Energetic, structural and dynamic evaluation of HIV-1 proteases

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. August 2014.Human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), remains a topic of global concern even though great strides have been made to combat the virus. The high replicative rate of the virus and recombination of the variety of viral strains complicate the treatment of AIDS. There has been an increasing prevalence of African HIV strains in the Americas and Europe. The viral protease (PR) is vital for the propagation of the virus; and thus, is a major target in antiviral therapy. The HIV-1 PR enzyme from the subtype C strain; which predominates in sub- Saharan Africa, has been greatly under-investigated in comparison to the protease from the subtype B strain which predominates in North America and Europe. Enzyme activity data which were part of this work suggested that the South African HIV-1 subtype C protease (CSA PR) displays improved substrate turnover in comparison to the subtype B PR. Thermodynamics and inhibition kinetics of drug binding showed that the C-SA PR is less susceptible to certain clinically-used protease inhibitors when compared to the subtype B PR. A crystal structure of the C-SA PR was solved and showed no difference to the global structure of the subtype B PR. Molecular dynamics simulations showed that the C-SA PR exhibits a wider range of open conformations. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) was performed to elucidate the mechanism of reduced drug susceptibility displayed by the C-SA PR. HDX-MS data provided insights into the basis of the increased preference for open conformers displayed by the C-SA PR and the stability of the terminal dimer interface which is a target in protease inhibition

Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9

The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant 1XTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No dierences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and 1XTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors.The Department of Science and Innovation (DSI), South African Medical Research Council– Strategic Health Innovation Partnership (SAMRC SHIP), National Research Foundation (NRF), Council for Scientific and Industrial Research (CSIR), and the CSIR: Young Researcher Establishment Fund.http://www.frontiersin.org/Plant_Sciencedm2022Plant Production and Soil Scienc

SWATH-MS based proteomic profiling of pancreatic ductal adenocarcinoma tumours reveals the interplay between the extracellular matrix and related intracellular pathways.

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