SUMMARY The UbiB protein kinase-like (PKL) family is widespread—comprising one-quarter of microbial PKLs and five human homologs—yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. While COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates—functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.
Lafora disease (LD), a progressive form of inherited epilepsy, is associated with widespread neurodegeneration and the formation of polyglucosan bodies in the neurons. Laforin, a protein phosphatase, and malin, an E3 ubiquitin ligase, are two of the proteins that are defective in LD. We have shown recently that laforin and malin (referred together as LD proteins) are recruited to aggresome upon proteasomal blockade, possibly to clear misfolded proteins through the ubiquitin-proteasome system (UPS). Here we test this possibility using a variety of cytotoxic misfolded proteins, including the expanded polyglutamine protein, as potential substrates. Laforin and malin, together with Hsp70 as a functional complex, suppress the cellular toxicity of misfolded proteins, and all the three members of this complex are required for this function. Laforin and malin interact with misfolded proteins and promote their degradation through the UPS. LD proteins are recruited to the polyglutamine aggregates and reduce the frequency of aggregate-positive cells. Taken together, our results suggest that the malin-laforin complex is a novel player in the neuronal response to misfolded proteins and could be potential therapeutic targets for neurodegenerative disorders associated with cytotoxic proteins.
Background:Earlier reports indicate that O-GlcNAcylation might be protective in neurodegenerative disorders. Results: Suppressing O-GlcNAcylation modulates autophagy to enhance the viability of neuronal cells expressing cytotoxic mutant huntingtin exon 1 protein (mHtt). Conclusion: O-GlcNAcylation regulates the clearance of mHtt by modulating the fusion of autophagosomes with lysosomes. Significance: This regulatory mechanism emerges as a novel therapeutic strategy for Huntington disease.
Synaptic transmission requires that vesicles filled with neurotransmitter molecules be docked to the plasma membrane by the SNARE protein complex. The SNARE complex applies attractive forces to overcome the long-range repulsion between the vesicle and membrane. To understand how the balance between the attractive and repulsive forces defines the equilibrium docked state we have developed a model that combines the mechanics of vesicle/membrane deformation with an apparently new coarse-grained model of the SNARE complex. The coarse-grained model of the SNARE complex is calibrated by comparison with all-atom molecular dynamics simulations as well as by force measurements in laser tweezer experiments. The model for vesicle/membrane interactions includes the forces produced by membrane deformation and hydration or electrostatic repulsion. Combining these two parts, the coarse-grained model of the SNARE complex with membrane mechanics, we study how the equilibrium docked state varies with the number of SNARE complexes. We find that a single SNARE complex is able to bring a typical synaptic vesicle to within a distance of ~3 nm from the membrane. Further addition of SNARE complexes shortens this distance, but an overdocked state of >4–6 SNAREs actually increases the equilibrium distance.
Membrane curvature of a biological cell is actively involved in various fundamental cell biological functions. It has been discovered that membrane curvature and binding of peripheral membrane proteins follow a symbiotic relationship. The exact mechanism behind this interplay of protein binding and membrane curvature has not yet been properly understood. To improve understanding of the mechanism, we study curvature sorting of proteins in a model system consisting of a tether pulled from a giant unilamellar vesicle using mechanical-thermodynamic models. The concentration of proteins bound to the membrane changes significantly due to curvature. This has also been observed in experiments by other researchers. We also find that there is a phase transition based on protein concentration and we discuss the coexistence of phases and stability of solutions. Furthermore, when sorting is favorable, the increase in protein concentration stabilizes the tether in the sense that less pulling force is required to maintain the tether. A similar mechanism may be in place, when motor proteins pull tethers from donor membranes.
Lafora disease (LD), an inherited and fatal neurodegenerative disorder, is characterized by increased cellular glycogen content and the formation of abnormally branched glycogen inclusions, called Lafora bodies, in the affected tissues, including neurons. Therefore, laforin phosphatase and malin ubiquitin E3 ligase, the two proteins that are defective in LD, are thought to regulate glycogen synthesis through an unknown mechanism, the defects in which are likely to underlie some of the symptoms of LD. We show here that laforin's subcellular localization is dependent on the cellular glycogen content and that the stability of laforin is determined by the cellular ATP level, the activity of 5=-AMP-activated protein kinase, and the affinity of malin toward laforin. By using cell and animal models, we further show that the laforin-malin complex regulates cellular glucose uptake by modulating the subcellular localization of glucose transporters; loss of malin or laforin resulted in an increased abundance of glucose transporters in the plasma membrane and therefore excessive glucose uptake. Loss of laforin or malin, however, did not affect glycogen catabolism. Thus, the excessive cellular glucose level appears to be the primary trigger for the abnormally higher levels of cellular glycogen seen in LD. Glucose is an essential metabolite in living systems. However, the regulatory roles of glucose in cellular physiological pathways and the mechanisms by which cells respond to changes in the intracellular levels of glucose are not fully understood (26). Dysregulation in these processes is thought to underlie the pathology of a few disorders that are associated with cytoplasmic glycogen inclusions (50). One such disorder is Lafora disease (LD), a heritable and fatal neurodegenerative disorder characterized by progressive myoclonus epilepsy and other neurological deficits, including ataxia and dementia (17,41). A hallmark of LD is the presence of Lafora bodies-insoluble and abnormally branched intracellular glycogen inclusions called polyglucosan-in neurons, muscle, liver, and other tissues (16,17,51,52). LD is caused by defects in the gene EPM2A, which encodes a dual-specificity protein phosphatase named laforin, or the NHLRC1 gene, which encodes an E3 ubiquitin ligase named malin (6,15,20,32). Laforin harbors a carbohydrate-binding domain (CBD) that binds to glycogen and Lafora bodies, both in vitro and in vivo (5,18,49). Thus, a role for laforin in carbohydrate metabolism and in the disposition of Lafora bodies was proposed (5, 18, 49). Besides Lafora bodies, glycogen content has also been found at higher levels in animals that were deficient for laforin or malin (11,43). Intriguingly, the glycogen reserve in LD animal models shows a higher phosphate content (11, 43), and laforin has been shown to dephosphorylate glycogen (43,44). A recent report suggested that glycogen phosphorylation possibly represents an error in a catalytic step in glycogen synthesis and that its removal by laforin could be a damage control mechanism (...
Loss of laforin, a protein phosphatase involved in Lafora disease (LD), results in the activation of serum/glucocorticoid-induced kinase 1 (SGK1), increased cellular glycogen accumulation, and autophagy defects. Inhibition of SGK1 restores normal glycogen content and autophagy defects in a cellular model of LD.
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