Characterization of Nascent Chain Binding Complex

;​R o o t E n t y F 6 * D6 @ C o m p o b j b W o r d d o c u m e n t O b j e c t P O O l 6 * 6 * D6 D6​F Microsoft Word 6.0文档MSWordDoc。6;Oh +' 0 $ H l D H R:\WWUSER\TEMPLATE\NORMAL。DOT marcia steinberg marcia steinberg @ ？6 @ e = e " " " " " " " L L L L L L L n L C x | | | | | | | # ？e V T 4 " | | | | | | " " | x | | | | " | " | 6 " " " " | | 4 | 9421946韦尔奇关于多肽在细胞内合成过程中的局部环境。成熟多肽是在（共翻译）合成过程中还是在（翻译后）合成完成后开始采用其最终折叠构象一直存在争议，但尚未完全解决。此外，尚不清楚新生多肽的折叠是否独立发生，或者是否有其他成分协助折叠过程。由于发现了一类被称为分子伴侣的蛋白质，这些问题正在被重新审视，它们似乎参与了蛋白质成熟的各个方面。虽然不传递折叠过程的任何特定信息，但分子伴侣似乎通过最小化蛋白质聚集的可能性来参与蛋白质成熟，这是蛋白质物质总体浓度高的细胞中一个严重和潜在的问题。P.I.表明，在蛋白质合成过程中，成熟多肽与一种或多种细胞质成分结合，这些成分统称为新生链结合复合物（NCBC）。一旦适当数量的氨基酸合成完成，以提供“蛋白质结构域”的折叠信息，新生多肽的折叠将开始，伴随NCBC的释放。P.I.已经确定了一种成分，70kda热休克蛋白（hsp70）是NCBC的一部分，但有证据表明其他蛋白质也与新生多肽相互作用。各种不同的方法将用于纯化和表征NCBC成分。此外，其他分子伴侣（其中P.I.有合适的抗体）存在于NCBC的可能性将被解决。一旦对NCBC成分达成共识，针对新的NCBC成分的抗体将被提高并用于促进其纯化。此外，针对NCBC成分的抗体，以及纯化的NCBC蛋白本身，将用于兔网织红细胞的消耗/重构实验，以确定对蛋白质成熟的影响。预计这些研究将导致对与大多数（如果不是全部的话）新合成蛋白质相互作用和促进成熟的不同成分的更彻底的理解。最近的见解导致了关于蛋白质如何合成和获得正确折叠和生物活性构象的全新概念。20世纪五六十年代进行的经典实验提出，蛋白质折叠是一个自发的过程，仅由多肽链中存在的氨基酸线性序列决定，不需要任何辅助成分的帮助。许多实验室的工作现在已经确定了一类蛋白质机制，称为分子伴侣，它似乎在帮助细胞内的其他蛋白质采用正确折叠构象方面起着密切的作用。在本文所述的研究中，P.I.将继续识别和表征相关成分，包括与蛋白质折叠相互作用并促进蛋白质折叠的伴侣家族成员。最后，使用针对新生链结合蛋白制备的抗体，P.I.将有效地从兔网织细胞中去除这些成分，并确定这种去除对特定蛋白质成熟的影响。总而言之，这样的工作将使我们更好地了解细胞内的蛋白质如何获得最终正确折叠的构象。*** ;@ ....()()))()() ;如果我是你，我是你，我是你，我是你，我是你。

; R o o t E n t r y F 6*D6 @ C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l 6*D6 6*D6 F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT marcia steinberg marcia steinberg @ (?6 @ e = e " " " " " " " L L L L L d n L C x | | | | | | | # ? e V T 4 " | | | | | | " " | x | | | | " | " | 6 " " " " | | 4 | 9421946 Welch Relatively little is known regarding the local environment of a polypeptide during the course of its synthesis in the cell. Whether the maturing polypeptide begins to adopt its final folded conformation either during (co translationally) or after (post translationally) completion of its synthesis has been debated, but not entirely resolved. In addition, whether folding of the nascent polypeptide occurs independently, or whether other components assist in the folding process is unknown. These sorts of questions are being revisited owing to the identification of a class of proteins, referred to as molecular chaperones, which appear to participate in various aspects of protein maturation. While not conveying any specific information for the folding process, molecular chaperones appear to participate in protein maturation by minimizing the possibility of protein aggregation, a serious and potential problem in the cell where the overall concentration of proteinacious material is high. The P.I. suggests that during the course of protein synthesis the maturing polypeptide becomes engaged with one or more cytosolic components referred to collectively as the nascent chain binding complex (NCBC). Once synthesis of a suitable number of amino acids has been completed to provide for the folding information of a "protein domain", folding of the nascent polypeptide would commence, concomitant with NCBC release. The P.I. has already identified one component, the 70 kDa heat shock protein (hsp 70) as being part of the NCBC, but has evidence that other proteins also interact with nascent polypeptides. A variety of different approaches will be used to purify and characterize NCBC components. In addition the possibility that other molecular chaperones (to which the P.I. has suitable antibodies) are present within the NCBC will be addressed. Once a consensus of NCBC components has been arrived at, antibodies to novel NCBC components will be raised and used to facilitate their purification. In addition antibodies to NCBC components, as well as the purified NCBC proteins themselves, will be used in depletion/reconstitution experiments in rabbit reticulocyte Iysates to ascertain the effects on protein maturation. It is anticipated that these studies will lead to a more thorough understanding of the different components involved in interacting with and facilitating the maturation of most, if not all, newly synthesized proteins. %%% Recent insights have led to entirely new concepts regarding how proteins are synthesized and acquire their properly folded and biologically active conformation. Classical experiments performed in the 1950's and 60's led to the proposal that protein folding was a spontaneous process, dictated on ly by the linear sequence of amino acids present within the polypeptide chain, and not requiring the help of any accessory components. Work from many laboratories now have identified a class of protein machinery, referred to as molecular chaperones, which appear to play an intimate role in helping other proteins inside the cell adopt their properly folded conformation. In studies described herein, the P.I. will continue to identify and characterize relevant components, including members of the chaperone family, which interact with and facilitate protein folding. Finally, using antibodies prepared against the nascent chain binding proteins the P.I. will effectively remove such components from the rabbit reticulocyte and determine the consequences of such removal on the maturation of particular proteins. Such work, in sum, will provide us with a better picture of how proteins inside the cell acquire their final properly folded conformation. *** ; @ ....()()))()() ; S u m m a r y I n f o r m a t i o n (