14-3-3蛋白家族及其临床应用研究进展

14-3-3蛋白家族是一组高度保守的可溶性酸性蛋白质,分子量在28～33kD之间,广泛分布于各种真核生物之中。该蛋白能够特异地结合含有磷酸化丝氨酸或苏氨酸的肽段,参与多种信号转导途径。14-3-3蛋白调节着许多重要细胞生命活动,如:新陈代谢、细胞周期、细胞生长发育、细胞的存活和凋亡以及基因转录,该蛋白家族异常与疾病的发生密切相关,尤其是14-3-3蛋白在脑脊液中的分布与一些神经系统疾病密切相关。14-3-3蛋白已成为一些疾病的临床诊断指标,其作为疾病治疗的靶点也在研究之中。主要阐述了14-3-3蛋白的结构、功能、及其在疾病治疗中的应用。     

14-3-3 proteins as potential therapeutic targets

The 14-3-3 family of phosphoserine/phosphothreonine-binding proteins dynamically regulates the activity of client proteins in various signaling pathways that control diverse physiological and pathological processes. In response to environmental cues, 14-3-3 proteins orchestrate the highly regulated flow of signals through complex networks of molecular interactions to achieve well-controlled physiological outputs, such as cell proliferation or differentiation. Accumulating evidence now supports the concept that either an abnormal state of 14-3-3 protein expression, or dysregulation of 14-3-3/client protein interactions, contributes to the development of a large number of human diseases. In particular, clinical investigations in the field of oncology have demonstrated a correlation between upregulated 14-3-3 levels and poor survival of cancer patients. These studies highlight the rapid emergence of 14-3-3 proteins as a novel class of molecular target for potential therapeutic intervention. The current status of 14-3-3 modulator discovery is discussed.     

14-3-3 proteins in neurodegeneration

Among the first reported functions of 14-3-3 proteins was the regulation of tyrosine hydroxylase (TH) activity suggesting a possible involvement of 14-3-3 proteins in Parkinson's disease. Since then the relevance of 14-3-3 proteins in the pathogenesis of chronic as well as acute neurodegenerative diseases, including Alzheimer's disease, polyglutamine diseases, amyotrophic lateral sclerosis and stroke has been recognized. The reported function of 14-3-3 proteins in this context are as diverse as the mechanism involved in neurodegeneration, reaching from basal cellular processes like apoptosis, over involvement in features common to many neurodegenerative diseases, like protein stabilization and aggregation, to very specific processes responsible for the selective vulnerability of cellular populations in single neurodegenerative diseases.Here, we review what is currently known of the function of 14-3-3 proteins in nervous tissue focussing on the properties of 14-3-3 proteins important in neurodegenerative disease pathogenesis.     

14-3-3 Protein, Total Tau and Phosphorylated Tau in Cerebrospinal Fluid of Patients with Creutzfeldt-Jakob Disease and Neurodegenerative Disease in Japan

1.Sporadic Creutzfeldt-Jakob disease (CJD) is a rapidly progressive and fatal disease. Patients with CJD usually become akinetic mutism within approximately 6 months. In addition, clinical signs and symptoms at early stage of sporadic CJD may not be easy to distinguish from other neurodegenerative diseases by neurological findings. However, diagnostic biochemical parameters including 14-3-3 protein, S100, neuron-specific enorase in cerebrospinal fluid (CSF) have been used as diagnostic markers, elevated titers of these markers can also be observed in CSF in other neurodegenerative diseases. Therefore, we examined other biochemical markers to discriminate CJD from other neurodegenerative diseases in CSF. 2.We analyzed CSF samples derived from 100 patients with various neurodegenerative disorders by Western blot of 14-3-3 protein, quantification of total tau (t-tau) protein, and phosphorylated tau (p-tau) protein. All patients with CJD in this study showed positive 14-3-3 protein and elevated t-tau protein (>1000 pg/mL) in CSF. We also detected positive 14-3-3 protein bands in two patients in non-CJD group (patients with dementia of Alzheimer's type; DAT) and also detected elevated t-tau protein in three patients in non-CJD group. Elevated t-tau protein levels were observed in two patients with DAT and in one patient with cerevrovascular disease in acute phase. 3.To distinguish patients with CJD from non-CJD patients with elevated t-tau protein in CSF, we compared the ratio of p-tau and t-tau proteins. The p-/t-tau ratio was dramatically and significantly higher in DAT patients rather than in CJD patients. 4.Therefore, we concluded that the assay of t-tau protein may be useful as 1st screening and the ratio of p-tau protein/t-tau protein would be useful as 2nd screening to discriminate CJD from other neurodegenerative diseases.     

Parkin-mediated K63-linked polyubiquitination: a signal for targeting misfolded proteins to the aggresome-autophagy pathway

Pathological inclusions containing misfolded proteins are a prominent feature common to many age-related neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. In cultured cells, when the production of misfolded proteins exceeds the capacity of the chaperone refolding system and the ubiquitin-proteasome degradation pathway, misfolded proteins are actively transported along microtubules to pericentriolar inclusions called aggresomes. The aggresomes sequester potentially toxic misfolded proteins and facilitate their clearance by autophagy. The molecular mechanism(s) that targets misfolded proteins to the aggresome-autophagy pathway is mostly unknown. Our recent work identifies parkin-mediated K63-linked polyubiquitination as a signal that couples misfolded proteins to the dynein motor complex via the adaptor protein histone deacetylase 6 and thereby promotes sequestration of misfolded proteins into aggresomes and subsequent clearance by autophagy. Our findings provide insight into the mechanisms underlying aggresome formation and suggest that parkin and K63-linked polyubiquitination may play a role in the autophagic clearance of misfolded proteins.     

14-3-3 proteins in neurological disorders

14-3-3 proteins were originally discovered as a family of proteins that are highly expressed in the brain. Through interactions with a multitude of binding partners, 14-3-3 proteins impact many aspects of brain function including neural signaling, neuronal development and neuroprotection. Although much remains to be learned and understood, 14-3-3 proteins have been implicated in a variety of neurological disorders based on evidence from both clinical and laboratory studies. Here we will review previous and more recent research that has helped us understand the roles of 14-3-3 proteins in both neurodegenerative and neuropsychiatric diseases.     

14-3-3蛋白参与植物应答非生物胁迫的研究进展

14-3-3蛋白是一种在真核生物细胞中普遍存在且高度保守的蛋白。该蛋白在大多数物种中由一个基因家族编码,并以同源或异源二聚体的形式存在。不同的14-3-3蛋白同工型具有不同的细胞特异性,可通过识别特异的磷酸化或非磷酸化序列与靶蛋白相互作用。14-3-3蛋白在植物生长和发育的各个方面都起重要作用。本文主要围绕植物14-3-3蛋白的种类、结构、磷酸化或非磷酸化识别序列及其响应干旱、冷冻、盐碱、营养和机械胁迫等的分子机制研究进展进行综述。     

Modulation of subfamily B/R4 RGS protein function by 14-3-3 proteins

Regulator of G protein signalling (RGS) proteins are primarily known for their ability to act as GTPase activating proteins (GAPs) and thus attenuate G protein function within G protein-coupled receptor (GPCR) signalling pathways. However, RGS proteins have been found to interact with additional binding partners, and this has introduced more complexity to our understanding of their potential role in vivo. Here, we identify a novel interaction between RGS proteins (RGS4, RGS5, RGS16) and the multifunctional protein 14-3-3. Two isoforms, 14-3-3β and 14-3-3ε, directly interact with all three purified RGS proteins and data from in vitro steady state GTP hydrolysis assays show that 14-3-3 inhibits the GTPase activity of RGS4 and RGS16, but has limited effects on RGS5 under comparable conditions. Moreover in a competitive pull-down experiment, 14-3-3ε competes with Go for RGS4, but not for RGS5. This mechanism is further reinforced in living cells, where 14-3-3ε sequesters RGS4 in the cytoplasm and impedes its recruitment to the plasma membrane by G protein. Thus, 14-3-3 might act as a molecular chelator, preventing RGS proteins from interacting with G, and ultimately prolonging the signal transduction pathway. In conclusion, our findings suggest that 14-3-3 proteins may indirectly promote GPCR signalling via their inhibitory effects on RGS GAP function.     

Ataxin-3 Regulates Aggresome Formation of Copper-Zinc Superoxide Dismutase (SOD1) by Editing K63-linked Polyubiquitin Chains

Polyubiquitination of misfolded proteins, especially K63-linked polyubiquitination, is thought to be associated with the formation of inclusion bodies. However, it is not well explored whether appropriate editing of the different types of ubiquitin linkages by deubiquitinating enzymes (DUBs) affects the dynamics of inclusion bodies. In this study, we report that a specific DUB, ataxin-3, is required for the efficient recruitment of the neurodegenerative disease-associated protein copper-zinc superoxide dismutase (SOD1) to aggresomes. The overexpression of ataxin-3 promotes mutant SOD1 aggresome formation by trimming K63-linked polyubiquitin chains. Moreover, knockdown of ataxin-3 decreases mutant SOD1 aggresome formation and increases cell death induced by mutant SOD1. Thus, our data suggest that the sequestration of misfolded SOD1 into aggresomes, which is driven by ataxin-3, plays an important role in attenuating protein misfolding-induced cell toxicity.     

Isoform pattern of 14-3-3 proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease

Two-dimensional polyacrylamide gel electrophoresis of CSF has been used in the diagnosis of Creutzfeldt-Jakob disease (CJD). One of the two diagnostic protein spots was identified as isoform(s) of the 14-3-3 family of abundant brain proteins. This has led to the development of one-dimensional 14-3-3 sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot, which is currently used to support the diagnosis of CJD. In the present study employing western blot analysis, we have identified the panel of 14-3-3 isoforms that appear in the CSF of 10 patients with CJD compared with 10 patients with other dementias. The results clearly show that the 14-3-3 isoforms beta, gamma, epsilon, and eta are present in the CSF of patients with CJD and can be used to differentiate other dementias. 14-3-3eta also gave a baseline signal in all patients with other dementias, including six patients with Alzheimer's disease. The presence of 14-3-3eta in the CSF of a patient with herpes simplex encephalitis was particularly noteworthy. This study has determined that isoform-specific 14-3-3 antibodies against beta, gamma, and epsilon should be considered for the neurochemical differentiation of CJD from other neurodegenerative diseases.     

14-3-3蛋白对植物发育的调控作用

14-3-3蛋白是高度保守并在真核生物中普遍存在的一类调节蛋白。不同的14-3-3蛋白同工型具有不同的细胞特异性, 并通过识别特异的磷酸化序列与靶蛋白相互作用, 被称为蛋白质与蛋白质相互作用的桥梁蛋白。在植物生长发育过程中, 14-3-3蛋白通过与其它蛋白的相互作用参与多种植物激素信号转导、各种代谢调控、物质运输和光信号应答等调控过程。该文主要对近年来有关14-3-3蛋白在植物生长发育中的调控作用, 特别是14-3-3蛋白参与调控植物激素信号转导等方面的研究进展进行综述。     

Aggresomes——错误折叠蛋白的一种普遍细胞反应

通常,细胞中的错误折叠蛋白质会被蛋白酶体降解。但是在一定的病理和生理条件下,一些错误折叠蛋白质几乎不被降解,反而可以形成蛋白质聚集体。研究表明,许多疾病,如神经退行性疾病的发病机理与错误折叠蛋白质在细胞内的聚集体沉积有关。这些蛋白质聚集体可以通过微管上动力蛋白依赖的逆行性运输形式传送、聚集,最终形成aggresomes。早新的报道还指出,蛋白质聚集体能直接损伤泛素—蛋白酶体系统的功能,从而引起细胞的调控紊乱和细胞死亡。     

Anoxic preconditioning up-regulates 14-3-3 protein expression in neonatal rat cardiomyocytes through extracellular signal-regulated protein kinase 1/2

Anoxic preconditioning (APC) attenuates myocardial injury caused by ischemia/reperfusion. The protective mechanisms of APC involve up-regulation of the protective proteins and inhibition of apoptosis. 14-3-3 protein, as a molecular chaperone, plays an important role in regulating cell survival and apoptosis. However, the role of 14-3-3 protein in cardioprotection of APC and the pathways determining 14-3-3 protein expression during APC are not clear. In this work, Western blotting analysis was used to detect the 14-3-3 protein expression and activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in cardiomyocytes subjected to anoxia-reoxygenation injury with and without APC and control. The cardiomyocytes from APC group were more resistant to injury induced by anoxia-reoxygenation and had much stronger phosphorylation of ERK1/2 than the control. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in cardiomyocytes induced by APC. The results indicate that APC up-regulates 14-3-3 protein expression through the ERK1/2 signaling pathways.     

神经变性构象病及其分子基础

构象病的概念被广泛用于命名与蛋白质的构象异常相关的疾病。随着生命科学的进步,人们对神经变性疾病发病的分子机制有了较好的认识,发现几乎所有的此类疾病,诸如阿尔采末病（AD）、帕金森病（PD）、亨廷顿病（HD）以及朊蛋白病（PrD）等都具有一个共同的特征,即病变细胞中蓄积有大量错误折叠并易于聚合的蛋白质,这符合构象病的特点,所以又派生了神经变性构象病的新概念。近年来,人们在神经变性构象病的蛋白质错误折叠和聚合以及其细胞毒性方面的认识越来越走向深入,这将对寻找有效的治疗方法起到极大的推动作用。     

Interaction between 14-3-3β and PrP influences the dimerization of 14-3-3 and fibrillization of PrP106–126

Proteins of the 14-3-3 family are universal participate in multiple cellular processes. However, their exact role in the pathogenesis of prion diseases remains unclear. In this study, we proposed that human PrP was able to form molecular complex with 14-3-3β. The domains responsible for the interactions between PrP and 14-3-3β were mapped at the segments of amino acid (aa) residues 106–126 within PrP and aa 1–38 within 14-3-3β. Homology modeling revealed that the key aa residues for molecular interaction were D22 and D23 in 14-3-3β as well as K110 in PrP. Mutations in these aa residues inhibited the interaction between the two proteins in vitro. Our results also showed that recombinant PrP encouraged 14-3-3β dimer formation, whereas PrP106–126 peptide inhibited it. Recombinant 14-3-3β disaggregated the mature PrP106–126 fibrils in vitro. Moreover, the PrP–14-3-3 protein complexes were observed in the brain tissues of normal and scrapie agent 263 K infected hamsters. Colocalization of PrP and 14-3-3 was seen in the cytoplasm of human neuroblastoma cell line SH-SY5Y, as well as human cervical cancer cell line HeLa transiently expressing full-length human PrP. Our current data suggest the neuroprotection of PrPC and neuron damage caused by PrPSc may be associated with their functions of 14-3-3 dimerization regulation.     

Post-translational modification of 14-3-3 isoforms and regulation of cellular function

14-3-3 is now well established as a family of dimeric proteins that can modulate interaction between proteins involved in a wide range of functions. In many cases, these proteins show a distinct preference for a particular isoform(s) of 14-3-3 and in many cases a specific repertoire of dimer formation influences the particular proteins that 14-3-3 interact. Well over 200 proteins have been shown to interact with 14-3-3. The purpose of this review is to give an overview of the recently identified post-translational modifications of 14-3-3 isoforms and how this regulates function, interaction, specificity of dimerisation between isoforms and cellular location of target proteins. The association between 14-3-3 and its targets usually involves phosphorylation of the interacting protein which has been the subject of many reviews and discussion of this is included in other reviews in this series. However, it is now realised that in some cases the phosphorylation and a number of other, novel covalent modifications of 14-3-3 isoforms may modulate interaction and dimerisation of 14-3-3. Since this aspect is now emerging to be of major importance in the mechanism of regulation by 14-3-3 isoforms and has not been the focus of previous reviews, this will be detailed here.     

14-3-3 proteins activate a plant calcium-dependent protein kinase (CDPK)

Plants and protozoa contain a unique family of calcium-dependent protein kinases (CDPKs) which are defined by the presence of a carboxyl-terminal calmodulin-like regulatory domain. We present biochemical evidence indicating that at least one member of this kinase family can be stimulated by 14-3-3 proteins. Isoform CPK-1 from the model plant Arabidopsis thaliana was expressed as a fusion protein in E. coli and purified. The calcium-dependent activity of this recombinant CPK-1 was shown to be stimulated almost twofold by three different 14-3-3 isoforms with 50% activation around 200 nM. 14-3-3 proteins bound to the purified CPK-1, as shown by binding assays in which either the 14-3-3 or CPK-1 were immobilized on a matrix. Both the 14-3-3 binding and activation of CPK-1 were specifically disrupted by a known 14-3-3 binding peptide LSQRQRSTpSTPNVHMV (IC₅₀=30 μM). These results raise the question of whether 14-3-3 can modulate the activity of CDPK signal transduction pathways in plants.     

Cytoplasmic dynein/dynactin mediates the assembly of aggresomes

Aggresomes are pericentrosomal cytoplasmic structures into which aggregated, ubiquitinated, misfolded proteins are sequestered. Misfolded proteins accumulate in aggresomes when the capacity of the intracellular protein degradation machinery is exceeded. Previously, we demonstrated that an intact microtubule cytoskeleton is required for the aggresome formation [Johnston et al., 1998: J. Cell Biol. 143:1883-1898]. In this study, we have investigated the involvement of microtubules (MT) and MT motors in this process. Induction of aggresomes containing misfolded DeltaF508 CFTR is accompanied by a redistribution of the retrograde motor cytoplasmic dynein that colocalizes with aggresomal markers. Coexpression of the p50 (dynamitin) subunit of the dynein/dynactin complex prevents the formation of aggresomes, even in the presence of proteasome inhibitors. Using in vitro microtubule binding assays in conjunction with immunogold electron microscopy, our data demonstrate that misfolded DeltaF508 CFTR associate with microtubules. We conclude that cytoplasmic dynein/dynactin is responsible for the directed transport of misfolded protein into aggresomes. The implications of these findings with respect to the pathogenesis of neurodegenerative disease are discussed.     

土生隐球酵母14-3-3蛋白耐铝能力的研究

14-3-3蛋白家族是由多个高度保守的成员构成的调节性蛋白质家族,它们主要以磷酸化的形式与伴侣蛋白相互作用,并能够以多种方式来影响靶蛋白。通过构建14-3-3蛋白原核表达载体,纯化重组蛋白获得14-3-3蛋白抗体。为了验证14-3-3蛋白基因在耐铝中的作用,构建14-3-3酵母表达载体,得到14-3-3过表达酵母菌株。在5mmol/L铝浓度下,转基因酵母比对照酵母长势好,这表明14-3-3蛋白通过促进生长赋予酵母对铝胁迫的耐受性。     

Purification, Properties, and Immunohistochemical Localisation of Human Brain 14-3-3 Protein

Abstract: A protein has been purified from human brain that appears to be the human equivalent of bovine 14-3-3 protein. On polyacrylamide gel electrophoresis the protein migrates as a faster major component, termed 14-3-3-2 protein, and a slower minor component, termed 14-3-3-1 protein, which consists of approximately 12% of the total protein. Both 14-3-3-1 and 14-3-3-2 have a native molecular weight of approximately 67,000. 14-3-3-2 appears to have the subunit composition (αβ; 14-3-3-1 has the composition ββ. Peptide mapping with Stuphvlococcus aureus V8 proteinase shows that α and β subunits are unrelated but the β and β' subunits show some common peptides. Immunoperoxidase labelling shows that 14-3-3 is localised in neurones in the human cerebral cortex. 14-3-3 shows no enolase, creatine kinase, triose phosphate isomerase, ATPase, cyclic nucleotide-dependent protein kinase, or purine nucleoside phosphorylase activity. 14-3-3 does not bind calcium and does not appear to be related to calmodulin, calcineurin, tubulin, neurofilament proteins, clathrin-associated proteins, or tropomyosin. The functional significance of this neuronal protein remains obscure.