Self-Organization and Regulation of Intrinsically Disordered Proteins with Folded N-Termini

How do mostly disordered proteins coordinate the specific assembly of very large signal transduction protein complexes? A newly emerging hypothesis may provide some clues towards a molecular mechanism

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

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

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

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

Requirements for immune recognition and processing of factor VIII by antigen-presenting cells

Generation of inhibitory antibodies upon repeated FVIII infusion represents a major complication in hemophilia care. Professional antigen presenting cells (APCs) are crucial for orchestration of humoral immune responses. APCs are capable of internalizing soluble as well as particulate antigens through various mechanisms resulting in loading of antigen-derived peptides on MHC class I or II for presentation to T cells. This review highlights how FVIII is recognized and processed by APCs. The significance and contribution of candidate receptors involved in FVIII uptake by APC are discussed. Recent findings defining the repertoire of FVIII peptides presented on MHC class II are addressed. Studies in murine models of hemophilia A suggest that modulation of APC function can reduce inhibitor formation. Based on this we anticipate that modulation of FVIII uptake by APCs may yield novel therapeutic approaches for treatment or prevention of inhibitor formation in patients with hemophilia A. (C) 2011 Elsevier Ltd. All rights reserve

Limited promiscuity of HLA-DRB1 presented peptides derived of blood coagulation factor VIII.

The formation of inhibitory antibodies directed against coagulation factor VIII (FVIII) is a severe complication in the treatment of hemophilia A patients. The induction of anti-FVIII antibodies is a CD4(+) T cell-dependent process. Activation of FVIII-specific CD4(+) T cells is dependent on the presentation of FVIII-derived peptides on MHC class II by antigen-presenting cells. Previously, we have shown that FVIII-pulsed human monocyte-derived dendritic cells can present peptides from several FVIII domains. In this study we show that FVIII peptides are presented on immature as well as mature dendritic cells. In immature dendritic cells half of the FVIII-loaded MHC class II molecules are retained within the cell, whereas in LPS-matured dendritic cells the majority of MHC class II/peptide complexes is present on the plasma membrane. Time-course studies revealed that presentation of FVIII-derived peptides was optimal between 12 and 24 hours after maturation but persisted for at least 96 hours. We also show that macrophages are able to internalize FVIII as efficiently as dendritic cells, however FVIII was presented on MHC class II with a lower efficiency and with different epitopes compared to dendritic cells. In total, 48 FVIII core-peptides were identified using a DCs derived of 8 different donors. Five HLA-promiscuous FVIII peptide regions were found - these were presented by at least 4 out of 8 donors. The remaining 42 peptide core regions in FVIII were presented by DCs derived from a single (30 peptides) or two to three donors (12 peptides). Overall, our findings show that a broad repertoire of FVIII peptides can be presented on HLA-DR

The macrophage mannose receptor promotes uptake of ADAMTS13 by dendritic cells

ADAMTS13 is a plasma metalloproteinase that regulates platelet adhesion and aggregation by cleaving ultra-large VWF multimers on the surfaces of endothelial cells. Autoantibodies directed against ADAMTS13 prohibit the processing of VWF multimers, initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic purpura. The formation of autoantibodies depends on the activation of CD4(+) T cells. This process requires immune recognition, endocytosis, and subsequent processing of ADAMTS13 into peptides that are presented on MHC class II molecules to CD4(+) T cells by dendritic cells (DCs). In the present study, we investigated endocytosis of recombinant ADAMTS13 by immature monocyte-derived DCs using flow cytometry and confocal microscopy. After incubation of fluorescently labeled ADAMTS13 with DCs, significant uptake of ADAMTS13 was observed. Endocytosis of ADAMTS13 was completely blocked by the addition of EGTA and mannan. ADAMTS13 endocytosis was decreased in the presence of a blocking mAb directed toward the macrophage mannose receptor (MR). Furthermore, siRNA silencing of MR reduced the uptake of ADAMTS13 by DCs. In addition, in vitro binding studies confirmed the interaction of ADAMTS13 with the carbohydrate recognition domains of MR. The results of the present study indicate that sugar moieties on ADAMTS13 interact with MR, thereby promoting its endocytosis by APCs. (Blood. 2012;119(16):3828-3835