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首页> 外文期刊>AIDS Research and Human Retroviruses >Using antiubiquitin antibodies to probe the ubiquitination state within rhTRIM5α cytoplasmic bodies
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Using antiubiquitin antibodies to probe the ubiquitination state within rhTRIM5α cytoplasmic bodies

机译：使用抗泛素抗体探测rhTRIM5α细胞质体内的泛素化状态

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摘要

The first line of defense protecting rhesus macaques from HIV-1 is the restriction factor rhTRIM5α, which recognizes the capsid core of the virus early after entry and normally blocks infection prior to reverse transcription. Cytoplasmic bodies containing rhTRIM5α have been implicated in the ubiquitin-proteasome pathway, but the specific roles these structures play remain uncharacterized. Here, we examine the ubiquitination status of cytoplasmic body proteins. Using antibodies specific for different forms of ubiquitin, we show that ubiquitinated proteins are present in cytoplasmic bodies, and that this localization is altered after proteasome inhibition. A decrease in polyubiquitinated proteins localizing to cytoplasmic bodies was apparent after 1 h of proteasome inhibition, and greater differences were seen after extended proteasome inhibition. The decrease in polyubiquitin conjugates within cytoplasmic bodies was also observed when deubiquitinating enzymes were inhibited, suggesting that the removal of ubiquitin moieties from polyubiquitinated cytoplasmic body proteins after extended proteasome inhibition is not responsible for this phenomenon. Superresolution structured illumination microscopy revealed finer details of rhTRIM5α cytoplasmic bodies and the polyubiquitin conjugates that localize to these structures. Finally, linkage-specific polyubiquitin antibodies revealed that K48-linked ubiquitin chains localize to rhTRIM5α cytoplasmic bodies, implicating these structures in proteasomal degradation. Differential staining of cytoplasmic bodies seen with different polyubiquitin antibodies suggests that structural changes occur during proteasome inhibition that alter epitope availability. Taken together, it is likely that rhTRIM5α cytoplasmic bodies are involved in recruiting components of the ubiquitin-proteasome system to coordinate proteasomal destruction of a viral or cellular protein(s) during restriction of HIV-1.
机译：保护猕猴免受HIV-1侵害的第一道防线是限制因子rhTRIM5α，该限制因子可在进入后早期识别病毒的衣壳核心，并通常在逆转录之前阻止感染。含有rhTRIM5α的细胞质体已参与泛素-蛋白酶体途径，但这些结构所起的特定作用仍未鉴定。在这里，我们检查细胞质体蛋白的泛素化状态。使用特异性针对不同形式的泛素的抗体，我们显示泛素化的蛋白质存在于细胞质体中，并且蛋白酶体抑制后这种定位发生了改变。蛋白酶体抑制1 h后，定位于细胞质体的多泛素化蛋白减少，并且蛋白酶体抑制延长后，差异更大。当去泛素化酶被抑制时，还观察到胞质体内多聚泛素结合物的减少，这表明在延长的蛋白酶体抑制作用后，从多泛素化的细胞质体蛋白中去除泛素部分并不是这种现象的原因。超分辨率结构照明显微镜显示了rhTRIM5α细胞质体和定位在这些结构上的聚泛素结合物的更详细信息。最后，连接特异性多聚泛素抗体显示，K48连接的泛素链位于rhTRIM5α细胞质体，将这些结构牵连到蛋白酶体降解中。用不同的多泛素抗体观察到的胞质体的差异染色表明，在蛋白酶体抑制过程中发生了结构变化，从而改变了表位的利用率。两者合计，rhTRIM5α细胞质体可能参与招募泛素-蛋白酶体系统的成分，以协调在限制HIV-1期间蛋白酶体对病毒或细胞蛋白的破坏。

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