Structural Basis For The Regulatory Role Of The PPxY Motifs In The Thioredoxin-interacting Protein TXNIP

TXNIP (thioredoxin-interacting protein) negatively regulates the antioxidative activity of thioredoxin and participates in pleiotropic cellular processes. Its deregulation is linked to various human diseases, including diabetes, acute myeloid leukaemia and cardiovascular diseases. The E3 ubiquitin ligase Itch (Itchy homologue)polyubiquitinates TXNIP to promote its degradation via the ubiquitin-proteasome pathway, and this Itch-mediated polyubiquitination of TXNIP is dependent on the interaction of the four WW domains of Itch with the two PPxY motifs of TXNIP.However, the molecular mechanism of this interaction of TXNIP with Itch remains elusive. In the present study, we found that each of the four WW domains of Itch exhibited different and weakly binding affinities for TXNIP. The binding ability between the two-tandem WW domain of Itch and the two PPxY motifs of TXNIP was significantly enhanced, indicating that the multivalent binding mode might be a molecular mechanism that regulates the selectivity and affinity of WW and PPxY motif interactions.The results also show that in vivo Itch may identify and bind the TXNIP tandem PPxY motifs through tandem WW domains such as (WW1-WW2, WW3-WW4,etc.). In addition, tyrosine residue phosphorylation (PPx (pY)) in the PPxY motif will destroy the interaction of the PPxY motif and Itch, and bind proteins containing SH2 domains such as SHP2, Src, Vav2 and the like, suggesting that phosphorylation state of the PPxY motif tyrosine residue is selected as a molecular switch for its binding partner,thereby regulating the degradation and function of TXNIP. Finally, we analyzed the high-resolution complex structures of the WW3-WW4-PPCY of Itch, the SH2-PPT (pY)of SHP2 and the SH2-PPC (pY) of Vav2 by protein crystallization and X-ray diffraction,which suggests the third and fourth WW domains of Itch simultaneously recognize the two PPxY motifs of TXNIP, supporting the multivalent binding pattern between Itch and TXNIP, whereas phosphorylation of tyrosine residues destroys the interaction between the WW domain and PPxY motif, but is necessary to mediate the interaction of SH2 domain and the PPx (pY) motif.