Formation of heterodimers of human-immunodeficiency-virus-type-1 reverse transcriptase by recombination of separately purified subunits.

Human-immunodeficiency-virus-type-1 reverse transcriptase exists in virions as a heterodimer of a M(r) 66,000 subunit and its C-terminally truncated form of M(r) 51,000, but, when expressed as a recombinant M(r) 66,000 protein, a mixture of heterodimers and homodimers results which co-purify by most conventional techniques. We describe a method of hydrophobic chromatography which gives baseline separation of these two forms of the protein. This method has been applied to purify heterodimers formed by recombination of separately expressed and purified M(r) 66,000 and 51,000 subunits, resulting in significantly more homogeneous heterodimer preparations. The recombined heterodimer showed similar kinetic properties and RNase H activity to the standard heterodimer and a specific activity significantly higher than the original homodimer of the M(r) 66,000 protein. Heterodimers having greater asymmetry have also been prepared by recombining Mr 66,000 subunits containing single-point or deletion mutations, with wild-type M(r) 51,000 subunits, and the resulting heterodimers analysed

HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis.

Bacterially expressed recombinant HIV-1 reverse transcriptase is active as both a homodimer of Mr 66,000 subunits and a heterodimer of Mr 66,000 and 51,000 subunits. The heterodimer is formed by cleavage of a C-terminal fragment from one Mr 66,000 polypeptide, which occurs during purification and crystallization of reverse transcriptase. Thus, crystals obtained from purified Mr 66,000 polypeptide preparations consisted of an apparently equimolar mixture of Mr 66,000 and 51,000 polypeptides, which were apparently analogous to the Mr 66,000 and 51,000 polypeptides detected in HIV-infected cells and in virions. Limited proteolysis of the homodimer with alpha-chymotrypsin also resulted in cleavage to a stable Mr 66,000/51,000 mixture, and proteolysis with trypsin resulted in the transient formation of some Mr 51,000 polypeptide. These results are consistent with the reverse transcriptase molecule having a protease-sensitive linker region following a structured domain of Mr 51,000. Further digestion with trypsin resulted in cleavage of the Mr 51,000 polypeptide after residue 223, yielding peptides of apparent Mr 29,000 and 30,000. A minor peptide of Mr 40,000 was also produced by cleavage of the Mr 66,000 polypeptide after residue 223. About half the original Mr 66,000 polypeptides remained resistant to proteolysis and existed in complex with the above peptides in solution. During both chymotrypsin and trypsin digestion there was an increase in the reverse transcriptase activity caused by a doubling of Vmax with little change in Km for dTTP.(ABSTRACT TRUNCATED AT 250 WORDS

Structural characterization of HIV reverse transcriptase: a target for the design of specific virus inhibitors.

The reverse transcriptase (RT) of HIV is an important target for chemotherapy as demonstrated by the effective treatment of AIDS patients with zidovudine, a potent inhibitor of RT. Structural studies of HIV RT were therefore undertaken with a view to designing more effective inhibitors. To obtain sufficient quantities of enzyme for these studies the reverse transcriptase gene of HIV was cloned into a high level expression plasmid yielding reverse transcriptase at a level of 10% of the total Escherichia coli proteins. Monoclonal antibodies to RT were raised in mice and have been used to purify the enzyme by immunoaffinity chromatography. Crystallization of the enzyme has been achieved and studies are underway to determine its three-dimensional structure. In addition, carboxy-terminal truncated mutants were prepared by inserting stop codons into the gene at appropriate sites. The proteins expressed were analysed for RT and RNase H activity and used for mapping RT epitopes. This, together with previous data on site-directed mutagenesis of conserved regions of HIV RT has helped to map some of the structural and functional regions of the enzyme

Incorporating human rights into the corporate domain: due diligence, impact assessment and integrated risk management

Business and human rights are often thought to be antithetical, but as societal expectations on companies have grown, it has become increasingly important for businesses to understand and act upon their legal and moral obligations to respect human rights. The authors of this paper begin by charting the evolution of the rights paradigm and its incorporation into the corporate sphere of influence. Second, the concept of human rights due diligence is examined, owing to its prominence in John Ruggie’s ‘Protect, Respect and Remedy’ framework. Human Rights Impact Assessments, as an emerging due diligence tool, warrant further attention, theorization and critique. Finally, it is suggested that human rights due diligence could be consolidated within existing corporate risk management systems. Reframing human rights in the context of social and business risks may provide a path forncompanies to understand the need for human rights due diligence by linking rights considerations with business concerns

Functional and structural insights into Sarcolipin, a regulator of the Sarco-endoplasmic reticulum Ca2+-ATPases

International audienceSarcolipin (SLN), a transmembrane peptide from sarcoplasmic reticulum, is one of the major proteins involved in the muscle contraction/relaxation process. A number of enzymological studies have underlined its regulatory role in connection with the SERCA1a activity. Indeed, SLN folds as a unique transmembrane helix and binds to SERCA1a in a groove close to transmembrane helices M2, M6, and M9, as proposed initially by cross-linking experiments and recently detailed in the 3D structures of the SLN–Ca2+-ATPase complex. In addition, association of SLN with SERCAs may depend on its phosphorylation. SLN possesses a peculiar C-terminus (RSYQY) critical for the regulation of the ATPases. This luminal tail appears to be essential for addressing SLN to the ER membrane. Moreover, we recently demonstrated that some SLN isoforms are acylated on cysteine 9, a feature which remained unnoticed so far even in the recent crystal structures of the SLN–SERCA1a complex. The removal of the fatty acid chain was shown to increase the activity of the membrane-embedded Ca2+-ATPase by about 20 %. The exact functional and structural role of this post-translational modification is presently unknown. Recent data are in favor of a key regulator role of SLN in muscle-based thermogenesis in mammals. The possible link of SLN to heat production could occur through an uncoupling of the SERCA1a-mediated ATP hydrolysis from calcium transport. Considering those particular features and the fact that SLN is not expressed at the same level in different tissues, the role of SLN and its exact mechanism of regulation remain sources of interrogation