Comparison of the Proteome of Huh7 Cells Transfected with Hepatitis B Virus Subgenotype A1, with or without G1862T.

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.

Comparative Proteomic Analysis of Huh7 Cells Transfected with Sub-Saharan African Hepatitis B Virus (Sub)genotypes Reveals Potential Oncogenic Factors.

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.

Mag-Net: Rapid enrichment of membrane-bound particles enables high coverage quantitative analysis of the plasma proteome.

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 of unfractionated plasma. We have developed an 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 collect results for >37,000 peptides from >4,000 plasma proteins with high precision. Using this analytical pipeline on a small cohort of patients with neurodegenerative disease and healthy age-matched controls, we discovered 204 proteins that differentiate (q-value < 0.05) patients with Alzheimer's disease dementia (ADD) from those without ADD. Our method also discovered 310 proteins that were different between Parkinson's disease and those with either ADD or healthy cognitively normal individuals. Using machine learning we were able to distinguish between ADD and not ADD with a mean ROC AUC = 0.98 ± 0.06.

Proteomic analysis identifies dysregulated proteins and associated molecular pathways in a cohort of gallbladder cancer patients of African ancestry.

BACKGROUND: Gallbladder cancer (GBC) is a lethal cancer with a poor prognosis. The lack of specific 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 profiling. A project-specific 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 identified dysregulated proteins help in understanding GBC molecular mechanisms in our patient group. Furthermore, the alteration of specific proteins in both tissue and plasma samples suggests their potential utility as biomarkers of GBC in this sample cohort.

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 ΔXTFT)-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 ΔXTFT 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.

Transcriptome and proteome of the corm, leaf and flower of Hypoxis hemerocallidea (African potato).

The corm of Hypoxis hemerocallidea, commonly known as the African potato, is used in traditional medicine to treat several medical conditions such as urinary infections, benign prostate hyperplasia, inflammatory conditions and testicular tumours. The metabolites contributing to the medicinal properties of H. hemerocallidea have been identified in several studies and, more recently, the active terpenoids of the plant were profiled. However, the biosynthetic pathways and the enzymes involved in the production of the terpene metabolites in H. hemerocallidea have not been characterised at a transcriptomic or proteomic level. In this study, total RNA extracted from the corm, leaf and flower tissues of H. hemerocallidea was sequenced on the Illumina HiSeq 2500 platform. A total of 143,549 transcripts were assembled de novo using Trinity and 107,131 transcripts were functionally annotated using the nr, GO, COG, KEGG and SWISS-PROT databases. Additionally, the proteome of the three tissues were sequenced using LC-MS/MS, revealing aspects of secondary metabolism and serving as data validation for the transcriptome. Functional annotation led to the identification of numerous terpene synthases such as nerolidol synthase, germacrene D synthase, and cycloartenol synthase amongst others. Annotations also revealed a transcript encoding the terpene synthase phytoalexin momilactone A synthase. Differential expression analysis using edgeR identified 946 transcripts differentially expressed between the three tissues and revealed that the leaf upregulates linalool synthase compared to the corm and the flower tissues. The transcriptome as well as the proteome of Hypoxis hemerocallidea presented here provide a foundation for future research.

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.