期刊
MOLECULAR & CELLULAR PROTEOMICS
卷 9, 期 4, 页码 705-718出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/mcp.M800390-MCP200
关键词
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资金
- National Institutes of Health through the Emory Alzheimer's Disease Center [P50AG025688]
- National Institutes of Health through the Emory Neuroscience NINDS Core Facilities [P30NS055077]
- National Institutes of Health through the Emory Neuroscience NINDS Core Facilities and Training [T32NS007480, F30NS057902]
- American Cancer Society [RSG-09-181-01]
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [T32GM008169] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [P30NS055077, T32NS007480, F30NS057902] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON AGING [P50AG025688] Funding Source: NIH RePORTER
Transactive response (TAR) DNA-binding protein 43 (TDP-43) is a major protein component within ubiquitin-positive inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Although TDP-43 is a nuclear DNA/RNA-binding protein, in pathological conditions, TDP-43 has been reported to redistribute to the cytoplasm where it is cleaved and forms insoluble, ubiquitinated, and phosphorylated inclusions. Here we present a cellular model in which full-length human TDP-43 or a splicing isoform (TDP-S6) that lacks the C terminus is overexpressed in a human cell line and mouse primary neurons. Whereas recombinant and endogenous TDP-43 was primarily localized in the nucleus, the shorter TDP-S6 formed highly insoluble cytoplasmic and nuclear inclusions reminiscent of disease-specific pathology. Western blot analysis of detergent-insoluble extracts showed an increase in high molecular weight immunoreactive species for TDP-S6 compared with TDP-43, consistent with ubiquitination or ubiquitin-like modifications. We used a multiplex stable isotope labeling with amino acids in cell culture approach to compare the detergent-insoluble proteome from mock-, TDP-43-, and TDP-S6-transfected cells. TDP-S6 overexpression caused a concomitant increase in both ubiquitin (Ub) and the small Ub-like modifier2/ 3 (SUMO-2/3) within the insoluble proteome. Similarly, full-length TDP-43 overexpression also resulted in the elevation of SUMO-2/3. Immunofluorescence showed strong co-localization of endogenous Ub with both cytoplasmic and nuclear TDP-S6 inclusions, whereas SUMO2/3 was co-localized mainly with the nuclear inclusions. Quantitative mass spectrometry further revealed that mixed Lys-48 and Lys-63 polyUb linkages were associated with the TDP insoluble fractions. Together our data indicate that expression of a TDP-43 splice variant lacking a C terminus recapitulates many of the cellular and biochemical features associated with disease pathology and that the interplay of ubiquitination and SUMOylation may have an important role in TDP-43 regulation. Molecular & Cellular Proteomics 9: 705-718, 2010.
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