Distribution of unipolar brush cells and other calretinin immunoreactive components in the mammalian cerebellar cortex

We have compared the distribution of unipolar brush cells (UBCs) in the cerebellum of Brazilian opossum (Monodelphis domestica), mouse, guinea pig, rabbit, cat, and Rhesus monkey, using an antiserum to calretinin which is present in those cells. The morphology and calretinin staining intensity of the UBCs remains constant across species. As a general trend, in all species studied, UBCs are particularly enriched in the vestibulocerebellum. Interspecies differences, however, were noted in the distribution of UBCs across other regions of the cerebellar cortex. A major variation involves the extent of the UBC-rich region of the ventral portion of the paraflocculus. The distribution of UBCs in non-vestibular vermal folia also varies substantially. UBCs are deployed in more or less distinct parasagittal zones in the vermis of the opossum, rabbit, cat, and macaque. The density of UBCs decreases progressively from medial to lateral portions of the same folium and is lowest in the lateral, posterior portions of the cerebellar hemispheres (crus II) and in the dorsal portion of the paraflocculus. In cat and macaque, the decrease in the density of UBCs across the intermediate cortex is more gradual than in the other species. The data indicate that the UBCs play a more prominent role in the modulation of sensorimotor transformations in carnivores and primates than in smaller mammals and should not be considered a vestigial form of neuron. In addition to the UBCs, calretinin antibody distinctly stains the following neurons in different species: granule cells and parallel fibers in all species except rabbit and cat; Golgi cells, especially in rat and macaque; Lugaro-like cells, especially in mouse, rat, and macaque; basket cells in macaque; subsets of mossy fibers in all species; and subsets of climbing fibers in all species but guinea pig. Usually, the distribution of UBCs is related to that of calretinin stained granule cells and mossy fibers.     

Tinnitus,Unipolar Brush Cells,and Cerebellar Glutamatergic Function in an Animal Model

Unipolar brush cells (UBCs) are excitatory interneurons found in the dorsal cochlear nucleus (DCN) and the granule cell layer of cerebellar cortex, being particularly evident in the paraflocculus (PFL) and flocculus (FL). UBCs receive glutamatergic inputs and make glutamatergic synapses with granule cells and other UBCs. It has been hypothesized that UBCs comprise local networks of tunable feed-forward amplifiers. In the DCN they might also participate in feed-back amplification of signals from higher auditory centers. Recently it has been shown that UBCs, in the vestibulocerebellum and DCN of adult rats, express doublecortin (DCX), previously considered a marker of newborn and migrating neurons. In an animal model, both the DCN, and more recently the PFL, have been implicated in contributing to the sensation of acoustic-exposure-induced tinnitus. These studies support the working hypothesis that tinnitus emerges after loss of peripheral sensitivity because inhibitory processes homeostatically down regulate, and excitatory processes up regulate. Here we report the results of two sequential experiments that examine the potential role of DCN and cerebellar UBCs in tinnitus, and the contribution of glutamatergic transmission in the PFL. In Experiment 1 it was shown that adult rats with psychophysical evidence of tinnitus induced by a single unilateral high-level noise exposure, had elevated DCX in the DCN and ventral PFL. In Experiment 2 it was shown that micro-quantities of glutamatergic antagonists, delivered directly to the PFL, reversibly reduced chronically established tinnitus, while similarly applied glutamatergic agonists induced tinnitus-like behavior in non-tinnitus controls. These results are consistent with the hypothesis that UBC up regulation and enhanced glutamatergic transmission in the cerebellum contribute to the pathophysiology of tinnitus.     

Metabotropic glutamate receptors are associated with non-synaptic appendages of unipolar brush cells in rat cerebellar cortex and cochlear nuclear complex

Unipolar brush cells (UBCs) are a class of small neurons that are densely concentrated in the granular layers of the vestibulocerebellar cortex and dorsal cochlear nucleus. The UBCs form giant synapses with individual mossy fibre rosettes on the dendrioles which make up their brush formations and are provided with numerous, unusual non-synaptic appendages. In accord with the glutamatergic nature of mossy fibres, our previous post-embedding immunocytochemical studies indicated that various ionotropic glutamate receptor subunits are localized at the post-synaptic densities of the giant synapses, whereas the non-synaptic appendages are immunonegative. On the contrary, the metabotropic glutamate receptors mGluR1α and mGluR2/3 are situated at the non-synaptic appendages and are lacking at the post-synaptic densities. Other authors, however, have shown that antibodies to these metabotropic receptors stain both appendages and post-synaptic densities. In the present study, we have re-evaluated the distribution of metabotropic glutamate receptors in the UBCs of the cerebellum and the cochlear nuclear complex by light and electron microscopic pre-embedding immunocytochemistry with subtype-specific antibodies. We confirm that UBCs dendritic brushes are densely immunostained by antibody to mGluR1α particularly in the cerebellum and that antibody to mGluR2/3 labels at least a percentage of the UBC brushes in both the cerebellum and cochlear nuclei. At the ultrastructural level, it appears that mGluR1α and mGluR2/3 immunoreactivities are not associated with the post-synaptic densities of the giant mossy fibre–UBC synapses, but instead are concentrated on the non-synaptic appendages of the cerebellar UBCs. The non-synaptic appendages, therefore, may be an important avenue for regulating the excitability of UBCs and mediating glutamate effects on their still unknown intracellular signal transduction cascades. We also show that the pre-synaptic densities of UBC dendrodendritic junctions are mGluR2/3 positive. As previously demonstrated, antibodies to mGluR1 α and mGluR2/3 label subsets of Golgi cells. Antibody to mGluR5 does not stain UBCs in the cerebellum and cochlear nucleus and reveals the somatodendritic compartment of Golgi cells situated in the core of the cerebellar granular layer, whilst cochlear nucleus Golgi cells are mGluR5 negative.     

Selective ubiquitylation of p21 and Cdt1 by UBCH8 and UBE2G ubiquitin-conjugating enzymes via the CRL4Cdt2 ubiquitin ligase complex

CRL4(Cdt2) is a cullin-based E3 ubiquitin ligase that promotes the ubiquitin-dependent proteolysis of various substrates implicated in the control of cell cycle and various DNA metabolic processes such as DNA replication and repair. Substrates for CRL4(Cdt2) E3 ubiquitin ligase include the replication licensing factor Cdt1 and the cyclin-dependent kinase (Cdk) inhibitor p21. Inhibition of this E3 ligase leads to serious abnormalities of the cell cycle and cell death. The ubiquitin-conjugating enzyme (UBC) involved in this important pathway, however, remains unknown. By a proteomic analysis of Cdt2-associated proteins and an RNA interference-based screening approach, we show that CRL4(Cdt2) utilizes two different UBCs to target different substrates. UBCH8, a member of the UBE2E family of UBCs, ubiquitylates and promotes the degradation of p21, both during the normal cell cycle and in UV-irradiated cells. Importantly, depletion of UBCH8 by small interfering RNA (siRNA) increases p21 protein level, delays entry into S phase of the cell cycle, and suppresses the DNA damage response after UV irradiation. On the other hand, members of the UBE2G family of UBCs (UBE2G1 and UBE2G2) cooperate with CRL4(Cdt2) to polyubiquitylate and degrade Cdt1 postradiation, an activity that is critical for preventing origin licensing in DNA-damaged cells. Finally, we show that UBCH8, but not UBE2G1 or UBE2G2, is required for CRL4(Cdt2)-mediated ubiquitylation and degradation of the histone H4 lysine 20 monomethyltransferase Set8, a previously identified CRL4(Cdt2) substrate, as well as for CRL4(Cdt2)-dependent monoubiquitylation of PCNA in unstressed cells. These findings identify the UBCs required for the activity of CRL4(Cdt2) on multiple substrates and demonstrate that different UBCs are involved in the selective ubiquitylation of different substrates by the same E3 complex.     

The Wnts

Background

The eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively. There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment. We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. We also present a phylogenetic analysis of UBCs.

Results

The C. elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins. RNAi shows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homolog of yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifier SUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifier Rub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless. RNAi of ubc-20, an ortholog of yeast UBC1, results in a low frequency of arrested larval development. A phylogenetic analysis of C. elegans, Drosophila and human UBCs shows that this protein family can be divided into 18 groups, 13 of which include members from all three species. The activating enzymes and the ubiquitin-like proteins NED-8 and SUMO are required for embryogenesis.

Conclusions

The number of UBC genes appears to increase with developmental complexity, and our results suggest functional overlap in many of these enzymes. The ubiquitin-like proteins NED-8 and SUMO and their corresponding activating enzymes are required for embryogenesis.     

The adipokine Retnla deficiency increases responsiveness to cardiac repair through adiponectin-rich bone marrow cells

Resistin-like alpha (Retnla) is a member of the resistin family and known to modulate fibrosis and inflammation. Here, we investigated the role of Retnla in the cardiac injury model. Myocardial infarction (MI) was induced in wild type (WT), Retnla knockout (KO), and Retnla transgenic (TG) mice. Cardiac function was assessed by echocardiography and was significantly preserved in the KO mice, while worsened in the TG group. Angiogenesis was substantially increased in the KO mice, and cardiomyocyte apoptosis was markedly suppressed in the KO mice. By Retnla treatment, the expression of p21 and the ratio of Bax to Bcl2 were increased in cardiomyocytes, while decreased in cardiac fibroblasts. Interestingly, the numbers of cardiac macrophages and unsorted bone marrow cells (UBCs) were higher in the KO mice than in the WT mice. Besides, phosphorylated histone H3(+) cells were more frequent in bone marrow of KO mice. Moreover, adiponectin in UBCs was notably higher in the KO mice compared with WT mice. In an adoptive transfer study, UBCs were isolated from KO mice to transplant to the WT infarcted heart. Cardiac function was better in the KO-UBCs transplanted group in the WT-UBCs transplanted group. Taken together, proliferative and adiponectin-rich bone marrow niche was associated with substantial cardiac recovery by suppression of cardiac apoptosis and proliferation of cardiac fibroblast.Subject terms: Apoptosis, Interleukins, Acute inflammation, Heart failure     

Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis

Attachment of ubiquitin to substrate proteins is catalyzed by the three enzymes E1, E2 (ubiquitin conjugating [UBC]), and E3 (ubiquitin ligase). Forty-one functional proteins with a UBC domain and active-site cysteine are predicted in the Arabidopsis (Arabidopsis thaliana) genome, which includes four that are predicted or shown to function with ubiquitin-like proteins. Only nine were previously characterized biochemically as ubiquitin E2s. We obtained soluble protein for 22 of the 28 uncharacterized UBCs after expression in Escherichia coli and demonstrated that 16 function as ubiquitin E2s. Twelve, plus three previously characterized ubiquitin E2s, were also tested for the ability to catalyze ubiquitination in vitro in the presence of one of 65 really interesting new gene (RING) E3 ligases. UBC22, UBC19-20, and UBC1-6 had variable levels of E3-independent activity. Six UBCs were inactive with all RINGs tested. Closely related UBC8, 10, 11, and 28 were active with the largest number of RING E3s and with all RING types. Expression analysis was performed to determine whether E2s or E3s were expressed in specific organs or under specific environmental conditions. Closely related E2s show unique patterns of expression and most express ubiquitously. Some RING E3s are also ubiquitously expressed; however, others show organ-specific expression. Of all the organs tested, RING mRNAs are most abundant in floral organs. This study demonstrates that E2 diversity includes examples with broad and narrow specificity toward RINGs, and that most ubiquitin E2s are broadly expressed with each having a unique spatial and developmental pattern of expression.     

Yeast ubiquitin-conjugating enzymes involved in selective protein degradation are essential for cell viability.

Ubiquitin-mediated proteolysis is a major pathway for selective protein degradation in eukaryotic cells. This proteolysis pathway involves the processive covalent attachment of ubiquitin to proteolytic substrates and their subsequent degradation by a specific ATP-dependent protease complex. We have cloned the genes and characterized the function of ubiquitin-conjugating enzymes (UBCs) from the yeast Saccharomyces cerevisiae. UBC1, UBC4 and UBC5 enzymes were found to mediate selective degradation of short-lived and abnormal proteins. These enzymes have overlapping functions and constitute a UBC subfamily essential for growth. UBC1 is specifically required at early stages of growth after germination of spores. UBC4 and UBC5 enzymes generate high molecular weight ubiquitin-protein conjugates and comprise a major ubiquitin-conjugation activity in yeast cells. Moreover, these enzymes are central components of the cellular stress response.     

Identification of the structural and functional human homolog of the yeast ubiquitin conjugating enzyme UBC9.

Ubiquitin conjugating enzymes (UBCs) are a family of proteins directly involved in ubiquitination of proteins. Ubiquitination is known to be involved in control of a variety of cellular processes, including cell proliferation, through the targeting of key regulatory proteins for degradation. The ubc9 gene of the yeast Saccharomyces cerevisiae (Scubc9) is an essential gene which is required for cell cycle progression and is involved in the degradation of S phase and M phase cyclins. We have identified a human homolog of Scubc9 (termed hubc9) using the two hybrid screen for proteins that interact with the human papillomavirus type 16 E1 replication protein. The hubc9 encoded protein shares a very high degree of amino acid sequence similarity with ScUBC9 and with the homologous hus5+ gene product of Schizosaccharomyces pombe. Genetic complementation experiments in a S.cerevisiae ubc9ts mutant reveal that hUBC9 can substitute for the function of ScUBC9 required for cell cycle progression.     

Functional and genetic interactions of TOR in the budding yeast Saccharomyces cerevisiae with myosin type II-deficiency (myo1Δ)

Background

Protein misfolding and subsequent aggregation are hallmarks of several human diseases. The cell has a variety of mechanisms for coping with misfolded protein stress, including ubiquitin-mediated protein degradation. In fact, the presence of ubiquitin at protein aggregates is a common feature of protein misfolding diseases. Ubiquitin conjugating enzymes (UBCs) are part of the cascade of enzymes responsible for the regulated attachment of ubiquitin to protein substrates. The specific UBC used during ubiquitination can determine the type of polyubiquitin chain linkage, which in turn plays an important role in determining the fate of the ubiquitinated protein. Thus, UBCs may serve an important role in the cellular response to misfolded proteins and the fate of protein aggregates.

Results

The Q82 strain of C. elegans harbors a transgene encoding an aggregation prone tract of 82 glutamine residues fused to green fluorescent protein (Q82::GFP) that is expressed in the body wall muscle. When measured with time-lapse microscopy in young larvae, the initial formation of individual Q82::GFP aggregates occurs in approximately 58 minutes. This process is largely unaffected by a mutation in the C. elegans E1 ubiquitin activating enzyme. RNAi of ubc-22, a nematode homolog of E2-25K, resulted in higher pre-aggregation levels of Q82::GFP and a faster initial aggregation rate relative to control. Knockdown of ubc-1 (RAD6 homolog), ubc-13, and uev-1 did not affect the kinetics of initial aggregation. However, RNAi of ubc-13 decreases the rate of secondary growth of the aggregate. This result is consistent with previous findings that aggregates in young adult worms are smaller after ubc-13 RNAi. mCherry::ubiquitin becomes localized to Q82::GFP aggregates during the fourth larval (L4) stage of life, a time point long after most aggregates have formed. FLIP and FRAP analysis indicate that mCherry::ubiquitin is considerably more mobile than Q82::GFP within aggregates.

Conclusions

These data indicate that initial formation of Q82::GFP aggregates in C. elegans is not directly dependent on ubiquitination, but is more likely a spontaneous process driven by biophysical properties in the cytosol such as the concentration of the aggregating species. The effect of ubiquitination appears to be most significant in later, secondary aggregate growth.     

Characterization of the Ubiquitin-Conjugating Enzyme Gene Family in Rice and Evaluation of Expression Profiles under Abiotic Stresses and Hormone Treatments

Ubiquitin-conjugating enzyme E2s (UBCs), which catalyze the transfer of ubiquitin to substrate or E3 ligases, are key enzymes in ubiquitination modifications of target proteins. However, little is known about the knowledge of UBC gene family in rice. In this study, a total of 39 UBC encoding genes, which all contained an UBC domain with a cysteine active site, were identified in the rice genome. These were classified into fifteen distinct subfamilies based upon their sequence similarity and phylogenetic relationships. A subset of 19 OsUBC genes exhibited chromosomal duplication; 4 and 15 OsUBC genes were tandemly and segmentally duplicated, respectively. Comprehensive analyses were performed to investigate the expression profiles of OsUBC genes in various stages of vegetative and reproductive development using data from EST, Microarrays, MPSS, and real-time PCR. Many OsUBC genes exhibited abundant and tissue-specific expression patterns. Moreover, 14 OsUBCs were found to be differentially expressed under treatments with drought, or salt stresses. The expression analysis after treatments with IAA, 6-BA, GA and ABA indicated that almost all OsUBC genes were responsive to at least two of the four hormones. Several genes were significantly down-regulated under all of the hormone treatments, and most of the genes reduced by 6-BA were also reduced by GA. This study will facilitate further studies of the OsUBC gene family and provide useful clues for functional validation of OsUBCs in rice.     

Ubiquitination of the glycosomal matrix protein receptor PEX5 in Trypanosoma brucei by PEX4 displays novel features

Trypanosomatids contain peroxisome-like organelles called glycosomes. Peroxisomal biogenesis involves a cytosolic receptor, PEX5, which, after its insertion into the organellar membrane, delivers proteins to the matrix. In yeasts and mammalian cells, transient PEX5 monoubiquitination at the membrane serves as the signal for its retrieval from the organelle for re-use. When its recycling is impaired, PEX5 is polyubiquitinated for proteasomal degradation. Stably monoubiquitinated TbPEX5 was detected in cytosolic fractions of Trypanosoma brucei, indicative for its role as physiological intermediate in receptor recycling. This modification's resistance to dithiothreitol suggests ubiquitin conjugation of a lysine residue. T. brucei PEX4, the functional homologue of the ubiquitin-conjugating (UBC) enzyme responsible for PEX5 monoubiquitination in yeast, was identified. It is associated with the cytosolic face of the glycosomal membrane, probably anchored by an identified putative TbPEX22. The involvement of TbPEX4 in TbPEX5 ubiquitination was demonstrated using procyclic ?PEX4 trypanosomes. Surprisingly, glycosomal matrix protein import was only mildly affected in this mutant. Since other UBC homologues were upregulated, it might be possible that these have partially rescued PEX4's function in PEX5 ubiquitination. In addition, the altered expression of UBCs, notably of candidates involved in cell-cycle control, could be responsible for observed morphological and motility defects of the ?PEX4 mutant.     

Members of two gene families encoding ubiquitin-conjugating enzymes, AtUBC1-3 and AtUBC4-6, fromArabidopsis thaliana are differentially expressed

Covalent attachment of ubiquitin to other intracellular proteins is essential for many physiological processes in eukaryotes, including selective protein degradation. Selection of proteins for ubiquitin conjugation is accomplished, in part, by a group of enzymes designated E2s or ubiquitin-conjugating enzymes (UBCs). At least six types of E2s have been identified in the plantArabidopsis thaliana; each type is encoded by a small gene family. Previously, we described the isolation and characterization of two three-member gene families, designatedAtUBC1-3 andAtUBC4-6, encoding two of these E2 types. Here, we investigated the expression patterns, of theAtUBC1-3 andAtUBC4-6 genes by the histochemical analysis of transgenicArabidopsis containing the corresponding promoters fused to the -glucuronidase-coding region. Staining patterns showed that these genes are active in many stages of development and some aspects of cell death, but are not induced by heat stress. Within the two gene families, individual members exhibited both overlapping and complementary expression patterns, indicating that at least one member of each gene family is expressed in most cell types and at most developmental stages. Different composite patterns of expression were observed between theAtUBC1-3 andAtUBC4-6 families, suggesting distinct biochemical and/or physiological functions for the encoded E2s inArabidopsis.