Myofiber-type-dependent 'boulder' or 'multitudinous pebble' formations across distinct amylopectinoses.

At least five enzymes including three E3 ubiquitin ligases are dedicated to glycogen's spherical structure. Absence of any reverts glycogen to a structure resembling amylopectin of the plant kingdom. This amylopectinosis (polyglucosan body formation) causes fatal neurological diseases including adult polyglucosan body disease (APBD) due to glycogen branching enzyme deficiency, Lafora disease (LD) due to deficiencies of the laforin glycogen phosphatase or the malin E3 ubiquitin ligase and type 1 polyglucosan body myopathy (PGBM1) due to RBCK1 E3 ubiquitin ligase deficiency. Little is known about these enzymes' functions in glycogen structuring. Toward understanding these functions, we undertake a comparative murine study of the amylopectinoses of APBD, LD and PGBM1. We discover that in skeletal muscle, polyglucosan bodies form as two main types, small and multitudinous ('pebbles') or giant and single ('boulders'), and that this is primarily determined by the myofiber types in which they form, 'pebbles' in glycolytic and 'boulders' in oxidative fibers. This pattern recapitulates what is known in the brain in LD, innumerable dust-like in astrocytes and single giant sized in neurons. We also show that oxidative myofibers are relatively protected against amylopectinosis, in part through highly increased glycogen branching enzyme expression. We present evidence of polyglucosan body size-dependent cell necrosis. We show that sex influences amylopectinosis in genotype, brain region and myofiber-type-specific fashion. RBCK1 is a component of the linear ubiquitin chain assembly complex (LUBAC), the only known cellular machinery for head-to-tail linear ubiquitination critical to numerous cellular pathways. We show that the amylopectinosis of RBCK1 deficiency is not due to loss of linear ubiquitination, and that another function of RBCK1 or LUBAC must exist and operate in the shaping of glycogen. This work opens multiple new avenues toward understanding the structural determinants of the mammalian carbohydrate reservoir critical to neurologic and neuromuscular function and disease.

Laforin targets malin to glycogen in Lafora progressive myoclonus epilepsy.

Glycogen is the largest cytosolic macromolecule and is kept in solution through a regular system of short branches allowing hydration. This structure was thought to solely require balanced glycogen synthase and branching enzyme activities. Deposition of overlong branched glycogen in the fatal epilepsy Lafora disease (LD) indicated involvement of the LD gene products laforin and the E3 ubiquitin ligase malin in regulating glycogen structure. Laforin binds glycogen, and LD-causing mutations disrupt this binding, laforin-malin interactions and malin's ligase activity, all indicating a critical role for malin. Neither malin's endogenous function nor location had previously been studied due to lack of suitable antibodies. Here, we generated a mouse in which the native malin gene is tagged with the FLAG sequence. We show that the tagged gene expresses physiologically, malin localizes to glycogen, laforin and malin indeed interact, at glycogen, and malin's presence at glycogen depends on laforin. These results, and mice, open the way to understanding unknown mechanisms of glycogen synthesis critical to LD and potentially other much more common diseases due to incompletely understood defects in glycogen metabolism.

Glycogen synthase downregulation rescues the amylopectinosis of murine RBCK1 deficiency.

Longer glucan chains tend to precipitate. Glycogen, by far the largest mammalian glucan and the largest molecule in the cytosol with up to 55 000 glucoses, does not, due to a highly regularly branched spherical structure that allows it to be perfused with cytosol. Aberrant construction of glycogen leads it to precipitate, accumulate into polyglucosan bodies that resemble plant starch amylopectin and cause disease. This pathology, amylopectinosis, is caused by mutations in a series of single genes whose functions are under active study toward understanding the mechanisms of proper glycogen construction. Concurrently, we are characterizing the physicochemical particularities of glycogen and polyglucosans associated with each gene. These genes include GBE1, EPM2A and EPM2B, which respectively encode the glycogen branching enzyme, the glycogen phosphatase laforin and the laforin-interacting E3 ubiquitin ligase malin, for which an unequivocal function is not yet known. Mutations in GBE1 cause a motor neuron disease (adult polyglucosan body disease), and mutations in EPM2A or EPM2B a fatal progressive myoclonus epilepsy (Lafora disease). RBCK1 deficiency causes an amylopectinosis with fatal skeletal and cardiac myopathy (polyglucosan body myopathy 1, OMIM# 615895). RBCK1 is a component of the linear ubiquitin chain assembly complex, with unique functions including generating linear ubiquitin chains and ubiquitinating hydroxyl (versus canonical amine) residues, including of glycogen. In a mouse model we now show (i) that the amylopectinosis of RBCK1 deficiency, like in adult polyglucosan body disease and Lafora disease, affects the brain; (ii) that RBCK1 deficiency glycogen, like in adult polyglucosan body disease and Lafora disease, has overlong branches; (iii) that unlike adult polyglucosan body disease but like Lafora disease, RBCK1 deficiency glycogen is hyperphosphorylated; and finally (iv) that unlike laforin-deficient Lafora disease but like malin-deficient Lafora disease, RBCK1 deficiency's glycogen hyperphosphorylation is limited to precipitated polyglucosans. In summary, the fundamental glycogen pathology of RBCK1 deficiency recapitulates that of malin-deficient Lafora disease. Additionally, we uncover sex and genetic background effects in RBCK1 deficiency on organ- and brain-region specific amylopectinoses, and in the brain on consequent neuroinflammation and behavioural deficits. Finally, we exploit the portion of the basic glycogen pathology that is common to adult polyglucosan body disease, both forms of Lafora disease and RBCK1 deficiency, namely overlong branches, to show that a unified approach based on downregulating glycogen synthase, the enzyme that elongates glycogen branches, can rescue all four diseases.

LUBAC: a new player in polyglucosan body disease.

Altered protein ubiquitination is associated with the pathobiology of numerous diseases; however, its involvement in glycogen metabolism and associated polyglucosan body (PB) disease has not been investigated in depth. In PB disease, excessively long and less branched glycogen chains (polyglucosan bodies, PBs) are formed, which precipitate in different tissues causing myopathy, cardiomyopathy and/or neurodegeneration. Linear ubiquitin chain assembly complex (LUBAC) is a multi-protein complex composed of two E3 ubiquitin ligases HOIL-1L and HOIP and an adaptor protein SHARPIN. Together they are responsible for M1-linked ubiquitination of substrates primarily related to immune signaling and cell death pathways. Consequently, severe immunodeficiency is a hallmark of many LUBAC deficient patients. Remarkably, all HOIL-1L deficient patients exhibit accumulation of PBs in different organs especially skeletal and cardiac muscle resulting in myopathy and cardiomyopathy with heart failure. This emphasizes LUBAC's important role in glycogen metabolism. To date, neither a glycogen metabolism-related LUBAC substrate nor the molecular mechanism are known. Hence, current reviews on LUBAC's involvement in glycogen metabolism are lacking. Here, we aim to fill this gap by describing LUBAC's involvement in PB disease. We present a comprehensive review of LUBAC structure, its role in M1-linked and other types of atypical ubiquitination, PB pathology in human patients and findings in new mouse models to study the disease. We conclude the review with recent drug developments and near-future gene-based therapeutic approaches to treat LUBAC related PB disease.

Targeting Gys1 with AAV-SaCas9 Decreases Pathogenic Polyglucosan Bodies and Neuroinflammation in Adult Polyglucosan Body and Lafora Disease Mouse Models.

Many adult and most childhood neurological diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency, and specificity hurdles. We applied CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric disease. Adult polyglucosan body disease is a neurodegenerative disease resembling amyotrophic lateral sclerosis. Lafora disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene, Gys1, responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body disease and two mouse models of Lafora disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, abnormal glycogen accumulation, and polyglucosan bodies, as well as ameliorations of neuroinflammatory markers in all three models. Our work represents proof of principle for virally delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological diseases.

Lafora disease - from pathogenesis to treatment strategies.

Lafora disease is a severe, autosomal recessive, progressive myoclonus epilepsy. The disease usually manifests in previously healthy adolescents, and death commonly occurs within 10 years of symptom onset. Lafora disease is caused by loss-of-function mutations in EPM2A or NHLRC1, which encode laforin and malin, respectively. The absence of either protein results in poorly branched, hyperphosphorylated glycogen, which precipitates, aggregates and accumulates into Lafora bodies. Evidence from Lafora disease genetic mouse models indicates that these intracellular inclusions are a principal driver of neurodegeneration and neurological disease. The integration of current knowledge on the function of laforin-malin as an interacting complex suggests that laforin recruits malin to parts of glycogen molecules where overly long glucose chains are forming, so as to counteract further chain extension. In the absence of either laforin or malin function, long glucose chains in specific glycogen molecules extrude water, form double helices and drive precipitation of those molecules, which over time accumulate into Lafora bodies. In this article, we review the genetic, clinical, pathological and molecular aspects of Lafora disease. We also discuss traditional antiseizure treatments for this condition, as well as exciting therapeutic advances based on the downregulation of brain glycogen synthesis and disease gene replacement.

Characterization of Staphylococcus epidermidis Polynucleotide phosphorylase and its interactions with ribonucleases RNase J1 and RNase J2.

Polynucleotide phosphorylase catalyzes both 3'-5' exoribonuclease and polyadenylation reactions. The crystal structure of Staphylococcus epidermidis PNPase revealed a bound phosphate in the PH2 domain of each protomer coordinated by three adjacent serine residues. Mutational analysis suggests that phosphate coordination by these serine residues is essential to maintain the catalytic center in an active conformation. We note that PNPase forms a complex with RNase J1 and RNase J2 without substantially altering either exo-ribonuclease or polyadenylation activity of this enzyme. This decoupling of catalytic activity from protein-protein interactions suggests that association of these endo- or exo-ribonucleases with PNPase could be more relevant for cellular localization or concerted targeting of structured RNA for recycling.

Six-Transmembrane Epithelial Antigen of Prostate 1 (STEAP1) Has a Single b Heme and Is Capable of Reducing Metal Ion Complexes and Oxygen.

STEAP1, six-transmembrane epithelial antigen of prostate member 1, is strongly expressed in several types of cancer cells, particularly in prostate cancer, and inhibition of its expression reduces the rate of tumor cell proliferation. However, the physiological function of STEAP1 remains unknown. Here for the first time, we purified a mammalian (rabbit) STEAP1 at a milligram level, permitting its high-quality biochemical and biophysical characterizations. We found that STEAP1 likely assembles as a homotrimer and forms a heterotrimer when co-expressed with STEAP2. Each STEAP1 protomer binds one heme prosthetic group that is mainly low-spin with a pair of histidine axial ligands, with small portions of high-spin and P450-type heme. In its ferrous state, STEAP1 is capable of reducing transition metal ion complexes of Fe3+ and Cu2+. Ferrous STEAP1 also reacts readily with O2 through an outer sphere redox mechanism. Kinetics with all three substrates are biphasic with ∼80 and ∼20% for the fast and slow phases, respectively, in line with its heme heterogeneity. STEAP1 retained a low level of bound FAD during purification, and the binding equilibrium constant, KD, was ∼30 µM. These results highlight STEAP as a novel metal reductase and superoxide synthase and establish a solid basis for further research into understanding how STEAP1 activities may affect cancer progression.

The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport.

The phosphoenolpyruvate:carbohydrate phosphotransferase systems are found in bacteria, where they play central roles in sugar uptake and regulation of cellular uptake processes. Little is known about how the membrane-embedded components (EIICs) selectively mediate the passage of carbohydrates across the membrane. Here we report the functional characterization and 2.55-Å resolution structure of a maltose transporter, bcMalT, belonging to the glucose superfamily of EIIC transporters. bcMalT crystallized in an outward-facing occluded conformation, in contrast to the structure of another glucose superfamily EIIC, bcChbC, which crystallized in an inward-facing occluded conformation. The structures differ in the position of a structurally conserved substrate-binding domain that is suggested to play a central role in sugar transport. In addition, molecular dynamics simulations suggest a potential pathway for substrate entry from the periplasm into the bcMalT substrate-binding site. These results provide a mechanistic framework for understanding substrate recognition and translocation for the glucose superfamily EIIC transporters.

Ubiquitin interacts with the Tollip C2 and CUE domains and inhibits binding of Tollip to phosphoinositides.

A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endosomal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways.

The C2 domain of Tollip, a Toll-like receptor signalling regulator, exhibits broad preference for phosphoinositides.

TLRs (Toll-like receptors) provide a mechanism for host defence immune responses. Activated TLRs lead to the recruitment of adaptor proteins to their cytosolic tails, which in turn promote the activation of IRAKs (interleukin-1 receptor-associated kinases). IRAKs act upon their transcription factor targets to influence the expression of genes involved in the immune response. Tollip (Toll-interacting protein) modulates IRAK function in the TLR signalling pathway. Tollip is multimodular, with a conserved C2 domain of unknown function. We found that the Tollip C2 domain preferentially interacts with phosphoinositides, most notably with PtdIns3P (phosphatidylinositol 3-phosphate) and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), in a Ca2+-independent manner. However, NMR analysis demonstrates that the Tollip C2 domain binds Ca2+, which may be required to target the membrane interface. NMR and lipid-protein overlay analyses suggest that PtdIns3P and PtdIns(4,5)P2 share interacting residues in the protein. Kinetic studies reveal that the C2 domain reversibly binds PtdIns3P and PtdIns(4,5)P2, with affinity values in the low micromolar range. Mutational analysis identifies key PtdIns3P- and PtdIns(4,5)P2-binding conserved basic residues in the protein. Our findings suggest that basic residues of the C2 domain mediate membrane targeting of Tollip by interaction with phosphoinositides, which contribute to the observed partition of the protein in different subcellular compartments.

Backbone (1)H, (15)N, and (13)C resonance assignments and secondary structure of the Tollip CUE domain.

The Toll-interacting protein (Tollip) is a negative regulator of the Toll-like receptor (TLR)-mediated inflammation response. Tollip is a modular protein that contains an Nterminal Tom1-binding domain (TBD), a central conserved domain 2 (C2), and a C-terminal coupling of ubiquitin to endoplasmic reticulum degradation (CUE) domain. Here, we report the sequence-specific backbone (1)H, (15)N, and (13)C assignments of the human Tollip CUE domain. The CUE domain was found to be a stable dimer as determined by size-exclusion chromatography and molecular crosslinking studies. Analysis of the backbone chemical shift data indicated that the CUE domain exhibits three helical elements corresponding to 52% of the protein backbone. Circular dichroism spectrum analysis confirmed the helical nature of this domain. Comparison of the location of these helical regions with those reported for yeast CUE domains suggest differences in length for all helical elements. We expect the structural analysis presented here will be the foundation for future studies on the biological significance of the Tollip CUE domain, its molecular interactions, and the mechanisms that modulate its function during the inflammatory response.