Publications by authors named "Mark D Gorrell"

82 Publications

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

Protein Expr Purif 2021 May 30;181:105833. Epub 2021 Jan 30.

Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia. Electronic address:

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 His-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 IC 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.
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http://dx.doi.org/10.1016/j.pep.2021.105833DOI Listing
May 2021

DPP4 Inhibitor Sitagliptin Enhances Lymphocyte Recruitment and Prolongs Survival in a Syngeneic Ovarian Cancer Mouse Model.

Cancers (Basel) 2021 Jan 27;13(3). Epub 2021 Jan 27.

Centre for Cancer Research, Hudson Institute of Medical Research, Clayton 3168, Australia.

Immunity plays a key role in epithelial ovarian cancer (EOC) progression with a well-documented correlation between patient survival and high intratumoral CD8+ to T regulatory cell (Treg) ratios. We previously identified dysregulated DPP4 activity in EOCs as a potentially immune-disruptive influence contributing to a reduction in CXCR3-mediated T-cell infiltration in solid tumours. We therefore hypothesized that inhibition of DPP4 activity by sitagliptin, an FDA-approved inhibitor, would improve T-cell infiltration and function in a syngeneic ID8 mouse model of EOC. Daily oral sitagliptin at 50 mg/kg was provided to mice with established primary EOCs. Sitagliptin treatment decreased metastatic tumour burden and significantly increased overall survival and was associated with significant changes to the immune landscape. Sitagliptin increased overall CXCR3-mediated CD8+ T-cell trafficking to the tumour and enhanced the activation and proliferation of CD8+ T-cells in tumour tissue and the peritoneal cavity. Substantial reductions in suppressive cytokines, including CCL2, CCL17, CCL22 and IL-10, were also noted and were associated with reduced CD4+ CD25+ Foxp3+ Treg recruitment in the tumour. Combination therapy with paclitaxel, however, typical of standard-of-care for patients in palliative care, abolished CXCR3-specific T-cell recruitment stimulated by sitagliptin. Our data suggest that sitagliptin may be suitable as an adjunct therapy for patients between chemotherapy cycles as a novel approach to enhance immunity, optimise T-cell-mediated function and improve overall survival.
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http://dx.doi.org/10.3390/cancers13030487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865851PMC
January 2021

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

Molecules 2020 Nov 18;25(22). Epub 2020 Nov 18.

Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia.

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.
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http://dx.doi.org/10.3390/molecules25225392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698748PMC
November 2020

Hypoxia Regulates DPP4 Expression, Proteolytic Inactivation, and Shedding from Ovarian Cancer Cells.

Int J Mol Sci 2020 Oct 30;21(21). Epub 2020 Oct 30.

Department of Molecular and Translational Sciences, Monash University, Clayton, VIC 3168, Australia.

The treatment of ovarian cancer has not significantly changed in decades and it remains one of the most lethal malignancies in women. The serine protease dipeptidyl peptidase 4 (DPP4) plays key roles in metabolism and immunity, and its expression has been associated with either pro- or anti-tumour effects in multiple tumour types. In this study, we provide the first evidence that DPP4 expression and enzyme activity are uncoupled under hypoxic conditions in ovarian cancer cells. Whilst we identified strong up-regulation of mRNA expression under hypoxic growth, the specific activity of secreted DPP4 was paradoxically decreased. Further investigation revealed matrix metalloproteinases (MMP)-dependent inactivation and proteolytic shedding of DPP4 from the cell surface, mediated by at least MMP10 and MMP13. This is the first report of uncoupled DPP4 expression and activity in ovarian cancer cells, and suggests a previously unrecognized, cell- and tissue-type-dependent mechanism for the regulation of DPP4 in solid tumours. Further studies are necessary to identify the functional consequences of DPP4 processing and its potential prognostic or therapeutic value.
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http://dx.doi.org/10.3390/ijms21218110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672561PMC
October 2020

Deletion of fibroblast activation protein provides atheroprotection.

Cardiovasc Res 2021 Mar;117(4):1060-1069

Center for Molecular Cardiology, University of Zurich, Wagistrasse 12, CH-8952 Schlieren, Switzerland.

Aims: Fibroblast activation protein (FAP) is upregulated at sites of tissue remodelling including chronic arthritis, solid tumours, and fibrotic hearts. It has also been associated with human coronary atherosclerotic plaques. Yet, the causal role of FAP in atherosclerosis remains unknown. To investigate the cause-effect relationship of endogenous FAP in atherogenesis, we assessed the effects of constitutive Fap deletion on plaque formation in atherosclerosis-prone apolipoprotein E (Apoe) or low-density lipoprotein receptor (Ldlr) knockout mice.

Methods And Results: Using en face analyses of thoraco-abdominal aortae and aortic sinus cross-sections, we demonstrate that Fap deficiency decreased plaque formation in two atherosclerotic mouse models (-46% in Apoe and -34% in Ldlr knockout mice). As a surrogate of plaque vulnerability fibrous cap thickness was used; it was increased in Fap-deficient mice, whereas Sirius red staining demonstrated that total collagen content remained unchanged. Using polarized light, atherosclerotic lesions from Fap-deficient mice displayed increased FAP targets in terms of enhanced collagen birefringence in plaques and increased pre-COL3A1 expression in aortic lysates. Analyses of the Stockholm Atherosclerosis Gene Expression data revealed that FAP expression was increased in human atherosclerotic compared to non-atherosclerotic arteries.

Conclusions: Our data provide causal evidence that constitutive Fap deletion decreases progression of experimental atherosclerosis and increases features of plaque stability with decreased collagen breakdown. Thus, inhibition of FAP expression or activity may not only represent a promising therapeutic target in atherosclerosis but appears safe at the experimental level for FAP-targeted cancer therapies.
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http://dx.doi.org/10.1093/cvr/cvaa142DOI Listing
March 2021

COVID-19 and comorbidities: A role for dipeptidyl peptidase 4 (DPP4) in disease severity?

J Diabetes 2020 Sep 27;12(9):649-658. Epub 2020 May 27.

Centenary Institute and The University of Sydney Faculty of Medicine and Health, Sydney, Australia.

The coronavirus disease 2019 (COVID-19) pandemic is caused by a novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), similar to SARS-CoV and Middle East respiratory syndrome (MERS-CoV), which cause acute respiratory distress syndrome and case fatalities. COVID-19 disease severity is worse in older obese patients with comorbidities such as diabetes, hypertension, cardiovascular disease, and chronic lung disease. Cell binding and entry of betacoronaviruses is via their surface spike glycoprotein; SARS-CoV binds to the metalloprotease angiotensin-converting enzyme 2 (ACE2), MERS-CoV utilizes dipeptidyl peptidase 4 (DPP4), and recent modeling of the structure of SARS-CoV-2 spike glycoprotein predicts that it can interact with human DPP4 in addition to ACE2. DPP4 is a ubiquitous membrane-bound aminopeptidase that circulates in plasma; it is multifunctional with roles in nutrition, metabolism, and immune and endocrine systems. DPP4 activity differentially regulates glucose homeostasis and inflammation via its enzymatic activity and nonenzymatic immunomodulatory effects. The importance of DPP4 for the medical community has been highlighted by the approval of DPP4 inhibitors, or gliptins, for the treatment of type 2 diabetes mellitus. This review discusses the dysregulation of DPP4 in COVID-19 comorbid conditions; DPP4 activity is higher in older individuals and increased plasma DPP4 is a predictor of the onset of metabolic syndrome. DPP4 upregulation may be a determinant of COVID-19 disease severity, which creates interest regarding the use of gliptins in management of COVID-19. Also, knowledge of the chemistry and biology of DPP4 could be utilized to develop novel therapies to block viral entry of some betacoronaviruses, potentially including SARS-CoV-2.
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http://dx.doi.org/10.1111/1753-0407.13052DOI Listing
September 2020

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

Sci Rep 2019 05 13;9(1):7292. Epub 2019 May 13.

Centenary Institute, The University of Sydney Faculty of Medicine and Health, Sydney, New South Wales, Australia.

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 (DPP9) 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 DPP9-reconstituted mice. In secondary chimeric mice, cells of DPP9-origin cells displayed enhanced engraftment compared to WT. However, we observed no differences in myeloid or lymphoid lineage reconstitution between WT and DPP9 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.
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http://dx.doi.org/10.1038/s41598-019-43739-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6513830PMC
May 2019

Animal models for hepatocellular carcinoma.

Biochim Biophys Acta Mol Basis Dis 2019 05 10;1865(5):993-1002. Epub 2018 Aug 10.

Centenary Institute, The University of Sydney, Newtown, New South Wales, 2042, Australia; The University of Sydney Faculty of Medicine and Health, New South Wales, 2006, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbadis.2018.08.009DOI Listing
May 2019

Non-Invasive Fluorescent Monitoring of Ovarian Cancer in an Immunocompetent Mouse Model.

Cancers (Basel) 2018 Dec 31;11(1). Epub 2018 Dec 31.

School of Health and Biomedical Sciences, RMIT University, Bundoora 3083, Australia.

Ovarian cancers (OCs) are the most lethal gynaecological malignancy, with high levels of relapse and acquired chemo-resistance. Whilst the tumour⁻immune nexus controls both cancer progression and regression, the lack of an appropriate system to accurately model tumour stage and immune status has hampered the validation of clinically relevant immunotherapies and therapeutic vaccines to date. To address this need, we stably integrated the near-infrared phytochrome iRFP720 at the genomic locus of ID8 mouse OC cells. Intrabursal ovarian implantation into C57BL/6 mice, followed by regular, non-invasive fluorescence imaging, permitted the direct visualization of tumour mass and distribution over the course of progression. Four distinct phases of tumour growth and dissemination were detectable over time that closely mimicked clinical OC progression. Progression-related changes in immune cells also paralleled typical immune profiles observed in human OCs. Specifically, we observed changes in both the CD8+ T cell effector (Teff):regulatory (Treg) ratio, as well as the dendritic cell (DC)-to-myeloid derived suppressor cell (MDSC) ratio over time across multiple immune cell compartments and in peritoneal ascites. Importantly, iRFP720 expression had no detectible influence over immune profiles. This new model permits non-invasive, longitudinal tumour monitoring whilst preserving host⁻tumour immune interactions, and allows for the pre-clinical assessment of immune profiles throughout disease progression as well as the direct visualization of therapeutic responses. This simple fluorescence-based approach provides a useful new tool for the validation of novel immuno-therapeutics against OC.
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http://dx.doi.org/10.3390/cancers11010032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356411PMC
December 2018

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

Mol Cell Proteomics 2019 01 26;18(1):65-85. Epub 2018 Sep 26.

From the ‡Centenary Institute, the University of Sydney, Locked Bag No.6, Newtown, New South Wales, 2042, Australia;; §Sydney Medical School, the University of Sydney Faculty of Medicine and Health, New South Wales, 2006, Australia;; ‖Charles Perkins Centre, the University of Sydney, New South Wales, 2006, Australia;. Electronic address:

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 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.
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http://dx.doi.org/10.1074/mcp.RA118.001046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317473PMC
January 2019

An Atypical Parvovirus Drives Chronic Tubulointerstitial Nephropathy and Kidney Fibrosis.

Cell 2018 10 13;175(2):530-543.e24. Epub 2018 Sep 13.

Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW 2050, Australia; Discipline of Dermatology, Faculty of Medicine and Health, University of Sydney, NSW 2006, Australia; Department of Dermatology, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia; Department of Dermatology, Medical University of Vienna, Vienna 1090, Austria. Electronic address:

The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed "mouse kidney parvovirus" (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.
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http://dx.doi.org/10.1016/j.cell.2018.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800251PMC
October 2018

Multiple liver insults synergize to accelerate experimental hepatocellular carcinoma.

Sci Rep 2018 07 6;8(1):10283. Epub 2018 Jul 6.

Centenary Institute, The University of Sydney, Newtown, New South Wales, 2042, Australia.

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.
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http://dx.doi.org/10.1038/s41598-018-28486-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6035229PMC
July 2018

Differential chemokine receptor expression and usage by pre-cDC1 and pre-cDC2.

Immunol Cell Biol 2018 11 20;96(10):1131-1139. Epub 2018 Jul 20.

Centenary Institute, Newtown, NSW, Australia.

Conventional dendritic cells (cDCs) are continuously replenished by bone marrow-derived precursors called pre-DCs, which traffic through the blood to peripheral tissues. Pre-DCs are a heterogeneous population that includes cDC subset-committed progenitors, namely pre-cDC1 and pre-cDC2, which give rise to mature cDC1 and cDC2, respectively. Regulation of pre-DC subset trafficking is thought to aid the host response to immune challenge. However, the molecular cues regulating pre-cDC1 versus pre-cDC2 trafficking toward peripheral sites during homeostasis and disease remain elusive. Here, we report that pre-cDC1 but not pre-cDC2 express the T helper type 1-associated chemokine receptor CXCR3. Moreover, we identify a cell-intrinsic role for CXCR3 in the trafficking of pre-cDC1 to melanoma tumors but not to non-inflamed organs. We also show that tumor cDC1 numbers can be increased pharmacologically by targeting dipeptidyl peptidase-4 (CD26), a negative regulator of CXCR3 ligands. Our findings demonstrate that pre-cDC1 trafficking is regulated distinctly from pre-cDC2, which is relevant for our understanding of the DC lineage in the context of cancer and inflammation.
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http://dx.doi.org/10.1111/imcb.12186DOI Listing
November 2018

Letter to Editor.

Dev Biol 2018 07 14;439(1). Epub 2017 Dec 14.

Centenary Institute and the Medical School of The University of Sydney, Sydney, New South Wales 2006, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.ydbio.2017.12.009DOI Listing
July 2018

Inhibition of human kynurenine aminotransferase isozymes by estrogen and its derivatives.

Sci Rep 2017 12 14;7(1):17559. Epub 2017 Dec 14.

Group in Biomolecular Structure and Informatics, Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia.

The kynurenine aminotransferase (KAT) enzymes are pyridoxal 5'-phosphate-dependent homodimers that catalyse the irreversible transamination of kynurenine into kynurenic acid (KYNA) in the tryptophan metabolic pathway. Kynurenic acid is implicated in cognitive diseases such as schizophrenia, and several inhibitors have been reported that selectively target KAT-II as it is primarily responsible for kynurenic acid production in the human brain. Not only is schizophrenia a sexually dimorphic condition, but women that have schizophrenia have reduced estrogen levels in their serum. Estrogens are also known to interact in the kynurenine pathway therefore exploring these interactions can yield a better understanding of the condition and improve approaches in ameliorating its effects. Enzyme inhibitory assays and binding studies showed that estradiol disulfate is a strong inhibitor of KAT-I and KAT-II (IC: 291.5 μM and 26.3 μM, respectively), with estradiol, estradiol 3-sulfate and estrone sulfate being much weaker (IC > 2 mM). Therefore it is possible that estrogen levels can dictate the balance of kynurenic acid in the brain. Inhibition assay results and modelling suggests that the 17-sulfate moiety in estradiol disulfate is very important in improving its potency as an inhibitor, increasing the inhibition by approximately 10-100 fold compared to estradiol.
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http://dx.doi.org/10.1038/s41598-017-17979-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730616PMC
December 2017

Circulating fibroblast activation protein activity and antigen levels correlate strongly when measured in liver disease and coronary heart disease.

PLoS One 2017 5;12(6):e0178987. Epub 2017 Jun 5.

Department of Molecular Hepatology, Centenary Institute, Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia.

Background And Aim: Circulating fibroblast activation protein (cFAP) is a constitutively active enzyme expressed by activated fibroblasts that has both dipeptidyl peptidase and endopeptidase activities. We aimed to assess the correlation between cFAP activity and antigen levels and to compare variations in levels.

Methods: In plasma of 465 control individuals, 368 patients with coronary heart disease (CHD) and 102 hepatitis C virus (HCV) infected patients with severe liver disease before and after liver transplant, cFAP activity levels were measured with a newly developed cFAP activity assay. In the same samples, cFAP antigen levels were measured using a commercially available cFAP ELISA. Correlation analyses between activity and antigen levels were performed by calculating Pearson's correlation coefficient (ρ). Additionally, normal ranges, determinants and differences between cohorts and between anticoagulants were investigated.

Results: cFAP activity and antigen levels significantly correlated in controls (ρ: 0.660, p<0.001) and in CHD patients (ρ: 0.709, p<0.001). cFAP activity and antigen levels in the HCV cohort were significantly lower in the samples taken after liver transplantation (p<0.001) and normalized toward levels of healthy individuals. Furthermore, cFAP activity and antigen levels were higher in men and significantly associated with body mass index. Also, cFAP activity and antigen levels were higher in EDTA plasma as compared to the levels in citrated plasma from the same healthy individuals.

Conclusions: For analyzing cFAP levels, either activity levels or antigen levels can be measured to investigate differences between individuals. However, it is of importance that blood samples are collected in the same anticoagulant.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178987PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5459491PMC
September 2017

Fibroblast activation protein is dispensable in the anti-influenza immune response in mice.

PLoS One 2017 3;12(2):e0171194. Epub 2017 Feb 3.

Immune Imaging Program, Centenary Institute for Cancer Medicine and Cell Biology, Newtown, New South Wales, Australia.

Fibroblast activation protein alpha (FAP) is a unique dual peptidase of the S9B serine protease family, being capable of both dipeptidyl peptidase and endopeptidase activities. FAP is expressed at low level in healthy adult organs including the pancreas, cervix, uterus, submaxillary gland and the skin, and highly upregulated in embryogenesis, chronic inflammation and tissue remodelling. It is also expressed by cancer-associated stromal fibroblasts in more than 90% of epithelial tumours. FAP has enzymatic and non-enzymatic functions in the growth, immunosuppression, invasion and cell signalling of tumour cells. FAP deficient mice are fertile and viable with no gross abnormality, but little data exist on the role of FAP in the immune system. FAP is upregulated in association with microbial stimulation and chronic inflammation, but its function in infection remains unknown. We showed that major populations of immune cells including CD4+ and CD8+ T cells, B cells, dendritic cells and neutrophils are generated and maintained normally in FAP knockout mice. Upon intranasal challenge with influenza virus, FAP mRNA was increased in the lungs and lung-draining lymph nodes. Nonetheless, FAP deficient mice showed similar pathologic kinetics to wildtype controls, and were capable of supporting normal anti-influenza T and B cell responses. There was no evidence of compensatory upregulation of other DPP4 family members in influenza-infected FAP-deficient mice. FAP appears to be dispensable in anti-influenza adaptive immunity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171194PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291439PMC
August 2017

High resolution crystal structures of human kynurenine aminotransferase-I bound to PLP cofactor, and in complex with aminooxyacetate.

Protein Sci 2017 04 6;26(4):727-736. Epub 2017 Mar 6.

Group in Biomolecular Structure and Informatics, Faculty of Pharmacy, University of Sydney, Sydney, New South Wales, Australia.

In this study, we report two high-resolution structures of the pyridoxal 5' phosphate (PLP)-dependent enzyme kynurenine aminotransferase-I (KAT-I). One is the native structure with the cofactor in the PLP form bound to Lys247 with the highest resolution yet available for KAT-I at 1.28 Å resolution, and the other with the general PLP-dependent aminotransferase inhibitor, aminooxyacetate (AOAA) covalently bound to the cofactor at 1.54 Å. Only small conformational differences are observed in the vicinity of the aldimine (oxime) linkage with which the PLP forms the Schiff base with Lys247 in the 1.28 Å resolution native structure, in comparison to other native PLP-bound structures. We also report the inhibition of KAT-1 by AOAA and aminooxy-phenylpropionic acid (AOPP), with IC50s of 13.1 and 5.7 μM, respectively. The crystal structure of the enzyme in complex with the inhibitor AOAA revealed that the cofactor is the PLP form with the external aldimine linkage. The location of this oxime with the PLP, which forms in place of the native internal aldimine linkage of PLP of the native KAT-I, is away from the position of the native internal aldimine, with the free Lys247 substantially retaining the orientation of the native structure. Tyr101, at the active site, was observed in two conformations in both structures.
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http://dx.doi.org/10.1002/pro.3119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368072PMC
April 2017

Circulating fibroblast activation protein and dipeptidyl peptidase 4 in rheumatoid arthritis and systemic sclerosis.

Int J Rheum Dis 2018 Nov 19;21(11):1915-1923. Epub 2016 Dec 19.

Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia.

Aim: To quantify circulating fibroblast activation protein (cFAP) and dipeptidyl peptidase 4 (cDPP4) protease activities in patients with rheumatoid arthritis (RA), systemic sclerosis (SSc), and a control group with mechanical back pain and to correlate plasma levels with disease characteristics.

Methods: Plasma was collected from patients with RA (n = 73), SSc (n = 37) and control subjects (n = 26). DPP4 and FAP were quantified using specific enzyme activity assays.

Results: Median cDPP4 was significantly lower in the RA group (P = 0.02), and SSc group (P = 0.002) compared with controls. There were no significant differences in median cFAP between the three groups. DPP4 and FAP demonstrated a negative correlation with inflammatory markers and duration of disease. There were no associations with disease subtypes in RA, including seropositive and erosive disease. Decreased cDPP4 was found in SSc patients with myositis. Plasma FAP was lower in RA patients receiving prednisone (P = 0.001) or leflunomide (P = 0.04), but higher with biologic agents (P = 0.01). RA patients receiving leflunomide also had decreased cDPP4 (P = 0.014). SSc patients receiving prednisone (P = 0.02) had lower cDPP4 but there was no association with cFAP.

Conclusions: No association was found between cFAP and RA or SSc. Plasma DPP4 was decreased in RA and SSc when compared with controls. cDPP4 and cFAP correlated negatively with inflammatory markers and there were no significant correlations with disease characteristics in this RA cohort.
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http://dx.doi.org/10.1111/1756-185X.13031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476518PMC
November 2018

The pro-fibrotic role of dipeptidyl peptidase 4 in carbon tetrachloride-induced experimental liver injury.

Immunol Cell Biol 2017 05 30;95(5):443-453. Epub 2016 Nov 30.

Centenary Institute and Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia.

Liver fibrosis is a progressive pathological process involving inflammation and extracellular matrix deposition. Dipeptidyl peptidase 4 (DPP4), also known as CD26, is a cell surface glycoprotein and serine protease. DPP4 binds to fibronectin, can inactivate specific chemokines, incretin hormone and neuropeptides, and influences cell adhesion and migration. Such properties suggest a pro-fibrotic role for this peptidase but this hypothesis needs in vivo examination. Experimental liver injury was induced with carbon tetrachloride (CCl) in DPP4 gene knockout (gko) mice. DPP4 gko had less liver fibrosis and inflammation and fewer B cell clusters than wild type mice in the fibrosis model. DPP4 inhibitor-treated mice also developed less liver fibrosis. DNA microarray and PCR showed that many immunoglobulin (Ig) genes and some metabolism-associated transcripts were differentially expressed in the gko strain compared with wild type. CCl-treated DPP4 gko livers had more IgM and IgG intrahepatic lymphocytes, and fewer CD4, IgD and CD21 intrahepatic lymphocytes. These data suggest that DPP4 is pro-fibrotic in CCl-induced liver fibrosis and that the mechanisms of DPP4 pro-fibrotic action include energy metabolism, B cells, NK cells and CD4 cells.
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http://dx.doi.org/10.1038/icb.2016.116DOI Listing
May 2017

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

Biol Chem 2016 09;397(9):837-56

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.
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http://dx.doi.org/10.1515/hsz-2016-0174DOI Listing
September 2016

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

Cancer Lett 2017 02 1;387:106-113. Epub 2016 Apr 1.

Centenary Institute and Sydney Medical School, University of Sydney, Sydney, New South Wales 2006 Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.canlet.2016.03.047DOI Listing
February 2017

Dipeptidyl peptidase 9 enzymatic activity influences the expression of neonatal metabolic genes.

Exp Cell Res 2016 Mar 28;342(1):72-82. Epub 2016 Feb 28.

Centenary Institute and Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia. Electronic address:

The success of dipeptidyl peptidase 4 (DPP4) inhibition as a type 2 diabetes therapy has encouraged deeper examination of the post-proline DPP enzymes. DPP9 has been implicated in immunoregulation, disease pathogenesis and metabolism. The DPP9 enzyme-inactive (Dpp9 gene knock-in; Dpp9 gki) mouse displays neonatal lethality, suggesting that DPP9 enzyme activity is essential in neonatal development. Here we present gene expression patterns in these Dpp9 gki neonatal mice. Taqman PCR arrays and sequential qPCR assays on neonatal liver and gut revealed differential expression of genes involved in cell growth, innate immunity and metabolic pathways including long-chain-fatty-acid uptake and esterification, long-chain fatty acyl-CoA binding, trafficking and transport into mitochondria, lipoprotein metabolism, adipokine transport and gluconeogenesis in the Dpp9 gki mice compared to wild type. In a liver cell line, Dpp9 knockdown increased AMP-activated protein kinase phosphorylation, which suggests a potential mechanism. DPP9 protein levels in liver cells were altered by treatment with EGF, HGF, insulin or palmitate, suggesting potential natural DPP9 regulators. These gene expression analyses of a mouse strain deficient in DPP9 enzyme activity show, for the first time, that DPP9 enzyme activity regulates metabolic pathways in neonatal liver and gut.
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http://dx.doi.org/10.1016/j.yexcr.2016.02.020DOI Listing
March 2016

Neuropeptide Y is a physiological substrate of fibroblast activation protein: Enzyme kinetics in blood plasma and expression of Y2R and Y5R in human liver cirrhosis and hepatocellular carcinoma.

Peptides 2016 Jan 24;75:80-95. Epub 2015 Nov 24.

Centenary Institute of Cancer Medicine and Cell Biology, Sydney Medical School, The University of Sydney, NSW 2006, Australia. Electronic address:

Fibroblast activation protein (FAP) is a dipeptidyl peptidase (DPP) and endopeptidase that is weakly expressed in normal adult human tissues but is greatly up-regulated in activated mesenchymal cells of tumors and chronically injured tissue. The identities and locations of target substrates of FAP are poorly defined, in contrast to the related protease DPP4. This study is the first to characterize the physiological substrate repertoire of the DPP activity of endogenous FAP present in plasma. Four substrates, neuropeptide Y (NPY), peptide YY, B-type natriuretic peptide and substance P, were analyzed by mass spectrometry following proteolysis in human or mouse plasma, and by in vivo localization in human liver tissues with cirrhosis and hepatocellular carcinoma (HCC). NPY was the most efficiently cleaved substrate of both human and mouse FAP, whereas all four peptides were efficiently cleaved by endogenous DPP4, indicating that the in vivo degradomes of FAP and DPP4 differ. All detectable DPP-specific proteolysis and C-terminal processing of these neuropeptides was attributable to FAP and DPP4, and plasma kallikrein, respectively, highlighting their combined physiological significance in the regulation of these neuropeptides. In cirrhotic liver and HCC, NPY and its receptor Y2R, but not Y5R, were increased in hepatocytes near the parenchymal-stromal interface where there is an opportunity to interact with FAP expressed on nearby activated mesenchymal cells in the stroma. These novel findings provide insights into the substrate specificity of FAP, which differs greatly from DPP4, and reveal a potential function for FAP in neuropeptide regulation within liver and cancer biology.
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http://dx.doi.org/10.1016/j.peptides.2015.11.004DOI Listing
January 2016

Identification of novel dipeptidyl peptidase 9 substrates by two-dimensional differential in-gel electrophoresis.

FEBS J 2015 Oct 3;282(19):3737-57. Epub 2015 Aug 3.

Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Australia.

Dipeptidyl peptidase 9 (DPP9) is a member of the S9B/DPPIV (DPP4) serine protease family, which cleaves N-terminal dipeptides at an Xaa-Pro consensus motif. Cytoplasmic DPP9 has roles in epidermal growth factor signalling and in antigen processing, whilst the role of the recently discovered nuclear form of DPP9 is unknown. Mice lacking DPP9 proteolytic activity die as neonates. We applied a modified 2D differential in-gel electrophoresis approach to identify novel DPP9 substrates, using mouse embryonic fibroblasts lacking endogenous DPP9 activity. A total of 111 potential new DPP9 substrates were identified, with nine proteins/peptides confirmed as DPP9 substrates by MALDI-TOF or immunoblotting. Moreover, we also identified the dipeptide Val-Ala as a consensus site for DPP9 cleavage that was not recognized by DPP8, suggesting different in vivo roles for these closely related enzymes. The relative kinetics for the cleavage of these nine candidate substrates by DPP9, DPP8 and DPP4 were determined. This is the first identification of DPP9 substrates from cells lacking endogenous DPP9 activity. These data greatly expand the potential roles of DPP9 and suggest different in vivo roles for DPP9 and DPP8.
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http://dx.doi.org/10.1111/febs.13371DOI Listing
October 2015

Kynurenine Aminotransferases and the Prospects of Inhibitors for the Treatment of Schizophrenia.

Curr Med Chem 2015 ;22(24):2902-18

Faculty of Pharmacy A15, The University of Sydney, Sydney NSW 2006, Australia.

Schizophrenia is a complex neuropsychiatric disorder with limited treatment options and highly debilitating symptoms, leading to poor personal, social, and occupational outcomes for an afflicted individual. Our current understanding of schizophrenia suggests that dopaminergic and glutamatergic systems have a significant role in the pathogenesis of the disease. Kynurenic acid, an endogenous glutamate antagonist, is found in elevated concentrations in the prefrontal cortex and cerebrospinal fluid of patients with schizophrenia, and this affects neurotransmitter release in a similar manner to previously observed psychotomimetic agents, such as phencyclidine, underlining the molecular basis to its link in schizophrenia pathophysiology. Kynurenic acid is a breakdown product of tryptophan degradation, through a transamination process mediated by kynurenine aminotransferase (KAT) enzymes. There are four KAT homologues reported, all of which are pyridoxal 5'- phosphate-dependent enzymes. All four KAT isoforms have been analysed structurally and biochemically, however the most extensive research is on KAT-I and KAT-II. These two enzymes have been targeted in structure-based drug design as a means of normalising raised kynurenic acid levels. The most potent KAT-I inhibitors and KAT-II inhibitors include phenylhydrazone hexanoic acid derivatives and a pyrazole series of compounds, respectively. KAT inhibitors have been shown to be effective in reducing kynurenic acid production, with accompanying changes in neurotransmitter release and pro-cognitive effects seen in animal studies. This review will discuss the characteristics pertaining to the different KAT isoforms, and will highlight the development of significant KAT inhibitors. KAT inhibitors have great potential for therapeutic application and represent a novel way in treating schizophrenia.
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http://dx.doi.org/10.2174/0929867322666150608094054DOI Listing
May 2016

Dipeptidyl peptidase 9 subcellular localization and a role in cell adhesion involving focal adhesion kinase and paxillin.

Biochim Biophys Acta 2015 Feb 5;1853(2):470-80. Epub 2014 Dec 5.

Centenary Institute and Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia. Electronic address:

Dipeptidyl peptidase 9 (DPP9) is a ubiquitously expressed member of the DPP4 gene and protease family. Deciphering the biological functions of DPP9 and its roles in pathogenesis has implicated DPP9 in tumor biology, the immune response, apoptosis, intracellular epidermal growth factor-dependent signaling and cell adhesion and migration. We investigated the intracellular distribution of DPP9 chimeric fluorescent proteins and consequent functions of DPP9. We showed that while some DPP9 is associated with mitochondria, the strongest co-localization was with microtubules. Under steady state conditions, DPP9 was not seen at the plasma membrane, but upon stimulation with either phorbol 12-myristate 13-acetate or epidermal growth factor, some DPP9 re-distributed towards the ruffling membrane. DPP9 was seen at the leading edge of the migrating cell and co-localized with the focal adhesion proteins, integrin-β1 and talin. DPP9 gene silencing and treatment with a DPP8/DPP9 specific inhibitor both reduced cell adhesion and migration. Expression of integrin-β1 and talin was decreased in DPP9-deficient and DPP9-enzyme-inactive cells. There was a concomitant decrease in the phosphorylation of focal adhesion kinase and paxillin, indicating that DPP9 knockdown or enzyme inhibition suppressed the associated adhesion signaling pathway, causing impaired cell movement. These novel findings provide mechanistic insights into the regulatory role of DPP9 in cell movement, and may thus implicate DPP9 in tissue and tumor growth and metastasis.
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http://dx.doi.org/10.1016/j.bbamcr.2014.11.029DOI Listing
February 2015

Circulating dipeptidyl peptidase-4 activity correlates with measures of hepatocyte apoptosis and fibrosis in non-alcoholic fatty liver disease in type 2 diabetes mellitus and obesity: A dual cohort cross-sectional study.

J Diabetes 2015 Nov 22;7(6):809-19. Epub 2014 Dec 22.

Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia.

Background: Intrahepatic expression of dipeptidyl peptidase-4 (DPP4), and circulating DPP4 (cDPP4) levels and its enzymatic activity, are increased in non-alcoholic fatty liver disease (NAFLD) and in type 2 diabetes mellitus and/or obesity. DPP4 has been implicated as a causative factor in NAFLD progression but few studies have examined associations between cDPP4 activity and NAFLD severity in humans. This study aimed to examine the relationship of cDPP4 activity with measures of liver disease severity in NAFLD in subjects with diabetes and/or obesity.

Methods: cDPP4 was measured in 106 individuals with type 2 diabetes who had transient elastography (Cohort 1) and 145 individuals with morbid obesity who had liver biopsy (Cohort 2). Both cohorts had caspase-cleaved keratin-18 (ccK18) measured as a marker of apoptosis.

Results: Natural log increases in cDPP4 activity were associated with increasing quartiles of ccK18 (Cohorts 1 and 2) and with median liver stiffness ≥10.3 kPa (Cohort 1) and significant fibrosis (F ≥ 2) on liver biopsy (Cohort 2).

Conclusions: In diabetes and/or obesity, cDPP4 activity is associated with current apoptosis and liver fibrosis. Given the pathogenic mechanisms by which DPP4 may progress NAFLD, measurement of cDPP4 activity may have utility to predict disease progression and DPP4 inhibition may improve liver histology over time.
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http://dx.doi.org/10.1111/1753-0407.12237DOI Listing
November 2015

Homology modeling of human kynurenine aminotransferase III and observations on inhibitor binding using molecular docking.

Cent Nerv Syst Agents Med Chem 2014 ;14(1):2-9

Faculty of Pharmacy A15, The University of Sydney, Sydney NSW 2006, Australia.

Kynurenine aminotransferase (KAT) isozymes are responsible for catalyzing the conversion of kynurenine (KYN) to kynurenic acid (KYNA), which is considered to play a key role in central nervous system (CNS) disorders, including schizophrenia. The levels of KYNA in the postmortem prefrontal cortex and in the Cerebrospinal fluid (CSF) of schizophrenics are greater than normal brain. A basic strategy to decrease kynurenic acid levels is to promote the inhibition of the biosynthetic KAT isozymes. As there is no crystallographic model for human kynurenine aminotransferase III (KAT III), therefore, homology modeling has been performed based on the Mus musculus kynurenine aminotransferase III crystal structure (PDB ID: 3E2Y) as a template, and the model of the human KAT III was refined and optimized with molecular dynamics simulations. Further evaluation of the model quality was accomplished by investigating the interaction of KAT III inhibitors with the modeled enzyme. Such interactions were determined employing the AutoDock 4.2 program using the MGLTools 1.5.6 package. The most important interactions for the binding of the inhibitors, which are probably also central components of the active site of KAT III, were identified as Ala134, Tyr135, Lys 280, Lys 288, Thr285 and Arg429, which provide hydrogen bond interactions. Additionally, Tyr135 and Arg429 have good electrostatic interactions with inhibitors consistent with these residues also being essential for inhibition of the enzyme activity. We expect that this model and these docking data will be a useful resource for the rational design of novel drugs for treating neuropathologies.
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http://dx.doi.org/10.2174/1871524914666140416095523DOI Listing
July 2015

A rare variant in human fibroblast activation protein associated with ER stress, loss of enzymatic function and loss of cell surface localisation.

Biochim Biophys Acta 2014 Jul 6;1844(7):1248-59. Epub 2014 Apr 6.

Centenary Institute, Camperdown, NSW, Australia; Sydney Medical School, University of Sydney, NSW 2006, Australia; Royal Prince Alfred Hospital, Camperdown, NSW, Australia. Electronic address:

Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the β propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the β-propeller domain, inactive, unfolded FAP can be tolerated by cells.
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http://dx.doi.org/10.1016/j.bbapap.2014.03.015DOI Listing
July 2014