Recent advances in protein methylation: Enzymatic methylation of nucleic acid binding proteins

Summary Heterogeneous nuclear RNP protein A1, one of the major proteins in hnRNP particle (precursor for mRNA), is known to be post-translationally arginine-methylatedin vivo on residues 193, 205, 217 and 224 within the RGG box, the motif postulated to be an RNA binding domain. Possible effect of N^G-arginine methyl-modification in the interaction of protein A1 to nucleic acid was investigated. The recombinant hnRNP protein A1 wasin vitro methylated by the purified nuclear protein/histone-specific protein methylase I (S-adenosylmethionine:protein-arginine N-methyltransferase) stoichiometrically and the relative binding affinity of the methylated and the unmethylated protein A1 to nucleic acid was compared: Differences in their binding properties to ssDNA-cellulose, pI values and trypsin sensitivities in the presence and absence of MS2-RNA all indicate that the binding property of hnRNP protein A1 to single-stranded nucleic acid has been significantly reduced subsequent to the methylation. These results suggest that posttranslational methyl group insertion to the arginine residue reduces protein-RNA interaction, perhaps due to interference of H-bonding between guanidino nitrogen arginine and phosphate RNA.Abbreviations hnRNP heterogeneous ribonucleoprotein particle - AdoMet S-adenosyl-L-methionine - AdoHcy S-adenosyl-L-homocysteine - MBP myelin basic protein - HMG high mobility group - ss single stranded     

Crystal structure of rubredoxin from Pyrococcus furiosus at 0.95 Å resolution, and the structures of N-terminal methionine and formylmethionine variants of Pf Rd. Contributions of N-terminal interactions to thermostability

The high-resolution crystal structure of the small iron-sulfur protein rubredoxin (Rd) from the hyperthermophilic archeon Pyrococcus furiosus (Pf) is reported in this paper, together with those of its methionine ([_0M]Pf Rd) and formylmethionine (f[_0M]Pf Rd) variants. These studies were conducted to assess the consequences of the presence or absence of a salt bridge between the amino terminal nitrogen of Ala1 and the side chain of Glu14 to the structure and stability of this rubredoxin. The structure of wild-type Pf Rd was solved to a resolution of 0.95?Å and refined by full-matrix least-squares techniques to a crystallographic agreement factor of 12.8% [F>2σ(F) data, 25?617 reflections], while those of the [_0M]Pf and f[_0M]Pf Rd variants were solved at slightly lower resolutions (1.1?Å, R=11.5%, 17?213 reflections; 1.2?Å, R=13.7%, 12?478 reflections, respectively). The quality of the data was such that about half of the hydrogen atoms of the protein were clearly visible. All three structures were ultimately refined using the program SHELXL-93 with anisotropic atomic displacement parameters for all non-hydrogen protein atoms, and calculated hydrogen positions included but not refined. In this paper we also report thermostability data for all three forms of Pf Rd, and show that they follow the sequence wild-type >[_0M]Pf>formyl[_0M]Pf. Comparison of the three Pf Rd structures in the N-terminal region show that the structures of wild-type Pf Rd and f[_0M]Pf are rather similar, while that of [_0M]Pf Rd shows a number of additional hydrogen bonds involving the extra methionine group. While the salt bridge between the Ala1 amino group and the Glu14 carboxylate is not the primary determinant of the thermostability of Pf Rd, alterations to the amino terminus do have a moderate influence on the thermostability of this protein.     

The level of expression of a protein kinase C gene may be an important component of the patterning process in Hydra

 Several studies have provided strong, but indirect evidence that signalling through pathways involving protein kinase C (PKC) plays an important role in morphogenesis and patterning in Hydra. We have cloned a gene (HvPKC2) from Hydra vulgaris which encodes a member of the nPKC subfamily. In adult polyps, HvPKC2 is expressed at high levels in two locations, the endoderm of the foot and the endoderm of the hypostomal tip. Increased expression of HvPKC2 is an early event during head and foot regeneration, with the rise in expression being restricted to the endodermal cells underlying the regenerating ends. No upregulation is observed if regenerates are cut too close to the head to form a foot. Elevated expression of HvPKC2 is also observed in the endoderm underlying lithium-induced ectopic feet. A dynamic and complex pattern of expression is seen in developing buds. Regeneration of either head or foot is accompanied by an increase in the amount of PKC in both soluble and particulate fractions. An increase in the fraction of PKC activity which is membrane-bound is specifically associated with head regeneration. Taken together these data suggest that patterning of the head and foot in Hydra is controlled in part by the level of HvPKC2 expression, whilst head formation is accompanied by an in vivo activation of both calcium-dependent and independent PKC isoforms. Received: 10 July 1997 / Accepted: 8 November 1997     

中国株丙型肝炎病毒（HCV）结构区蛋白在昆虫细胞中的表达及加工

利用杆状病毒表达系统在昆虫细胞中表达了完整的中国河北株丙丙型肝炎病毒结构蛋白。免疫印迹实验结果显示,表达产物中有一系列分子量不同、可以与ＨＣＶ抗体阳性病人血清反应的蛋白,表明结构蛋白被宿主细胞蛋白酶切割与加工,相应分别为２０ｋＤ的核心蛋白、３２ｋＤ糖基化的Ｅ１蛋白４０ｋＤ的未糖化的Ｅ２蛋白和７０ｋＤ糖基化的Ｅ２蛋白,另有８０ｋＤ及１００ｋＤ的两组前体蛋白。利用表达产物检测慢性ＨＣＶ感染者血清,发现     

可食性蛋白质酶水解过程中形成苦味原因的探讨

本文探讨了天然蛋白质水解过程中形成苦味的原因。     

Contamination of a dredged material disposal site La Rochelle Bay,France .The use of the acetylcholinesterase activity of Mytilus edulis L. as a biomarker of pesticides: the need for a critical approach

A single aspect of the toxic impact of a dredged material disposal site located near a mussel-farming zone was followed for eight months. Acetylcholinesterase activity (AChE) of Mytilus edulis was investigated as a biomarker for possible contamination by neurotoxic compounds (carbamates and/or organophosphorous pesticides). Our observations showed that the enzymatic activities (including AChE) of these harbour mussels were decreased in sites directly and indirectly influenced (according to hydrodynamic conditions) by the dumping of dredged sediments, suggesting possible contamination by pesticides. The strong correlations observed between AChE activity and growth parameters (length and weight) seems to show, however, that the enzyme activity is also indirectly controlled through growth restriction, which may imply limitation of the development of the nervous system in juveniles. The concentration of total proteins, as well as the spawning process also seem to disturb the assessment of AChE activity. These field observations clearly indicate that the use of this enzyme activity as a biomarker should proceed with caution. For example, the seasonal variability of such activity should be taken into account in a biomonitoring programme.     

Pressure enhances thermal stability of DNA polymerase from three thermophilic organisms

DNA polymerases derived from three thermophilic microorganisms, Pyrococcus strain ES4, Pyrococcus furiosus, and Thermus aquaticus, were stabilized in vitro by hydrostatic pressure at denaturing temperatures of 111°C, 107.5°C, and 100°C (respectively). Inactivation rates, as determined by enzyme activity measurements, were measured at 3, 45, and 89 MPa. Half-lives of P. strain ES4, P. furiosus, and T. aquaticus DNA polymerases increased from 5.0, 6.9, and 5.2 minutes (respectively) at 3 MPa to 12, 36, and 13 minutes (respectively) at 45 MPa. A pressure of 89 MPa further increased the half-lives of P. strain ES4 and T. aquaticus DNA polymerases to 26 and 39 minutes, while the half-life of P. furiosus DNA polymerase did not increase significantly from that at 45 MPa. The decay constant for P. strain ES4 and T. aquaticus polymerases decreased exponentially with increasing pressure, reflecting an observed change in volume for enzyme inactivation of 61 and 73 cm³/mol, respectively. Stabilization by pressure may result from pressure effects on thermal unfolding or pressure retardation of unimolecular inactivation of the unfolded state. Regardless of the mechanism, pressure stabilization of proteins could explain the previously observed extension of the maximum temperature for survival of P. strain ES4 and increase the survival of thermophiles in thermally variable deep-sea environments such as hydrothermal vents. Received: September 12, 1997 / Accepted: February 24, 1998     

人造血干/祖细胞多态性的研究:Ⅺ.人正常骨髓CD34~ 造血细胞周期动力学相关大分子DNA、RNA和PRO含量的分析

DNA、RNA和PRO的合成、积累及相互关系是调控细胞周期动力学最主要的三个参数。同时检测这些组成部分能够更精确细致地评判细胞的周期动力学特征。本文探索了人正常骨髓CD34~ 造血细胞周期动力学相关大分子DNA、RNA和PRO的含量,以便认识CD34~ 造血细胞周期动力学的特征。用新型CIMS-100免疫磁性分离系统高效富集人骨髓CD34~ 造血细胞,经FCM及APAAP鉴定,富集的CD34~ 造血细胞的纯度达90～95%。随之采用碘化丙啶(PI)、派若宁Y(PY)及异硫氰荧光素(FITC)分别进行标记DNA、RNA和PRO并在FCM上检测。结果表明,DNA、RNA和PRO在CD34~ 造血细胞中的含量明显低于单个核细胞,分别仅占后者的34±3%、48±21%及62±14%。结合我们以往的结果,我们认为CD34~ 造血细胞的确是一独特的体细胞群,不仅表现在重建造血与免疫学功能上,而且表现在细胞周期动力学上。据我们所知,这是目前国际上首次有关人CD34~ 造血细胞周期动力学相关大分子的系统分析报道,提供了大多数CD34~ 造血细胞处于静止期的直接证据。     

Dynamic and structural properties of glucose oxidase enzyme

The catalytic oxidation of β-D-glucose by the enzyme glucose oxidase involves a redox change of the flavin coenzyme. The structure and the dynamics of the two extreme glucose oxidase forms were studied by using infrared absorption spectroscopy of the amide I′ band, tryptophan fluorescence quenching and hydrogen isotopic exchange. The conversion of FAD to FADH₂ does not change the amount of α-helix present in the protein outer shell, but reorganises a fraction of random coil to β-sheet structure. The dynamics of the protein interior vary with the redox states of the flavin without affecting the motions of the structural elements near the protein surface. From the structure of glucose oxidase given by X-ray crystallography, these results suggest that the dynamics of the interface between the two monomers are involved in the catalytic mechanism. Received: 27 December 1996 / Accepted: 18 July 1997