蛋白转导域内在化机制的研究进展

蛋白转导域（protein transduction domain,PTD）可以携带外源生物大分子进入细胞,在分子生物学、细胞生物学的基础研究及生物技术应用中,都展示出良好的前景,应用广泛,但机制不甚明确。已知的PTD均有其关键的特定氨基酸存在和较强的正电荷分布,并具有独特的二级结构及空间构象,这些特殊的结构特征对其内在化机制起决定作用。目前认为巨胞饮作用是PTD入胞的主要机制,PTD在经过细胞表面糖胺聚糖紧密结合快速作用及电荷作用后,由脂筏蛋白介导的巨胞饮作用内在化,然后巨胞饮体脂质双层破裂,使蛋白转导域．大分子释放入胞浆及胞核。     

Preparation and biological properties of native and recombinant ciliary neurotrophic factor

CNTF (ciliary neurotrophic factor), purified from rabbit sciatic nerves by a relatively simple procedure, is bioactive in tissue culture at low picomolar concentration and appears as a doublet on polyacrylamide gel electrophoresis (PAGE). In these nerves, CNTF accounts for more than one-half of the survival-promoting activity on ciliary neurons. The concentration of CNTF in rabbit sciatic nerves is estimated to be 5 nmol/kg, more than 1000 times higher than would seem to be required to support neurons if the neurotrophic factor were homogeneously distributed. With recombinant DNA technology, rat CNTF has been synthesized in Escherichia coli, purified without denaturating agents, and found to be bioactive at a slightly lower concentration than CNTF extracted from rabbit sciatic nerves. After radioiodination, CNTF retains biological activity but is not specifically internalized and retrogradely transported in motor and sensory axons. In peripheral nerves, ciliary neurotrophic factor differs biologically from nerve growth factor (NGF) by its much higher tissue concentration and apparent lack of internalization by peripheral nerve axons. © 1992 John Wiley & Sons, Inc.     

Short- and long-term effects of ciliary neurotrophic factor on androgen-sensitive motoneurons in the lumbar spinal cord

Motoneuron death in the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN) of the lumbar spinal cord is androgen regulated. As a result, many more SNB and DLN motoneurons die in perinatal female rats than in males, whereas treatment of newborn females with androgen results in a permanent sparing of the motoneurons and their target muscles. We previously observed that a neurotrophic molecule, ciliary neurotrophic factor (CNTF), also arrests the death of SNB motoneurons and their target musculature, at least in the short term. The present study compares the short- and long-term consequences of perinatal CNTF treatment on motoneuron number in the SNB, the DLN, and the retrodorsolateral nucleus (RDLN), a motor pool in the lower lumbar cord that does not exhibit hormone-regulated cell death. Female pups were treated with CNTF or vehicle alone from embryonic day 22 through postnatal day 6 (P6). Motoneuron number in each nucleus was then determined immediately after treatment on P7, or 10 weeks later (P77). CNTF treatment significantly elevated motoneuron number in the SNB and DLN on P7; the volume of SNB target muscles on P7 was also greater in the CNTF-treated group. These effects were transient, however, as motoneuron number and ratings of muscle size were not different in CNTF- and vehicle-treated females on P77. Perinatal CNTF treatment did not alter cell number in the RDLN at either age. The finding that effects of CNTF on SNB and DLN motoneuron number are short lived contrasts with the permanent effects of early androgen treatment, and has implications for molecular models of the actions of androgen and neurotrophic factors on the developing spinal cord. © 1996 John Wiley & Sons, Inc.     

睫状神经营养因子对应激引起大鼠海马神经元损伤的保护作用及其机制的研究

本实验用Ｎｉｓｓｌ染色法、Ｂｉｅｌｓｃｈｏｗｓｋｙ－Ｇｒｏｓ－Ｌａｗｒｅｎｔｊｅｗ染色法、常规透射电镜、行为活动测定、双侧海马微量给药、海马神经元原代培养、活细胞连续照相、全细胞膜片钳记录、细胞内游离Ｃａ＾２＋浓度测定及Ｐ５３蛋白免疫组化测定等方法,观察了睫状神经营养因子（ＣＮＴＦ）对应激引起动物行为变化和海马神经元形态学变化的影响,探讨了ＣＮＴＦ的部分作用机制。结果表明,急性应激不引起大鼠海马神     

Ciliary neurotrophic factor upregulates ubiquitin-proteasome components in a rat model of neuronal injury

Neuronal injury triggers the release of ciliary neurotrophic factor (CNTF), promoting local neuronal repair but producing systemic effects of anorexia and lean body weight loss. Due to the rapid rate of systemic protein loss stimulated by CNTF, we hypothesized involvement of the hepatic ubiquitin-proteasome proteolytic (UPP) pathway in CNTF-induced proteolysis. To assess the role of central CNTF in systemic UPP regulation, we measured hepatic UPP mRNA and proteasome activity in a rat model of neuronal injury and determined alterations induced by intracerebroventricular (ICV) administration of CNTF-neutralizing antibody or additional exogenous CNTF. We also assessed proteolytic parameters and nutritional status by measuring caloric intake, body weight, and protein levels. We produced neuronal injury by implanting a lateral ventricle cannula and giving daily ICV saline bolus injections, which increased hepatic 20S proteasome mRNA and enzymatic activity while reducing caloric intake, body weight, and protein levels compared to controls. Administration of ICV anti-CNTF antibodies (but not control antibodies) prevented these effects. Addition of exogenous CNTF augmented the weight loss along with the increases in 20S proteasome mRNA and proteolytic activity induced by neuronal injury. We conclude that CNTF decreases lean body weight through a combination of appetite inhibition and UPP pathway activation.     

Inducers of oxidative stress block ciliary neurotrophic factor activation of Jak/STAT signaling in neurons

Generation of reactive oxygen species (ROS) with the accumulation of oxidative damage has been implicated in neurodegenerative disease and in the degradation of nervous system function with age. Here we report that ROS inhibit the activity of ciliary neurotrophic factor (CNTF) in nerve cells. Treatment with hydrogen peroxide (H(2)O(2)) as a generator of ROS inhibited CNTF-mediated Jak/STAT signaling in all cultured nerve cells tested, including chick ciliary ganglion neurons, chick neural retina, HMN-1 motor neuron hybrid cells, and SH-SY5Y and BE(2)-C human neuroblastoma cells. H(2)O(2) treatment of non-neuronal cells, chick skeletal muscle and HepG2 hepatoma cells, did not inhibit Jak/STAT signaling. The H(2)O(2) block of CNTF activity was seen at concentrations as low as 0.1 mm and within 15 min, and was reversible upon removal of H(2)O(2) from the medium. Also, two other mediators of oxidative stress, nitric oxide and rotenone, inhibited CNTF signaling. Treatment of neurons with H(2)O(2) and rotenone also inhibited interferon-gamma-mediated activation of Jak/STAT1. Depleting the intracellular stores of reduced glutathione by treatment of BE(2)-C cells with nitrofurantoin inhibited CNTF activity, whereas addition of reduced glutathione protected cells from the effects of H(2)O(2). These results suggest that disruption of neurotrophic factor signaling by mediators of oxidative stress may contribute to the neuronal damage observed in neurodegenerative diseases and significantly affect the utility of CNTF-like factors as therapeutic agents in preventing nerve cell death.     

Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain

A number of studies have shown that a short peptide, the protein transduction domain (PTD) derived from the HIV-1 Tat protein (Tat-PTD) improved cellular uptake in vitro and distribution in vivo of recombinant proteins bearing such PTDs when administered systemically. To investigate the effects of Tat-PTD addition on the subcellular localization of the lysosomal enzyme galactocerebrosidase (GALC, EC 3.2.2.46) and with a view towards designing improved therapeutic strategies for Krabbe disease (globoid cell leukodystrophy), mouse GALC was tagged C-terminally with the Tat-PTD. Compared with unmodified GALC, GALC bearing a Tat-PTD, a myc epitope and 6 consecutive His residues [GALC-TMH (Tat-PTD, a myc epitope and 6 consecutive His residues)] was found to be secreted more efficiently. Also, GALC-TMH was found to be taken up by cells both via mannose-6-phosphate receptor (M6PR)-mediated endocytosis as well as by M6PR-independent mechanisms. GALC-TMH displayed increased M6PR-independent uptake in fibroblasts derived from twitcher mice (a murine model of globoid cell leukodystrophy) and in neurons derived from the mouse brain cortex compared with GALC lacking a Tat-PTD. Immunocytochemical analyses revealed that Tat-modified GALC protein co-localized in part with the lysosome-associated membrane protein-1. Complete correction of galactosylceramide accumulation was achieved in twitcher mouse fibroblasts lacking GALC activity following addition of GALC-TMH. Therefore, GALC-TMH not only maintained the features of the native GALC protein including enzymatic function, intracellular transport and location, but also displayed more efficient cellular uptake.     

Brain-derived neurotrophic factor shows a protective effect and improves recovery of the ERG b-wave response in light-damage

We investigated the neuroprotective effects of brain-derived neurotrophic factor (BDNF) and its influence on the functional recovery of the retina following light-induced retinal damage by electroretinogram (ERG). Rats were exposed to constant fluorescent light for 2, 5, 7, or 14 days, then returned to a cyclic light environment for 14 days. The result indicated that BDNF had few effects on the a-wave amplitude, but there was a statistically significant difference in the b-wave amplitudes between BDNF-treated and control eyes from day 0-14 of the recovery period following 2 days of light exposure (p < 0.05). Our findings suggest that BDNF not only protects the retinal neuronal function but also enhances the recovery from retinal light damage.     

Brain-derived neurotrophic factor regulates LYN kinase–mediated myosin light chain kinase activation to modulate nonmuscle myosin II activity in hippocampal neurons

Myosins belong to a large superfamily of actin-dependent molecular motors. Nonmuscle myosin II (NM II) is involved in the morphology and function of neurons, but little is known about how NM II activity is regulated. Brain-derived neurotrophic factor (BDNF) is a prevalent neurotrophic factor in the brain that encourages growth and differentiation of neurons and synapses. In this study, we report that BDNF upregulates the phosphorylation of myosin regulatory light chain (MLC2), to increases the activity of NM II. The role of BDNF on modulating the phosphorylation of MLC2 was validated by using Western blotting in primary cultured hippocampal neurons. This result was confirmed by injecting BDNF into the dorsal hippocampus of mice and detecting the phosphorylation level of MLC2 by Western blotting. We further perform coimmunoprecipitation assay to confirm that this process depends on the activation of the LYN kinase through binding with tyrosine kinase receptor B, the receptor of BDNF, in a kinase activity-dependent manner. LYN kinase subsequently phosphorylates MLCK, further promoting the phosphorylation of MLC2. Taken together, our results suggest a new molecular mechanism by which BDNF regulates MLC2 activity, which provides a new perspective for further understanding the functional regulation of NM II in the nervous system.     

All‐trans retinoic acid upregulates the expression of ciliary neurotrophic factor in retinal pigment epithelial cells

Retinopathy of prematurity, a leading cause of visual impairment in low birth‐weight infants, remains a crucial therapeutic challenge. Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that promotes rod and cone photoreceptor survival and cone outer segment regeneration in the degenerating retina. Ciliary neurotrophic factor expression is regulated by many factors such as all‐trans retinoic acid (ATRA). In this study, we found that ATRA increased CNTF expression in mouse retinal pigment epithelial (RPE) cells in a dose‐ and time‐dependent manner, and PKA signaling pathway is necessary for ATRA‐induced CNTF upregulation. Furthermore, we showed that ATRA promoted CNTF expression through CREB binding to its promoter region. In addition, CNTF levels were decreased in serum of retinopathy of prematurity children and in retinal tissue of oxygen‐induced retinopathy mice. In mouse RPE cells cultured with high oxygen, CNTF expression and secretion were decreased, but could be recovered after treatment with ATRA. In conclusion, our data suggest that ATRA administration upregulates CNTF expression in RPE cells.     

Expression and androgen regulation of the ciliary neurotrophic factor receptor (CNTFRα) in muscles and spinal cord

We have previously observed that ciliary neurotrophic factor (CNTF) can prevent the degeneration of androgen-sensitive perineal motoneurons and their target muscles, the bulbocavernosus and levator ani (BC/LA), in perinatal female rats. Response to CNTF is dependent on the expression of the alpha component of the CNTF receptor (CNTFRα). In the present study, we examined the developmental profile and androgen regulation of CNTFRα gene expression in BC/LA muscle, thigh muscle, and lumbosacral spinal cord. CNTFRα mRNA was abundantly expressed in the BC/LA and thigh around the time of birth; expression declined progressively after birth and remained low into adulthood. In contrast, CNTFRα message remained high in the lumbosacral spinal cord throughout development. Androgen regulation of CNTFRα expression was examined in prenatal animals by administering the androgen receptor blocker hydroxyflutamide from embryonic days E18 through E21. Four days of androgen deprivation caused a significant up-regulation of CNTFRα mRNA in the BC/LA, thigh, and spinal cord of male fetuses. After castration in adulthood, CNTFRα expression in the BC/LA transiently increased, then decreased below control levels. Expression of CNTFRα in thigh muscles and the lumbosacral spinal cord was not affected by adult castration. Thus, the perineal muscles and motoneurons are potential sites of direct CNTF action, and expression of the CNTFRα gene is modulated by androgen, especially in the androgen-sensitive perineal muscles. Transient up-regulation of CNTFRα following castration or androgen receptor blockade may represent a protective response designed to counteract the muscle atrophy normally induced by androgen withdrawal. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 217–225, 1998     

The excitoprotective effect of N-methyl-D-aspartate receptors is mediated by a brain-derived neurotrophic factor autocrine loop in cultured hippocampal neurons

The neuroprotective effect and molecular mechanisms underlying preconditioning with N-methyl-D-aspartate (NMDA) in cultured hippocampal neurons have not been described. Pre-incubation with subtoxic concentrations of the endogenous neurotransmitter glutamate protects vulnerable neurons against NMDA receptor-mediated excitotoxicity. As a result of physiological preconditioning, NMDA significantly antagonizes the neurotoxicity resulting from subsequent exposure to an excitotoxic concentration of glutamate. The protective effect of glutamate or NMDA is time- and concentration-dependent, suggesting that sufficient agonist and time are required to establish an intracellular neuroprotective state. In these cells, the TrkB ligand, brain-derived neurotrophic factor (BDNF) attenuates glutamate toxicity. Therefore, we tested the hypothesis that NMDA protects neurons via a BDNF-dependent mechanism. Exposure of hippocampal cultures to a neuroprotective concentration of NMDA (50 microM) evoked the release of BDNF within 2 min without attendant changes in BDNF protein or gene expression. The accumulated increase of BDNF in the medium is followed by an increase in the phosphorylation (activation) of TrkB receptors and a later increase in exon 4-specific BDNF mRNA. The neuroprotective effect of NMDA was attenuated by pre-incubation with a BDNF-blocking antibody and TrkB-IgG, a fusion protein known to inhibit the activity of extracellular BDNF, suggesting that BDNF plays a major role in NMDA-mediated survival. These results demonstrate that low level stimulation of NMDA receptors protect neurons against glutamate excitotoxicity via a BDNF autocrine loop in hippocampal neurons and suggest that activation of neurotrophin signaling pathways plays a key role in the neuroprotection of NMDA.     

Use of a fusion protein to obtain crystals suitable for X-ray analysis: crystallization of a GST-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF.

Crystals of glutathione-S-transferase (GST)-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF, were obtained under crystallization conditions similar to those for GST. Preliminary X-ray crystallographic analysis revealed that crystals of the GST-fused protein belong to space group P6(1)22 or P6(5)22 with unit cell dimensions a = b = 140.4 A, c = 93.5 A and gamma = 120 degrees, having one molecule in the crystallographic asymmetric unit. The crystals diffract to 2.5 A resolution. The cell dimensions are related to those of GST crystals thus far reported. Crystallization of the DNA-binding domain that was cleaved from the fused protein by thrombin was also carried out using several methods under numerous conditions, but efforts to produce well-ordered large crystals were unsuccessful. A possible application of GST-fusion proteins for small target proteins or domains to obtain crystals suitable for X-ray structure determination is proposed.     

Cloning, expression, purification and functional characterization of the oligomerization domain of Bcr–Abl oncoprotein fused to the cytoplasmic transduction peptide

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to localize in the cytoplasmic compartment and is, therefore, applicable for cytoplasmic targeting. The Bcr–Abl fusion protein, playing major causative role in chronic myeloid leukemia (CML), is a cytoplasmic oncoprotein that contains an N-terminus oligomerization domain (OD) mediating homodimerization of Bcr–Abl proteins, and an intact OD in Bcr–Abl is required both for the activation of its transforming activity and tyrosine kinase. Therefore, disrupting Bcr–Abl oligomerization represents a potential therapeutic strategy for inhibiting Bcr–Abl oncogenicity. In this study, we explored the possible homodimerization-disrupting and tyrosine kinase inhibiting effect of the transduction of OD in Bcr–Abl positive K562 cells. By expressing in Escherichia coli a CTP-OD-HA fusion protein followed by Ni⁺–NTA affinity purification, immunoblot identification and enterokinase cleavage, we showed that the CTP-OD-HA protein was structurally and functionally active in that it potently transduced and primarily localized into the cytoplasmic compartment, heterodimerized with Bcr–Abl, and potently inhibited the phospho-tyrosine pathways of Bcr–Abl oncoprotein at a low concentration of 4 μM. These results delineate strategies for the expression and purification of therapeutic molecules for intracytoplasmic protein based therapeutics and the CTP-OD-HA-mediated killing strategy could be explored as a promising anti-leukemia agent or an adjuvant to the conventional therapeutic modalities in chronic myeloid leukemia, such as in vitro purging.     

Expression of CNTF/LIF‐receptor components and activation of STAT3 signaling in axotomized facial motoneurons: Evidence for a sequential postlesional function of the cytokines

Several lines of evidence suggest that ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are important for the survival and regeneration of axotomized motoneurons. To investigate the role of CNTF/LIF signaling in regenerative responses of motoneurons, we studied the expression of the three receptor components, CNTF receptor α (CNTFRα), LIF receptor β (LIFRβ), and gp130, and the activation of the STAT3 signal transduction pathway in the rat facial nucleus following peripheral nerve transection. As shown by in situ hybridization and immunoblotting, axotomy resulted in a rapid down‐regulation of CNTFRα mRNA expression within 24 h and a concomitant massive up‐regulation of LIFRβ mRNA and protein in the lesioned motoneurons. The altered mRNA levels were maintained for 3 weeks but had returned back to control levels by 6 weeks postlesion after successful regeneration. In contrast, mRNA levels remained in the lesioned state during the 6‐week period studied, when regeneration was prevented by nerve resection. Significant lesion‐induced changes in gp130 mRNA levels were not detectable. Rapid (within 24 h) and sustained (for at least 5 days) activation of STAT3 in axotomized facial motoneurons was revealed by demonstrating the phosphorylation and nuclear translocation of the protein using immunocytochemistry and immunoblotting. In agreement with previous studies showing a complementary regulation of CNTF and LIF in the lesioned facial nerve, our observations on the postlesional regulation of CNTF/LIF receptor components in the facial nucleus indicate a direct and sequential action of the two neurotrophic proteins on axotomized facial motoneurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 559–571, 1999     

Neurotrophic and neuroprotective effects of the neuregulin glial growth factor-2 on dopaminergic neurons in rat primary midbrain cultures

Glial growth factor-2 (GGF2) and other neuregulin (NRG) isoforms have been shown to play important roles in survival, migration, and differentiation of certain neural and non-neural cells. Because midbrain dopamine (DA) cells express the NRG receptor, ErbB4, the present study examined the potential neurotrophic and/or neuroprotective effects of GGF2 on cultured primary dopaminergic neurons. Embryonic day 14 rat mesencephalic cell cultures were maintained in serum-free medium and treated with GGF2 or vehicle. The number of tyrosine hydroxylase-positive (TH+) neurons and high-affinity [3H]DA uptake were assessed at day in vitro (DIV) 9. Separate midbrain cultures were treated with 100 ng/mL GGF2 on DIV 0 and exposed to the catecholamine-specific neurotoxin 6-hydroxydopamine (6-OHDA) on DIV 4. GGF2 treatment significantly increased DA uptake, the number of TH+ neurons, and neurite outgrowth when compared to the controls in both the serum-free and the 6-OHDA-challenged cultures. Furthermore, three NRG receptors were detected in the midbrain cultures by western blot analysis. Immunostaining for glial fibrillary acidic protein revealed that GGF2 also weakly promoted mesencephalic glial proliferation in the midbrain cultures. These results indicate that GGF2 is neurotrophic and neuroprotective for developing dopaminergic neurons and suggest a role for NRGs in repair of the damaged nigrostriatal system that occurs in Parkinson's disease.