植物重金属转运蛋白研究进展

土壤中的有毒重金属不仅对植物有害,也可通过食物链危害人类和动物的健康.重金属转运蛋白在植物吸收、抵抗重金属的复杂机制中起着关键作用.植物重金属转运蛋白分为吸收蛋白和排出蛋白,其中,吸收蛋白转运必需重金属进入细胞,同时也会因为必需重金属的缺乏或离子之间的竞争而运载有毒重金属;排出蛋白是一类解毒蛋白,可将过量的或有毒的重金属逆向转运出细胞,或区室化于液泡中.目前,细胞内多种重金属转运蛋白基因的转录水平与重金属离子积累之间的联系已被揭示,并分离克隆出诸多相关蛋白家族成员.本文综述了近年来发现并鉴定的主要重金属转运蛋白的金属亲和性、器官表达特异性及细胞内定位等的研究进展.     

Roles for mannose-6-phosphate receptors in lysosomal enzyme sorting, IGF-II binding and clathrin-coat assembly

Two related transmembrane proteins in mammalian cells bind the mannose 6-phosphate recognition marker present on all soluble lysosomal hydrolases. Recent studies of cells that express only one or neither of these proteins have shed light not only on their function in directing lysosomal enzymes into the endocytic pathway but also on their critical role in transport vesicle formation in the trans Golgi network. One of these proteins also binds insulin-like growth factor II (IGF-II) and may be an important part of the IGF-dependent system that regulates development.     

Intrinsically disordered regions have specific functions in mitochondrial and nuclear proteins

Proteins in general consist not only of globular structural domains (SDs), but also of intrinsically disordered regions (IDRs), i.e. those that do not assume unique three-dimensional structures by themselves. Although IDRs are especially prevalent in eukaryotic proteins, the functions are mostly unknown. To elucidate the functions of IDRs, we first divided eukaryotic proteins into subcellular localizations, identified IDRs by the DICHOT system that accurately divides entire proteins into SDs and IDRs, and examined charge and hydropathy characteristics. On average, mitochondrial proteins have IDRs more positively charged than SDs. Comparison of mitochondrial proteins with orthologous prokaryotic proteins showed that mitochondrial proteins tend to have segments attached at both N and C termini, high fractions of which are IDRs. Segments added to the N-terminus of mitochondrial proteins contain not only signal sequences but also mature proteins and exhibit a positive charge gradient, with the magnitude increasing toward the N-terminus. This finding is consistent with the notion that positively charged residues are added to the N-terminus of proteobacterial proteins so that the extended proteins can be chromosomally encoded and efficiently transported to mitochondria after translation. By contrast, nuclear proteins generally have positively charged SDs and negatively charged IDRs. Among nuclear proteins, DNA-binding proteins have enhanced charge tendencies. We propose that SDs in nuclear proteins tend to be positively charged because of the need to bind to negatively charged nucleotides, while IDRs tend to be negatively charged to interact with other proteins or other regions of the same proteins to avoid premature proteasomal degradation.     

Location of Sulfate-binding Protein in Salmonella typhimurium

A method is described for location of proteins in bacteria. It depends upon two techniques. One technique is the inactivation of the protein by a reagent which is incapable of penetrating the bacterial membrane (permeability barrier). Proteins inside this membrane cannot be inactivated unless the cells are disrupted; proteins on or outside the membrane can be inactivated. The second technique depends upon inactivation of the protein by specific antibody. Antibody should not penetrate the external bacterial wall, and therefore should only inactivate proteins that are on the wall surface. Thus, proteins can be localized inside the membrane, in the wall-membrane area, or outside the wall. One reagent developed for use with the first technique is diazo-7-amino-1,3-naphthalene-disulfonate. It inactivated beta-galactoside transport, but not beta-galactosidase of intact Escherichia coli. Similarly, it inactivated sulfate binding and transport but not uridine phosphorylase activity of Salmonella typhimurium. This indicates that the sulfate-binding protein is on or outside the cell membrane, and that uridine phosphorylase is inside the cell. The organic mercurial compounds used also showed that the sensitive parts of the sulfate and alpha-methylglucoside transport systems are less reactive than the sensitive part of the beta-galactoside system. Antibody to the sulfate-binding protein inactivated the purified protein but did not inactivate this protein when intact bacteria were employed. Thus, it appears that the sulfate-binding protein does not protrude outside the cell wall. The conclusion that the binding protein is located in the wall-membrane region is supported by its release upon spheroplast formation or osmotic shock, and also by its ability to combine with sulfate in bacteria which cannot transport sulfate into the cell.     

Head and flagella subcompartmental proteomic analysis of human spermatozoa

Subcellular proteomics not only deepens our knowledge of what proteins are present within cells, but also opens our understanding as to where those proteins reside. Given the highly differentiated, cross‐linked state of spermatozoa, such studies have proven difficult to perform. In this study we have fractionated spermatozoa into two components, consisting of either the head or flagellar region. Following SDS‐PAGE, 1 mm slices were digested and used for LC‐MS/MS analysis. In total, 1429 proteins were identified with 721 proteins being exclusively found in the tail and 521 exclusively in the head. Not only is this the largest reported proteomic analysis of human spermatozoa, but also it has provided novel insights into the compartmentalization of proteins, particularly receptors, never previously reported to be present in this cell type.     

核定位信号及其分析策略

真核细胞核膜上的核孔复合体 (nuclear pore complex, NPC) 是细胞核内外进行物质交换的主要通道, 分子量较小的化合物可自由通过NPC或采取被动扩散的方式进入细胞核, 而分子量为50 kD以上的蛋白质则只能通过主动转运进入细胞核. 以这种方式进入细胞核的 蛋白质必须在其氨基酸序列上拥有特殊的核定位信号(nuclear localization signal, NLS)以被相应的核转运蛋白(karyopherins) 识别. 核定位信号具有多样性, 包括经典核定位信号(classical NLS,cNLS), 内输蛋白β2识别的核定位信号（又称PY模体-NLS）和其它类型的NLS. 每一类NLS具有相似的特征, 但并不具有完全保守的氨基酸组成. 不同的NLS, 往往对应着各不相同的核输入机制. 而对同一蛋白质来说, 也可能同时拥有几个功能性的NLS. 研究核定位信号一方面可以帮助揭示新的大分子物质核转运机制, 另一方面也有助于发现一些蛋白质的新功能. 本文对常见NLS的分类进行了总结, 并介绍了两种常用的NLS预测软件及鉴定NLS的一般策略.     

MxiG, a membrane protein required for secretion of Shigella spp. Ipa invasins: involvement in entry into epithelial cells and in intercellular dissemination

Entry of Shigella flexneri into epithelial cells involves secretory proteins, the lpa proteins, and their dedicated secretion apparatus, the Mxi—Spa translocon, which is encoded by the mxi and spa operons. We have characterized the mxiG gene that is located at the proximal part of the mxi operon. Inactivation of mxiG abolished lpa secretion, which indicates that MxiG is an essential component of the Mxi-Spa translocon. Immunoblotting analysis of membrane fractions suggests that the 42 kDa MxiG protein is associated with both the inner and outer membranes. Taking advantage of the complementation of the mxiG mutant by a plasmid carrying a wild-type copy of mxiG (which restored lpa secretion, entry into HeLa cells, and cell-to-cell spread) we mutagenized the mxiG gene carried by the complementing plasmid to replace the RGD motif of MxiG by RAD. This mutation ( mxiG *), which had no effect on the stability of the protein, did not affect lpa secretion in vitro or entry into HeLa cells, but impaired intercellular dissemination. Therefore, MxiG and possibly proteins secreted by the Mxi-Spa translocon are involved not only in entry but also in spread of shigella between epithelial cells.     

Proteomic characterization of specific minor proteins in the human milk casein fraction

Human milk contains many bioactive proteins that are likely to support the early development of the newborn. The aim of this study was to identify whether there are specific minor proteins associated with the human milk casein micelle prepared by the acid precipitation method. Protein identification was performed by liquid chromatography tandem mass spectrometry analysis. Eighty-two proteins were identified in the casein micelle, 18 of which are not present in their whey compartment. Thirty-two of these proteins specifically associated with the casein micelle have not previously been identified in human milk or colostrum. Proteins involved in immune function comprised the major part (28%) of total proteins, and another significant part is involved in metabolism/energy production (22%). Most of the proteins were of extracellular or cytoplasmic origin (accounting for 50 and 29%, respectively). This study indicates that various soluble proteins should be considered as part of the casein compartment, prepared by the acid precipitation method. The data provide new insight not only into the proteomic profile of the human milk casein micelle and its physiological significance, but also into the proper proportion of casein and casein-associated proteins to use in infant formula.     

Biogenesis and exocytosis of Weibel-Palade bodies

Vascular endothelial cells contain typical, elongated vesicles, the so-called Weibel-Palade bodies, which serve as a storage compartment for von Willebrand factor (VWF), a plasma protein that plays an essential role in controlling the adhesion and aggregation of platelets at sites of vascular injury. Upon activation of endothelial cells by agonists such as thrombin, epinephrine or histamine, the Weibel-Palade bodies fuse with the plasma membrane and release their contents into the blood circulation. This process provides an adequate means by which endothelial cells can actively participate in controlling the arrest of bleeding upon vascular damage. Besides VWF, Weibel-Palade bodies contain a subset of other proteins, including interleukin-8 (IL-8), P-selectin and endothelin. Similar to VWF, these proteins are transported to the outside of the cell upon stimulation and may control local or systemic biological effects, including inflammatory and vasoactive responses. Apparently, endothelial cells are able to create a storage pool for a variety of bioactive molecules which can be mobilised upon demand. Endothelial cells that are deficient of VWF synthesis are not only unable to form Weibel-Palade bodies, but also lack the ability to store IL-8 or P-selectin or release these proteins in a regulated manner. It thus appears that VWF not only plays a prominent role in controlling primary haemostasis, but also may modulate inflammatory processes through its ability to target inflammatory mediators to the regulated secretion pathway of the endothelium.     

Current thoughts on the phosphatidylinositol transfer protein family

Monomeric transport of lipids is carried out by a class of proteins that can shield a lipid from the aqueous environment by binding the lipid in a hydrophobic cavity. One such group of proteins is the phosphatidylinositol transfer proteins (PITP) that can bind phosphatidylinositol and phosphatidylcholine and transfer them from one membrane compartment to another. PITPs are found in both unicellular and multicellular organisms but not bacteria. In mice and humans, the PITP domain responsible for lipid transfer is found in five proteins, which can be classified into two classes based on sequence. Class I PITPs comprises two family members, alpha and beta, small 35 kDa proteins with a single PITP domain which are ubiquitously expressed. Class IIA PITPs (RdgBalphaI and II) are larger proteins possessing additional domains that target the protein to membranes and are only able to bind lipids but not mediate transfer. Finally, Class IIB PITP (RdgBbeta) is similar to Class I in size (38 kDa) and is also ubiquitously expressed. Class III PITPs, exemplified by the Sec14p family, are found in yeast and plants but are unrelated in sequence and structure to Class I and Class II PITPs. In this review we discuss whether PITP proteins are passive transporters or are regulated proteins that are able to couple their transport and binding properties to specific biological functions including inositol lipid signalling and membrane turnover.     

Purification and characterization of a novel calcium-dependent serine proteinase secreted from malignant hamster embryo fibroblast Nil2C2

A novel serine proteinase was purified from the conditioned medium of malignant hamster embryo fibroblasts, Nil2C2. The molecular weight of the purified enzyme was estimated to be 88,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. The enzyme was split into two subunits (Mr 66,000 and 33,000) with a reducing agent. The enzyme hydrolyzed not only synthetic peptides which are susceptible to trypsin digestion but also extracellular matrix proteins such as type I and IV collagen, fibronectin and gelatin. For the digestion of these proteins, Ca2+ at millimolar concentrations was essential but Ca2+ or chelators did not affect the esterase activity for synthetic peptides. The proteolytic activity was inhibited by diisopropyl fluorophosphate (DFP) and also by phenylmethylsulfonyl fluoride. DFP was shown to bind to the 33 kDa subunit, indicating that the catalytic machinery of the enzyme is located in this subunit.     

SecY alterations that impair membrane protein folding and generate a membrane stress

We report on a class of Escherichia coli SecY mutants that impair membrane protein folding. The mutants also up-regulate the Cpx/sigma(E) stress response pathways. Similar stress induction was also observed in response to a YidC defect in membrane protein biogenesis but not in response to the signal recognition particle-targeting defect or in response to a simple reduction in the abundance of the translocon. Together with the previous contention that the Cpx system senses a protein abnormality not only at periplasmic and outer membrane locations but also at the plasma membrane, abnormal states of membrane proteins are postulated to be generated in these secY mutants. In support of this notion, in vitro translation, membrane integration, and folding of LacY reveal that mutant membrane vesicles allow the insertion of LacY but not subsequent folding into a normal conformation recognizable by conformation-specific antibodies. The results demonstrate that normal SecY function is required for the folding of membrane proteins after their insertion into the translocon.     

Topogenesis of peroxisomal proteins does not require a functional cytoplasm-to-vacuole transport

Folded and even oligomeric proteins can be imported from the cytosol into vacuoles and into peroxisomes. Pro-aminopeptidase I (prAPI) oligomerizes into a dodecamer and is imported into the vacuole via the cytoplasm-to-vacuole transport (cvt) pathway. How peroxisomes accommodate folded proteins is completely unknown. Peroxisome biogenesis and cvt do not only share the import of folded protein complexes but also show mechanistic parallels such as the employment of ubiquitin conjugation systems. In search for a genetic overlap, selected cvt and autophagocytosis (atg) mutants were tested for defects in peroxisome biogenesis. Most of the mutants did not exhibit a mislocalization of peroxisomal matrix proteins to the cytosol which would be typical of a defect in the peroxisome biogenesis. However, two mutants, deltaatg14 and deltacvt4/vam6, displayed a general growth defect and deltacvt8/vps41 showed cytosolic mislocalization not only of peroxisomal but also of mitochondrial proteins, indicating a more general defect in organelle biogenesis. Our data do not provide evidence for a genetic overlap of the import pathway for peroxisomal proteins and the cvt pathway.     

Carbon recycling into de novo lipogenesis is a major pathway in neonatal metabolism of linoleate and alpha-linolenate.

Recent reports indicate that recycling of the beta-oxidized carbon skeleton of linoleate and alpha-linolenate into newly synthesized cholesterol and fatty acids in the brain is quantitatively significant in both suckling rats and pre- and postnatally in rhesus monkeys. The recycling appears to occur via ketones which are not only readily produced from these 18 carbon polyunsaturates but are also the main lipogenic precursors for the developing mammalian brain. Since the neonatal rat brain appears not to acquire cholesterol or long chain saturated or monounsaturated fatty acids from the circulation, ketones and ketogenic precursors seem to be crucial for normal brain synthesis of these lipids. Cholesterol is plentiful in brain membranes and it has also been discovered to be the essential lipid adduct of the 'hedgehog' family of proteins, the appropriate expression of which determines normal embryonic tissue patterning and neurological development. Insufficient cholesterol or inappropriate expression of 'sonic hedgehog' has major adverse neurodevelopmental consequences typified in humans by Smith-Lemli-Optiz syndrome. Hence, we propose that the importance of alpha-linolenate and linoleate for normal neural development arises not only from being precursors to longer chain polyunsaturates incorporated into neuronal membranes but, perhaps equally importantly, by being ketogenic precursors needed for in situ brain lipid synthesis.     

Changes in protein phosphorylation in the eye of Aplysia associated with circadian rhythm regulation by serotonin

Serotinin (5-HT) shifts the phase of the circadian oscillator of the eye of Aplysia californica in a phase dependent manner. This indicates that 5-HT acts, either directly or through some intermediaries, on a component of the oscillator. Since our goal is to identify the components of the oscillator, we are following the pathway through which 5-HT has its effect on the rhythm. The effect of 5-HT on the rhythm has been shown to be mediated by an increase in intracellular cyclic adenosine-3′,5′-monophosphate (cAMP). The most likely action of cAMP is to activate cAMP-dependent protein kinase. Therefore, we used two-dimensional polyacrylamide gel electrophoresis to investigate changes in ³²P labelled phosphoproteins which occur with 5-HT and other treatements. Fourteen proteins showed increased incorporation of ³²P when eyes were exposed to treatments of 5-HT from CT 06 to 12. Two proteins showed decreased incorporation. 8-bt-cAMP mimicked all but one of the increases and both decreases in incorporation produced by 5-HT. 8-bt-cAMP increased incorporation into three additional proteins and decreased incorporation into three additional proteins and decreased incorporation into three other that were not affected by 5-HT. Incorporation into one protein was increased by 5-HT but decreased by 8-bt-cAMP. By comparison, light, which has little or no effect on the rhythm at this phase, only affected one protein. The protein increased by light was also increased by 5-HT. Tetradecanoic phorbol acetate (TPA), administered during CT 06-12, also had little effect on the rhythm at this phase. TPA increased incorporation into twenty proteins and decreased incorporatoin into three. Seven of the increased proteins were also increased by 5-HT. Some of the phosphoproteins altered by 5-HT and cAMP analogue may mediate the effect of 5-HT on the circadian rhythm.     

Recombination nodules in plants

The molecular events of recombination are thought to be catalyzed by proteins present in recombination nodules (RNs). Therefore, studying RN structure and function should give insights into the processes by which meiotic recombination is regulated in eukaryotes. Two types of RNs have been identified so far, early (ENs) and late (LNs). ENs appear at leptotene and persist into early pachytene while LNs appear in pachytene and remain into early diplotene. ENs and LNs can be distinguished not only on their time of appearance, but also by such characteristics as shape and size, relative numbers, and association with unsynapsed and/or synapsed chromosomal segments. The function(s) of ENs is not clear, but they may have a role in searching for DNA homology, synapsis, gene conversion and/or crossing over. LNs are well correlated with crossing over. Here, the patterns of ENs and LNs during prophase I in plants are reviewed.     

GFP-like proteins stably accumulate in lysosomes

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its GFP variants (Aequorea GFPs), and more recently the novel GFP-like proteins from Anthozoa have greatly advanced our technologies for fluorescently labeling cells, organelles, and proteins. It has been shown, however, that some GFP-like proteins have a tendency to oligomerize and aggregate. Transfection of GFP-like proteins into cultured mammalian cells results in bright punctate structures, which are thought to be cytosolic protein aggregates. In this study, we demonstrate that these structures are not cytosolic aggregates but lysosomes that have accumulated the GFP-like proteins. Our biochemical and immunocytochemical experiments have revealed that certain GFP-like proteins expressed in the cytosol enter lysosomes possibly by an autophagy-related mechanism, but retain their fluorescence because of resistance not only to acidity but also to lysosomal proteases.     

Natures antibiotics: the potential of antimicrobial peptides as new drugs

Animals and plants make a variety of substances to prevent potentially lethal infections. These include small antibiotic proteins, or peptides, which target bacteria, fungi and viruses. Research into these peptides not only give us an insight into how we naturally prevent infections, but can also provide us with new drugs to treat the ever-increasing danger of infections caused by antibiotic-resistant bacteria.