L55P Transthyretin Accelerates Subunit Exchange and Leads to Rapid Formation of Hybrid Tetramers

Abstract

Transthyretin is a tetrameric protein associated with the commonest form of systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry, we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, L55P. Wild-type subunit exchange occurs via both monomers and dimers, whereas exchange via dimers is the dominant mechanism for the L55P variant. Because patients with the L55P mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and L55P tetramers. We found that hybrid tetramers containing two or three L55P subunits dominate in the early stages of the reaction. Surprisingly, we also found that, in the presence of L55P transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidosis.

Impact of Deuteration on the Assembly Kinetics of Transthyretin Monitored by Native Mass Spectrometry and Implications for Amyloidoses

“…16 hours after the reaction onset, the MS data also indicated the presence of 2H2D and 1H3D hetero‐tetramers (Figure S4 A). The peaks that could be assigned to the 3H1D hetero‐tetramer were not resolvable from the other signals, as previously reported 14. In the second experiment, 4H and pure CNTTR (4CN) samples were prepared and monitored over 10 days (Figure 3 C and 3D).…”

“…While this study shows consistent and reproducible effects of protein and solvent deuteration on TTR tetramer stability, isotope effects such as these are generally rather subtle and unpredictable. It should be noted that the macromolecular isotope effect described here for DTTR is relatively small by comparison with the effects of serious aggressive mutations such as TTR‐L55P (associated with FAP) where a very much faster subunit exchange has been reported 14. In the overall context of this work, where the faster subunit exchange, decreased tetramer stability, and enhanced fibril formation of the deuterated TTR show similar trends to that observed for L55P, it is interesting to speculate that the use of deuteration may provide a useful probe of the factors underlying amyloidoses associated with TTR and other proteins.…”

“…The latter study was used as a reference, based on the assumption that 13 C‐ and 15 N‐labeling of proteins is not generally thought to introduce significant isotope effects, as noted in previous work where a similar labeling strategy was used to investigate subunit exchange between WT and the L55P mutant of TTR 14. Given these types of mixture, the various possible TTR tetramers resulting from the combination of pure HTTR (4H) and pure DTTR (4D) samples are summarized in Figure S3.…”

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

“…The dynamics of the L7/L12 proteins were investigated using an MS strategy that has proven successful to elucidate the subunit exchange dynamics of other protein complexes (30)(31)(32)(33). Specifically, ribosomes are uniformly labeled with stable isotopes ( 13 C and 15 N) to provide sufficient mass differences to resolve heterocomplexes formed with wild-type ribosomes.…”

Influence of coulombic repulsion on the dissociation pathways and energetics of multiprotein complexes in the gas phase

“…Consequently, the characterization of protein assemblies, their structure, stability, and biological function represent important analytical objectives. Mass spectrometry (MS), with its speed, sensitivity, and specificity, combined with electrospray ionization (ES), is an established tool for detecting specific multiprotein complexes in solution [1], the real time monitoring their assembly/disassembly [2] and subunit exchange reactions [3,4], and identifying and quantifying their interactions with other biopolymers, as well as other ligands and cofactors [5]. Several comprehensive reviews of the applications of ES/MS to protein complexes have appeared, [1,[5][6][7][8][9], including a recent review by Robinson and coworkers [8].…”