导读：本室黄粤教授课题组研究论文“CDK5RAP 3, a UFL1 substrate adaptor , is crucial for liver development” 于2019年1月25日发表于《Development》(doi:10.1242/dev.169235)。

 文章链接：http://dev.biologists.org/content/146/2/dev169235.long 

     蛋白分子的泛素和类泛素（UBLs）修饰参与调节许多的生物学功能。UFM1系统是一类新发现的类泛素修饰，它已被证实参与了哺乳动物的造血系统发育，但其广泛的生物学功能和作用机制尚待深入研究。以前有报道称 CDK5RAP3 蛋白可能是 Ufm1系统的一个底物分子，而且它的缺失会严重影响斑马鱼的胚胎外包和原肠胚形成，但其是否在哺乳动物发育中发挥作用尚未见报道。本研究中，我们发现CDK5RAP3在小鼠的肝脏发育和肝脏功能完善中发挥关键作用。Cdk5rap3基因组成性敲除 (KO) 小鼠胚胎发育晚期死亡，肝脏严重发育不全，成肝细胞增殖延迟且分化能力严重缺陷。肝细胞特异的Cdk5rap3基因敲除 (CKO) 小鼠在离乳后陆续死亡，有严重的低血糖和脂代谢异常。CDK5RAP3的缺失在肝细胞中引起内质网应激，并激活了UPR（unfolded prote in responses）。我们证实在肝细胞中CDK5RAP3和UFL1（Ufm1系统的E3连接酶）之间存在直接相互作用；而CDK5RAP3的缺失会显著改变肝细胞中的Ufm1系统的底物谱，表明CDK5RAP3是这种类泛素修饰的一个底物适配器。总的来说，我们的研究首次报道了CDK5RAP3是Ufm1系统的一个重要的调节者，并且首次揭示了Ufm1系统参与哺乳动物的肝脏发育。

 [摘要]：Protein modification by ubiquitin and ubiquitin-like proteins (UBLs) regulates numerous biological functions. The UFM1 system, a novel UBL conjugation system, is implicated in mouse development and hematopoiesis. However, its broad biological functions and working mechanisms remain largely elusive. CDK5RAP3, a possible ufmylation substrate, is essential for epiboly and gastrulation in zebrafish. Herein, we report a crucial role of CDK5RAP3 in liver development and hepatic functions. Cdk5rap3 knockout mice displayed prenatal lethality with severe liver hypoplasia, as characterized by delayed proliferation and compromised differentiation. Hepatocyte-specific Cdk5rap3 knockout mice suffered post-weaning lethality, owing to serious hypoglycemia and impaired lipid metabolism. Depletion of CDK5RAP3 triggered endoplasmic reticulum stress and activated unfolded protein responses in hepatocytes. We detected the in vivo interaction of CDK5RAP3 with UFL1, the defined E3 ligase in ufmylation. Notably, loss of CDK5RAP3 altered the ufmylation profile in liver cells, suggesting that CDK5RAP3 serves as a novel substrate adaptor for this UBL modification. Collectively, our study identifies CDK5RAP3 as an important regulator of ufmylation and suggests the involvement of ufmylation in mammalian development. 

      黄粤课题组的杨瑞博士（2017年毕业）和王焕民硕士（2018年毕业）为该研究论文的共同第一作者，黄粤研究员和贾玉艳副研究员为本文通讯作者。本所的张业、常永生课题组，中科院生物物理所的朱明昭课题组、国家蛋白质科学中心的徐平课题组参与了本研究。该项目得到了科技部重点研发计划（2016YFA0100103）、中国医学科学院医学与健康科技创新工程（2016-I2M-3-002，2017-I2M-1-008）及国家自然科学基金（31501176和91231111）等项目的资助。

Figure 1. CDK5RAP3 plays an important role in liver development via regulating Ufmylation.

(A) Schematic representation of wild-type, Cdk5rap3^tm1a and Cdk5rap3^tm1b alleles. The restriction enzyme digestion sites and probe hybridization locations for Southern Blot are labeled.

(B) The number of embryos and pups from timed mating of Cdk5rap3^tm1b/+ mice.

(C) Representative photographs of E16.5 wild-type and KO embryos, and their corresponding livers. The marks on the ruler scale are 1 mm apart.

(D) Immunostaining using anti-PCNA, anti-CK19 and anti-HNF4ɑ antibodies on E16.5 wild-type and KO liver sections. Scale bar: 50 μm.

(E) Survival profile of CKO ( n=20) and control ( n=20) mice.

(F) Top: 1-month-old CKO and control (Con) mice. Bottom: whole-mount livers from the above mice. The marks on the ruler scale are 1 mm apart.

(G) Representative images of Hematoxylin and Eosin (H&E) staining, PAS staining and Oil red O staining are shown for 1-month-old CKO and control livers. Scale bar: 50 μm.

(H) List of the candidate target proteins identified by mass spectrometry.

(I) Identification of UFL1 as a CDK5RAP3-binding protein. E16.5 wild-type liver lysates were subject to co-IP with IgG or anti-CDK5RAP3 antibody followed by Western Blot. CDK5RAP3 and UFL1 bands are indicated by arrows.

(J) UFM1 conjugates in E16.5 KO and wild-type embryos were detected by Western Blot using anti-UFM1 antibody.

(K) Left: expression of p-PERK, eIF2α and its activated form (p-eIF2α), ATF4, and Chop in 1-month-old CKO and control livers. Right: expression of ER stress sensor IRE1 α, unspliced XBP-1 and its spiced form [XBP-1(s)] in 1-month-old CKO and control livers.

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