神经颗粒素（Neurogranin，Ng）是一种新发现的由78个氨基酸组成的脑特异性蛋白，主要分布于人类或动物的大脑皮层、海马和嗅球等脑区的神经突触后。作为Calpacitin蛋白家族中的一员，Ng是蛋白激酶C的天然作用底物及钙调蛋白（CaM）的储库。在生理状态下，Ng与CaM结合形成复合体，而在蛋白激酶C或氧化剂的作用下，Ng可被磷酸化、氧化及谷胱甘肽化等化学修饰，降低其与CaM的亲和力，从而参与对CaM及CaM2激活的蛋白酶，如CaM2依赖性NO合酶、CaM2依赖性蛋白激酶II（CaMKII）及CaM2依赖性腺苷酸环化酶的调节。同时，由于CaM2依赖性蛋白酶大多参与长时程增强（LTP）和长时程抑制（LTD）的诱导，并且Ng的基因表达和蛋白质合成与神经元的突触形成、分化同步，因此，Ng可能在学习、记忆、神经系统发育（可塑性）等生理性变化中具有重要作用。此外，一些研究表明，Ng还可能参与甲状腺机能减退、睡眠剥夺、衰老及脑低氧预适应等病理生理学变化所造成的神经系统功能的改变。

S-Nitrosoglutathione (GSNO) undergoes spontaneous degradation that generates several nitrogen-containing compounds and oxidized glutathione derivatives. We identified glutathione sulfonic acid, glutathione disulfide S-oxide (GS (O) SG), glutathione disulfide S-dioxide, and GSSG as the major decomposition products of GSNO. Each of these compounds and GSNO were tested for their efficacies to modify rat brain neurogranin/RC3 (Ng) and neuromodulin/GAP-43 (Nm). Among them, GS (O) SG was found to be the most potent in causing glutathiolation of both proteins; four glutathiones were incorporated into the four Cys residues of Ng, and two were incorporated into the two Cys residues of Nm. Ng and Nm are two in vivo substrates of protein kinase C; their phosphorylations by protein kinase C attenuate the binding affinities of both proteins for calmodulin. When compared with their respective unmodified forms, the glutathiolated Ng was a poorer substrate and glutathiolated Nm a better substrate for protein kinase C. Glutathiolation of these two proteins caused no change in their binding affinities for calmodulin. Treatment of [35S] cysteine-labeled rat brain slices with xanthine/xanthine oxidase or a combination of xanthine/xanthine oxidase with sodium nitroprusside resulted in an increase in cellular level of GS (O) SG. These treatments, as well as those by other oxidants, all resulted in an increase in thiolation of proteins; among them, thiolation of Ng was positively identified by immunoprecipitation. These results show that GS (O) SG is one of the most potent glutathiolating agents generated upon oxidative stress.

Neurogranin (NG) binding of calmodulin (CaM) at its IQ domain is sensitive to Ca2+ concentration and to modifications by protein kinase C (PKC) and oxidants. The PKC phosphorylation site of NG is within the IQ domain whereas the four oxidant-sensitive Cys residues are outside this region. These Cys residues were oxidized forming two pairs of intramolecular disulfides, and could also be glutathiolated by S-nitrosoglutathione resulting in the incorporation of four glutathiones per NG. Circular dichroism (CD) showed that modification of NG by phosphorylation, oxidation forming intramolecular disulfides, or glutathiolation did not affect the R-helical content of this protein. Mutation of the four Cys residues [Cys (-)-NG] to Gly and Ser did not affect the R-helical content either. Interaction of CaM with the reduced (red), glutathiolated (GS)-, or Cys(-)-NG in the Ca2+-free solution resulted in an increase in the R-helicity determined by their CD spectra, but relatively little change was seen with the oxidized NG (ox-NG) or phosphorylated NG (PO4-NG). The binding affinities between the various modified forms of NG and CaM were determined by CD spectrometry and sedimentation equilibrium: their affinities were Cys (-)-NG > red-NG, GS-NG > ox-NG > PO4-NG. Unlike Cys (-)-, red-, and GS-NG, neither ox-nor PO4-NG bound to a CaM-affinity column. Thus, both oxidation of NG to form intramolecular disulfides and phosphorylation of NG by PKC are effective in modulating the intracellular level of CaM. These results indicate that modification of NG to form intramolecular disulfides outside the IQ domain provides an alternative mechanism for regulation of its binding affinity to CaM.

探讨cPKCs特定亚型在脑低氧预适应形成过程中的作用，借助已建立的小鼠整体低氧预适应模型，应用SDS-PAGE和Western bolt等生化技术，并结合Gel Doc成像系统，半定量检测脑组织内cPKCα和γ的膜转位水平和蛋白表达量。结果发现，随低氧暴露次数（低氧2-4次）增加，在小鼠海马和皮层组织内cPKCγ膜转位水平增高显著（p<0.05，n=6）；然而，cPKCα的膜转位水平和cPKCα及γ的蛋白表达量的变化均不显著。提示，cPKCγ的激活可能参与了脑低氧预适应的形成过程。

Previous studies have shown that the level of total conventional protein kinase C (cPKC) membrane translocation (activation) was increased in brain of hypoxic preconditioned mice. In order to find out which isoform of cPKC may participate in the development of cerebral hypoxic preconditioning (HPC), we used Western bolt and immunohistochemistry to observe the effects of repetitive hypoxic exposure (H1-H6, n=6 for each group) on the level of cPKC isoform-specific protein expression and its membrane translocation in cortex and hippocampus of mice. We found that levels of cPKC βII and γ membrane translocation were increased significantly (p<0.05 versus normoxic H0 group, n=6) in response to repetitive hypoxic exposure (H1-H4) at early phase of hypoxic preconditioning, but no significant changes of cPKC α and βI membrane translocation while cPKC α, βI, βII and γ protein expression were found both in hippocampus and cortex. In addition, an extensive subcellular redistribution of cPKC βII and γ was detected by immunohistochemistry staining in cortex after repetitive hypoxic exposures (H3). However, a significant decrease in the expression of cPKC γ protein (p<0.05 versus H0 group) was found only in cortex of delayed hypoxic preconditioned mice (H5-6). These results suggested that the activation of cPKC βII and γ may be involved in the early phase of cerebral hypoxic preconditioning and the changes in cPKC γ protein expression may participate in the development of late phase of cerebral hypoxic preconditioning, as well as selective vulnerability to hypoxia both in cortex and hippocampus.