TAILIEUCHUNG - Báo cáo khoa học: Growth-associated protein of 43 kDa (GAP-43) is cleaved nonprocessively by the 20S proteasome

Purified, nonubiquitinated growth-associated protein of 43 kDa (GAP-43) was attacked by purified reticulocyte 20S proteasomebutnotby 12 N-terminally labelled GAP-43 fragments that could be resolved by SDS/PAGE. Inhibitor experiments suggested that proteasomeb1 activity yielded the resolved bands and that proteasome b5 activity generated nonresolvable frag-ments. Processive degradation, yielding only nonresolvable fragments, therefore did not occur. | Eur. J. Biochem. 271 2480-2493 2004 FEBS 2004 doi Growth-associated protein of 43 kDa GAP-43 is cleaved nonprocessively by the 20S proteasome John B. Denny Department of Ophthalmology University of Texas Health Science Center at San Antonio San Antonio TX USA Purified nonubiquitinated growth-associated protein of 43 kDa GAP-43 was attacked by purified reticulocyte 20S proteasome but not by the 26S proteasome. Cleavage yielded 12 N-terminally labelled GAP-43 fragments that could be resolved by SDS PAGE. Inhibitor experiments suggested that proteasome b1 activity yielded the resolved bands and that proteasome P5 activity generated nonresolvable fragments. Processive degradation yielding only nonresolvable fragments therefore did not occur. Most of the resolved fragments co-migrated with fragments formed in the reticulocyte lysate translation mixture used for GAP-43 synthesis which suggested that the fragments were also produced in the translation mixture by the endogenous reticulocyte lysate proteasome. Consistent with this idea the addition of proteasome inhibitors to translation mixtures blocked fragment production. Ubiquitinated GAP-43 appeared to be the source of the fragments in the presence of ATP and nonubiquitinated GAP-43 the source in the absence of ATP. The results therefore suggest that the lack of processing seen with the 20S proteasome is not an artefact arising from the way in which the 20S proteasome was purified. In one purification protocol the GAP-43 fragments formed in translation mixtures co-purified with full-length GAP-43. These fragments were digested to nonresolvable products upon addition of purified 20S proteasome. Addition of calmodulin or G-actin blocked the consumption of both full-length GAP-43 and the co-purified GAP-43 fragments. This showed that the resolved fragments can re-enter the proteasome and be cleaved to nonresolvable products indicating that the lack of processivity is not a result of their

• Báo cáo khoa học
• Report medicine
• traditional medicine
• scientific reports
• biochemical studies
• environmental medicine
• Crystal structure
• medical knowledge
• medical research
• analysis of cytoplasmic
• oxidation
• bacteria
• hemoglobin
• medicine
• cell proliferation
• breast cancer
• chemical reaction
• gastric cancer
• hormone medicine
• tumor cells