美國purdue（普渡大學(xué)）和日本京都大學(xué)的一項合作研究表明兩種與控制植物生長過程有關(guān)的蛋白質(zhì)可能解釋為什么人類細胞會將化療藥物驅(qū)逐出去,。

        這項研究首次證明人類中與多藥物抗性蛋白相似的蛋白質(zhì)能夠?qū)⒁环N植物生長激素移動到細胞中,。由于這種叫做PGP（P-glycoproteins,，P糖蛋白）的植物蛋白與人類的影響化療藥物效果的P-糖蛋白很相似（生物谷注：PGP是引起腫瘤多重耐藥性MDR的主要蛋白之一）,，因此發(fā)現(xiàn)控制這種植物蛋白活性的方法就可能有助于開發(fā)出能降低癌癥藥物使用劑量的治療方法。研究的發(fā)現(xiàn)分別發(fā)表在11月的Plant  Cell雜志和10月的Plant雜志上。 （圖為Angus Murphy)

        這兩項研究揭示出與這個過程相關(guān)的兩個蛋白質(zhì)家族之間的一種鮮為人知的關(guān)系。這些蛋白通過合作，將植物生長素分子運送通過細胞膜,。在人類中，相似的蛋白質(zhì)能夠幫助細胞排除像癌癥藥物這樣的毒素,。

        這兩項研究的發(fā)現(xiàn)使研究人員能確定出決定細胞是否攝取不同分子例如癌癥藥物的蛋白質(zhì),。

        在Plant雜志的研究中，Murphy和蘇黎世大學(xué)的同事首次證明PGP1（來自擬南芥的一種P-糖蛋白）直接將生長素運出植物細胞,，并且還能將激素從酵母和哺乳動物細胞中運送出去,。在Plant  Cell雜志上發(fā)表的研究中，他們發(fā)現(xiàn)了另外一種PGP蛋白質(zhì)能夠?qū)⑸L素運入細胞,。

        人類和植物中的這類多藥物抗性PGP都是屬于一種叫做ATP結(jié)合cassette（ABC,，ATP-bingding  cassette）蛋白家族，它們就好像貨車一樣為細胞解毒,、在細胞間傳遞信號以影響生化反應(yīng)并調(diào)節(jié)那些反應(yīng),。

        另一類叫做PIN1的轉(zhuǎn)運蛋白也可能是一種傳送機，但似乎只是充當副手而不是生長素的運送“卡車”,。這些發(fā)現(xiàn)揭示出PIN和PGP可以在長距離的生長素運輸中共同協(xié)作,。

        在Plant  Journal上的這項研究中，Murphy和同事發(fā)現(xiàn)PGP1和PGP19能夠?qū)⑸L素移出細胞。而在Plant  Cell上的文章中,，Murphy的研究組發(fā)現(xiàn)PGP4的功能恰好相反,，它促進生長素進入細胞并增加被轉(zhuǎn)運的量。這些研究首次證明分子的攝入和釋放由PGP轉(zhuǎn)運子蛋白和PIN助理蛋白之間的反應(yīng)介導(dǎo),。

  此前Murphy作為該領(lǐng)域的頂尖科學(xué)家,，其相關(guān)研究還發(fā)表在Science，PNAS和annual系列的雜志上,，見附錄,。

相關(guān)報道：

生物谷：本篇報道的重點在于如何將不同的領(lǐng)域的研究結(jié)合起來。植物的PGP在植物中的作用與動物中的作用是完全不同的,，但實際上在作用機制上卻是相似的,。因此這啟發(fā)了科學(xué)家們通過研究它們在植物中的機制，從而試圖解決癌癥治療中的MDR問題,。這種科學(xué)的聯(lián)想有可能會帶動某種領(lǐng)域的突破或飛躍,。這也是科學(xué)的跳躍式的思維方式。

有關(guān)抗癌機理和藥物研究,，十分多,。如抗癌藥物的研究方向包括化學(xué)合成抗癌藥物，中草藥提取抗癌成分,，癌癥疫苗的研制，化療藥的增敏劑等,。尤其是近年來植物藥中抗癌成分越來越令人刮目（其實以前有很多抗癌藥物就與植物有關(guān),，如長春新堿，喜樹堿,，紫杉醇等）,。這里特引用近年來相關(guān)植物藥抗癌的研究，也許能給大家?guī)硪恍﹩⑹?。在傳統(tǒng)中醫(yī)藥中,，有許多經(jīng)典的抗癌中藥，如半枝蓮,，白花蛇舌草,，白毛藤等。尤其是半枝蓮早年國內(nèi)研究表明,，它并不具有抗癌作用,。近年來國外研究表明，它的抗腫瘤機制與一般的藥物完全不同,，而且效果非常良好,。

·抗腫瘤藥物市場
·世界首例“餓死腫瘤療法”抗癌新藥問世
·福建：抗癌藥K—22申報國家一類新藥
·美國NCI：H2AX基因p53基因協(xié)同抗癌
·酵母研究揭示出抗癌藥物的分子作用機理
·抗癌新藥“槐定堿”獲新藥證書
·我國抗癌中藥康萊特在俄上市
·尿中提取物治療腫瘤的抗癌新藥尿多酸肽注射液在安徽問世
·俄羅斯合成具抗癌作用天然卟啉藥物
·中醫(yī)藥抗癌并非只盯住“瘤”
·美醫(yī)學(xué)專家從灌木中找到純天然抗癌物質(zhì)“M4N”
·美中合作研制青蒿素抗癌藥物
·俄研制出抗癌植物藥劑
·澳科學(xué)家發(fā)現(xiàn)菠蘿葉含抗癌成分
·英國專家研究發(fā)現(xiàn)復(fù)方萬年青膠囊的主要成份抗癌療效強大
·臺灣公司開發(fā)出中草藥抗癌復(fù)方
·臺灣發(fā)現(xiàn)具有抗癌性的原生種山藥
·臺灣中草藥抗癌研究獲突破進展
·美研究稱蒜香草根葉可以抗癌
·FDA批準抗癌藥紫杉醇注射液
·英國專家研究發(fā)現(xiàn)中草藥半枝蓮可令癌組織枯死
·生物谷綜述：有關(guān)半枝蓮研究最新進展
·白花蛇舌草注射液

附錄：

本文原始出處：

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以前相關(guān)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.

作者主頁和實驗室主頁：

http://www.hort.purdue.edu/hort/people/faculty/murphy.shtml 

http://www.hort.purdue.edu/hort/research/murphy/mainpage.htm

聯(lián)系方式

 Angus Murphy
Associate Professor
Ph.D.

Area of Interest: Auxin-related growth, Herbicide metabolism, Metal tolerance and accumulation

E-mail: [email protected] [email protected] 

研究興趣

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.