Molecular basis of Pirh2-mediated p53 ubiquitylation

Abstract

Pirh2 (p53-induced RING-H2 domain protein, also known as Rchy1), is an E3 ubiquitin ligase involved in a negative-feedback loop with p53. Using NMR spectroscopy we show that Pirh2 is a unique cysteine-rich protein comprising three modular domains. The protein binds nine zinc ions using a variety of zinc coordination schemes including a RING domain and a novel left-handed β-spiral in which three zinc ions align three consecutive small β-sheets in an interleaved fashion. We demonstrate that Pirh2-p53 interaction is dependent on the C-terminal Zn-binding module of Pirh2 which binds to the tetramerization domain (TET) of p53. As a result, Pirh2 ubiquitylates preferably the tetrameric form of p53 in vitro and in vivo, suggesting that Pirh2 regulates protein turnover of the transcriptionally active form of p53 in the cell.

Identification and Characterization of Two Novel Isoforms of Pirh2 Ubiquitin Ligase That Negatively Regulate p53 Independent of RING Finger Domains

“…7C shows the results of these experiments, demonstrating that the endogenous p53 from the wild-type p53-expressing HCT116 cells interacts with recombinant GST-Pirh2A and the Pirh2B isoforms but not with Pirh2C. While this manuscript was in revision, a structural study by Sheng et al (35) found that Pirh2 associated with p53 via contacts with the Pirh2 N and C termini. Using various synthetic deletions, one of which corresponded to the region deleted in Pirh2C, the authors concluded that primary binding is dependent upon a C-terminal region of full-length Pirh2 and that the Pirh2 N terminus harbors only weak p53 binding potential (35).…”

“…Given that the two proteins bind disparate regions of p53 (11,35), this outcome could be the result of paralleled ubiquitination of p53, and this could be true for Pirh2A that harbors intrinsic E3 ligase activity. However, our results indicate that both Pirh2B and Pirh2C lack ubiquitin ligase activity and Pirh2C has a diminished ability to bind p53.…”

“…However, in the case of MDM2, in addition to ubiquitination/degradation, it is well accepted that p53 inhibition also involves a physical interaction at the N terminus of p53 to block its association with the basal transcription machinery (36 -38). Because Pirh2 is known to bind to the central DNA binding and tetramerization domains of p53 (11,35), it is possible that the full-length Pirh2A and its novel isoforms may interfere with the ability of p53 to bind to DNA (4) and hence regulate p53 function independent of their ubiquitin ligase activities. Further in depth studies will help to better elucidate how Pirh2A and the two novel isoforms elicit their ubiquitin-dependent and/or independent effects on the tumor suppressor p53.…”

“…While this manuscript was in revision, a structural study by Sheng et al (35) found that Pirh2 associated with p53 via contacts with the Pirh2 N and C termini. Using various synthetic deletions, one of which corresponded to the region deleted in Pirh2C, the authors concluded that primary binding is dependent upon a C-terminal region of full-length Pirh2 and that the Pirh2 N terminus harbors only weak p53 binding potential (35). Their results appear to correlate well with our findings and provide an explanation for the weak p53 binding that we have noted for C-terminally deleted Pirh2C.…”

Pirh2

“…2 Studies of the interaction between Pirh2 and p53 showed that Pirh2 preferentially interacts with and leads to the degradation of p53 in its active tetrameric form rather than its monomeric form. 12 Despite the data showing Pirh2 ubiquitylation of p53 and other key cellular proteins, the in vivo functions of this E3 ligase remained elusive. Recently, we reported that in contrast to Mdm2 knockouts, Pirh2 −/− mice were viable and displayed normal development.…”