The HIV protease plays a major role in the life cycle of the virus and has long been a target in antiviral therapy. Resistance of HIV protease to protease inhibitors (PIs) is problematic for the effective treatment of HIV infection. The South African HIV-1 subtype C protease (C-SA PR), which contains eight polymorphisms relative to the consensus HIV-1 subtype B protease, was expressed in Escherichia coli, purified, and crystallized. The crystal structure of the C-SA PR was resolved at 2.7 Å, which is the first crystal structure of a HIV-1 subtype C protease that predominates in Africa. Structural analyses of the C-SA PR in comparison to HIV-1 subtype B proteases indicated that polymorphisms at position 36 of the homodimeric HIV-1 protease may impact on the stability of the hinge region of the protease, and hence the dynamics of the flap region. Molecular dynamics simulations showed that the flap region of the C-SA PR displays a wider range of movements over time as compared to the subtype B proteases. Reduced stability in the hinge region resulting from the absent E35-R57 salt bridge in the C-SA PR, most likely contributes to the increased flexibility of the flaps which may be associated with reduced susceptibility to PIs. An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:36

In 2012, 25 million people [71% of global human immunodeficiency virus (HIV) infection] were estimated to be living with HIV in sub-Saharan Africa. Of these, approximately 1.6 million were new infections and 1.2 million deaths occurred. South Africa alone accounted for 31% of HIV/acquired immunodeficiency syndrome (AIDS) deaths in sub-Saharan Africa. This disturbing statistic indicates that South Africa remains the epicenter of the HIV/AIDS pandemic, compounded by the fact that only 36% of HIV-positive patients in South Africa have access to antiretroviral (ARV) treatment. Drug resistance mutations have emerged, and current ARVs show reduced efficacy against non-B subtypes. In addition, several recent studies have shown an increased prevalence of non-B African HIV strains in the Americas and Europe. Therefore, the use of ARVs in a non-B HIV-1 subtype context requires further investigation. HIV-1 subtype C protease, found largely in sub-Saharan Africa, has been under-investigated when compared with the subtype B protease, which predominates in North America and Europe. This review, therefore, focuses on HIV-1 proteases from B and C subtypes.

Urine provides a diverse source of information related to a patient’s health status and is ideal for clinical proteomics due to its ease of collection. To date, most methods for the preparation of urine samples lack the throughput required to analyze large clinical cohorts. To this end, we developed a novel workflow, urine-HILIC (uHLC), based on an on-bead protein capture, clean-up, and digestion without the need for bottleneck processing steps such as protein precipitation or centrifugation. The workflow was applied to an acute kidney injury (AKI) pilot study. Urine from clinical samples and a pooled sample was subjected to automated sample preparation in a KingFisher™ Flex magnetic handling station using the novel approach based on MagReSyn^® HILIC microspheres. For benchmarking, the pooled sample was also prepared using a published protocol based on an on-membrane (OM) protein capture and digestion workflow. Peptides were analyzed by LCMS in data-independent acquisition (DIA) mode using a Dionex Ultimate 3000 UPLC coupled to a Sciex 5600 mass spectrometer. The data were searched in Spectronaut™ 17. Both workflows showed similar peptide and protein identifications in the pooled sample. The uHLC workflow was easier to set up and complete, having less hands-on time than the OM method, with fewer manual processing steps. Lower peptide and protein coefficient of variation was observed in the uHLC technical replicates. Following statistical analysis, candidate protein markers were filtered, at ≥8.35-fold change in abundance, ≥2 unique peptides and ≤1% false discovery rate, and revealed 121 significant, differentially abundant proteins, some of which have known associations with kidney injury. The pilot data derived using this novel workflow provide information on the urinary proteome of patients with AKI. Further exploration in a larger cohort using this novel high-throughput method is warranted.

Forkhead box (FOX) proteins are a ubiquitously expressed family of transcription factors that regulate the development and differentiation of a wide range of tissues in animals. The FOXP subfamily members are the only known FOX proteins capable of forming domain-swapped forkhead domain (FHD) dimers. This is proposed to be due to an evolutionary mutation (P539A) that lies in the FHD hinge loop, a key region thought to fine-tune DNA sequence specificity in the FOX transcription factors. Considering the importance of the hinge loop in both the dimerisation mechanism of the FOXP FHD and its role in tuning DNA binding, a detailed investigation into the implications of mutations within this region could provide important insight into the evolution of the FOX family. Isothermal titration calorimetry and hydrogen exchange mass spectroscopy were used to study the thermodynamic binding signature and changes in backbone dynamics of FOXP2 FHD DNA binding. Dual luciferase reporter assays were performed to study the effect that the hinge-loop mutation has on FOXP2 transcriptional activity in vivo. We demonstrate that the change in dynamics of the hinge-loop region of FOXP2 alters the energetics and mechanism of DNA binding highlighting the critical role of hinge loop mutations in regulating DNA binding characteristics of the FOX proteins.