Amyloid formation is associated with structural changes of native polypeptides to monomeric intermediate states and their self-assembly into insoluble aggregates. Characterizations of the amyloidogenic intermediate state are, therefore, of great importance in understanding the early stage of amyloidogenesis. Here, we present NMR investigations of the structural and dynamic properties of the acid-unfolded amyloidogenic intermediate state of the phosphatidylinositol 3-kinase (PI3K) SH3 domain-a model peptide. The monomeric amyloidogenic state of the SH3 domain studied at pH 2.0 (35°C) was shown to be substantially disordered with no secondary structural preferences.15 N NMR relaxation experiments indicated that the unfolded polypeptide is highly flexible on a subnanosecond timescale when observed under the amyloidogenic condition (pH 2.0, 35°C). However, more restricted motions were detected in residues located primarily in the b-strands as well as in a loop in the native fold. In addition, nonnative long-range interactions were observed between the residues with the reduced flexibility by paramagnetic relaxation enhancement (PRE) experiments. These indicate that the acid-unfolded state of the SH3 domain adopts a partly folded conformation through nonnative longrange contacts between the dynamically restricted residues at the amyloid-forming condition.Keywords: amyloids; PI3K SH3; NMR; dynamics; amyloidogenic intermediate; long-range interactions; PRE Interest in the unfolded and partly folded states of proteins has been growing because of their important roles in a variety of biological processes, including cellular signaling and transcriptional activation (Shortle 1993;Dyson and Wright 2005). In addition, it is widely accepted that partly structured states of proteins are involved in early events in the formation of amyloid, which underlies a number of debilitating human diseases such as Alzheimer's and Parkinson's diseases (Dobson 2003). Thus, characterization of the unfolded and partially folded states of proteins is of central importance in understanding the molecular mechanism of the biological processes as well as the amyloid diseases. Multinuclear, multidimensional NMR spectroscopy has proven to be a powerful technique to investigate site-specific structural and dynamic properties of nonnative states of proteins (Shortle 1996;Dyson and Wright 2004;Redfield 2004 Abbreviations: NMR, nuclear magnetic resonance; HSQC, heteronuclear single-quantum coherence; PI3K SH3, phosphatidylinositol 3-kinase Src homology 3; R 2 , transverse relaxation rate; NOE, nuclear Overhauser effect; R 1r , relaxation rate in the rotating frame; H/D, hydrogen/deuterium; PRE, paramagnetic relaxation enhancement; MTSL, (1-oxyl-2,2,5,5-tetramethyl-D3-pyrroline-3-methyl) methanethiosulfonate; UV, ultraviolet; CD, circular dichroism.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi
Much has been learned regarding factors that specify joint placement, but less is known regarding how these molecular instructions are translated into functional joint tissues. Previous studies have shown that the matrix chondroitin sulfate proteoglycan, versican, exhibits a similar pattern of expression in the embryonic joint rudiment of chick and mouse suggesting conserved function during joint development. In the present study versican’s importance in developing joints was investigated by specific inhibition of its expression in the early joint interzone, tissue that gives rise to articular cartilages and joint cavity. In ovo microinjection of adenoviral shRNA constructs into the HH25 chick wing was employed to silence endogenous versican protein in developing appendicular joints. Results showed statistically significant (12–14%) reduction of non-chondrogenic elbow joint interzone area in whole mount specimens at HH36 in response to versican knockdown. Attenuated expression of key versican-associated molecules including hyaluronan, tenascin, CD44, and link protein was also noted by histochemical and immunohistochemical analysis. Versican knockdown also lowered collagen II expression in presumptive articular chondrocytes indicating possible delay in chondrogenesis. Results suggest that versican functions interactively with other matrix/cell surface molecules to facilitate establishment or maintenance of early joint interzone structure.
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