Screening and Functional Analysis of Cellular Proteins Interacting with Chicken (Gallus gallus) Speckle-type POZ Protein
WANG Yan-Bi, ZHAO Cai-Qin, TANG Hong, ZHOU Lei, HAN Yi-Fan, XING Jing-Ru, SHI Hai-Ying, DUAN Zhi-Qiang*
Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education/Key Laboratory of Animal Genetics, Breeding and Reproduction of Guizhou Province/College of Animal Science, Guizhou University, Guiyang 550025, China
Abstract：The speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) is a member of Cullin 3 family of E3 ubiquitin ligase, which promotes the ubiquitination and degradation of substrate by interacting with substrate protein, thus affecting the biological function of substrate protein. In this study, the recombinant eukaryotic expression vector pCMV-HA-SPOP of chicken (Gallus gallus) SPOP gene was constructed, and
pCMV-HA-SPOP and empty vector pCMV-HA were transfected into DF-1 respectively, and the total cellular
proteins were extracted. Co-immunoprecipitation (Co-IP) combined with mass spectrometry was used to
screen and identify the cellular proteins interacting with SPOP protein in chickens, and GO function
annotation, KEGG signaling pathway and protein interaction network analysis were carried out. Furthermore,
the interaction between chicken SPOP protein and the selected cell protein 26S proteasome non ATPase
regulatory subunit 11 (PSMD11) was verified by fluorescence co-localization and Co-IP test. The results
showed that the recombinant protein HA-SPOP was correctly expressed in cells, and mainly located in nucleus
and cytoplasm. A total of 158 cellular proteins interacting with chicken SPOP protein were screened by Co-IP
combined mass spectrometry, which mainly distributed in nucleus, cytoskeleton and cytoplasm, and
participated in biological processes such as enzyme activity, nucleic acid binding, protein process, and signal
pathways such as metabolism, ribosome composition and biological regulation. There were complex
interaction networks among cellular proteins interacting with chicken SPOP protein, among which proteasome
activator complex subunit 3 (PSME3), PSMD11 and H2A histone family, member Z (H2AFZ) may have direct
interaction with chicken SPOP protein. The results of fluorescence co-localization and Co-IP assay showed
that chicken SPOP protein could interact with PSMD11 protein and alter its intracellular localization. This
study provides research foundation for further exploring the biological function of chicken SPOP protein.
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