We report the first account of a comparative analysis of the binding affinities of nine FDA-approved drugs against subtype B as well as the South African subtype C HIV PR (C-SA). A standardized protocol was used to generate the inhibitor/C-SA PR complexes with the relative positions of the inhibitors taken from the corresponding X-ray structures for subtype B complexes. The dynamics and stability of these complexes were investigated using molecular dynamics calculations. Average relative binding free energies for these inhibitors were calculated from the molecular dynamics simulation using the molecular mechanics generalized Born surface area method. The calculated energies followed a similar trend to the reported experimental binding free energies. Postdynamic hydrogen bonding and electrostatic interaction analysis of the inhibitors with both subtypes reveal similar interactions. Most inhibitors show slightly weaker binding affinities for C-SA PR. Molecular dynamics studies demonstrated increased flap movement for C-SA PR, which can perhaps explain the weaker affinities. This study serves as a standardized platform for optimizing the design of future more potent HIV C-SA PR inhibitors.
Phosphopeptide enrichment is an essential step in large-scale, quantitative phosphoproteomics by mass spectrometry. Several phosphopeptide affinity enrichment techniques exist, such as Immobilized Metal ion Affinity Chromatography (IMAC) and Metal Oxide Affinity Chromatography (MOAC). We compared Zirconium(IV) IMAC (Zr-IMAC) magnetic microparticles to more commonly used Titanium(IV) IMAC (Ti-IMAC) and TiO2 magnetic microparticles for phosphopeptide enrichment from simple and complex protein samples prior to phosphopeptide sequencing and characterization by mass spectrometry (LC-MS/MS). We optimized sample-loading conditions to increase phosphopeptide recovery for Zr-IMAC, Ti-IMAC and TiO2 based workflows by 22%, 24% and 35% respectively. The optimized protocol resulted in improved performance of Zr-IMAC over Ti-IMAC and TiO2 as well as HPLC-based Fe(III)-IMAC with up to 23% more identified phosphopeptides. The different enrichment chemistries showed a high degree of overlap but also differences in phosphopeptide selectivity and complementarity. We conclude that Zr-IMAC improves phosphoproteome coverage and recommend that this complementary and scalable affinity enrichment method is more widely used in biological and biomedical studies of cell signaling and the search for biomarkers. Data are available via ProteomeXchange with identifier PXD018273.
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.
Since its identification, HIV has continued to have a detrimental impact on the lives of millions of people throughout the world. The protease of HIV is a major target in antiviral treatment. The South African HIV–1 subtype C (C–SA) protease displays weaker binding affinity for some clinically approved protease inhibitors in comparison with the HIV–1 subtype B protease. The heavy HIV burden in sub‐Saharan Africa, where subtype C HIV–1 predominates, makes this disparity a topic of great interest. In light of this, the enzyme activity and affinity of protease inhibitors for the subtype B and C–SA proteases were determined. The relative vitality, indicating the selective advantage of polymorphisms, of the C–SA protease relative to the subtype B protease in the presence of ritonavir and darunavir was four‐ and tenfold greater, respectively. Dynamic differences that contribute to the reduced drug susceptibility of the C–SA protease were investigated by performing hydrogen–deuterium exchange/mass spectrometry (HDX/MS) on unbound subtype B and C–SA proteases. The reduced propensity to form the E35–R57 salt bridge, and alterations in the hydrophobic core of the C–SA protease, are proposed to affect the anchoring of the flexible flaps, resulting in an increased proportion of the fully open flap conformation. HDX/MS data suggested that the N–terminus of both proteases is less stable than the C–terminus of the proteases, thus explaining the increased efficacy of dimerization inhibitors targeted toward the C–terminus of HIV proteases. As far as we are aware, this is the first report on assessment of HIV protease dynamics using HDX/MS.
Membrane-bound particles in plasma are composed of exosomes, microvesicles, and apoptotic bodies and represent ~1-2% of the total protein composition. Proteomic interrogation of this subset of plasma proteins augments the representation of tissue-specific proteins, representing a 'liquid biopsy,' while enabling the detection of proteins that would otherwise be beyond the dynamic range of liquid chromatography-tandem mass spectrometry in unfractionated plasma. We have developed a one-step enrichment strategy (Mag-Net) using hyper-porous strong-anion exchange magnetic microparticles to sieve membrane-bound particles from plasma. The Mag-Net method is robust, reproducible, inexpensive, and requires <100 μL plasma input. Coupled to a quantitative data-independent mass spectrometry analytical strategy, we demonstrate that we can routinely collect results for >37,000 peptides from >4,000 plasma proteins with high precision. We demonstrate excellent quantitative accuracy and analytical reproducibility of the protocol.
Pancreatic cancer accounts for 2.8% of new cancer cases worldwide and is projected to become the second leading cause of cancer-related deaths by 2030. Patients of African ancestry appear to be at an increased risk for pancreatic ductal adenocarcinoma (PDAC), with more severe disease and outcomes. The purpose of this study was to map the proteomic and genomic landscape of a cohort of PDAC patients of African ancestry. Thirty tissues (15 tumours and 15 normal adjacent tissues) were obtained from consenting South African PDAC patients. Optimisation of the sample preparation method allowed for the simultaneous extraction of high-purity protein and DNA for SWATH-MS and OncoArray SNV analyses. We quantified 3402 proteins with 49 upregulated and 35 downregulated proteins at a minimum 2.1 fold change and FDR adjusted p-value (q-value) � 0.01 when comparing tumour to normal adjacent tissue. Many of the upregulated proteins in the tumour samples are involved in extracellular matrix formation (ECM) and related intracellular pathways. In addition, proteins such as EMIL1, KBTB2, and ZCCHV involved in the regulation of ECM proteins were observed to be dysregulated in pancreatic tumours. Downregulation of pathways involved in oxygen and carbon dioxide transport were observed. Genotype data showed missense mutations in some upregulated proteins, such as MYPN, ESTY2 and SERPINB8. Approximately 11% of the dysregulated proteins, including ISLR, BP1, PTK7 and OLFL3, were predicted to be secretory proteins. These findings help in further elucidating the biology of PDAC and may aid in identifying future plausible markers for the disease.
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.
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