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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. [ABSTRACT FROM AUTHOR]

Lipid accumulation in hepatocytes can lead to non-alcoholic fatty liver disease (NAFLD), which can progress to non-alcoholic steatohepatitis (NASH) and Type 2 diabetes (T2D). Hormone-initiated release of Ca2+ from the endoplasmic reticulum (ER) stores and subsequent replenishment of these stores by Ca2+ entry through SOCs (store-operated Ca2+ channels; SOCE) plays a critical role in the regulation of liver metabolism. ER Ca2+ homoeostasis is known to be altered in steatotic hepatocytes. Whether store-operated Ca2+ entry is altered in steatotic hepatocytes and the mechanisms involved were investigated. Lipid accumulation in vitro was induced in cultured liver cells by amiodarone or palmitate and in vivo in hepatocytes isolated from obese Zucker rats. Rates of Ca2+ entry and release were substantially reduced in lipid-loaded cells. Inhibition of Ca2+ entry was associated with reduced hormone-initiated intracellular Ca2+ signalling and enhanced lipid accumulation. Impaired Ca2+ entry was not associated with altered expression of stromal interaction molecule 1 (STIM1) or Orai1. Inhibition of protein kinase C (PKC) reversed the impairment of Ca2+ entry in lipid-loaded cells. It is concluded that steatosis leads to a substantial inhibition of SOCE through a PKC-dependent mechanism. This enhances lipid accumulation by positive feedback and may contribute to the development of NASH and insulin resistance. [ABSTRACT FROM AUTHOR]

Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine isotopic labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease. [ABSTRACT FROM AUTHOR]

Abstract: N-terminal truncation of chemokines by proteases including dipeptidyl peptidase (DP) IV significantly alters their biological activity; generally ablating cognate G-protein coupled receptor engagement and often generating potent receptor antagonists. DP8 is a recently recognised member of the prolyl oligopeptidase gene family that includes DPIV. Since DPIV is known to process chemokines we surveyed 27 chemokines for cleavage by DP8. We report DP8 cleavage of the N-terminal two residues of IP10 (CXCL10), ITAC (CXCL11) and SDF-1 (CXCL12). This has implications for DP8 substrate specificity. Chemokine cleavage and inactivation may occur in vivo upon cell lysis and release of DP8 or in the inactivation of internalized chemokine/receptor complexes. [Copyright &y& Elsevier]

The protease degradome is defined as the complete repertoire of proteases and inhibitors, and their nonfunctional homologs present in a cell, tissue or organism at any given time. We review the tissue distribution of virtually the entire degradome in 23 different human tissues and 6 ovarian cancer cell lines. To do so, we developed the CLIP-CHIP™, a custom microarray based on a 70-mer oligonucleotide platform, to specifically profile the transcripts of the entire repertoire of 473 active human proteases, 156 protease inhibitors and 92 non-proteolytically active homologs known at the design date using one specific 70-mer oligonucleotide per transcript. Using the CLIP-CHIP™ we mapped the expression profile of proteases and their inhibitors in 23 different human tissues and 6 ovarian cancer cell lines in 104 sample datasets. Hierarchical cluster analysis showed that expression profiles clustered according to their anatomic locations, cellular composition, physiologic functions, and the germ layer from which they are derived. The human ovarian cancer cell lines cluster according to malignant grade. 110 proteases and 42 inhibitors were tissue specific (1 to 3 tissues). Of these 110 proteases 69% (74) are mainly extracellular, 30% (34) intracellular and 1% intramembrane. Notably, 35% (197/565) of human proteases and 30% (47/156) of inhibitors were ubiquitously expressed in all 23 tissues; 27% (155) of proteases and 21% (32) of inhibitors were broadly expressed in 4–20 tissues. Our datasets provide a valuable resource for the community of baseline protease and inhibitor relative expression in normal human tissues and can be used for comparison with diseased tissue, e.g. ovarian cancer, to decipher pathogenesis, and to aid drug development. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John. [ABSTRACT FROM AUTHOR]

The release of Ca 2 + from the endoplasmic reticulum (ER) and subsequent replenishment of ER Ca 2 + by Ca 2 + entry through store-operated Ca 2 + channels (SOCE) play critical roles in the regulation of liver metabolism by adrenaline, glucagon and other hormones. Both ER Ca 2 + release and Ca 2 + entry are severely inhibited in steatotic hepatocytes. Exendin-4, a slowly-metabolised glucagon-like peptide-1 (GLP-1) analogue, is known to reduce liver glucose output and liver lipid, but the mechanisms involved are not well understood. The aim of this study was to determine whether exendin-4 alters intracellular Ca 2 + homeostasis in steatotic hepatocytes, and to evaluate the mechanisms involved. Exendin-4 completely reversed lipid-induced inhibition of SOCE in steatotic liver cells, but did not reverse lipid-induced inhibition of ER Ca 2 + release. The action of exendin-4 on Ca 2 + entry was rapid in onset and was mimicked by GLP-1 or dibutyryl cyclic AMP. In steatotic liver cells, exendin-4 caused a rapid decrease in lipid (half time 6.5 min), inhibited the accumulation of lipid in liver cells incubated in the presence of palmitate plus the SOCE inhibitor BTP-2, and enhanced the formation of cyclic AMP. Hormone-stimulated accumulation of extracellular glucose in glycogen replete steatotic liver cells was inhibited compared to that in non-steatotic cells, and this effect of lipid was reversed by exendin-4. It is concluded that, in steatotic hepatocytes, exendin-4 reverses the lipid-induced inhibition of SOCE leading to restoration of hormone-regulated cytoplasmic Ca 2 + signalling. The mechanism may involve GLP-1 receptors, cyclic AMP, lipolysis, decreased diacylglycerol and decreased activity of protein kinase C. [ABSTRACT FROM AUTHOR]