Yi-chen Chen

The correlation between neurotoxicity, aggregative ability and secondary structure studied by sequence truncated Aβ peptides

Aggregated β‐amyloid (Aβ) peptides are neurotoxic and cause neuronal death both in vitro and in vivo. Although the formation of a β‐sheet structure is usual required to form aggregates, the relationship between neurotoxicity and the Aβ sequence remains unclear. To explore the correlation between Aβ sequence, secondary structure, aggregative ability, and neurotoxicity, we utilized both full‐length and fragment‐truncated Aβ peptides. Using a combination of spectroscopic and cellular techniques, we demonstrated that neurotoxicity and aggregative ability are correlated while the relationship between these characteristics and secondary structure is not significant. The hydrophobic C‐terminus, particularly the amino acids of 17–21, 25–35, and 41–42, is the main region responsible for neurotoxicity and aggregation. Deleting residues 17–21, 25–35 or 41–42 significantly reduced the toxicity. On the other hand, truncation of the peptides at either residues 22–24 or residues 36–40 had little effect on toxicity and aggregative ability. While the N‐terminal residues 1–16 may not play a major role in neurotoxicity and aggregation, a lack of N‐terminal fragment Aβ peptide, (e.g. Aβ17–35), does not display the neurotoxicity of either full‐length or 17–21, 25–35 truncated Aβ peptides.

The effect of PKA-phosphorylation on the structure of inhibitor-1 studied by NMR spectroscopy

Inhibitor-1 is an acid- and heat-stable protein. It can be turned into a potent inhibitor of protein phosphatase-1 (PP1) after phosphorylation at Thr35 by c-AMP-dependent protein kinase (PKA). Although it has been known that pre-phosphorylation is essential for inhibition of PP1, the structure-function relationship of Thr(35)-phosphorylated inhibitor-1, such as whether or not PKA-phosphorylation pre-triggers conformational changes in inhibitor-1, remains unclear. In this study, we performed structural characterization of Thr(35)-phosphoroylated inhibitor-1 by using multi-dimensional heternuclear NMR spectroscopy. The result of structural comparison between Thr(35)-phosphoroylated and non-phosphorylated inhibitor-1 indicated that PKA-phosphorylation has no significant effect on the global conformation of free-state inhibitor-1. This finding may support the inference that regulation of the interactions between inhibitor-1 and PP1 through PKA-phosphorylation mainly depends on the phosphate group instead of phosphorylation-induced conformational change.

Structural and biochemical characterization of inhibitor‐1α

Inhibitor‐1α is one of the isoforms of human protein phosphatase inhibitor‐1. It is a product of alternative splicing of inhibitor‐1 gene and lacks 51 internal amino acids from residue 84 to 134 of inhibitor‐1. Here we have characterized the structural and biochemical properties of inhibitor‐1α. Structural analysis of recombinant inhibitor‐1α by NMR spectroscopy revealed that inhibitor‐1α adopts a predominantly random coil conformation. Excluding the region from residue 84 to 134 of inhibitor‐1, the structural features of inhibitor‐1 and inhibitor‐1α are almost the same as each other. The IC50 value of inhibitor‐1α in inhibition of Protein phosphatase‐1 (PP1) is comparable to that of inhibitor‐1, indicating that inhibitor‐1α is a potent inhibitor of PP1 when Thr‐35 is phosphorylated by PKA. For phosphorylation by PKA and dephosphorylation by protein phosphatase‐1, ‐2A, and ‐2B, the measured kinetic parameters of inhibitor‐1α are very close to those of inhibitor‐1. Taken together, these results suggest that inhibitor‐1α preserves the structure of inhibitor‐1, the PP1 inhibitory activity and the functional specificities toward phosphorylation by PKA and dephosphorylation by protein phosphatase‐1, ‐2A, and ‐2B. Proteins 2007.

Phosphorylation by glycogen synthase kinase of inhibitor-2 does not change its structure in free state

Inhibitor‐2 (I2) is a thermostable protein that specifically binds to the catalytic subunit of protein phosphatase‐1 (PP1), resulting in the formation of the inactive holoenzyme, ATP‐Mg‐dependent phosphatase. Phosphorylation of I2 at Thr‐72 by glycogen synthase kinase‐3 (GSK‐3) results in activation of the phosphatase, suggesting that kinase action triggers conformational change in the complex. In this paper, we characterize the effect of GSK‐3 phosphorylation on the structure of free state I2[1–172] by nuclear magnetic resonance and circular dichroism spectroscopy, and show that phosphorylation has no significant effect on its conformation. We conclude that the conformational changes of ATP‐Mg‐dependent phosphatase induced by GSK‐3 phosphorylation must depend on the interactions between PP1 and I2.