The active site sulfhydryl of aconitase is not required for catalytic activity

Previous reports have demonstrated that aconitase has a single reactive sulfhydryl at or near the active site (Johnson, P. G., Waheed, A., Jones, L., Glaid, A. J., and Gawron, O. (1977) Biochem. Biophys. Res. Commun. 74, 384-389). On the basis of experiments with phenacyl bromide in which enzyme activity was abolished while substrate afforded protection, it was concluded that this group was an essential sulfhydryl. We have further examined the reactivity of this group and confirmed the result that, when reagents with bulky groups (e.g. N-ethylmaleimide or phenacyl bromide) modify the protein at the reactive sulfhydryl, activity is lost. However, when smaller groups, e.g. the SCH3 from methylmethanethiosulfonate or the CH2CONH2 from iodoacetamide, are introduced, there is only partial (50%) or no loss of activity. Experiments were performed to obtain evidence that these reagents are modifying the same residue. Methylmethanethio-sulfonate-treated enzyme showed an increase in the Km for citrate from 200 to 330 microM. EPR spectra were taken of the reduced N-ethylmaleimide- and iodoacetamide-modified enzyme in the presence of substrate. The former gave a spectrum typical of the substrate-free enzyme, while the spectrum of the latter was identical to enzyme with bound substrate. We, therefore, conclude that modification of this sulfhydryl affects activity by interfering with the binding of substrate to the active site and is not essential in the catalytic process.     

Drosophila Dystrophin is required for integrity of the musculature

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene and is characterized by progressive muscle wasting. The highly conserved dystrophin gene encodes a number of protein isoforms. The Dystrophin protein is part of a large protein assembly, the Dystrophin glycoprotein complex, which stabilizes the muscle membrane during contraction and acts as a scaffold for signaling molecules. How the absence of Dystrophin results in the onset of muscular dystrophy remains unclear. Here, we have used transgenic RNA interference to examine the roles of the Drosophila Dystrophin isoforms in muscle. We previously reported that one of the Drosophila Dystrophin orthologs, the DLP2 isoform, is not required to maintain muscle integrity, but plays a role in neuromuscular homeostasis by regulating neurotransmitter release. In this report, we show that reduction of all Dystrophin isoform expression levels in the musculature does not apparently affect myogenesis or muscle attachment, but results in progressive muscle degeneration in larvae and adult flies. We find that a recently identified Dystrophin isoform, Dp117, is expressed in the musculature and is required for muscle integrity. Muscle fibers with reduced levels of Dp117 display disorganized actin-myosin filaments and the cellular hallmarks of necrosis. Our results indicate the existence of at least two possibly separate roles of dystrophin in muscle, maintaining synaptic homeostasis and preserving the structural stability of the muscle.     

Lysine 144, a ubiquitin attachment site in HIV-1 Nef, is required for Nef-mediated CD4 down-regulation

Nef is a HIV-1 accessory protein critical for the replication of the virus and the development of AIDS. The major pathological activity of Nef is the down-regulation of CD4, the primary receptor of HIV-1 infection. The mechanism underlying Nef-mediated CD4 endocytosis and degradation remains incompletely understood. Since protein ubiquitination is the predominant sorting signal in receptor endocytosis, we investigated whether Nef is ubiquitinated. The in vivo ubiquitination assay showed that both HIV-1 and SIV Nef proteins expressed in Jurkat T cells and 293T cells were multiple ubiquitinated by ubiquitin-His. The lysine-free HIV-1 Nef mutant (Delta10K) generated by replacing all 10 lysines with arginines was not ubiquitinated and the major ubiquitin-His attachment sites in HIV-1 Nef were determined to be lysine 144 (di-ubiquitinated) and lysine 204 (mono-ubiquitinated). Lysine-free HIV-1 Nef was completely inactive in Nef-mediated CD4 down-regulation, so was the Nef mutant with a single arginine substitution at K144 but not at K204. A mutant HIV-1 provirion NL4-3 with a single arginine substitution in Nef at K144 was also inactive in Nef-mediated CD4 down-regulation. Lysine-free Nef mutant reintroduced with lysine 144 (DeltaK10 + K144) was shown active in CD4 down-regulation. These data suggest that ubiquitination of Nef, particularly diubiquitination of the lysine 144, is necessary for Nef-mediated CD4 down-regulation.