br Experimental Procedure br Acknowledgment This work
Acknowledgment This work was supported by Ministry of Education, Singapore (MoE Tier-2) grant (R-154-000-625-112) and R154-000-A72-114 (AcRF Tier 1 grant) respectively. We acknowledge the Advanced Photon Source Beamline 24-ID-C, Argonne National Laboratory, USA. D.N. is a graduate scholar in receipt of a research scholarship from NUS. Author Contributions: J.S. conceived and designed the study. D.N. performed all the experiments. D.N. and J.S. analyzed the data and wrote the paper. Conflict of Interest Statement: The authors declare that they have no conflicts of interest.
Introduction Lafora disease (LD; OMIM254780, ORPHA501) is a rare form of progressive myoclonus epilepsy, first described in 1911 by the Spanish neurologist Gonzalo R. Lafora (Lafora and Glueck, 1911). The first symptoms of the disease appear at early adolescence, producing myoclonus, tonic-clonic seizures and epileptic crises, and a progressive dementia, apraxia, aphasia and visual loss (Monaghan and Delanty, 2010). Histologically, the most characteristic hallmark of LD is the accumulation of aberrant glycogen forms named Lafora bodies (LBs) (Minassian, 2001). However, at present, it is still not clear whether the accumulation of LBs is the cause of the disease or a consequence of previous alterations. In the vast majority of cases, the disease is produced by mutations either in the EPM2A gene, which encodes the dual specificity phosphatase laforin (Minassian et al., 1998, Serratosa et al., 1999), or in the EPM2B gene, encoding the E3 ubiquitin ligase malin (Chan et al., 2003). Patients with mutations in either EPM2A or EPM2B DMH-1 are histologically and neurologically indistinguishable, suggesting that both proteins are involved in the same physiological pathways (Gómez-Abad et al., 2005). It has been described that laforin and malin form a functional complex in which laforin targets malin to the appropriate substrates for their ubiquitination. Several substrates of the malin–laforin complex have been described (Romá-Mateo et al., 2012), including proteins involved in glycogen synthesis. Thereby, the loss of either laforin or malin results in a dysregulation of glycogen metabolism, leading to the formation of LBs (Lohi et al., 2005, Rubio-Villena et al., 2013, Solaz-Fuster et al., 2008, Worby et al., 2008). Other processes have been shown to be affected by mutations in laforin and malin. For instance, the malin–laforin complex participates in the clearance of misfolded proteins through the ubiquitin-proteasome pathway: dysfunction of the malin–laforin complex results in a reduction of proteasomal activity leading to increased sensitivity to endoplasmic reticulum stress (Garyali et al., 2009, Vernia et al., 2009). In addition, a clear impairment in autophagy has also been described both in malin and laforin knock-out mice (Aguado et al., 2010, Criado et al., 2012), as well as the appearance of oxidative stress (Romá-Mateo et al., 2015a). Taken together, these evidences suggest a broad impairment of the protein clearance systems when the malin–laforin complex is not functional, indicating that there are more processes implicated in the disease other than glycogen synthesis and accumulation (Romá-Mateo et al., 2015b). Ubiquitination is a highly conserved process in eukaryotic organisms, which depends on the sequential activity of three different enzymes: an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and an ubiquitin ligase (E3). E1s use ATP to bind a molecule of ubiquitin to the Cys residue in their active site. Then, this ubiquitin is transferred to the Cys residue of an E2. The third step depends on the type of E3 involved: E3s with a HECT domain incorporate themselves the ubiquitin from the E2, before transferring it to the substrate. However, E3s with a RING domain, like malin, act as scaffolds bringing together the substrate and an activated E2, so the ubiquitin can be transferred directly from the E2 to a Lys residue of the substrate (Ye and Rape, 2009). In this type of E3 ubiquitin ligases, the specific combination of the E2 and the E3 determines the topology and length of the ubiquitin chains conjugated to the substrate [see (Deshaies and Joazeiro, 2009, Metzger et al., 2014, Ye and Rape, 2009) for review]. The function of the ubiquitin chains depends on their topology, which might either label proteins for proteasomal degradation or target them to participate in alternative cellular functions (DNA repair, cell signaling, etc.) (Deshaies and Joazeiro, 2009, Ye and Rape, 2009). For instance, poly-ubiquitin chains linked through Lys63 direct the substrate to autophagy (Pickart and Fushman, 2004).