Cavities in protein–DNA and protein–RNA interfaces

An analysis of cavities present in protein–DNA and protein–RNA complexes is presented. In terms of the number of cavities and their total volume, the interfaces formed in these complexes are akin to those in transient protein–protein heterocomplexes. With homodimeric proteins protein–DNA interfaces may contain cavities involving both the protein subunits and DNA, and these are more than twice as large as cavities involving a single protein subunit and DNA. A parameter, cavity index, measuring the degree of surface complementarity, indicates that the packing of atoms in protein–protein/DNA/RNA is very similar, but it is about two times less efficient in the permanent interfaces formed between subunits in homodimers. As within the tertiary structure and protein–protein interfaces, protein–DNA interfaces have a higher inclination to be lined by β-sheet residues; from the DNA side, base atoms, in particular those in minor grooves, have a higher tendency to be located in cavities. The larger cavities tend to be less spherical and solvated. A small fraction of water molecules are found to mediate hydrogen-bond interactions with both the components, suggesting their primary role is to fill in the void left due to the local non-complementary nature of the surface patches.     

Purification of MAP–kinase protein complexes and identification of candidate components by XL–TAP–MS

The purification of low-abundance protein complexes and detection of in vivo protein–protein interactions in complex biological samples remains a challenging task. Here, we devised crosslinking and tandem affinity purification coupled to mass spectrometry (XL–TAP–MS), a quantitative proteomics approach for analyzing tandem affinity-purified, crosslinked protein complexes from plant tissues. We exemplarily applied XL–TAP–MS to study the MKK2–Mitogen-activated protein kinase (MPK4) signaling module in Arabidopsis thaliana. A tandem affinity tag consisting of an in vivo-biotinylated protein domain flanked by two hexahistidine sequences was adopted to allow for the affinity-based isolation of formaldehyde–crosslinked protein complexes under fully denaturing conditions. Combined with ¹⁵N stable isotopic labeling and tandem MS we captured and identified a total of 107 MKK2–MPK4 module-interacting proteins. Consistent with the role of the MPK signaling module in plant immunity, many of the module-interacting proteins are involved in the biotic and abiotic stress response of Arabidopsis. Validation of binary protein–protein interactions by in planta split-luciferase assays and in vitro kinase assays disclosed several direct phosphorylation targets of MPK4. Together, the XL–TAP–MS approach purifies low abundance protein complexes from biological samples and discovers previously unknown protein–protein interactions.

XL–TAP–MS: a novel technique that allows purification of crosslinked, low abundant protein complexes from plant tissues under denatured conditions and detection of in vivo protein–protein interactions.     

UPA, a universal protein array system for quantitative detection of protein–protein, protein–DNA, protein–RNA and protein–ligand interactions

Protein–protein interactions have been widely used to study gene expression pathways and may be considered as a new approach to drug discovery. Here I report the development of a universal protein array (UPA) system that provides a sensitive, quantitative, multi-purpose, effective and easy technology to determine not only specific protein–protein interactions, but also specific interactions of proteins with DNA, RNA, ligands and other small chemicals. (i) Since purified proteins are used, the results can be easily interpreted. (ii) UPA can be used multiple times for different targets, making it economically affordable for most laboratories, hospitals and biotechnology companies. (iii) Unlike DNA chips or DNA microarrays, no additional instrumentation is required. (iv) Since the UPA uses active proteins (without denaturation and renaturation), it is more sensitive compared with most existing methods. (v) Because the UPA can analyze hundreds (even thousands on a protein microarray) of proteins in a single experiment, it is a very effective method to screen proteins as drug targets in cancer and other human diseases.     

Structure of the human RAD17–RFC clamp loader and 9–1–1 checkpoint clamp bound to a dsDNA–ssDNA junction

The RAD9–RAD1–HUS1 (9–1–1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or resection of DNA double strand breaks. Loaded at the 5′-recessed end of a dsDNA–ssDNA junction by the RAD17–RFC clamp loader complex, the phosphorylated C-terminal tail of the RAD9 subunit of 9–1–1 engages with the mediator scaffold TOPBP1 which in turn activates the ATR kinase, localised through the interaction of its constitutive partner ATRIP with RPA-coated ssDNA. Using cryogenic electron microscopy (cryoEM) we have determined the structure of a complex of the human RAD17–RFC clamp loader bound to human 9–1–1, engaged with a dsDNA–ssDNA junction. The structure answers the key questions of how RAD17 confers specificity for 9–1–1 over PCNA, and how the clamp loader specifically recognises the recessed 5′ DNA end and fixes the orientation of 9–1–1 on the ssDNA.     

Evidence supporting the microbiota–gut–brain axis in a songbird

Recent research in mammals supports a link between cognitive ability and the gut microbiome, but little is known about this relationship in other taxa. In a captive population of 38 zebra finches (Taeniopygia guttata), we quantified performance on cognitive tasks measuring learning and memory. We sampled the gut microbiome via cloacal swab and quantified bacterial alpha and beta diversity. Performance on cognitive tasks related to beta diversity but not alpha diversity. We then identified differentially abundant genera influential in the beta diversity differences among cognitive performance categories. Though correlational, this study provides some of the first evidence of an avian microbiota–gut–brain axis, building foundations for future microbiome research in wild populations and during host development.     

Insights into innate immune activation via PS-ASO–protein–TLR9 interactions

Non-CpG PS-ASOs can activate the innate immune system, leading to undesired outcomes. This response can vary—in part—as a function of 2′modifications and sequence. Here we investigated the molecular steps involved in the varied effects of PS-ASOs on the innate immune system. We found that pro-inflammatory PS-ASOs require TLR9 signaling based on the experimental systems used. However, the innate immunity of PS-ASOs does not correlate with their binding affinity with TLR9. Furthermore, the innate immune responses of pro-inflammatory PS-ASOs were reduced by coincubation with non-inflammatory PS-ASOs, suggesting that both pro-inflammatory and non-inflammatory PS-ASOs can interact with TLR9. We show that the kinetics of the PS-ASO innate immune responses can vary, which we speculate may be due to the existence of alternative PS-ASO binding sites on TLR9, leading to full, partial, or no activation of the pathway. In addition, we found that several extracellular proteins, including HMGB1, S100A8 and HRG, enhance the innate immune responses of PS-ASOs. Reduction of the binding affinity by reducing the PS content of PS-ASOs decreased innate immune responses, suggesting that PS-ASO–protein complexes may be sensed by TLR9. These findings thus provide critical information concerning how PS-ASOs can interact with and activate TLR9.     

Time-Warp–Invariant Neuronal Processing

Fluctuations in the temporal durations of sensory signals constitute a major source of variability within natural stimulus ensembles. The neuronal mechanisms through which sensory systems can stabilize perception against such fluctuations are largely unknown. An intriguing instantiation of such robustness occurs in human speech perception, which relies critically on temporal acoustic cues that are embedded in signals with highly variable duration. Across different instances of natural speech, auditory cues can undergo temporal warping that ranges from 2-fold compression to 2-fold dilation without significant perceptual impairment. Here, we report that time-warp–invariant neuronal processing can be subserved by the shunting action of synaptic conductances that automatically rescales the effective integration time of postsynaptic neurons. We propose a novel spike-based learning rule for synaptic conductances that adjusts the degree of synaptic shunting to the temporal processing requirements of a given task. Applying this general biophysical mechanism to the example of speech processing, we propose a neuronal network model for time-warp–invariant word discrimination and demonstrate its excellent performance on a standard benchmark speech-recognition task. Our results demonstrate the important functional role of synaptic conductances in spike-based neuronal information processing and learning. The biophysics of temporal integration at neuronal membranes can endow sensory pathways with powerful time-warp–invariant computational capabilities.     

Understanding the impact of correlation within pair‐bonds on Cormack–Jolly–Seber models

1. The Cormack–Jolly–Seber (CJS) model and its extensions have been widely applied to the study of animal survival rates in open populations. The model assumes that individuals within the population of interest have independent fates. It is, however, highly unlikely that a pair of animals which have formed a long‐term pairing have dissociated fates.
2. We examine a model extension which allows animals who have formed a pair‐bond to have correlated survival and recapture fates. Using the proposed extension to generate data, we conduct a simulation study exploring the impact that correlated fate data has on inference from the CJS model. We compute Monte Carlo estimates for the bias, range, and standard errors of the parameters of the CJS model for data with varying degrees of survival correlation between mates. Furthermore, we study the likelihood ratio test of sex effects within the CJS model by simulating densities of the deviance. Finally, we estimate the variance inflation factor c^ for CJS models that incorporate sex‐specific heterogeneity.
3. Our study shows that correlated fates between mated animals may result in underestimated standard errors for parsimonious models, significantly deflated likelihood ratio test statistics, and underestimated values of c^ for models taking sex‐specific effects into account.
4. Underestimated standard errors can result in lowered coverage of confidence intervals. Moreover, deflated test statistics will provide overly conservative test results. Finally, underestimated variance inflation factors can lead researchers to make incorrect conclusions about the level of extra‐binomial variation present in their data.