SUMMARY Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled, due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease.

Highlights d De novo synthesized glycogen accumulates in the nucleus of non-small cell lung cancers d Nuclear glycogen provides a carbon pool for histone acetylation d Nuclear glycogenolysis is dependent on translocation of glycogen phosphorylase d Glycogen phosphorylase translocation is regulated by the E3 ubiquitin ligase malin

Scaffold proteins play a critical role in controlling the activity of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Shoc2 is a leucine-rich repeat scaffold protein that acts as a positive modulator of ERK1/2 signaling. However, the precise mechanism by which Shoc2 modulates the activity of the ERK1/2 pathway is unclear. Here we report the identification of the E3 ubiquitin ligase HUWE1 as a binding partner and regulator of Shoc2 function. HUWE1 mediates ubiquitination and, consequently, the levels of Shoc2. Additionally, we show that both Shoc2 and HUWE1 are necessary to control the levels and ubiquitination of the Shoc2 signaling partner, RAF-1. Depletion of HUWE1 abolishes RAF-1 ubiquitination, with corresponding changes in ERK1/2 pathway activity occurring. Our results indicate that the HUWE1-mediated ubiquitination of Shoc2 is the switch that regulates the transition from an active to an inactive state of the RAF-1 kinase. Taken together, our results demonstrate that HUWE1 is a novel player involved in regulating ERK1/2 signal transmission through the Shoc2 scaffold complex.

Aim: To explore the anticancer activity of a novel BRD4 protein degrader ARV-825 (ARV) and its nanoformulation development (ARV-NP) for treatment of pancreatic cancer. Materials & methods: ARV-NP were prepared using nanoprecipitation method and characterized for their physicochemical properties and various anticancer cell culture assays. Results: ARV-NP (89.63 ± 16.39 nm) demonstrated good physical stability, negligible hemolysis and improved half-life of ARV. ARV-NP showed significant cytotoxicity, apoptosis and anticlonogenic effect in pancreatic cancer cells. Significant downregulation of target proteins BRD4, c-Myc, Bcl-2 and upregulation of apoptotic marker cleaved caspase-3 was observed. Most importantly, ARV-NP treatment significantly inhibited the cell viability of 3D tumor spheroids of pancreatic cancer. Conclusion: ARV-NP represents a novel therapeutic strategy for pancreatic cancer.

Manganese (Mn) is neurotoxic: the underlying mechanisms have not been fully elucidated. L: -Buthionine-(S,R)-sulfoximine (BSO) is an irreversible inhibitor of gamma-glutamylcysteine synthetase, an important enzyme in glutathione (GSH) synthesis. To test the hypothesis that BSO modulates Mn toxicity, we investigated the effects of treatment of U-87 or SK-N-SH cells with MnCl(2), BSO, or MnCl(2) plus BSO. We monitored cell viability using MTT assay, staining with HO-33342 to assess live and/or apoptotic cells, and staining with propidium iodide (PI) to assess necrotic cells; we also measured cellular glutathione. Our results indicate decreased viability in both cell types when treated with MnCl(2) or BSO: Mn was more toxic to SK-N-SH cells, whereas BSO was more toxic to U-87 cells. Because BSO treatment accentuated Mn toxicity in both cell lines, GSH may act to combat Mn toxicity. Thus, further investigation in oxidative stress mediated by glutathione depletion will unravel new Mn toxicity mechanism(s).

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.

SYNOPSIS 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.

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