美国purdue（普渡大学）和日本京都大学的一项合作研究表明两种与控制植物生长过程有关的蛋白质可能解释为什么人类细胞会将化疗药物驱逐出去。
这项研究首次证明人类中与多药物抗性蛋白相似的蛋白质能够将一种植物生长激素移动到细胞中。由于这种叫做PGP（P-glycoproteins，P糖蛋白）的植物蛋白与人类的影响化疗药物效果的P-糖蛋白很相似（生物谷注：PGP是引起肿瘤多重耐药性MDR的主要蛋白之一），因此发现控制这种植物蛋白活性的方法就可能有助于开发出能降低癌症药物使用剂量的治疗方法。研究的发现分别发表在11月的Plant Cell杂志和10月的Plant杂志上。 （图为Angus Murphy)
这两项研究揭示出与这个过程相关的两个蛋白质家族之间的一种鲜为人知的关系。这些蛋白通过合作，将植物生长素分子运送通过细胞膜。在人类中，相似的蛋白质能够帮助细胞排除像癌症药物这样的毒素。
这两项研究的发现使研究人员能确定出决定细胞是否摄取不同分子例如癌症药物的蛋白质。
在Plant杂志的研究中，Murphy和苏黎世大学的同事首次证明PGP1（来自拟南芥的一种P-糖蛋白）直接将生长素运出植物细胞，并且还能将激素从酵母和哺乳动物细胞中运送出去。在Plant Cell杂志上发表的研究中，他们发现了另外一种PGP蛋白质能够将生长素运入细胞。
人类和植物中的这类多药物抗性PGP都是属于一种叫做ATP结合cassette（ABC，ATP-bingding cassette）蛋白家族，它们就好像货车一样为细胞解毒、在细胞间传递信号以影响生化反应并调节那些反应。
另一类叫做PIN1的转运蛋白也可能是一种传送机，但似乎只是充当副手而不是生长素的运送“卡车”。这些发现揭示出PIN和PGP可以在长距离的生长素运输中共同协作。
在Plant Journal上的这项研究中，Murphy和同事发现PGP1和PGP19能够将生长素移出细胞。而在Plant Cell上的文章中，Murphy的研究组发现PGP4的功能恰好相反，它促进生长素进入细胞并增加被转运的量。这些研究首次证明分子的摄入和释放由PGP转运子蛋白和PIN助理蛋白之间的反应介导。
此前Murphy作为该领域的顶尖科学家，其相关研究还发表在Science，PNAS和annual系列的杂志上，见附录。
相关报道：
生物谷：本篇报道的重点在于如何将不同的领域的研究结合起来。植物的PGP在植物中的作用与动物中的作用是完全不同的，但实际上在作用机制上却是相似的。因此这启发了科学家们通过研究它们在植物中的机制，从而试图解决癌症治疗中的MDR问题。这种科学的联想有可能会带动某种领域的突破或飞跃。这也是科学的跳跃式的思维方式。
有关抗癌机理和药物研究，十分多。如抗癌药物的研究方向包括化学合成抗癌药物，中草药提取抗癌成分，癌症疫苗的研制，化疗药的增敏剂等。尤其是近年来植物药中抗癌成分越来越令人刮目（其实以前有很多抗癌药物就与植物有关，如长春新碱，喜树碱，紫杉醇等）。这里特引用近年来相关植物药抗癌的研究，也许能给大家带来一些启示。在传统中医药中，有许多经典的抗癌中药，如半枝莲，白花蛇舌草，白毛藤等。尤其是半枝莲早年国内研究表明，它并不具有抗癌作用。近年来国外研究表明，它的抗肿瘤机制与一般的药物完全不同，而且效果非常良好。
·抗肿瘤药物市场
·世界首例“饿死肿瘤疗法”抗癌新药问世
·福建：抗癌药K—22申报国家一类新药
·美国NCI：H2AX基因p53基因协同抗癌
·酵母研究揭示出抗癌药物的分子作用机理
·抗癌新药“槐定碱”获新药证书
·我国抗癌中药康莱特在俄上市
·尿中提取物治疗肿瘤的抗癌新药尿多酸肽注射液在安徽问世
·俄罗斯合成具抗癌作用天然卟啉药物
·中医药抗癌并非只盯住“瘤”
·美医学专家从灌木中找到纯天然抗癌物质“M4N”
·美中合作研制青蒿素抗癌药物
·俄研制出抗癌植物药剂
·澳科学家发现菠萝叶含抗癌成分
·英国专家研究发现复方万年青胶囊的主要成份抗癌疗效强大
·台湾公司开发出中草药抗癌复方
·台湾发现具有抗癌性的原生种山药
·台湾中草药抗癌研究获突破进展
·美研究称蒜香草根叶可以抗癌
·FDA批准抗癌药紫杉醇注射液
·英国专家研究发现中草药半枝莲可令癌组织枯死
·生物谷综述：有关半枝莲研究最新进展
·白花蛇舌草注射液
附录：
本文原始出处：
Kazuyoshi Terasaka, Joshua J. Blakeslee, Boosaree Titapiwatanakun, Wendy A. Peer, Anindita Bandyopadhyay, Srinivas N. Makam, Ok Ran Lee, Elizabeth L. Richards, Angus S. Murphy, Fumihiko Sato, and Kazufumi Yazaki .PGP4, an ATP Binding Cassette P-Glycoprotein, Catalyzes Auxin Transport in Arabidopsis thaliana Roots .Plant Cell 2005 17: 2922-2939
Geisler M, Blakeslee JJ, Bouchard R, Lee OR, Vincenzetti V, Bandyopadhyay A, Titapiwatanakun B, Peer WA, Bailly A, Richards EL, Ejendal KF, Smith AP, Baroux C, Grossniklaus U, Muller A, Hrycyna CA, Dudler R, Murphy AS, Martinoia E. Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1.
Plant J. 2005 Oct;44(2):179-94
Murphy以前相关的研究
Li J, Yang H, Peer WA, Richter G, Blakeslee J, Bandyopadhyay A, Titapiwantakun B, Undurraga S, Khodakovskaya M, Richards EL, Krizek B, Murphy AS, Gilroy S, Gaxiola R.Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ development.
Science. 2005 Oct 7;310(5745):121-5
Murphy AS, Bandyopadhyay A, Holstein SE, Peer WA. Endocytotic cycling of PM proteins.
Annu Rev Plant Biol. 2005;56:221-51.
Baxter IR, Young JC, Armstrong G, Foster N, Bogenschutz N, Cordova T, Peer WA, Hazen SP, Murphy AS, Harper JF. A plasma membrane H+-ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana.
Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2649-54.
作者主页和实验室主页：
http://www.hort.purdue.edu/hort/people/faculty/murphy.shtml
http://www.hort.purdue.edu/hort/research/murphy/mainpage.htm
联系方式
Angus Murphy
Associate Professor
Ph.D.
Area of Interest: Auxin-related growth, Herbicide metabolism, Metal tolerance and accumulation
E-mail: murphy@purdue.edu murphy@purdue.edu
研究兴趣
The cellular basis of auxin transport
Plant form is dependent on the establishment of polarity: growth takes place in apical regions of roots and shoots in response to basic developmental programming which is then modulated by environmental cues. Plants can also undergo tropic growth in order to adapt to changes in light, orientation, or surface contact. However, even when tropic responses alter the direction of growth, the overall polarity of the plant remains intact.
Biochemical and physiological evidence suggests that the polarity of plant growth is regulated at the cellular level and involves components of the cytoskeleton, plasma membrane, and cell wall. Plant cells must therefore possess mechanisms to asymmetrically direct proteins to specific cell surfaces. These mechanisms appear to be regulated by developmental and environmental cues.
Auxin, or indole acetic acid (IAA), is an essential, multifunctional plant hormone that influences virtually every aspect of plant growth and development. Although auxin-dependent growth is evident in all plant tissues, it is synthesized primarily in apical regions of the shoot and is then transported in a polar fashion to other sites. When auxin reaches the root apex, it is redistributed away from the root tip through cortical and epidermal tissues. In tropic growth, auxin is diverted laterally to one side of the plant stem or root. As a result, the cells in that portion of the stem or root below the point of redistribution elongate. The result is bending toward light, gravitational pull, or a potential point of attachment.
Auxin is taken up into cells by diffusion augmented by a proton co-transporter, but can only exit from cells via basally-localized efflux carriers. Mutants deficient in auxin transport generally display aberrant morphology. Auxin is thus thought to maintain cellular polarity and, as a result, its own asymmetric transport mechanism. The genes that encode the auxin efflux facilitators have been identified and are generally referred to as PIN genes, for the pin-formed phenotype resulting from mutations of these genes. Biochemical evidence suggests that the PIN proteins effectively facilitate auxin transport only when functionally associated with other proteins. A number of proteins that appear to modulate PIN-associated auxin transport have recently been identified.
MDR (p-glycoprotein) modulators of auxin transport
Bosl Noh, Edgar Spalding, and I recently reported that polar auxin transport in the upper inflorescences and hypocotyls of Arabidopsis requires a transporter encoded by the multidrug-resistance-like gene AtMDR1 (Plant Cell 13: 2441). The auxin transport inhibitor, 1-naphthylphthalamic acid (NPA), binds tightly and specifically to AtMDR1. Basipetal auxin transport in seedling hypocotyls of atmdr1 null mutants is reduced to 15% of wild type. More recent experiments have also demonstrated that, in Arabidopsis, the AtMDR1 protein interacts with an immunophilin named TWD (for twisted dwarf, the phenotype resulting from disruption of the gene). Recent experiments conducted in the laboratories of Dr. Burkhard Schulz at the University of T焍ingen and Dr. Enrico Martinoia at the University of Zurich suggest that MDR-like proteins function is some tissues as an alternate efflux system and in other tissues as a regulator of the PIN proteins.
Surprisingly, researchers in the Spalding lab at the University of Wisconsin found that the atmdr1 mutation results in faster and increased gravitropic and phototropic responses in hypocotyls compared to wild type. This result was unexpected because other mutations that impair polar auxin transport also impair gravitropism (e.g. atpin2). Exaggerated differential growth resulting from loss of AtMDR1 is further manifested as enhanced nutation in etiolated hypocotyls and in more root waving, but these phenotypes are only evident when the most closely related homologue, AtPGP1, is also mutated, as might be expected from the overlapping tissue-specific expression of AtPGP1 and AtMDR1 reported previously. One possible interpretation of the exaggerated curvatures found in the atmdr1 mutation is that a higher density of PIN-associated auxin efflux channels along the basal wall of hypocotyl cells affects basipetal auxin flow, are more uniformly distributed in atmdr1 mutants. Such a lack of asymmetric PIN localization may at once enhance lateral auxin conductance and reduce the basipetal movement. We are currently localizing a number of PIN proteins using biochemical and immunohistochemical techniques to determine whether MDR proteins interact with AtPIN1, but not other PIN proteins, to enhance basipetal transport. Ablation of MDRs would then result in enhanced auxin retention and consequent enhanced lateral efflux.
An asymmetric targeting mechanism for transport proteins
Recent cellular localization studies have shown that PIN1 cycles between the plasma membrane and an internal compartment in membrane vesicles associated with actin cytoskeletal fibers. The role of actin in this process may be to provide tracks for vesicle movement and to fix the efflux carriers in a specific location after delivery to the membrane surface. When chemical agents are used to disrupt cytoskeletal tracking, auxin transport inhibitors prevent relocalization of PIN proteins on the plasma membrane. This suggests that the proteins that bind auxin transport inhibitors may provide a bridge between the efflux carriers and the actin network used to transport and localize these complexes. Rapid vesicular cycling is now thought to redistribute carriers to a new site when auxin transport polarity is changed by environmental stimuli, such as light or gravity. Therefore, direct analysis of the proteins that bind auxin efflux inhibitors, and examination of endogenous molecules, such as flavonoids, that may regulate auxin efflux in vivo is crucial to understanding how the PIN cycling apparatus functions.
There is a striking similarity between the cycling of PIN-associated auxin transporters and the mechanism that mediates the movement of glucose transporters to the plasma membrane in mammalian insulin-responsive tissues. In those tissues, when blood glucose levels rise, an insulin-induced signaling cascade causes endomembrane vesicles containing the GLUT4 glucose transporter to be dispatched asymmetrically to the plasma membrane. Change in protein phosphorylation states activates some components of GLUT4 secretory vesicles (GSVs) and deactivates anchoring components that normally repress movement. The net result is relocation of transporters from internal compartments to docking sites on the plasma membrane.
Many of the components of the mammalian GLUT4 inducible vesicle secretion mechanism have orthologs in Arabidopsis, a number of which have been directly or indirectly implicated in the regulation of auxin transport and/or the asymmetric distribution of the PIN1 protein. For example, mutations in kinase and phosphatase genes homologous to their mammalian GSV counterparts results in growth defects, altered auxin transport, and altered sensitivity to auxin transport inhibitors. Other Arabidopsis proteins known to associate with the PIN proteins or to be implicated in auxin transport are also homologs of important components of the GLUT4 cycling mechanism. One of the most important of these may be the apparent Arabidopsis counterpart of the mammalian Insulin Responsive Aminopeptidase (IRAP), which is essential for mammalian GLUT4 cycling. IRAP and its Arabidopsis homolog, AtAPM1, have a high degree of sequence similarity, have similar membrane orientations and enzymatic activities, and undergo unique processing of their carboxy-terminal domains when secreted to the plasma membrane. Recently, we have shown that treatment of Arabidopsis seedlings with IRAP inhibitors results in delocalization of PIN1 from the plasma membrane and strong localization of AtAPM1 to the basal ends of auxin-conducting cells. Natural flavonoid inhibitors of AtAPM1 have been found to alter PIN1 localization as well.
Insulin signaling is a key component of vesicle targeting in the GLUT4 localization system. For vesicle mediated targeting of IAA transport proteins to be truly parallel to the GLUT4 model, it is necessary to ask what signal(s) might control the localization of auxin transport proteins. The simplest possibility is that auxin acts as the signal to stimulate its own transport. Auxin has been reported to stimulate IAA transport and is generally thought to be required for the establishment of both embryonic polarity and auxin transport pathways themselves.
The focus of the research in my lab is to dissect the interactions of the potential components of the PIN vesicular cycling apparatus in Arabidopsis. We are currently analyzing the localization of PIN proteins in mutants lacking various components of the vesicular cycling mechanism in order to better understand the asymmetric targeting of membrane proteins and polar growth in plants. We are complementing the localization studies with biochemical assays of protein-protein interactions. It is my hope that these experiments will help us determine the applicability of the GLUT4 cycling model to plant growth and development.
Figure 1. The mammalian GLUT4 asymmetric vesicular targeting mechanism as a model for localization of the auxin efflux carrier. A vesicular cycling mechanism similar to the mammalian insulin-inducible GLUT4 glucose transporter trafficking system is suggested by recent studies of PIN protein localization and protein interactions with auxin transport inhibitors. Sequence homologies and analogous functions of many of the protein components of the two systems further suggest parallel mechanisms. An external signal (hormone binding) triggers a phosphatidylinositol / phosphorylation cascade that activates asymmetric vesicular trafficking by 1) causing relocation of an inhibitory ARF-GEF protein (GRP1 or GNOM) from an endomembrane compartment to PIP3 -enriched plasma membranes and 2) phosphorylating both a vesicular aminopeptidase (IRAP or AtAPM1) and the Vamp2 adaptor protein. Vesicles then traffic on actin filaments to a Munc18c / Keule plasma membrane docking site where Vamp2 interacts with syntaxin 4 / Knolle to initiate vesicle fusion. Endocytotic vesicles enriched in dynamin and b adaptin traffic back to the endosomal compartment in a similar actin-dependent fashion. PI3K, phosphatidylinositol-3 kinase; PKC, protein kinase C; PID, PINOID; PKB, protein kinase B /AKT; PP2a, phosphatase 2a; RCN1, root curling in NPA-1 PP2a; Vamp2, vesicle associated membrane protein 2 (v-SNARE2); IRAP, insulin responsive aminopeptidase; AtAPM1, Arabidopsis thaliana microsomal aminopeptidase; GRP1 ARF-GEF, general receptor for phosphoinositides ADP ribosylation factor-guanine nucleotide exchange factor; Munc18c, mammalian homolog of unc18c; FKB506BP, FKB506-binding immunophilin; TWD, Twisted Dwarf. From Muday and Murphy (2002) Plant Cell 14: 293-299.
Amide herbicide metabolism
A byproduct of the auxin transport research in my lab has been the dissection of amidase activities in plant tissues that hydrolyze amide herbicides like Alanap. My lab is exploring the metabolism of amide herbicides in planta to determine 1) the extent to which their carcinogenic breakdown products are retained in horticultural crops and 2) whether these compounds enhance susceptibility to plant pathogens. Additionally, we are exploring use of plant and microbe combinations to remediate soils contaminated with either amide herbicides or their polycyclic aromatic hydrocarbon breakdown products.
Evolution of metal tolerance and hyperaccumulation
Metal tolerance mechanisms in plant are often thought to have evolved serendipitously as a result of adaptations to desiccation, competition, or herbivory. Hyperaccumulation appears to be the result of loss of function mutations in tolerant plants in which plants can no longer sense that metal accumulation has exceeded normal limits. We are exploring two subspecies of Arabidopsis lyrata as a model for comparative studies of metal accumulation. One subspecies, found growing at a zinc mine site in Eastern Pennsylvania is an accumulator of zinc and cadmium, the other is not. The two subspecies are now being analyzed at the molecular level, and have already been found to have different forms of Metal Tolerance Protein genes already implicated in metal tolerance.
