ORCID Profile
0000-0003-4030-2575
Current Organisation
Colorado State University
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Publisher: Elsevier BV
Date: 2021
Publisher: Springer Science and Business Media LLC
Date: 22-01-2019
Publisher: Elsevier BV
Date: 05-1995
Abstract: The proteasome plays a central role in ubiquitin-dependent and -independent proteolysis in eukaryotic cells. The hawkmoth proteasome was purified from larval body wall and characterized with respect to substrate specificity, sensitivity to protease inhibitors, and cross-reactivity with monoclonal antibodies (mAbs) raised against human placenta proteasome. Leupeptin selectively inhibited the trypsin-like activity (T-L) and N-ethylmaleimide inhibited both T-L and chymotrypsin-like activities, whereas 0.02% sodium dodecyl sulfate stimulated the peptidylglutamyl peptide hydrolase, branched-chain amino acid preferring, and caseinolytic activities 20-, 18-, and 3.8-fold, respectively. All four peptidase activities were inhibited by 3,4-dichloroisocoumarin. One-dimensional immunoblot analysis showed that the level and subunit composition of the proteasome varied between tissues. The relative levels of proteasome were high in intersegmental muscle and ovary, lower in Malpighian tubule, male accessory gland, and ventral nerve cord, and lowest in flight muscle and fat body. The tissues differed in the relative amount of a 41-kDa doublet a 22-kDa subunit was present only in the male accessory gland. Two-dimensional polyacrylamide gel electrophoresis showed that the hawkmoth proteasome contained at least 26 subunits, compared with 28 subunits in lobster. Immunological analysis using four subunit-specific mAbs identified the putative homologs of the human zeta, C2, C3, and C8 alpha-type subunits in the hawkmoth and lobster enzymes. Two of the four mAbs reacted with three or more of the hawkmoth subunits and three of the mAbs reacted with two or more of the lobster subunits. In addition, two other mAbs that recognize epitopes shared by a number of alpha-type subunits indicated that at least 15 (lobster) or 16 (hawkmoth) subunits were alpha-type. These results suggest that much of the subunit complexity of the arthropod proteasomes is a consequence of extensive post-translational modifications.
Publisher: Elsevier BV
Date: 06-1995
Publisher: Elsevier BV
Date: 11-2020
Publisher: Wiley
Date: 16-04-2002
DOI: 10.1002/JEZ.10081
Abstract: The closer muscle of large-clawed decapod crustaceans undergoes a proecdysial (premolt) atrophy to facilitate withdrawal of the appendage at ecdysis. This atrophy involves the activation of both calcium-dependent (calpains) and ubiquitin (Ub) roteasome-dependent proteolytic systems that break down proteins to reduce muscle mass. Moreover, the large slow-twitch (S(1)) fibers undergo a greater atrophy than the small slow-tonic (S(2)) fibers. Both polyUb mRNA and Ub-protein conjugates increase during claw muscle atrophy. In this study in situ hybridization and RT-PCR were used to determine the temporal and spatial expression of polyUb and alpha-actin. A cDNA encoding the complete sequence of lobster muscle alpha-actin was characterized a probe synthesized from the cDNA provided a positive control for optimizing RT-PCR and in situ hybridization. PolyUb was expressed at low levels in claw closer muscle from anecdysial (intermolt) land crab. By early proecdysis (premolt stage D(0)), polyUb mRNA levels increased in medial fibers that insert along the midline of the apodeme, with greater expression in S(1) than S(2), while levels remained low in peripheral fibers. By late proecdysis, polyUb mRNA decreased in central fibers, while mRNA increased in peripheral S(1) fibers. In contrast, alpha-actin was expressed in lobster claw muscles at relatively constant levels during the intermolt cycle. These results suggest that Ub roteasome-dependent proteolysis contributes to enhanced turnover of myofibrillar proteins during claw closer muscle atrophy. Furthermore, atrophy is not synchronous within the muscle it begins in medial fibers and then progresses peripherally.
Location: United States of America
No related grants have been discovered for Donald Mykles.