Study On The Formation Of Aggregates Of The Autophagy Receptor Protein P62/ref(2)P In The Silkworm, Bombyx Mori

The host protein,p62,namely sequestosome 1,is a well-known cellular autophagic adapter,and plays an essential role in clearance of protein aggregates,damaged mitochondria,midbody rings,peroxisomes and invading microbes.Previously,the most striking observation was that over-expression of p62 led to remarkable decrease in polyhedrin expression and polyhedra production in the nucleus of infected cells,suggesting a restriction activity of p62 against infection of Bombyx mori nucleopolyhedrovirus（BmNPV）.As a paralog of silkworm p62,the protein ref（2）P displayed some characteristics similar to those of p62.In this study,expession of fusion protein eGFP-p62 in Escherichia coli BL21（DE3）,binding of p62 with the viral protein polyhedrin,and molecular basis of ref（2）P to aggregate in the cytoplasm,were mainly investigated.The results were showed as follows.1.Expression of p62 in E.coli BL21（DE3）The coding regions of eGFP and p62 were cloned into vector pET-30 a,and transformed into E.coli BL21（DE3）cells.The fusion protein eGFP-p62 was expressed by adding isopropyl β-D-thiogalactoside（IPTG）,and was purified.A mouse polyclonal antibody was generated..2.Sequence analyses of silkworm p62 and ref（2）PUsing the amino acid sequence of human p62（NP₀03891.1）as the search sequence,BLASTP analysis was performed in the silkworm protein database.The silkworm genome contains only one p62 gene（NCBI GeneID: 101736823）but to express two proteins,i.e.p62（NCBI protein_id: XP₀04932349.1）and ref（2）P（NCBI protein_id: XP₀12551153.1）.Compared to p62,ref（2）P lacks the PB1 domain.The other amino acid sequences of these two proteins（including ZZ,LIR,UBA and other domains）are completely identical,implying that these two proteins share some common characteristics.The transient expression vectors to express p62 and ref（2）P fused to the C-terminus of eGFP or mCherry were constructed.Fusion proteins eGFP-p62 and eGFP-ref（2）P were expressed in BmN cells and observed under a Leica TCS SP7 confocal laser scanning microscope to be localized mainly in the cytoplasm to form IBs.When mCherry-p62 and eGFP-ref（2）P were co-expressed,these two fusion proteins were co-localized in the cytoplasm to form IBs.3.The mechanism of p62/ref（2）P to form IBsSeveral hydrophobic residues in two amino acid patches（M1 and M2）of ref（2）P were mutated to alanines.Fusion proteins eGFP-ref（2）P（M1-A）and eGFP-ref（2）P（M2-A）were expressed in BmN cells.A decrease in the number of cells containing IBs formed by eGFP-ref（2）P（M1-A）or eGFP-ref（2）P（M2-A）was observed.Moreover,these hydrophobic residues in M1 and M2 patches were mutated to express a double patch mutated protein eGFP-ref（2）P（M1/M2-A）.A dramatical decrease in the number of eGFP-ref（2）P（M1/M2-A）IBs,and a diffuse distribution pattern in the cytoplasm were observed in eGFP-ref（2）P（M1/M2-A）-transfected BmN cells.It was inferred that the hydrophobic residues in M1 patch bind to those in M2 patch to form IBs,which will be of significant interest in future studies.It was reported that K8 and D69 were key residues for human p62 to form IBs.The corresponding residues K7 and D69,together with these hydrophobic ones in M1 and M2 patches of silkworm p62,were mutated,to construct a vector expressing fusion protein eGFP-p62（K8/D59/M1/M2-A）.In most transfected BmN cells,no IBs were formed and the distribution of eGFP-p62（K8/D59/M1/M2-A）became diffuse in the cytoplasm.4.The binding of polyhedrin to p62 or ref（2）P（1）Based on the analysis of the ref（2）P domains,a series of truncated mutants were constructed and co-expressed with polyhedrin-mCherry.The viral polyhedrin is a protein harboring a nuclear localization signal,thereby localized mainly in the nucleus.When co-expressed with eGFP-ref（2）P or eGFP-p62,polyhedrin-mCherry was co-localized with eGFP-ref（2）P or eGFP-p62 in the cytoplasm to form IBs.A ref（2）P mutant lacking the ubiquitin-associated domain（UBA）was constructed and co-expressed with polyhedrin-mCherry in BmN cells.The fluorescence observation showed that polyhedrin-mCherry was co-localized mainly in the nucleus,whereas eGFP-ref（2）P（ΔUBA）was distributed evenly in the cytoplasm;Hence no co-localization pattern between eGFP-ref（2）P（ΔUBA）and polyhedrin-mCherry was detected.The hydrophobic M2 patch in UBA was mutated and co-expressed with polyhedrin-mCherry,and similar observations were obtained.（2）A series of truncated mutants of polyhedrin,i.e.,polyhedrin（1-28）,polyhedrin（1-63）,polyhedrin（1-95）,polyhedrin（1-112）,polyhedrin（1-129）,polyhedrin（1-189）,polyhedrin（1-211）and polyhedrin（1-232）were constructed and transiently expressed.It was found that polyhedrin（1-28）,polyhedrin（1-63）,polyhedrin（1-95）,polyhedrin（1-112）,polyhedrin（1-129）and polyhedrin（1-232）were mainly distributed in the nucleus,and polyhedrin（1-189）and polyhedrin（1-211）were located in the cytoplasm to form IBs,whereas polyhedrin（1-28）was dispersed evenly between the nucleus and the cytoplasm.When co-expressed with eGFP-ref（2）P oreGFP-p62,these polyhedrin truncations were co-localized with eGFP-ref（2）P or eGFP-p62 in the cytoplasm to form IBs.The polyhedrin（1-28）is currently known the shortest peptide to be co-localizable with ref（2）P or p62.