Background. Tau hyperphosphorylation and aggregation to form intracellular neurofibrillar tangles is prevalent in a number of tauopathies. Thus there is current interest in the mechanisms involved in Tau clearance. It was recently reported that Tau can be degraded by an aminopeptidase known as the puromycin sensitive aminopeptidase (PSA). Until now PSA has been reported to only cleave peptides, with the largest reported substrates having 30-50 amino acids. We have studied this unique PSA cleavage reaction using a number of different PSA preparations. Results. An N-terminally His tagged-PSA was expressed and purified from Sf9 insect cells. Although this PSA preparation cleaved Tau, product analysis with N and C terminal Tau antibodies coupled with mass spectrometry showed an endoproteolytic cleavage atypical for an aminopeptidase. Furthermore, the reaction was not blocked by the general aminopeptidase inhibitor bestatin or the specific PSA inhibitor puromycin. In order to test whether Tau hydrolysis might be caused by a protease contaminant the enzyme was expressed in E. coli as glutathione S-transferase and maltose binding protein fusion proteins or in Sf9 cells as a C-terminally His-tagged protein. After purification to near homogeneity none of these other recombinant forms of PSA cleaved Tau. Further, Tau-cleaving activity and aminopeptidase activities derived from the Sf9 cell expression system were separable by molecular sieve chromatography. When tested in a cellular context we again failed to see a PSA dependent cleavage of Tau. A commercial preparation of a related aminopeptidase, aminopeptidase N, also exhibited Tau cleaving activity, but this activity could also be separated from aminopeptidase activity. Conclusion. It is concluded that PSA does not directly cleave Tau.
|State||Published - 2010|
Bibliographical noteFunding Information:
Protein bands were identified by mass spectrometry at the University of Kentucky Proteomics core within the Center of Structure Biology. This facility is supported in part by grant P20RR020171 from the NIH NCRR. Briefly, gel bands were excised from SDS-PAGE, digested with Sigma proteomics grade trypsin, and alkylated with iodoacetamide. Samples were then analyzed with an Applied Biosystems 4800 MALDI TOF/TOF Proteomics Analyzer. MALDI MS spectra were used for analyzing Tau products. For unknown proteins, MS/MS spectra were used to search against the Uniprot_-sprot_20100125+ contams.fasta database through Protein Pilot (Applied Biosystems) Ver. 2.0, Rev. 50861.
This research was supported by grants from the National Institute on Drug Abuse, grant DA02243 and from the National Center for Research Resources (NCRR), grant P20RR02017. We would like to thank Dr. Jeff Kuret (Ohio State University) for supplying the Tau expressing HEK 293 cells and Dr. Mike Hutton (Mayo Clinic, Jacksonville, Fl.) for generously providing us Tau clones. We thank Dr. Carol Beach for conducting the mass spectrometry analysis at the University of Kentucky Proteomics Core facility, which is supported in part by National Center for Research Resources grant P20RR02017.
ASJC Scopus subject areas
- Molecular Biology
- Clinical Neurology
- Cellular and Molecular Neuroscience