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Molecular dissection of established and proposed members of the Op18/Stathmin family of tubulin binding proteins
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

My initial aim was a functional analysis of the conserved Op18/stathmin family of microtubule-regulators, which includes the ubiquitous cytosolic Op18 protein and the neural membrane-attached RB3 and SCG10 proteins. The solved X-ray structure has shown that these proteins form a complex with tubulin -heterodimers via two imperfect helical repeats, which result in two head-to-tail aligned heterodimers in a tandem-tubulin complex. We have analyzed GTP exchange and GTP hydrolysis at the two exchangeable GTP-binding sites (E-site) within the tandem-tubulin complex. A comparison of Op18, RB3 and SCG10 proteins indicates that Op18/Stathmin family proteins have evolved to maintain the two heterodimers in a configuration that restrains the otherwise potent GTPase productive interactions facilitated by the head-to-head alignment of heterodimers in protofilaments. We concluded from these studies that tubulin heterodimers in complex with Op18/stathmin family members are subject to allosteric effects that prevent futile cycles of GTP hydrolysis.

To understand the significance of the large differences in tubulin affinity of Op18, RB3 and SCG10, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell-surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. We showed that, in contrast to CD2-Op18, both the CD2-SCG10 and CD2-RB3 chimeras sequester tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels. However, all three CD2-chimeras, including the tubulin sequestration-incompetent CD2-Op18, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during the interphase, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules.

Sm16/SmSLP (Stathmin-Like Protein) has been identified as a protein released during skin penetration of the Schistosoma mansoni parasite. This protein has been ascribed both anti-inflammatory activities and a functional similarity with the conserved cytosolic tubulin-binding protein stathmin/Op18. However, our studies refuted any functional similarity with stathmin/Op18 and we found instead that Sm16/SmSLP is a lipid bilayer binding protein that is taken up by cells through endocytosis.

To study immuno-modulatory properties of Sm16/SmSLP, we designed an engineered version with decreased aggregation propensity, thus facilitating expression and purification of a soluble Sm16 /SmSLP protein from the eukaryotic organism Pichia pastoris. Determination of the hydrodynamic parameters revealed that both the recombinant and native Sm16/SmSLP is a ~9-subunits oligomer. The recombinant protein was found to have no effect on T lymphocyte activation, cell proliferation or the basal level of cytokine production of whole human blood or monocytic cells. Interestingly, however, recombinant Sm16 was found to potently inhibit the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and Poly(I:C). Since Sm16 specifically inhibits degradation of the IRAK1 signaling protein in LPS stimulated monocytes, it seems likely that inhibition is exerted proximal to the TLR-complex.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Medicinska fakulteten) , 2009. , 47 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1234
Keyword [en]
Op18/Stathmin, Sm16, tubulin, microtubule, schistosoma mansoni
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1949ISBN: 978-91-7264-705-3 (print)OAI: oai:DiVA.org:umu-1949DiVA: diva2:142536
Public defence
2009-01-09, Major grove, 6L, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2008-12-18 Created: 2008-12-18 Last updated: 2010-03-25Bibliographically approved
List of papers
1. Deciphering the cellular functions of the Op18/stathmin family of microtubule-regulators by plasma membrane-targeted localization
Open this publication in new window or tab >>Deciphering the cellular functions of the Op18/stathmin family of microtubule-regulators by plasma membrane-targeted localization
2003 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, Vol. 14, no 9, 3716-3729 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-3708 (URN)
Available from: 2008-12-18 Created: 2008-12-18Bibliographically approved
2. Functional dissection of GTP hydrolysis and exchange within the ternary complex of tubulin heterodimers and Op18/stathmin family members
Open this publication in new window or tab >>Functional dissection of GTP hydrolysis and exchange within the ternary complex of tubulin heterodimers and Op18/stathmin family members
2003 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 278, no 19, 16651-16657 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-3709 (URN)
Available from: 2008-12-18 Created: 2008-12-18Bibliographically approved
3. The Schistosoma mansoni protein SM16/SmSLP/SmSPO-1 is a membrane-binding protein that lacks the proposed microtubule-regulatory activity
Open this publication in new window or tab >>The Schistosoma mansoni protein SM16/SmSLP/SmSPO-1 is a membrane-binding protein that lacks the proposed microtubule-regulatory activity
Show others...
2007 (English)In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 156, no 2, 225-234 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-3710 (URN)17913257 (PubMedID)
Available from: 2008-12-18 Created: 2008-12-18 Last updated: 2011-03-30Bibliographically approved
4. The Schistosoma mansoni protein Sm16/SmSLP/SmSPO-1 assembles into a nine-subunit oligomer with potential To inhibit Toll-like receptor signaling.
Open this publication in new window or tab >>The Schistosoma mansoni protein Sm16/SmSLP/SmSPO-1 assembles into a nine-subunit oligomer with potential To inhibit Toll-like receptor signaling.
Show others...
2009 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 77, no 3, 1144-1154 p.Article in journal (Refereed) Published
Abstract [en]

The Sm16/SmSLP/SmSPO-1 (Sm16) protein is secreted by the parasite Schistosoma mansoni during skin penetration and has been ascribed immunosuppressive activities. Here we describe the strategy behind the design of a modified Sm16 protein with a decreased aggregation propensity, thus facilitating the expression and purification of an Sm16 protein that is soluble in physiological buffers. The Stokes radii and sedimentation coefficients of recombinant and native proteins indicate that Sm16 is an approximately nine-subunit oligomer. Analysis of truncated Sm16 derivatives showed that both oligomerization and binding to the plasma membrane of human cells depend on multiple C-terminal regions. For analysis of immunomodulatory activities, Sm16 was expressed in Pichia pastoris to facilitate the preparation of a pyrogen/endotoxin-free purified protein. Recombinant Sm16 was found to have no effect on T-lymphocyte activation, cell proliferation, or the basal level of cytokine production by whole human blood or monocytic cells. However, Sm16 exerts potent inhibition of the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and poly(I:C) while being less efficient at inhibiting the response to the TLR ligand peptidoglycan or a synthetic lipopeptide. Since Sm16 specifically inhibits the degradation of the IRAK1 signaling protein in LPS-stimulated monocytes, our findings indicate that inhibition is exerted proximal to the TLR complex.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-32760 (URN)10.1128/IAI.01126-08 (DOI)19124604 (PubMedID)
Available from: 2010-03-24 Created: 2010-03-24 Last updated: 2016-08-22Bibliographically approved

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