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Real-Time Monitoring of Apoptosis by Caspase-3-Like Protease Induced FRET Reduction Triggered by Amyloid Aggregation
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institute, 17176 Stockholm, Sweden.
The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institute, 17176 Stockholm, Sweden.
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2008 (English)In: Experimental Diabetes Research, ISSN 1687-5214, E-ISSN 1687-5303, Vol. 2008, no 865850Article in journal (Refereed) Published
Abstract [en]

Amyloid formation is cytotoxic and can activate the caspase cascade. Here, we monitor caspase-3-like activity as reduction of fluorescence resonance energy transfer (FRET) using the contstruct pFRET2-DEVD containing enhanced cyan fluorescent protin (EYFP) linked by the caspase-3 specific cleavage site residues DEVD. Beta-TC-6 cells were transfected, and the fluoorescence was measured at 440 nm excitation and 535 nm (EYFP) and 480 nm (ECFP) emission wavelength. Cells were incubated with recombinant pro lset Amyloid Polypeptide (rec prolAPP) or the processing metabolites of prolAPP; the N-terminal flanking peptide withIAPP (recN+IAPP); IAPP with the C-terminal flanking peptied (recIAPP+C) and lslet Amyloid Polypeptide (recIAPP). Peptides were added in solubilized from (50 mu M) or as performed amyloid-like fibrils, or as a combination of these. FRET was measured and incubation with a mixture of solubilized peptide and performed fibrils resulted in loss of FRET and apoptosis was determined to occurein cells incubated with recproIAPP (49%), recN+IAPP (46%), recIAPP (72%) and recIAPP+C (59%). These results show that proIAPP and the processing intermediates reside the same cell toxic capacity as IAPP, and they can all have a central role in the reduction of beta-cell number in type 2 diabetes.

Place, publisher, year, edition, pages
2008. Vol. 2008, no 865850
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-19669DOI: 10.1155/2008/865850OAI: oai:DiVA.org:liu-19669DiVA: diva2:227295
Note
Original Publication: Johan F Paulsson, Sebastian Schultz, Martin Kohler, Ingo Leibiger, Per-Olof Berggren and Gunilla Westermark, Real-Time Monitoring of Apoptosis by Caspase-3-Like Protease Induced FRET Reduction Triggered by Amyloid Aggregation, 2008, EXPERIMENTAL DIABETES RESEARCH, (2008), 865850. http://dx.doi.org/10.1155/2008/865850 Copyright: Authors Available from: 2009-08-28 Created: 2009-07-10 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Studies on Islet Amyloid Polypeptide Aggregation: From Model Organism to Molecular Mechanisms
Open this publication in new window or tab >>Studies on Islet Amyloid Polypeptide Aggregation: From Model Organism to Molecular Mechanisms
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The proper folding of a protein into its defined three--‐dimensional structure is one of the many fundamental challenges a cell encounters. A number of tightly controlled pathways have evolved to assist in the proper folding of a protein, but also to aid in the removal of misfolded proteins. Despite the presence of these pathways accumulation of misfolded proteins can still occur. Amyloid deposits consist of misfolded proteins with a characteristic highly ordered fibrillar structure that will exert affinity for the amyloid dye Congo red and has a unique X-ray diffraction pattern. Currently 27 different proteins have been identified as amyloid forming proteins in human, however the exact role of amyloid in the pathogenesis of the connected disease is most often unclear.

Islet amyloid is made up of the beta cell derived hormone islet amyloid polypeptide (IAPP) and is associated with the development of type 2 diabetes. Propagation of IAPP-fibrils is believed to be one important cause of the pancreatic beta cell death detected in patients with type 2 diabetes. IAPP is a naturally occurring polypeptide hormone stored and secreted together with insulin. IAPP and insulin arise from posttranslational processing of their biological inactive precursors proIAPP and proinsulin. In addition to human, cat and monkey IAPP will form amyloid deposits in conditions resembling human type 2 diabetes. However, IAPP from mouse and rat do not form amyloid as a result of the differences in amino acid sequence.

My main research goal was to establish a unique model system suitable to study the effects of proIAPP and IAPP aggregation. I selected Drosophila melanogaster due to its many suitable characteristics as a model organism and its superior genetic toolbox. I have demonstrated that over--‐expression of hproIAPP and hIAPP in the central nervous system (CNS) results in aggregate formation in the brain and neighbouring fat body. Consistent with previous studies, expression of mIAPP does not result in the formation of aggregates. To investigate the intracellular effects of hproIAPP and hIAPP aggregation on a specific population of neurons, we targeted the expression of these peptides specifically to 16 neurons in the brain, the pdf- neurons. These pdf-neurons are divided into 2 clusters of 8 cells per brain hemisphere. First I showed that expression of aggregation prone hIAPP and hproIAPP resulted in significant death of the 8 cells, whereas expression of mIAPP had no such effect. In efforts to pinpoint the mechanisms behind the observed cell death I demonstrated that hproIAPP and hIAPP both pass the ERs quality control for protein folding and that the initiated cell death does not occur through classical apoptosis. Instead, selective autophagy is activated by hIAPP and hproIAPP. This activation counteracts the usually neuro-protective effects of autophagy and contributes to cell death. Strikingly, I also showed that Aâ, the amyloid protein implicated in Alzheimer’s disease, does not exhibit any intracellular toxicity when expressed in pdf-cells. This supports the existence of separate toxic pathways for different amyloid proteins.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2011. 100 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1254
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-70094 (URN)9789173930994 (ISBN)
Public defence
2011-09-16, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2011-08-18 Created: 2011-08-18 Last updated: 2017-12-12Bibliographically approved
2. Proislet Amyloid Polypeptide (proIAPP): Impaired Processing is an Important Factor in Early Amyloidogenesis in Type 2 Diabetes
Open this publication in new window or tab >>Proislet Amyloid Polypeptide (proIAPP): Impaired Processing is an Important Factor in Early Amyloidogenesis in Type 2 Diabetes
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Amyloid is defined as extracellular protein aggregates with a characteristic fibrillar ultra-structure, Congo red affinity and a unique x-ray diffraction pattern. At present, 25 different human amyloid fibril proteins have been identified, and amyloid aggregation is associated with pathological manifestations such as Alzheimer’s disease, spongiform encephalopathy and type 2 diabetes. Amyloid aggregation triggers apoptosis by incorporation of early oligomers in cellular membranes, causing influx of ions. Amyloid is the only visible pathological islet alteration in subjects with type 2 diabetes, and islet amyloid polypeptide (IAPP) is the major islet amyloid fibril component. IAPP is produced by beta-cells and co-localized with insulin in the secretory granules. Both peptides are synthesised as pro-molecules and undergo proteolytic cleavage by the prohormone convertase 1/3 and 2. Although IAPP is the main amyloid constituent, both proIAPP and proIAPP processing intermediates have been identified in islet amyloid.

The aim of this thesis was to study the role of impaired processing of human proIAPP in early islet amyloidogenesis. Five cell lines with individual processing properties were transfected with human proIAPP and expression, aggregation and viability were studied. Cells unable to process proIAPP into IAPP or to process proIAPP at the N-terminal processing site accumulated intracellular amyloid-like aggregates and underwent apoptosis. Further, proIAPP immunoreactivity was detected in intracellular amyloid-like aggregates in betacells from transgenic mice expressing human IAPP and in transplanted human beta-cells. ProIAPP was hypothesized to act as a nidus for further islet amyloid deposition, and to investigate this theory, amyloid-like fibrils produced from recombinant IAPP, proIAPP and insulin C-peptide/A-chain were injected in the tail vein of transgenic mice expressing the gene for human IAPP. Pancreata were recovered after 10 months and analysed for the presence of amyloid. Both IAPP and proIAPP fibrils but not des-31,32 proinsulin fibrils, caused an increase in affected islets and also an increase of the amyloid amount. This finding demonstrates a seeding capacity of proIAPP on IAPP fibrillogenesis. IAPP has been known for some time to trigger apoptosis in cultured cells, and a novel method for real time detection of apoptosis in beta-cells was developed. Aggregation of recombinant proIAPP and proIAPP processing intermediates were concluded to be inducers of apoptosis as potent as IAPP fibril formation.

From the results of this study, a scenario for initial islet amyloidogenesis is proposed. Initial amyloid formation occurs intracellularly as a result of alterations in beta-cell processing capacity. When the host cell undergoes apoptosis intracellular proIAPP amyloid becomes extracellular and can act as seed for further islet amyloid deposition.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2006. 74 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 967
Keyword
Biosynthesis amyloid, Genetics amyloid, Metabolism amyloid, Islets of Langerhans, Proprotein convertases, Posttranslation protein processing
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:liu:diva-8243 (URN)91-85643-59-9 (ISBN)
Public defence
2006-11-24, Berzeliussalen, Campus US, Linköpings Universitet, Linköping, 13:15 (English)
Opponent
Supervisors
Available from: 2007-02-01 Created: 2007-02-01 Last updated: 2012-01-04Bibliographically approved

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