The rational parameterisation theorem for multisite post-translational modification systems

Post-translational modification of proteins plays a central role in cellular regulation but its study has been hampered by the exponential increase in substrate modification forms (“modforms”) with increasing numbers of sites. We consider here biochemical networks arising from post-translational modification under mass-action kinetics, allowing for multiple substrates, having different types of modification (phosphorylation, methylation, acetylation, etc.) on multiple sites, acted upon by multiple forward and reverse enzymes (in total number L), using general enzymatic mechanisms. These assumptions are substantially more general than in previous studies. We show that the steady-state modform concentrations constitute an algebraic variety that can be parameterised by rational functions of the L free enzyme concentrations, with coefficients which are rational functions of the rate constants. The parameterisation allows steady states to be calculated by solving L algebraic equations, a dramatic reduction compared to simulating an exponentially large number of differential equations. This complexity collapse enables analysis in contexts that were previously intractable and leads to biological predictions that we review. Our results lay a foundation for the systems biology of post-translational modification and suggest deeper connections between biochemical networks and algebraic geometry

Differentiated, promoter-specific response of [4Fe-4S] NsrR DNA-binding to reaction with nitric oxide

NsrR is an iron-sulfur cluster protein that regulates the nitric oxide (NO) stress response of many bacteria. NsrR from Streptomyces coelicolor regulates its own expression and that of only two other genes, hmpA1 and hmpA2, which encode HmpA enzymes predicted to detoxify NO. NsrR binds promoter DNA with high affinity only when coordinating a [4Fe-4S] cluster. Here we show that reaction of [4Fe-4S] NsrR with NO affects DNA-binding differently depending on the gene promoter. Binding to the hmpA2 promoter was abolished at ~2 NO per cluster, while for the hmpA1 and nsrR promoters, ~4 and ~8 NO molecules, respectively, were required to abolish DNA binding. Spectroscopic and kinetic studies of the NO reaction revealed a rapid, multi-phase, non-concerted process involving up to 8 – 10 NO molecules per cluster, leading to the formation of several iron-nitrosyl species. A distinct intermediate was observed at ~2 NO per cluster, along with two further intermediates at ~4 and ~6 NO. The NsrR nitrosylation reaction was not significantly affected by DNA-binding. These results show that NsrR regulates different promoters in response to different concentrations of NO. Spectroscopic evidence indicates that this is achieved by different NO-FeS complexes

Dyads, Triads and Consumer Treachery: When Interpersonal Connections Guard Against Brand Cheating

Consumers develop committed and meaningful relationships with brands, yet still sometimes buy or use options that compete directly with these ‘relationship partners’, an activity that might be understood as a form of cheating or infidelity. Using data from three studies, we assess whether so-called triadic brand relationships – those that implicate an interpersonal third party (i.e., some form of interpersonal bond) – safeguard against cheating. We find compared to dyadic brand relationships that implicate only the consumer and the brand, triadic brand relationships protect against emotional and behavioral cheating by virtue of reinforcing expectations of consumer’s exclusive behavior and monogamy within the relationship

Diffusion as a Ruler: Modeling Kinesin Diffusion as a Length Sensor for Intraflagellar Transport

An important question in cell biology is whether cells are able to measure size, either whole cell size or organelle size. Perhaps cells have an internal chemical representation of size that can be used to precisely regulate growth, or perhaps size is just an accident that emerges due to constraint of nutrients. The eukaryotic flagellum is an ideal model for studying size sensing and control because its linear geometry makes it essentially one-dimensional, greatly simplifying mathematical modeling. The assembly of flagella is regulated by intraflagellar transport (IFT), in which kinesin motors carry cargo adaptors for flagellar proteins along the flagellum and then deposit them at the tip, lengthening the flagellum. The rate at which IFT motors are recruited to begin transport into the flagellum is anticorrelated with the flagellar length, implying some kind of communication between the base and the tip and possibly indicating that cells contain some mechanism for measuring flagellar length. Although it is possible to imagine many complex scenarios in which additional signaling molecules sense length and carry feedback signals to the cell body to control IFT, might the already-known components of the IFT system be sufficient to allow length dependence of IFT? Here we investigate a model in which the anterograde kinesin motors unbind after cargo delivery, diffuse back to the base, and are subsequently reused to power entry of new IFT trains into the flagellum. By mathematically modeling and simulating such a system, we are able to show that the diffusion time of the motors can in principle be sufficient to serve as a proxy for length measurement. We found that the diffusion model can not only achieve a stable steady-state length without the addition of any other signaling molecules or pathways, but also is able to produce the anticorrelation between length and IFT recruitment rate that has been observed in quantitative imaging studies

Amplification free detection of Herpes Simplex Virus DNA

Amplification-free detection of nucleic acids in complex biological samples is an important technology for clinical diagnostics, especially in the case where the detection is quantitative and highly sensitive. Here we present the detection of a synthetic DNA sequence from Herpes Simplex Virus-1 within swine cerebrospinal fluid (CSF), using a sandwich-like, magnetic nanoparticle pull-down assay. Magnetic nanoparticles and fluorescent polystyrene nanoparticles were both modified with DNA probes, able to hybridise either end of the target DNA, forming the sandwich-like complex which can be captured magnetically and detected by fluorescence. The concentration of the target DNA was determined by counting individual and aggregated fluorescent nanoparticles on a planar glass surface within a fluidic chamber. DNA probe coupling for both nanoparticles was optimized. Polystyrene reporter nanoparticles that had been modified with amine terminated DNA probes were also treated with amine terminated polyethylene glycol, in order to reduce non-specific aggregation and target independent adhesion to the magnetic particles. This way, a limit of detection for the target DNA of 0.8 pM and 1 pM could be achieved for hybridisation buffer and CSF respectively, corresponding to 0.072 and 0.090 femtomoles of target DNA, in a volume of 0.090 mL

Different types of soluble fermentable dietary fibre decrease food intake, body weight gain and adiposity in young adult male rats

We thank Donna Wallace and the Rowett Animal House staff for the daily care of experimental rats, body weight and food intake measurements and MRI scanning, Vivien Buchan and Donna Henderson of the Rowett Analytical Department for proximate analyses and SCFA GC, and Andrew Chappell for conducting the beta-glucan analysis. This research was funded by the Scottish Government’s Rural and Environment Science and Analytical Services Division.Peer reviewedPublisher PD

NsrR from Streptomyces coelicolor is a nitric oxide-sensing [4Fe-4S] cluster protein with a specialized regulatory function

The Rrf2 family transcription factor NsrR controls expression of genes in a wide range of bacteria in response to nitric oxide (NO). The precise form of the NO-sensing module of NsrR is the subject of controversy because NsrR proteins containing either [2Fe-2S] or [4Fe-4S] clusters have been observed previously. Optical, Mössbauer, resonance Raman spectroscopies and native mass spectrometry demonstrate that Streptomyces coelicolor NsrR (ScNsrR), previously reported to contain a [2Fe-2S] cluster, can be isolated containing a [4Fe-4S] cluster. ChIP-seq experiments indicated that the ScNsrR regulon is small, consisting of only hmpA1, hmpA2, and nsrR itself. The hmpA genes encode NO-detoxifying flavohemoglobins, indicating that ScNsrR has a specialized regulatory function focused on NO detoxification and is not a global regulator like some NsrR orthologues. EMSAs and DNase I footprinting showed that the [4Fe-4S] form of ScNsrR binds specifically and tightly to an 11-bp inverted repeat sequence in the promoter regions of the identified target genes and that DNA binding is abolished following reaction with NO. Resonance Raman data were consistent with cluster coordination by three Cys residues and one oxygen-containing residue, and analysis of ScNsrR variants suggested that highly conserved Glu-85 may be the fourth ligand. Finally, we demonstrate that some low molecular weight thiols, but importantly not physiologically relevant thiols, such as cysteine and an analogue of mycothiol, bind weakly to the [4Fe-4S] cluster, and exposure of this bound form to O2 results in cluster conversion to the [2Fe-2S] form, which does not bind to DNA. These data help to account for the observation of [2Fe-2S] forms of NsrR