Effects of spatial heterogeneity on bacterial genetic circuits

Intracellular spatial heterogeneity is frequently observed in bacteria, where the chromosome occupies part of the cell’s volume and a circuit’s DNA often localizes within the cell. How this heterogeneity affects core processes and genetic circuits is still poorly understood. In fact, commonly used ordinary differential equation (ODE) models of genetic circuits assume a well-mixed ensemble of molecules and, as such, do not capture spatial aspects. Reaction-diffusion partial differential equation (PDE) models have been only occasionally used since they are difficult to integrate and do not provide mechanistic understanding of the effects of spatial heterogeneity. In this paper, we derive a reduced ODE model that captures spatial effects, yet has the same dimension as commonly used well-mixed models. In particular, the only difference with respect to a well-mixed ODE model is that the association rate constant of binding reactions is multiplied by a coefficient, which we refer to as the binding correction factor (BCF). The BCF depends on the size of interacting molecules and on their location when fixed in space and it is equal to unity in a well-mixed ODE model. The BCF can be used to investigate how spatial heterogeneity affects the behavior of core processes and genetic circuits. Specifically, our reduced model indicates that transcription and its regulation are more effective for genes located at the cell poles than for genes located on the chromosome. The extent of these effects depends on the value of the BCF, which we found to be close to unity. For translation, the value of the BCF is always greater than unity, it increases with mRNA size, and, with biologically relevant parameters, is substantially larger than unity. Our model has broad validity, has the same dimension as a well-mixed model, yet it incorporates spatial heterogeneity. This simple-to-use model can be used to both analyze and design genetic circuits while accounting for spatial intracellular effects.Abstract FigureHighlightsIntracellular spatial heterogeneity modulates the effective association rate constant of binding reactions through a binding correction factor (BCF) that fully captures spatial effectsThe BCF depends on molecules size and location (if fixed) and can be determined experimentallySpatial heterogeneity may be detrimental or exploited for genetic circuit designTraditional well-mixed models can be appropriate despite spatial heterogeneityStatement of significanceA general and simple modeling framework to determine how spatial heterogeneity modulates the dynamics of gene networks is currently lacking. To this end, this work provides a simple-to-use ordinary differential equation (ODE) model that can be used to both analyze and design genetic circuits while accounting for spatial intracellular effects. We apply our model to several core biological processes and determine that transcription and its regulation are more effective for genes located at the cell poles than for genes located on the chromosome and this difference increases with regulator size. For translation, we predict the effective binding between ribosomes and mRNA is higher than that predicted by a well-mixed model, and it increases with mRNA size. We provide examples where spatial effects are significant and should be considered but also where a traditional well-mixed model suffices despite severe spatial heterogeneity. Finally, we illustrate how the operation of well-known genetic circuits is impacted by spatial effects.

Immunoactivity of self-assembled antibodies investigated by atomic force microscopy

We investigated self-assembly such as hexamerization and two-dimensional crystallization of immunoglobulin G (IgG) molecules on mica by atomic force microscopy. We also estimated the association rate constant of the self-assembled IgG antibodies.

Comparison Of Plasma Kallikrein Inhibition By The Endogenous C1-Inhibitor Versus DX-2930, a Monoclonal Antibody Inhibitor

Dysregulated plasma kallikrein proteolytic activity leads to edematous attacks in hereditary angioedema (HAE) and has been associated with inflammation and thrombosis. Plasma kallikrein (pKal) is a serine protease that circulates as prekallikrein, a zymogen, which, together with factor XII (FXII) and high molecular weight kininogen (HMWK), constitutes the contact system. Activation of the contact system following assembly of FXII, HMWK, and prekallikrein on a negatively charged surface promotes inflammation via the generation of bradykinin and triggers intrinsic pathway coagulation via formation of activated coagulation factor XIa. Normal hemostasis appears not to be mediated by the contact system as individuals deficient in contact system proteins are not at risk for bleeding. However, the contact system has been shown to be pathologically activated by agents that include misfolded proteins, platelet polyphosphate, and implanted devices. Therefore, pharmacologic modulation of the contact system may attenuate thrombosis and inflammation without disrupting normal hemostasis. C1 inhibitor (C1-INH) is a serpin and a key endogenous, protein-based, inhibitor of pKal activity. HAE is caused by autosomal dominant mutations in the C1-INH gene resulting in functional protein levels that are approximately 30% or less than normal (16-33 mg/dL or 1.6-3.3 μM). Prekallikrein is present in plasma at a concentration of approximately 500 nM and it has been estimated that only 30-110 nM is converted to active pKal during an HAE attack. This study investigates the requirement for super-stoichiometric amounts of endogenous C1-INH to adequately regulate pKal activity. In vitro enzyme inhibition kinetics experiments with purified proteins show that the need for high concentrations of C1-INH is likely due to its relatively slow association rate constant (1.7 x 104 M-1s-1). In contrast, DX-2930, a human monoclonal antibody inhibitor of pKal being developed for prophylactic treatment of HAE, potently inhibited pKal (Ki = 125 pM) with a faster association rate constant (3.4 x 106 M-1s-1). Contact activation was observed in human plasma activated by the addition of ellagic acid and monitored using a pKal-selective synthetic peptide substrate. Consistent with the data obtained using purified proteins, the apparent IC50 observed upon adding exogenous C1-INH to normal human plasma was approximately 100-fold higher than that of DX-2930. Using a Western blot assay to monitor endogenous HMWK cleavage in activated plasma we similarly observed that stoichiometric additions of DX-2930 were sufficient to prevent HMWK proteolysis by active pKal; whereas significantly higher concentrations of C1-INH (e.g. 1 µM) were required to block HMWK proteolysis. Active pKal can bind endothelial cells via interactions between the non-catalytic domain of pKal with HMWK, which binds receptors (urokinase receptor, cytokeratin 1, and the globular C1q receptor) present on endothelial cells. Cell bound pKal is likely to be a physiologically relevant form of the enzyme and may provide an explanation for attack localization in HAE. In this study, active pKal was assembled in vitro on cultured human umbilical vein endothelial cells (HUVEC) and binding of a range of concentrations of either biotinylated C1-INH or biotinylated DX-2930 was observed using streptavidin-horseradish peroxidase as detection. The data obtained demonstrates that C1-INH bound cell-associated pKal with > 200-fold less potency than DX-2930. Regarding protease inhibition specificity, while DX-2930 did not inhibit any of 20 tested serine proteases at a concentration of 1 µM, C1-INH is known to inhibit multiple serine proteases. This study demonstrates that effective regulation of pKal activity requires high concentrations of C1-INH (≥ 1 µM), which are necessary to drive the kinetics of this second order, irreversible interaction. These high inhibitory concentrations of C1-INH match the normal range and provide a potential kinetic mechanism for why HAE attacks can occur at C1-INH levels that exceed expected levels of activated pKal. Furthermore, the broad specificity of C1-INH towards other proteases that could be activated during disease could sufficiently deplete C1-INH levels and thereby lead to dysregulated pKal activity. Disclosures: Sexton: Dyax Corp: Employment. Kenniston:Dyax Corp: Employment. Faucette:Dyax Corp: Employment. Nixon:Dyax Corp: Employment. TenHoor:Dyax Corp: Employment. Chyung:Dyax Corp: Employment. Adelman:Dyax Corp: Employment.

Slow-binding inhibitors of prolyl oligopeptidase with different functional groups at the P1 site

POP (prolyl oligopeptidase) specifically hydrolyses a number of small proline-containing peptides at the carboxy end of the proline residue and POP inhibitors have been shown to have cognition-enhancing properties. It has been noted that certain functional groups at the P1 site of the inhibitor, which correspond to the substrate residue on the N-terminal side of the bond to be cleaved, increase the inhibitory potency. However, detailed mechanistic and kinetic analysis of the inhibition has not been studied. In the present study, we examined the effect of different functional groups at the P1 site of the parent inhibitor isophthalic acid bis-(L-prolylpyrrolidine) amide on the binding kinetics to POP. Addition of CHO, CN or COCH2OH groups to the P1 site increased the inhibitory potency by two orders of magnitude (Ki=11.8–0.1 nM) and caused a clear slow-binding inhibition. The inhibitor containing a CHO group had the lowest association rate constant, kon=(2.43±0.12)×105 M−1·s−1, whereas the inhibitor with a CN group exhibited the fastest binding, kon=(12.0±0.08)×105 M−1·s−1. In addition, the dissociation rate was found to be crucially dependent on the type of the functional group. Compounds with COCH2OH and CHO groups had much longer half-lives of dissociation (over 5 h) compared with the compound with the CN group (25 min), although the Ki values of the compounds were relatively similar. A possibility to optimize the duration of inhibition by changing the functional group at the P1 site is important when planning therapeutically useful POP inhibitors.

Hemoglobin in Frankia, a Nitrogen-Fixing Actinomycete

ABSTRACT Frankia strain CcI3 grown in culture produced a hemoglobin which had optical absorption bands typical of a hemoglobin and a molecular mass of 14.1 kDa. Its equilibrium oxygen binding constant was 274 nM, the oxygen dissociation rate constant was 56 s−1, and the oxygen association rate constant was 206 μM−1 s−1.