Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY

The protein (Escherichia coli CheY) that controls the direction of flagellar rotation during bacterial chemotaxis has been shown to be phosphorylated on the aspartate 57 residue. The residue phosphorylated is present within a conserved sequence in every member of a family of bacterial regulatory proteins. The phosphorylation is transient, with a much shorter half-life than that expected of a simple acyl phosphate intermediate, indicating that the sequence and conformation of the protein is designed to achieve a rapid hydrolysis. The CheY-phosphate linkage can be reductively cleaved by sodium borohydride. High-performance tandem mass-spectrometric analysis of proteolytic peptides derived from [3H]borohydride-reduced phosphorylated CheY protein was used to identify the position of phosphorylation. Mutants with altered aspartate 57 exhibited no chemotaxis. When aspartate 13, another conserved residue, was changed, greatly reduced chemotaxis was observed, suggesting an important role for aspartate 13. The rate-determining step of chemotactic signaling is governed by the kinetics of formation and hydrolysis of the CheY protein phosphoaspartate bond. The CheY protein apparently functions as a protein phosphatase that possesses a transient covalent intermediate. Transient phosphorylation of an aspartate residue is an effective mechanism for producing a biochemical signal with a short concentration-independent half-life. The duration of the signal can be controlled by small structural elements within the phosphorylated protein.     

Identification of glycoinositol phospholipid linked and truncated forms of the scrapie prion protein

Analysis of carboxy-terminal peptides derived from endoproteinase Lys-C digests of the scrapie isoform of the hamster prion protein revealed that the majority of the molecules are glycoinositol phospholipid linked through ethanolamine attached at serin-231. However, approximately 15% of PrPSc had a carboxy-terminal peptide that ends at glycine-228. It is intriguing that this glycine is part of the PrP sequence Gly-Arg-Arg, which is an established target sequence for the proteolysis and release of bioactive peptides from larger precursors. The mechanism of formation, as well as the role of the truncated carboxy terminus in the dissemination and neuropathology of scrapie, remains to be determined.     

Targeting CoV-2 spike RBD and ACE-2 interaction with flavonoids of Anatolian propolis by in silico and in vitro studies in terms of possible COVID-19 therapeutics

Propolis is a multi-functional bee product rich in polyphenols. In this study, the inhibitory effect of Anatolian propolis against SARS-coronavirus-2 (SARS-CoV-2) was investigated in vitro and in silico. Raw and commercial propolis samples were used, and both samples were found to be rich in caffeic acid, p-coumaric acid, ferulic acid, t-cinnamic acid, hesperetin, chrysin, pinocembrin, and caffeic acid phenethyl ester (CAPE) at HPLC-UV analysis. Ethanolic propolis extracts (EPE) were used in the ELISA screening test against the spike S1 protein (SARS-CoV-2): ACE-2 interaction for in vitro study. The binding energy values of these polyphenols to the SARS-CoV-2 spike and ACE-2 protein were calculated separately with a molecular docking study using the AutoDock 4.2.6 program. In addition, the pharmacokinetics and drug-likeness properties of these eight polyphenols were calculated according to the SwissADME tool. The binding energy value of pinocembrin was highest in both receptors, followed by chrysin, CAPE, and hesperetin. Based on the in silico modeling and ADME (absorption, distribution, metabolism, and excretion) behaviors of the eight polyphenols, the compounds exhibited the potential ability to act effectively as novel drugs. The findings of both studies showed that propolis has a high inhibitory potential against the Covid-19 virus. However, further studies are now needed.     

Three-Dimensional Trabecular Alignment Model

The Shear-slip Mesh Update Method (SSMUM) is being used in flow simulations involving large but regular displacements of one or more boundaries of the computational domain. We follow up the earlier discussion of the method with notes on practical implementation aspects. In order to establish a benchmark problem for this class of flow problems, we define and report results from a two-dimensional viscous flow around a rotating stirrer in a square chamber. The application potential of the method is demonstrated in the context of biomedical design problem, as we perform an analysis of blood flow in a centrifugal left ventricular assist device, or blood pump, which involves a rotating impeller in a non-axisymmetric housing.     

Rearrangements within human spliceosomes captured after exon ligation

In spliceosomes, dynamic RNA/RNA and RNA/protein interactions position the pre-mRNA substrate for the two chemical steps of splicing. Not all of these interactions have been characterized, in part because it has not been possible to arrest the complex at clearly defined states relative to chemistry. Previously, it was shown in yeast that the DEAD/H-box protein Prp22 requires an extended 3′ exon to promote mRNA release from the spliceosome following second-step chemistry. In line with that observation, we find that shortening the 3′ exon blocks cleaved lariat intron and mRNA release in human splicing extracts, which allowed us to stall human spliceosomes in a new post-catalytic complex (P complex). In comparison to C complex, which is blocked at a point following first-step chemistry, we detect specific differences in RNA substrate interactions near the splice sites. These differences include extended protection across the exon junction and changes in protein crosslinks to specific sites in the 5′ and 3′ exons. Using selective reaction monitoring (SRM) mass spectrometry, we quantitatively compared P and C complex proteins and observed enrichment of SF3b components and loss of the putative RNA-dependent ATPase DHX35. Electron microscopy revealed similar structural features for both complexes. Notably, additional density is present when complexes are chemically fixed, which reconciles our results with previously reported C complex structures. Our ability to compare human spliceosomes before and after second-step chemistry has opened a new window to rearrangements near the active site of spliceosomes, which may play roles in exon ligation and mRNA release.     

Activity-dependent Protein Dynamics Define Interconnected Cores of Co-regulated Postsynaptic Proteins

Synapses are highly dynamic structures that mediate cell–cell communication in the central nervous system. Their molecular composition is altered in an activity-dependent fashion, which modulates the efficacy of subsequent synaptic transmission events. Whereas activity-dependent trafficking of individual key synaptic proteins into and out of the synapse has been characterized previously, global activity-dependent changes in the synaptic proteome have not been studied.To test the feasibility of carrying out an unbiased large-scale approach, we investigated alterations in the molecular composition of synaptic spines following mass stimulation of the central nervous system induced by pilocarpine. We observed widespread changes in relative synaptic abundances encompassing essentially all proteins, supporting the view that the molecular composition of the postsynaptic density is tightly regulated. In most cases, we observed that members of gene families displayed coordinate regulation even when they were not known to physically interact.Analysis of correlated synaptic localization revealed a tightly co-regulated cluster of proteins, consisting of mainly glutamate receptors and their adaptors. This cluster constitutes a functional core of the postsynaptic machinery, and changes in its size affect synaptic strength and synaptic size. Our data show that the unbiased investigation of activity-dependent signaling of the postsynaptic density proteome can offer valuable new information on synaptic plasticity.Excitatory synaptic transmission is the primary mode of cell–cell communication in the central nervous system. The efficacy of synaptic transmission is highly regulated, and alterations in the strength of synaptic signaling within networks of neurons provide a mechanism for learning and memory storage, as well as for overall network stability. Modulation of synapse efficacy can occur through alterations in the structure and composition of the postsynaptic spine. The synaptic abundance of several molecules has been shown to be regulated in response to activity (1).The levels of individual proteins at postsynaptic spines are regulated through multiple processes. Active transport mechanisms exist and have been well characterized for AMPA-type glutamate receptors (AMPA-Rs)¹ via either insertion into the synapse or tighter association with the postsynaptic density (PSD) following lateral diffusion within the cell membrane (2). In addition to AMPA-Rs, other proteins known to be subject to activity-dependent regulation include calcium calmodulin-dependent protein kinase II alpha and beta, NMDA-type glutamate receptors (NMDA-Rs), and proteosome subunits (3–5). Synaptic protein content is dysregulated in a number of neuropsychiatric and neurodegenerative diseases, including Alzheimer''s disease and fragile X mental retardation (6–8).Most studies reported thus far have focused on a small number of selected molecules in individual experiments using a subset of synapses. Whereas learning and memory rely on the differential response of individual synapses to their specific input patterns, overall network excitability has to be maintained by homeostatic means. This homeostasis is governed by multiple pathways, and very little is known about the principles that regulate synaptic protein content across large numbers of synapses and neurons. The contributions of individual pathways and the interactions among them are largely unknown.In order to explore synaptic dynamics with a global view, we took advantage of a chemically induced mass stimulation protocol to stimulate synapses broadly throughout the central nervous system. We employed mass spectrometry and isotopically encoded isobaric peptide tagging with the iTRAQ reagent to quantify changes in the abundance of 893 proteins (9). We then analyzed changes in the relative abundance of these proteins at 0, 10, 20, and 60 min after the onset of stimulation.We observed evidence of the coordinated activation of synaptic protein groups, thereby identifying functional core complexes within the PSD. We demonstrate that adopting a quantitative systems biology approach provides insight allowing for a new level of analysis of synaptic function.