Dipeptidyl Peptidase-4-Mediated Fibronectin Processing Evokes a Profibrotic Extracellular Matrix.

Fibronectin serves as a platform to guide and facilitate deposition of collagen and fibrillin microfibrils. During development of fibrotic diseases, altered fibronectin deposition in the extracellular matrix (ECM) is generally an early event. After this, dysregulated organization of fibrillins and fibrillar collagens occurs. Because fibronectin is an essential orchestrator of healthy ECM, perturbation of its ECM-organizational capacity may be involved in development of fibrosis. To investigate this, we employed recessive dystrophic epidermolysis bullosa as a disease model with progressive, severe dermal fibrosis. Fibroblasts from donors with recessive dystrophic epidermolysis bullosa in 2-dimensional and 3-dimensional cultures displayed dysregulated fibronectin deposition. Our analyses revealed that increase of profibrotic dipeptidyl peptidase-4-positive fibroblasts coincides with altered fibronectin deposition. Dipeptidyl peptidase-4 inhibitors normalized deposition of fibronectin and subsequently of fibrillin microfibrils and collagen I. Intriguingly, proteomics and inhibitor and mutagenesis studies disclosed that dipeptidyl peptidase-4 modulates ECM deposition through the proteolysis of the fibronectin N-terminus. Our study provides mechanistic insights into the observed profibrotic activities of dipeptidyl peptidase-4 and extends the understanding of fibronectin-guided ECM assembly in health and disease.

Diversity of amyloid beta peptide actions.

Fibril formation by amyloidogenic proteins and peptides is considered the cause of a number of incurable diseases. One of the most known amyloid diseases is Alzheimer's disease (AD). Traditionally, amyloidogenic beta peptides Aß40 and Aß42 (Aßs) are considered as main causes of AD and the foremost targets in AD fight. The main efforts in pharmacology are aimed at reducing Aßs concentration to prevent their accumulation, aggregation, formation of senile plaques, neuronal death, and neurodegeneration. However, a number of publications have demonstrated certain beneficial physiological effects of Aßs. Simultaneously, it is indicated that the effects of Aßs turn into pathological due to the development of certain diseases in the body. The accumulation of C- and N-terminal truncated Aßs under diverse conditions is supposed to play a role in AD development. The significance of transformation of glutamate residue at positions 3 or 11 of Aßs catalyzed by glutaminyl cyclase making them more degradation resistant, hydrophobic, and prone to aggregation, as well as the participation of dipeptidyl peptidase IV in these transformations are discussed. The experimental data presented confirm the maintenance of physiological, nonaggregated state of Aßs by plant preparations. In conclusion, this review suggests that in the fight against AD, instead of removing Aßs, preference should be given to the treatment of common diseases. Glutaminyl cyclase and dipeptidyl peptidase IV can be considered as targets in AD treatment. Flavonoids and plant preparations that possess antiamyloidogenic propensity are proposed as beneficial neuroprotective, anticancer, and antidiabetic food additives.

Citrullination of adenosine deaminase impairs its binding to dipeptidyl peptidase IV.

The presence of citrullinated adenosine deaminase (ADA) was reported in the synovial fluids of rheumatoid arthritis individuals. This work reports the effects of ADA citrullination on the formation/stabilization of ADA complex with dipeptidyl peptidase IV (DPPIV). The electrophoretic mobility of in vivo citrullinated ADA was diminished compared to the native one. The biosensor binding study demonstrated approximately four-fold lower affinity of both in vivo and in vitro citrullinated ADAs to DPPIV (KD = 161 ± 51.3 and 171 ± 52.2 nM, respectively) compared with wild ADA (KD = 38 ± 9.4 nM). These results were confirmed by examining the ADA interaction with DPPIV using size-exclusion chromatography and fluorescence anisotropy methods. The computational modeling of Arg142 â†’ Cit142 modification in ADA showed a local structural rearrangement and a less favorable binding affinity to DPPIV. According to these observations, citrullinated ADA being a possible target triggering autoimmunity hinders also the formation of ADA-DPPIV complex, essential in immune system function.

Adenosine deaminase - A target for new piperazine derivatives.

The level of adenosine deaminase (ADA) activity increases in pathological effusions. Therefore, the concentration of its substrate, anti-inflammatory adenosine, decreases, thereby aggravating inflammation. Hence, the quest for ADA inhibiting compounds is an actual problem in medicine and pharmacology. This work describes the inhibition of bovine ADA by new synthesized piperazine compounds. 15 compounds were screened; IC50 values for 5 more potent ones of them were between 3.4 and 98.6 µg/ml. The inhibition of activity of intracellular and ecto- forms of ADA by the most effective "compound 1" was of competitive nature. For these two forms of enzyme, the inhibition constants, Ki (1.5 and 115 µM) and IC50 values (6.5 and 480 µM), respectively, differed by nearly two orders. The constant of bimolecular interaction KSV between "compound 1" and the tryptophan residues in ADA was estimated in fluorescence quenching study as of 0.145 ± 0.027 µM. Finally, the molecular interactions between "compound 1" and the bovine enzyme ADA were highlighted through molecular docking studies.

Concerted action of dipeptidyl peptidase IV and glutaminyl cyclase results in formation of pyroglutamate-modified amyloid peptides in vitro.

Compelling evidence suggests a crucial role of amyloid beta peptides (Aß(1-40/42)) in the etiology of Alzheimer's disease (AD). The N-terminal truncation of Aß(1-40/42) and their modification, e.g. by glutaminyl cyclase (QC), is expected to enhance the amyloid toxicity. In this work, the MALDI-TOF mass spectrometry application proved N-terminal cleavage of Aß(1-40/42) by purified dipeptidyl peptidase IV (DPPIV) in vitro observed earlier. The subsequent transformation of resulted Aß(3-40/42) to pE-Aß(3-40/42) in QC catalyzed glutamate cyclization was manifested. Hence, consecutive conversion of Aß(1-40/42) by DPPIV and QC can be assumed as a potential mechanism of formation of non-degrading pyroglutamated pE-Aß(3-40/42), which might accumulate and contribute to AD progression. The in vitro acceleration of Aß(1-40) aggregation in the simultaneous presence of DPPIV and QC was shown also.

Interaction of dipeptydil peptidase IV with amyloid peptides.

UNLABELLED: The aggregates of amyloid beta peptides (Aßs) are regarded as one of the main pathological hallmarks of Alzheimer's disease (AD). An imbalance between the rates of synthesis and clearance of Aßs is considered to be a possible cause for the onset of AD. Dipeptidyl peptidases II and IV (DPPII and DPPIV) are serine proteases removing N-terminal dipeptides from polypeptides and proteins with proline or alanine on the penultimate position. Alanine is an N-terminal penultimate residue in Аßs, and we presumed that DPPII and DPPIV could cleave them. The results of present in vitro research demonstrate for the first time the ability of DPPIV to truncate the commercial Aß40 and Aß42 peptides, to hinder the fibril formation by them and to participate in the disaggregation of preformed fibrils of these peptides. The increase of absorbance at 334 nm due to complex formation between primary amines with o-phtalaldehyde was used to show cleaving of Aß40 and Aß42. The time-dependent increase of the quantity of primary amines during incubation of peptides in the presence of DPPIV suggested their truncation by DPPIV, but not by DPPII. The parameters of the enzymatic breakdown by DPPIV were determined for Aß40 (Km=37.5 µM, kcat/Km=1.7×10(3)M(-1)sec(-1)) and Aß42 (Km=138.4 µM, kcat/Km=1.90×10(2)M(-1)sec(-1)). The aggregation-disaggregation of peptides was controlled by visualization on transmission electron microscope and by Thioflavin-T fluorescence on spectrofluorimeter and fluorescent microscope. DPPIV hindered the peptide aggregation/fibrillation during 3-4 days incubation in 20mM phosphate buffer, pH 7.4, 37°C by 50-80%. Ovalbumin, BSA and DPPII did not show this effect. In the presence of DPPIV, the preformed fibrils were disaggregated by 30-40%. CONCLUSION: for the first time it was shown that the Aß40 and Aß42 are substrates of DPPIV. DPPIV prohibits the fibrillation of peptides and promotes disaggregation of their preformed aggregates.

Proline-rich cytokine from neurosecretory granules: a new natural substrate for dipeptidyl peptidase IV.

A proline-rich cytokine from neurosecretory granules of bovine neurohypophysis, 15 amino acids containing PRP-1 (Ala-Gly-Ala-Pro-Glu-Pro-Ala-Glu-Pro-Ala-Gln-Pro-Gly-Val-Tyr), had been demonstrated as a unique regulator of activity of neurons, strong antibacterial agent, and mediator of the hypothalamus-neurohypophysis-bone marrow-thymus axis, which participates in hematopoietic stem cells differentiation. In the present work it was shown that this neuropeptide represents a new natural substrate for Dipeptidyl Peptidase IV (DPPIV). The time-dependent increase of primary amines quantity in the assay mixture of PRP-1 and DPPIV has been observed allowing to conclude, that DPPIV catalyses the enzymatic reaction of PRP-1 cleavage. The amount of primary amines in the assay mixtures was evaluated using o-phtaldialdehyde dye. The gel-filtration and paper electrophoresis analyses proved this conclusion. The catalytic parameters of catalyzed by DPPIV enzymatic reaction of PRP-1 cleavage were determined as: V(max) = 1.27 ± 0.11 nmol/min and K(m) = 0.38 ± 0.1 mM.

Influence of dipeptidyl peptidase IV on enzymatic properties of adenosine deaminase.

The importance of ADA (adenosine deaminase) in the immune system and the role of its interaction with an ADA-binding cell membrane protein dipeptidyl peptidase IV (DPPIV), identical to the activated immune cell antigen, CD26, has attracted the interest of researchers for many years. To investigate the specific properties in the structure-function relationship of the ADA/DPPIV-CD26 complex, its soluble form, identical to large ADA (LADA), was isolated from human blood serum, human pleural fluid and bovine kidney cortex. The kinetic constants (Km and Vmax) of LADA and of small ADA (SADA), purified from bovine lung and spleen, were compared using adenosine (Ado) and 2'-deoxyadenosine (2'-dAdo) as substrates. The Michaelis constant, Km, evidences a higher affinity of both substrates (in particular of more toxic 2'-dAdo) for LADA and proves the modulation of toxic nucleoside neutralization in the extracellular medium due to complex formation between ADA and DPPIV-CD26. The values of Vmax are significantly higher for SADA, but the efficiency, Vmax/Km, in LADA-catalyzed 2'-dAdo deamination is higher than that in Ado deamination. The interaction of all enzyme preparations with derivatives of adenosine and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was studied. 1-DeazaEHNA and 3-deazaEHNA demonstrate stronger inhibiting activity towards LADA, the DPPIV-CD26-bound form of ADA. The observed differences between the properties of the two ADA isoforms may be considered as a consequence of SADA binding with DPPIV-CD26. Both SADA and LADA indicated a similar pH-profile of adenosine deamination reaction with the optimum at pHs 6.5-7.5, while the pH-profile of dipeptidyl peptidase activity of the ADA/DPPIV-CD26 complex appeared in a more alkaline region.