Altered Activity Profiles Research Articles

Identification of activity-based biomarkers for early-stage pancreatic tumors in blood using single-molecule enzyme activity screening

Single-molecule enzyme activity-based enzyme profiling (SEAP) is a methodology to globally analyze protein functions in living samples at the single-molecule level. It has been previously applied to detect functional alterations in phosphatases and glycosidases. Here, we expand the potential for activity-based biomarker discovery by developing a semi-automated synthesis platform for fluorogenic probes that can detect various peptidases and protease activities at the single-molecule level. The peptidase/protease probes were prepared on the basis of a 7-amino-4-methylcoumarin fluorophore. The introduction of a phosphonic acid to the core scaffold made the probe suitable for use in a microdevice-based assay, while phosphonic acid served as the handle for the affinity separation of the probe using Phos-tag. Using this semi-automated scheme, 48 fluorogenic probes for the single-molecule peptidase/protease activity analysis were prepared. Activity-based screening using blood samples revealed altered single-molecule activity profiles of CD13 and DPP4 in blood samples of patients with early-stage pancreatic tumors. The study shows the power of single-molecule enzyme activity screening to discover biomarkers on the basis of the functional alterations of proteins.

Impact of aging on meningeal gene expression

BackgroundThe three-layered meninges cover and protect the central nervous system and form the interface between cerebrospinal fluid and the brain. They are host to a lymphatic system essential for maintaining fluid dynamics inside the cerebrospinal fluid-filled subarachnoid space and across the brain parenchyma via their connection to glymphatic structures. Meningeal fibroblasts lining and traversing the subarachnoid space have direct impact on the composition of the cerebrospinal fluid through endocytotic uptake as well as extensive protein secretion. In addition, the meninges are an active site for immunological processes and act as gatekeeper for immune cells entering the brain. During aging in mice, lymphatic drainage from the brain is less efficient contributing to neurodegenerative processes. Aging also affects the immunological status of the meninges, with increasing numbers of T cells, changing B cell make-up, and altered macrophage complement.MethodsWe employed RNASeq to measure gene expression and to identify differentially expressed genes in meninges isolated from young and aged mice. Using Ingenuity pathway, GO term, and MeSH analyses, we identified regulatory pathways and cellular functions in meninges affected by aging.ResultsAging had profound impact on meningeal gene expression. Pathways related to innate as well as adaptive immunity were affected. We found evidence for increasing numbers of T and B lymphocytes and altered activity profiles for macrophages and other myeloid cells. Furthermore, expression of pro-inflammatory cytokine and chemokine genes increased with aging. Similarly, the complement system seemed to be more active in meninges of aged mice. Altered expression of solute carrier genes pointed to age-dependent changes in cerebrospinal fluid composition. In addition, gene expression for secreted proteins showed age-dependent changes, in particular, genes related to extracellular matrix composition and organization were affected.ConclusionsAging has profound effects on meningeal gene expression; thereby affecting the multifaceted functions meninges perform to maintain the homeostasis of the central nervous system. Thus, age-dependent neurodegenerative processes and cognitive decline are potentially in part driven by altered meningeal function.

The zinc-binding motif in tankyrases is required for the structural integrity of the catalytic ADP-ribosyltransferase domain.

Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and are considered promising drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyltransferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on the activity, stability and structure of human tankyrases. We generated mutants of human tankyrase (TNKS) 1 and TNKS2, abolishing the zinc-binding capabilities, and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive, probably due to introduction of major structural defects, the TNKS2 mutant remained active and displayed an altered activity profile compared to the wild-type.

Cytomegalovirus Latent Infection is Associated with an Increased Risk of COVID-19-Related Hospitalization.

Some risk factors for severe coronavirus disease 2019 (COVID-19) have been identified, including age, race, and obesity. However, 20%–50% of severe cases occur in the absence of these factors. Cytomegalovirus (CMV) is a herpesvirus that infects about 50% of all individuals worldwide and is among the most significant nongenetic determinants of immune system. We hypothesized that latent CMV infection might influence the severity of COVID-19. Our analyses demonstrate that CMV seropositivity is associated with more than twice the risk of hospitalization due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Immune profiling of blood and CMV DNA quantitative polymerase chain reaction in a subset of patients for whom respiratory tract samples were available revealed altered T-cell activation profiles in absence of extensive CMV replication in the upper respiratory tract. These data suggest a potential role for CMV-driven immune perturbations in affecting the outcome of SARS-CoV-2 infection and may have implications for the discrepancies in COVID-19 severity between different human populations.

NAD(H)-mediated tetramerization controls the activity of Legionella pneumophila phospholipase PlaB

The virulence factor PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires' disease. It is a highly active phospholipase exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal α/β-hydrolase domain expanded by two noncanonical two-stranded β-sheets, β-6/β-7 and β-9/β-10. The C-terminal domain reveals a fold displaying a bilobed β-sandwich with a hook structure required for dimer formation and structural complementation of the enzymatic domain in the neighboring monomer. This highlights the dimer as the active form. Δβ-9/β-10 mutants showed a decrease in the tetrameric fraction and altered activity profiles. The variant also revealed restricted binding to membranes resulting in mislocalization and bacterial lysis. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetramer and concomitantly reduced activity. Together, these data reveal structural elements and an unprecedented NAD(H)-mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The allosteric regulatory process identified here is suited to fine tune PlaB in a way that protects Legionella pneumophila from self-inflicted lysis while ensuring its activity at the pathogen-host interface.

Discovery of gramicidin A analogues with altered activities by multidimensional screening of a one-bead-one-compound library

Gramicidin A (1) is a peptide antibiotic that disrupts the transmembrane ion concentration gradient by forming an ion channel in a lipid bilayer. Although long used clinically, it is limited to topical application because of its strong hemolytic activity and mammalian cytotoxicity, likely arising from the common ion transport mechanism. Here we report an integrated high-throughput strategy for discovering analogues of 1 with altered biological activity profiles. The 4096 analogue structures are designed to maintain the charge-neutral, hydrophobic, and channel forming properties of 1. Synthesis of the analogues, tandem mass spectrometry sequencing, and 3 microscale screenings enable us to identify 10 representative analogues. Re-synthesis and detailed functional evaluations find that all 10 analogues share a similar ion channel function, but have different cytotoxic, hemolytic, and antibacterial activities. Our large-scale structure-activity relationship studies reveal the feasibility of developing analogues of 1 that selectively induce toxicity toward target organisms.

Increased T Cell Differentiation and Cytolytic Function in Bangladeshi Compared to American Children.

During the first 5 years of life, children are especially vulnerable to infection-related morbidity and mortality. Conversely, the Hygiene Hypothesis suggests that a lack of exposure to infectious agents early in life could explain the increasing incidence of allergies and autoimmunity in high-income countries. Understanding these phenomena, however, is hampered by a lack of comprehensive, direct immune monitoring in children with differing degrees of microbial exposure. Using mass cytometry, we provide an in-depth profile of the peripheral blood mononuclear cells (PBMCs) of children in regions at the extremes of exposure: the San Francisco Bay Area, USA and an economically poor district of Dhaka, Bangladesh. Despite variability in clinical health, functional characteristics of PBMCs were similar in Bangladeshi and American children at 1 year of age. However, by 2–3 years of age, Bangladeshi children's immune cells often demonstrated altered activation and cytokine production profiles upon stimulation with PMA-ionomycin, with an overall immune trajectory more in line with American adults. Conversely, immune responses in children from the US remained steady. Using principal component analysis, donor location, ethnic background, and cytomegalovirus infection status were found to account for some of the variation identified among samples. Within Bangladeshi 1-year-olds, stunting (as measured by height-for-age z-scores) was found to be associated with IL-8 and TGFβ expression in PMA-ionomycin stimulated samples. Combined, these findings provide important insights into the immune systems of children in high vs. low microbial exposure environments and suggest an important role for IL-8 and TGFβ in mitigating the microbial challenges faced by the Bangladeshi children.

Discovery and structure of the antimicrobial lasso peptide citrocin

We report the identification of citrocin, a 19-amino acid-long antimicrobial lasso peptide from the bacteria Citrobacter pasteurii and Citrobacter braakii We refactored the citrocin gene cluster and heterologously expressed it in Escherichia coli We determined citrocin's NMR structure in water and found that is reminiscent of that of microcin J25 (MccJ25), an RNA polymerase-inhibiting lasso peptide that hijacks the TonB-dependent transporter FhuA to gain entry into cells. Citrocin has moderate antimicrobial activity against E. coli and Citrobacter strains. We then performed an in vitro RNA polymerase (RNAP) inhibition assay using citrocin and microcin J25 against E. coli RNAP. Citrocin has a higher minimal inhibition concentration than microcin J25 does against E. coli but surprisingly is ∼100-fold more potent as an RNAP inhibitor. This suggests that citrocin uptake by E. coli is limited. We found that unlike MccJ25, citrocin's activity against E. coli relied on neither of the two proton motive force-linked systems, Ton and Tol-Pal, for transport across the outer membrane. The structure of citrocin contains a patch of positive charge consisting of Lys-5 and Arg-17. We performed mutagenesis on these residues and found that the R17Y construct was matured into a lasso peptide but no longer had activity, showing the importance of this side chain for antimicrobial activity. In summary, we heterologously expressed and structurally and biochemically characterized an antimicrobial lasso peptide, citrocin. Despite being similar to MccJ25 in sequence, citrocin has an altered activity profile and does not use the same outer-membrane transporter to enter susceptible cells.

Abstract A209: Targeting immune cell-specific sphingosine-1-phosphate receptor 4 to restore antitumor immunity resulting in improved therapy response

Abstract The inflammatory tumor microenvironment is a critical element of tumor development and progression, as well as a parameter predicting the response to cancer treatment. An increased abundance of suppressive immune cells such as tumor-associated macrophages (TAMs) and regulatory T-cells (Tregs) appears thereby as a limiting factor for a successful treatment due to the inhibition of tumoricidal lymphocytes such as cytotoxic CD8+ T-cells. The bioactive lipid sphingosine-1-phosphate (S1P), which signals mainly through its five G-protein-coupled receptors (S1PR1-5), emerges as an important regulator of carcinogenesis that promotes tumor growth, angiogenesis and metastasis, among others, by affecting tumor immunity. We investigated S1PR4, which is exclusively expressed by immune cells, as a potential regulator of antitumor immunity. Using a transgenic mouse model of spontaneous invasive ductal mammary carcinoma (PyMT mice) and the AOM/DSS model of colitis-associated cancer, we show that S1PR4 deficiency (S1PR4-/-) leads to a significant decrease in lung metastasis and a delay of tumor growth in case of the PyMT breast cancer model and reduced colon tumor incidence and size in S1PR4-/- mice subjected to the AOM/DSS model. S1PR4-/- PyMT tumors contained macrophages with an altered activation profile, characterized by reduced IL-10, IL-1β and IL-6 production. Likely as a consequence, S1PR4-/- mice showed a less immunosuppressive lymphocyte infiltrate composed of reduced Tregs and Th17 cells, but increased cytotoxic CD8+ T-cell numbers. Interestingly, AOM/DSS-treated colons of S1PR4-/- mice also displayed a marked increase in CD8+ intestinal epithelial lymphocytes, underlining the negative impact of S1PR4 signaling on CD8+ T-cell abundance during tumor development. Next, we asked whether increased CD8+ T-cell numbers in S1PR4-/- tumors would increase the efficacy of chemotherapy or immunotherapy. Indeed, S1PR4-/- PyMT mice exhibited a significantly improved response to treatment with doxorubicin (DXR), characterized by the absence of relapse, whereas WT PyMT tumors relapsed after DXR treatment. This enhanced tumor control was strictly CD8+ T-cell dependent, as shown by experiments utilizing CD8 antibody depletion in combination with DXR treatment. Finally, using a MC38 colon adenocarcinoma model, where MC38 cells were engrafted into the flanks of WT and S1PR4-/- mice, we demonstrate a synergistic effect of S1PR4 depletion and anti-PD-1 immunotherapy reflected by reduced tumor growth and an increased survival rate of tumor-bearing S1PR4-/- compared to WT mice. A similar effect of additional tumor growth reduction by combinatorial anti-PD-1 treatment was seen in S1PR4-/- PyMT mice compared to their WT counterpart, although less pronounced compared to the MC38 allograft model. Taken together, our data indicate that S1PR4 depletion restores antitumor immunity by, among other mechanisms, increasing CD8+ T-cell abundance, translating into enhanced response to chemotherapy and anti-PD-1 immunotherapy. Thus, we reveal the potential of S1PR4 as a target to improve cancer therapy. Citation Format: Catherine Olesch, Evelyn Sirait-Fischer, Bernhard Brüne, Andreas Weigert. Targeting immune cell-specific sphingosine-1-phosphate receptor 4 to restore antitumor immunity resulting in improved therapy response [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A209.

The guanine nucleotide exchange factor Vav3 modulates oligodendrocyte precursor differentiation and supports remyelination in white matter lesions.

The tightly controlled processes of myelination and remyelination require the participation of the cytoskeleton. The reorganization of the cytoskeleton is controlled by small GTPases of the RhoA family. Here, we report that Vav3, a Rho GTPase regulating guanine nucleotide exchange factor (GEF) is involved in oligodendrocyte maturation, myelination and remyelination. When Vav3 was eliminated by genetic recombination, oligodendrocyte precursor cell (OPC) differentiation toward mature oligodendrocytes was accelerated. In contrast, Vav3-deficient oligodendrocytes displayed a reduced capacity to myelinate synthetic microfibers in vitro. Furthermore, remyelination was impaired in Vav3 knockout cerebellar slice cultures that were demyelinated by the addition of lysolecithin. In agreement with these observations, remyelination was compromised when the cuprizone model of myelin lesion was performed in Vav3-deficient mice. When Vav3-deficient oligodendrocytes were examined with Förster resonance energy transfer (FRET)-based biosensors, an altered activation profile of RhoA GTPases was revealed on the cellular level, which could be responsible for an impaired remyelination. Taken together, this study highlights Vav3 as a novel regulator of oligodendrocyte maturation and remyelination, suggesting that manipulation of the Vav3-dependent signaling pathway could help to improve myelin repair.

Systems analysis of metabolism in platelet concentrates during storage in platelet additive solution.

Platelets (PLTs) deteriorate over time when stored within blood banks through a biological process known as PLT storage lesion (PSL). Here, we describe the refinement of the biochemical model of PLT metabolism, iAT-PLT-636, and its application to describe and investigate changes in metabolism during PLT storage. Changes in extracellular acetate and citrate were measured in buffy coat and apheresis PLT units over 10 days of storage in the PLT additive solution T-Sol. Metabolic network analysis of these data was performed alongside our prior metabolomics data to describe the metabolism of fresh (days 1-3), intermediate (days 4-6), and expired (days 7-10) PLTs. Changes in metabolism were studied by comparing metabolic model flux predictions of iAT-PLT-636 between stages and between collection methods. Extracellular acetate and glucose contribute most to central carbon metabolism in PLTs. The anticoagulant citrate is metabolized in apheresis-stored PLTs and is converted into aconitate and, to a lesser degree, malate. The consumption of nutrients changes during storage and reflects altered PLT activation profiles following their collection. Irrespective of the collection method, a slowdown in oxidative phosphorylation takes place, consistent with mitochondrial dysfunction during PSL. Finally, the main contributors to intracellular ammonium and NADPH are highlighted. Future optimization of flux through these pathways provides opportunities to address intracellular pH changes and reactive oxygen species, which are both of importance to PSL. The metabolic models provide descriptions of PLT metabolism at steady state and represent a platform for future PLT metabolic research.

Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase

The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic β-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or β-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible cspD2 promoter in the genetically tractable Haloarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-β-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (kcat/Km) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both Km and kcat (three substitutions) or either Km or kcat (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme.

Young adult binge drinkers have immunophenotypic changes in peripheral polymorphonuclear cells and monocytes

ABSTRACTBackground: High alcohol intake on weekends (binge drinking) is more frequent in young adults, who could undergo early liver damage. Alcohol-induced liver damage is characterized by polymorphonuclear cell (PMN) infiltration, which can be represented in the peripheral blood by altered trafficking and activation profiles. Objective: To evaluate the PMN trafficking and activation immunophenotypic profiles in people with a binge drinking pattern. Methods: People with binge drinking (n = 18, 8 females) or at low risk (n = 16, 13 females) based on their AUDIT and HEPCA scores were studied. Hematic biometry and liver enzyme tests were conducted. Peripheral blood leukocytes were stained for CCR5, CCR4, and CXCR4 (trafficking) and CD69 and CD127 (activation). PMNs and monocytes were analyzed by FACS. The data were analyzed using the T-test and Mann–Whitney’s U-test for contrasts and principal component and Fuzzy C means analyses for clustering, with p < 0.05 considered significant. Results: Compared to the low-risk group, the binge group showed higher CCR5 expression on PMNs, decreases in the CD69 percentage and positive PMNs per microliter, and decreased CXCR4 expression on monocytes. Six immunophenotypical clusters were identified, all of which were distributed following the CCR5 and CXCR4 main vectors. Conclusion: Young adult binge drinkers have differential PMN trafficking and activation immunophenotypes, which could be related to the initial onset of alcoholic liver disease and a systemic inflammatory state in response to their alcohol consumption pattern. These findings could lead to the future development of an early diagnostic tool.

Use of muscle synergies and wavelet transforms to identify fatigue during squatting

The objective of this study was to supplement continuous wavelet transforms with muscle synergies in a fatigue analysis to better describe the combination of decreased firing frequency and altered activation profiles during dynamic muscle contractions. Nine healthy young individuals completed the dynamic tasks before and after they squatted with a standard Olympic bar until complete exhaustion. Electromyography (EMG) profiles were analyzed with a novel concatenated non-negative matrix factorization method that decomposed EMG signals into muscle synergies. Muscle synergy analysis provides the activation pattern of the muscles while continuous wavelet transforms output the temporal frequency content of the EMG signals. Synergy analysis revealed subtle changes in two-legged squatting after fatigue while differences in one-legged squatting were more pronounced and included the shift from a general co-activation of muscles in the pre-fatigue state to a knee extensor dominant weighting post-fatigue. Continuous wavelet transforms showed major frequency content decreases in two-legged squatting after fatigue while very few frequency changes occurred in one-legged squatting. It was observed that the combination of methods is an effective way of describing muscle fatigue and that muscle activation patterns play a very important role in maintaining the overall joint kinetics after fatigue.

Altered activity profile of a tertiary silanol analog of multi-targeting nuclear receptor modulator T0901317

We report the design, synthesis, and physicochemical/biological evaluation of novel silanol derivative 6 (sila-T) as a silanol analog of multi-target nuclear receptor modulator T0901317 (5). Compound 6 showed intermediate hydrophobicity between the corresponding alcohol 13 and perfluoroalcohol 5. While 5 exhibited potent activities toward liver X receptor α and β, farnesoid X receptor, pregnane X receptor (PXR) and retinoic acid receptor-related orphan receptor (ROR)γ, silanol 6 exhibited activity only toward PXR and RORs. Incorporation of silanol instead of perfluoroalcohol is a promising option for developing novel target-selective, biologically active compounds.

New products for the treatment of haemophilia.

Current developments in haemophilia therapy are directed at two therapeutic targets: reduction of injection frequency and reduction or bypassing of inhibitors. A variety of new molecules addressing these aims are now completing clinical trials and are ready to enter clinical use. First amongst these are modified Factor VIII (FVIII) and Factor IX (FIX) molecules with extended half-lives. FIX modifications have achieved 5-fold prolongation of half-life whilst effects on FVIII have been more modest, at less than two-fold. We now face the problem of integrating these into clinical practice. Other approaches have generated chemically modified FVIII molecules with altered activation profiles. An alternative way of correcting the haemophilia defect is to reduce the activity of natural anticoagulants in an attempt to restore the balance of haemostasis. These methods are also giving promising results but, as with all new approaches, it will be some while before they all find their place in practice.

Fine Tuning of a Type 1 Interferon Antagonist.

Type I interferons are multi-potent cytokines that serve as first line of defense against viruses and other pathogens, posses immunomudolatory functions and elicit a growth inhibitory response. In recent years it has been shown that interferons are also detrimental, for example in lupus, AIDS, tuberculosis and cognitive decline, highlighted the need to develop interferon antagonists. We have previously developed the antagonist IFN-1ant, with much reduced binding to the IFNAR1 receptor and enhanced binding to IFNAR2. Here, we further tune the IFN-1ant by producing three additional antagonists based on IFN-1ant but with altered activity profiles. We show that in all three cases the antiproliferative activity of interferons is blocked and the induction of gene transcription of immunomudolatory and antiproliferative associated genes are substantially decreased. Conversely, each of the new antagonists elicits a different degree of antiviral response, STAT phosphorylation and related gene induction. Two of the new antagonists promote decreased activity in relation to the original IFN-1ant, while one of them promotes increased activity. As we do not know the exact causes of the detrimental effects of IFNs, the four antagonists that were produced and analyzed provide the opportunity to investigate the extent of antagonistic and agonistic activity optimal for a given condition.

Conserved residues of the Pro103-Arg115 loop are involved in triggering the allosteric response of the Escherichia coli ADP-glucose pyrophosphorylase.

The synthesis of glycogen in bacteria and starch in plants is allosterically controlled by the production of ADP-glucose by ADP-glucose pyrophosphorylase. Using computational studies, site-directed mutagenesis, and kinetic characterization, we found a critical region for transmitting the allosteric signal in the Escherichia coli ADP-glucose pyrophosphorylase. Molecular dynamics simulations and structural comparisons with other ADP-glucose pyrophosphorylases provided information to hypothesize that a Pro103-Arg115 loop is part of an activation path. It had strongly correlated movements with regions of the enzyme associated with regulation and ATP binding, and a network analysis showed that the optimal network pathways linking ATP and the activator binding Lys39 mainly involved residues of this loop. This hypothesis was biochemically tested by mutagenesis. We found that several alanine mutants of the Pro103-Arg115 loop had altered activation profiles for fructose-1,6-bisphosphate. Mutants P103A, Q106A, R107A, W113A, Y114A, and R115A had the most altered kinetic profiles, primarily characterized by a lack of response to fructose-1,6-bisphosphate. This loop is a distinct insertional element present only in allosterically regulated sugar nucleotide pyrophosphorylases that could have been acquired to build a triggering mechanism to link proto-allosteric and catalytic sites.

Alterations in blood leukocytes of G551D-bearing cystic fibrosis patients undergoing treatment with ivacaftor

BackgroundIvacaftor improves clinical outcome by potentiation of mutant G551D CFTR. Due to the presence of CFTR in monocytes and polymorphonuclear neutrophils (PMNs), we hypothesized that ivacaftor may impact leukocyte activation. MethodsWe examined blood leukocytes from G551D CF subjects prior to and at one and six months after receiving ivacaftor. Blood leukocytes from ivacaftor-naïve G551D, F508del, and healthy controls were also treated with ivacaftor ex vivo to assess mutation-specific effects. ResultsCompared to healthy controls, G551D CF subjects had significantly higher expression of active CD11b on PMNs and of CD63 on monocytes, which were normalized by in vivo ivacaftor treatment. Ex vivo exposure to ivacaftor of blood cells from G551D, but not F508del and healthy subjects, resulted in changes in CXCR2 and CD16 expression on PMNs. ConclusionsIn vivo and ex vivo exposure of G551D CF leukocytes to ivacaftor resulted in an altered activation profile, suggesting mutation-specific leukocyte modulation.

166 Increase in TILRR Expression in Areas of Vascular Inflammation Alters NF-KB Control Through Cytoskeletal Release of the Inhibitor IKBA – Interdisciplinary Analysis Using Predictive Agent Based Modelling

IntroductionInterleukin-1 (IL-1) and its signalling receptor (IL-1RI) are well known as central regulators of vascular repair and injury. Specifically, activation of the NF-kB pathway and its control of inflammatory and...