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1.  Laforin, a protein with many faces: glucan phosphatase, adapter protein, et alii 
The FEBS journal  2012;280(2):525-537.
Lafora disease (LD) is a rare, fatal neurodegenerative disorder characterized by the accumulation of glycogen-like inclusions in the cytoplasm of cells from most tissues of affected patients. 100 years since the first description of these inclusions, the molecular bases underlying the processes involved in LD physiopathology are finally being elucidated. The main cause for the disease relies on the activity of two proteins, the dual specificity phosphatase laforin and the E3-ubiquitin ligase malin, that form a functional complex. Laforin is unique in humans since it is comprised of a carbohydrate binding module attached to a cysteine-based catalytic dual specificity phosphatase domain. Laforin directly dephosphorylates glycogen, but other proteinaceous substrates, if existent, have remained elusive. Recently, an emerging set of laforin binding partners apart from malin have been described, suggestive of laforin roles unrelated to its catalytic activity. Further investigations based on different transgenic mice models have shown that the laforin-malin complex is also involved in other cellular processes such as response to ER stress and misfolded proteins clearance by the lysosomal pathway. However, controversial data and some missing links still make difficult to assess the concrete relationship between glycogen deregulation and neuronal damage leading to the fatal symptoms observed in LD patients, such as myoclonic seizures and epilepsy. Consequently, clinical treatments are far from being achieved. In the present review, we focus on the knowledge of laforin biology not only as a glucan phosphatase, but also as an adaptor protein involved in several physiological pathways.
PMCID: PMC3371293  PMID: 22364389
Laforin; malin; glucan phosphatase; Lafora disease; Lafora bodies; glycogen; autophagy; ER stress
2.  Deciphering the role of malin in the Lafora progressive myoclonus epilepsy 
IUBMB life  2012;64(10):801-808.
Lafora disease (LD) is a fatal, autosomal recessive neurodegenerative disorder that results in progressive myoclonus epilepsy. A hallmark of LD is the accumulation of insoluble, aberrant glycogen-like structures called Lafora bodies. LD is caused by mutations in the gene encoding the E3 ubiquitin ligase malin or the glucan phosphatase laforin. Although LD was first described in 1911, its symptoms are still lacking a consistent molecular explanation and consequently a cure is far from being achieved. Some data suggest that malin forms a functional complex with laforin. This complex promotes the ubiquitination of proteins involved in glycogen metabolism and misregulation of pathways involved in this process results in Lafora body formation. In addition, recent results obtained from both cell culture and LD mouse models have highlighted a role of the laforin-malin complex in the regulation of ER-stress and protein clearance pathways. These results suggest that LD should be considered as a novel member of the group of protein clearance diseases such as Parkinson’s, Huntington’s, or Alzheimer’s, in addition to being a glycogen metabolism disease. Herein, we review the latest results concerning the role of malin in LD and attempt to decipher its function.
PMCID: PMC3458166  PMID: 22815132
Laforin; malin; glucan phosphatase; Lafora disease; Lafora bodies; glycogen; autophagy; ER stress; Ubiquitination; E3-ubiquitin ligase
3.  Dimerization of the Glucan Phosphatase Laforin Requires the Participation of Cysteine 329 
PLoS ONE  2013;8(7):e69523.
Laforin, encoded by a gene that is mutated in Lafora Disease (LD, OMIM 254780), is a modular protein composed of a carbohydrate-binding module and a dual-specificity phosphatase domain. Laforin is the founding member of the glucan-phosphatase family and regulates the levels of phosphate present in glycogen. Multiple reports have described the capability of laforin to form dimers, although the function of these dimers and their relationship with LD remains unclear. Recent evidence suggests that laforin dimerization depends on redox conditions, suggesting that disulfide bonds are involved in laforin dimerization. Using site-directed mutagenesis we constructed laforin mutants in which individual cysteine residues were replaced by serine and then tested the ability of each protein to dimerize using recombinant protein as well as a mammalian cell culture assay. Laforin-Cys329Ser was the only Cys/Ser mutant unable to form dimers in both assays. We also generated a laforin truncation lacking the last three amino acids, laforin-Cys329X, and this truncation also failed to dimerize. Interestingly, laforin-Cys329Ser and laforin-Cys329X were able to bind glucans, and maintained wild type phosphatase activity against both exogenous and biologically relevant substrates. Furthermore, laforin-Cys329Ser was fully capable of participating in the ubiquitination process driven by a laforin-malin complex. These results suggest that dimerization is not required for laforin phosphatase activity, glucan binding, or for the formation of a functional laforin-malin complex. Cumulatively, these results suggest that cysteine 329 is specifically involved in the dimerization process of laforin. Therefore, the C329S mutant constitutes a valuable tool to analyze the physiological implications of laforin’s oligomerization.
PMCID: PMC3724922  PMID: 23922729
4.  Erratum to 
Autophagy  2012;8(7):1163.
PMCID: PMC3429560
Lafora disease; autophagy; glycogen metabolism; laforin; malin; neurodegeneration
5.  Laforin, a dual specificity protein phosphatase involved in Lafora disease, is phosphorylated at Ser25 by AMP-activated protein kinase 
Biochemical Journal  2011;439(2):265-275.
Lafora progressive myoclonus epilepsy (Lafora disease; LD) is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others showed that laforin and malin form a functional complex that regulates multiple aspects of glycogen metabolism, and that the interaction between laforin and malin is enhanced by conditions activating AMP-activated protein kinase (AMPK). Here, we demonstrate that laforin is a phosphoprotein, as indicated by two-dimensional electrophoresis, and we identify Ser25 as the residue involved in this modification. We also show that Ser25 is phosphorylated both in vitro and in vivo by AMPK. Lastly, we demonstrate that this residue plays a critical role for both the phosphatase activity and the ability of laforin to interact with itself and with previously established binding partners. Our data suggest that phosphorylation of laforin-Ser25 by AMPK provides a mechanism to modulate the interaction between laforin and malin. Regulation of this complex is necessary to maintain normal glycogen metabolism. Importantly, Ser25 is mutated in some Lafora disease patients (S25P), and our results begin to elucidate the mechanism of disease in these patients.
PMCID: PMC3299407  PMID: 21728993
Laforin; AMPK; phosphorylation; alanine scanning mutagenesis; protein-protein interaction; glucan-phosphatase
6.  The PRINTS database: a fine-grained protein sequence annotation and analysis resource—its status in 2012 
The PRINTS database, now in its 21st year, houses a collection of diagnostic protein family ‘fingerprints’. Fingerprints are groups of conserved motifs, evident in multiple sequence alignments, whose unique inter-relationships provide distinctive signatures for particular protein families and structural/functional domains. As such, they may be used to assign uncharacterized sequences to known families, and hence to infer tentative functional, structural and/or evolutionary relationships. The February 2012 release (version 42.0) includes 2156 fingerprints, encoding 12 444 individual motifs, covering a range of globular and membrane proteins, modular polypeptides and so on. Here, we report the current status of the database, and introduce a number of recent developments that help both to render a variety of our annotation and analysis tools easier to use and to make them more widely available.
Database URL:
PMCID: PMC3326521  PMID: 22508994
7.  Laforin, a Dual Specificity Phosphatase Involved in Lafora Disease, Is Present Mainly as Monomeric Form with Full Phosphatase Activity 
PLoS ONE  2011;6(8):e24040.
Lafora Disease (LD) is a fatal neurodegenerative epileptic disorder that presents as a neurological deterioration with the accumulation of insoluble, intracellular, hyperphosphorylated carbohydrates called Lafora bodies (LBs). LD is caused by mutations in either the gene encoding laforin or malin. Laforin contains a dual specificity phosphatase domain and a carbohydrate-binding module, and is a member of the recently described family of glucan phosphatases. In the current study, we investigated the functional and physiological relevance of laforin dimerization. We purified recombinant human laforin and subjected the monomer and dimer fractions to denaturing gel electrophoresis, mass spectrometry, phosphatase assays, protein-protein interaction assays, and glucan binding assays. Our results demonstrate that laforin prevalently exists as a monomer with a small dimer fraction both in vitro and in vivo. Of mechanistic importance, laforin monomer and dimer possess equal phosphatase activity, and they both associate with malin and bind glucans to a similar extent. However, we found differences between the two states' ability to interact simultaneously with malin and carbohydrates. Furthermore, we tested other members of the glucan phosphatase family. Cumulatively, our data suggest that laforin monomer is the dominant form of the protein and that it contains phosphatase activity.
PMCID: PMC3162602  PMID: 21887368
8.  Lafora disease E3-ubiquitin ligase malin is related to TRIM32 at both the phylogenetic and functional level 
Malin is an E3-ubiquitin ligase that is mutated in Lafora disease, a fatal form of progressive myoclonus epilepsy. In order to perform its function, malin forms a functional complex with laforin, a glucan phosphatase that facilitates targeting of malin to its corresponding substrates. While laforin phylogeny has been studied, there are no data on the evolutionary lineage of malin.
After an extensive search for malin orthologs, we found that malin is present in all vertebrate species and a cephalochordate, in contrast with the broader species distribution previously reported for laforin. These data suggest that in addition to forming a functional complex, laforin and perhaps malin may also have independent functions. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32, which belongs to the tripartite-motif containing family of proteins. We present experimental evidence that both malin and TRIM32 share some substrates for ubiquitination, although they produce ubiquitin chains with different topologies. However, TRIM32-specific substrates were not reciprocally ubiquitinated by the laforin-malin complex.
We found that malin and laforin are not conserved in the same genomes. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32. The latter result suggests a common origin for malin and TRIM32 and provides insights into possible functional relationships between both proteins.
PMCID: PMC3160408  PMID: 21798009
AMPK; malin; TRIM32; E3 ubiquitin ligase; phylogeny; Lafora disease

Results 1-8 (8)