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. 2011 Apr 6;6(4):e18517.
doi: 10.1371/journal.pone.0018517.

Ubiquitin fold modifier 1 (UFM1) and its target UFBP1 protect pancreatic beta cells from ER stress-induced apoptosis

Katleen Lemaire  1 , Rodrigo F Moura, Mikaela Granvik, Mariana Igoillo-Esteve, Hans E Hohmeier, Nico Hendrickx, Christopher B Newgard, Etienne Waelkens, Miriam Cnop, Frans Schuit
Affiliations

Ubiquitin fold modifier 1 (UFM1) and its target UFBP1 protect pancreatic beta cells from ER stress-induced apoptosis

Katleen Lemaire et al. PLoS One. .

Abstract

UFM1 is a member of the ubiquitin like protein family. While the enzymatic cascade of UFM1 conjugation has been elucidated in recent years, the biological function remains largely unknown. In this report we demonstrate that the recently identified C20orf116, which we name UFM1-binding protein 1 containing a PCI domain (UFBP1), and CDK5RAP3 interact with UFM1. Components of the UFM1 conjugation pathway (UFM1, UFBP1, UFL1 and CDK5RAP3) are highly expressed in pancreatic islets of Langerhans and some other secretory tissues. Co-localization of UFM1 with UFBP1 in the endoplasmic reticulum (ER) depends on UFBP1. We demonstrate that ER stress, which is common in secretory cells, induces expression of Ufm1, Ufbp1 and Ufl1 in the beta-cell line INS-1E. siRNA-mediated Ufm1 or Ufbp1 knockdown enhances apoptosis upon ER stress. Silencing the E3 enzyme UFL1, results in similar outcomes, suggesting that UFM1-UFBP1 conjugation is required to prevent ER stress-induced apoptosis. Together, our data suggest that UFM1-UFBP1 participate in preventing ER stress-induced apoptosis in protein secretory cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression profile of Ufm1 in differentmouse tissues.
A Ufm1 mRNA expression in 19 different mouse tissues and MIN6 cells, measured via microarray (probe set 1449263_at), n≥3, B Ufm1 mRNA expression in islets from mice which were fasted for 20 hours or fed a normal diet, n≥3, * p = 0.02, C UFM1 protein expression in the same mouse tissues. Immunodetection was done with a UFM1 specific antibody. Both free UFM1 (←) and UFM1 conjugates (*) are shown. An equal amount of protein was loaded on gel. Both GAPDH and β-actin were used as control, since no protein is equally expressed in all tissues. Representative immunoblot is shown.
Figure 2
Figure 2. UFM1 interacts with UFBP1 and CDK5RAP3.
A GST pull down (UFM1(G) and CDK5RAP3) with in vitro T7 transcribed/translated 35S-labelled UFBP1, UFBP129–314, UFBP11–219 CDK5RAP3, UFL1 and BiP. Lane 1 shows the starting labeled proteins used for the pull down experiment (input) and a schematic overview of the used UFBP1 constructs. A GST-antibody was used for immunoblotting (WB), B co-immunoprecipitation with BiP, UFL1 and UFM1 specific antibodies, C Schematic overview of the protein interactions of UFM1 demonstrated in mouse in this manuscript, D Evolutionary conservation of UFBP1 (2600009E05Rik). Protein sequence in Mm (Musmusculus), Rn (Rattusnovergicus), Hs (Homo sapiens), Dr (Daniorerio), Gg (Gallus gallus), Tn (Tetraodonnigroviridis), Fr (Fuguribripes), Ag (Anopheles gambiae), Ce (Caenorhabditiselegans), Dm (Drosophila melanogaster) and At (Arabidopsis thaliana). Alignment was performed using ClustalW. The signal peptide of UFBP1 is boxed in grey, the nuclear localization signal is depicted in green, the PCI domain is boxed in black and lysine268 (K268) is shown in red, E Presence of UFM1 conjugates at the same height and with the same cellular localization as UFBP1 (*). MIN6 cell lysates were separated in 5 different fractions: N = nuclear and whole cell fraction (770×g), M = heavy mitochondrial fraction (2330×g), L = light mitochondrial, peroxisomal and lysosomal fraction (13,000×g), P = cell membrane fraction (100,000×g) and S = cytosolic and large protein complexes supernatant (100,000×g).
Figure 3
Figure 3. mRNA expression of Ufbp1, Ufl1 and Cdk5Rap3 in 23 different mouse tissues and MIN6 cells, measured via microarray (probe set 1434702_at, 1429008_at and 1423067_at, respectively).
UFBP1 protein expression measured in the same tissue panel. A UFBP1 specific antibody was used, and the same amount of protein was loaded as in Figure 1C.
Figure 4
Figure 4. UFBP1 and UFM1 are co-localized in the ER.
A INS1-832/13 cells transfected with different eGFP constructs as indicated on the picture. INS1-832/13 cells (B) and Hela cell (C) co-transfected with mRFP-UFM1 and UFBP1-eGFP, D Overview of INS1-832/13 cells co-transfected with mRFP-UFM1 and UFBP1-eGFP, UFBP129–314-eGFP or UFBP11–214-eGFP, as depicted. Cells were also stained with an ER-tracker (blue). Pictures were taken with a 63× objective on a Zeiss confocal microscope, E UFM1 and UFBP1 expression in different MIN6 cellular fractions, similar as in Figure 2E. Different cellular markers were used: GDH, mitochondrial marker; LAMP2, lysosomal marker; BiP, ER marker and HSPA8, cytosolic marker. F UFM1 expression after cellular fractionation of MIN6 cells overexpressing UFM1, UFBP1, both UFM1 and UFBP1 or UFM1G83A, as depicted (*, unprocessed; ∧, processed).
Figure 5
Figure 5. Ufm1, Ufbp1and Ufl1 expressionis induced upon ER stress.
A mRNA expression level of Ufm1 (A), Ufbp1(B) or Ufl1 (C) after exposure of INS-1E cells to 25 µM CPA, 1 µM thapsigargin, 1 µg/ml brefeldin A, 5 µg/ml cycloheximide, 30 µM H2O2, 0.5 mMoleate and 0.5 mMpalmitate for 14 hours, measured via qPCR and normalized for GAPDH (Ufm1 and Ufbp1) or actin (Ufl1). Data are means±SEM, n = 4–6, *, p≤0.05; **, p≤0.01, paired student t-test, D UFM1 and UFBP1 protein expression is induced in cells exposed for 14 hours to CPA. Actin is shown as a control for protein loading, E UFM1-UFBP1 conjugates. Incubation of MIN6 or INS1 cells with ER stressors (25 µM CPA, 14h, INS1 cells; 1 µM thapsigargin (Tg), 1h, MIN6 cells) or proteasome inhibitor (100 µM MG115, 2h, INS1 cells) decreased conjugation, while incubation with a translational inhibitor (10 mg/l cycloheximide (CHX), 2h, MIN6 cells) increased the conjugate formation. * = UFM1−UFBP1 conjugate, 10 kDa = free UFM1.
Figure 6
Figure 6. UFM1 and UFBP1 are involved in ER stress induced apoptosis.
INS-1E cells were treated with the ER stressors oleate, palmitate, cyclopiazonic acid (CPA) and brefeldin A or the ER stress-independent apoptosis inducers cycloheximide and H2O2, and silenced forUfm1 (A), Ufbp1 (B) or Ufl1 (C). Apoptosis was evaluated by Hoechst/PI staining. Data are means±SEM of 3–7 independent experiments. Paired student t test: *, p≤0.05; **, p≤0.01; ***, p≤0.001 compared to control treatment; #, p≤0.05; ##, p≤0.01 silenced cells compared to siControl cells, D Caspase 3 activation in Ufm1 and Ufbp1 silenced cells after treatment with the ER stressors CPA and brefeldin A (Bref). The densitometric quantification of the immunoblots is shown in the lower panel. Cleaved Caspase-3 signal was normalized for α-tubulin expression. The results are means ± SEM of 2–3 independent experiments. In the assays, the respective controls contain the vehicles ethanol and DMSO.
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References

    1. Tatsumi K, Sou YS, Tada N, Nakamura E, Iemura S, et al. A novel type of E3 ligase for the Ufm1 conjugation system. J Biol Chem. 2010;285:5417–5427. - PMC - PubMed
    1. Welchman RL, Gordon C, Mayer RJ. Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nat Rev Mol Cell Biol. 2005;6:599–609. - PubMed
    1. Hochstrasser M. Evolution and function of ubiquitin-like protein-conjugation systems. Nat Cell Biol. 2000;2:E153–157. - PubMed
    1. Komatsu M, Chiba T, Tatsumi K, Iemura S, Tanida I, et al. A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier. Embo J. 2004;23:1977–1986. - PMC - PubMed
    1. Kang SH, Kim GR, Seong M, Baek SH, Seol JH, et al. Two novel ubiquitin-fold modifier 1 (Ufm1)-specific proteases, UfSP1 and UfSP2. J Biol Chem. 2007;282:5256–5262. - PubMed

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