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Purification and Characterization of the Nerve Growth Factor R221W mutant causing Insensitivity to Pain
Umeå University, Faculty of Medicine, Department of Medical Biosciences.
Umeå University, Faculty of Medicine, Department of Medical Biosciences.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

 

We have previously identified a homozygous missense (R221W) mutation in the NGFβ gene which causes insensitivity to pain in patients. The mutation impairs the secretion of NGF and the majority of the protein accumulates as proNGF. NGF mediates its function by binding and activating the TrkA and p75 receptors and is important for the survival of the sensory and sympathetic neurons as well as the cholinergic neurons of the basal forebrain. However, the R221W mutation seems to discriminate between these types of neurons as it is the sensory neurons that are mainly affected in the patients. A second human NGFβ mutation causes a more severe form of pain insensitivity with additional anhidrosis and cognitive dysfunctions in affected patients which is also seen in patients with mutations in the gene encoding the TrkA receptor. Because R221W NGF cause a less severe phenotype we hypothesised that the mutation mainly affects the p75 interaction which is also strengthened by the fact that the substitution is located in a region known to interact with p75. In this report, we show that R221W NGF is able to bind and activate TrkA at a level comparable to wild-type NGF in cells stably expressing TrkA while the activation of the downstream target ERK1/2 is impaired in cells that co-express TrkA and p75. We also describe the effects of the mutation in terms of expression and purification properties from E.coli which indicate the likelihood that eukaryotic folding machinery is needed for correct folding of R221W NGF.

Keyword [en]
NGF, TrkA, p75, Pain insensitivity, protein
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:umu:diva-51117OAI: oai:DiVA.org:umu-51117DiVA: diva2:475825
Available from: 2012-01-11 Created: 2012-01-11 Last updated: 2012-01-20
In thesis
1. The use of monogenic disease to study basal and disease associated mechanisms with focus on NGF dependent pain insensitivity and ISCU myopathy
Open this publication in new window or tab >>The use of monogenic disease to study basal and disease associated mechanisms with focus on NGF dependent pain insensitivity and ISCU myopathy
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Monogenic diseases make excellent models for the study of gene functions and basal cellular mechanisms in humans. The aim of this thesis was to elucidate how genetic mutations affect the basal cellular mechanisms in the monogenic diseases Nerve growth factor (NGF) dependent pain insensitivity and Iron-Sulphur cluster assembly protein U (ISCU) myopathy.

NGF dependent pain insensitivity is a rare genetic disorder with clinical manifestations that include insensitivity to deep pain, development of Charcot joints, and impaired temperature sensation but with no effect on mental abilities. The disease is caused by a missense mutation in the NGFβ gene causing a drastic amino acid substitution (R221W) in a well-conserved region of the protein. NGF is secreted in limited amounts by its target tissues and is important for the development and maintenance of the cholinergic forebrain neurons as well as the sensory and sympathetic neurons. To reveal the underlying mechanisms of disease we performed functional studies of the mutant NGF protein. We could show that mutant NGF was unable to induce differentiation of PC12 cells as a consequence of impaired secretion. Furthermore, mutant NGF had different intracellular localisation compared to normal NGF and resided mostly in its unprocessed form proNGF. Mature NGF and proNGF have different binding properties to the receptors TrkA and p75. Individuals with mutations in TRKA are, aside from pain insensitive mentally affected; therefore it has been proposed that the R221W mutation mainly affects the interaction with p75. In agreement with this, we could show that R221W NGF was able to bind and activate TrkA whereas the interaction with p75 was impaired as compared to normal NGF.

ISCU myopathy is a monogenic disease where the affected patients suffer from severe exercise intolerance resulting in muscle cramps and sometimes severe lactic acidosis. The disease is caused by a point mutation in the last intron of the Iron sulphur cluster assembly gene, ISCU, resulting in the inclusion of a part of the intron in the mRNA. ISCU functions as a scaffold protein in the assembly of iron-sulphur (Fe-S) clusters important for electron transport in Kreb’s cycle and the respiratory chain. We have shown that ISCU is vital in mammals since complete knock-down of Iscu in mice results in early embryonic death. The deletion of ISCU homologous in lower organisms has also been shown fatal. In spite this central role in energy metabolism the disease is restricted to the patient’s skeletal muscles while other energy demanding organs seem unaffected. To address this contradiction we examined if tissue-specific differences in the splicing of mutant ISCU could explain the muscle-specific phenotype. We could show that the splicing pattern did, indeed, differ with more incorrectly spliced ISCU in muscle compared to other tissues. This was accompanied by a decrease in Fe-S containing proteins in muscle, while no decrease was observed in other tissues. Alternative splicing is more common then previously thought and may depend upon interacting factors and/or differences in the surrounding milieu. To reveal plausible mechanisms involved in the tissue-specific splicing we identified nuclear factors that interacted with the region where the mutation was located. Five interacting factors were identified, out of which three affected the splicing of ISCU. PTBP1 was shown to repress the incorrect splicing while IGF2BP1 and RBM39 repressed the formation of normal transcript and could also counteract the effect of PTBP1. IGF2BP1 was the only factor that showed higher affinity to the mutant sequence making it a possible key factor in the incorrect splicing of the mutant ISCU gene.

Together, these results offer important insights into the cellular mechanisms causing these diseases. We found impaired secretion and inaccurate sorting of NGF to be cellular mechanisms contributing to NGF dependent pain insensitivity while tissue-specific splicing of ISCU was found to be the event contributing to the phenotype of ISCU myopathy.

Place, publisher, year, edition, pages
Umeå: Umea university, Department of Medical Biosciences, 2012. 46 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1463
Keyword
monogenic, disease, NGF, receptor, pain insensitivity, ISCU, myopathy, splicing
National Category
Other Basic Medicine
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:umu:diva-51140 (URN)978-91-7459-326-6 (ISBN)
Public defence
2012-02-10, Betula, By 6M, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-01-20 Created: 2012-01-11 Last updated: 2012-01-20Bibliographically approved

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Larsson, ElinHolmberg, Monica

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