Characterization of ATP-dependent proteolysis in embryos of the brine shrimp, Artemia franciscana

Under anoxia, embryos of Artemia franciscana enter a state of quiescence. During this time protein synthesis is depressed, and continued degradation of proteins could jeopardize the ability to recover from quiescence upon return to favorable conditions. In this study, we developed an assay for monitoring ATP/ubiquitin-dependent proteolysis in order to establish the presence of this degradation mechanism in A. franciscana embryos, and to describe some characteristics that may regulate its function during anoxia-induced quiescence. For lysates experimentally depleted of adenylates, supplementation with ATP and ubiquitin stimulated protein degradation rates by 92 ± 17% (mean ± SE) compared to control rates. The stimulation by ATP was maximal at concentrations ≥11 μmol · l⁻¹. In the presence of ATP and ubiquitin, ubiquitin-conjugated proteins were produced by lysates during the course of the 4-h assays, as detected by Western blotting. Acute acidification of lysates to values approximating the intracellular pH observed under anoxia completely inhibited ATP/ubiquitin-dependent proteolysis. Depressed degradation was also observed under conditions where net ATP hydrolysis occurred. These results suggest that ATP/ubiquitin-dependent proteolysis is markedly inhibited under cellular conditions promoted by anoxia. Inhibition of proteolysis during quiescence may be one critical factor that increases macromolecular stability, which may ultimately govern the duration of embryo survival under anoxia. Accepted: 2 November 1999     

Synthetic constructs functionally mimicking ribonuclease A

The mechanism of hydrolysis of RNA substrates—diribonucleoside monophosphate CpA and decaribonucleotide UUCAUGUAAA—by chemical constructs functionally mimicking ribonuclease A was studied. It is shown that RNA cleavage by chemical RNases 2L2 and 2D3 proceeds similar to the RNase A-induced RNA hydrolysis through 2′,3′-cyclophosphate as an intermediate product. A comparison of hydrolyses of CpA in water and D₂O revealed an isotope effect (K _H/K _D=2.28), which implies acid-base catalysis at the limiting stage of the reaction. Two feasible mechanisms of RNA hydrolysis by chemical RNases (linear and adjacent) are discussed.     

Create and preserve: Proteostasis in development and aging is governed by Cdc48/p97/VCP

The AAA-ATPase Cdc48 (also called p97 or VCP) acts as a key regulator in proteolytic pathways, coordinating recruitment and targeting of substrate proteins to the 26S proteasome or lysosomal degradation. However, in contrast to the well-known function in ubiquitin-dependent cellular processes, the physiological relevance of Cdc48 in organismic development and maintenance of protein homeostasis is less understood. Therefore, studies on multicellular model organisms help to decipher how Cdc48-dependent proteolysis is regulated in time and space to meet developmental requirements. Given the importance of developmental regulation and tissue maintenance, defects in Cdc48 activity have been linked to several human pathologies including protein aggregation diseases. Thus, addressing the underlying disease mechanisms not only contributes to our understanding on the organism-wide function of Cdc48 but also facilitates the design of specific medical therapies. In this review, we will portray the role of Cdc48 in the context of multicellular organisms, pointing out its importance for developmental processes, tissue surveillance, and disease prevention. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L.

In this study, we performed cloning and expression analysis of six putative sucrose transporter genes, designated TcSUT1, TcSUT2, TcSUT3, TcSUT4, TcSUT5 and TcSUT6, from the cacao genotype ‘TAS-R8’. The combination of cDNA and genomic DNA sequences revealed that the cacao SUT genes contained exon numbers ranging from 1 to 14. The average molecular mass of all six deduced proteins was approximately 56 kDa (range 52 to 66 kDa). All six proteins were predicted to exhibit typical features of sucrose transporters with 12 trans-membrane spanning domains. Phylogenetic analysis revealed that TcSUT2 and TcSUT4 belonged to Group 2 SUT and Group 4 SUT, respectively, and the other TcSUT proteins were belonging to Group 1 SUT. Real-time PCR was conducted to investigate the expression pattern of each member of the SUT family in cacao. Our experiment showed that TcSUT1 was expressed dominantly in pods and that, TcSUT3 and TcSUT4 were highly expressed in both pods and in bark with phloem. Within pods, TcSUT1 and TcSUT4 were expressed more in the seed coat and seed from the pod enlargement stage to the ripening stage. TcSUT5 expression sharply increased to its highest expression level in the seed coat during the ripening stage. Expression pattern analysis indicated that TcSUT genes may be associated with photoassimilate transport into developing seeds and may, therefore, have an impact on seed production.