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Unfolded protein response and enpl-1 depletion sensitize C. elegans to the anti-cancer drug cisplatin
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery. (Peter Naredi)
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
(English)Manuscript (preprint) (Other academic)
National Category
Basic Medicine
Research subject
Developmental Biology
Identifiers
URN: urn:nbn:se:umu:diva-60952OAI: oai:DiVA.org:umu-60952DiVA: diva2:564941
Available from: 2012-11-06 Created: 2012-11-05 Last updated: 2013-07-08Bibliographically approved
In thesis
1. New modifiers of insulin signalling identified by interaction screens with ASNA-1 in C. elegans
Open this publication in new window or tab >>New modifiers of insulin signalling identified by interaction screens with ASNA-1 in C. elegans
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Insulin is a hormone released by the pancreatic beta cells in response to elevated levels of nutrients in the blood. Insulin triggers the uptake of glucose, fatty acids and amino acids into the liver, adipose tissue and muscles. Genes regulating insulin signalling are thus of vital importance for metabolic homeostasis and for preventing the development of diabetes. This thesis aims to identify new modifiers of insulin signalling, while carrying out functional studies of a homolog to human arsenite translocating ATPase, ASNA1. ASNA1 activates the insulin signalling pathway and promotes insulin secretion in mammalian cell lines and in Caenorhabditis elegans. A second aim is to better understand how ASNA1 and its interactors regulate sensitivity to the chemotherapeutic drug, cisplatin. Results: Regulators of insulin/IGF signalling (IIS) in C. elegans were identified based on the Larval arrest arrest aspect of the asna-1 depletion phenotype. Sixty-five genes were selected by virtue of their predicted interaction with ASNA-1 and screened for asna-1-like larval arrest upon inactivation of the genes . mrps-2, mrps-10, mrpl-43 encoding mitochondrial ribosomal protein subunits, and enpl-1 encoding an ER chaperone, GRP94 homolog were identified as the genes which when inactivated caused larval arrest without any associated feeding defects. IIS was weaker and insulin secretion was defective in these knockdown animals. ENPL-1 and ASNA-1 proteins interacted with one another both ex vivo and in vitro. ASNA-1 protein and mRNA level swere greatly reduced in enpl-1 mutants and enpl-1(-);asna-1(-) double-mutant worms displayed synthetic lethality. Overexpression of the insulins INS-4 and DAF-28 caused partial rescue of the germline phenotype of enpl-1 mutants, indicating that the phenotype of enpl-1 mutants was due at least in part to insufficient insulin levels. Studies of enpl-1 mutants also helped to understand the role of asna-1 in cisplatin sensitivity. The unfolded protein response (UPR) was induced in asna-1 and enpl-1 knockdown animals. enpl-1 mutants displayed higher sensitivity to cisplatin, when compared to asna-1 mutants and this correlated to higher UPR in enpl-1 knockdown animals. Pharmacological induction of the UPR in intrinsically cisplatin resistant wildtype worms also resulted in increased cisplatin sensitivity. This suggests that manipulation of ENPL-1 levels or of the UPR could enhance the anti-tumoral effects of cisplatin based cancer therapy. With a yeast two hybridscreen 27 putative physical interactors of ASNA-1 were identified. Amongst these candidate swas smn-1, which encodes survival of motor neuron protein homolog. RNAi knockdown of smn-1 caused a larval arrest phenotype similar to asna-1 depleted animals and smn-1 positively regulated IIS, like asna-1. Defects in IIS may be at the level of insulin release because neuropeptide secretion was impaired upon smn-1 knockdown. Further in vitro binding studies showed that SMN-1 and ASNA-1 interacted and inactivation of smn-1 in asna-1 mutants resulted in decreased viability. This implies that SMN-1 is another modifier of ASNA-1 and also a new component in IIS. Conclusion: With a directed RNAi screen and a yeast two hybrid screen several interactors of ASNA-1 that are also IIS modifiers were identified. ENPL-1 and SMN-1 are both involved in insulin release. We also found that induction of the UPR in enpl-1 and asna-1 mutants is a possible mechanism for increased sensitivity to cisplatin.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2012. 75 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1530
Keyword
Insulin, C. elegans, ASNA1, Cisplatin, GRP94, unfolded protein response, SMN1
National Category
Medical and Health Sciences
Research subject
Developmental Biology
Identifiers
urn:nbn:se:umu:diva-60949 (URN)978-91-7459-508-6 (ISBN)
Public defence
2012-12-10, Sal B, 9tr, Norrlands universitetssjukhus (NUS), Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-11-09 Created: 2012-11-05 Last updated: 2012-11-09Bibliographically approved

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Natarajan, BalasubramanianHemmingsson, OskarKao, GautamNaredi, Peter

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