p38MAPK (SB203580), COX-1 (indomethacin), and glycoprotein I

    Oxidation enhances human serum albumin thermal stability and changes the routes of amyloid fibril formation.

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    Oxidative damages are linked to several aging-related diseases and are among the chemical pathways determining protein degradation. Specifically, interplay of oxidative stress and protein aggregation is recognized to have a link to the loss of cellular function in pathologies like Alzheimer's and Parkinson's diseases. Interaction between protein and reactive oxygen species may indeed induce small changes in protein structure and lead to the inhibition/modification of protein aggregation process, potentially determining the formation of species with different inherent toxicity. Understanding the temperate relationship between these events can be of utmost importance in unraveling the molecular basis of neurodegeneration. In this work, we investigated the effect of hydrogen peroxide oxidation on Human Serum Albumin (HSA) structure, thermal stability and aggregation properties. In the selected conditions, HSA forms fibrillar aggregates, while the oxidized protein undergoes aggregation via new routes involving, in different extents, specific domains of the molecule. Minute variations due to oxidation of single residues affect HSA tertiary structure leading to protein compaction, increased thermal stability, and reduced association propensity
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