ResearchSpace :: Browsing by Author "Naicker, Previn"

Browsing by Author "Naicker, Previn"

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Boar seminal plasma proteomic profiling for biomarker discovery
(2022-12) Mokwena, Pateswana W; Stoychev, Stoyan H; Buthelezi, Sindisiwe G; Naicker, Previn; Ramukhithi, FV; Lehloenya, KC
As an important technology in the swine industry, artificial insemination is of great significance in the preservation of elite breeds and the improvement of the utilisation rate of elite breeds, which makes the quality of preserved pig semen particularly critical. This abstract mainly evaluates a proteomic method to profile boar seminal plasma of different breeds and evaluate potential candidate biomarkers. It has considerable practical value, new technical methods, reasonable test steps, and important theoretical reference value for the test results. In this study, various sample preparation methods were compared (acetone precipitation [urea and SDS resuspension] vs non precipitated) to develop a standard workflow that can be used for large proteomics studies; the optimal method (non-precipitated) was then tested on a pilot study that was composed of seminal plasma from eight boars belonging to three different breeds (two Large White, three Kolbroek, and three Windsnyer). Briefly, proteins from seminal plasma were digested on an automated FlexDuo system using trypsin. The peptides were pefractionated into six fractions using the hydrophilic interaction liquid chromatography/strong anion exchange method. The fractionated peptides were then analysed on the Evosep One connected to a 6600 TripleTOF Sciex mass spectrometer. A data-independent acquisition method was used. Spectronaut software was used for protein identifications and differential protein analysis. A total of 1,314 peptides were identified across runs from 233 protein groups. The proteins identified covered a wide range of biological functions related to sperm function, such as AQN-1, AWN, and SOD. To mention a few candidate proteins of interest, superoxide dismutase, glutathione transferase, ß hexosaminidase, and the prostaglandin isomerase were diferentially expressed among breeds and also in individual boars within the same breed. The in-solution method used in this study is semi-automated and can be used for high throughput biomarker and validation studies.
Candida parapsilosis cell wall proteome characterization and effectiveness against hematogenously disseminated candidiasis in a murine model
(2023-03) Gong, X; Srivastava, V; Naicker, Previn; Khan, A; Ahmad, A
Candida parapsilosis poses huge treatment challenges in the clinical settings of South Africa, and often causes infections among immunocompromised patients and underweight neonates. Cell wall proteins have been known to play vital roles in fungal pathogenesis, as these are the first points of contact toward environments, the host, and the immune system. This study characterized the cell wall immunodominant proteins of pathogenic yeast C. parapsilosis and evaluated their protective effects in mice, which could add value in vaccine development against the rising C. parapsilosis infections. Among different clinical strains, the most pathogenic and multidrug-resistant C. parapsilosis isolate was selected based on their susceptibility towards antifungal drugs, proteinase, and phospholipase secretions. Cell wall antigens were prepared by ß-mercaptoethanol/ammonium bicarbonate extraction from selected C. parapsilosis strains. Antigenic proteins were identified using LC–MS/MS, where 933 proteins were found, with 34 being immunodominant. The protective effect of the cell wall immunodominant proteins was observed by immunizing BALB/c mice with cell wall protein extracts. After the immunization and booster, the BALC/c mice were challenged with a lethal dose of C. parapsilosis. In vivo results demonstrated increased survival rates and lower fungal burden in vital organs in the immunized mice compared to the unimmunized mice, thereby confirming the immunogenic property of cell wall-associated proteins of C. parapsilosis. Therefore, these results advocated the potential of these cell wall proteins to act as biomarkers for the development of diagnostic assays and/or vaccines against infections caused by C. parapsilosis.
Candida parapsilosis cell wall proteome characterization and effectiveness against hematogenously disseminated candidiasis in a murine model
(2023-03) Gong, X; Srivastava, V; Naicker, Previn; Khan, A; Ahmad, A
Candida parapsilosis poses huge treatment challenges in the clinical settings of South Africa, and often causes infections among immunocompromised patients and underweight neonates. Cell wall proteins have been known to play vital roles in fungal pathogenesis, as these are the first points of contact toward environments, the host, and the immune system. This study characterized the cell wall immunodominant proteins of pathogenic yeast C. parapsilosis and evaluated their protective effects in mice, which could add value in vaccine development against the rising C. parapsilosis infections. Among different clinical strains, the most pathogenic and multidrug-resistant C. parapsilosis isolate was selected based on their susceptibility towards antifungal drugs, proteinase, and phospholipase secretions. Cell wall antigens were prepared by ß-mercaptoethanol/ammonium bicarbonate extraction from selected C. parapsilosis strains. Antigenic proteins were identified using LC–MS/MS, where 933 proteins were found, with 34 being immunodominant. The protective effect of the cell wall immunodominant proteins was observed by immunizing BALB/c mice with cell wall protein extracts. After the immunization and booster, the BALC/c mice were challenged with a lethal dose of C. parapsilosis. In vivo results demonstrated increased survival rates and lower fungal burden in vital organs in the immunized mice compared to the unimmunized mice, thereby confirming the immunogenic property of cell wall-associated proteins of C. parapsilosis. Therefore, these results advocated the potential of these cell wall proteins to act as biomarkers for the development of diagnostic assays and/or vaccines against infections caused by C. parapsilosis.
Comparative Proteomic Analysis of Huh7 Cells Transfected with Sub-Saharan African Hepatitis B Virus (Sub)genotypes Reveals Potential Oncogenic Factors
(2024-06) Padarath, Kiyasha; Deroubaix, A; Naicker, Previn; Stoychev, S; Kramvis, A
In sub-Saharan Africa (SSA), the (sub)genotypes A1, D3, and E of the hepatitis B virus (HBV) prevail. Individuals infected with subgenotype A1 have a 4.5-fold increased risk of HCC compared to those infected with other (sub)genotypes. The effect of (sub)genotypes on protein expression and host signalling has not been studied. Mass spectrometry was used to analyse the proteome of Huh7 cells transfected with replication-competent clones. Proteomic analysis revealed significantly differentially expressed proteins between SSA (sub)genotypes. Different (sub)genotypes have the propensity to dysregulate specific host signalling pathways. Subgenotype A1 resulted in dysregulation within the Ras pathway. Ras-associated protein, RhoC, was significantly upregulated in cells transfected with subgenotype A1 compared to those transfected with other (sub)genotypes, on both a proteomic (>1.5-fold) and mRNA level (p < 0.05). Two of the main cellular signalling pathways involving RHOC, MAPK and PI3K/Akt/mTOR, regulate cell growth, motility, and survival. Downstream signalling products of these pathways have been shown to increase MMP2 and MMP9 expression. An extracellular MMP2 and MMP9 ELISA revealed a non-significant increase in MMP2 and MMP9 in the cells transfected with A1 compared to the other (sub)genotypes (p < 0.05). The upregulated Ras-associated proteins have been implicated as oncoproteins in various cancers and could contribute to the increased hepatocarcinogenic potential of A1.
Comparison of the proteome of Huh7 Cells transfected with Hepatitis B Virus Subgenotype A1, with or without G1862T
(2024-07) Padarath, A; Deroubaix, A; Naicker, Previn; Stoychev, S
HBeAg is a non-structural, secreted protein of hepatitis B virus (HBV). Its p25 precursor is post-translationally modified in the endoplasmic reticulum. The G1862T precore mutation leads to the accumulation of P25 in the endoplasmic reticulum and activation of unfolded protein response. Using mass spectrometry, comparative proteome profiling of Huh-7 cells transfected with wildtype (WT) or G1862T revealed significantly differentially expressed proteins resulting in 12 dysregulated pathways unique to WT-transfected cells and 7 shared between cells transfected with either WT or G1862T. Except for the p38 MAPK signalling pathway, WT showed a higher number of DEPs than G1862T-transfected cells in all remaining six shared pathways. Two signalling pathways: oxidative stress and cell cycle signalling were differentially expressed only in cells transfected with G1862T. Fifteen pathways were dysregulated in G1862T-transfected cells compared to WT. The 15 dysregulated pathways were involved in the following processes: MAPK signalling, DNA synthesis and methylation, and extracellular matrix organization. Moreover, proteins involved in DNA synthesis signalling (replication protein A (RPA) and DNA primase (PRIM2)) were significantly upregulated in G1862T compared to WT. This upregulation was confirmed by mRNA quantification of both genes and immunofluorescent confocal microscopy for RPA only. The dysregulation of the pathways involved in these processes may lead to immune evasion, persistence, and uncontrolled proliferation, which are hallmarks of cancer.
Covalent binding of human two-domain CD4 to an HIV-1 subtype C SOSIP.664 trimer modulates its structural dynamics
(2022-07) Tumba, NL; Naicker, Previn; Stoychev, Stoyan H; Killick, MA; Owen, GR; Papathanasopoulos, MA
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) mediates host cell infection by binding to the cellular receptor CD4. Recombinant Env bound to CD4 has been explored for its potential as an HIV vaccine immunogen as receptor binding exposes otherwise shielded, conserved functional sites. Previous preclinical studies showed an interchain disulphide linkage facilitated between Env and 2dCD4S60C generates an immunogenic complex that elicits potent, broadly neutralizing antibodies (bNAbs) against clinically relevant HIV-1. This study investigated conformational dynamics of 2dCD4WT and 2dCD4S60C bound to an HIV-1C SOSIP.664 Env trimer using hydrogen-deuterium exchange mass spectrometry. The Env:2dCD4S60C complex maintains key contact residues required for MHCII and Env/gp120 binding and the residues encompassing Ibalizumab's epitope. Important residues remaining anchored, with an increased flexibility in surrounding regions, evidenced by the higher exchange seen in flanking residues compared to Env:2dCD4WT. While changes in Env:2dCD4S60C dynamics in domain 1 were moderate, domain 2 exhibited greater variation. Lack of stability-inducing H-bonds in these allosteric sites suggest the improved immunogenicity of Env:2dCD4S60C result from exposed CD4 residues providing diverse/novel antigenic targets for the development of potent, broadly neutralizing Ibalizumab-like antibodies.
The forkhead domain hinge-loop plays a pivotal role in DNA binding and transcriptional activity of FOXP2
(De Gruyter, 2018-07) Morris, G; Stoychev, Stoyan H; Naicker, Previn; Dirr, HW; Fanucchi, Stephanie
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.
Inhibition of the complement pathway induces cellular proliferation and migration in pancreatic ductal adenocarcinoma
(2024-02) Nsingwane, Z; Naicker, Previn; Omoshoro-Jones, J; Devar, J; Smith, M; Candy, G; Augustine, TN; Nweke, EE
Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a growing incidence and mortality despite novel therapeutic strategies. Its aggressiveness and difficulty in treatment suggest the need for a better understanding of associated molecular mechanisms that could be targeted for treatment. The complement signalling pathway may play diverse roles in PDAC by eliciting an immune response, inducing inflammatory responses, and elevating pathways linked to chemoresistance. However, their role in the progression of PDAC is not fully understood. This study aimed to identify potential immune response-related targets in a group of patients. Thirty tissue samples (tumours and corresponding normal tissues) were obtained from 15 PDAC patients, 34 plasma samples from 25 PDAC patients, six patients with chronic pancreatitis, and three healthy control participants. Targeted pathway-specific polymerase chain reaction (PCR) analysis was conducted to determine the gene expression profiles of immune-response related genes. The circulating levels of complement proteins C3 and C5 were further investigated. Pharmacological inhibition of the complement pathway in MIAPaCa-2 pancreatic cancer cell lines was performed, and the effect was assessed by cell proliferation, cell migration, and cell cycle assays. Finally, Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) was performed to identify potential molecular mechanisms during inhibition. The results identified C3 as overly expressed in early PDAC compared to later stages in plasma (p = 0.047). Pharmacological inhibition of the complement pathway led to increased cell growth (p < 0.0001), proliferation (p = 0.001) and migration (p = 0.002) in vitro. Proteomic analysis implicated several proteins, such as the mitochondrial and histone proteins, that could play a role in inducing this phenotype. Complement C3 and C5 are elevated in PDAC samples compared to healthy ones. Furthermore, the inhibition of the complement pathway was shown in vitro to result in a more aggressive phenotype by stimulating cellular growth, proliferation, and migration, indicating the involvement of complement C3 and C5 in tumour progression. This study helps to delineate further the role of the complement pathway in PDAC progression.
A look at emerging therapeutic targets for gallbladder cancer: A multi-omics approach
(Springer Nature, 2023-01) Baichan, P; Naicker, Previn; Devar, J; Smith, M; Nweke, EE; Shukla, VK; Pandey, M; Dixit, R
Gallbladder cancer (GBC) is the most frequent biliary tract cancer (including cancers of the intra- and extrahepatic biliary tree). Like many biliary tract carcinomas, GBC is identified by late diagnosis, poor prognosis, and ineffective treatment. Surgery remains the most effective management strategy. Nevertheless, the 5-year survival rate of GBC ranges from approximately 0 to 12%, thus better treatment modalities are required. Over recent years, a multi-omics approach has been implored in the discovery of therapeutic biomarkers. In this chapter, we review known chemotherapeutic drugs used in GBC treatment. Then, we extensively analyze the discovery of several proteins, genes, microRNAs, mutations, metabolites, and microbes whose expressions are dysregulated in GBC. Importantly, we highlight their potential use as therapeutic biomarkers due to their functions in GBC progression. Lastly, we review emerging strategies such as immunotherapy and their potential of improving patient survival.
Proteomic analysis identifies dysregulated proteins and associated molecular pathways in a cohort of gallbladder cancer patients of African ancestry
(2023-03) Baichan, P; Naicker, Previn; Augustine, TN; Smith, M; Candy, G; Devar, J; Nweke, EE
Background Gallbladder cancer (GBC) is a lethal cancer with a poor prognosis. The lack of specifc and sensitive biomarkers results in delayed diagnosis with most patients presenting at late stages of the disease. Furthermore, there is little known about the molecular mechanisms associated with GBC, especially in patients of African ancestry. This study aimed to determine dysregulated proteins in South African GBC patients to identify potential mechanisms of the disease progression and plausible biomarkers. Methods Tissues (27 GBC, 13 Gallstone disease, and 5 normal tissues) and blood plasma (54 GBC and 73 Benign biliary pathology) were obtained from consenting patients. Protein extraction was performed on all tissues and liquid chromatography-mass spectrometry was used for proteomic profling. A project-specifc spectral library was built using the Pulsar search algorithm. Principal component and Spearman’s rank correlation analyses were performed using PAST (V4.07b). Pathway and Network analyses were conducted using REACTOME (v3.7) and stringAPP (v1.7.0), respectively. Results In the tissue sample group, there were 62 and 194 dysregulated proteins in GBC compared to normal and gallstone groups, respectively. In the plasma group, there were 33 altered proteins in GBC compared to the benign biliary pathology group. We found 9 proteins (APOA1, APOA2, RET4, TTR, HEMO, HBB, HBA, PIGR, and APOE) to be commonly dysregulated in both tissue and plasma. Furthermore, a subset analysis demonstrated that 2 proteins, S100A8 and S100A9, were downregulated in GBC patients with GD history compared to those without. Pathway analysis showed that the dysregulated proteins in GBC patients were enriched in pathways involved in smooth muscle contraction, metabolism, ECM organization, and integrin cell surface interactions. Conclusion The identifed dysregulated proteins help in understanding GBC molecular mechanisms in our patient group. Furthermore, the alteration of specifc proteins in both tissue and plasma samples suggests their potential utility as biomarkers of GBC in this sample cohort.
Proteomic analysis identifies dysregulated proteins in albuminuria: A South African pilot study
(2024-08) Khoza, S; George, JA; Naicker, Previn; Stoychev , SH; Fabian, J; Govender, Ireshyn S
Albuminuria may precede decreases in the glomerular filtration rate (GFR) and both tests are insensitive predictors of early stages of kidney disease. Our aim was to characterise the urinary proteome in black African individuals with albuminuria and well-preserved GFR from South Africa. This case-controlled study compared the urinary proteomes of 52 normoalbuminuric (urine albumin: creatinine ratio (uACR) < 3 mg/mmol) and 56 albuminuric (uACR ≥ 3 mg/mmol) adults of black African ethnicity. Urine proteins were precipitated, reduced, alkylated, digested, and analysed using an Evosep One LC (Evosep Biosystems, Odense, Denmark) coupled to a Sciex 5600 Triple-TOF (Sciex, Framingham, MA, USA) in data-independent acquisition mode. The data were searched on SpectronautTM 15. Differentially abundant proteins (DAPs) were filtered to include those with a ≥2.25-fold change and a false discovery rate ≤ 1%. Receiver–operating characteristic curves were used to assess the discriminating abilities of proteins of interest. Pathway analysis was performed using Enrichr software. As expected, the albuminuric group had higher uACR (7.9 vs. 0.55 mg/mmol, p < 0.001). The median eGFR (mL/min/1.73 m2) showed no difference between the groups (111 vs. 114, p = 0.707). We identified 80 DAPs in the albuminuria group compared to the normoalbuminuria group, of which 59 proteins were increased while 21 proteins were decreased in abundance. We found 12 urinary proteins with an AUC > 0.8 and a p < 0.001 in the multivariate analysis. Furthermore, an 80-protein model was developed that showed a high AUC ˃ 0.907 and a predictive accuracy of 91.3% between the two groups. Pathway analysis found that the DAPs were involved in insulin growth factor (IGF) functions, innate immunity, platelet degranulation, and extracellular matrix organization. In albuminuric individuals with a well-preserved eGFR, pathways involved in preventing the release and uptake of IGF by insulin growth factor binding protein were significantly enriched. These proteins are indicative of a homeostatic imbalance in a variety of cellular processes underlying renal dysfunction and are implicated in chronic kidney disease.
Protocol for high-throughput semi-automated label-free- or TMT-based phosphoproteome profiling
(2023-09) Koenig, C; Martinez-Val, A; Naicker, Previn; Stoychev, S; Jordaan, J; Olsen, JV
Tandem mass tags data-dependent acquisition (TMT-DDA) as well as data-independent acquisition-based label-free quantification (LFQ-DIA) have become the leading workflows to achieve deep proteome and phosphoproteome profiles. We present a modular pipeline for TMT-DDA and LFQ-DIA that integrates steps to perform scalable phosphoproteome profiling, including protein lysate extraction, clean-up, digestion, phosphopeptide enrichment, and TMT-labeling. We also detail peptide and/or phosphopeptide fractionation and pre-mass spectrometry desalting and provide researchers guidance on choosing the best workflow based on sample number and input.
SWATH-MS based proteomic profiling of Pancreatic Ductal Adenocarcinoma tumours reveals the interplay between the extracellular matrix and related intracellular pathways
(medRxiv, 2020-06) Nweke, EE; Naicker, Previn; Aaron, S; Stoychev, Stoyan J; Tabb, DL; Jones, OJ; Smith, MD; Candy, GP
Pancreatic cancer accounts for 2.8% of new cancer cases worldwide and is projected to become by 2030 the second leading cause of cancer-related deaths. Patients of African ancestry appear to be at an increased risk for pancreatic ductal adenocarcinoma (PDAC), with worse severity 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. Proteins such as EMIL1, ZCCHV and KBTB2 involved in the regulation of ECM proteins were observed to be dysregulated in pancreatic tumours. Approximately 11% of the dysregulated proteins, including ISLR, BP1, PTK7 and OLFL3, were predicted to be secretory proteins. Additionally, we identified missense mutations in some upregulated proteins, such as MYPN, ESTY2 and SERPINB8. These findings help in further elucidating the biology of PDAC and may aid in identifying future plausible markers for the disease.
Targeting gallbladder cancer: a pathway based perspective
(Springer, 2020-02) Baichan, P; Naicker, Previn; Devar, JWS; Smith, M; Candy, GP; Nweke, E
Gallbladder cancer (GBC) has a poor prognosis with a 5-year survival rate suggesting the need for more effective treatment strategies. Studying the cross-talk of several pathways involved in crucial cellular and biological processes such as cell growth, proliferation, migration and apoptosis would prove beneficial in identifying key players of GBC progression and targeting them. This review highlights several pathways known to be dysregulated in GBC onset and progression and describes known and potential targets. Within these pathways, there are proteins involved in the signalling cascade, which may be targeted as potential biomarkers and drug targets. Furthermore, the cross-talk of these pathways is investigated in the context of GBC and the implications thereof. A better understanding of the pathways involved in GBC pathogenesis will aid clinicians in the prognosis, diagnosis and treatment of patients. There are significant clinical implications of GBC pathway-based studies as they permit the understanding of onset and progression of the disease.
Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9
(2022-08) Singh, Advaita A; Pillay, Priyen; Naicker, Previn; Alexandre, Kabamba B; Malatji, Kanyane; Mach, L; Steinkellner, H; Vorster, J; Chikwamba, Rachel K; Tsekoa, Tsepo L
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 XTFT)-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 differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and XTFT 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.
Unraveling the interplay between the leucine zipper and forkhead domains of FOXP2: Implications for DNA binding, stability and dynamics
(2024-05) Perumal, CM; Thulo, M; Buthelezi, Sindisiwe G; Naicker, Previn; Stoychev, Stoyan; Lahki, A; Fanucchi, S
FOXP2 is a transcription factor associated with speech and language. Like other FOX transcription factors, it has a DNA binding region called the forkhead domain (FHD). This domain can exist as a monomer or a domain swapped dimer. In addition to the FHD, the leucine zipper region (LZ) of FOXP2 is also believed to be associated with both DNA binding and oligomerization. To better understand the relationship between DNA binding and oligomerization of FOXP2, we investigated its structure, stability and dynamics, focusing specifically on the FHD and the LZ. We did this by using two constructs: one containing the isolated FHD and one containing both the LZ and the FHD (LZ-END).We demonstrate in this work, that while the FHD maintains a monomeric form that is capable of binding DNA, the LZ-END undergoes a dynamic transition between oligomeric states in the presence of DNA. Our findings suggest that FOXP2's LZ domain influences DNA binding affinity through a change in oligomeric state. We show through hydrogen exchange mass spectroscopy that certain parts of the FHD and interlinking region become less dynamic when in the presence of DNA, confirming DNA binding and oligomerization in these regions. Moreover, the detection of a stable equilibrium intermediate state during LZ-END unfolding supports the idea of cooperation between these two domains. Overall, our study sheds light on the interplay between two FOXP2 domains, providing insight into the protein's ability to respond dynamically to DNA, and enriching our understanding of FOXP2's role in gene regulation.
Urine-HILIC: Automated sample preparation for bottom-up urinary proteome profiling in clinical proteomics
(2023-09) Govender, Ireshyn S; Mokoena, Rethabile; Stoychev, Stoyan; Naicker, Previn
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.
Zirconium(IV)-IMAC for phosphopeptide enrichment in phosphoproteomics
(2020-04) Diez, IA; Govender, Ireshyn S; Naicker, Previn; Stoychev, Stoyan H; Jordaan, J; Jensen, ON
Phosphopeptide enrichment is an essential step in large-scale, quantitative phosphoproteomics studies 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 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. The performance of Zr-IMAC was enhanced by 19-22% to recover up to 5173 phosphopeptides from 200 µg of protein extract from HepG2/C3A cells, making Zr-IMAC the preferred method for phosphopeptide enrichment in this study. Ti-IMAC and TiO2 performance were also optimized to improve phosphopeptide numbers by 28% and 35%, respectively. Furthermore, Zr-IMAC based phosphoproteomics in the magnetic microsphere format identified 23% more phosphopeptides than HPLC-based Fe(III)-IMAC for same sample amount (200 µg), thereby adding 37% more uniquely identified phosphopeptides. We conclude that Zr-IMAC improves phosphoproteome coverage and recommend that this affinity enrichment method should be more widely used in biological and biomedical studies of cell signalling and in the search for disease-biomarkers.

Browsing by Author "Naicker, Previn"

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