(F-221) Studying Effects of Disease-related Mutations in Progranulin Using Molecular Dynamics Simulations and Circular Dichroism

Introduction:: Progranulin is a secreted glycoprotein of 75-80 kDa, containing 7.5 granulin modules (p,G,F,B,A,C,D and E) which are encoded by the GRN gene. Mutations within the GRN gene have been linked to the development of frontotemporal lobar degeneration (FTLD)1. FTLD is an early-onset dementia syndrome characterized by progressive decline in behavior or language. So far, the effect of disease-related mutations on the structural characteristics and unique cysteine-cysteine pairing pattern of granulin modules is not well understood. Our goal was to study disease related mutations that introduce a new cysteine to human granulin sequence.


Materials and Methods:: Using molecular dynamics simulations (MDS), we studied the effects of the W541C1, a cysteine-introducing mutation, in granulin module E on stability and disulfide bond patterns. For this, we used the known structure of the zebra fish granulin (pdb # 6cku), a stack of four β-hairpins stabilized by a total of six disulfide bonds, which has high homology to granulin E. The 1000 ns MDS were run for the wild-type and the mutated proteins, either with all six disulfide bonds intact or with two of them reduced. The polypeptide structure visualization was done in UCSF Chimera3 which was also used for introducing the W541C mutation in silico. The MDS was performed using AMBER182. We cloned, expressed, and tested multiple redox conditions for refolding of granulin E and A. To study the refolding process of the purified proteins, we used circular dichroism (CD).

Results, Conclusions, and Discussions:: First, we studied the propensity of disulfide bonds in the wild-type protein. Then we compared structures and bond formation for wild-type and the mutated proteins. Using the developed purification conditions, we were able to compare refolding of the wild-type and the W541C mutated granulins and observe the destabilizing effect of the mutations on the granulin structure.

Our CD and MDS results suggested that formation of the disulfide bond between C10 and C26 is disfavored due to the mutation. This work will help characterize the structure/function relationships in human granulins. By understanding the effects of the mutations and how it compromises the functionality of human granulin we will be able to detect and combat FTLD in its early stages accurately.




Acknowledgements (Optional): :

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< Gijselinck I, Broeckhoven C, Cruts M. HUMU, 2008. Vol 29, Issue 12. Page 1373-1386

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< !Case Da,Ben-Shalom IY, Brozell SR, Cerutti DS, Cheatham TE. AMBER 2018, University of California, San Francisco.2018

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< !Pettersen EF, Goddard TD, Huang CC,Couch GS, Greenblatt DM, Meng EC, Ferrin TE. Comput Chem, 2004. Vol Oct; 25(13). Page 1605-12