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1.
Cell ; 175(2): 530-543.e24, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30220458

RESUMO

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.


Assuntos
Nefrite Intersticial/virologia , Parvovirus/isolamento & purificação , Parvovirus/patogenicidade , Animais , Austrália , Progressão da Doença , Feminino , Fibrose/patologia , Fibrose/virologia , Humanos , Rim/metabolismo , Rim/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nefrite Intersticial/fisiopatologia , América do Norte , Infecções por Parvoviridae/metabolismo
2.
Am J Transplant ; 2024 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-39059585

RESUMO

Bile duct regeneration is hypothesized to prevent biliary strictures, a leading cause of morbidity after liver transplantation. Assessing the capacity for biliary regeneration may identify grafts as suitable for transplantation that are currently declined, but this has been unfeasible until now. This study used long-term ex situ normothermic machine perfusion (LT-NMP) to assess biliary regeneration. Human livers that were declined for transplantation were perfused at 36 °C for up to 13.5 days. Bile duct biopsies, bile, and perfusate were collected throughout perfusion, which were examined for features of injury and regeneration. Biliary regeneration was defined as new Ki-67-positive biliary epithelium following severe injury. Ten livers were perfused for a median duration of 7.5 days. Severe bile duct injury occurred in all grafts, and biliary regeneration occurred in 70% of grafts. Traditional biomarkers of biliary viability such as bile glucose improved during perfusion but this was not associated with biliary regeneration (P > .05). In contrast, the maintenance of interleukin-6 and vascular endothelial growth factor-A levels in bile was associated with biliary regeneration (P = .017 for both cytokines). This is the first study to demonstrate biliary regeneration during LT-NMP and identify a cytokine signature in bile as a novel biomarker for biliary regeneration during LT-NMP.

3.
EMBO Rep ; 23(10): e54136, 2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-35912982

RESUMO

N-terminal sequences are important sites for post-translational modifications that alter protein localization, activity, and stability. Dipeptidyl peptidase 9 (DPP9) is a serine aminopeptidase with the rare ability to cleave off N-terminal dipeptides with imino acid proline in the second position. Here, we identify the tumor-suppressor BRCA2 as a DPP9 substrate and show this interaction to be induced by DNA damage. We present crystallographic structures documenting intracrystalline enzymatic activity of DPP9, with the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in its active site. Intriguingly, DPP9-depleted cells are hypersensitive to genotoxic agents and are impaired in the repair of DNA double-strand breaks by homologous recombination. Mechanistically, DPP9 targets BRCA2 for degradation and promotes the formation of RAD51 foci, the downstream function of BRCA2. N-terminal truncation mutants of BRCA2 that mimic a DPP9 product phenocopy reduced BRCA2 stability and rescue RAD51 foci formation in DPP9-deficient cells. Taken together, we present DPP9 as a regulator of BRCA2 stability and propose that by fine-tuning the cellular concentrations of BRCA2, DPP9 alters the BRCA2 interactome, providing a possible explanation for DPP9's role in cancer.


Assuntos
Reparo do DNA , Dipeptidil Peptidases e Tripeptidil Peptidases , Aminopeptidases , DNA , Dano ao DNA , Dipeptídeos , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Prolina , Rad51 Recombinase/genética , Serina
4.
Molecules ; 27(3)2022 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-35163991

RESUMO

A diet-induced non-alcoholic fatty liver disease (NAFLD) model causing obesity in rodents was used to examine whether sitagliptin and gliclazide therapies have similar protective effects on pathological liver change. METHODS: Male mice were fed a high-fat diet (HFD) or standard chow (Chow) ad libitum for 25 weeks and randomly allocated to oral sitagliptin or gliclazide treatment for the final 10 weeks. Fasting blood glucose and circulating insulin were measured. Inflammatory and fibrotic liver markers were assessed by qPCR. The second messenger ERK and autophagy markers were examined by Western immunoblot. F4/80, collagens and CCN2 were assessed by immunohistochemistry (IHC). RESULTS: At termination, HFD mice were obese, hyperinsulinemic and insulin-resistant but non-diabetic. The DPP4 inhibitor sitagliptin prevented intrahepatic induction of pro-fibrotic markers collagen-IV, collagen-VI, CCN2 and TGF-ß1 and pro-inflammatory markers TNF-α and IL-1ß more effectively than sulfonylurea gliclazide. By IHC, liver collagen-VI and CCN2 induction by HFD were inhibited only by sitagliptin. Sitagliptin had a greater ability than gliclazide to normalise ERK-protein liver dysregulation. CONCLUSION: These data indicate that sitagliptin, compared with gliclazide, exhibits greater inhibition of pro-fibrotic and pro-inflammatory changes in an HFD-induced NAFLD model. Sitagliptin therapy, even in the absence of diabetes, may have specific benefits in diet-induced NAFLD.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Gliclazida/farmacologia , Inflamação/prevenção & controle , Cirrose Hepática/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Fosfato de Sitagliptina/farmacologia , Animais , Hipoglicemiantes/farmacologia , Inflamação/etiologia , Inflamação/patologia , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia
5.
Protein Expr Purif ; 181: 105833, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33524496

RESUMO

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.


Assuntos
Endopeptidases , Proteínas de Membrana , Animais , Endopeptidases/biossíntese , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Células Sf9 , Spodoptera
6.
Mol Cell Proteomics ; 18(1): 65-85, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30257879

RESUMO

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.


Assuntos
Fibroblastos/citologia , Gelatinases/genética , Gelatinases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteômica/métodos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Adipocinas/sangue , Adipocinas/química , Aminoácido Oxirredutases/sangue , Aminoácido Oxirredutases/química , Animais , Técnicas de Cultura de Células , Linhagem Celular , Quimiocina CXCL5/sangue , Quimiocina CXCL5/química , Endopeptidases , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Humanos , Fator Estimulador de Colônias de Macrófagos/sangue , Fator Estimulador de Colônias de Macrófagos/química , Camundongos , Mapas de Interação de Proteínas , Proteólise , Especificidade por Substrato
7.
Int J Mol Sci ; 21(21)2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-33143089

RESUMO

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 DPP4 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.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Hipóxia/fisiopatologia , Neoplasias Ovarianas/patologia , Peptídeo Hidrolases/metabolismo , Proteólise , Serina Endopeptidases/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Transdução de Sinais
8.
Molecules ; 25(22)2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33218025

RESUMO

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.


Assuntos
Enzima de Conversão de Angiotensina 2/isolamento & purificação , Dipeptidil Peptidase 4/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Enzima de Conversão de Angiotensina 2/biossíntese , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Clonagem Molecular , Dipeptidil Peptidase 4/biossíntese , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Humanos , Cinética , Modelos Moleculares , Plasmídeos/química , Plasmídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Glicoproteína da Espícula de Coronavírus/biossíntese , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Spodoptera , Ressonância de Plasmônio de Superfície
9.
Immunol Cell Biol ; 96(10): 1131-1139, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29920767

RESUMO

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.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Receptores de Quimiocinas/genética , Animais , Quimiotaxia/imunologia , Dipeptidil Peptidase 4/metabolismo , Melanoma , Melanoma Experimental , Camundongos , Camundongos Knockout , Receptores CXCR3/genética , Receptores CXCR3/metabolismo , Receptores de Quimiocinas/metabolismo
10.
Immunol Cell Biol ; 95(5): 443-453, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27899813

RESUMO

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 (CCl4) 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. CCl4-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 CCl4-induced liver fibrosis and that the mechanisms of DPP4 pro-fibrotic action include energy metabolism, B cells, NK cells and CD4+ cells.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Cirrose Hepática/enzimologia , Cirrose Hepática/patologia , Fígado/enzimologia , Fígado/lesões , Animais , Tetracloreto de Carbono , Linhagem Celular , Inibidores da Dipeptidil Peptidase IV/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Leucócitos/patologia , Fígado/patologia , Cirrose Hepática/genética , Camundongos , Camundongos Knockout , Fenótipo , Baço/patologia , Regulação para Cima
11.
Exp Cell Res ; 342(1): 72-82, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26930324

RESUMO

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.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Adenilato Quinase/metabolismo , Adipocinas/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Ativação Enzimática , Fator de Crescimento Epidérmico/fisiologia , Expressão Gênica , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Insulina/fisiologia , Metabolismo dos Lipídeos , Fígado/enzimologia , Camundongos Transgênicos , Ácido Palmítico/farmacologia
12.
Biochim Biophys Acta ; 1853(2): 470-80, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25486458

RESUMO

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-ß1 and talin. DPP9 gene silencing and treatment with a DPP8/DPP9 specific inhibitor both reduced cell adhesion and migration. Expression of integrin-ß1 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.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Paxilina/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Colágeno/farmacologia , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Família de Proteínas EGF/farmacologia , Fibronectinas/farmacologia , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Humanos , Integrina beta1/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Fosforilação/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Talina/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
13.
Biol Chem ; 397(9): 837-56, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27410463

RESUMO

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.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Descoberta de Drogas/métodos , Espectrometria de Massas/métodos , Animais , Humanos , Ligação Proteica , Proteômica
14.
Biochim Biophys Acta ; 1844(7): 1248-59, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24717288

RESUMO

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 ß 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 ß-propeller domain, inactive, unfolded FAP can be tolerated by cells.


Assuntos
Braquidactilia/genética , Surdez/genética , Estresse do Retículo Endoplasmático/genética , Degradação Associada com o Retículo Endoplasmático/genética , Gelatinases/genética , Gelatinases/metabolismo , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Anormalidades da Boca/genética , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Anormalidades Dentárias/genética , Substituição de Aminoácidos , Apoptose , Western Blotting , Estudos de Casos e Controles , Membrana Celular/metabolismo , Células Cultivadas , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Endopeptidases , Chaperona BiP do Retículo Endoplasmático , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Pele/citologia , Pele/metabolismo , Frações Subcelulares
15.
Dev Biol ; 439(1): 1, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29248439
16.
Biochim Biophys Acta Mol Basis Dis ; 1870(3): 167029, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38325224

RESUMO

High fructose diets are associated with an increased risk of liver cancer. Previous studies in mice suggest increased lipogenesis is a key mechanism linking high fructose diets to liver tumour growth. However, these studies administered fructose to mice at supraphysiological levels. The aim of this study was to determine whether liver tumour growth and lipogenesis were altered in mice fed fructose at physiological levels. To test this, we injected male C57BL/6 mice with the liver carcinogen diethylnitrosamine and then fed them diets without fructose or fructose ranging from 10 to 20 % total calories. Results showed mice fed diets with ≥15 % fructose had significantly increased liver tumour numbers (2-4-fold) and total tumour burden (∼7-fold) vs mice fed no-fructose diets. However, fructose-associated tumour burden was not associated with lipogenesis. Conversely, unbiased metabolomic analyses revealed bile acids were elevated in the sera of mice fed a 15 % fructose diet vs mice fed a no-fructose diet. Using a syngeneic ectopic liver tumour model, we show that ursodeoxycholic acid, which decreases systemic bile acids, significantly reduced liver tumour growth in mice fed the 15 % fructose diet but not mice fed a no-fructose diet. These results point to a novel role for systemic bile acids in mediating liver tumour growth associated with a high fructose diet. Overall, our study shows fructose intake at or above normal human consumption (≥15 %) is associated with increased liver tumour numbers and growth and that modulating systemic bile acids inhibits fructose-associated liver tumour growth in mice.


Assuntos
Ácidos e Sais Biliares , Neoplasias Hepáticas , Humanos , Camundongos , Masculino , Animais , Frutose/efeitos adversos , Camundongos Endogâmicos C57BL , Neoplasias Hepáticas/induzido quimicamente
17.
Nutrients ; 16(1)2023 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-38201904

RESUMO

There are no ideal non-invasive tests for assessing the severity of liver fibrosis in people with metabolic dysfunction-associated steatotic liver disease (MASLD) and class 3 obesity, where body habitus often makes imaging technically challenging. This study aimed to assess the applicability and diagnostic performance of two-dimensional shear wave elastography (2D-SWE), alongside several serum-based liver fibrosis scoring methods, in individuals with class 3 obesity. A cross-sectional study was conducted in patients aged ≥18 years and with a body mass index (BMI) ≥ 40 kg/m2 who were participants in a publicly funded multidisciplinary weight management program in South Western Sydney. The 2D-SWE was performed using the ElastQ Imaging (EQI) procedure with the Phillips EPIQ Elite series ultrasound. An EQI Median value of ≥6.43 kPa was taken as a cutoff score for significant fibrosis, and the scan was considered valid when the liver EQI IQR/Med value was <30%. The Fibrosis-4 (FIB-4) index, AST-to-platelet ratio index (APRI), NAFLD fibrosis score (NFS), and circulating fibroblast activation protein index (FAP index) were calculated from fasting blood samples. The participants (n = 116; 67.2% female) were aged 47.2 ± 12.9 years, with BMI 54.5 ± 11.0 kg/m2. EQI Median values were obtained for 97.4% (113/116) of the 2D-SWE scans, and 91.4% (106/116) of the scans were considered valid. The EQI Median values exhibited a moderately positive correlation with the FIB-4 index (r = 0.438; p < 0.001) and a weakly positive correlation with the APRI (r = 0.388; p < 0.001), NFS (r = 0.210; p = 0.036) and FAP index (r = 0.226; p = 0.020). All liver fibrosis scores were positively correlated with one another. Among those referred for a liver biopsy based on the 2D-SWE and serum scores, half (11/22) underwent liver biopsy, and their 2D-SWE scores exhibited 72.7% accuracy (sensitivity: 71.4%; specificity: 75%) in detecting significant fibrosis. Our results show that 2D-SWE is a feasible, non-invasive test to assess liver fibrosis among people with class 3 obesity. Further research is needed to assess how 2D-SWE can be used alongside existing serum-based risk scores to reliably detect significant fibrosis, which would potentially reduce the need for invasive liver biopsy.


Assuntos
Técnicas de Imagem por Elasticidade , Adulto , Humanos , Feminino , Adolescente , Masculino , Estudos Transversais , Fatores de Risco , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/etiologia , Obesidade/complicações
18.
Am J Pathol ; 178(3): 1134-44, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21356365

RESUMO

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and is activated by collagens. Transcriptional profiling of cirrhosis in human liver using a DNA array and quantitative PCR detected elevated mRNA expression of DDR1 compared with that in nondiseased liver. The present study characterized DDR1 expression in cirrhotic and nondiseased human liver and examined the cellular effects of DDR1 expression. mRNA expression of all five isoforms of DDR1 was detected in human liver, whereas DDR1a demonstrated differential expression in liver with hepatitis C virus and primary biliary cirrhosis compared with nondiseased liver. In addition, immunoblot analysis detected shed fragments of DDR1 more readily in cirrhotic liver than in nondiseased liver. Inasmuch as DDR1 is subject to protease-mediated cleavage after prolonged interaction with collagen, this differential expression may indicate more intense activation of DDR1 protein in cirrhotic compared with nondiseased liver. In situ hybridization and immunofluorescence localized intense DDR1 mRNA and protein expression to epithelial cells including hepatocytes at the portal-parenchymal interface and the luminal aspect of the biliary epithelium. Overexpression of DDR1a altered hepatocyte behavior including increased adhesion and less migration on extracelular matrix substrates. DDR1a regulated extracellular expression of matrix metalloproteinases 1 and 2. These data elucidate DDR1 function pertinent to cirrhosis and indicate the importance of epithelial cell-collagen interactions in chronic liver injury.


Assuntos
Cirrose Hepática/enzimologia , Cirrose Hepática/patologia , Fígado/enzimologia , Fígado/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Adolescente , Adulto , Adesão Celular , Linhagem Celular , Movimento Celular , Receptor com Domínio Discoidina 1 , Feminino , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Adulto Jovem
19.
Bioorg Med Chem Lett ; 22(4): 1579-81, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22281190

RESUMO

Herein we report 6-ethoxy-6-oxo-5-(2-phenylhydrazono) hexanoic acid and 3-(2-carboxyethyl)-1H-indole-2-carboxylic acid derivatives as synthetically accessible leads for human kynurenine aminotransferase-I (KAT-I) inhibitors. In total, 12 compounds were synthesized and their biological activities were determined using the HPLC-UV based KAT-I inhibition assay. Of the 12 compounds synthesized, 10 were found to inhibit human KAT-I and the most active compound was found to be 5-(2-(4-chlorophenyl) hydrazono)-6-ethoxy-6-oxohexanoic acid (9a) with an IC(50) of 19.8 µM.


Assuntos
Caproatos/síntese química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Hidrazinas/síntese química , Modelos Moleculares , Transaminases/antagonistas & inibidores , Caproatos/química , Caproatos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Hidrazinas/química , Hidrazinas/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Esquizofrenia/tratamento farmacológico
20.
Clin Transl Gastroenterol ; 13(1): e00452, 2022 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-35060938

RESUMO

INTRODUCTION: Dipeptidyl peptidase (DPP)-4 is part of a larger family of proteases referred to as DPPs. DPP4 has been suggested as a possible biomarker for inflammatory bowel disease (IBD). Circulating DPP4 (cDPP4) enzyme activity was investigated as a potential biomarker for IBD. In addition, DPP enzyme activity and gene expression were quantified in colonic tissue of patients with IBD and non-IBD. METHODS: In study 1, DPP enzyme activity was quantified in plasma samples from 220 patients with IBD (Crohn's disease [CD] n = 130 and ulcerative colitis [UC] n = 90) and non-IBD controls (n = 26) using a colorimetric assay. In study 2, tissue and plasma samples were collected from 26 patients with IBD and 20 non-IBD controls. Plasma C-reactive protein (CRP) was quantified in all patients. Colonic DPP4, DPP8, DPP9, and fibroblast activation protein (FAP) gene expression was determined by quantitative polymerase chain reaction. cDPP and cFAP enzyme activity was also measured. Sensitivity and specificity were determined by receiver operating characteristic curve analysis. RESULTS: In study 1, total cDPP activity was found to differentiate patients with CD with active disease (n = 18) from those in remission (n = 19; sensitivity 78% and specificity 63%). In study 2, total cDPP and cFAP activity was 28% and 48% lower in patients with elevated CRP (>10 mg/L), respectively, compared with patients with normal CRP. Gene expression of DPP4, FAP, and DPP8 was also significantly higher in colonic biopsies from patients with IBD compared with non-IBD patients (P < 0.05). DISCUSSION: Our findings implicate the DPP enzyme family in intestinal inflammation and suggest future biomarker applications to differentiate the pathophysiological aspects of IBD.


Assuntos
Colite Ulcerativa , Doença de Crohn , Doenças Inflamatórias Intestinais , Biomarcadores , Proteína C-Reativa/análise , Colite Ulcerativa/diagnóstico , Doença de Crohn/diagnóstico , Humanos , Doenças Inflamatórias Intestinais/diagnóstico
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