Biomedical subjects

Chuantao Jiang

Publications and source records attributed to Chuantao Jiang.

6 recordsLinked to original sources

PubMed2007

Isomers of human alpha-synuclein stabilized by disulfide bonds exhibit distinct structural and aggregative properties.

The discovery of three mutants in the -synuclein (alphaSyn) gene and the identification of alphaSyn as the major component of Lewy body have opened a new field for understanding the pathogenesis of Parkinson's disease (PD). AlphaSyn is a natively unfolded protein with unknown function and unspecified conformational heterogeneity. In this study, we introduce four Ser/Ala --> Cys mutations at positions 9, 42, 69, and 89 in human wild-type alphaSyn (wt-alphaSyn) and two PD-associated alphaSyn mutants, A30P-alphaSyn and A53T-alphaSyn. This allows expression of three alphaSyn mutants, wt-alphaSyn(4C), A30P-alphaSyn(4C), and A53T-Syn(4C). Subsequent oxidative folding enables each alphaSyn(4C) mutant to form three partially stabilized two-disulfide isomers, designated as alphaSyn(2SS), that are amenable to further isolation and characterization. These alphaSyn mutants exhibit the following properties. (a) A30P-alphaSyn(4C) exhibits a lower folding flexibility than wt-alphaSyn(4C) and A53T-alphaSyn(4C). (b) All three alphaSyn(4C) mutants, like wt-alphaSyn, exhibit a predominant structure of random coil. However, wt-alphaSyn(2SS) adopts an alpha-helical conformation, whereas A30P-alphaSyn(2SS) and A53T-alphaSyn(2SS) take on significant beta-sheet structure. (c) A30P-alphaSyn(2SS) shows a stronger tendency to aggregate than A53T-alphaSyn(2SS) and wt-alphaSyn(2SS). (d) Three isolated isomers of wt-alphaSyn(2SS) exhibit a propensity for forming oligomers different yet enhanced versus that for wt-alphaSyn. These data together substantiate the notion that under physiological conditions, human alphaSyn exists as diverse conformational isomers which exhibit distinct propensities for aggregation and fibril formation.

Amino Acid Sequence↗

PubMed2005

Isomers of epidermal growth factor with Ser --> Cys mutation at the N-terminal sequence: isomerization, stability, unfolding, refolding, and structure.

The structure of human epidermal growth factor (EGF, 53 amino acids) comprises three distinct loops (A, B, and C) connected correspondingly by the three native disulfide bonds, Cys(6)-Cys(20), Cys(14)-Cys(31), and Cys(33)-Cys(42). The connection of Cys(6) and Cys(20) forming the N-terminal A loop is essential for the biological activity of EGF [Barnham et al. (1998) Protein Sci. 7, 1738-1749] and has also been shown to represent a major kinetic trap in the oxidative folding of EGF [Chang et al. (2001) J. Biol. Chem. 276, 4845-4852]. To further understand the chemical nature of this kinetic trap, we have prepared three EGF mutants each with a single Ser --> Cys mutation at Ser residues (Ser(2), Ser(4), and Ser(9)) flanking Cys(6). This allows competition between Cys(6) and mutated Cys(2), Cys(4), and Cys(9) to link with Cys(20) and to form EGF isomers containing different sizes of the A loop. The results show that, in the cases of EGF(S2C) and EGF(S4C), native Cys(6)-Cys(20) is favored over Cys(2)-Cys(20) and Cys(4)-Cys(20) by 4.5- and 9-fold, respectively, in the state of equilibrium. However, in the case of EGF(S9C), a non-native Cys(9)-Cys(20) is thermodynamically more stable than the native Cys(6)-Cys(20) by a free-energy difference (DeltaG degrees ) of 1.12 kcal/mol. Implications of these data in the formation of kinetic trap of EGF folding are discussed. Stabilized isomers of EGF were further generated from denaturation of wild-type and mutant EGF via the method of disulfide scrambling. Properties of these diverse isomers of EGF, including their isomerization, stability, unfolding, refolding, and disulfide structures, are described in this paper.

Amino Acid Sequence↗

PubMed2005

Unfolding and breakdown of insulin in the presence of endogenous thiols.

Native insulin denatures and unfolds in the presence of thiol catalyst via disulfide scrambling (isomerization). It undergoes two transient non-native conformational isomers, followed by an irreversible breakdown of the protein to form oxidized A- and B-chain. Denaturation and breakdown of native insulin may occur under physiological conditions. At 37 degrees C, pH 7.4, and in the presence of cysteine (0.2 mM), native insulin decomposes with a pseudo first order kinetic of 0.075 h(-1). At 50 degrees C, the rate increases by 5-fold. GdnCl and urea induced denaturation of insulin follows the same mechanism. These results demonstrate that stability and unfolding pathway of insulin in the presence of endogenous thiol differ fundamentally from its reversible denaturation observed in the absence of thiol, in which native disulfide bonds of insulin were kept intact during the process of denaturation.

Animals↗

PubMed2005

Neuronal differentiation of NTE-deficient embryonic stem cells.

Organophosphates induce neurological disorders. One of the enzymes inhibited by these compounds is neuropathy target esterase (NTE). In vitro, inhibition of NTE activity by organophosphates is correlated with inhibition of neurite initiation and reduction of neurite length, supporting the hypothesis that organophosphate-induced neurological disorders are caused by inhibition of NTE activity. However, there is no direct evidence for the involvement of NTE in organophosphate-induced impairment of neurites in vitro. To examine the role of NTE, we have generated NTE-deficient mouse embryonic stem cells. These cells can differentiate into neuron-like cells. Although NTE-deficient cells exhibited a delay in neurite initiation in vitro, both the proportion of neuron-like cells which initiated neurites and the elongation of these neurites occurred at the normal rate. These results demonstrate that NTE activity is not required for neurite initiation or elongation per se, but is essential for the optimal rate of neurite initiation.

Animals↗

PubMed2005

Age-dependent dopaminergic dysfunction in Nurr1 knockout mice.

The Nurr1 gene, which codes for a transcriptional factor in the nuclear receptor superfamily, plays an important role in the development of the mesencephalic dopaminergic (DAergic) system. To study the age-dependent effects of Nurr1 expression in maintaining mature nigrostriatal DAergic neuronal function, we examined motor behaviors, determined nigrostriatal dopamine (DA) levels and the number of nigral DAergic neurons, and measured the expression of several DAergic neuron-associated genes in heterozygous Nurr1-deficient (Nurr1+/-) and wild-type mice of different ages. In contrast to the same-aged, wild-type mice, old Nurr1+/- mice (>15 months) had a significant decrease in both rotarod performance and locomotor activities, suggesting a motor impairment that is analogous to parkinsonian deficit. Furthermore, the abnormal motor behaviors in old Nurr1+/- mice were associated with decreased DA levels in the striatum, decreased number of DAergic neurons in the nigra, and reduced expression of Nurr1 and DA transporter in the nigra. Our data indicate that Nurr1 plays an important role in the functional maintenance and survival of nigral DAergic neurons and suggest that the Nurr1+/- mouse is a useful animal model to study the pathogenesis of Parkinson disease (PD) and to explore disease-modifying strategies.

Aging↗

PubMedDate not supplied

Dopaminergic properties and experimental anti-parkinsonian effects of IPX750 in rodent models of Parkinson disease.

With a view toward improving the neural bioavailability of administered dopaminergic compounds, including dopamine, synthetic efforts have been directed toward enhancing the brain bioavailability of these compounds by accessing cellular sugar transport systems with stereoselective dopaminergic drugs. While synthesis and chemistry of the resultant class of compounds has recently been described in US Patent No. 6,548,484, the associated biologic properties have not previously been reported. One member of this new class, IPX-750, is a pro-drug dopamine-gluconamine designed to retain stereospecificity of binding at: glucose transporters (GLUT 1/GLUT 3 and intestinal Na/glucose co-transporters SGLT1), dopamine transporter (DAT); and, dopaminergic receptors of the D1/D2 families. Designed to be cleavable by tissue amidases, results reported here show that intact IPX-750 pro-drug retains dopaminergic agonist binding and biologic activities both in vitro and in vivo. IPX-750, like dopamine, exhibited predominant D5/D1 binding specificity with lower binding activity at D2. As expected, binding was highly stereo-specific, ie, IPX-760, a benzamide differing in just a hydrogen atom and keto oxygen from IPX-750, bound with 6-fold lower activity at D5. In cell culture, activation resulted from binding of IPX-750 at D1 or D5 in transfected cells was measured by increased intracellular cAMP. Interestingly, considering prior reported in vitro toxicity of dopamine oxidized and metabolic product dopamine, no evidence of in vitro toxicity was observed at up to 72 hrs in cell cultures at the EC50 of IPX-750 for increasing intracellular cAMP. IPX-750 was evaluated in the Parkinson's disease animal models, including MPTP mouse model, the 6-hydroxydopamine (6-OHDA) rat model and the Nurr1(+/-) knockout mouse model. In MPTP-lesioned and Nurr1+/- knockout mice, IPX-750 significantly increased Rota-rod time. In 6-OHDA-lesioned rats, IPX-750 significantly decreased apomorphine (APO)-induced rotation. Worthy of note, after cessation of IPX-750 treatments the anti-parkinsonian activity in MPTP-lesioned and Nurr1+/- mice required about 2 weeks to washout, suggesting a possible biologic reservoir of drug. In addition, after eight weeks of twice daily administration of 20 mg/kg IPX-750, mice did not show statistical difference in the total number of TH-positive neurons in substantia nigra (SN). These combined results suggest (i) that stereo-specific glycoconjugation may be an effective method to improve penetrability of drugs through the blood brain barrier; (ii) treatment with bioavailable IPX-750 in vitro did not show evidence for neurotoxicity; and, (iii) IPX-750 possesses dopaminergic properties and exerts anti-parkinsonian effects in three different PD rodent models, suggesting therapeutic potential for this new class of drugs in treating dopamine deficiency diseases.

Animals↗