Mesenchymal stem cells (MSCs) have therapeutic potential in various diseases, including myocardial infarction. Recently, the importance of the therapeutic effects of secreted factors from MSCs is increasing, but their identification has not progressed. In this study, we uncover the secreted protein profiles of MSCs derived from bone marrow, adipose tissue and dental pulp. Shotgun proteome analysis of conditioned MSC media by mass spectrometry identified 1533 proteins totally and 124 secreted proteins commonly produced among all three MSCs. The commonly secreted factors include already well-known factors whose functions are linked to MSCs' biological effects, such as CTGF, SERPINE1, TGFB1, DKK3 and MYDGF, and also include newly identified factors whose roles are not well investigated, for example AIMP1, CLEC11A, GAS6, HDGF, INHBA, and PCSK5. Computational biological pathway analysis revealed that these common factors strongly relate to tissue regeneration pathways such as angiogenesis, migration, and inflammatory response. Further analysis showed enrichment of ossification, sprouting, and organ survival factors, suggesting connection to the functions closely related to MSCs' therapeutic effects. This list of commonly secreted proteins could provide a reliable resource of biological factors which explain various effects of MSCs and would be useful for identifying new therapeutic factors produced from MSCs.

ABSTRACT:Olmesartan medoxomil (OM) is a prodrug-type angiotensin II type 1 receptor antagonist. The OM-hydrolyzing enzyme responsible for prodrug bioactivation was purified from human plasma through successive column chromatography and was molecularly identified through N-terminal amino acid sequencing, which resulted in a sequence of 20 amino acids identical to that of human paraoxonase 1 (PON1). Two recombinant allozymes of human PON1 (PON1 192QQ and PON1 192RR ) were constructed and were clearly demonstrated to hydrolyze OM; hydrolysis by the latter allozyme was slightly faster than that by the former. In addition, we evaluated the contribution of PON1 to OM bioactivation in human plasma. Enzyme kinetic studies demonstrated that OM was hydrolyzed more effectively by the recombinant PON1 proteins than by purified albumin. The OM-hydrolyzing activities of the recombinant PON1 proteins and diluted plasma were greatly reduced in the absence of calcium ions. Immunoprecipitation with anti-PON1 IgG completely abolished the OM-hydrolyzing activity in human plasma, whereas the activity was partially inhibited with anti-albumin IgG. The distribution pattern of the OM-hydrolyzing activity in human serum lipoprotein fractions and lipoprotein-deficient serum was examined and showed that most of the OM-hydrolyzing activity was located in the high-density lipoprotein fraction, with which PON1 is closely associated. In conclusion, we identified PON1 as the OM-bioactivating hydrolase in human plasma on a molecular basis and demonstrated that PON1, but not albumin, plays a major role in OM bioactivation in human plasma.

Objective: Enhancement of LCAT (lecithin:cholesterol acyltransferase) activity has possibility to be beneficial for atherosclerosis. To evaluate this concept, we characterized our novel, orally administered, small-molecule LCAT activator DS-8190a, which was created from high-throughput screening and subsequent derivatization. We also focused on its mechanism of LCAT activation and the therapeutic activity with improvement of HDL (high-density lipoprotein) functionality. Approach and Results: DS-8190a activated human and cynomolgus monkey but not mouse LCAT enzymes in vitro. DS-8190a was orally administered to cynomolgus monkeys and dose dependently increased LCAT activity (ca. 2.1-fold in 3 mg/kg group on day 7), resulting in HDL cholesterol elevation without drastic changes of non-HDL cholesterol. Atheroprotective effects were then evaluated using Ldl-r KO × hLcat Tg mice fed a Western diet for 8 weeks. DS-8190a treatment achieved significant reduction of atherosclerotic lesion area (48.3% reduction in 10 mg/kg treatment group). Furthermore, we conducted reverse cholesterol transport study using Ldl-r KO × hLcat Tg mice intraperitoneally injected with J774A.1 cells loaded with [ 3 H]-cholesterol and confirmed significant increases of [ 3 H] count in plasma (1.4-fold) and feces (1.4-fold on day 2 and 1.5-fold on day3) in the DS-8190a–treated group. With regard to the molecular mechanism involved, direct binding of DS-8190a to human LCAT protein was confirmed by 2 different approaches: affinity purification by DS-8190a–immobilized beads and thermal shift assay. In addition, the candidate binding site of DS-8190a in human LCAT protein was identified by photoaffinity labeling. Conclusions: This study demonstrates the potential of DS-8190a as a novel therapeutic for atherosclerosis. This is also the first report describing that a small-molecule direct LCAT activator achieved HDL cholesterol elevation in monkey and reduction of atherosclerotic lesion area with enhanced HDL function in rodent.

RNF43 is an oncogenic RING finger protein overexpressed in colorectal cancer. To dissect its biological functions, we explored RNF43-interacting proteins by pull-down assay and MS. We identified a heterodimer, p54nrb and PSF, as RNF43's binding partners and confirmed their physical interaction in vivo by the co-immunoprecipitation experiment. Immunofluorescence analysis revealed that co-expression of PSF relocates RNF43 from the nuclear periphery to the nucleoplasm. Thus, proteomic identification of RNF43-associated proteins sheds light on its dynamic interaction network in nuclear events.

Molecular identification of endogenous enzymes and biologically active substances from complex biological sources remains a challenging task, and although traditional biochemical purification is sometimes regarded as outdated, it remains one of the most powerful methodologies for this purpose. While biochemical purification usually requires large amounts of starting material and many separation steps, we developed an advanced method named “proteomic correlation profiling” in our previous study. In proteomic correlation profiling, we first fractionated biological material by column chromatography, and then calculated each protein's correlation coefficient between the enzyme activity profile and protein abundance profile determined by proteomics technology toward fractions. Thereafter, we could choose possible candidates for the enzyme among proteins with a high correlation value by domain predictions using informatics tools. Ultimately, this streamlined procedure requires fewer purification steps and reduces starting materials dramatically due to low required purity compared with conventional approaches. To demonstrate the generality of this approach, we have now applied an improved workflow of proteomic correlation profiling to a drug metabolizing enzyme and successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as a phosphatase of CS-0777 phosphate (CS-0777-P), a selective sphingosine 1-phosphate receptor 1 modulator with potential benefits in the treatment of autoimmune diseases including multiple sclerosis, from human kidney extract. We identified ALPL as a candidate protein only by the 200-fold purification and only from 1 g of human kidney. The identification of ALPL as CS-0777-P phosphatase was strongly supported by a recombinant protein, and contribution of the enzyme in human kidney extract was validated by immunodepletion and a specific inhibitor. This approach can be applied to any kind of enzyme class and biologically active substance; therefore, we believe that we have provided a fast and practical option by combination of traditional biochemistry and state-of-the-art proteomic technology.

CS-917 (MB06322) is a selective small compound inhibitor of fructose 1,6-bisphosphatase (FBPase), which is expected to be a novel drug for the treatment of type 2 diabetes by inhibiting gluconeogenesis. CS-917 is a bisamidate prodrug and activation of CS-917 requires a two-step enzyme catalyzed reaction. The first-step enzyme, esterase, catalyzes the conversion of CS-917 into the intermediate form (R-134450) and the second-step enzyme, phosphoramidase, catalyzes the conversion of R-134450 into the active form (R-125338). In this study, we biochemically purified the CS-917 esterase activity in monkey small intestine and liver. We identified cathepsin A (CTSA) and elastase 3B (ELA3B) as CS-917 esterases in the small intestine by mass spectrometry, whereas we found CTSA and carboxylesterase 1 (CES1) in monkey liver. We also purified R-134450 phosphoramidase activity in monkey liver and identified sphingomyelin phosphodiesterase, acid-like 3A (SMPADL3A), as an R-134450 phosphoramidase, which has not been reported to have any enzyme activity. Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes. Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level. This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs.

A fungus, Thelonectria discophora SANK 18292 (JCM 30947), produces nectrisine that has a nitrogen-containing heterocyclic 5-membered ring acting as a glycosidase inhibitor. Our previous study showed the possibility that 4-amino-4-deoxyarabinitol was enzymatically converted to nectrisine but the enzyme was not known. In order to characterize the enzyme, which is designated as NecC, it was purified from the fungus using ammonium sulfate precipitation and anion exchange chromatography. Liquid chromatography-tandem mass spectrometry analysis of NecC tryptic digests revealed partial NecC protein sequences. Subsequently, the partial DNA fragments were amplified by polymerase chain reaction with degenerate oligonucleotide primers and cloned. Then, necC complete genomic DNA was cloned by screening a genomic library of the fungus. Recombinant NecC also had NecC enzymatic activity, thus providing verification for the necC gene. NecC presumably belonged to the family of glucose methanol choline oxidoreductases, forming oligomers ranging approximately from 8 mer to 16 mer based on the results of native PAGE, and was also found to have a melting temperature of 57 °C, an optimal reaction condition of pH 7 at 30 °C, an activity inhibited by Cu2+ or ethylenediaminetetraacetic acid, and 4-amino-4-deoxyarabinitol as its preferred substrate. It was also indicated that not nectrisine but 4-amino-4-deoxyarabinitol was mainly extracted from the mycelium, and then was converted to nectrisine by the enzyme NecC in vitro. We believe that these findings are helpful to establish a nectrisine manufacturing process at large scale with the fungus.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0176-1) contains supplementary material, which is available to authorized users.

Loxoprofen (LX) is a prodrug-type non-steroidal anti-inflammatory drug which is used not only as an oral drug but also as a transdermal formulation. As a pharmacologically active metabolite, the trans-alcohol form of LX (trans-OH form) is generated after oral administration to humans. The objectives of this study were to evaluate the generation of the trans-OH form in human in vitro skin and to identify the predominant enzyme for its generation. In the permeation and metabolism study using human in vitro skin, both the permeation of LX and the formation of the trans-OH form increased in a time- and dose-dependent manner after the application of LX gel to the skin. In addition, the characteristics of permeation and metabolism of both LX and the trans-OH form were examined by a mathematical pharmacokinetic model. The K value was calculated to be 10.3 mm in the human in vitro skin. The predominant enzyme which generates the trans-OH form in human whole skin was identified to be carbonyl reductase 1 (CBR1) by immunodepletion using the anti-human CBR1 antibody. The results of the enzyme kinetic study using the recombinant human CBR1 protein demonstrated that the K and V values were 7.30 mm and 402 nmol/min/mg protein, respectively. In addition, it was found that no unknown metabolites were generated in the human in vitro skin. This is the first report in which LX is bioactivated to the trans-OH form in human skin by CBR1. Copyright © 2015 John Wiley & Sons, Ltd.