Research Article: RAIDD mutations underlie the pathogenesis of thin lissencephaly (TLIS)

Date Published: October 3, 2018

Publisher: Public Library of Science

Author(s): Hyun Ji Ha, Hyun Ho Park, Shawn B. Bratton.


Abnormal regulation of caspase-2-mediated neuronal cell death causes neurodegenerative diseases and defective brain development. PIDDosome is caspase-2 activating complex composed of PIDD, RAIDD, and caspase-2. Recent whole-exome sequencing study showed that the RAIDD mutations in the death domain (DD), including G128R, F164C, R170C, and R170H mutations, cause thin lissencephaly (TLIS) by reducing caspase-2-mediated neuronal apoptosis. Given that the molecular structure of the RAIDD DD:PIDD DD complex is available, in this study, we analyzed the molecular mechanisms underlying TLIS caused by the RAIDD TLIS variants by performing mutagenesis and biochemical assays.

Partial Text

The balance between cell proliferation and cell death is critical for normal development and homeostasis in multicellular organisms [1–4], and disruption of this balance leads to serious human diseases, such as cancers and neurodegenerative diseases [1, 5–8]. Apoptosis, a type of programmed cell death, is mediated by the sequential activation of caspases, a family of cysteine proteases that cleave specifically after aspartic acid residues [9, 10]. Caspases are divided into two classes according to their roles in apoptosis and their sequence of activation, namely, initiator caspases (including caspases 2, 8, 9, and 10) and effector caspases (including caspases 3 and 7) [10–13]. Initiator caspases are activated via the formation of huge molecular complexes, which can induce self-activation through proximity to the caspases. On the other hand, effector caspases are constitutive dimers and are activated upon cleavage by initiator caspases [14–16]. Caspase-8, -9, -1, and -2 are activated by the death-inducing signaling complex (DISC) [17], the apoptosome [18], the inflammasome [19, 20], and the PIDDosome [21], respectively, which are well-known molecular complexes required for the activation of initiator caspases.

PIDDosome formation followed by caspase-2 activation by proximity-induced self-cleavage is a critical step for programmed cell death in certain cell types, including neuronal cells. Higher oligomerization of macromolecules is considered as a critical event for various cellular signaling events [40–42]. Genotoxic stress is the most well-known trigger for caspase-2 activation. Considering the involvement of caspase-2 in neuronal cell death, blocking PIDDosome formation was suggested as an effective therapeutic intervention against neurodegenerative diseases caused by excessive neuronal cell death under certain conditions [32, 33]. A recent study that performed whole-genome sequencing of TLIS patients suggested the role of caspase-2 in the brain [33]. RAIDD mutations in the DD, namely, G128R, F164C, R170C, and R170H, were found to cause TLIS by reducing caspase-2-mediated neuronal apoptosis [33]. Given that all the TLIS-causing mutations are located in the RAIDD DD (protein interaction modules), the loss of the binding activity of RAIDD caused by mutations are likely to mediate disease pathogenesis. Previously, we solved the RAIDD DD: PIDD DD complex structure, identified all the interaction interfaces involved in complex formation [34]. In this study, we attempted to identify the effects of RAIDD DD mutations on caspase-2 activity and TLIS. Results of a mapping study showed that among the three TLIS-related residues (G128 located in the loop connecting H1 and H2, F164 located in H4, and R170 located in H4), G128 and R170 are localized on PPI (protein-protein interface), whereas F164 is located inside the six-helix bundle fold of RAIDD DD. The above results suggested that the G128R variant (located in the type III interface, which is formed between H3 of the first DD and the H1-H2 and the H3-H4 connecting loops of the second DD) and the R170C or H variants (located in the type I interface, which is formed by between H4 of the first DD and H5-H6 loop and H6 helix of the second DD), disrupted the interactions with PIDD. In turn, impaired interactions with PIDD inhibited PIDDosome formation, which is required for caspase-2 activation. On the other hand, the F164C variant potentially leads to incorrect folding of RAIDD DD because F164 is located in the helix bundle and is responsible for the formation of hydrophobic clusters with neighboring hydrophobic resides. Results of mutagenesis, size exclusion chromatography, and native PAGE experiments demonstrated that two R170 TLIS variants, R170C and R170H, lost their ability to bind PIDD DD. The F164C variant was extremely unstable, while the G128R variant was not expressed. Taken together, our current in vitro findings supported the notion that two RAIDD-TLIS variants, namely, R170C and R170H, cause defective interactions with PIDD. The F164C variant was found to be caused to the loss of stability of RAIDD, although cell experiments indicated that RAIDD-TLIS variants retained the ability to interact with PIDD [33]. If RAIDD-TLIS variants still bind to PIDD, there might be alternative mechanism required for caspase-2 activation in the pathogenesis of TLIS. Recent studies showed that LUBAC is essential for embryogenesis by preventing cell death and OTULIN limits cell death by deubiqutination LUBAC [43, 44]. It will be interesting to examine the tentative involvement of LUBAC and OTULIN in the RAIDD mutation-mediated TLIS pathogenesis. Although, further studies are required to explain the discrepancy between the in vitro and in vivo results, the current findings suggested that the RAIDD-TLIS variants lost its capacity to interact to PIDD or, at least, reduced the binding affinity to PIDD.




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