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Early-onset torsion dystonia (EOTD) is a neurological disorder characterized by involuntary and sustained muscle contractions that can lead to paralysis and abnormal posture. EOTD is associated with the deletion of a glutamate (ΔE) in torsinA, an endoplasmic reticulum (ER) resident AAA+ ATPase. To date, the effect of ΔE on torsinA and the reason that this mutation results in EOTD are unclear. Moreover, there are no specific therapeutic options to treat EOTD. To define the underlying biochemical defects associated with torsinAΔE and to uncover factors that might be targeted to offset defects associated with torsinAΔE, we developed a yeast torsinA expression system and tested the roles of ER chaperones in mediating the folding and stability of torsinA and torsinAΔE. We discovered that the ER lumenal Hsp70, BiP, an associated Hsp40, Scj1, and a nucleotide exchange factor, Lhs1, stabilize torsinA and torsinAΔE. BiP also maintained torsinA and torsinAΔE solubility. Mutations predicted to compromise specific torsinA functional motifs showed a synthetic interaction with the ΔE mutation and destabilized torsinAΔE, suggesting that the ΔE mutation predisposes torsinA to defects in the presence of secondary insults. In this case, BiP was required for torsinAΔE degradation, consistent with data that specific chaperones exhibit either pro-degradative or pro-folding activities. Finally, using two independent approaches, we established that BiP stabilizes torsinA and torsinAΔE in mammalian cells. Together, these data define BiP as the first identified torsinA chaperone, and treatments that modulate BiP might improve symptoms associated with EOTD. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid type B (GABAB) receptors are important for slow synaptic inhibition in the CNS. The efficacy of inhibition is directly related to the stability of cell surface receptors. For GABAB receptors, heterodimerization between R1 and R2 subunits is critical for cell surface expression and signaling, but how this determines the rate and extent of receptor internalization is unknown. Here, we insert a high affinity a-bungarotoxin binding site into the N terminus of the R2 subunit and reveal its dominant role in regulating the internalization of GABAB receptors in live cells. To simultaneously study R1a and R2 trafficking, a new α-bungarotoxin binding site-labeling technique was used, allowing α-bungarotoxin conjugated to different fluorophores to selectively label R1a and R2 subunits. This approach demonstrated that R1a and R2 are internalized as dimers. In heterologous expression systems and neurons, the rates and extents of internalization for R1aR2 heteromers and R2 homomers are similar, suggesting a regulatory role for R2 in determining cell surface receptor stability. The fast internalization rate of R1a, which has been engineered to exit the endoplasmic reticulum, was slowed to that of R2 by truncating the R1a C-terminal tail or by removing a dileucine motif in its coiled-coil domain. Slowing the rate of internalization by co-assembly with R2 represents a novel role for GPCR heterodimerization whereby R2 subunits, via their C terminus coiled-coil domain, mask a dileucine motif on R1a subunits to determine the surface stability of the GABAB receptor. [ABSTRACT FROM AUTHOR]

Early onset dystonia is commonly associated with the deletion of one of a pair of glutamate residues (ΔE302/303) near the C terminus of torsinA, a member of the AAA+ protein family (ATPases associated with a variety of cellular activities) located in the endoplasmic reticulum lumen. The functional consequences of the disease-causing mutation, ΔE, are not currently understood. By contrast to other AAA+ proteins, torsin proteins contain two conserved cysteine residues in the C-terminal domain, one of which is located in the nucleotide sensor II motif. Depending on redox status, an ATP hydrolysis mutant of torsinA interacts with lamina-associated polypeptide 1 (LAP1) and lumenal domain like LAP1 (LULL1). Substitution of the cysteine in sensor II diminishes the redox-regulated interaction of torsinA with these substrates. Significantly, the dystonia-causing mutation, ΔE, alters the ability of torsinA to mediate the redox-regulated interactions with LAP1 and LULL1. Limited proteolysis experiments reveal redox- and mutation-dependent changes in the local conformation of torsinA as a function of nucleotide binding. These results indicate that the cysteine-containing sensor II plays a critical role in redox sensing and the nucleotide and partner binding functions of torsinA and suggest that loss of this function of torsinA contributes to the development of DYT1 dystonia. [ABSTRACT FROM AUTHOR]

Parkinson disease and other ɑ-synucleinopathies are characterized by the deposition of intraneuronal ɑ-synuclein (ɑSyn) inclusions. A significant fraction (about 15%) of ɑSyn in these pathological structures are truncated forms that have a much higher propensity than the full-length ɑSyn to form aggregates in vitro. However, little is known about the role of truncated ɑSyn species in pathogenesis or the means by which they are generated. Here, we have provided an in vitro mechanistic study demonstrating that truncated ɑSyns induce rapid aggregation of full-length protein at substoichiometric ratios. Co-overexpression of truncated ɑSyn with full-length protein increases cell vulnerability to oxidative stress in dopaminergic SH-SY5Y cells. These results suggest a precipitating role for truncated ɑSyn in the pathogenesis of diseases involving ɑSyn aggregation. In this regard, the A53T mutation found in some cases of familial Parkinson disease exacerbates the accumulation of insoluble ɑSyns that correlates with the onset of pathology in transgenic mice expressing human ɑSyn-A53T mutant. The caspase-like activity of the 20 S proteasome produces truncated fragments similar to those found in patients and animal models from degradation of unstructured ɑSyn. We propose a model in which incomplete degradation of ɑSyn, especially under overloaded proteasome capacity, produces highly amyloidogenic fragments that rapidly induce the aggregation of full-length protein. These aggregates in turn reduce proteasome activity, leading to further accumulation of fragmented and full-length ɑSyns, creating a vicious cycle of cytotoxicity. This model has parallels in other neurodegenerative diseases, such as Huntington disease, where coaggregation of poly(Q) fragments with full-length protein has been observed. [ABSTRACT FROM AUTHOR]

HCO3- secretion is a vital activity in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing epithelia. However, the role of CFTR in this activity is not well understood. Simultaneous measurements of membrane potential and pHi and/or current in CFTRexpressing Xenopus oocytes revealed dynamic control of CFTR Cl-/HCO3- permeability ratio, which is regulated by external Cl- (Clo-). Thus, reducing external Cl- from 110 to 0-10 mM resulted in the expected increase in membrane potential, but with no corresponding OH- or HCO3- influx. Approximately 3-4 min after reducing Clo- to 0 mM, an abrupt switch in membrane potential occurs that coincided with an increased rates of OH- and HCO3- influx. The switch in membrane permeability to OH-/HCO3- can also be recorded as a leftward shift in the reversal potential. Furthermore, an increased rate of OH- influx in response to elevating pHo to 9.0 was observed only after the switch in membrane potential. The time to switch increased to 11 min at Clo- of 5 mM. Conversely, re-addition of external Cl- after the switch in membrane potential did not stop HCO3- influx, which continued for about 3.9 min after Cl- addition. Importantly, addition of external Cl- to cells incubated in Cl--free medium never resulted in HCO3- efflux. Voltage and current clamp experiments showed that the delayed HCO3- transport is electrogenic. These results indicate that CFTR exists in two conformations, a Cl- only and a Cl- and OH-/HCO3- permeable state. The switch between the states is controlled by external Cl-. Accordingly, a different tryptic pattern of CFTR was found upon digestion in Cl--containing and Cl--free media. The physiological significance of these finding is discussed in the context of HCO3- secretion by tissues such as the pancreas and salivary glands. [ABSTRACT FROM AUTHOR]

The multipass membrane protein APH-1, found in the γ-secretase complex together with presenilin, nicastrin, and PEN-2, is essential for Notch signaling in Caenorhabditis elegans embryos and is required for intramembrane proteolysis of Notch and β-amyloid precursor protein in mammalian and Drosophila cells. In C. elegans, a mutation of the conserved transmembrane Gly123 in APH-1 (mutant or28) leads to a notch/glp-1 loss-of-function phenotype. In this study, we show that the corresponding mutation in mammalian APH-1aL (G122D) disrupts the physical interaction of APH-1aL with hypoglycosylated immature nicastrin and the presenilin holoprotein as well as with mature nicastrin, presenilin, and PEN-2. The G122D mutation also reduced γ-secretase activity in intramembrane proteolysis of membrane-tethered Notch. Moreover, we found that the conserved transmembrane Gly122, Gly126, and Gly130 in the fourth transmembrane region of mammalian APH-1aL are part of the membrane helix-helix interaction GXXXG motif and are essential for the stable association of APH1aL with presenilin, nicastrin, and PEN-2. These findings suggest that APH-1 plays a GXX G-dependent scaffolding role in both the initial assembly and subsequent maturation and maintenance of the active γ-secretase complex. [ABSTRACT FROM AUTHOR]

The most common defect in cystic fibrosis, deletion of phenylalanine from position 508 of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), decreases the trafficking of this protein to the cell surface membrane. Previous studies have shown that low temperature and high concentrations of glycerol or trimethylamine N-oxide can partially counteract the processing defect of ΔF508 CFTR. The present study investigates whether physiologically relevant concentrations of organic solutes, accumulated by cotransporter proteins, can rescue the misprocessing of ΔF508 CFTR. Myoinositol alone or myoinositol, betaine, and taurine given sequentially increased the processing of core-glycosylated, endoplasmic reticulum-arrested ΔF508 CFTR into the fully glycosylated form of CFTR in IB3 cells or NIH 3T3 cells stably expressing ΔF508 CFTR. Pulsechase experiments using transiently transfected COS7 cells demonstrated that organic solutes also increased the processing of the core-glycosylated form of green fluorescent protein-ΔF508 CFTR. Moreover, the prolonged half-life of the complex-glycosylated form of GFP-ΔF508 CFTR suggests that this treatment stabilized the mature form of the protein. In vitro studies of purified NBD1 stability and aggregation showed that myoinositol stabilized both the ΔF508 and wild type CFTR and inhibited ΔF508 misfolding. Most significantly, treatment of CF bronchial airway cells with these transportable organic solutes restores cAMP-stimulated single channel activity of both CFTR and outwardly rectifying chloride channel in the cell surface membrane and also restores a forskolin-stimulated macroscopic [sup 36]Cl[sup -] efflux. We conclude that organic solutes can repair CFTR functions by enhancing the processing of ΔF508 CFTR to the plasma membrane by stabilizing the complex-glycosylated form of ΔF508 CFTR. [ABSTRACT FROM AUTHOR]