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th
PCISBIO Day – 11 July 2016
http://www.itqb.unl.pt/pcisbio2016/
Developing nanobodies to manipulate protein:protein interactions
in
neurodegenerative diseases
Joana S. Cristóvão and Cláudio M. Gomes*
Faculdade de Ciências Universidade de Lisboa, Biosystems and Integrative
Sciences Institute
http://folding.fc.ul.pt [email protected]
Insoluble β-amyloid peptide (Aβ) deposits formed in the synaptic milieu, chronic activation of
glial cells and inflammation are consistent features in Alzheimer’s disease (AD) and strong
candidates for the initiation of this process. S100B is one of the numerous pro-inflammatory
molecules produced by astrocytes, which is up regulated in AD and is found associated with
plaques [1].
S100B is a small dimeric protein whose structure and functional regulatory interactions with
other proteins, including the multi-ligand receptor RAGE. S100B has a broad range of
cellular concentrations (from nano to micromolar) that explains why S100B is found as
functional tetramers, hexamers and octamers, which enhance RAGE functional activation
[2]. Many of the S100B mediated interactions are modulated by calcium-binding through EF
hand motifs and by zinc- and copper-binding, to a site located at the dimer interface. These
facts and our recent observation that S100 proteins have intrinsic β-aggregation propensity
[3] have prompted us to investigate S100B self-assembly reactions and co-aggregation
phenomena involving other abundant neuronal proteins implicated in AD pathology, such as
the amyloid β peptide.
Along these lines, it became critical to develop tools to modulate the formation of S100B
assemblies; among such reagents are nanobodies, which are conformation sensitive
antibodies. In addition, these high-affinity single-domain antibodies derived from the atypical
immunoglobulins from Camelidae have a small size (~15 kDa) and can be expressed in
bacterial expression systems. Through Instruct, we have successfully accessed the
Nanobodies4Instruct nanobody production facility with the goal to develop these molecules
against S100B oligomers. These tools will be critical to establish future structural
mechanistic studies and cellular assays that will uncover new roles of S100B oligomers in
brain processes and AD neurodegeneration.
1. Cristóvão, J. S.; Santos, R.; Gomes, C. M., Metals and Neuronal Metal Binding Proteins Implicated
in Alzheimer's Disease. Oxid Med Cell Longev 2016, 2016, 9812178.
2. Fritz, G.; Botelho, H. M.; Morozova-Roche, L. A.; Gomes, C. M., Natural and amyloid self-assembly
of S100 proteins: structural basis of functional diversity. Febs J 2010, 277, (22), 4578-90.
3. Carvalho, S. B.; Botelho, H. M.; Leal, S. S.; Cardoso, I.; Fritz, G.; Gomes, C. M., Intrinsically
Disordered and Aggregation Prone Regions Underlie beta-Aggregation in S100 Proteins. PLoS One
2013, 8, (10), e76629.
Acknowledgements:
Research in the Gomes laboratory is supported by research grants from FCT/MCTES (PTDC/NEUNMC/2138/2014) and by the Bial Foundation (Ref 343/14). Instruct is acknowledged for support
through an access grant (to CMG) and Internship support (to JSC). J.Steyaert and E. Pardon
(Nanobodies4Instruct, Vrije Universiteit Brussel) are gratefully acknowledged for support and
collaboration in the nanobody production. CMG is a recipient of a Consolidation level Investigador
FCT (FCT/MCTES IF/01046/2014)