Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B

Nuclear factor kappa B (NF-kappa B), which was first detected by its binding to the kappa B site in the immunoglobulin kappa-gene enhancer, is important for the regulated expression of the kappa-gene and is partly responsible for the induction in appropriate cells of interleukin-2 (IL-2), IL-2 alpha receptor, beta-interferon and serum amyloid A protein. NF-kappa B is present as a nuclear DNA-binding protein in B lymphocytes and mature macrophages, but is found in the cytoplasm of many cells in a form unable to bind to DNA. The cytoplasmic form is bound to an inhibitor protein, I kappa B, from which it can be released in vitro by deoxycholate and other agents. Activation of cells by various agents, notably the phorbol esters that stimulate protein kinase C (PKC), leads to dissociation in vivo of the NF-kappa B/I kappa B complex and migration of NF-kappa B to the nucleus. Therefore, it acts as a second messenger system, transducing activation signals from the cytoplasm to the nucleus. To elucidate the mechanism of signal transfer, we have used an in vitro system in which addition of purified protein kinases to a partially purified NF-kappa B/I kappa B complex leads to the activation of the DNA-binding activity of NF-kappa B. Using gel retardation assays we found that PKC, cyclic AMP-dependent protein kinase (PKA) and a haem-regulated eIF-2 kinase (HRI) could activate NF-kappa B in vitro, whereas casein kinase II was ineffective. To determine the target for the protein kinases we purified and characterized both NF-kappa B and I kappa B and found that I kappa B is phosphorylated and inactivated in the presence of PKC and HRI but not PKA.     

TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2

The I kappa B kinase (IKK), consisting of the IKK1 and IKK2 catalytic subunits and the NEMO (also known as IKK gamma) regulatory subunit, phosphorylates I kappa B proteins, targeting them for degradation and thus inducing activation of NF-kappa B (reviewed in refs 1, 2). IKK2 and NEMO are necessary for NF-kappa B activation through pro-inflammatory signals. IKK1 seems to be dispensable for this function but controls epidermal differentiation independently of NF-kappa B. Previous studies suggested that NF-kappa B has a function in the growth regulation of epidermal keratinocytes. Mice lacking RelB or I kappa B alpha, as well as both mice and humans with heterozygous NEMO mutations, develop skin lesions. However, the function of NF-kappa B in the epidermis remains unclear. Here we used Cre/loxP-mediated gene targeting to investigate the function of IKK2 specifically in epidermal keratinocytes. IKK2 deficiency inhibits NF-kappa B activation, but does not lead to cell-autonomous hyperproliferation or impaired differentiation of keratinocytes. Mice with epidermis-specific deletion of IKK2 develop a severe inflammatory skin disease, which is caused by a tumour necrosis factor-mediated, alpha beta T-cell-independent inflammatory response that develops in the skin shortly after birth. Our results suggest that the critical function of IKK2-mediated NF-kappa B activity in epidermal keratinocytes is to regulate mechanisms that maintain the immune homeostasis of the skin.     

Bcl-2家族蛋白和乙肝病毒x蛋白在肝癌组织中的表达和意义

应用免疫组织化学方法检测了34例肝癌组织及其相对应的癌旁组织,探讨了Bcl-2家族中七种基因(包括促凋亡基因Bak、Bad、Bid、Bax和Bcl-xs及抑凋亡基因Bcl-2、Bcl-w)和乙肝病毒三种抗原(包括HBsAg、HBcAg和HBxAg)在肝癌组织中的表达及意义,结果显示:在肝癌组织中HBsAg、HBcAg和HBxA的阳性率分别为58.8％、26.5％和76.5％、Bcl-2七种蛋白的阳性率分别为58.8％(Bak)、55.9％(Bad)、44.1％(Bid)、41.2％(Bax)、29.4％(Bcl-xs)、35.3％(Bcl-w)和41.2％(Bcl-2)。这七种Bcl-2蛋白的表达均位于肝癌细胞的胞浆,多呈弥漫性分布,少数阳性颗粒呈散在性分布,研究发现,Bcl-2家族中抑凋亡基因Bcl-w和Bcl-2在癌组织中表达的阳性率明显高于癌旁组织(P相似文献   

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.     

Ankyrin and spectrin associate with voltage-dependent sodium channels in brain

The segregation of voltage-dependent sodium channels to specialized regions of the neuron is crucial for propagation of an action potential. Studies of their lateral mobility indicate that sodium channels are freely mobile on the neuronal cell body but are immobile at the axon hillock, presynaptic terminal and at focal points along the axon. To elucidate the mechanisms that regulate sodium channel topography and mobility, we searched for specific proteins from the brain that associate with sodium channels. Here we show that sodium channels labelled with 3H-saxitoxin (STX) are precipitated in the presence of exogenous brain ankyrin by anti-ankyrin antibodies and that 125I-labelled ankyrin binds with high affinity to sodium channels reconstituted into lipid vesicles. The cytoplasmic domain of the erythrocyte anion transporter competes for the latter interaction. Neither the neuronal GABA (gamma-aminobutyric acid) receptor channel complex nor the dihydropyridine (DHP) receptor bind brain ankyrin. The results indicate that brain ankyrin links the voltage-dependent sodium channel to the underlying cytoskeleton and may help to maintain axolemmal membrane heterogeneity and control sodium channel mobility.     

Nanospring behaviour of ankyrin repeats

Ankyrin repeats are an amino-acid motif believed to function in protein recognition; they are present in tandem copies in diverse proteins in nearly all phyla. Ankyrin repeats contain antiparallel alpha-helices that can stack to form a superhelical spiral. Visual inspection of the extrapolated structure of 24 ankyrin-R repeats indicates the possibility of spring-like behaviour of the putative superhelix. Moreover, stacks of 17-29 ankyrin repeats in the cytoplasmic domains of transient receptor potential (TRP) channels have been identified as candidates for a spring that gates mechanoreceptors in hair cells as well as in Drosophila bristles. Here we report that tandem ankyrin repeats exhibit tertiary-structure-based elasticity and behave as a linear and fully reversible spring in single-molecule measurements by atomic force microscopy. We also observe an unexpected ability of unfolded repeats to generate force during refolding, and report the first direct measurement of the refolding force of a protein domain. Thus, we show that one of the most common amino-acid motifs has spring properties that could be important in mechanotransduction and in the design of nanodevices.     

Hereditary spherocytosis associated with deletion of human erythrocyte ankyrin gene on chromosome 8

Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic anaemias. HS red cells from both autosound dominant and recessive variants are spectrin-deficient, which correlates with the severity of the disease. Some patients with recessive HS have a mutation in the spectrin alpha-2 domain (S.L.M. et al., unpublished observations), and a few dominant HS patients have an unstable beta-spectrin that is easily oxidized, which damages the protein 4.1 binding site and weakens spectrin-actin interactions. In most patients, however, the cause of spectrin deficiency is unknown. The alpha- and beta-spectrin loci are on chromosomes 1 and 14 respectively. The only other genetic locus for HS is SPH2, on the short arm of chromosome 8 (8p11). This does not correspond to any of the known loci of genes for red cell membrane proteins including protein 4.1 (1p36.2-p34), the anion exchange protein (AE1, band 3; 17q21-qter), glycophorin C (2q14-q21), and beta-actin (7pter-q22). Human erythrocyte ankyrin, which links beta-spectrin to the anion exchange protein, has recently been cloned. We now show that the ankyrin gene maps to chromosome 8p11.2, and that one copy is missing from DNA of two unrelated children with severe HS and heterozygous deletions of chromosome 8 (del(8)(p11-p21.1)). Affected red cells are also ankyrin-deficient. The data suggest that defects or deficiency or ankyrin are responsible for HS at the SPH2 locus.     

黄瓜离体雌核发育早期的特异蛋白研究

通过改进的IEF-SDS-PAGE技术研究了黄瓜雌核发育早期的特异蛋白质表达,并发现了11个特异多肽点,其中3个(A 1~A 3)可能是开启分裂基因表达的一类反式作用因子,起到抑制该基因表达作用.8个(B 1~B 8)可能是启动雌配子分裂密切相关的基因表达产物,以上特异蛋白在多次重复中均显示出良好的重复性,因此认为,这些特异蛋白可能对黄瓜雌核发育早期的启动起重要作用.     

Knowledge-based prediction of protein structures and the design of novel molecules

Prediction of the tertiary structures of proteins may be carried out using a knowledge-based approach. This depends on identification of analogies in secondary structures, motifs, domains or ligand interactions between a protein to be modelled and those of known three-dimensional structures. Such techniques are of value in prediction of receptor structures to aid the design of drugs, herbicides or pesticides, antigens in vaccine design, and novel molecules in protein engineering.     

Triphenyltin inhibition of the proteasome activity and its influence on substrate protein levels in nerve cells

Triphenyltin (TPT) widely exists as environmental pollutant, but its toxicity towards nerve cells and the related mechanism remain unclear. In this research, SHSY-5Y cells were exposed to TPT, and the cellular proteasome activity, Iκb proteins, Bax and α-synuclein levels were investigated. The results show that TPT can inhibit the cellular proteasome activity, and result in the accumulation of ubiquitinized proteins. TPT exposure can change the protein levels of IκB, Bax, and α-synuclein, affect NFκB pathwa...