Expression and Functional Characterization of the Human Ether-à-go-go-Related Gene (<Emphasis Type="Italic">HERG</Emphasis>) K<Superscript>+</Superscript> Channel Cardiac Splice Variant in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Oocytes

HERG C_Cardiac, a C-terminal splice variant of the human ether-à-go-go-related gene (HERG A), was identified and found to be 100% homologous to HERG_USO. Real-time polymerase chain reaction data indicated that in the human heart HERG C_Cardiac mRNA was expressed eight times more than HERG A, whereas in human ventricular tissue it was expressed six times more than HERG A. A HERG C_Cardiac-green fluorescence protein (GFP) construct was heterologously expressed in Xenopus oocytes. Confocal micrographs revealed that HERG C_Cardiac was mainly expressed in the plasma membrane. HERG C_Cardiac channel expressed in oocytes produced slower inactivating outward currents and faster deactivating tail currents than those of HERG A channel. Equal amounts of HERG A and HERG C_Cardiac cRNA coinjected into oocytes formed intermediate HERG A + HERG C_Cardiac heteromultimers, which was reconfirmed by immunoprecipitation experiments with a HERG A N-terminal antibody. These heteromultimers had different inactivation, deactivation and activation kinetics from those of HERG A and HERG C_Cardiac channels. HERG A + HERG C_Cardiac heteromultimers significantly reduced the model action potential mean amplitude and increased the fast and slow inactivation τ values of the action potential repolarization phase, suggesting involvement of HERG A and HERG C_Cardiac heteromultimers in modulation of the refractory interval.     

Amino acid pairing at the N- and C-termini of helical segments in proteins

A systematic survey was carried out in an unbiased sample of 815 protein chains with a maximum of 20% homology selected from the Protein Data Bank, whose structures were solved at a resolution higher than 1.6 A and with a R-factor lower than 25%. A set of 5556 subsequences with alpha-helix or 3(10)-helix motifs was extracted from the protein chains considered. Global and local propensities were then calculated for all possible amino acid pairs of the type (i, i + 1), (i, i + 2), (i, i + 3), and (i, i + 4), starting at the relevant helical positions N1, N2, N3, C3, C2, C1, and N-int (interior positions), and also at the first nonhelical positions in both termini of the helices, namely, N-cap and C-cap. The statistical analysis of the propensity values has shown that pairing is significantly dependent on the type of the amino acids and on the position of the pair. A few sequences of three and four amino acids were selected and their high prevalence in helices is outlined in this work. The Glu-Lys-Tyr-Pro sequence shows a peculiar distribution in proteins, which may suggest a relevant structural role in alpha-helices when Pro is located at the C-cap position. A bioinformatics tool was developed, which updates automatically and periodically the results and makes them available in a web site.     

Protein structure modeling indicates hexahistidine-tag interference with enzyme activity

Unusual kinetic characteristics of tropinone reductase, an enzyme in the family of short chain dehydrogenases, prompted to investigate a possible impact of the hexahistidine affinity tag on catalytic properties. Comparison of enzymes from Solanum dulcamara, Solanaceae, tagged at the N-terminus or at the C-terminus revealed that the C-terminally tagged form was functionally impaired. Protein modeling indicated that the hexahistidine tag attached at the C-terminus but not at the N-terminus of the polypeptide can interfere with the active site by steric or electrostatic interactions. In consequence, protein modeling is suggested before enzyme expression with affinity tags to estimate possible interactions of affinity tags with the active center.     

Selective Glutaraldehyde-Mediated Coupling of Proteins to the 3′-Adenine Terminus of Polymerase Chain Reaction Products

Attachment of proteins to the 3′ end of DNA increases stability of the DNA in serum and retards clearance of DNA by major organs, thereby enhancing in vivo half-life and therapeutic potential of DNA. Unfortunately, the length of DNA molecules that can be produced with 3 ′ modifications by solid-phase synthesis for protein attachment is limited to 45–60 nucleotides due to uncertainties about sequence fidelity for longer oligonucleotides. Here we describe selective covalent coupling of proteins or other molecules to the 3′-adenine overhang of unlabeled and fluorophore-labeled double-stranded polymerase chain reaction products putatively at the N⁶ position of adenine using 2.5% glutaraldehyde at pH 6.0 and 4°C for at least 16 h. Gel mobility shift analyses and fluorescence analyses of the shifted bands supported conjugate formation between double-stranded polymerase chain reaction products and β2-microglobulin. In addition, blunt-ended DNA ladder fragments treated with glutaraldehyde at 4°C showed no evidence of DNA–DNA or DNA–protein conjugate formation. With the present cold glutaraldehyde technique, longer DNA–3′-protein conjugates might be easily mass-produced. The protein portion of a DNA–3′-protein conjugate could possess functionality as well, such as receptor binding for cell entry, cytotoxicity, or opsonization.     

Biologically active and C-amidated hinnavinII-38-Asn produced from a Trx fusion construct in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn (HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15–20% of the total cell proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography, and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus.     

截短型人乳头瘤病毒58型L1蛋白的表达及其体外生物活性研究

利用PCR技术克隆截短型HPV58 L1基因并重组入杆状病毒表达系统穿梭质粒pFastBac-Htb,通过转座反应,将目的基因片段重组入杆状病毒基因组,分离重组的Bacmid DNA, 并转染Sf-9昆虫细胞,收集被转染的Sf-9细胞,提取细胞蛋白,SDS-PAGE检测可见在大约58Kda处出现一新生蛋白条带,Western blot 证实为HPV58L1蛋白。用ProBondTM纯化系统纯化所表达的蛋白。小鼠红细胞凝集试验证实纯化的蛋白可介导小鼠红细胞凝集,透射电镜观察证实纯化蛋白可自组装成VLP。结果表明昆虫杆状病毒表达系统可高效表达截短型HPV58L1蛋白,纯化后的截短型HPV58L1蛋白在体外可自组装VLP,并具有介导小鼠红细胞凝集的生物学活性。     

Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli

Adenoregulin is a 33 amino acid antimicrobial peptide isolated from the skin of the arboreal frog Phyllomedusa bicolor. Natural adenoregulin is synthesized with an amidated valine residue at C-terminus and shows lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. A synthetic gene for adenoregulin (ADR) with an additional amino acid glutamine at C-terminus was cloned into pET32a vector to allow expression of ADR as a Trx fusion protein in Escherichia coli BL21(DE3). The resulting expression level of the fusion protein could reach up to 20% of the total cell proteins. The fusion protein could be purified effectively by Ni2+-chelating chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant ADR displayed antimicrobial activity similar to that of the synthetic ADR reported earlier. Comparing the antimicrobial activities of the recombinant adenoregulin with C-amidated terminus to that without an amidated C-terminus, we found that the amide of glutamine at C-terminus of ADR improved its potency on certain microorganisms such as Tritirachium album and Saccharomyces cerevisiae.     

Parallel folding pathways in the SH3 domain protein

The transition-state ensemble (TSE) is the set of protein conformations with an equal probability to fold or unfold. Its characterization is crucial for an understanding of the folding process. We determined the TSE of the src-SH3 domain protein by using extensive molecular dynamics simulations of the Go model and computing the folding probability of a generated set of TSE candidate conformations. We found that the TSE possesses a well-defined hydrophobic core with variable enveloping structures resulting from the superposition of three parallel folding pathways. The most preferred pathway agrees with the experimentally determined TSE, while the two least preferred pathways differ significantly. The knowledge of the different pathways allows us to design the interactions between amino acids that guide the protein to fold through the least preferred pathway. This particular design is akin to a circular permutation of the protein. The finding motivates the hypothesis that the different experimentally observed TSEs in homologous proteins and circular permutants may represent potentially available pathways to the wild-type protein.     

Inhibition of apoptosis via CHIP-mediated proteasomal degradation of TAp73α

TAp73, a homologous of tumor suppressor p53, regulates apoptosis in a p53-independent manner and its suppressive as well as stimulatory role in promoting angiogenesis has been reported. It exists in multiple isoforms which varies structurally in their N-terminus and C-terminus region and crucial interplay among them guides the decision of cell survival and death. As molecular chaperones control both stability and degradation of TAp73, selective regulation of p73 isoforms has implication upon developing new therapeutic for hypoxic tumor. We have discovered that under DNA damage carboxy terminus Hsp70 interacting protein (CHIP's) antiapoptotic function is displayed via its E3 ligase activity that inhibits exclusively TAp73α-mediated apoptosis in cancer cell. The decrease in TAp73α level by CHIP as it is supported by increased ubiquitination pattern is reverted back by sh-CHIP. Further, the transactivation of p53-downstream apoptotic genes BAX, PUMA and PIG3 by TAp73α is also shown to be subsequently inhibited by CHIP. The tetratricopeptide TPR-domain of CHIP in its amino-terminus interacts with the carboxy-terminus of TAp73α and ΔNp73α and as a result, U-BOX domain of CHIP in the carboxy-terminus is able to ubiquitinate TAp73α for proteasomal degradation. Due to lack of C-terminus in TAp73β, CHIP fails to interact with and degrade it. In conclusion, we have thus uncovered for the first time a novel mechanism of chaperone-assisted regulation of p73 stability as well as its apoptotic functions by CHIP that might be utilized to develop new anticancer strategies.     

STRUCTURAL CHARACTERIZATION OF THE TERMINAL DOMAINS OF LINEAR PLASMID‐LIKE DNA FROM THE GREEN ALGA ERNODESMIS (CHLOROPHYTA)1

Terminal regions of linear plasmid‐like DNA molecules from chloroplasts of the green alga Ernodesmis verticillata (Kützing) Børgesen were cloned and structurally characterized. Phosphorylation experiments with polynucleotide kinase indicated the presence of a 5′‐phosphate, but the data did not reveal any protective protein(s) at the 5′ end. To characterize the 3′ end of these molecules, homopolymer‐ (poly(G)‐) tailed molecules were annealed to and cloned into a linearized vector that was poly(C) tailed with terminal deoxynucleotidyl transferase. Sequencing analyses verified the heterogeneous nature of the molecules. Two distinct clones displayed extensive terminal inverted repeats (TIRs) at the 3′ end (94 and 433 nt). Shorter TIRs (3–6 nt) were identified at the 3′ end of most clones, which may serve to protect the ends. In fact, exonuclease III and λ exonuclease digested the plasmid‐like DNAs only after heat denaturation, signifying that conformational changes due to such treatment potentially make the 3′ and 5′ ends (respectively) susceptible to degradation. Multiple tandem and direct repeats were evident near the 3′ ends. A consensus sequence of 18+ nt was discovered in nearly every clone opposite the poly(G) tail, suggesting that this sequence has structural and/or functional significance. Pair‐wise sequence comparisons and the presence of repeats indicated that these novel molecules may be highly recombinant.