A Structural Biology Study Of SPSB2 Inhibitory Peptides And Identifying The Taget Proteins Of SPSB3

The SPRY domain and SOCS box-containing proteins are a family of four proteins that consist of a SPRY domain and a SOCS box.SPSB1,2,and 4 bind to the N-terminus of inducible nitric oxide synthase(iNOS)via the SPRY domain and target iNOS for ubiquitination and proteasomal degradation.Inhibiting of the SPSB2-iNOS interaction can increase the cellular NO production and may enhance pathogen or cancer cell killing.In this study,inhibitory peptides aiming to disrupting SPSB2-iNOS interaction were designed and characterized using biophysical and structural approaches.On the other hand,cellular target proteins of SPSB3 remain elusive.Thus,experiments were carried out to identify SPSB3-interacting proteins.Firstly,unlabeled and 15N-labeled recombinant proteins of the SPRY domain of human SPSB2 were expressed and purified.The binding of an iNOS N-terminal peptide(K9)and a cyclic 8 mer inhibitory peptide(cR8)was analyzed using nuclear magnetic resonance(NMR)and isothermal titration calorimetry(ITC).Binding affinity of the K9 peptide for SPSB2 SPRY domain was 10 + 1 nM,whereas that of the cR8 peptide was 699 + 104 nM.Structures of the SPSB2-K9 complex and the SPSB2-cR8 complex were solved using X-ray crystallography.We have also found that,an iNOS N-terminal peptide either exogenously overexpressed or as a cell-penetrating peptide,significantly increased the NO production in RAW264.7 cells.In addition,we identified a number of SPSB3 interacting proteins by co-immunoprecipitation and mass spectrometry analysis.These potential SPSB3 target proteins are to be validated in future work.In summary,we have revealed the structural basis for the regulation of iNOS by SPSB proteins and have provided novel structural and biological data of certain SPSB-inhibitory peptides.Furthermore,our findings will help to elucidate the biological functions of SPSB3.