pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

Abstract

Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4th_FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4th_FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities.

Instantaneous fibrillation of egg white proteome with ionic liquid and macromolecular crowding

“…It has been concluded that the rate and efficiency of proteins fibrillation are ruled by the proteins hydrophobic-hydrophilic nature, secondary structure changes, possibility of establishing electrostatic, πˑˑˑπ and hydrogenbonding interactions, among others, which can be potentiated by fibrillation agents [12][13][14][15][16][17][18][19][20][21] . Two major mechanisms of fibrils formation have been proposed: (i) seedling of fibrils in the form of misfolded monomeric intermediates, showing a lag phase in the kinetics curve of fibrils formation 22 ; and (ii) formation of misfolded oligomer intermediates as seeds, in which a lag phase is absent in the kinetics curve 23 . Accordingly, when the goal is to induce proteins fibrillation, fibrillation agents exhibiting both polar and nonpolar domains are the best option since they can induce the formation of misfolded oligomer intermediates, while assisting in fast fibrillation by surpassing the lag phase.…”

Effect of position-specific single-point mutations and biophysical characterization of amyloidogenic peptide fragments identified from lattice corneal dystrophy patients

“…This region is clinically important as more than 16 different mutations have been reported and many of them alter the overall net charge of the protein, which leads to the formation of amyloid deposits in the cornea. We recently reported a 23-residue peptide from this region (E 611 PVAEPDIMATNGVVHVITNVLQ 633 ) that forms amyloid fibrils under physiological conditions [ 26 , 27 ]. Thus, we synthesized five different peptides from clinically significant mutations for the present study ( Table 2 ).…”

“…Mutants associated with corneal dystrophies exhibit tissue damage in disease conditions. We have shown previously [ 27 ] that β-oligomeric aggregates of TGFBIp mutants display cytotoxicity in HCSF cells. Analysis of the cytotoxicity of peptides on corneal fibroblasts by label-free xCELLigence in both the amyloidogenic and the prefibrillar forms indicated considerable differences in the inhibition of cell proliferation between the two states, although no cytotoxic effect was observed.…”

Fascinating Fasciclins: A Surprisingly Widespread Family of Proteins that Mediate Interactions between the Cell Exterior and the Cell Surface

“…In many cases of this disease, mutated HsTgfbi forms aggregates in the corneal stroma. This is due to the thermal and pH instability of the protein and to its high abundance in the cornea [ 85 ]; however, the condition might not help us to understand HsTgfbi function. A complete Tgfbi gene knockout in mice caused neither corneal abnormalities nor any other major defects [ 86 ], suggesting compensation by other ECM molecules.…”