How do SUMO proteins be attached to targets?
What's the input and output of PKC-θ?
How does immunological synaspe form? What's its function?
导读上一部分的最后我说要有问题问你，现在这些问题们可是来了，如果你读懂了上部分要求读的内容，回答这些问题应该是不在话下的，同时这些问题的答案也会帮助你更好地理解之后的内容。Frankly speaking, 这就是我们文献阅读会准备的重要部分——提思考题，这里我先提了几个，之后大家都是要提的哦～还有，上一篇导读中我用一些资料为大家解释了一些重要的概念/背景知识，今后找出哪些概念需要搜集资料、搜集整理的过程以及即将在本篇中出现的一些东西都将会是大家的工作，要做好心里准备哟。
只是分割线而已
答出了上面的问题，接下来就可以正式进入Results部分了。这篇文章的Results足足有5页，里面有大量的技术细节，初读者要是陷进去可就麻烦了，所以我们先来大致整理一下Results到底得出了哪些结论——
Sumoylation of PKC-θ upon TCR stimulation
首先当然是确认TCR-induced sumoylation of the kinase PKC-θ这件事确实发生了这里用到了SDS-PAGE电泳技术判断分子量（SDS-PAGE是分子生物学最重要的实验方法之一，随手搜索就能搜出大量资料，我就不越俎代庖了）等方法来确认SUMO1在TCR、CD28 costimulated之后确实SUMOylate了PKC-θ，以及确证和SUMO1很像的另一个SUMO亚家族SUMO3不参与这件事，说明其特异性。
确认了会发生SUMOylation，那么发生在哪呢？
Lys325 and Lys506 are major PKC-θ-sumoylation sites
这一段基本就是通过替换氨基酸残基后检测SUMOylation的方法寻找target site（检测的方法各位可以试着自己看看先，看不懂也没关系），最后
this would suggest that sumoylation of PKC-θ was conserved during evolution and thus has an important regulatory role. That conclusion was supported by our finding that amphioxus PKC-δ, a common ancestor of both PKC-δ and PKC-θ, was also modified by SUMO1 conjugation.
在哪也知道了，那么这个SUMOylation修饰究竟有什么功能呢？
T cell activation and TH2 differentiation require PKC-θ sumoylation
这里用到了上一段实验得到的一个工具——PKC-θ(2KR)，也就是把Lys325 and Lys506两个SUMOylation位点都突变掉的PKC-θ，这样的PKC-θ就没法被SUMO化了，结果发现原来的信号传递不再存在了，so
sumoylation of PKC-θ at Lys325 and Lys506 was essential for its downstream signaling functions in T cells.
These results provided evidence that SUMO conjugation of PKC-θ was essential for its physiological function.
接下来是
PIASxβ is a SUMO E3 ligase for PKC-θ
鉴定了在这个具体通路中发生作用的SUMO E3 ligase, 个人认为跟前后关系不太大，看看就好。
PKC-θ–CD28’s colocalization to the cSMAC requires sumoylation
重头戏来了，为啥子PKC-θ非得被SUMO化才能在这好好发挥作用呢？是因为这样才有催化活性吗？
Ectopic expression of SENP1 inhibited the TCR-induced PKC-θ activity but not the basal PKC-θ activity in an in vitro kinase assay (i.e., in the absence of cofactors to reflect the in vivo PKC-θ activity in the absence of in vitro stimulation) of PKC-θ immunoprecipitated from T cells.
When we performed the in vitro kinase assay in the presence of phosphatidylserine plus PMA, the catalytic activity of PKC-θ was induced similarly regardless of whether PKC-θ was immunoprecipitated from T cells ectopically expressing SENP1 or from T cells not ectopically expressing SENP1 and whether the cells were stimulated with SEE-pulsed APCs or not.
看起来好像不是呢（PS: SENP1前文有提到，是一种SUMO protease）。那是因为啥呢？
Because PKC-θ is the only PKC isoform stably localized in the cSMAC of the immunological synapse following antigen stimulation, and because this unique localization is required for productive T cell activation, we next investigated whether sumoylation-deficient PKC-θ mutants could be properly targeted to the cSMAC in primary human naive CD4+ T cells stimulated with SEE-pulsed APCs.
于是就继续PKC-θ(2KR)大法好
Quantitative analysis of the experiments above (Fig. 4a) confirmed that PKCθ-2KR was significantly more diffuse than wild-type PKC-θ in the cells stimulated with SEE-pulsed APCs (Fig. 4c).
………………
Together these results indicated that sumoylation of PKC-θ targeted it to the cSMAC and that this might be a mechanism for regulating its function.
那么，仅仅是PKC-θ本身的定位吗？
Sumoylation mediates PKC-θ’s association with CD28 and filamin A
还记得那副抽象画吗？
synapse抽象画
正常的synapse中，PKC-θ应该集中分布在TCR周围，而filamin A则应该在外周，这样的定位依赖于PKC-θ’s association with CD28 and filamin A，而这样的association依赖于PKC-θ的SUMO化，无法SUMO化的PKC-θ无法正确引导synapse的形成，也就无法正常行使它的历史免疫使命了。这一段具体的实验步骤比较多但是技术不算复杂，大家可以在有时间的时候耐着性子慢慢阅读。
依然只是分割线而已
好了，Results部分到此就大致整理完毕了，现在各位可以尝试做一件事来验证自己是不是真的理解了Results的大意了——回去自己把Abstract读懂吧！
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