Cellular ubiquitin pool dynamics and homeostasis

Ubiquitin (Ub) is a versatile signaling molecule that plays important roles in a variety of cellular processes. Cellular Ub pools, which are composed of free Ub and Ub conjugates, are in dynamic equilibrium inside cells. In particular, increasing evidence suggests that Ub homeostasis, or the maintenance of free Ub above certain threshold levels, is important for cellular function and survival under normal or stress conditions. Accurate determination of various Ub species, including levels of free Ub and specific Ub chain linkages, have become possible in biological specimens as a result of the introduction of the proteomic approach using mass spectrometry. This technology has facilitated research on dynamic properties of cellular Ub pools and has provided tools for in-depth investigation of Ub homeostasis. In this review, we have also discussed the consequences of the disruption of Ub pool dynamics and homeostasis via deletion of polyubiquitin genes or mutations of deubiquitinating enzymes. The common consequence was a reduced availability of free Ub and a significant impact on the function and viability of cells. These observations further indicate that the levels of free Ub are important determinants for cellular protection. [BMB Reports 2014; 47(9): 475-482]     

Energetics and specificity of interactions within Ub.Uev.Ubc13 human ubiquitin conjugation complexes

Lys(63)-linked polyubiquitin (poly-Ub) chains appear to play a nondegradative signaling and/or recruitment role in a variety of key eukaryotic cellular processes, including NF-kappaB signal transduction and DNA repair. A protein heterodimer composed of a catalytically active ubiquitin-conjugating enzyme (Ubc13) and its homologue (Mms2 or Uev1a) forms a catalytic scaffold upon which a noncovalently associated acceptor Ub and thiolester-linked donor Ub are oriented such that Lys(63)-linked poly-Ub chain synthesis is facilitated. In this study, we have used (1)H-(15)N nuclear magnetic resonance spectroscopy, in combination with isothermal titration calorimetry, to determine the thermodynamics and kinetics of the interactions between various components of the Lys(63)-linked poly-Ub conjugation machinery. Mms2 and Uev1a interact in vitro with acceptor Ub to form 1/1 complexes with macroscopic dissociation constants of 98 +/- 15 and 213 +/- 14 microM, respectively, and appear to bind Ub in a similar fashion. Interestingly, the Mms2.Ubc13 heterodimer associates with acceptor Ub in a 1/1 complex and binds with a dissociation constant of 28 +/- 6 microM, significantly stronger than the binding of Mms2 alone. Furthermore, a dissociation constant of 49 +/- 7 nM was determined for the interaction between Mms2 and Ubc13 using isothermal titration calorimetry. In connection with previous structural studies for this system, the thermodynamics and kinetics of acceptor Ub binding to the Mms2.Ubc13 heterodimer described in detail in this study will allow for a more thorough rationalization of the mechanism of formation of Lys(63)-linked poly-Ub chains.     

Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase

Ubiquitin (Ub)-protein ligases (E3s) frequently modify their substrates with multiple Ub molecules in the form of a polyubiquitin (poly-Ub) chain. Although structurally distinct poly-Ub chains (linked through different Ub lysine (Lys) residues) can confer different fates on target proteins, little is known about how E3s select the Lys residue to be used in chain synthesis. Here, we used a combination of mutagenesis, biochemistry, and mass spectrometry to map determinants of linkage choice in chain assembly catalyzed by KIAA10, an HECT (Homologous to E6AP C-Terminus) domain E3 that synthesizes K29- and K48-linked chains. Focusing on the Ub molecule that contributes the Lys residue for chain formation, we found that specific surface residues adjacent to K48 and K29 are critical for the usage of the respective Lys residues in chain synthesis. This direct mechanism of linkage choice bears similarities to the mechanism of substrate site selection in sumoylation catalyzed by Ubc9, but is distinct from the mechanism of chain linkage selection used by the Mms2/Ubc13 (Ub E2 variant (UEV)/E2) complex.     

Regulation of polyubiquitin genes to meet cellular ubiquitin requirement

Ubiquitin (Ub) is one of the proteins that are highly conserved from yeast to humans. It is an essential core unit of the well-defined post-translational modification, called ubiquitination, which is involved in a variety of biological processes. In meta-zoans, Ub is encoded by two monoubiquitin genes and two polyubiquitin genes, in which a single Ub is fused to a ribosomal protein or Ub coding units are arranged in tandem repeats. In mice, polyubiquitin genes (Ubb and Ubc) play a pivotal role to meet the requirement of cellular Ub pools during embryonic development. In addition, expression levels of polyubiquitin genes are increased to adapt to environmental stimuli such as oxidative, heat-shock, and proteotoxic stress. Several researchers have reported about the perturbation of Ub pools through genetic alteration or exogenous Ub delivery using diverse model systems. To study Ub pool changes in a physiologically relevant manner, changing Ub pools via the regulation of endogenous polyubiquitin gene expression has recently been introduced. Furthermore, to understand the regulation of polyubiquitin gene expression more precisely, cis-acting elements and trans-acting factors, which are regulatory components of polyubiquitin genes, have been analyzed. In this review, we discuss how the role of polyu-biquitin genes has been studied during the past decade, es-pecially focusing on their regulation.     

The mouse polyubiquitin gene UbC is essential for fetal liver development, cell-cycle progression and stress tolerance

UbC is one of two stress-inducible polyubiquitin genes in mammals and is thought to supplement the constitutive UbA genes in maintaining cellular ubiquitin (Ub) levels during episodes of cellular stress. We have generated mice harboring a targeted disruption of the UbC gene. UbC(-/-) embryos die between embryonic days 12.5 and 14.5 in utero, most likely owing to a severe defect in liver cell proliferation. Mouse embryonic fibroblasts from UbC(-/-) embryos exhibit reduced growth rates, premature senescence, increased apoptosis and delayed cell-cycle progression, with slightly, but significantly, decreased steady-state Ub levels. UbC(-/-) fibroblasts are hypersensitive to proteasome inhibitors and heat shock, and unable to adequately increase Ub levels in response to these cellular stresses. Most, but not all of the UbC(-/-) phenotypes can be rescued by providing additional Ub from a poly hemagglutinin-tagged Ub minigene expressed from the Hprt locus. We propose that UbC is regulated by a process that senses Ub pool dynamics. These data establish that UbC constitutes an essential source of Ub during cell proliferation and stress that cannot be compensated by other Ub genes.     

Genetic inactivation of Kcnj16 identifies Kir5.1 as an important determinant of neuronal PCO2/pH sensitivity

The molecular identity of ion channels which confer PCO(2)/pH sensitivity in the brain is unclear. Heteromeric Kir4.1/Kir5.1 channels are highly sensitive to inhibition by intracellular pH and are widely expressed in several brainstem nuclei involved in cardiorespiratory control, including the locus coeruleus. This has therefore led to a proposed role for these channels in neuronal CO(2) chemosensitivity. To examine this, we generated mutant mice lacking the Kir5.1 (Kcnj16) gene. We show that although locus coeruleus neurons from Kcnj16((+/+)) mice rapidly respond to cytoplasmic alkalinization and acidification, those from Kcnj16((-/-)) mice display a dramatically reduced and delayed response. These results identify Kir5.1 as an important determinant of PCO(2)/pH sensitivity in locus coeruleus neurons and suggest that Kir5.1 may be involved in the response to hypercapnic acidosis.     

Increased isoprostane and prostaglandin are prominent in neurons in Alzheimer disease

Parkinson's disease is the most common movement disorder characterized by dopaminergic dysfunction and degeneration. Loss-of-function mutations in the DJ-1 gene have been linked to autosomal recessive forms of early-onset familial Parkinson's disease. DJ-1 is thought to play roles in protection of cells against oxidative stress and in maintenance of the normal dopaminergic function in the nigrostriatal pathway. Here we investigate the consequence of both DJ-1 inactivation and aging in mice. We found that DJ-1-/- mice at the age of 24–27 months have normal numbers of dopaminergic neurons in the substantia nigra and normal levels of dopamine and its major metabolites in the striatum. The number of noradrenergic neurons in the locus coeruleus is also unchanged in DJ-1-/- mice. Moreover, there is no accumulation of oxidative damage or inclusion bodies in aged DJ-1-/- brains. Together, these results indicate that loss of DJ-1 function alone is insufficient to cause nigral degeneration and oxidative damage in the life span of mice.     

Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis

Individual ubiquitin (Ub)-protein ligases (E3s) cooperate with specific Ub-conjugating enzymes (E2s) to modify cognate substrates with polyubiquitin chains. E3s belonging to the Really Interesting New Gene (RING) and Homologous to E6-Associated Protein (E6AP) C-Terminus (HECT) domain families utilize distinct molecular mechanisms. In particular, HECT E3s, but not RING E3s, form a thiol ester with Ub before transferring Ub to the substrate lysine. Here we report that different HECT domain E3s can employ distinct mechanisms of polyubiquitin chain synthesis. We show that E6AP builds up a K48-linked chain on its HECT cysteine residue, while KIAA10 builds up K48- and K29-linked chains as free entities. A small region near the N-terminus of the conserved HECT domain helps to bring about this functional distinction. Thus, a given HECT domain can specify both the linkage of a polyubiquitin chain and the mechanism of its assembly.     

Prostacyclin (PGI2) induces rapid depletion of cyclic AMP levels in brain nuclei of rats

Intravenous injection of prostacyclin (100 micrograms/kg) in rats resulted in a decrease of systolic blood pressure within 2 minutes. Concentrations of cAMP in 15 brain regions and nuclei were determined by radioimmunoassay. In lower brain stem nuclei, such as the nucleus of the solitary tract and the lateral reticular nucleus (A1 and C1 catecholaminergic cell groups) cAMP levels were depleted significantly, while in others, including the locus coeruleus and the periaqueductal central gray, cAMP levels did not show any alterations. Levels of cAMP were also depleted in some of the hypothalamic nuclei (periventricular, anterior hypothalamic, ventromedial), and in cerebral cortical areas. Lowered cAMP levels in brain areas might indicate lower cellular activity in cells participating in baroreceptor control mechanisms.     

Human Cdc34 employs distinct sites to coordinate attachment of ubiquitin to a substrate and assembly of polyubiquitin chains

The Cdc34 E2 ubiquitin (Ub) conjugating enzyme catalyzes polyubiquitination of a substrate recruited by the Skp1-Cullin 1-F-box protein-ROC1 E3 Ub ligase. Using mutagenesis studies, we now show that human Cdc34 employs distinct sites to coordinate the transfer of Ub to a substrate and the assembly of polyubiquitin chains. Mutational disruption of the conserved charged stretch (residues 143 to 153) or the acidic loop residues D102 and D103 led to accumulation of monoubiquitinated IkappaBalpha while failing to yield polyubiquitin chains, due to a catalytic defect in Ub-Ub ligation. These results suggest an ability of human Cdc34 to position the attacking Ub for assembly of polyubiquitin chains. Analysis of Cdc34N85Q and Cdc34S138A revealed severe defects of these mutants in both poly- and monoubiquitination of IkappaBalpha, supporting a role for N85 in stabilizing the oxyanion and in coordinating, along with S138, the attacking lysine for catalysis. Finally, Cdc34S95D and Cdc34(E108A/E112A) abolished both poly- and monoubiquitination of IkappaBalpha. Unexpectedly, the catalytic defects of these mutants in di-Ub synthesis can be rescued by fusion of a glutathione S-transferase moiety at E2's N terminus. These findings support the hypothesis that human Cdc34 S95 and E108/E112 are required to position the donor Ub optimally for catalysis, in a manner that might depend on E2 dimerization.     

Perturbation of the hematopoietic system during embryonic liver development due to disruption of polyubiquitin gene Ubc in mice

Disruption of the polyubiquitin gene Ubc leads to a defect in fetal liver development, which can be partially rescued by increasing the amount of ubiquitin. However, it is still not known why Ubc is required for fetal liver development and the nature of the defective cell types responsible for embryonic lethality have not been characterized. In this study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We found that Ubc was highly expressed in the embryonic liver, and the proliferation capacity of fetal liver cells was reduced in Ubc(-/-) embryos. Specifically, Ubc was most highly expressed in hematopoietic cells, and the proliferation capacity of hematopoietic cells was significantly impaired in Ubc(-/-) embryos. While hematopoietic cell and hematopoietic stem cell (HSC) frequency was maintained in Ubc(-/-) embryos, the absolute number of these cells was diminished because of reduced total liver cell number in Ubc(-/-) embryos. Transplantations of fetal liver cells into lethally irradiated recipient mice by non-competitive and competitive reconstitution methods indicated that disruption of Ubc does not significantly impair the intrinsic function of fetal liver HSCs. These findings suggest that disruption of Ubc reduces the absolute number of HSCs in embryonic livers, but has no significant effect on the autonomous function of HSCs. Thus, the lethality of Ubc(-/-) embryos is not the result of intrinsic HSC failure.     

Efferent ACTH-IR opiocortin projections from nucleus tractus solitarius: a hypothalamic deafferentation study

The distribution of opiocortin (OR-ir) immunoreactive fibers was examined immunocytochemically throughout the brain in rats following surgical isolation of the arcuate opiocortin-ir neuronal pool in the medial basal hypothalamus (MBH). Fibers which emanate from this pool were completely severed and thus eliminated from the rest of the brain, leaving intact those which can be identified immunocytochemically as opiocortin-ir projections from the medullary pool located in the nucleus tractus solitarius (NTS). These studies reveal a unique organizational pattern of proopiomelanocortin (POMC) peptidergic neuronal systems and demonstrate that several pontine and medullary regions receive projections from both the hypothalamic (arcuate) and medullary (NTS) opiocortin-ir perikarya. Comparative analyses of deafferented and control brains reveal that certain brainstem autonomic centers such as parabrachial (PB), locus coeruleus (LC), nucleus paragiganticellularis (PGi) are recipients of fibers which emanate from both arcuate and NTS opiocortin-ir perikarya. Areas which receive projections from arcuate opiocortin-ir neurons alone include forebrain and hypothalamic nuclei as well as the periaqueductal grey.     

Specific Neurochemical Derangements of Brain Projecting Neurons in Apolipoprotein E-Deficient Mice

Abstract: Apolipoprotein E (apoE)-deficient mice provide a useful system for studying the role of apoE in neuronal maintenance and repair. Previous studies revealed specific memory impairments in these mice that are associated with presynaptic derangements in projecting forebrain cholinergic neurons. In the present study we examined whether dopaminergic, noradrenergic, and serotonergic projecting pathways of apoE-deficient mice are also affected and investigated the mechanisms that render them susceptible. The densities of nerve terminals of forebrain cholinergic projections were monitored histochemically by measurements of acetylcholinesterase activity, whereas those of the dopaminergic nigrostriatal pathway, the noradrenergic locus coeruleus cortical projection, and the raphe-cortical serotonergic tract were measured autoradiographically using radioligands that bind specifically to the respective presynaptic transporters of these neuronal tracts. The results obtained revealed that synaptic densities of cholinergic, noradrenergic, and serotonergic projections in specific brain regions of apoE-deficient mice are markedly lower than those of controls. Furthermore, the extent of presynaptic derangement within each of these tracts was found to be more pronounced the further away the nerve terminal is from its cell body. In contrast, the nerve terminal density of the dopaminergic neurons that project from the substantia nigra to the striatum was unaffected and was similar to that of the controls. The rank order of these presynaptic derangements at comparable distances from the respective cell bodies was found to be septohippocampal cholinergic > nucleus basalis cholinergic > locus coeruleus adrenergic > raphe serotonergic ? nigrostriatal dopaminergic, which interestingly is similar to that observed in Alzheimer's disease. These results suggest that two complementary factors determine the susceptibility of brain projecting neurons to apoE deficiency: pathway-specific differences and the distance of the nerve terminals from their cell body.     

Neuronal expression of mutations affecting primarily glia in the mouse: studies in the dysmyelinated and convulsive mutant quaking

1. Various aspects of the noradrenergic system in the brain of the dysmyelinating convulsive mutant mice quaking have been examined. 2. Determination of the endogenous contents of noradrenaline and its metabolite 3-methoxy 4-hydroxyphenyl-ethyleneglycol (MOPEG), as well as measurement of the electrically-evoked release of (3H)-noradrenaline shows an increased noradrenergic activity in the brain of the mutants, when compared to non convulsive controls of the same strain. 3. Ontogenic development of alpha adrenergic receptors indicate that an increased density of alpha-2 sites precedes the appearance of the first convulsions by approximately one week. 4. Anatomical determination of the number of noradrenergic neuronal cell bodies in the locus coeruleus shows a hyperplasia of this nucleus in the mutants. 5. Electrolytic coagulation of the locus coeruleus inhibits the convulsions of the quaking mice. 6. These results suggest that an alteration of the embryonic differentiation of the locus coeruleus, which gives rise to the majority of brain noradrenergic neurons, provokes a hyperactivity of this neuronal system, thereby triggering the convulsions of the quaking mutant mice. 7. The possible involvement of other neurotransmitter systems in the convulsions of these mutants, together with the nature of the relationship between neuronal abnormalities and dysmyelination phenomenon, are discussed.     

Diurnal levels of Fos immunoreactivity are elevated within hypocretin neurons in lactating mice

The hypocretins modulate arousal via actions across multiple terminal fields. Thus, alterations in hypocretin neurotransmission may contribute to altered sleep patterns observed during lactation. This study examined whether lactation is associated with alterations in the number of hypocretin neurons and in diurnal Fos-immunoreactivity within hypocretin neurons in female mice. Alterations in Fos-immunoreactivity were also examined within two hypocretin terminal regions; the medial preoptic area and the locus coeruleus. Fos-immunoreactivity was increased within hypocretin neurons and the medial preoptic area in lactating females. No differences were observed in the number of hypocretin neurons or in Fos-immunoreactivity within the locus coeruleus.     

Mutant ubiquitin found in Alzheimer's disease causes neuritic beading of mitochondria in association with neuronal degeneration

A dinucleotide deletion in human ubiquitin (Ub) B messenger RNA leads to formation of polyubiquitin (UbB)+1, which has been implicated in neuronal cell death in Alzheimer's and other neurodegenerative diseases. Previous studies demonstrate that UbB+1 protein causes proteasome dysfunction. However, the molecular mechanism of UbB+1-mediated neuronal degeneration remains unknown. We now report that UbB+1 causes neuritic beading, impairment of mitochondrial movements, mitochondrial stress and neuronal degeneration in primary neurons. Transfection of UbB+1 induced a buildup of mitochondria in neurites and dysregulation of mitochondrial motor proteins, in particular, through detachment of P74, the dynein intermediate chain, from mitochondria and decreased mitochondria-microtubule interactions. Altered distribution of mitochondria was associated with activation of both the mitochondrial stress and p53 cell death pathways. These results support the hypothesis that neuritic clogging of mitochondria by UbB+1 triggers a cascade of events characterized by local activation of mitochondrial stress followed by global cell death. Furthermore, UbB+1 small interfering RNA efficiently blocked expression of UbB+1 protein, attenuated neuritic beading and preserved cellular morphology, suggesting a potential neuroprotective strategy for certain neurodegenerative disorders.     

Transgenic mice engineered to target Cre/loxP-mediated DNA recombination into catecholaminergic neurons

To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We crossbred TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of "floxed" genes in catecholaminergic neurons.     

Neuronal Activation in the Central Nervous System of Rats in the Initial Stage of Chronic Kidney Disease-Modulatory Effects of Losartan and Moxonidine

The effect of mild chronic renal failure (CRF) induced by 4/6-nephrectomy (4/6NX) on central neuronal activations was investigated by c-Fos immunohistochemistry staining and compared to sham-operated rats. In the 4/6 NX rats also the effect of the angiotensin receptor blocker, losartan, and the central sympatholyticum moxonidine was studied for two months. In serial brain sections Fos-immunoreactive neurons were localized and classified semiquantitatively. In 37 brain areas/nuclei several neurons with different functional properties were strongly affected in 4/6NX. It elicited a moderate to high Fos-activity in areas responsible for the monoaminergic innervation of the cerebral cortex, the limbic system, the thalamus and hypothalamus (e.g. noradrenergic neurons of the locus coeruleus, serotonergic neurons in dorsal raphe, histaminergic neurons in the tuberomamillary nucleus). Other monoaminergic cell groups (A5 noradrenaline, C1 adrenaline, medullary raphe serotonin neurons) and neurons in the hypothalamic paraventricular nucleus (innervating the sympathetic preganglionic neurons and affecting the peripheral sympathetic outflow) did not show Fos-activity. Stress- and pain-sensitive cortical/subcortical areas, neurons in the limbic system, the hypothalamus and the circumventricular organs were also affected by 4/6NX. Administration of losartan and more strongly moxonidine modulated most effects and particularly inhibited Fos-activity in locus coeruleus neurons. In conclusion, 4/6NX elicits high activity in central sympathetic, stress- and pain-related brain areas as well as in the limbic system, which can be ameliorated by losartan and particularly by moxonidine. These changes indicate a high sensitivity of CNS in initial stages of CKD which could be causative in clinical disturbances.     

Increased muscle ubiquitin mRNA levels in gastric cancer patients

The intramuscular ATP-dependent ubiquitin (Ub)-proteasome proteolytic system is hyperactivated in experimental cancer cachexia. The present study aimed at verifying whether the expression of the muscle Ub mRNA is altered in patients with cancer. Total muscle RNA was extracted using the guanidinium isothiocyanate/phenol/chloroform method from rectus abdominis biopsies obtained intraoperatively from 20 gastric cancer (GC) patients and 10 subjects with benign abdominal diseases (CON) undergoing surgery. Ub mRNA levels were measured by northern blot analysis. Serum soluble tumor necrosis factor receptor (sTNFR) was measured by ELISA. Ub mRNA levels (arbitrary units, means +/- SD) were 2,345 +/- 195 in GC and 1,162 +/- 132 in CON (P = 0.0005). Ub mRNA levels directly correlated with disease stage (r = 0.608, P = 0.005), being 1,945 +/- 786 in stages I and II, 2,480 +/- 650 in stage III, and 3,799 +/- 66 in stage IV. Ub mRNA and sTNFR did not correlate with age and nutritional parameters. This study confirms experimental data indicating an overexpression of muscle Ub mRNA in cancer cachexia. Lack of correlation with nutritional status suggests that Ub activation in human cancer is an early feature that precedes any clinical sign of cachexia.