Cell cycle-dependent 3D distribution of telomeres and telomere repeat-binding factor 2 (TRF2) in HaCaT and HaCaT-myc cells

Telomeres are specialized structures at the ends of the chromosomes that, with the help of proteins--such as the telomere repeat-binding factor TRF2 -, form protective caps which are essential for chromosomal integrity. Investigating the structure and three-dimensional (3D) distribution of the telomeres and TRF2 in the nucleus, we now show that the telomeres of the immortal HaCaT keratinocytes are distributed in distinct non-overlapping territories within the inner third of the nuclear space in interphase cells, while they extend more widely during mitosis. TRF2 is present at the telomeres at all cell cycle phases. During mitosis additional TRF2 protein concentrates all around the chromosomes. This change in staining pattern correlates with a significant increase in TRF2 protein at the S/G2 transition as seen in Western blots of synchronized cells and is paralleled by a cell cycle-dependent regulation of TRF2 mRNA, arguing for a specific role of TRF2 during mitosis. The distinct territorial localization of telomeres is abrogated in a HaCaT variant that constitutively expresses c-Myc--a protein known to contribute to genomic instability. These cells are characterized by overlapping telomere territories, telomeric aggregates (TAs), that are accompanied by an overall irregular telomere distribution and a reduced level in TRF2 protein. These TAs which are readily detectable in interphase nuclei, are similarly present in mitotic cells, including cells in telophase. Thus, we propose that TAs, which subsequently also cluster their respective chromosomes, contribute to genomic instability by forcing an abnormal chromosome segregation during mitosis.     

Expression, purification, and molecular characterization of Plasmodium falciparum FK506-binding protein 35 (PfFKBP35)

The immunosuppressive drug FK506 binds its targets FK506-binding protein (FKBP) family and modulates cellular processes. Recent studies demonstrated that FK506 shows anti-malaria effects. Newly identified FK506-binding protein 35 from Plasmodium falciparum (PfFKBP35) is assumed to be the molecular target of FK506 in the parasite. Currently, molecular and structural basis of growth inhibition of the parasite by FK506 remains unclear. In this study, to examine characteristics of PfFKBP35 and also understand its molecular mechanism of the inhibition by FK506, we have cloned, expressed, and purified the full-length PfFKBP35 and its FK506-binding domain (FKBD). We demonstrate that the full-length PfFKBP35 and the FKBD were properly folded, and suitable for biochemical and biophysical studies. PfFKBP35 showed a basal activity in inhibiting the phosphatase activity of calcineurin in the absence of FK506, but the presence of FK506 greatly enhanced its calcineurin-inhibitory activity. Our NMR data indicate that the FKBD binds FK506 with a high affinity.     

Tay1 protein, a novel telomere binding factor from Yarrowia lipolytica

Inspection of the complete genome of the yeast Yarrowia lipolytica for the presence of genes encoding homologues of known telomere-binding proteins surprisingly revealed no counterparts of typical yeast Myb domain-containing telomeric factors including Rap1 or Taz1. Instead, we identified a gene, YALIOD10923g, encoding a protein containing two Myb domains, exhibiting a high degree of similarity to the Myb domain of human telomeric proteins TRF1 and TRF2 and homologous to an essential fission yeast protein Mug152 whose expression is elevated during meiosis. The protein, which we named Tay1p (telomere-associated in Yarrowia lipolytica 1), was purified for biochemical studies. Using a model Y. lipolytica telomere, we demonstrate that the protein preferentially binds to Y. lipolytica telomeric tracts. Tay1p binds along the telomeric tract as dimers and larger oligomers, and it is able to remodel the telomeric DNA into both looped structures and synaptic complexes of two model telomere DNAs. The ability of Tay1p to induce dimerization of telomeres in vitro goes in line with its oligomeric nature, where each oligomer can employ several Myb domains to form intermolecular telomere clusters. We also provide experimental evidence that Tay1p may be associated with Y. lipolytica telomeres in vivo. Together with its homologues from Schizosaccharomyces pombe and several basidiomycetous fungi (Sánchez-Alonso, P., and Guzman, P. (2008) Fungal Genet. Biol. 45, S54-S62), Tay1p constitutes a novel family of putative telomeric factors whose analysis may be instrumental in understanding the function and evolution of double-stranded DNA telomeric proteins.     

Splicing factor poly(rC)-binding protein 1 is a novel and distinctive tumor suppressor

A lot of evidence has been found on the link between tumorigenesis and the aberrant expression of splicing factors. A number of splicing factors have been reported to be either oncogenic or overexpressed in cancer cells. However, splicing factors can also play negative roles in tumorigenesis. In the current review, we focus on splicing factor poly(rC)-binding protein 1 (PCBP1), a novel tumor suppressor that is characterized by downregulation in many cancer types and shows inhibition of tumor formation and metastasis. Notably, the messenger RNA levels of PCBP1 are not significantly decreased in most cancer types. In fact, PCBP1 protein is often degraded or shows a loss-of-function through phosphorylation in cancer cells. PCBP1 is highly homologous to its family member, PCBP2. Interestingly, PCBP2 appears to be an oncogenic splicing factor. A growing body of evidence has shown that PCBP1 regulates alternative splicing, translation, and RNA stability of many cancer-related genes. Taking together, PCBP1 has distinctive tumor suppressive functions, and increasing PCBP1 expression may represent a new approach for cancer treatment.     

Expression,purification and characterization of a vascular endothelial growth factor fusion protein

Objectives

To prepare recombinant tPep-(vascular endothelial growth factor) VEGF-B and assess its biological activity.

Results

This new VEGF fusion protein was constructed using a targeting peptide and prepared using E.coli. The tPep-VEGF-B was refolded from inclusion bodies and purified using affinity chromatography. Its bioactivity was determined in vitro using proliferation assay and wounding healing assay, and in vivo in zebrafish. By using the optimized downstream process, recombinant tPep-VEGF-B can be obtained with a purity of >90 % and a yield of 80 mg protein/l culture medium. The refolded protein is highly effective in promoting cell migration in vitro and in enhancing angiogenesis in vivo.

Conclusion

We have constructed a new VEGF fusion protein with potential therapeutic application in treating metabolic diseases.

     

Identification and characterization of RPK118, a novel sphingosine kinase-1-binding protein

Sphingosine kinase (SPHK) is a key enzyme catalyzing the formation of sphingosine 1 phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through intracellular as well as extracellular mechanisms. However, the molecular mechanism of the intracellular actions of SPP remains unclear. Here we have cloned a novel sphingosine kinase-1 (SPHK1)-binding protein, RPK118, by yeast two-hybrid screening. RPK118 contains several functional domains whose sequences are homologous to other known proteins including the phox homology domain and pseudokinase 1 and 2 domains and is shown to be a member of an evolutionarily highly conserved gene family. The pseudokinase 2 domain of RPK118 is responsible for SPHK1 binding as judged by yeast two-hybrid screening and immunoprecipitation studies. RPK118 is also shown to co-localize with SPHK1 on early endosomes in COS7 cells expressing both recombinant proteins. Furthermore, RPK118 specifically binds to phosphatidylinositol 3-phosphate. These results strongly suggest that RPK118 is a novel SPHK1-binding protein that may be involved in transmitting SPP-mediated signaling into the cell.     

Expression, purification and characterization of murine Dkk1 protein

Dickkopf-1 (Dkk1) protein is a secreted inhibitor of canonical Wnt signaling and modulates that pathway during embryonic development. It is also implicated in several diseases and hence Dkk1 is a potential target for therapeutic intervention. In the present study 6His-tagged Dkk1 expression and secretion was assessed in five mammalian cell types. Only FreeStyle 293-F cells showed significant Dkk1 protein expression in culture medium. High and stable expression of the Dkk1 protein was obtained from a selected stable FreeStyle 293-F clone 3F8, that grows in suspension in serum-free medium. The 3F8 clone showed a high Dkk1 production level (10 mg/L) for up to 2 months of culture. A one step purification procedure resulting in large amounts of highly pure and active Dkk1 protein was developed. Purified Dkk1 binds its receptors LRP5 and LRP6, and is able to dose dependently inhibit canonical Wnt signaling. Recombinant Dkk1 is glycosylated, but this modification is not essential for its biological activity. In summary, an abundant source of pure and functionally active Dkk1 protein is developed that will support the identification of inhibitors such as neutralizing antibodies that could find therapeutic use.     

Expression, purification and crystallization of the human UL16-binding protein ULBP1

UL16-binding proteins (ULBPs) are markers of cellular stress which are upregulated on the surface of virus-infected and tumor cells. Recognition of ULBP1 by the activating receptor NKG2D on the surface of cytotoxic natural killer (NK) and T cells promotes lysis of cells expressing ULBP1 and is an important mechanism of immune surveillance. We report a robust method for the generation of large quantities of crystal-grade recombinant ULBP1 protein. The extracellular portion of human ULBP1 was cloned into a T7 expression vector for expression in Escherichia coli. Unpaired cysteines in the sequence which are predicted not to be involved in the intramolecular disulfide bond formation were mutated to serine. ULBP1 was expressed in E. coli BL21 (DE3) pLysS cells as inclusion bodies. Purified inclusion bodies were solubilized by denaturation in guanidine, and refolded by slow dilution. The refolded protein was purified by size exclusion gel filtration and anion exchange chromatography. Furthermore, we have identified conditions optimal for the crystallization of this protein and have obtained initial diffraction data to 4.6? from these crystals.     

Control of human telomere length by TRF1 and TRF2

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.     

Expression, purification, and characterization of a novel methyl parathion hydrolase

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.     

Dynamics of TRF1 organizing a single human telomere

Chromosome stability is primarily determined by telomere length. TRF1 is the core subunit of shelterin that plays a critical role in telomere organization and replication. However, the dynamics of TRF1 in scenarios of telomere-processing activities remain elusive. Using single-molecule magnetic tweezers, we here investigated the dynamics of TRF1 upon organizing a human telomere and the protein-DNA interactions at a moving telomeric fork. We first developed a method to obtain telomeres from human cells for directly measuring the telomere length by single-molecule force spectroscopy. Next, we examined the compaction and decompaction of a telomere by TRF1 dimers. TRF1 dissociates from a compacted telomere with heterogenous loops in ∼20 s. We also found a negative correlation between the number of telomeric loops and loop sizes. We further characterized the dynamics of TRF1 at a telomeric DNA fork. With binding energies of 11 k_BT, TRF1 can modulate the forward and backward steps of DNA fork movements by 2–9 s at a critical force of F_1/2, temporarily maintaining the telomeric fork open. Our results shed light on the mechanisms of how TRF1 organizes human telomeres and facilitates the efficient replication of telomeric DNA. Our work will help future research on the chemical biology of telomeres and shelterin-targeted drug discovery.     

Expression and purification of the fusion protein HMGB1Abox-TMD1, a novel HMGB1 antagonist

High mobility group box chromosomal protein 1 (HMGB1) is a lethal mediator of systemic inflammation, and its A box domain is isolated as an antagonist of HMGB1. To enhance its expression level and its anti-HMGB1 effect, the A box cDNA was coupled with the sequence encoding lectin-like domain of thrombomodulin (TMD1). The fusion DNA fragment was ligated into the prokaryotic expression vector pQE-80L to construct the recombinant plasmid pQE80L-A/TMD1. The plasmid was then transformed into Escherichia coli DH5α, and the recombinant fusion protein A/TMD1 was expressed at 37°C for 4 h, with induction by IPTG at the final concentration of 0.2 mM. The expression level of the fusion protein was up to 40% of the total cellular protein. The fusion protein was purified by Ni-NTA chromatography and the purity was about 95%. After passing over a polymyxin B column to remove any contaminating lipopolysaccharides, the purified protein was tested for its anti-inflammatory activity. Our data show that A/TMD1 significantly inhibits HMGB1-induced TNF-α release and might be useful in treating HMGB1-elevated sepsis.     

具有抗血小板聚集功能的新型葡激酶突变体的表达、纯化及性质

[目的]为解决溶栓后再栓塞问题,构建N-端含RGD(Arg-Gly-Asp)序列的葡激酶双功能突变体.研究突变体的表达和纯化,并进行性质分析.[方法]将突变后的葡激酶突变体序列连入pBV220质粒,转化大肠杆菌BL21进行表达.阳离子交换、凝胶过滤和阴离子交换三步层析法纯化表达产物,采用溶圈法对纯化产物进行生物学活性测定,并测定纯化产物对血小板聚集的抑制效应.[结果]PAGE扫描结果显示,葡激酶突变体蛋白在大肠杆菌BL21中的表达量约占菌体蛋白总量的40%～50%;三步层析纯化后,HPLC测定其纯度可达95%.酪蛋白凝胶板溶圈法测得其比活性分别为10.8×104和11.0×104HU/mg,与野生型葡激酶活性相当;且具有明显的抗血小板聚集活性,血小板聚集仪测定其血小板聚集抑制率分别为10.72%和19.71%,明显高于野生型葡激酶血小板聚集抑制率.本实验利用pBV220载体高效表达了葡激酶突变体基因,得到了高纯度、高活性的突变体蛋白,为葡激酶生产产业化和临床应用奠定了良好的基础.     

Expression, purification and characterization of recombinant mouse translation initiation factor eIF4E as a dihydrofolate reductase (DHFR) fusion protein

One of the earliest steps in translation initiation is recognition of the mRNA cap structure (m7GpppX) by the initiation factor eIF4E. Studies of interactions between purified eIF4E and its binding partners provide important information for understanding mechanisms underlying translational control in normal and cancer cells. Numerous impediments of the available methods used for eIF4E purification led us to develop a novel methodology for obtaining fractions of eIF4E free from undesired by-products. Herein we report methods for bacterial expression of eIF4E tagged with mutant dihydrofolate reductase (DHFR) followed by isolation and purification of the DHFR–eIF4E protein by using affinity and anion exchange chromatography. Fluorescence quenching experiments indicated the cap-analog, 7MeGTP, bound to DHFR–eIF4E and eIF4E with a dissociation constant (K_d) of 6 ± 5 and 10 ± 3 nM, respectively. Recombinant eIF4E and DHFR–eIF4E were both shown to significantly enhance in vitro translation in dose dependent manner by 75% at 0.5 μM. Nevertheless increased concentrations of eIF4E and DHFR–eIF4E significantly inhibited translation in a dose dependent manner by a maximum at 2 μM of 60% and 90%, respectively. Thus, we have demonstrated that we have developed an expression system for fully functional recombinant eIF4E. We have also shown that the fusion protein DHFR–eIF4E is functional and thus may be useful for cell based affinity tag studies with fluorescently labeled trimethoprim analogs.     

Expression, purification, and characterization of protein kinase C-epsilon

Of the recently described members of the protein kinase C (PKC) family (-delta, -epsilon, -zeta), no detailed properties of the purified enzymes have been presented. Here we describe the expression of PKC-epsilon in insect cells using a baculovirus vector. The recombinant enzyme has been purified to homogeneity by sequential chromatography on DEAE-cellulose, serine-Sepharose, Mono Q, and Superose 12; the protein shows a molecular mass of 90 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PKC-epsilon is dependent upon phospholipid and diacylglycerol (or phorbol esters) for activity and displays a pattern of specificity for these effectors similar to other PKC isotypes. Similarly, inhibition of PKC-epsilon by staurosporine and H-7 parallels inhibition of other PKC isotypes. However, unlike PKC-alpha, -beta, and -gamma, PKC-epsilon shows no dependence upon Ca2+. Furthermore, the substrate specificity of PKC-epsilon is quite different from other characterized PKCs. The importance of functional diversity within the PKC family is discussed.     

TAT-Cytoglobin融合蛋白的原核表达、纯化及生物活性分析

旨在利用细胞穿膜肽TAT的穿膜作用和细胞珠蛋白(Cytoglobin,Cygb)的抗衰老、抗纤维化的功能,将二者通过基因工程的手段融合在一起,以期获得能够穿透细胞屏障的Cygb。通过两次重叠PCR技术获得了TAT-Cygb DNA,将其插入原核表达载体pET22b质粒中,转化至大肠杆菌BL-21,筛选出可表达TAT-Cygb融合蛋白的大肠杆菌工程菌株。经乳糖诱导表达TAT-Cygb,CM阳离子交换层析(CM Sepharose Fast Flow Protocol)获得纯度高达95%的TAT-Cygb融合蛋白,分子量约23 kDa。生物活性实验显示,TAT-Cygb过氧化物酶比活力达到(422.30±0.36)U/mg。TAT-Cygb预处理的Hacat细胞可免受H2O2氧化应激的损伤(RGR=100%),同时TAT-Cygb可治疗已被H2O2氧化损伤的细胞(RGR=98%),与Cygb处理组相比具有显著差异(RGR=79%)。该研究首次成功利用大肠杆菌表达系统表达了可穿透细胞膜的、有生物活性的TAT-Cygb融合蛋白,为继续开展Cygb在抗衰老、抗纤维化和抗癌领域的研究奠定了基础。     

CAMDI, a novel disrupted in schizophrenia 1 (DISC1)-binding protein, is required for radial migration

Centrosomes play a crucial role in the directed migration of developing neurons. However, the underlying mechanism is poorly understood. This study has identified a novel disrupted in schizophrenia 1 (DISC1)-interacting protein, named CAMDI after coiled-coil protein associated with myosin II and DISC1, which translocates to the centrosome in a DISC1-dependent manner. Knockdown of CAMDI by shRNA revealed severely impaired radial migration with disoriented centrosomes. A yeast two-hybrid screen identified myosin II as a binding protein of CAMDI. CAMDI interacts preferentially with phosphomyosin II and induces an accumulation of phosphomyosin II at the centrosome in a DISC1-dependent manner. Interestingly, one single nucleotide polymorphism of the CAMDI gene (R828W) is identified, and its gene product was found to reduce the binding ability to phosphomyosin II. Furthermore, mice with overexpression of R828W in neurons exhibit an impaired radial migration. Our findings indicate that CAMDI is required for radial migration probably through DISC1 and myosin II-mediated centrosome positioning during neuronal development.