An improved production and purification protocol for recombinant soluble human fibroblast activation protein alpha.

Fibroblast activation protein alpha (FAP) is a cell-surface expressed type II glycoprotein that has a unique proteolytic activity. FAP has active soluble forms that retain the extracellular portion but lack the transmembrane domain and cytoplasmic tail. FAP expression is normally very low in adult tissue but is highly expressed by activated fibroblasts in sites of tissue remodelling. Thus, FAP is a potential biomarker and pharmacological target in liver fibrosis, atherosclerosis, cardiac fibrosis, arthritis and cancer. Understanding the biological significance of FAP by investigating protein structure, interactions and activities requires reliable methods for the production and purification of abundant pure and stable protein. We describe an improved production and purification protocol for His6-tagged recombinant soluble human FAP. A modified baculovirus expression construct was generated using the pFastBac1 vector and the gp67 secretion signal to produce abundant active soluble recombinant human FAP (residues 27-760) in insect cells. The FAP purification protocol employed ammonium sulphate precipitation, ion exchange chromatography, immobilised metal affinity chromatography and ultrafiltration. High purity was achieved, as judged by gel electrophoresis and specific activity. The purified 82 kDa FAP protein was specifically inhibited by a FAP selective inhibitor, ARI-3099, and was inhibited by zinc with an IC50 of 25 µM. Our approach could be adopted for producing the soluble portions of other type II transmembrane glycoproteins to study their structure and function.

Identification of Novel Natural Substrates of Fibroblast Activation Protein-alpha by Differential Degradomics and Proteomics.

Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2.

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

Dipeptidyl peptidase 9 substrates and their discovery: current progress and the application of mass spectrometry-based approaches.

The enzyme members of the dipeptidyl peptidase 4 (DPP4) gene family have the very unusual capacity to cleave the post-proline bond to release dipeptides from the N-terminus of peptide/protein substrates. DPP4 and related enzymes are current and potential therapeutic targets in the treatment of type II diabetes, inflammatory conditions and cancer. Despite this, the precise biological function of individual dipeptidyl peptidases (DPPs), other than DPP4, and knowledge of their in vivo substrates remains largely unknown. For many years, identification of physiological DPP substrates has been difficult due to limitations in the available tools. Now, with advances in mass spectrometry based approaches, we can discover DPP substrates on a system wide-scale. Application of these approaches has helped reveal some of the in vivo natural substrates of DPP8 and DPP9 and their unique biological roles. In this review, we provide a general overview of some tools and approaches available for protease substrate discovery and their applicability to the DPPs with a specific focus on DPP9 substrates. This review provides comment upon potential approaches for future substrate elucidation.

Differential Effects of 'Vaping' on Lipid and Glucose Profiles and Liver Metabolic Markers in Obese Versus Non-obese Mice.

Tobacco smoking increases the risk of metabolic disorders due to the combination of harmful chemicals, whereas pure nicotine can improve glucose tolerance. E-cigarette vapour contains nicotine and some of the harmful chemicals found in cigarette smoke at lower levels. To investigate how e-vapour affects metabolic profiles, male Balb/c mice were exposed to a high-fat diet (HFD, 43% fat, 20kJ/g) for 16weeks, and e-vapour in the last 6weeks. HFD alone doubled fat mass and caused dyslipidaemia and glucose intolerance. E-vapour reduced fat mass in HFD-fed mice; only nicotine-containing e-vapour improved glucose tolerance. In chow-fed mice, e-vapour increased lipid content in both blood and liver. Changes in liver metabolic markers may be adaptive responses rather than causal. Future studies can investigate how e-vapour differentially affects metabolic profiles with different diets.

DPP9: Comprehensive In Silico Analyses of Loss of Function Gene Variants and Associated Gene Expression Signatures in Human Hepatocellular Carcinoma.

Dipeptidyl peptidase (DPP) 9, DPP8, DPP4 and fibroblast activation protein (FAP) are the four enzymatically active members of the S9b protease family. Associations of DPP9 with human liver cancer, exonic single nucleotide polymorphisms (SNPs) in DPP9 and loss of function (LoF) variants have not been explored. Human genomic databases, including The Cancer Genome Atlas (TCGA), were interrogated to identify DPP9 LoF variants and associated cancers. Survival and gene signature analyses were performed on hepatocellular carcinoma (HCC) data. We found that DPP9 and DPP8 are intolerant to LoF variants. DPP9 exonic LoF variants were most often associated with uterine carcinoma and lung carcinoma. All four DPP4-like genes were overexpressed in liver tumors and their joint high expression was associated with poor survival in HCC. Increased DPP9 expression was associated with obesity in HCC patients. High expression of genes that positively correlated with overexpression of DPP4, DPP8, and DPP9 were associated with very poor survival in HCC. Enriched pathways analysis of these positively correlated genes featured Toll-like receptor and SUMOylation pathways. This comprehensive data mining suggests that DPP9 is important for survival and that the DPP4 protease family, particularly DPP9, is important in the pathogenesis of human HCC.

Dipeptidyl Peptidase Inhibition Enhances CD8 T Cell Recruitment and Activates Intrahepatic Inflammasome in a Murine Model of Hepatocellular Carcinoma.

The mRNA expression of the dipeptidyl peptidase 4 (DPP4) gene family is highly upregulated in human hepatocellular carcinoma (HCC) and is associated with poor survival in HCC patients. Compounds that inhibit the DPP4 enzyme family, such as talabostat and ARI-4175, can mediate tumour regression by immune-mediated mechanisms that are believed to include NLRP1 activation. This study investigated the expression and activity of the DPP4 family during the development of HCC and evaluated the efficacy of ARI-4175 in the treatment of early HCC in mice. This first report on this enzyme family in HCC-bearing mice showed DPP9 upregulation in HCC, whereas intrahepatic DPP8/9 and DPP4 enzyme activity levels decreased with age. We demonstrated that ARI-4175 significantly lowered the total number of macroscopic liver nodules in these mice. In addition, ARI-4175 increased intrahepatic inflammatory cell infiltration, including CD8+ T cell numbers, into the HCC-bearing livers. Furthermore, ARI-4175 activated a critical component of the inflammasome pathway, caspase-1, in these HCC-bearing livers. This is the first evidence of caspase-1 activation by a pan-DPP inhibitor in the liver. Our data suggest that targeting the DPP4 enzyme family may be a novel and effective approach to promote anti-tumour immunity in HCC via caspase-1 activation.

Animal models for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) represents ~90% of all cases of primary liver cancer and occurs predominantly in patients with underlying chronic liver disease and cirrhosis. Establishing appropriate animal models for HCC is required for basic and translational studies, especially the models that can recapitulate one of the human disease settings. Current animal models can be categorized as chemically-induced, genetically-engineered, xenograft, or a combination of these with each other or with a metabolic insult. A single approach to resemble human HCC in animals is not sufficient. Combining pathogenic insults in animal models may more realistically recapitulate the multiple etiologic agents occurring in humans. Combining chemical injury with metabolic disorder or alcohol consumption in mice reduces the time taken to hepatocarcinogenesis. Genetically-engineering weak activation of HCC-promoting pathways combined with disease-specific injury models will possibly mimic the pathophysiology of human HCC in distinct clinical settings.

Fibroblast activation protein in liver fibrosis.

Fibroblast activation protein (FAP) belongs to the dipeptidyl peptidase IV (DPP4; CD26) gene family. Other related genes in this family of enzyme include DPP4, 8 and 9. The FAP serine protease has the rare property of both dipeptidyl peptidase and endopeptidase activities capable of cleaving the post-proline bond at two or more residues from the N-terminus. FAP is involved in a variety of biological processes but its expression in healthy tissues is low. In contrast, FAP is significantly elevated in pathological conditions such as at sites of tissue remodelling and repair. Its differential pattern of expression in diseases supports the emerging concept for FAP as a potential disease biomarker as well as a useful therapeutic target for drug intervention. This review summarizes the current knowledge of FAP, particularly its diagnostic and pathological significance in liver fibrosis.

Immune regeneration in irradiated mice is not impaired by the absence of DPP9 enzymatic activity.

The ubiquitous intracellular protease dipeptidyl peptidase 9 (DPP9) has roles in antigen presentation and B cell signaling. To investigate the importance of DPP9 in immune regeneration, primary and secondary chimeric mice were created in irradiated recipients using fetal liver cells and adult bone marrow cells, respectively, using wild-type (WT) and DPP9 gene-knockin (DPP9S729A) enzyme-inactive mice. Immune cell reconstitution was assessed at 6 and 16 weeks post-transplant. Primary chimeric mice successfully regenerated neutrophils, natural killer, T and B cells, irrespective of donor cell genotype. There were no significant differences in total myeloid cell or neutrophil numbers between DPP9-WT and DPP9S729A-reconstituted mice. In secondary chimeric mice, cells of DPP9S729A-origin cells displayed enhanced engraftment compared to WT. However, we observed no differences in myeloid or lymphoid lineage reconstitution between WT and DPP9S729A donors, indicating that hematopoietic stem cell (HSC) engraftment and self-renewal is not diminished by the absence of DPP9 enzymatic activity. This is the first report on transplantation of bone marrow cells that lack DPP9 enzymatic activity.

Multiple liver insults synergize to accelerate experimental hepatocellular carcinoma.

The urgent unmet need for hepatocellular carcinoma (HCC) therapies is addressed here by characterising a novel mouse model of HCC in the context of ongoing liver damage and overnutrition. Male C57Bl/6J mice were treated with diethylnitrosamine (DEN) and thioacetamide (TAA), and some were provided with an atherogenic high fat diet (HFD). Inflammation, steatosis, fibrosis, 87 genes, liver lesions and intratumoural leukocyte subsets were quantified up to 24 weeks of age. Adding HFD to DEN/TAA increased fibrosis, steatosis and inflammation, and the incidence of both HCC and non-HCC dysplastic lesions. All lesions contained α-SMA positive fibroblasts. Macrophage marker F4/80 was not significantly different between treatment groups, but the macrophage-associated genes Arg-1 and Cd47 were differentially expressed. Fibrosis, cancer and cell death associated genes were upregulated in DEN/TAA/HFD livers. Fewer Kupffer cells and plasmacytoid dendritic cells were in tumours compared to control liver. In conclusion, combining a hepatotoxin with an atherogenic diet produced more intrahepatic tumours, dysplastic lesions and fibrosis compared to hepatotoxin alone. This new HCC model provides a relatively rapid means of examining primary HCC and potential therapies in the context of multiple hepatotoxins including those derived from overnutrition.

Hepatocellular carcinoma: Mouse models and the potential roles of proteases.

Primary liver cancer is the second most common cause of mortality from cancer. The most common models of hepatocellular carcinoma, which use a chemical and/or metabolic insult, xenograft, or genetic manipulation, are discussed in this review. In the tumour microenvironment lymphocytes, fibroblasts, endothelial cells and antigen presenting cells are important determinants of cell fate. These cells make a range of proteases that modify the biological activity of other proteins, particularly extracellular matrix proteins that alter cell migration of tumour cells, fibroblasts and leucocytes, and chemokines that alter leucocyte migration. The DPP4 family of post-proline peptidase enzymes modifies cell movement and the activities of many bioactive molecules including growth factors and chemokines.