Differential Involvement of the Npl4 Zinc Finger Domains of SHARPIN and HOIL-1L in Linear Ubiquitin Chain Assembly Complex-Mediated Cell Death Protection

Abstract

The linear ubiquitin chain assembly complex (LUBAC) participates in NF-κB activation and cell death protection. Loss of any of the three LUBAC subunits (catalytic HOIP, accessory HOIL-1L, or accessory SHARPIN subunit) leads to distinct phenotypes in mice and human. cpdm mice (chronic proliferative dermatitis in mice [cpdm]) that lack SHARPIN exhibit chronic inflammatory phenotypes, whereas HOIL-1L knockout mice exhibit no overt phenotypes, despite sharing highly homologous ubiquitin-like (UBL) and Npl4 zinc finger (NZF) domains. Here, we intercrossed mice lacking HOIL-1L and SHARPIN and found that reduction of HOIL-1L in cpdm mice exacerbated inflammatory phenotypes without affecting characteristic features of cpdm disease, whereas reduction of SHARPIN in HOIL-1L knockout mice provoked no overt phenotypes. Hence, loss of SHARPIN and reduction of LUBAC triggers cpdm phenotypes. We found that the NZF domain of SHARPIN, but not that of HOIL-1L, is critical for effective protection from programmed cell death by enhancing the recruitment of LUBAC to the activated TNFR complex. The binding activity to K63-linked ubiquitin chains that the NZF domain of SHARPIN, but not that of HOIL-1L, possesses appears to be involved in the recruitment. Thus, selective recognition of ubiquitin chains by NZFs in LUBAC underlies the regulation of LUBAC function.

ACKNOWLEDGMENTS

S.S. is supported by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science.

We thank Keiko Okamoto-Furuta and Haruyasu Kohda (Division of Electron Microscopic Study, Center for Anatomical Studies, Graduate School of Medicine, Kyoto University) for skillful technical assistance in electron microscopy. Preparation of the paraffin-embedded sections was supported by the Anatomic Pathology Center of the Graduate School of Medicine of Kyoto University. We thank Toshio Kitamura (the Institute of Medical Science, University of Tokyo) for providing the pMXs-IP retroviral mammalian expression plasmid. We thank Yukiko Takeda, Katsuhiro Sasaki, and Erik Walinda for insightful discussions and Tomoko Nakagawa, Ai Himeno, and Sachiko Asano for assistance with animal care and genotyping of mice.

S.S., H.F., Y.S., K.F, and K.I. designed the research; S.S. and K.I. wrote the manuscript; S.S. and H.F. performed the experiments and prepared the figures; and T.T. performed the pathology analyses.

We declare no financial conflict of interests in association with this study.