The porcine reproductive and respiratory syndrome virus (PRRSV) has three major structural proteins which designated as GP4, GP5, and M. They have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. However, the PRRSV structural proteins are produced in low levels in the infected cells because it forms insoluble protein and possesses several transmembrane regions. To overcome this problem, we fused the GP4, GP5, and M with SUMO (Small ubiquitin-related modifier), and expressed the fused gene in Bm5 cells and silkworm larvae. Expression of the proteins were analyzed by 12% SDS-PAGE and western blotting using 6xHis tag and porcine anti-PRRSV antibodies. In results, SUMO fused proteins were expressed at a high level in Bm5 cells. The levels of protein using the silkworm larvae is higher than that using Bm5 cells. The fused protein was purified by Ni-NTA affinity chromatography. This study demonstrated that SUMO, when fused with PRRSV structural proteins, was able to promote its soluble expression. This may be a better method to produce PRRSV structural proteins for vaccine development.

Arabidopsis E3 SUMO ligase SIZ1 (AtSIZ1) controls vegetative growth and development including responses to nutrient deficiency and environment stresses. Here, we analyzed the effect of AtSIZ1 on the stability and amount of seed proteins. Proteomic analysis showed that the amount of three major nutrient reservoir proteins, CRUCIFERIN (CRU) 1, 2 and 3, were decreased in siz1-2 mutants. However, quantitative real-time RT-PCR showed that transcript levels of CRU1, 2 and 3 genes were rather significantly higher in siz1-2 mutants than wild-type plants. Yeast two hybrid analysis revealed that AtSIZ1 interacts with CRU1, CRU2 and CRU3, strongly suggesting that CRU1, 2 and 3 proteins are sumoylated by AtSIZ1. In addition, the analysis of amino acid composition by HPLC showed that the contents of amino acids were a bit high in siz1-2 mutants. Our data indicate that AtSIZ1 plays an important function for accumulation of seed storage proteins through its ligase activity.

Gene expression is regulated by DNA and histone methylation by DNA and histone methyltransferases, respectively. In animal system, DNA methyltransferase with CG methylation activity is modified by SUMO conjugation and then its activity was increased, which means that the activity of DNA methyltransferase is modulated by posttranslational modification. so Chromatin remodeling is a new concept for expression of controlling of gene function. We thus analyzed the effect of E3 SUMO ligase AtSIZ1 in CMT3 (chromometnylase 3)-mediated genome methylation by next-generation sequencing (NGS), methyl binding domain MeDIP-sequencing and gene analysis using siz1-2 and cmt3 mutants. we carried out CG-enrich analysis by MeDIP sequencing revealed that the methylation level of the genome including transposons was significantly low in siz1-2 mutants compared to wild-type. Result showed the genes regulated by methylation, that genes related of embryo and root development, cellulose metabolism, and post-translational modifications. All of our data indicate that the methyltransfearse activity of CMT3 may be able to be regulated by AtSIZ1 and thereby CMT3-mediated gene expression and plant development also can be controlled by E3 SUMO ligase activity. Besides, our data also suggest that ammonium (NH4+) can stimulate AtSIZ1- and CMT3- mediated DNA methylation.

단백질의 기능을 다양화하기 위한 하나의 방법으로 전사 후 단백질 변형(post-translational modification)을 통해 단백질 활성이 조절된다. 또한, 최근 단백질의 sumoylation에 의한 활성 변화와 조절기전에 관해 많은 연구가 진행되어 여러 종류의 small ubiquitin-like protein(Ubl)이 밝혀졌다. 그 중 SUMO-1(small ubiquitin-related modifier 1)에 의한 sumoylation을 통해 androgen receptor(AR)가 modification 되어진다. Sumoylation은 protein을 targeting하고, 단백질을 안정화시키며, 전사를 활성화시키는 다양한 역할을 가지고 있다. 본 연구에서는 부정소에서 sumo-1 발현에 미치는 남성호르몬의 기능을 규명하고자 하였다. 본 연구에서는 생후 8주인 수컷 생쥐를 이용하여 정소가 제거 되지 않은 생쥐(Sham)와 정소를 제거한 생쥐(ORX), 정소를 제거한 후 1주간 Dihydrotestosterone(DHT)를 subcutaneous에 주사한 생쥐(ORX+DHT) 등으로부터 적출한 부정소를 이용하였다. 면역조직화학법을 이용하여 부정소 내 SUMO-1의 발현 부위와 정량적 RT-PCR를 통해 SUMO-1 mRNA 발현을 두부, 체부, 미부로 나누어 분석하였다. 또한, Western blot을 이용하여 부정소 내 sumo-1에 의한 단백질 패턴을 확인하였다. 생쥐 부정소에서 SUMO-1 immunoreactivity는 Sham군보다 ORX군에서 강하게 발현되었으며, ORX+DHT군에서 다시 감소하는 것으로 확인되었다. 특히, ORX군에서 두부보다는 체부와 미부에서 강하게 발현된 것을 확인하였다. 정량적 RT-PCR에 의하면 두부와 체부 부정소에서는 Sham군에 비해 ORX군에서 SUMO-1 mRNA 발현이 유의적으로 감소하였다. 하지만 미부에서는 변화가 나타나지 않았다. 또한, sham군보다 ORX+DHT군 미부에서 SUMO-1 mRNA 발현 유의성이 증가하였지만 두부와 체부에서는 변화를 나타내지 않았다. ORX+DHT군에서 SUMO-1 mRNA 발현이 ORX군에 비해 모든 부위에서 유의적으로 증가하였다. Western blot에서는 각 부정소에서 단백질 발현 패턴이 다른 것을 확인하였고 특히 sham군에서 발현된 단백질은 ORX군에서는 발현되지 않았으며 ORX군에서 발현된 단백질은 sham군에서는 발현되지 않았다. 부정소에서 sumo-1 전사와 단백질량은 음의 상관성을 보였으며 sumo-1의 전사는 두부, 체부가 유사하고 미부는 이들과 다른 양상을 보였다. 각 부정소에서 단백질 발현 패턴이 상이한 것을 알 수 있었고 남성호르몬에 의해서 ORX군에서 감소하고 증가한 단백질들을 전반적으로 회복시키는 것으로 사료된다. ORX군 상피조직의 핵에서 sumo-1 증가는 AR의 감소를 수반하는 것으로 보아 상피세포에서 AR등과 같은 전사인자의 분해를 촉진하는 것으로 추측된다. 반대로 AR이 감소한 경우 남성호르몬에 의한 AR의 전사활성 감수성을 증가시키기 위한 기작으로 추측할 수 있는 것으로 사료된다.

Post-translational covalent modifications by small molecules or peptides remodel target proteins. One such modification, made by ubiquitin or small ubiquitin-related modifier (SUMO), is a rapidly expanding field in cell signaling pathways. Ubiquitin attachment controls the turnover and degradation of target proteins while SUMO conjugation regulates their activity and function. Recent studies report many examples of cross-talk between ubiquitin and SUMO pathways, indicating that the boundary is no longer clear. Here, we review recent progress concerning how ubiquitin and SUMO participate in new regulatory roles in plant cell, and how ubiquitination and sumoylation control plant growth and development.