The histone H3-H4 tetramer is a copper reductase enzyme

2020年7月发表在 Science 上

摘要：真核生物组蛋白的 H3-H4 四聚物的 H3-H3' 二聚表面有一个可能的铜离子结合位点，这个位点的功能未知。真核生物出现时正伴随着全球oxygenation，氧含量的升高对于细胞对铜的利用产生了威胁。结合这一事实，组蛋白可能对于细胞内的铜稳态起作用。研究者们发现，重组的 Xenopus laevis H3-H4 四聚体是一种能结合二价铜离子、将其还原为一价铜离子的氧化还原酶（in vitro）。对可能的活性位点的 loss- and gain-of-function mutation 能够相应地影响铜离子结合和酶活性，同时影响细胞内一价铜离子浓度以及依赖铜离子的线粒体呼吸作用，以及酵母 Saccharomyces cerevisiae 的 Sod1 功能。因此，组蛋白 H3-H4 四聚物的功能并不仅仅是染色质 compaction 和表观调控，它还能生成 biousable Cu1+。

真核生物组蛋白是从古生菌的组蛋白样蛋白进化而来。古生菌没有细胞核，其组蛋白类似于真核生物 H3-H4 四聚物。古生菌没有明显的表观遗传调控能力，也不需要压缩基因组来适应细胞核，这些我们认为的组蛋白功能它都不需要，那它的这种蛋白质是用来做什么的呢？可能有其他的功能。

Fig. 1. Recombinant X. laevis histone H3-H4 tetramer interacts with cupric ions. (A)Left: X. laevis (Xl) nucleosome core particle structure [Protein Data Bank (PDB) 1KX5] (38). The box delineates the H3-H3′ interface. Right: Interface residues H3H113 and H3C110 are shown. (B) Alignment of the C-terminal region of S. cerevisiae (S. c.)and Homo sapiens (H. s.) histone H3 and archaeal [Methanothermus fervidus (M. f.)] histones. (C)Left: UV-visible absorbance spectrum of the Xl H3-H4 tetramer incubated with or without Cu2+. Inset: Differential absorbance compared to tetramer without Cu2+. Right: Buffer-corrected differential absorbance of the indicated Xl tetramers. AU,absorbanceunits;eq.,equivalents.(D) Representative ITC titration profile of the Xl H3-H4 tetramer. Circles are experimental data, and the line is the fitted curve. Average dissociation constant (KD), enthalpy change (DH), and stoichiometry (N) ± SD of the H3-H4 tetramer-Cu2+ complex calculated from three experiments are shown. Single-letterabbreviationsfor theaminoacidresiduesare as follows:A,Ala;C,Cys;D,Asp;E,Glu;F,Phe;G,Gly;H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

A 图所示为 H3 组蛋白二聚面上的铜离子结合位点，两个关键氨基酸残基的保守性如 B 图所示。作者在后面的研究里就是对 110 位的半胱氨酸、113 位的组氨酸进行了突变。

C 图左图是将不同浓度铜离子和 H3-H4 四聚体共同孵育，光吸收反映出形成了配体。右边两个图则是反应 110 位的半胱氨酸突变为丙氨酸后结合铜离子能力变差（酵母中的功能没有人类中的好，见B图）。

Fig. 2. The X. laevis H3-H4 tetramer catalyzes reduction of cupric ions. (A) Photographic representation of in vitro Cu2+ reduction assay. The yellow color is due to NC2-Cu1+ complex formation. (B) Progress curves of Cu2+ reduction with 1 mM of tetramer, heated tetramer, or buffer in presence of 30 mM TCEP and 1 mM CuCl2-10 mM Tricine. Lines and shading represent the mean ± SD of three to five assays. (C to F) Same as (B) but with the indicated amount of (C) TCEP or (D) CuCl2; or with (E) 1 mM of the indicated tetramers or (F) 5 mMof the indicated tetramers in the presence of 100 mM TCEP and 0.5 mM CuCl2-ADA pH 8.

体外实验，检测一价铜离子的浓度上升（反应铜离子还原酶活性）。野生型表现最好，突变型都不那么好，有的好些有的差些。

Fig. 3. The yeast H3-H4 tetramer potentially is a copper reductase. (A and B) Buffer-corrected differential absorbance of the indicated yeast tetramers. (C) Progress curves of Cu2+ reduction with 5 mMoftetramers in the presence of 100 mM TCEP and 0.5 mM CuCl2-ADA pH 8.

酵母中的 H3-H4 四聚体是潜在的铜还原酶。AB 图反应的是在不同的铜离子浓度下光吸收的变化程度。C 图是一价铜离子浓度随时间的上升。

Fig. 4. H3H113 regulates Cu1+-dependent transcriptional activities of Mac1 and Cup2. (A and B) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses of Mac1 target gene (39) expression relative to WT and normalized to ACT1 expression. The (A) boxes and (B) bars show means from four independent experiments (dots) in YPD with or without CuSO4. Baseline copper concentration in YPD is ~1 mM. (C) Intracellular copper content of exponentially growing strains. Bars are means ± SD from three to six experiments. (D) Schematic representation of the p(CUP1)-GFP reporter system. (E and F) Average flow cytometry distributions of cells containing the p(CUP1)-GFP plasmid grown in SC lacking uracil (SC-ura) with or without BCS or CuSO4 from five or six experiments. Baseline copperconcentration in SC is ~0.25 mM. *P ≤ 0.05; ***P ≤ 0.001; ns, not significant.

A图中的一列基因是 Mac1 转录因子（会被亚铜离子抑制的一种转录因子）调控表达的基因，通过 RT-qPCR 研究这些基因的表达，在有铜离子和没有铜离子的情况下突变体的这些基因表达出现了很大不同，说明原本在缺乏铜离子的情况下这些基因也可以通过某种稳态机制表达，而突变体失去了这一稳态机制。研究者们还敲出了 ctr1 基因，它编码铜离子通道蛋白（但是据说主要是转运亚铜离子），发现敲不敲 在野生型和突变型之间都有明显区别。C 图则是证明了在敲除、不敲除 ctr1 的情况下，胞内的铜含量没有明显差别，这是排除了一个可能的干扰因素。

D-F 图则是用了另一种受铜离子调控的转录因子 CUP2 接上一个荧光蛋白基因进行报告。

Fig. 5. H3H113 is required for utilization of copper for mitochondrial respiration and Sod1 function. (A and B) Oxygen consumption assays of cells incubated for (A) 18 or (B) 4 hours in liquid YPEG (yeast extract peptone ethanol glycerol) with or without CuSO4. Baseline copper concentration in YPEG is ~1 mM. Bars show means in (A) linear and (B) log2 scale ± SD from three experiments. Bars in (A) are scaled to mitochondrial DNA contents. (C) Cytochrome c oxidase assays of cells incubated for 4 hours in liquid YPEG with or without CuSO4. Bars show means in log2 scale ± SD from three experiments. N.D., not detectable. (D) Spot test assays in media with or without CuSO4.(E) Intracellular copper content of cells grown in YPEG with or without CuSO4. Bars show means ± SD from three to six experiments. #The ctr1D strains, which do not grow in nonfermentable media, were incubated in YPEG for 12 hours and assessed for metal content for reference. (F) Representative Sod1 activity (top) and Sod1 disulfide-bond assays (bottom) from three experiments for cells grown in SC with or without CuSO4. Relative signal intensities are indicated (bottom numbers are the ratio of oxidized to total Sod1). (G) Same as (F) but for cells grown in minimal medium with or without CuSO4. Baseline copperconcentration in minimal medium is ~0.25 mM.*P ≤ 0.05; **P ≤ 0.01; ns, not significant.

这里展示的是 H3-H4 四聚体对线粒体呼吸作用的影响，AB 图的指标是氧气消耗量，C 图指标是细胞色素 b 的活性。敲除 ctr1 后，在铜离子浓度很低和很高时野生型和突变型之间都没有明显差别（因为很低的时候就大家都没有，很高的时候可能即使突变型功能没那么好，也通过浓度给它补上了），在铜离子浓度适中的时候表现出了明显的区别。

F-G 图看的是对超氧化物歧化酶 Sod1 的影响.正常情况下这个酶要保持氧化态，缺亚铜离子的时候会是还原态。突变型在缺铜的时候 Sod1 活性就不太行。

【总结】

文章从细胞核内、细胞质、线粒体三个角度，证明了 H3-H4 四聚体对于铜离子稳态的重要性以及对生理生化过程的重要性。

在我们熟知的折叠染色质、调控转录之外，组蛋白竟然还可以直接作为酶参与细胞生化过程，是不是很新奇呢。