Loss of polyubiquitin gene Ubb leads to metabolic and sleep abnormalities in mice.

AIMS: Ubiquitin performs essential roles in a myriad of signalling pathways required for cellular function and survival. Recently, we reported that disruption of the stress-inducible ubiquitin-encoding gene Ubb reduces ubiquitin content in the hypothalamus and leads to adult-onset obesity coupled with a loss of arcuate nucleus neurones and disrupted energy homeostasis in mice. Neuropeptides expressed in the hypothalamus control both metabolic and sleep behaviours. In order to demonstrate that the loss of Ubb results in broad hypothalamic abnormalities, we attempted to determine whether metabolic and sleep behaviours were altered in Ubb knockout mice. METHODS: Metabolic rate and energy expenditure were measured in a metabolic chamber, and sleep stage was monitored via electroencephalographic/electromyographic recording. The presence of neurodegeneration and increased reactive gliosis in the hypothalamus were also evaluated. RESULTS: We found that Ubb disruption leads to early-onset reduced activity and metabolic rate. Additionally, we have demonstrated that sleep behaviour is altered and sleep homeostasis is disrupted in Ubb knockout mice. These early metabolic and sleep abnormalities are accompanied by persistent reactive gliosis and the loss of arcuate nucleus neurones, but are independent of neurodegeneration in the lateral hypothalamus. CONCLUSIONS: Ubb knockout mice exhibit phenotypes consistent with hypothalamic dysfunction. Our data also indicate that Ubb is essential for the maintenance of the ubiquitin levels required for proper regulation of metabolic and sleep behaviours in mice.

Effect of ubiquitin expression on neuropathogenesis in a mouse model of familial amyotrophic lateral sclerosis.

The ubiquitin-proteasome system (UPS) is a central component in the cellular defence against potentially toxic protein aggregates. UPS dysfunction is linked to the pathogenesis of both sporadic and inherited neurodegenerative diseases, including dominantly inherited familial amyotrophic lateral sclerosis (fALS). To investigate the role of the UPS in fALS pathogenesis, transgenic mice expressing mutant G9 3A Cu,Zn superoxide dismutase (SOD1) were crossed with transgenic mice expressing epitope tagged, wild-type or dominant-negative mutant ubiquitin (Ub(K48R)). RNase protection assays were used to confirm expression of the Ub transgenes in spinal cord and ubiquitin transgene levels were estimated to account for 9-12% of total ubiquitin. Mice expressing the G9 3A transgene exhibited neurological symptoms and histopathological changes typical of this model irrespective of ubiquitin transgene status. Impaired rotarod performance was observed in all G9 3A transgenics by 7 weeks of age irrespective of ubiquitin genotype. The presence of wild-type or mutant ubiquitin transgenes resulted in a small but significant delay in the onset of clinical symptoms and mild acceleration of disease progression, without influencing overall survival. These data suggest that relatively small changes in ubiquitin expression can influence the development of neurodegenerative disease and are consistent with a neuroprotective role for the UPS.

Specificity in intracellular protein aggregation and inclusion body formation.

Protein aggregation is widely considered to be a nonspecific coalescence of misfolded proteins, driven by interactions between solvent-exposed hydrophobic surfaces that are normally buried within a protein's interior. Accordingly, abnormal interactions between misfolded proteins with normal cellular constituents has been proposed to underlie the toxicity associated with protein aggregates in many neurodegenerative disorders. Here we have used fluorescence resonance energy transfer and deconvolution microscopy to investigate the degree to which unrelated misfolded proteins expressed in the same cells coaggregate with one another. Our data reveal that in cells, protein aggregation exhibits exquisite specificity even among extremely hydrophobic substrates expressed at very high levels.

Impairment of the ubiquitin-proteasome system by protein aggregation.

Intracellular deposition of aggregated and ubiquitylated proteins is a prominent cytopathological feature of most neurodegenerative disorders. Whether protein aggregates themselves are pathogenic or are the consequence of an underlying molecular lesion is unclear. Here, we report that protein aggregation directly impaired the function of the ubiquitin-proteasome system. Transient expression of two unrelated aggregation-prone proteins, a huntingtin fragment containing a pathogenic polyglutamine repeat and a folding mutant of cystic fibrosis transmembrane conductance regulator, caused nearly complete inhibition of the ubiquitin-proteasome system. Because of the central role of ubiquitin-dependent proteolysis in regulating fundamental cellular events such as cell division and apoptosis, our data suggest a potential mechanism linking protein aggregation to cellular disregulation and cell death.

Aggresomes, inclusion bodies and protein aggregation.

Intracellular and extracellular accumulation of aggregated protein are linked to many diseases, including ageing-related neurodegeneration and systemic amyloidosis. Cells avoid accumulating potentially toxic aggregates by mechanisms including the suppression of aggregate formation by molecular chaperones and the degradation of misfolded proteins by proteasomes. Once formed, aggregates tend to be refractory to proteolysis and to accumulate in inclusion bodies. This accumulation has been assumed to be a diffusion-limited process, but recent studies suggest that, in animal cells, aggregated proteins are specifically delivered to inclusion bodies by dynein-dependent retrograde transport on microtubules. This microtubule-dependent inclusion body is called an aggresome.

Conformational disease.

A large and diverse number of diseases are now recognized as 'conformational diseases', caused by adoption of non-native protein conformations that lead to aggregation. The recent conference, 'Alpha1-antitrypsin deficiency and other conformational diseases', held in Airlie, Virginia, USA (27-30 June, 2000) focused on some of the common pathways by which cells protect themselves from toxicity associated with protein misfolding and aggregation.

Formation of high molecular weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis.

Deposition of aggregated protein into neurofilament-rich cytoplasmic inclusion bodies is a common cytopathological feature of neurodegenerative disease. How-or indeed whether-protein aggregation and inclusion body formation cause neurotoxicity are presently unknown. Here, we show that the capacity of superoxide dismutase (SOD) to aggregate into biochemically distinct, high molecular weight, insoluble protein complexes (IPCs) is a gain of function associated with mutations linked to autosomal dominant familial amyotrophic lateral sclerosis. SOD IPCs are detectable in spinal cord extracts from transgenic mice expressing mutant SOD several months before inclusion bodies and motor neuron pathology are apparent. Sequestration of mutant SOD into cytoplasmic inclusion bodies resembling aggresomes requires retrograde transport on microtubules. These data indicate that aggregation and inclusion body formation are mechanistically and temporally distinct processes.

Aggresomes and Russell bodies. Symptoms of cellular indigestion?

All cells are equipped with a proteolytic apparatus that eliminates damaged, misfolded and incorrectly assembled proteins. The principal engine of cytoplasmic proteolysis, the 26S proteasome, requires that substrates be unfolded to gain access to the active site; consequently, it is relatively ineffective at degrading aggregated proteins. Cellular indigestion occurs when the production of aggregation-prone proteins exceeds the cell's (or organelle's) capacity to eliminate them. Cellular pathways that resolve this indigestion exist, but appear to have limited capacities. Russell bodies and aggresomes are manifestations of cellular indigestion in the endoplasmic reticulum and cytoplasmic compartments, respectively, and are often associated with disease.

The role of multiubiquitination in dislocation and degradation of the alpha subunit of the T cell antigen receptor.

Unassembled alpha subunits of the T cell receptor (TCRalpha) are degraded by proteasomes following their dislocation from the endoplasmic reticulum membrane. We previously demonstrated that a variant of TCRalpha lacking lysines (KalphaR) is degraded by this pathway with kinetics indistinguishable from those of the wild type protein (Yu, H., Kaung, G., Kobayashi, S., and Kopito, R. R. (1997) J. Biol. Chem. 272, 20800-20804), demonstrating that ubiquitination on lysines is not required for TCRalpha degradation by the proteasome. Here, we show that dislocation and degradation of TCRalpha and KalphaR are suppressed by dominant negative ubiquitin coexpression and by mutations in the ubiquitin activating enzyme, indicating that their degradation requires a functional ubiquitin pathway. A cytoplasmic TCRalpha variant that mimics a dislocated degradation intermediate was degraded 5 times more rapidly than full-length TCRalpha, suggesting that dislocation from the endoplasmic reticulum membrane is the rate-limiting step in TCRalpha degradation. We conclude that ubiquitination is required both for dislocation and for targeting TCRalpha chains to the proteasome.

Redox reagents and divalent cations alter the kinetics of cystic fibrosis transmembrane conductance regulator channel gating.

Gating of the cystic fibrosis Cl(-) channel requires hydrolysis of ATP by its nucleotide binding folds, but how this process controls the kinetics of channel gating is poorly understood. In the present work we show that the kinetics of channel gating and presumably the rate of ATP hydrolysis depends on the species of divalent cation present and the oxidation state of the protein. With Ca(2+) as the dominant divalent cation instead of Mg(2+), the open burst duration of the channel is increased approximately 20-fold, and this change is reversible upon washout of Ca(2+). In contrast, "soft" divalent cations such as Cd(2+) interact covalently with cystic fibrosis transmembrane conductance regulator (CFTR). These metals decrease both opening and closing rates of the channel, and the effects are not reversed by washout. Oxidation of CFTR channels with a variety of oxidants resulted in a similar slowing of channel gating. In contrast, reducing agents had the opposite effect, increasing both opening and closing rates of the channel. In cell-attached patches, CFTR channels exhibit both oxidized and reduced types of gating, raising the possibility that regulation of the redox state of the channel may be a physiological mode of control of CFTR channel activity.

Biosynthesis and degradation of CFTR.

Biosynthesis and Degradation of CFTR. Physiol. Rev. 79, Suppl.: S167-S173, 1999. - Many of the mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause cystic fibrosis interfere with the folding and biosynthetic processing of nascent CFTR molecules in the endoplasmic reticulum. Mutations in the cytoplasmic nucleotide binding domains, including the common allele DeltaF508, decrease the efficiency of CFTR folding, reduce the probability of its dissociation from molecular chaperones, and largely prevent its maturation through the secretory pathway to the plasma membrane. These mutant CFTR molecules are rapidly degraded by cytoplasmic proteasomes by a process that requires covalent modification by multiubiquitination. The effects of temperature and chemical chaperones on the intracellular processing of mutant CFTR molecules suggest that strategies aimed at increasing the folding yield of this protein in vivo may eventually lead to the development of novel therapies for cystic fibrosis.

Aggresomes: a cellular response to misfolded proteins.

Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.

Cytosolic pH regulates GCl through control of phosphorylation states of CFTR.

Our objective in this study was to determine the effect of changes in luminal and cytoplasmic pH on cystic fibrosis transmembrane regulator (CFTR) Cl- conductance (GCl). We monitored CFTR GCl in the apical membranes of sweat ducts as reflected by Cl- diffusion potentials (VCl) and transepithelial conductance (GCl). We found that luminal pH (5.0-8.5) had little effect on the cAMP/ATP-activated CFTR GCl, showing that CFTR GCl is maintained over a broad range of extracellular pH in which it functions physiologically. However, we found that phosphorylation activation of CFTR GCl is sensitive to intracellular pH. That is, in the presence of cAMP and ATP [adenosine 5'-O-(3-thiotriphosphate)], CFTR could be phosphorylated at physiological pH (6.8) but not at low pH (approximately 5.5). On the other hand, basic pH prevented endogenous phosphatase(s) from dephosphorylating CFTR. After phosphorylation of CFTR with cAMP and ATP, CFTR GCl is normally deactivated within 1 min after cAMP is removed, even in the presence of 5 mM ATP. This deactivation was due to an increase in endogenous phosphatase activity relative to kinase activity, since it was reversed by the reapplication of ATP and cAMP. However, increasing cytoplasmic pH significantly delayed the deactivation of CFTR GCl in a dose-dependent manner, indicating inhibition of dephosphorylation. We conclude that CFTR GCl may be regulated via shifts in cytoplasmic pH that mediate reciprocal control of endogenous kinase and phosphatase activities. Luminal pH probably has little direct effect on these mechanisms. This regulation of CFTR may be important in shifting electrolyte transport in the duct from conductive to nonconductive modes.

Cotranslational ubiquitination of cystic fibrosis transmembrane conductance regulator in vitro.

Ubiquitination is a covalent protein modification that can target proteins in eukaryotic cells for degradation by the 26 S proteasome. Substrates for this degradation pathway include abnormal proteins that arise from misfolding and/or mutation. How and when the ubiquitination machinery recognizes misfolded proteins and targets them for degradation remains largely unknown. We have previously shown that cystic fibrosis transmembrane conductance regulator (CFTR), is rapidly degraded in a ubiquitin-dependent fashion, without any detectable lag following its synthesis (Ward, C. L., and Kopito, R. R. (1994) J. Biol. Chem. 269, 25710-25718), suggesting that ubiquitination and protein synthesis may be temporally linked. In the present study, we have investigated the timing of CFTR ubiquitination relative to its translation in reticulocyte lysates containing 125I-ubiquitin. In synchronized, proteasome-inhibited lysates, translation of full-length CFTR chains was completed in approximately 30 min, whereas modification of CFTR with [125I]ubiquitin was evident by 20 min, indicating that ubiquitination precedes the completion of full-length polypeptide chains. Moreover, ubiquitin was also found to be transferred to nascent CFTR chains while attached to ribosomes. Together, these data establish that ubiquitination, which is widely assumed to be a post-translational event, can occur cotranslationally and suggest a role for ubiquitination early in protein biosynthesis.

Cytosolic degradation of T-cell receptor alpha chains by the proteasome.

The T-cell antigen receptor (TCR) is an hetero-oligomeric membrane complex composed of at least seven transmembrane polypeptide chains that has served as a model for the assembly and degradation of integral membrane proteins in the endoplasmic reticulum (ER). Unassembled TCRalpha chains fail to mature to the Golgi apparatus and are rapidly degraded by a non-lysosomal "ER degradation" pathway that has been proposed to be autonomous to the ER. In these studies we show that the degradation of core-glycosylated TCRalpha is blocked by N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) and lactacystin, implicating the proteasome in ER degradation. Either acute or chronic treatment of TCRalpha-transfected cells with proteasome inhibitors cause the core-glycosylated TCRalpha chains to progressively shift to an approximately 28-kDa form that lacks N-linked oligosaccharides and the N-terminal signal peptide. The susceptibility of this 28-kDa species to extravesicular protease indicates that it is not protected by the ER membrane and, hence, cytoplasmic. These data suggest a model in which TCRalpha chains that are translocated across the membrane, core-glycosylated, but fail to assemble are dislocated back to the cytoplasm for degradation by cytoplasmic proteasomes. Our data also suggest that covalent modification of TCRalpha with ubiquitin is not required for its degradation.

A cluster of cytoplasmic histidine residues specifies pH dependence of the AE2 plasma membrane anion exchanger.

Intracellular pH is maintained by a dynamic equilibrium balancing the opposing forces of proton loading and proton extrusion. By providing an efflux pathway for base, anion exchangers constitute a key component of the plasma membrane proton-loading machinery. The data in this paper identify a histidine-rich sequence within the cytoplasmic domain of the nonerythroid anion exchanger, AE2, that serves as an intracellular pH "sensor" that modulates anion exchange activity within the physiological range of cytoplasmic pH. These data reveal an interaction between the two major domains of the anion exchanger and suggest a novel substrate feedback mechanism by which intracellular protons directly control the activity of an acid-loading plasma membrane ion transporter.

Immunocytochemical localization of vacuolar H+-ATPase and Cl--HCO3- anion exchanger (erythrocyte band-3 protein) in avian osteoclasts: effect of calcium-deficient diet on polar expression of the H+-ATPase pump.

Osteoclasts attach to the bone surface and resorb bone by secreting protons into an isolated subosteoclastic compartment. Previous studies have shown the presence of a vacuolar type H+-ATPase, and a functional Cl--HCO3- anion exchanger in the osteoclast. In the present studies, using a monoclonal antibody to the 31-kDa subunit of H+-ATPase and a rabbit antiserum to the erythrocyte band-3 protein (Cl--HCO3- anion exchanger) we have immunocytochemically localized the respective pumps in bone sections obtained from chickens fed a normal or a calcium-deficient diet for 4 weeks. Our results indicate that although H+-ATPase is either evenly distributed throughout the osteoclast or is more polarized at its ruffled membrane juxtaposed to the bone surface, the band-3 protein immunoreactivity is always localized to the plasma membrane which is not attached to the bone surface (basolateral membrane). Four weeks of a calcium-deficient diet resulted in a significant increase in the percentage of osteoclasts that were polarized for the H+-ATPase pump at their ruffled membrane, and a trend toward increased total number of osteoclasts, although the latter did not reach statistical significance (P = 0.09). These changes were not accompanied by a significant increase in the intensity of staining for H+-ATPase. Band-3 protein immunoreactivity was always prominent, limited to the basolateral membrane, and did not alter with calcium-deficient diet or with changes in the degree of H+-ATPase polarization.

Failure of the cystic fibrosis transmembrane conductance regulator to conduct ATP.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel regulated by protein kinase A and adenosine triphosphate (ATP). Loss of CFTR-mediated chloride ion conductance from the apical plasma membrane of epithelial cells is a primary physiological lesion in cystic fibrosis. CFTR has also been suggested to function an an ATP channel, although the size of the ATP anion is much larger than the estimated size of the CFTR pore. ATP was not conducted through CFTR in intact organs, polarized human lung cell lines, stably transfected mammalian cell lines, or planar lipid bilayers reconstituted with CFTR protein. These findings suggest that ATP permeation through the CFTR is unlikely to contribute to the normal function of CFTR or to the pathogenesis of cystic fibrosis.