15 research outputs found
Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import
Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport
Human Papillomavirus 16 E5 Induces Bi-Nucleated Cell Formation By Cell-Cell Fusion
Human Papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes – E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, co-expression of HPV16 E6/E7 enhances the proliferation of these cells. Expression of HPV16 E6/E7 also increases bi-nucleated cell colony formation. These findings identify a new role for HPV16 E5 and support a model in which complementary roles of the HPV16 oncogenes lead to the induction of carcinogenesis
Regulation of Nrf2 by X Box-Binding Protein 1 in Retinal Pigment Epithelium
Normal function of the retinal pigment epithelium (RPE) is essential for maintaining the structural integrity of retinal photoreceptors and the visual process. Sustained oxidative damage of the RPE due to aging and other risk factors contributes to the development of age-related macular degeneration (AMD). The transcription factor NF-E2-related factor 2 (Nrf2) is a central regulator of cellular antioxidant and detoxification responses. Enhancing Nrf2 function protects RPE cells from oxidation-related apoptosis and cell death. Previously, we demonstrated that Nrf2 activation can be induced by endoplasmic reticulum (ER) stress; however, the mechanisms are not fully understood. In the present study, we examined the role of X box-binding protein 1 (XBP1), an ER stress-inducible transcription factor, in regulation of Nrf2 in the RPE. We found that RPE-specific XBP1 conditional knockout (cKO) mice exhibit a significant reduction in Nrf2 mRNA and protein levels, along with decreased expression of major Nrf2 target genes, in the RPE/choroid complex. Using primary RPE cells isolated from XBP1 cKO mice and human ARPE-19 cell line, we confirmed that loss of XBP1 gene or pharmacological inhibition of XBP1 splicing drastically reduces Nrf2 levels in the RPE. Conversely, overexpression of spliced XBP1 results in a modest but significant increase in cytosolic and nuclear Nrf2 protein levels without affecting the transcription of Nrf2 gene. Moreover, induction of ER stress by tunicamycin and thapsigargin markedly increases Nrf2 expression, which is abolished in cells pretreated with XBP1 splicing inhibitors 4ÎĽ8C and quinotrierixin. Mechanistic studies indicate that quinotrierixin reduces Nrf2 expression likely through inhibition of protein translation. Finally, we demonstrate that overexpression of Nrf2 protected RPE cells against oxidative injury but appeared to be insufficient to rescue from XBP1 deficiency-induced cell death. Taken together, our results indicate that XBP1 modulates Nrf2 activity in RPE cells and that XBP1 deficiency contributes to oxidative injury of the RPE
Loss of the ubiquitin conjugating enzyme UBE2E3 induces cellular senescence
Cellular senescence plays essential roles in tissue homeostasis as well as a host of diseases ranging from cancers to age-related neurodegeneration. Various molecular pathways can induce senescence and these different pathways dictate the phenotypic and metabolic changes that accompany the transition to, and maintenance of, the senescence state. Here, we describe a novel senescence phenotype induced by depletion of UBE2E3, a highly-conserved, metazoan ubiquitin conjugating enzyme. Cells depleted of UBE2E3 become senescent in the absence of overt DNA damage and have a distinct senescence-associated secretory phenotype, increased mitochondrial and lysosomal mass, an increased sensitivity to mitochondrial and lysosomal poisons, and an increased basal autophagic flux. This senescence phenotype can be partially suppressed by co-depletion of either p53 or its cognate target gene, p21CIP1/WAF1, or by co-depleting the tumor suppressor p16INK4a. Together, these data describe a direct link of a ubiquitin conjugating enzyme to cellular senescence and further underscore the consequences of disrupting the integration between the ubiquitin proteolysis system and the autophagy machinery. Keywords: UBE2E3, Senescence, Autophagy, Ubiquitin, Mitochondri
The Ubiquitin-Conjugating Enzyme, UbcM2, Is Restricted to Monoubiquitylation by a Two-Fold Mechanism That Involves Backside Residues of E2 and Lys48 of Ubiquitin
Proteins
can be modified on lysines (K) with a single ubiquitin
(Ub) or with polymers of Ub (polyUb). These different configurations
and their respective topologies are primary factors for determining
whether substrates are targeted to the proteasome for degradation
or directed to nonproteolytic outcomes. We report here on the intrinsic
ubiquitylation properties
of UbcM2 (UBE2E3/UbcH9), a conserved Ub-conjugating enzyme linked
to cell proliferation, development, and the cellular antioxidant defense
system. Using a fully recombinant ubiquitylation assay,
we show that UbcM2 is severely limited in its ability to synthesize
polyUb chains with wild-type Ub. Restriction to monoubiquitylation
is governed by multiple residues on the backside of the enzyme, far
removed from its active site, and by lysine 48 of Ub. UbcM2 with mutated
backside residues can synthesize K63-linked polyUb chains and to a
lesser extent K6- and K48-linked chains. Additionally, we identified
a single residue on the backside of the enzyme that promotes monoubiquitylation.
Together, these findings reveal that a combination of noncatalytic
residues within the Ubc catalytic core domain of UbcM2 as well as
a lysine(s) within Ub can relegate a Ub-conjugating enzyme to monoubiquitylate
its cognate targets despite having the latent capacity to construct
polyUb chains. The two-fold mechanism for restricting activity to
monoubiquitylation provides
added insurance that UbcM2 will not build polyUb chains on its substrates,
even under conditions of high local Ub concentrations
The Ubiquitin-Conjugating Enzyme, UbcM2, Is Restricted to Monoubiquitylation by a Two-Fold Mechanism That Involves Backside Residues of E2 and Lys48 of Ubiquitin
Proteins
can be modified on lysines (K) with a single ubiquitin
(Ub) or with polymers of Ub (polyUb). These different configurations
and their respective topologies are primary factors for determining
whether substrates are targeted to the proteasome for degradation
or directed to nonproteolytic outcomes. We report here on the intrinsic
ubiquitylation properties
of UbcM2 (UBE2E3/UbcH9), a conserved Ub-conjugating enzyme linked
to cell proliferation, development, and the cellular antioxidant defense
system. Using a fully recombinant ubiquitylation assay,
we show that UbcM2 is severely limited in its ability to synthesize
polyUb chains with wild-type Ub. Restriction to monoubiquitylation
is governed by multiple residues on the backside of the enzyme, far
removed from its active site, and by lysine 48 of Ub. UbcM2 with mutated
backside residues can synthesize K63-linked polyUb chains and to a
lesser extent K6- and K48-linked chains. Additionally, we identified
a single residue on the backside of the enzyme that promotes monoubiquitylation.
Together, these findings reveal that a combination of noncatalytic
residues within the Ubc catalytic core domain of UbcM2 as well as
a lysine(s) within Ub can relegate a Ub-conjugating enzyme to monoubiquitylate
its cognate targets despite having the latent capacity to construct
polyUb chains. The two-fold mechanism for restricting activity to
monoubiquitylation provides
added insurance that UbcM2 will not build polyUb chains on its substrates,
even under conditions of high local Ub concentrations