3 research outputs found
The contribution of F99 to the structure and function of South African HIV-1 subtype C protease
The HIV/AIDS still remains a global health challenge with 42 million people infected
with the virus. An alarming 70% of these people reside in sub-Saharan Africa with
HIV-1 subtype C being the most prevalent subtype in this region and globally. HIV-1
protease (PR) is an obligate homodimer which plays a pivotal role in the maturation
and hence propagation of the HI virus. Although successful developments on PR
active site inhibitors have been achieved, the major limiting factor has been the
emergence of HIV drug resistant strains. It has been postulated that
disruption/dissociation of the dimer interface may lead to an inactive enzyme. The
development of small molecules and peptides has been a major research area with the
key target being the N- and C-termini antiparallel β-sheet. This is due to its highly
conserved nature and because it consists of a cluster of amino acids that contribute
most of the binding energy and stability of the dimer interface. Hence it is referred to
as a ‘hot-spot’. Therefore, binding of protease inhibitors at this site could cause
destabilisation and/or dissociation of the enzyme. The terminal residue, F99, was
mutated to an alanine disrupting the presumed lock-and-key motif it forms and in turn
creating a cavity at the N- and C-termini antiparallel β-sheet. A second mutant,
W42F/F99A, was created for monitoring tertiary structural changes exclusively at the
N- and C-termini antiparallel β-sheet. The F99A and W42F/F99A, compared to the
wild-type, showed a higher expression yield and also migrated further when separated
using tricine SDS-PAGE. Wild-type protease CD spectra showed a minimum at 214
nm and a local maximum at 230 nm, while the mutants exhibited minima at 203 nm
and absence of the local maxima. A 50% higher fluorescence intensity and a 2 nm
red-shift for the mutants versus the wild-type was observed. According to SE-HPLC
data the relative molecular weight of the wild-type, F99A and W42F/F99A are 16.4
kDa, 20.7 kDa and 18.1 kDa, respectively. Although the thermal unfolding of all three
proteases was irreversible, the unfolding transition of the wild-type was clearly
defined between 55 °C and 63 °C. The F99A and W42F/F99A unfolding curves were
linear without clearly defined transition states. The specific activity of the F99A
(0.13 μmol/min/mg) amounted to a ten-fold reduction compared to the wild-type
(1.5 μmol/min/mg). The substrate binding affinity (KM) for the F99A was 41% lower
than the wild-type when 2 μM of protein was used. The Vmax and kcat values were about 30-fold and two-fold, respectively, higher for the
wild-type when compared to the F99A. Therefore, the tricine SDS-PAGE analysis,
secondary and tertiary structural characterisation and thermal denaturation curve
showed that the F99A mutation has altered the structure causing ‘partial’ unfolding of
the protein. But, the protein still maintained minute activity. The overlap between the
ANS binding spectra of the wild-type and variants suggests that the dimeric form still
exists
Characterisation of dysregulated proteins in macrophages infected with Mycobacterium smegmatis focusing on matrix metalloproteases and their effectors
Cavitation is a key facilitator in transmission of Mycobacterium tuberculosis (M. tb). Upregulation of matrix metalloproteases (MMPs) has been documented in patients with tuberculosis (TB), while their tissue inhibitor (TIMPs) levels remained the same. Animals which can develop cavities have well-conserved MMP-1 orthologs suggesting a pivotal role of MMP-1 in cavitation. The migration of immune cells to the site of infection and maturation of the granuloma is associated with MMP-9 expression. Our understanding of the phenotypic changes induced by Mycobacterium smegmatis (M. smeg) in THP-1 macrophages is central to understanding its avirulent nature especially its effects on MMPs. The aim of this study was to evaluate the role of MMP-1 and MMP-9, and their effectors in macrophages infected with M. smeg. Differentiated THP-1 monocytes were incubated in serum-free media with or without bacilli. Thereafter, the secretome and lysate were harvested at different time points. The activity of MMPs was analysed by zymography. The activity of MMP-1 and MMP-9 were specifically determined using an MMP-1 fluorogenic assay and a non-fluorogenic MMP-9 substrate monitored using the HPLC. Discovery proteomics was performed for the 18-hour time point with the use of mass spectrometry. The generated data was used to evaluate dysregulated proteins and those that act as upstream and downstream effectors of MMPs. The phenotypic changes induced by M. smeg were also analysed. In addition to that, the hosts’ response to lipoarabinomannan H37Rv (LAM) treatment was assessed by discovery proteomics and zymography. There was an increase in gelatinase activity of secreted MMP-9 which was maintained between the 1 and 18-hour time points. The fold difference in activity between uninfected and infected declined at 24 hours, and at the 72-hour time point the uninfected was slightly higher versus the infected. The data also suggests a switch in the proteolytic repertoire of the macrophages between the 6- and 18-hour time points to one that potentially generates the same degradation products as the uninfected macrophages. The intracellular gelatinase activity of MMP-9 (82 kDa) was not significantly altered by the M. smeg infection, in fact the activity was slightly higher in the uninfected in the 18 and 24-hour time points. In contrast to MMP-9, MMP-1 was secreted in the later time points and was significantly decreased by the infection. This supports the postulation that upregulation of MMP-1 is specific for M. tb infection. The proteomics data depict significant upregulation of MMP-9 in the lysate and secretome, while TIMP-1 was exclusively expressed and secreted by infected macrophages, validating the non-destructive ECM phenotype induced by M. smeg. The dysregulation of IL-1β and COX pathways were implicated in the overexpression of MMP-9, as well as tRNA aminoacylation in alternative splicing of MMPs. The GO enrichment of exosomes is postulated to play a role in the recycling of MMP-9. Intercellular communication is hypothesised to be delivered to neighbouring cells through exosomes carrying DNA, RNA, proteins and DNA/RNA binding proteins, and via signalling scaffolds formed by the 14-3-3 proteins amongst others. LAM treatment did not induce dysregulation in the activity of expressed and secreted MMP-9, however, TIMP-1 was upregulated explaining the lack of differential gelatinase activity between treated and non-treated macrophages. The C-type mannose receptor 2 (MRC2) and C-type lectin domain family 11 (CLEC11A) were only expressed by the LAM-treated macrophages and may partake in the recognition and uptake of M. tb. Interestingly, the data indicates the presence of chromatin in the secretome which may be responsible for the formation of extracellular traps (NETs) and facilitating the transport of LAM across the glomerular basal membrane (GBM) through exosomes. Inhibition of MMP activity by TIMPs could result in decreased aggregation of NETS (aggNETS) that trap the LAM from being transported by binding to the chromatin. This decreases the concentration of LAM in urine and means MMP inhibitors that chelate the active-site zinc could decrease the sensitivity of urine-LAM detection kits
A Review of the Nucleic Acid-Based Lateral Flow Assay for Detection of Breast Cancer from Circulating Biomarkers at a Point-of-Care in Low Income Countries
The current levels of breast cancer in African women have contributed to the high mortality rates among them. In South Africa, the incidence of breast cancer is also on the rise due to changes in behavioural and biological risk factors. Such low survival rates can be attributed to the late diagnosis of the disease due to a lack of access and the high costs of the current diagnostic tools. Breast cancer is asymptomatic at early stages, which is the best time to detect it and intervene to prevent high mortality rates. Proper risk assessment, campaigns, and access to adequate healthcare need to be prioritised among patients at an early stage. Early detection of breast cancer can significantly improve the survival rate of breast cancer patients, since therapeutic strategies are more effective at this stage. Early detection of breast cancer can be achieved by developing devices that are simple, sensitive, low-cost, and employed at point-of-care (POC), especially in low-income countries (LICs). Nucleic-acid-based lateral flow assays (NABLFAs) that combine molecular detection with the immunochemical visualisation principles, have recently emerged as tools for disease diagnosis, even for low biomarker concentrations. Detection of circulating genetic biomarkers in non-invasively collected biological fluids with NABLFAs presents an appealing and suitable method for POC testing in resource-limited regions and/or LICs. Diagnosis of breast cancer at an early stage will improve the survival rates of the patients. This review covers the analysis of the current state of NABLFA technologies used in developing countries to reduce the scourge of breast cancer