（如果你还不知道怎么突然要读起这篇文献来了，先戳这里看看吧）
手把手带读文献模块到了！接下来是各部分Results的思路整理、陌生概念解释和思考题，快抓起文献跟上我们的步伐开始读吧！
PS: 不记得各个分子在通路中的位置的时候就回来看看这幅图
Result 1 - Identification of USP38 as an Essential Negative Regulator of Type I IFN Signaling
Summary中提到：
Here we report that USP38 negatively regulates type I IFN signaling by targeting the active form of TBK1 for degradation in vitro and in vivo.
那么，Result 1就是要确认USP38识别是一个重要的负调节I型干扰素的信号。
为了确定去泛素化酶在抗病毒免疫性中的角色，研究者运用生物信息学方法进行了数据分析，找到了一种去泛素化酶——USP38：
we screened 81 genes encoding deubiquitinases（DUBs）and identified USP38 as a negative regulator of type I IFN signaling.
运算得来终觉浅，还是得做点实验
USP38 potently inhibited the activation of IFN-b by treatments of ...(lots of pathogen-associated molecular patterns) (Figure 1A).
也就是说，USP38可以强效抑制IFN-b的激活。
因为IFN-b激活时，需要合作激活 IRF3 和 NF-kB，于是我们想检验USP38是否直接影响IRF3信号。
we used an IFN- stimulated response element (ISRE) luciferase（荧光素酶） reporter to test whether USP38 directly affects IRF3 signaling.
结果发现 USP38 同样抑制 ISRE-luc 活性。接下来，
We assessed phosphorylation of IRF3 in 293T cells, expressing USP38 together with RIG-I(N), MDA5, MAVS, STING-cGAS, or TRIF.
并发现 USP38 抑制所有这些先天免疫受体和接头蛋白诱导的 IRF3 活化。
进一步的 qPCR 分析表明，
the mRNA levels of IFNa4, IFNb, IFIT1, IFIT2, and ISG15 in USP38 ectopic expression cells were markedly reduced during VSV infection (Figure S1B)，
也就是 USP38 抑制干扰素刺激基因（ISGs）的表达。
为了确定生理条件下 USP38 的作用，
we efficiently knocked down USP38 in 293T cells, THP-1 cells, or human peripheral blood mononuclear cells (PBMCs，外周血细胞) (Figure S1C）.
荧光素酶测定分析表明，
USP38 knockdown could enhance the IC poly(I:C)-, poly(dA:dT)-, or VSV-EGFP-induced ISRE activation (Figure 1C).
与这一结果相一致，我们发现敲除 USP38 显著增强 IRF3 的磷酸化 (Figure 1D)。
为了证实这些发现，
we knocked down USP38 in THP-1 cells, 293T cells, A549 cells, or PBMCs, and we found that USP38 knockdown significantly increased the IFN-a4 and IFN-b expression, as well as IFN-b secretion induced by VSV-EGFP or HSV-1 infection (Figures 1E–1G and S1D–S1F).
进一步我们表明在这些细胞中的 USP38 敲除使他们抵抗 VSV EGFP 感染（Figure 1H, 1I, S1G, S1H）。
这些结果共同表明，USP38 负调节I型干扰素信号以及在人体各种细胞类型中的抗病毒免疫。
思考题：
这里用了哪几个实验验证 USP38 负调节I型干扰素信号？
Results 2 - USP38 Deficiency Enhances Antiviral In Vivo
首先我们回顾一下SUMMARY：
Knockdown or knockout of USP38 increases K48-linked ubiquitination and degradation of TBK1, thus enhancing type I IFN signing.
并且，在Result1中得出的结论：
……suggesting that USP38 inhibits the expression of IFN-stimulated genes(ISGs)…Collectively,these results suggest that USP38 negatively regulates type I IFN signing as well as antiviral immunity human cell types.
那么，作为条件对照，说明USP38 缺失对抗病毒免疫的作用是必要的.
尽管Result1中有：
Further we showed that USP38 knockdown **in this cells **rendered them resistant to VSV-EGFP infection.
但由于都是体外实验，所以这里进行体内实验.
于是，实验一，利用人工培育的USP38缺陷的小鼠进行细胞水平验证.
To determine the fuction of USP38 in primary cells,we generated USP38-deficient mice using a TAL effector nuclease (TALEN)-based system,and we prepared bone marrow-derived macrophages(BMMs) from wild-type(WT) and Usp38`/` mice. Next we infected those BMMs for 16 or 24 hr with the RNA virus VSV or the DNA virus HSV-1.
结果是：
USP38ˉ/ ˉ BMMs produce 2- to 4-fold more IFN-阝in responds to VSV or HSV-1 than WT BMMs.
…deletion of USP38 had a strong effect on the expression of many ISGs…as well as the pro-inflammatory cytokines TNF-∝and IL-6……
We detected significantly less VSV or HSV-1 in USP38ˉ/ˉ BMMs than in WT BMMs.These data indicate a negative role for USP38 in the sensing of both RNA and DNA viruses in mouse BMMs.
接着是实验二，在器官个体水平的实验，不过这次为了更好说明USP38的"functional significance", 使用了高浓度病毒VSV. 结果是：
器官水平：
……We found that VSV loads in tissue sample infected Usp38'/' mice were significantly lower than those from WT mice 24 hr post-infection.
个体水平：
Importantly, Usp38ˉ/ˉ mice were signifcantly more resistant to VSV infection in overall survival compared with WT mice.
于是自然得出结论：USP38 Deficiency Enhances Antiviral Immunity In Vivo
陌生概念：
plaque-forming units(PFU)：空斑形成单位，是一种测量每单位体积可以形成斑块的颗粒数量的办法；是一种功能性的测量，有缺陷或不影响目标细胞的病毒颗粒不产生斑块.
最后是思考题：
Why they challenged Usp38ˉ/ˉ mice with VSV(1x10^8 plaque-forming units(PFU)/g)？
Result 3 - USP38 Interacts with TBK1 after Viral Infection
为了确定和USP38相互作用的分子，研究者主要进行了几个实验。
实验一
we co-transfected 293T cells with RIG-I, MDA5, MAVS, TBK1, IKKi, or IRF3 together with increasing amounts of USP38 plus the IFN-β or ISRE luciferase reporter
and we found that USP38 inhibited activation of both luciferase reporters induced by RIG-I, MDA5, MAVS, TBK1, but not IKKi or IRF3 (Figure 3A).
将RIG-I、MAD-5、MAVS、TBK1、IKKi、IRF3与附带了了荧光蛋白酶的IFN-β和ISRE共同导入细胞，试验结果是USP38抑制了RIG-I、MAD-5、MAVS、TBK1诱导的荧光蛋白酶表达，但没有抑制由IKKi or IRF3诱导的表达。（荧光蛋白酶用于报告IFN-β or ISRE的表达）
实验二
Co-immunoprecipitation and immunoblot analyses revealed that USP38 specifically interacted with TBK1, but not MAVS, IKKi, IRF3, or IRF7 (Figure 3B).
免疫共沉淀和免疫印迹分析发现USP38与TBK1特异性相互作用。
实验三
confocal microscopic analysis using 293T cells transfected with USP38-GFP and TBK1-dsRFP showed that USP38 co-localized with TBK1 in the cytosol (Figure 3C).
对带有荧光蛋白的USP38与TBK1用共聚焦显微镜进行分析后，发现USP38与TBK1在胞液中共定位。
在这些实验之后，我们已经看出了USP38和TBK1之间确实有交易一些关系，于是我们进一步研究
我们要验证在生理条件下USP38与TBK1的配合。
we infected 293T cells, THP-1 cells, or PBMCs with VSV-EGFP, and we found that the interaction between USP38 and TBK1 was barely detectable in unstimulated cells (Figures 3D–3F).
我们发现在未感染的细胞中很少的USP38与TBK1共同作用的现象，在感染后的这三种细胞中均有显著的增加。
进一步研究它的分子机理，我们想知道USP38的哪个结构域负责它与TBK1的交易共同作用。
we constructed several deletion constructs of USP38. Co-immunoprecipitation and immunoblot analyses revealed that the N-terminal USP domain of USP38 interacted with TBK1, but its C-terminal domain showed no interaction with TBK1 (Figure 3G).
我们构建了一系列残缺的USP38，并让它们分别与TBK1共免疫沉淀，免疫印迹分析。
于是我们发现，N端USP结构域和TKB1有共同作用，而C端没有。
传说中的思考题环节:
实验一说明了什么？和这个result有什么联系？
怎么理解Figure 3C？
Result 4 - USP38 Specifically Degrades Active TBK1
我们要去研究USP38与TBK1相互作用的分子机制。
恩，如何研究呢？
In 293T cells transfected with FLAG-tagged TBK1 and HA-tagged IRF3, together with increasing doses of USP38, we found that the concentration of TBK1 proteinde creased considerably with increasing USP38 expression. However, the mRNA level of TBK1 remained unchanged (Figure 4A), suggesting that USP38 causes TBK1 protein degradation.
TBK1随USP38的增多而减少，但是TBK1的mRNA却不变，暗示了USP38导致TBK1降解。
To determine the specificity of the USP38-mediated TBK1 degradation, we used other USP family members USP3 and USP13 as a control, and we found that USP38, but not USP3 or USP13, induced TBK1 degradation (Figure S3A).
这里用了USP3 and USP13作为对照，证明只有USP38导致TBK1降解。
To determine whether USP38 causes the degradation of other proteins, we performed a similar experiment and found that USP38 specifically degraded TBK1, but not IKKi, IKK-a, or IKK-b(Figure 4B).
这里用IKKi, IKK-a or IKK-b做对照，说明只有TBK1被USP38控制降解
To determine whether USP38 degrades TBK1 through aubiquitin-protease pathway, we performed experiments in the presence of the proteasome inhibitor MG-132 or 3-Methyladenine (3-MA), and we found that MG-132, but not 3-MA, blocked the degradation of TBK1 (Figure 4C), suggesting that USP38-induced TBK1 degradation is dependent on a proteasome pathway.
这里用了蛋白酶的抑制剂，结果TBK1不降解了，说明USP38是通过蛋白酶达到控制TBK1的效果的。
Murine USP38(mUSP38) showed similarinhibitory function of human USP38 protein (Figure S3B), suggesting a conserved function of USP38 between mice and humans.
小鼠和人类的USP38功能相似，USP38相对保守。
To determine whether USP38 can mediate degradation ofendogenous TBK1 under physiological conditions, we transfected 293T cells with USP38, and we found that endogenous TBK1 protein was unchanged (Figure S3C).
很奇怪的是USP38并没有使外源的TBK1降解。于是问题来了。
We reasoned that USP38 may specifically target the activated form of TBK1 for degradation.
我们的猜测是USP38是在TBK1激活时才起作用。
于是设计实验。
Therefore, we generated an inactive mutant ofTBK1 with a substitution of alanine for the serine at position 172 (S172A), which abrogates its auto-phosphorylation, and then we co-transfected the 293T cells with USP38, WT TBK1, or TBK1 (S172A). Indeed, we found that USP38 could not interact with mutant TBK1 (S172A) and failed to promote it for degradation (Figures 4F and 4G); however, TBK1 constitutive active mutant (S172E) showed an increasing binding ability to USP38 compared to WT TBK1 (Figure 4F).
这里用了一个失活的TBK1，果然USP38不能使TBK1降解了。
Furthermore, we found that USP38 expression reduced TBK1 protein, but not TBK1 mRNA, after VSV infection compared with 293T cells transfected with the empty vector (EV) (Figures 4H andS3D). Conversely,USP38knockdown in THP-1 cells increased endogenous TBK1 protein levels in cells infected with VSVEGFP or transfected with IC poly(I:C), but not in uninfected cells (Figures 4I andS3E).
这里说的是USP38没有使TNK1的mRNA减少，而USP38 knockdown只在cells infected with VSVEGFP or transfected with IC poly中使TBK1增多，而在uninfected cells却不行。
Similar results were obtained with WT or Usp38/BMMs with VSV or HSV-1 infection (Figure 4J). These results suggest that USP38 specifically degrades the active form (p-TBK1) of TBK1 after viral infection.
嗯，通过这些实验数据证明了在病毒感染后USP38特异性的降低TBK1的活性。
思考题：
如何证明USP38是特异性调控TBK1的？
如何证明USP38是针对激活的TBK1的？
Result 5 - USP38 Midiates the K38-K48 Ubiquitination Transition of TBK1 on Lys670
在介绍这个result之前，回顾一下summary中的一句话
Knockdown or knockout of USP38 increases K33-linked ubiquitination, but it abrogates K48-linked ubiquitination and degrdation of TBK1, thus enhanceing type I IFN signaling.
而从题目中我们可以看出，result5正是从实验角度对summary的这一部分的阐述。现在进入正题。
因为之前的研究显示TBK1 在被病毒感染后 K48-linked and K63-linked ubiquitination都会发生，所以先验证了一下，发现
USP38 markedly increased K48-linked, but not K63-linked ubiquitination of TBK1
说明USP38的作用在这里确实是只管K-48
然后
Since the K48-linked ubiquitination of TBK1 is regulated by NLRP4, DTX4, or TRIP protiens
所以看一下是不是USP38通过调节这些因子来调节TBK1, 结果
We did not observe any apparent differences in the protein abundance of NLRP4, DTX4, or TRIP in the process or absence of USP38 after viral infection(Figures S4A-S4C)
于是就有了第一段结尾的结果
USP38 regulates K48-linked ubiquitination of TBK1 via a distinct mechanism.
考虑到USP38是DUB，于是他们开始思考USP38是如何通过去泛素化增加而不是减少TBK1 K48-linked ubiquitination
他们利用USP38的突变体C545A, H857A实验，发现突变体是不能使TBK1降解的，所以他们就得出了USP38的去泛素化活性是降解TBK1的前提这一结论。
基于以上实验他们开始思考USP38会移去和加上哪种ubiquitin chain，于是他们做了这样一个实验
We performed experiments using 293T cells expressing FLAG-TBK1, Myc-USP38, and different.types of HA-tagged ubiquitin.
从Figures S4D-S4F中他们发现了K33-linked ubiquitination 被移去了。再与利用突变体不能移去K33-linked ubiquitination of TBK1这一结果对比，说明K33-linked poly-ubiquitin chain on TBK1的去除是依赖于USP38的去泛素化活性的。
另外，他们还发现TBK1在病毒感染后会经历K48-和K33-linked ubiquitination，但是K48-linked ubiquitination是被促进的，所以他们想到了这个
USP38 may remove K33-linked ubiquitin chains and add K48-linked ubiquitin chains to TBK1 after viral infection.
如何在论证生理条件下会发生这样的过程呢？这里用到了mass spectrometry analysis(质谱分析)
we performed mass spectrometry analysis of ubiquitinated TBK1 from 293T cells or USP38-/-293T cells by VSV infection, using a similar strategy as previously described(2014)
结果发现
ubiquitinated TBK1 with K33 or K48 linkage was hardly detected without viral infection.
并且，作为对比
after VSV infection, the increased K33-linked ubiquitinated TBK1 as well as decreased K48-linked ubiquitinated TBK1 in USP38−/− cells were readily detected by mass spectrometry analysis, compared to WT cells
所以有了
USP38 is responsible for the cleavage of K33-linked ubiquitin chains of TBK1
更深一步的研究是基于 confocal microscopic analysis ，他们发现
USP38 was colocalized with K33-linked. ubiquitin chains inside the cells, while the co-localization between USP38 and K33-linked ubiquitin chains was enhanced after HSV-1 infection.
所有上面这些研究最终得出的结论就是
USP38 is responsible for cleaving K33-linked ubiquitin chains from TBK1
至于如何证明接上K44-linked poly-ubiquitination of TBK1 at Lys670的问题(从summary之前的图里面我们可以看到不论是移去还是接上都是在同一位点进行的)，首先是先前研究
NLRP4 induced the K48-linked poly-ubiquitination of TBK1 at Lys670, leading to its proteasomal degration.
而这次更深入的研究目的是介绍发现在同一位点进行移去和连接的实验原因
neither USP38 nor TRIP can degrate TBK1(K670R) mutants
TBK1 K670R mutant abrogates not only K48-linked ubiquitination but also K33-linked ubiquitination of TBK1
(实验数据在Figures 5J-S4G 及Figures 5K and S4H)
通过这两个实验结果他们得出了
K33-和K48-linked ubiquitin chains may conjugate to TBK1 at the same amino acid position(即Lys670)
那么如何证明这两个过程只在Lys670位进行呢？
we pulled down the WT TBK1 and TBK(K670R) mutant and checked the ratio of K33 and K48 poly-ubiquitin chains in the immunoprecitates by mass spectrometry analysis
发现在TBK1(K670R) 免疫共沉淀中没有K33和K48 poly-ubiquitin信号
所以！位点只有这一个
最后！他们研究了是否K33-linked ubiquitin of TBK1 is more stable than the K48-linked ubiquitinated TBK1
方法是we performed the cycloheximide(CHX) assay and pulled down K33-, K63-, or K48-ubiquitinated TBK1.
这样就发现他们的假设是正确的，也就是我们的结果
USP38 inhibits type I IFN signaling by removing K33-linked and promoting K48-linked poly-ubiquitination chains of TBK1 at Lys670
以上就是这个result的实验思路和结果的叙述。
那么！传说中的思考题时间到了！
USP38是如何通过K33-linked ubiquitination和K48-linked ubiquitination来控制TBK1的？
多组实验是如何一步步证明K38-linked ubiquitin chains被移去及K48-linked ubiquitin chains被接上是在同一位点进行的？
Result 6 - USP38 Promotes TBK1 Degradation in an NLRP4-Dependent Manner
这一段主要讲NLPR4在USP38 Promotes TBK1 Degradation中的重要性
首先有一个事实依据
Since both NLRP4 and USP38 degraded TBK1 by a similar mechanism that promoted the K48-linked ubiquitination of TBK1 at Lys670 （就是USP38与NLRP4作用效果类似）
然后提出猜想
NLRP4 and USP38 work together to regulate TBK1 stability.
之后用实验证明
第一个实验
we performed immunoprecipitation and immunoblot analysis in 293T cells expressing USP38, NLRP3, or NLRP4,
第一个实验发现
USP38 interacted with NLRP4, but not with NLRP3 (Figure 6A).
然后第二个实验验证交互的过程
To demonstrate the interaction between USP38 and NLRP4 in physiological conditions, we infected THP-1 cells or PBMCs with VSV-EGFP for the indicated time points,
第二个实验发现
and we observed the enhanced interaction between USP38 and NLRP4 after viral infection (Figures 6B and 6C). In addition, we found that the PYD and NOD domains of NLRP4 interacted with USP38 (Figure 6D), whereas the N-terminal domain of USP38 was essential for binding to NLRP4 (Figure 6E).
同时
Further experiments showed that USP38 was co-localized with TBK1 and NLRP4 (Figure S5A).
为了证实这些发现,又做了一些实验 (括号内为结论)
(1)we specifically knocked downNLRP4 and found that【 the interaction between USP38 and TBK1was markedly attenuated (Figure 6F).】
(2)we demonstrated that【 the dynamic interaction between USP38 and TBK1 was totally abrogated 】in NLRP4 small interfering RNA (siRNA)- treated THP-1 cells after viral infection (Figure 6G).
(3)【we also observed the reduced interaction between TBK1 and TRIP or DTX4】 when NLRP4 was knocked down (Figures S5B and S5C).
结果显示
These results suggest that USP38, TRIP, and DTX4 may bind to TBK1 in an NLRP4-dependent manner.
同时
Luciferase reporter assay further showed that the inhibition of ISRE-luc activity by USP38 could be completely relieved when NLRP4 was knocked down (Figures S5E and S5F).
综合以上所有
Taken together, these results suggest that NLRP4 is indispensable for the inhibitory function of USP38.
。。。。貌似只有一个概念需要解释
siRNA是一类约21~25nt的RNA分子，由Dicer(RNAaseⅢ家族中对双链RNA具有特异性的酶)加工而成，通过完全互补配对的方式与目标mRNA结合，引起其降解，从而导致靶基因的的沉默。
然后，问题来了
Which phenomenon has proved that the NLPR4 is essential?
Result 7 - Regulation of TBK1 Stability by the NLRP4 Signalosome
与本result 相关的introduction对应为：
Recently, we and others found that NLRP4/DTX4 and another E3 ligase TRAF-interacting protein (TRIP) induce the K48-linked poly-ubiquitination of TBK1, leading to its proteasomal degradation (Cui et al., 2012; Zhang et al., 2012)
所以这里就是要说NLRP4信号小体调节TBK1稳定性相关问题
开头说到core protein NLRP3, 比较地引出了USP38, DTX4 AND TRIP三者与TBK1 的相互作用依赖于NLRP4。因此，根据发现，我们猜想：
NLRP4, together with USP38 and E3 ligases,might form the NLRP4 signalosome to control TBK1 stability and type I IFN signaling。
接下来我们分3个部分实验验证了猜想：
TBK1和NLRP4信号小体成员间的相互作用
方法是：
infected Myc-TRIP- and GFP-DTX4-expressingcells with VSV, and then we immunoprecipitated different complexes from cells, collected at different time points post-infection with either TBK1 or NLRP4 antibodies.
通过免疫印迹分析结果发现：
NLRP4 and USP38 targeted to TBK1 at 6 hr post-infection, whereas DTX4 and TRIP interacted with TBK1 at a later time point (at 8 hr) (Figure 7A)
Importantly, USP38, but not DTX4 or TRIP, constitutively interacted with NLRP4 in unstimulated cells (Figure 7B).
Interaction between NLRP4 and USP38 was further increased after viral infection. Interactions between NLRP4 and TRIP or DTX4 were detected at 6 or 8 hr, respectively, post-infection (Figure 7B).
这些结果都显示了病毒入侵之后NLRP4信号小体的动态装配。
NLRP4信号转导体调节TBK1稳定性的分子机制
方法是：
generated NLRP4(-/-) and USP38(-/-) HEK293T cells
我们发现：
USP38(-/-）cells and NLRP4(-/-）cells showed increased ISRE luciferase activity, ISG expression, and antiviral immunity after viralinfection (Figures S6C–S6F).
有了这个，我们进一步就确定NLRP4和USP38在TBK1和NLRP4信号转导体相互作用的角色：
USP38 deficiency does not affect the interaction of TBK1 with NLRP4, TRIP, or DTX4 (Figure 7C). By contrast, in NLRP4(-/-) cells, the interactions of TBK1 with USP38, TRIP, or DTX4 were completely abrogated (Figure 7D).
结果表明，在NLRP4信号转导体中，NLRP4是一个链接USP38, TRIP和DTX4这三者与TBK1的关键蛋白，而TBK1与NLRP4, TRIP和DTX4的相互作用却不依赖于USP38.
NLRP4信号小体成员如何影响TBK1的ubiquitination
实验现象摆在这里：
observed increased levels of K33-linke dubiquitination and decreased levels of K48-linked ubiquitination of TBK1 in both USP38(-/-) and NLRP4(-/-) cells (Figures 7E and 7F).
还有：
TRIP or DTX4 deficiency only affected K48-linked ubiquitination, but not K33-linked ubiquitination, of TBK1 (Figures S7A and S7B).
结论告诉我们：
TBK1上K33-linked的泛素化的消除需要NLRP4和USP38,同时K48-linked的泛素化需要E3连接酶DTX4和TRIP。
还有有趣的一个现象：
although NLRP4 or TRIP still interacted with TBK1 in USP38(-/-) cells (Figure 7C), they could not cause TBK1 degradation in USP38(-/-) cells even after VSV infection or IC poly(I:C)treatment (Figures 7G,7H,S7C,and S7D).
相对应的是：
Overexpression of WT USP38, but not the USP38 (CA/ HA) mutant, restored the function of NLRP4 to degrade TBK1 in USP38(-/-) cells (Figure 7I)。
这就是说，对于NLRP4信号转导体降解TBK1的全过程，USP38的去泛素化是灰常关键的。
而且，我们发现在NLRP4(-/-)细胞中USP38或者TRIP都不能降解TBK1.
因此，result 7的结论是：
NLRP4, USP38, TRIP, and DTX4 worked cooperatively as a signalosome to control TBK1 ubiquitination transition and degradation, thus inhibiting IFN-b signaling.。
So here comes a QUESTION:
Whats the mechanism for these members of the NLRP4 signalosome to control TBK1 ubiquitination transition and degradation?
Discussion
终于我们来到了最后的部分——Discussion. 在这部分中崔老师（或者是他的学生？）简述了一下相关研究的情况，强调了这项研究的重要性，并根据这项研究本身和一些相关研究勾勒出了NLRP4信号小体调控TBK1以至于下游许多信号的全貌，并简述了本研究在癌症研究中的潜在应用。嗯，详细内容就照例自己读啦，反正大部分内容前面都已有提及。
最后，记得10月30日下午三点来北院101讲学厅听崔隽老师的亲自讲解哦～
