基因外显子连接序列与相应内含子序列的相互作用

剪接后的内含子与相应mRNA序列的相互作用在基因表达调控过程中起着非常重要的作用。基于27个物种的核糖核蛋白基因序列,采用Smith—Waterman局域比对方法得到外显子连接序列与相应内含子序列的最佳匹配片段,分析了外显子连接序列上的匹配频率分布和匹配片段的序列特征。发现一些低等真核生物EJC结合区域的匹配频率明显低于其它区域,所有物种EJC结合区域的序列构成呈现出相对低的结构序。最佳匹配片段的平均长度和配对率分布与siRNA和miRNA的结合特征相同。推测EJC和内含子在与外显子序列结合的过程中存在相互竞争和相互协作的关系,内含子中部序列在基因表达调控过程中起着重要的作用。     

蛋白质功能预测方法概述

蛋白质是生物体内最必需也是最通用的大分子,对它们功能的认识对于科学领域和农业领域的发展有着至关重要的作用。随着后基因组时代的发展,NCBI数据库中迅速涌现出大量不明结构与功能的蛋白质序列,这些蛋白质序列甚至一跃成了研究的热点。近几十年来蛋白质功能预测的方法不断被完善。由最初的仅基于蛋白质序列或3D结构信息的方法衍生出更多的基于序列相似性、基于结构基序、基于相互作用网络等新方法,这些新型方法采用新的算法、新的研究思路和技术手段,力求得到准确性与普遍性并存,能够被广泛应用的蛋白质功能预测方法。本文综述了近年来蛋白质功能预测的方法,并将这些研究方法分类归纳,各自阐明了每类方法的优缺点。     

分离自安徽土壤金龟子绿僵菌种群的遗传异质性研究

金龟子绿僵菌是昆虫种群自然控制和害虫生物防治中重要的虫生真菌,已被作为化学农药的替代品广泛应用于农林害虫的防治。其寄主范围和地理分布极广,因此种群结构也十分复杂。为明确安徽省土栖金龟子绿僵菌的种群遗传结构,采用ISSR分子标记技术对采集自安徽省不同地区土壤中的116株金龟子绿僵菌进行遗传异质性分析。分子数据显示,筛选出的8个引物共获得79个位点,其中多态位点比率为100%。不同地区种群的Nei’s基因多样性(H)为0.2796,Shannon信息指数(I)为0.4425,种群间存在一定程度的基因流(Nm=4.4282)和遗传分化(Gst=0.1015),种群内的基因多样度占总居群的89.85%,种群间占10.15%,表明安徽土栖金龟子绿僵菌的遗传变异主要来源于种群内,且种群内表现出较大的遗传变异。并采用UPGMA对所有供试材料进行聚类分析,得到7大类种群。结果表明,安徽土栖金龟子绿僵菌之间的亲缘关系与地理来源不存在相关性。     

Stem cells: Insights into the secretome

Stem cells have been considered as possible therapeutic vehicles for different health related problems such as cardiovascular and neurodegenerative diseases and cancer. Secreted molecules are key mediators in cell–cell interactions and influence the cross talk with the surrounding tissues. There is strong evidence supporting that crucial cellular functions such as proliferation, differentiation, communication and migration are strictly regulated from the cell secretome. The investigation of stem cell secretome is accumulating continuously increasing interest given the potential use of these cells in regenerative medicine. The scope of the review is to report the main findings from the investigation of stem cell secretome by the use of contemporary proteomics methods and discuss the current status of research in the field. This article is part of a Special Issue entitled: An Updated Secretome.     

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry

Human M-proinsulin was cleaved by trypsin at the R³¹R³²–E³³ and K⁶⁴R⁶⁵–G⁶⁶ bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K⁵⁹R⁶⁰–G⁶¹ bond but at the B/C junction cleavage occurred at the R³¹R³²–E³³ as well as the R³¹–R³²E³³ bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k_cat./K_m values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 10⁵ s^− 1 M^− 1) and the cleavage of the K²⁹–T³⁰ bond of M-insulin-RR (1.3 ± 0.07 × 10⁵ s^− 1 M^− 1). However, the K²⁹–T³⁰ bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k_cat./K_m values around 1000 s^− 1 M^− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K²⁹–T³⁰ bond becomes shielded, exposed then shielded again respectively.     

Evaluation of structural features in fungal cytochromes P450 predicted to rule catalytic diversification

Fungi belong to the large kingdom of lower eukaryotic organisms encompassing yeasts along with filamentous and dimorphic members. Microbial P450 enzymes have contributed to exploration of and adaptation to diverse ecological niches such as conversion of lipophilic compounds to more hydrophilic derivatives or degradation of a vast array of environmental toxicants. To better understand diversification of the catalytic behavior of fungal P450s, detailed insight into the molecular machinery steering oxidative attack on the distinctly structured endogenous and xenobiotic substrates is of preeminent interest. Based on a general, CYP102A1-related template the bulk of predicted substrate/inhibitor-binding determinants were shown to cluster near the distal heme face within the six known substrate recognition sites (SRSs) made up by the α-helical B′/F/G/I tetrad, the B′–C interhelical loop and strands of the β6-sheet, population density being highest in the structurally flexible SRS-1 and SRS-4 domains, showing a low degree of conservation. Reactivity toward ligands favorably coincides with the lipophilicity/hydrophilicity profile and bulkiness of critical amino acids acting as selective filters. Some decisive elements may also serve in maintenance of catalytic competence via their action as gatekeepers directing substrate access/positioning or stabilizers of the heme environment enabling dioxygen activation. Non-SRS residues seem to control spin state equilibria and attract redox partners by electrostatic forces. Of note, the inhibitory potency of azole-type fungicides is likely to arise from perturbation of the complex interplay of the mechanistic principles addressed above. Knowledge-supported exploitation of the topological data will be helpful in the manufacture of commodity/specialty chemicals as well as therapeutic agents. Also, engineered fungal P450s may be used to improve pollutant-specific bioremediation of contaminated soils.     

The heterodimerization of platelet-derived chemokines

Chemokines encompass a large family of proteins that act as chemoattractants and are involved in many biological processes. In particular, chemokines guide the migration of leukocytes during normal and inflammatory conditions. Recent studies reveal that the heterophilic interactions between chemokines significantly affect their biological activity, possibly representing a novel regulatory mechanism of the chemokine activities. The co-localization of platelet-derived chemokines in vivo allows them to interact. Here, we used nano-spray ionization mass spectrometry to screen eleven different CXC and CC platelet-derived chemokines for possible interactions with the two most abundant chemokines present in platelets, CXCL4 and CXCL7. Results indicate that many screened chemokines, although not all of them, form heterodimers with CXCL4 and/or CXCL7. In particular, a strong heterodimerization was observed between CXCL12 and CXCL4 or CXCL7. Compared to other chemokines, the main structural difference of CXCL12 is in the orientation and packing of the C-terminal alpha-helix in relation to the beta-sheet. The analysis of one possible structure of the CXCL4/CXCL12 heterodimer, CXC-type structure, using molecular dynamics (MD) trajectory reveals that CXCL4 may undergo a conformational transition to alter the alpha helix orientation. In this new orientation, the alpha-helix of CXCL4 aligns in parallel with the CXCL12 alpha-helix, an energetically more favorable conformation. Further, we determined that CXCL4 and CXCL12 physically interact to form heterodimers by co-immunoprecipitations from human platelets. Overall, our results highlight that many platelet-derived chemokines are capable of heterophilic interactions and strongly support future studies of the biological impact of these interactions.     

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Hydrogen peroxide reactivity and cyanide binding of the TriAla,TriVal, and TriLeu variants

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H₂O₂ compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H₂O₂ and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H₂O₂ and HCN while features that inhibit substrate ionization slow the reactions.     

Enhancement of thermal stability of chondroitinase ABC I by site-directed mutagenesis: An insight from Ramachandran plot

The application of chondroitinase ABC I (cABC I) in damaged nervous tissue is believed to prune glycosaminoglycan chains of proteoglycans, thereby facilitates axon regeneration. However, the utilization of cABC I as therapeutics is notably restricted due to its thermal instability. In the present study, we have explored the possibility of thermostabilization of cABC I through release of its conformational strain using Ramachandran plot information. In this regard, Gln140 with non-optimal φ and ψ values were replaced with Gly, Ala and Asn. The results indicated that Q140G and Q140A mutants were able to improve both activity and thermal stability of the enzyme while Q140N variant reduced the enzyme activity and destabilized it. Moreover, the two former variants displayed a remarkable resistance to trypsin degradation. Structural analysis of all mutants showed an increase in intrinsic fluorescence intensity and secondary structure content of Q140G and Q140A compared to the wild type which indicated more compact structure upon mutation. This investigation demonstrated that relief of conformational tension can be considered as a possible approach to increase the stability of the protein.     

Comparative analysis of inner cavities and ligand migration in non-symbiotic AHb1 and AHb2

This study reports a comparative analysis of the topological properties of inner cavities and the intrinsic dynamics of non-symbiotic hemoglobins AHb1 and AHb2 from Arabidopsis thaliana. The two proteins belong to the 3/3 globin fold and have a sequence identity of about 60%. However, it is widely assumed that they have distinct physiological roles. In order to investigate the structure–function relationships in these proteins, we have examined the bis-histidyl and ligand-bound hexacoordinated states by atomistic simulations using in silico structural models. The results allow us to identify two main pathways to the distal cavity in the bis-histidyl hexacoordinated proteins. Nevertheless, a larger accessibility to small gaseous molecules is found in AHb2. This effect can be attributed to three factors: the mutation Leu35(AHb1) → Phe32(AHb2), the enhanced flexibility of helix B, and the more favorable energetic profile for ligand migration to the distal cavity. The net effect of these factors would be to facilitate the access of ligands, thus compensating the preference for the fully hexacoordination of AHb2, in contrast to the equilibrium between hexa- and pentacoordinated species in AHb1. On the other hand, binding of the exogenous ligand introduces distinct structural changes in the two proteins. A well-defined tunnel is formed in AHb1, which might be relevant to accomplish the proposed NO detoxification reaction. In contrast, no similar tunnel is found in AHb2, which can be ascribed to the reduced flexibility of helix E imposed by the larger number of salt bridges compared to AHb1. This feature would thus support the storage and transport functions proposed for AHb2. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.