Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Mar 20;284(12):8083-92.
doi: 10.1074/jbc.M808064200. Epub 2008 Dec 26.

Potentiation of amyotrophic lateral sclerosis (ALS)-associated TDP-43 aggregation by the proteasome-targeting factor, ubiquilin 1

Sang Hwa Kim  1 , Yuling Shi, Keith A Hanson, Leah M Williams, Ryo Sakasai, Michael J Bowler, Randal S Tibbetts
Affiliations

Potentiation of amyotrophic lateral sclerosis (ALS)-associated TDP-43 aggregation by the proteasome-targeting factor, ubiquilin 1

Sang Hwa Kim et al. J Biol Chem. .

Abstract

TDP-43 (43-kDa TAR DNA-binding domain protein) is a major constituent of ubiquitin-positive cytoplasmic aggregates present in neurons of patients with fronto-temporal lobular dementia and amyotrophic lateral sclerosis (ALS). The pathologic significance of TDP-43 aggregation is not known; however, dominant mutations in TDP-43 cause a subset of ALS cases, suggesting that misfolding and/or altered trafficking of TDP-43 is relevant to the disease process. Here, we show that the presenilin-binding protein ubiquilin 1 (UBQLN) plays a role in TDP-43 aggregation. TDP-43 interacted with UBQLN both in yeast and in vitro, and the carboxyl-terminal ubiquitin-associated domain of UBQLN was both necessary and sufficient for binding to polyubiquitylated forms of TDP-43. Overexpression of UBQLN recruited TDP-43 to detergent-resistant cytoplasmic aggregates that colocalized with the autophagosomal marker, LC3. UBQLN-dependent aggregation required the UBQLN UBA domain, was mediated by non-overlapping regions of TDP-43, and was abrogated by a mutation in UBQLN previously linked to Alzheimer disease. Four ALS-associated alleles of TDP-43 also coaggregated with UBQLN, and the extent of aggregation correlated with in vitro UBQLN binding affinity. Our findings suggest that UBQLN is a polyubiquitin-TDP-43 cochaperone that mediates the autophagosomal delivery and/or proteasome targeting of TDP-43 aggregates.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
TDP-43 interacts with UBQLN. A, two-hybrid interaction between TDP-43 and UBQLN in yeast. Yeast were cotransformed with shuttle vectors encoding TDP-43 and UBQLN (or empty vector; (see “Experimental Procedures”). Growth on His-, Leu-, and Trp-deficient media and α-galactosidase activity was measured 1 week after cotransformation. Yeast were cotransformed with SV40 large T antigen (TAg) and p53 as a positive control. B, schematic depiction of UBQLN domains and deletion mutants used for TDP-43 binding assays. UBL (ubiquitin-like) and UBA (ubiquitin-associated) domains are shown. C, TDP-43 interacts with the UBQLN UBA domain. Purified GST fusions of full-length UBQLN or the indicated UBQLN deletion mutants were incubated with 500 μg of HEK 293T cell extract containing HA-TDP-43. Bound proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. For panels C and D, the “input” lane contains 1/20th of the total cell extract used for the pull-down experiments. Arrows indicate the positions of the various intact GST-UBQLN fusion proteins relative to lower molecular weight degradation products. D, the UBQLN UBA domain interacts with high molecular weight forms of TDP-43. HeLa cells were transfected with HA-TDP-43 plasmid DNA and soluble fractions incubated with the indicated GST fusion proteins. Bound proteins were analyzed by immunoblotting with α-HA and equal levels of fusion protein confirmed by immunoblotting with α-GST.
FIGURE 2.
FIGURE 2.
UBQLN stimulates TDP-43 aggregation. A, localization patterns of HA-TDP-43 in HeLa cells. HeLa cells were transfected with a HA-TDP-43 expression plasmid and then stained with α-TDP-43 antibodies 48 h later. Three common staining patterns are shown: diffuse-nuclear (top), aggregate-nuclear (middle), and aggregate-cytoplasmic (bottom). B, effects of UBQLN overexpression on TDP-43 localization. HeLa cells were cotransfected with HA-TDP-43 and Myc-UBQLN expression plasmids and then immunostained 48 h later with α-Myc (red) and α-TDP-43 (green) antibodies. Two types of TDP-43 localization patterns are shown. C, colocalization of endogenous TDP-43 with Myc-UBQLN. HeLa cells were transfected with a Myc-UBQLN expression plasmid and then immunostained with α-TDP-43 and α-Myc antibodies. D, overexpression of TDP-43 recruits endogenous UBQLN to cytoplasmic aggregates. HeLa cells were transfected with HA-TDP-43 and then stained with rabbit α-TDP-43 and mouseα-UBQLN antibodies. E, colocalization analysis of UBQLN and GFP-tagged TDP-43 fusions. HeLa cells were cotransfected with Myc-UBQLN and TDP-43 containing either a carboxyl-terminal (C-term) or amino-terminal (N-term) GFP tag. The transfected cells were then immunostained with α-Myc (red) to visualize Myc-UBQLN.
FIGURE 3.
FIGURE 3.
The UBA domain of UBQLN is required for its coaggregation with TDP-43. A, HeLa cells were cotransfected with HA-TDP-43 and either wild-type Myc-UBQLN (top panel) or Myc-UBQLN containing deletions in either the UBA (middle panel) or UBL (bottom panel) domains. The transfected cells were immunostained with α-Myc and α-TDP-43 antibodies. B, the AD-associated UBQLN-8i mutant does not coaggregate with TDP-43. HeLa cells were cotransfected with HA-TDP-43 and wild-type or exon 8-deleted (8i) UBQLN. The transfected cells were stained with α-TDP-43 and α-Myc antibodies.
FIGURE 4.
FIGURE 4.
UBQLN coaggregates with non-overlapping fragments of TDP-43. A, schematic depiction of TDP-43 deletion mutants. B, colocalization analysis of UBQLN with TDP-43 deletion mutants. HeLa cells were cotransfected with wild-type or mutant HA-TDP-43 plasmids and Myc-UBQLN and stained with α-TDP-43 and α-UBQLN antibodies. Representative images (×100) for each mutant are shown.
FIGURE 5.
FIGURE 5.
ALS-associated TDP-43 mutants coaggregate with UBQLN. A, ALS-associated TDP-43 mutants interact with the UBQLN UBA domain. GST-UBA pull-down assays were performed using HeLa cell extracts containing wild-type HA-TDP-43 or the indicated HA-TDP-43 point mutants. B, localization patterns of TDP-43 point mutants. HeLa cells were cotransfected with Myc-UBQLN and wild-type HA-TDP-43 or the indicated HA-TDP-43 point mutants. C, particle size analysis of transfected cells displaying HA-TDP-43 aggregates in panel B was established for the wild-type and mutant proteins. Approximately 150 aggregates were plotted for each sample (see “Experimental Procedures”). D, polyubiquitylation of wild-type and mutant TDP-43. Wild-type HA-TDP-43 or the indicated HA-TDP-43 point mutants were immunoprecipitated (IP) under denaturing conditions from extracts of transfected HeLa cells with α-TDP-43 antibodies followed by immunoblot using α-HA and α-Myc antibodies. The positions of molecular weight standards are indicated on the right side of the panel.
FIGURE 6.
FIGURE 6.
UBQLN targets TDP-43 to autophagosomes. A, UBQLN-TDP-43G37V aggregates are insoluble. HeLa cells were transfected with a fixed amount of plasmid DNA encoding HA-TDP-43G37V and increasing amounts of plasmid DNA encoding Myc-UBQLN. Empty pCMV-Myc vector was added so that a total of 5 μg of DNA was used for each transfection. HeLa cells were fractionated into detergent-soluble and -insoluble fractions, resolved by SDS-PAGE, and the levels of HA-TDP-43G37V, endogenous TDP-43, and Myc-UBQLN were measured by immunoblotting with α-TDP-43 and α-Myc antibodies. B, UBQLN decorates the periphery of TDP-43 aggregates. Confocal images of HeLa cells coexpressing Myc-UBQLN and wild-type TDP-43 (top) or TDP-43G37V (bottom). C, enlargement and pixel density analysis of the boxed aggregates shown in panel B. D, HeLa cells were cotransfected with HA-TDP-43, Myc-UBQLN, and GFP-LC3 and immunostained with α-HA (top) or α-Myc (bottom) antibodies. Representative images are shown. E, HeLa cells were treated with 50 μm concanamycin A (CMA) for 4 h and immunostained with α-TDP-43 and α-UBQLN antibodies.
FIGURE 7.
FIGURE 7.
Speculative model depicting the potential role of UBQLN in TDP-43 trafficking. In this model TDP-43 accumulates in the cytoplasm either due to impaired nuclear import (1), overexpression (2), or misfolding (3), possibly as a result of ALS-associated point mutations. After ubiquitylation of TDP-43 (4), cytosolic UBQLN binds (5) via the interaction of its UBA domain with ubiquitin on TDP-43. UBQLN potentially then aids in delivering accumulated TDP-43 to the proteasome (6) via its UBL domain. Alternatively, UBQLN may target TDP-43 to autophagosomes (7), where it is degraded by the lysosomal pathway (8).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mitsumoto, H., Chad, D. A., and Pioro, E. P. (1998) Amyotrophic Lateral Sclerosis, Davis, Philadelphia, PA
    1. Rosen, D. R., Siddique, T., Patterson, D., Figlewicz, D. A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J. P., Deng, H. X., et al. (1993) Nature 362 59-62 - PubMed
    1. Andersen, P. M., Sims, K. B., Xin, W. W., Kiely, R., O'Neill, G., Ravits, J., Pioro, E., Harati, Y., Brower, R. D., Levine, J. S., Heinicke, H. U., Seltzer, W., Boss, M., and Brown, R. H., Jr. (2003) Amyotroph. Lateral Scler. Other Motor Neuron Disord. 4 62-73 - PubMed
    1. Boillee, S., Vande Velde, C., and Cleveland, D. W. (2006) Neuron 52 39-59 - PubMed
    1. Cassina, P., Cassina, A., Pehar, M., Castellanos, R., Gandelman, M., de Leon, A., Robinson, K. M., Mason, R. P., Beckman, J. S., Barbeito, L., and Radi, R. (2008) J. Neurosci. 28 4115-4122 - PMC - PubMed

Publication types

MeSH terms