Aggregation of alpha-synuclein is thought to play a major role in the pathogenesis of Parkinson's disease (PD), which is characterized by the presence of intracytoplasmic Lewy bodies (LB) in the brain. alpha-Synuclein and its deletion mutants are largely unfolded proteins with random coil structures as revealed by CD spectra, fluorescence spectra, gel filtration chromatography, and ultracentrifugation. On the basis of its highly unfolded and flexible conformation, we have investigated the chaperone-like activity of alpha-synuclein in vitro. In our experiments, alpha-synuclein inhibited the aggregation of model substrates and protected the catalytic activity of alcohol dehydrogenase and rhodanese during heat stress. In addition, alpha-synuclein inhibited the initial aggregation of reduced/denatured lysozyme on the refolding pathway. Interestingly, deletion of the C-terminal regions led to the abolishment of chaperone activity, although largely unstructured conformations are maintained. Moreover, alpha-synuclein could inhibit the aggregation of various Escherichia coli cellular proteins during heat stress, and C-terminal deletion mutants could not provide any protection to these cellular proteins. Results with synthetic C-terminal peptides and C-terminal deletion mutants suggest that the second acidic repeat, (125)YEMPSEEGYQDYEPEA(140), is important for the chaperone activity of alpha-synuclein, and C-terminal deletion leads to the facilitated aggregation with the elimination of chaperone activity.

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Abstracta-Synuclein, a major constituent of Lewy bodies~LBs! in Parkinson's disease~PD!, has been implicated to play a critical role in synaptic events, such as neuronal plasticity during development, learning, and degeneration under pathological conditions, although the physiological function of a-synuclein has not yet been established. We here present biochemical evidence that recombinant a-synuclein has a chaperone-like function against thermal and chemical stress in vitro. In our experiments, a-synuclein protected glutathione S-transferase~GST! and aldolase from heatinduced precipitation, and a-lactalbumin and bovine serum albumin from dithiothreitol~DTT!-induced precipitation like other molecular chaperones. Moreover, preheating of a-synuclein, which is believed to reorganize the molecular surface of a-synuclein, increased the chaperone-like activity. Interestingly, in organic solvents, which promotes the formation of secondary structure, a-synuclein aggregated more easily than in its native condition, which eventually might abrogate the chaperone-like function of the protein. In addition, a-synuclein was also rapidly and significantly precipitated by heat in the presence of Zn 2ϩ in vitro, whereas it was not affected by the presence of Ca 2ϩ or Mg 2ϩ . Circular dichroism spectra confirmed that a-synuclein underwent conformational change in the presence of Zn 2ϩ . Taken together, our data suggest that a-synuclein could act as a molecular chaperone, and that the conformational change of the a-synuclein could explain the aggregation kinetics of a-synuclein, which may be related to the abolishment of the chaperonic-like activity.

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Most proteins are denatured by heat treatment, and the process is usually irreversible. However, some proteins, such as hyperthermophilic proteins are known to be stable even at the boiling temperature of water. We here describe a systematic investigation of thermal behavior of proteins by purifying and characterizing some heat-resistant proteins (HRPs) that are not aggregated upon heat treatment. Although most proteins were precipitated by boiling in a water bath, about 20 and 70 wt % of total proteins appeared to be heat-resistant in Jurkat T-cell lysates and human serum, respectively. We identified major HRPs from Jurkat T-cells and human serum by N-terminal amino acid sequencing and Western blot analysis. HRPs of 20 and 45 kDa (HRP20 and HRP45) were identified as alpha-synuclein and calreticulin, respectively, and HRPs of 60, 27, and 16 kDa (HRP60, HRP27, and HRP16) were identified as human serum fetuin, apolipoprotein A-I, and transthyretin, respectively. By a systematic investigation of the effect of heat on the secondary structure of the purified HRPs by circular dichroic spectroscopy, we observed four major types of thermal behavior, suggesting that the proteins could protect themselves through these pathways. Although our analysis is restricted to protein secondary structural changes, our data indicate that heat resistance of protein can be achieved in several different ways depending on the thermodynamic stability of native (N), unfolded (U), denatured (D), and intermediate (I) states.

␣-Synuclein, an acidic neuronal protein of 140 amino acids, is extremely heat-resistant and is natively unfolded. Recent studies have demonstrated that ␣-synuclein has chaperone activity both in vitro and in vivo, and that this activity is lost upon removing its C-terminal acidic tail. However, the detailed mechanism of the chaperone action of ␣-synuclein remains unknown. In this study, we investigated the molecular mechanism of the chaperone action of ␣-synuclein by analyzing the roles of its N-terminal and C-terminal domains. The N-terminal domain (residues 1-95) was found to bind to substrate proteins to form high molecular weight complexes, whereas the C-terminal acidic tail (residues 96 -140) appears to be primarily involved in solubilizing the high molecular weight complexes. Because the substrate-binding domain and the solubilizing domain for chaperone function are well separated in ␣-synuclein, the N-terminal-binding domain can be substituted by other proteins or peptides. Interestingly, the resultant engineered chaperone proteins appeared to display differential efficiency and specificity in terms of the chaperone function, which depended upon the nature of the binding domain. This finding implies that the C-terminal acidic tail of ␣-synuclein can be fused with other proteins or peptides to engineer synthetic chaperones for specific purposes.

In children born prematurely, the development of myopia is mainly influenced by anterior segment components, whereas hyperopia is mainly attributable to short AL. Astigmatism is primarily cornea-related. A combination of various optical components results in complicated refractive outcomes. The presence of ROP may be associated with significantly shorter ACD, thicker lens, and higher myopia and astigmatism. (ClinicalTrials.gov number, NCT01045616.).

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