Post-radiation xerostomia therapy with allogeneic mesenchymal stromal stem cells in patients with head and neck cancer: study protocol for phase I clinical trial.

BACKGROUND: Xerostomia is a common side effect of radiotherapy in patients with head and neck tumors that negatively affects quality of life. There is no known effective standard treatment for xerostomia. Here, we present the study protocol used to evaluate the safety and preliminary efficacy of allogeneic mesenchymal stromal stem cells (MSCs) derived from umbilical cord tissue. PATIENTS AND METHODS: Ten oropharyngeal cancer patients with post-radiation xerostomia and no evidence of disease recurrence 2 or more years after (chemo)irradiation (intervention group) and 10 healthy volunteers (control group) will be enrolled in this nonrandomized, open-label, phase I exploratory study. MSCs from umbilical cord tissue will be inserted under ultrasound guidance into both parotid glands and both submandibular glands of the patients. Toxicity of the procedure will be assessed according to CTCAE v5.0 criteria at days 0, 1, 5, 28, and 120. Efficacy will be assessed by measuring salivary flow and analyzing its composition, scintigraphic evaluation of MSC grafting, retention, and migration, and questionnaires measuring subjective xerostomia and quality of life. In addition, the radiological, functional, and morphological characteristics of the salivary tissue will be assessed before, at 4 weeks, and at 4 months after the procedure. In the control group subjects, only salivary flow rate and salivary composition will be determined. DISCUSSION: The use of allogeneic MSCs from umbilical cord tissue represents an innovative approach for the treatment of xerostomia after radiation. Due to the noninvasive collection procedure, flexibility of cryobanking, and biological advantages, xerostomia therapy using allogeneic MSCs from umbilical cord tissue may have an advantage over other similar therapies.

Substrate-dependent inactivation of recombinant paraoxonase 1 during catalytic dihydrocoumarin turnover and the protective properties of surfactants.

Human paraoxonase-1 (PON1) is the most studied member of the paraoxonases (PONs) family and catalyzes the hydrolysis of various substrates (lactones, aryl esters, and paraoxon). Numerous studies link PON1 to oxidative stress-related diseases such as cardiovascular disease, diabetes, HIV infection, autism, Parkinson's, and Alzheimer's, where the kinetic behavior of an enzyme is characterized by initial rates or by modern methods that obtain enzyme kinetic parameters by fitting the computed curves over the entire time-courses of product formation (progress curves). In the analysis of progress curves, the behavior of PON1 during hydrolytically catalyzed turnover cycles is unknown. Hence, progress curves for enzyme-catalyzed hydrolysis of the lactone substrate dihydrocoumarin (DHC) by recombinant PON1 (rePON1) were analyzed to investigate the effect of catalytic DHC turnover on the stability of rePON1. Although rePON1 was significantly inactivated during the catalytic DHC turnover, its activity was not lost due to the product inhibition or spontaneous inactivation of rePON1 in the sample buffers. Examination of the progress curves of DHC hydrolysis by rePON1 led to the conclusion that rePON1 inactivates itself during catalytic DHC turnover hydrolysis. Moreover, human serum albumin or surfactants protected rePON1 from inactivation during this catalytic process, which is significant because the activity of PON1 in clinical samples is measured in the presence of albumin.

Investigation of Paraoxonase-1 Genotype and Enzyme-Kinetic Parameters in the Context of Cognitive Impairment in Parkinson's Disease.

Cognitive impairment is a common non-motor symptom of Parkinson's disease (PD), which often progresses to PD dementia. PD patients with and without dementia may differ in certain biochemical parameters, which could thus be used as biomarkers for PD dementia. The enzyme paraoxonase 1 (PON1) has previously been investigated as a potential biomarker in the context of other types of dementia. In a cohort of PD patients, we compared a group of 89 patients with cognitive impairment with a group of 118 patients with normal cognition. We determined the kinetic parameters Km and Vmax for PON1 for the reaction with dihydrocoumarin and the genotype of four single nucleotide polymorphisms in PON1. We found that no genotype or kinetic parameter correlated significantly with cognitive impairment in PD patients. However, we observed associations between PON1 rs662 and PON1 Km (p < 10-10), between PON1 rs662 and PON1 Vmax (p = 9.33 × 10-7), and between PON1 rs705379 and PON1 Vmax (p = 2.21 × 10-10). The present study is novel in three main aspects. (1) It is the first study to investigate associations between the PON1 genotype and enzyme kinetics in a large number of subjects. (2) It is the first study to report kinetic parameters of PON1 in a large number of subjects and to use time-concentration progress curves instead of initial velocities to determine Km and Vmax in a clinical context. (3) It is also the first study to calculate enzyme-kinetic parameters in a clinical context with a new algorithm for data point removal from progress curves, dubbed iFIT. Although our results suggest that in the context of PD, there is no clinically useful correlation between cognitive status on the one hand and PON1 genetic and enzyme-kinetic parameters on the other hand, this should not discourage future investigation into PON1's potential associations with other types of dementia.

iFIT: An automated web tool for determining enzyme-kinetic parameters based on the high-curvature region of progress curves.

The area where progress curve exhibits maximum curvature contains the most information about kinetic parameters. To determine these parameters more accurately from progress curves, we propose an iterative approach that calculates the area of maximum curvature based on an estimate of kinetic parameters and then recalculates the parameters based on time-concentration data points within this area. Based on this algorithm, we developed a computer script called iFIT as a free web application at http://www.i-fit.si. The benefits of working with iFIT are that it decreases the importance of initial substrate concentration and the impact of certain side reactions on the final calculated kinetic parameters.

The Removal of Time-Concentration Data Points from Progress Curves Improves the Determination of Km: The Example of Paraoxonase 1.

Several approaches for determining an enzyme's kinetic parameter Km (Michaelis constant) from progress curves have been developed in recent decades. In the present article, we compare different approaches on a set of experimental measurements of lactonase activity of paraoxonase 1 (PON1): (1) a differential-equation-based Michaelis-Menten (MM) reaction model in the program Dynafit; (2) an integrated MM rate equation, based on an approximation of the Lambert W function, in the program GraphPad Prism; (3) various techniques based on initial rates; and (4) the novel program "iFIT", based on a method that removes data points outside the area of maximum curvature from the progress curve, before analysis with the integrated MM rate equation. We concluded that the integrated MM rate equation alone does not determine kinetic parameters precisely enough; however, when coupled with a method that removes data points (e.g., iFIT), it is highly precise. The results of iFIT are comparable to the results of Dynafit and outperform those of the approach with initial rates or with fitting the entire progress curve in GraphPad Prism; however, iFIT is simpler to use and does not require inputting a reaction mechanism. Removing unnecessary points from progress curves and focusing on the area around the maximum curvature is highly advised for all researchers determining Km values from progress curves.

A Multi-Omics Analysis of PON1 Lactonase Activity in Relation to Human Health and Disease.

Paraoxonase 1 (PON1) enzyme has antioxidative properties and is present in mammalian blood and several other body fluids. In blood, PON1 is usually integrated into the high-density lipoprotein (HDL) cholesterol. PON1 is a highly versatile enzyme displaying diverse functions such as arylesterase, lactonase, and paraoxonase, among others. PON1 activities are usually investigated with artificial substrates, for example, dihydrocoumarin and thiobutyl butyrolactone for lactonase activity. The PON1 enzyme activities measured with different substrates tend to be falsely assumed as being equivalent in the literature, although there are poor or weak correlations among the PON1 enzyme activities with different substrates. In addition, and despite our knowledge of the factors influencing PON1 paraoxonase and arylesterase activities, there is little knowledge of PON1 lactonase activity variations and attendant mechanisms. This is important considering further that the lactonase activity is the native activity of PON1. We report here a multi-omics analysis of PON1 lactonase activity. The influence of genetic variations, particularly of single nucleotide polymorphisms and epigenetic, proteomic, and lipidomic variations on PON1 lactonase activity are reviewed. In addition, the influence of various environmental, clinical, and demographic variables on PON1 lactonase activity is discussed. Finally, we examine the associations between PON1 lactonase activity and health states and common complex diseases such as atherosclerosis, dementias, obesity, and diabetes. To the best of our knowledge, this is the first multi-omics analysis of PON1 lactonase activity with an eye to future applications in basic life sciences and translational medicine and the nuances of critically interpreting PON1 function with lactones as substrates.

The Structure and Function of Paraoxonase-1 and Its Comparison to Paraoxonase-2 and -3.

Serum paraoxonase-1 (PON1) is the most studied member of the group of paraoxonases (PONs). This enzyme possesses three enzymatic activities: lactonase, arylesterase, and paraoxonase activity. PON1 and its isoforms play an important role in drug metabolism as well as in the prevention of cardiovascular and neurodegenerative diseases. Although all three members of the PON family have the same origin and very similar amino acid sequences, they have different functions and are found in different locations. PONs exhibit substrate promiscuity, and their true physiological substrates are still not known. However, possible substrates include homocysteine thiolactone, an analogue of natural quorum-sensing molecules, and the recently discovered derivatives of arachidonic acid-bioactive δ-lactones. Directed evolution, site-directed mutagenesis, and kinetic studies provide comprehensive insights into the active site and catalytic mechanism of PON1. However, there is still a whole world of mystery waiting to be discovered, which would elucidate the substrate promiscuity of a group of enzymes that are so similar in their evolution and sequence yet so distinct in their function.

Interactions of Paraoxonase-1 with Pharmacologically Relevant Carbamates.

Mammalian paraoxonase-1 hydrolyses a very broad spectrum of esters such as certain drugs and xenobiotics. The aim of this study was to determine whether carbamates influence the activity of recombinant PON1 (rePON1). Carbamates were selected having a variety of applications: bambuterol and physostigmine are drugs, carbofuran is used as a pesticide, while Ro 02-0683 is diagnostic reagent. All the selected carbamates reduced the arylesterase activity of rePON1 towards the substrate S-phenyl thioacetate (PTA). Inhibition dissociation constants (Ki), evaluated by both discontinuous and continuous inhibition measurements (progress curves), were similar and in the mM range. The rePON1 displayed almost the same values of Ki constants for Ro 02-0683 and physostigmine while, for carbofuran and bambuterol, the values were approximately ten times lower and two times higher, respectively. The affinity of rePON1 towards the tested carbamates was about 3-40 times lower than that of PTA. Molecular modelling of rePON1-carbamate complexes suggested non-covalent interactions with residues of the rePON1 active site that could lead to competitive inhibition of its arylesterase activity. In conclusion, carbamates can reduce the level of PON1 activity, which should be kept in mind, especially in medical conditions characterized by reduced PON1 levels.

Evaluation of the paraoxonase-1 kinetic parameters of the lactonase activity by nonlinear fit of progress curves.

Although paraoxonase-1 (PON1) activity has been demonstrated to be a reliable biomarker of various diseases, clinical studies have been based only on relative comparison of specific enzyme activities, which capture differences mainly due to (usually unknown) PON1 concentration. Hence, the aim of this report is to present for the first time the simple evaluation method for determining autonomous kinetic parameter of PON1 that could be also associated with polymorphic forms and diseases; i.e. the Michaelis constant which is enzyme concentration independent quantity. This alternative approach significantly reduces the number of experiments needed, and it yields the results with great accuracy.

Exploring the aryl esterase catalysis of paraoxonase-1 through solvent kinetic isotope effects and phosphonate-based isosteric analogues of the tetrahedral reaction intermediate.

Although a recent study of Debord et al. in Biochimie (2014; 97:72-77) described the thermodynamics of the catalysed hydrolysis of phenyl acetate by human paraoxonase-1, the mechanistic details along the reaction route of this enzyme remain unclear. Therefore, we briefly present the solvent kinetic isotope effects on the phenyl acetate esterase activity of paraoxonase-1 and its inhibition with the phenyl methylphosphonate anion, which is a stable isosteric analogue that mimics the high-energy tetrahedral intermediate on the hydroxide-promoted hydrolysis pathway. The data show normal isotope effects, while proton inventory analysis indicates that two protons contribute to the kinetic isotope effect. Coherently, moderate competitive inhibition with the phenyl methylphosphonate anion reveals that the rate-limiting transition state suboptimally resembles the tetrahedral intermediate. The implications of these findings can be attributed to two possible reaction mechanisms that might occur during the paraoxonase-1-catalysed hydrolysis of phenyl acetate.

(Poly)peptide-based therapy for diabetes mellitus: insulins versus incretins.

Insulin therapy remains the standard of care for achieving and maintaining adequate glycemic control, especially in hospitalized patients with critical and noncritical illnesses. Insulin therapy is more effective against elevated fasting glycaemia but less in the reduction of postprandial hyperglycaemia. It is associated with a high incidence of hypoglycemia and weight gain. Contrary, GLP-1 mimetic therapy improves postprandial glycaemia without the hypoglycaemia and weight gain associated with aggressive insulin therapy. Moreover, it has the potential to reduce cardiovascular related morbidity. However, its increased immunogenicity and severe gastrointestinal adverse effects present a huge burden on patients. Thus, a right combination of basal insulin which has lowering effect on fasting plasma glucose and GLP-1 mimetic with its lowering effect on postprandial plasma glucose with minimal gastrointestinal adverse effects, seems the right therapy choice from a clinical point of view for some diabetic patients. In this article, we discuss the pros and cons of the use of insulin analogues and GLP-1 mimetics that are associated with the treatment of type 2 diabetes.

Mono-ADP-ribosyltransferase as a potential pharmacological drug target in the GLP-1 based therapy of obesity and diabetes mellitus type 2.

Glucagon-like peptide-1 (GLP-1) based therapy is well established for treating diabetes mellitus type 2. Moreover, GLP-1 receptor agonists influence weight loss, and have potential for treating obesity. GLP-1 receptor agonists should be administered in low doses, together with drugs that potentiate insulin release, to avoid some minor side effects. We have focused on incretin hormones, especially GLP-1 and its analogues. Here we discuss the effect of the third intracellular loop-derived peptide of GLP-1 receptor on intracellular mono-ADP-ribosyltransferase and its role in regulating the receptor. We suggest that this intracellular mono-ADP-ribosyltransferase could constitute a possible novel pharmacological target in the treatment of diabetes mellitus type 2 and obesity.

Glucagon like-peptide-1 receptor is covalently modified by endogenous mono-ADP-ribosyltransferase.

Our previous study revealed a mono-ADP-ribosyltransferase mediated in vitro mono-ADP-ribosylation of IC(3) peptide, a peptide with sequence corresponded to third intracellular loop of glucagon like-peptide-1 (GLP-1) receptor. Furthermore, Arg(348) was shown to be modified amino acid residue although its mutation did not eliminate mono-ADP-ribosylation completely. In order to further study the signaling mechanisms of GLP-1 receptor, we took on lease a possibility that an alternative site of enzymatic modification exist so mono-ADP-ribosylation of Cys(341) was hypothesized. The results confirmed both Arg(348) and Cys(341) as a site of mono-ADP-ribosylation where Arg(348) is modified predominantly. Sum of mono-ADP-ribosylation rate of both single IC(3) mutants coincided with IC(3) rate. What is in vivo role of Cys(341) mono-ADP-ribosylation is entirely speculative but our study represents an important step toward a complete understanding of signaling via GLP-1 receptor.

Third intracellular loop of glucagon like-peptide-1 receptor is coupled with endogenous mono-ADP-ribosyltransferase - novel type of receptor regulation?

Our previous studies revealed the main role of the third intracellular loop (IC(3)) of glucagon-like peptide-1 receptor (GLP-1 receptor), in G-protein activation, where the presence or absence of agonist and the receptor phosphorylation seemed to be the only regulatory mechanisms. In order to further study the signaling mechanisms of GLP-1 receptor, we investigated the effect of the third intracellular loop-derived peptide on endogenous mono-ADP-ribosyltransferase mediated mono-ADP-ribosylation of G-proteins ß subunit in CHO cells. Results showed an inhibitory effect of IC(3) peptide on mono-ADP-ribosylation of ß subunit, obviously via the mechanism of competitive inhibition. Excluding the activity of this inhibitory mechanism via pertussis toxin-sensitive G proteins, the direct functional coupling of IC(3) of GLP-1 receptor and endogenous mono-ADP-ribosyltransferase was confirmed. We suggest that this arginine specific enzymatic posttranslational modification of third intracellular loop of GLP-1 receptor might represent a possible novel mechanism of receptor activity regulation and the pharmacological potential in treatment of diabetes mellitus type 2.

Structure, Function and Regulation of Group IV Phospholipase A2 Family.

The Group IV phospholipase A2 family is consisted of six intracellular enzymes. They catalyze hydrolysis of the sn-2 ester bond of glycerophospholipids, releasing fatty acid metabolites and lysophospholipids. Agonist-induced release of arachidonic acid for the production of eicosanoids by PLA2IValpha enzyme is important in regulating normal and pathological processes in a variety of target tissues. Here, we compare PLA2IValpha, and its paralogs ß, γ, δ, ε and ζ in term of of their structure, function and regulation.

Constructing glucagon like peptide-1 receptor fused with derivatives of GFP for visualizing protein-protein interaction in living cells.

The glucagon-like peptide-1 receptor (GLP-1 receptor) mediates important antidiabetogenic effects on peripheral tissues. It appears to be one of the most promising therapeutic targets for treatment of diabetes mellitus type 2. Surprisingly, very little is known about the cellular mechanisms that regulate receptor function in living cells. One of the approaches how to study receptor dynamics is by using tagged fluorescent proteins. In this study, YFP-tagged GLP-1 (YFP-GLP-1) receptor and CFP-tagged GLP-1 (CFP-GLP-1) receptor for visualizing protein-protein interaction in living cells were constructed and localized in CHO cells. Cells expressing YFP-GLP-1 and CFP-GLP-1 receptor showed characteristic GLP-1 mediated increase in cAMP, similar to cells expressing a wild type GLP-1 receptor. This means that both types of receptors are functional and localized in plasma membrane.

Functional characterization of N-terminally GFP-tagged GLP-1 receptor.

The glucagon-like peptide-1 receptor (GLP-1 receptor) mediates important effects on peripheral tissues and the central nervous system. It seems one of the most promising therapeutic targets for treatment of diabetes mellitus type 2. Surprisingly, very little is known about the cellular mechanisms that regulate its function in vivo. One of the approaches to study receptor dynamics, expression, or signaling is using GFP-tagged fluorescent proteins. In this study, we synthesized and characterized N-terminally GFP-tagged GLP-1 (GFP-GLP-1) receptor in CHO cells. We demonstrated that GFP-GLP-1 receptor is weakly expressed in the plasma membranes and is functionally coupled to adenylyl cyclase via heterotrimeric G-proteins, similarly as its wild type.

Role of cysteine 341 and arginine 348 of GLP-1 receptor in G-protein coupling.

We have demonstrated the ability of peptides derived from the third intracellular loop of GLP-1 receptor to differently modulate activity of four different types of G-proteins overexpressed in sf9 cells. In this respect, the involvement of Cys(341) in inhibition of G(s) and Cys(341) in activation of G(s) and in inhibition of G(i1,) G(o), and G(11), respectively, indicates their potential role in discrimination between different types of G-proteins. Moreover, these two amino acids from the third intracellular loop might represent an important novel targets for covalent modification by downstream regulators in signaling through GLP-1 receptor.

Galpha13 mediates activation of the cytosolic phospholipase A2alpha through fine regulation of ERK phosphorylation.

Heterotrimeric GTP-binding (G) proteins transduce hormone-induced signals to their effector enzymes, which include several phospholipases. In particular, the G(o)/G(i) and G(q) protein families have been shown to couple signaling to phospholipase A(2) (PLA(2)), phospholipase C, and phospholipase D, while the G(12)/G(13) family has been linked to the activation of small GTPases of the Rho family, and hence, to phospholipase D activation. Here, we demonstrate that in CHO cells, the G(12)/G(13) family is also able to activate cPLA(2)alpha, through the activation of RhoA and, subsequently, ERK1/2. Hormone-induced arachidonic acid release increased as a consequence of Galpha(13) overexpression, and was inhibited through inhibition of Galpha(13) signaling. The Galpha(13)-mediated cPLA(2)alpha activation was inhibited by pharmacological blockade of ERK1/2 with either U0126 or PD98059, and by RhoA inactivation with C3 toxin or a dominant-negative RhoA (N19RhoA), and was stimulated by the serine-threonine phosphatase inhibitor calyculin A. Our data thus identify a pathway of cPLA(2)alpha regulation that is initiated by thrombin and purinergic receptor activation, and that signals through Galpha(13), RhoA and ERK1/2, with the involvement of a calyculin-sensitive phosphatase.

Novel features of amphiphilic peptide Mas7 in signalling via heterotrimeric G-proteins.

Amphiphilic peptide Mas7, a structural analogue of mastoparan is a known activator of heterotrimeric Gi-proteins and its downstream effectors. This study investigated the functional interaction of Mas7 with a plasma membrane protein from CHO cells, the endogenous mono-ADP-ribosyltransferase. The substrate of endogenous mono-ADP-ribosyltransferase was the ADP-ribosylated protein with a molecular mass of 36 kDa, which corresponded to the beta subunit of heterotrimeric G-proteins. The effect of Mas7 on endogenous mono-ADP-ribosyltransferase activity was in the micromolar range with a maximal activation of 205% over the basal. In pertussis treated plasma membranes, it was found that the effect of Mas7 on endogenous mono-ADP-ribosyltransferase was partially blocked, which suggests the involvement of G-proteins, such as Gi or G0. In addition, an immunoassay was developed for the visualization of interaction between the a subunit and the betagamma dimer of G-protein on a Ni-NTA support. The physical interaction was tested of Mas7 with the heterotrimeric G-protein alphai2 subunit, which was overexpressed together with beta1gamma2-His6 subunits in sf9 cells. An interaction between Gi2 heterotrimer and Mas7 was not observed, which was not in accordance with previously reported results of mastoparan obtained for Gi-proteins from bovine brain. In conclusion, the signal is mediated from Mas7 to endogenous mono-ADP-ribosyltransferase via pertussis sensitive G-proteins. Furthermore, it is hypothesized that Gi2 G-proteins are not involved in the process.