Inactivating Pit-1 mutations alter subnuclear dynamics suggesting a protein misfolding and nuclear stress response.
Inactivating Pit-1 mutations alter subnuclear dynamics suggesting a protein misfolding and nuclear stress response.
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失活的 Pit-1 突变会改变亚核动力学，表明蛋白质错误折叠和核应激反应。
DOI：
10.1002/jcb.20028
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发表时间：
2004
影响因子：
4
通讯作者：
Mancini,MichaelA
中科院分区：
文献类型：
--
作者：
Sharp,ZDave;Stenoien,DavidL;Mancini,MaureenG;Ouspenski,IliaI;Mancini,MichaelA
Pit-1是一种POU-类核DNA结合转录因子，在垂体前叶个体发育中指定了三种实质细胞类型。使用荧光融合和活细胞成像，我们比较了野生型和失活Pit-1点突变的动态行为。光漂白后荧光恢复（FRAP）和真实的时间提取数据表明野生型Pit-1具有动态迁移率曲线，当从低到高表达时，t1/2s分别为5-7 s。在生物化学上，根据提取过程中的直接观察，Pit-1保留了约50%，表明与核结构的动态相互作用。对携带两种不同致弱突变的瞬时表达的Pit-1的分析表明，它们通常易位到细胞核，但表现出两种不同水平的迁移率，两者都与野生型Pit-1明显不同。在低表达水平下，PitW 261 C和PitA 158 P的t1/2s非常快（分别为0.3和0.6 s t1/2s）。在更高的表达水平下，与野生型Pit-1不同，两种突变蛋白都变得固定化和不溶性，并且与不溶性核基质完全断裂。相对于野生型，突变的Pit-1的过表达引起核应激反应，其通过热休克诱导的热休克蛋白70（Hsp 70）的水平增加和热休克因子-1的重组来指示。PitA 158 P相对于PitW 261 Cat低表达水平的降低的迁移率与其在低水平表达时部分活化的能力及其结合同源DNA的能力相关。在高表达水平下，较低的PitA 158 P活化与其固定和不溶性相关。这些数据表明特定的核内迁移率与Pit-1转录功能之间存在联系，可能是为了确保与染色质的充分相互作用，或者在非DNA结合的Pit-1的情况下，作为阻遏物相互作用（Scully和Rosenfeld [2002]：Science 295：2231-2235）。这些数据意味着失活突变可以导致核内分选远离转录相关途径，并且至少部分导致错误折叠的蛋白质途径。两者合计，谨慎的建议时，解释点（或其他）突变分析的反式激活因子的功能，作为新的区室化，特别是在表达水平的背景下，可能会云之间的区别定义功能分子结构域和错误折叠蛋白质的核内加工。© 2004 Wiley利斯公司
Pit‐1, a POU‐class nuclear DNA‐binding transcription factor, specifies three of the parenchymal cell types in anterior pituitary ontogeny. Using fluorescent fusions and live cell imaging, we have compared the dynamic behavior of wild‐type and inactivating Pit‐1 point mutations. Fluorescence recovery after photobleaching (FRAP) and real‐time extraction data indicate that wild‐type Pit‐1 has a dynamic mobility profile, with t1/2s∼ 5–7 s when expressed from low to high amounts, respectively. Biochemically, Pit‐1 is ∼50% retained according to direct observation during extraction, indicating a dynamic interaction with nuclear structure. An analysis of transiently expressed Pit‐1 carrying two different debilitating mutations reveals that they translocate normally to the nucleus, but exhibit two different levels of mobility, both clearly distinguishable from wild‐type Pit‐1. At low expression levels, the t1/2sof PitW261Cand PitA158Pare extremely rapid (0.3 and 0.6 s t1/2s, respectively). At higher expression levels, unlike wild‐type Pit‐1, both mutant proteins become immobilized and insoluble, and fractionate completely with the insoluble nuclear matrix. Relative to wild‐type, over expression of mutated Pit‐1 elicits a nuclear stress response indicated by increased levels of heat shock inducible heat shock protein 70 (Hsp70), and reorganization of heat shock factor‐1. The decreased mobility of PitA158Prelative to PitW261Cat low expression levels correlates with its ability to partially activate when expressed at low levels and its ability to bind cognate DNA. At high expression levels, lower PitA158Pactivation correlates with its immobilization and insolubility. These data suggest a link between specific rates of intranuclear mobility and Pit‐1 transcription function, perhaps to insure sufficient interactions with chromatin, or in the case of non‐DNA binding Pit‐1, interaction as a repressor (Scully and Rosenfeld [2002]: Science 295:2231–2235). These data imply inactivating mutations can lead to an intranuclear sorting away from transcription related pathways, and at least in part to a misfolded protein pathway. Taken together, caution is suggested when interpreting point (or other) mutational analyses of transactivator function, as new compartmentation, especially in the context of expression levels, may cloud the distinction between defining functional molecular domains and intranuclear processing of misfolded proteins. © 2004 Wiley‐Liss, Inc.