Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway

Substrates of a ubiquitin-dependent proteolytic system called the N-end rule pathway include proteins with destabilizing N-terminal residues. N-recognins, the pathway’s ubiquitin ligases, contain three substrate-binding sites. The type-1 site is specific for basic N-terminal residues (Arg, Lys, His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, Ile). We show here that the type-1/2 sites of UBR1, the sole N-recognin of the yeast Saccharomyces cerevisiae, are located in the first ~700 residues of the 1,950-residue UBR1. These sites are distinct in that they can be selectively inactivated by mutations, identified through a genetic screen. Mutations inactivating the type-1 site are in the previously delineated ~70 residue UBR motif characteristic of N-recognins. Fluorescence polarization and surface plasmon resonance were used to determine that UBR1 binds, with Kd of ~1 microM, to either type-1 or type-2 destabilizing N-terminal residues of reporter peptides, but does not bind to a stabilizing N-terminal residue such as Gly. A third substrate-binding site of UBR1 targets an internal degron of CUP9, a transcriptional repressor of peptide import. We show that the previously demonstrated in vivo dependence of CUP9 ubiquitylation on the binding of (cognate) dipeptides to the type-1/2 sites of UBR1 can be reconstituted in a completely defined in vitro system. We also found that purified UBR1 and CUP9 interact nonspecifically, and that specific binding (which involves, in particular, the binding by cognate dipeptides to the UBR1’s type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding

Biochemical and Genetic Studies of UBR3, a Ubiquitin Ligase with a Function in Olfactory and Other Sensory Systems

Our previous work identified E3 ubiquitin ligases, termed UBR1-UBR7, that contain the ~70-residue UBR box, a motif important for the targeting of N-end rule substrates. In this pathway, specific N-terminal residues of substrates are recognized as degradation signals by UBR box-containing E3s that include UBR1, UBR2, UBR4, and UBR5. The other E3s of this set, UBR3, UBR6, and UBR7, remained uncharacterized. Here we describe the cloning and analyses of mouse UBR3. The similarities of UBR3 to the UBR1 and UBR2 E3s of the N-end rule pathway include the RING and UBR domains. We show that HR6A and HR6B, the E2 enzymes that bind to UBR1 and UBR2, also interact with UBR3. However, in contrast to UBR1 and UBR2, UBR3 does not recognize N-end rule substrates. We also constructed UBR3-lacking mouse strains. In the 129SvImJ background, UBR3-/- mice died during embryogenesis, whereas the C57BL/6 background UBR3-/- mice exhibited neonatal lethality and suckling impairment that could be partially rescued by litter size reduction. The adult UBR3-/- mice had female-specific behavioral anosmia. Cells of the olfactory pathway were found to express beta-galactosidase (LacZ) that marked the deletion/disruption UBR3- allele. The UBR3-specific LacZ expression was also prominent in cells of the touch, vision, hearing, and taste systems, suggesting a regulatory role of UBR3 in sensory pathways, including olfaction. By analogy with functions of the UBR domain in the N-end rule pathway, we propose that the UBR box of UBR3 may recognize small compounds that modulate the targeting, by this E3, of its currently unknown substrates

Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China.

An in-depth annotation of the newly discovered coronavirus (2019-nCoV) genome has revealed differences between 2019-nCoV and severe acute respiratory syndrome (SARS) or SARS-like coronaviruses. A systematic comparison identified 380 amino acid substitutions between these coronaviruses, which may have caused functional and pathogenic divergence of 2019-nCoV

All-Trans Retinoic Acid Promotes TGF-β-Induced Tregs via Histone Modification but Not DNA Demethylation on Foxp3 Gene Locus

It has been documented all-trans retinoic acid (atRA) promotes the development of TGF-β-induced CD4(+)Foxp3(+) regulatory T cells (iTreg) that play a vital role in the prevention of autoimmune responses, however, molecular mechanisms involved remain elusive. Our objective, therefore, was to determine how atRA promotes the differentiation of iTregs.Addition of atRA to naïve CD4(+)CD25(-) cells stimulated with anti-CD3/CD28 antibodies in the presence of TGF-β not only increased Foxp3(+) iTreg differentiation, but maintained Foxp3 expression through apoptosis inhibition. atRA/TGF-β-treated CD4(+) cells developed complete anergy and displayed increased suppressive activity. Infusion of atRA/TGF-β-treated CD4(+) cells resulted in the greater effects on suppressing symptoms and protecting the survival of chronic GVHD mice with typical lupus-like syndromes than did CD4(+) cells treated with TGF-β alone. atRA did not significantly affect the phosphorylation levels of Smad2/3 and still promoted iTreg differentiation in CD4(+) cells isolated from Smad3 KO and Smad2 conditional KO mice. Conversely, atRA markedly increased ERK1/2 activation, and blockade of ERK1/2 signaling completely abolished the enhanced effects of atRA on Foxp3 expression. Moreover, atRA significantly increased histone methylation and acetylation within the promoter and conserved non-coding DNA sequence (CNS) elements at the Foxp3 gene locus and the recruitment of phosphor-RNA polymerase II, while DNA methylation in the CNS3 was not significantly altered.We have identified the cellular and molecular mechanism(s) by which atRA promotes the development and maintenance of iTregs. These results will help to enhance the quantity and quality of development of iTregs and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation

BAFF Promotes Th17 Cells and Aggravates Experimental Autoimmune Encephalomyelitis

BAFF, in addition to promoting B cell survival and differentiation, may affect T cells. The objective of this study was to determine the effect of BAFF on Th17 cell generation and its ramifications for the Th17 cell-driven disease, EAE.Th17 cells were increased in BAFF-Tg B6 (B6.BTg) mice and decreased in B6.Baff(-/-) mice. Th17 cells in B6.Baff(-/-) mice bearing a BAFF Tg (B6.Baff(-/-).BTg mice) were identical to those in B6.BTg mice, indicating that membrane BAFF is dispensable for Th17 cell generation as long as soluble BAFF is plentiful. In T + non-T cell criss-cross co-cultures, Th17 cell generation was greatest in cultures containing B6.BTg T cells and lowest in cultures containing B6.Baff(-/-) T cells, regardless of the source of non-T cells. In cultures containing only T cells, Th17 cell generation followed an identical pattern. CD4(+) cell expression of CD126 (IL-6R α chain) was increased in B6.BTg mice and decreased in B6.Baff(-/-) mice, and activation of STAT3 following stimulation with IL-6 + TGF-β was also greatest in B6.BTg cells and lowest in B6.Baff(-/-) cells. EAE was clinically and pathologically most severe in B6.BTg mice and least severe in B6.Baff(-/-) mice and correlated with MOG(35-55) peptide-induced Th17 cell responses.Collectively, these findings document a contribution of BAFF to pathogenic Th17 cell responses and suggest that BAFF antagonism may be efficacious in Th17 cell-driven diseases

Construction and Analysis of Mouse Strains Lacking the Ubiquitin Ligase UBR1 (E3α) of the N-End Rule Pathway

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, the UBR1-encoded ubiquitin ligase (E3) of the N-end rule pathway mediates the targeting of substrate proteins in part through binding to their destabilizing N-terminal residues. The functions of the yeast N-end rule pathway include fidelity of chromosome segregation and the regulation of peptide import. Our previous work described the cloning of cDNA and a gene encoding the 200-kDa mouse UBR1 (E3α). Here we show that mouse UBR1, in the presence of a cognate mouse ubiquitin-conjugating (E2) enzyme, can rescue the N-end rule pathway in ubr1Δ S. cerevisiae. We also constructed UBR1(−/−) mouse strains that lacked the UBR1 protein. UBR1(−/−) mice were viable and fertile but weighed significantly less than congenic +/+ mice. The decreased mass of UBR1(−/−) mice stemmed at least in part from smaller amounts of the skeletal muscle and adipose tissues. The skeletal muscle of UBR1(−/−) mice apparently lacked the N-end rule pathway and exhibited abnormal regulation of fatty acid synthase upon starvation. By contrast, and despite the absence of the UBR1 protein, UBR1(−/−) fibroblasts contained the N-end rule pathway. Thus, UBR1(−/−) mice are mosaics in regard to the activity of this pathway, owing to differential expression of proteins that can substitute for the ubiquitin ligase UBR1 (E3α). We consider these UBR1-like proteins and discuss the functions of the mammalian N-end rule pathway

Female Lethality and Apoptosis of Spermatocytes in Mice Lacking the UBR2 Ubiquitin Ligase of the N-End Rule Pathway

Substrates of the ubiquitin-dependent N-end rule pathway include proteins with destabilizing N-terminal residues. UBR1(−/−) mice, which lacked the pathway's ubiquitin ligase E3α, were viable and retained the N-end rule pathway. The present work describes the identification and analysis of mouse UBR2, a homolog of UBR1. We demonstrate that the substrate-binding properties of UBR2 are highly similar to those of UBR1, identifying UBR2 as the second E3 of the mammalian N-end rule pathway. UBR2(−/−) mouse strains were constructed, and their viability was found to be dependent on both gender and genetic background. In the strain 129 (inbred) background, the UBR2(−/−) genotype was lethal to most embryos of either gender. In the 129/B6 (mixed) background, most UBR2(−/−) females died as embryos, whereas UBR2(−/−) males were viable but infertile, owing to the postnatal degeneration of the testes. The gross architecture of UBR2(−/−) testes was normal and spermatogonia were intact as well, but UBR2(−/−) spermatocytes were arrested between leptotene/zygotene and pachytene and died through apoptosis. A conspicuous defect of UBR2(−/−) spermatocytes was the absence of intact synaptonemal complexes. We conclude that the UBR2 ubiquitin ligase and, hence, the N-end rule pathway are required for male meiosis and spermatogenesis and for an essential aspect of female embryonic development

Interleukin-1 as an injury signal mobilizes retinyl esters in hepatic stellate cells through down regulation of lecithin retinol acyltransferase.

Retinoids are mostly stored as retinyl esters in hepatic stellate cells (HSCs) through esterification of retinol and fatty acid, catalyzed by lecithin-retinol acyltransferase (LRAT). This study is designated to address how retinyl esters are mobilized in liver injury for tissue repair and wound healing. Initially, we speculated that acute inflammatory cytokines may act as injury signal to mobilize retinyl esters by down-regulation of LRAT in HSCs. By examining a panel of cytokines we found interleukin-1 (IL-1) can potently down-regulate mRNA and protein levels of LRAT, resulting in mobilization of retinyl esters in primary rat HSCs. To simulate the microenvironment in the space of Disse, HSCs were embedded in three-dimensional extracellular matrix, by which HSCs retaine quiescent phenotypes, indicated by up-regulation of LRAT and accumulation of lipid droplets. Upon IL-1 stimulation, LRAT expression went down together with mobilization of lipid droplets. Secreted factors from Kupffer cells were able to suppress LRAT expression in HSCs, which was neutralized by IL-1 receptor antagonist. To explore the underlying mechanism we noted that the stability of LRAT protein is not significantly regulated by IL-1, indicating the regulation is likely at transcriptional level. Indeed, we found that IL-1 failed to down-regulate recombinant LRAT protein expressed in HSCs by adenovirus, while transcription of endogenous LRAT was promptly decreased. Following liver damage, IL-1 was promptly elevated in a close pace with down-regulation of LRAT transcription, implying their causative relationship. After administration of IL-1, retinyl ester levels in the liver, as measured by LC/MS/MS, decreased in association with down-regulation of LRAT. Likewise, IL-1 receptor knockout mice were protected from injury-induced down-regulation of LRAT. In summary, we identified IL-1 as an injury signal to mobilize retinyl ester in HSCs through down-regulation of LRAT, implying a mechanism governing transition from hepatic injury to wound healing

Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China.

An in-depth annotation of the newly discovered coronavirus (2019-nCoV) genome has revealed differences between 2019-nCoV and severe acute respiratory syndrome (SARS) or SARS-like coronaviruses. A systematic comparison identified 380 amino acid substitutions between these coronaviruses, which may have caused functional and pathogenic divergence of 2019-nCoV