Biophysical and functional characterizations of recombinant RimI acetyltransferase from Mycobacterium tuberculosis

2019 ◽  
Vol 51 (9) ◽  
pp. 960-968
Author(s):  
Meijing Hou ◽  
Jie Zhuang ◽  
Shihui Fan ◽  
Huilin Wang ◽  
Chenyun Guo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Quantum Coherence ◽  
Protein Modification ◽  
Structural Information ◽  
Biochemical Properties ◽  
Structural Determination ◽  
Physiological Processes ◽  
High Quality Protein ◽  
Wide Range ◽  
Docking Approach

Abstract Nα-acetylation is a universal protein modification related to a wide range of physiological processes in eukaryotes and prokaryotes. RimI, an Nα-acetyltransferase in Mycobacterium tuberculosis, is responsible for the acetylation of the α-amino group of the N-terminal residue in the ribosomal protein S18. Despite growing evidence that protein acetylation may be correlated with the pathogenesis of tuberculosis, no structural information is yet available for mechanistically understanding the MtRimI acetylation. To enable structural studies for MtRimI, we constructed a serial of recombinant MtRimI proteins and assessed their biochemical properties. We then chose an optimal construct MtRimIC21A4-153 and expressed and purified the truncated high-quality protein for further biophysical and functional characterizations. The 2D 1H-15N heteronuclear single quantum coherence spectrum of MtRimIC21A4-153 exhibits wider chemical shift dispersion and favorable peak isolation, indicating that MtRimIC21A4-153 is amendable for further structural determination. Moreover, bio-layer interferometry experiments showed that MtRimIC21A4-153 possessed similar micromolar affinity to full-length MtRimI for binding the hexapeptide substrate Ala-Arg-Tyr-Phe-Arg-Arg. Enzyme kinetic assays also exhibited that MtRimIC21A4-153 had almost identical enzymatic activity to MtRimI, indicating insignificant influence of the recombinant variations on enzymatic functions. Furthermore, binding sites of the peptide were predicted by molecular docking approach, suggesting that this substrate binds to MtRimI primarily through electrostatic and hydrogen bonding interactions. Our results lay a foundation for the further structural determination and dynamics detection of MtRimI.

scholarly journals Role of Insulin in Health and Disease: An Update

2021 ◽  
Vol 22 (12) ◽  
pp. 6403
Author(s):  
Md Saidur Rahman ◽  
Khandkar Shaharina Hossain ◽  
Sharnali Das ◽  
Sushmita Kundu ◽  
Elikanah Olusayo Adegoke ◽  
...  
Keyword(s):  
Insulin Signaling ◽  
Basic Research ◽  
Future Research ◽  
Protective Effects ◽  
Glucose Levels ◽  
Physiological Processes ◽  
Blood Glucose Levels ◽  
Wide Range ◽  
Health And Disease

Insulin is a polypeptide hormone mainly secreted by β cells in the islets of Langerhans of the pancreas. The hormone potentially coordinates with glucagon to modulate blood glucose levels; insulin acts via an anabolic pathway, while glucagon performs catabolic functions. Insulin regulates glucose levels in the bloodstream and induces glucose storage in the liver, muscles, and adipose tissue, resulting in overall weight gain. The modulation of a wide range of physiological processes by insulin makes its synthesis and levels critical in the onset and progression of several chronic diseases. Although clinical and basic research has made significant progress in understanding the role of insulin in several pathophysiological processes, many aspects of these functions have yet to be elucidated. This review provides an update on insulin secretion and regulation, and its physiological roles and functions in different organs and cells, and implications to overall health. We cast light on recent advances in insulin-signaling targeted therapies, the protective effects of insulin signaling activators against disease, and recommendations and directions for future research.

scholarly journals Molecular recognition of surface-immobilized carbohydrates by a synthetic lectin

2014 ◽  
Vol 10 ◽  
pp. 1354-1364 ◽  
Author(s):  
Melanie Rauschenberg ◽  
Eva-Corrina Fritz ◽  
Christian Schulz ◽  
Tobias Kaufmann ◽  
Bart Jan Ravoo
Keyword(s):  
Molecular Recognition ◽  
Self Assembled Monolayers ◽  
Carbohydrate Binding ◽  
Air Oxidation ◽  
Acetylneuraminic Acid ◽  
Physiological Processes ◽  
Wide Range ◽  
Assembled Monolayers ◽  
Synthetic Carbohydrate ◽  
Derivatives Of

The molecular recognition of carbohydrates and proteins mediates a wide range of physiological processes and the development of synthetic carbohydrate receptors (“synthetic lectins”) constitutes a key advance in biomedical technology. In this article we report a synthetic lectin that selectively binds to carbohydrates immobilized in a molecular monolayer. Inspired by our previous work, we prepared a fluorescently labeled synthetic lectin consisting of a cyclic dimer of the tripeptide Cys-His-Cys, which forms spontaneously by air oxidation of the monomer. Amine-tethered derivatives of N-acetylneuraminic acid (NANA), β-D-galactose, β-D-glucose and α-D-mannose were microcontact printed on epoxide-terminated self-assembled monolayers. Successive prints resulted in simple microarrays of two carbohydrates. The selectivity of the synthetic lectin was investigated by incubation on the immobilized carbohydrates. Selective binding of the synthetic lectin to immobilized NANA and β-D-galactose was observed by fluorescence microscopy. The selectivity and affinity of the synthetic lectin was screened in competition experiments. In addition, the carbohydrate binding of the synthetic lectin was compared with the carbohydrate binding of the lectins concanavalin A and peanut agglutinin. It was found that the printed carbohydrates retain their characteristic selectivity towards the synthetic and natural lectins and that the recognition of synthetic and natural lectins is strictly orthogonal.

Micro-Scale Flow Simulations and Permeability Estimation of Cleat Networks in Coal

2016 ◽  
Vol 846 ◽  
pp. 42-47
Author(s):  
J. Busse ◽  
S. Galindo Torres ◽  
Alexander Scheuermann ◽  
L. Li ◽  
D. Bringemeier
Keyword(s):  
Gas Flow ◽  
Structural Information ◽  
Porous Matrix ◽  
Groundwater Levels ◽  
Micro Scale ◽  
Wide Range ◽  
Boltzmann Method ◽  
The Impact ◽  
Flow Experiments

Coal mining raises a number of environmental and operational challenges, including the impact of changing groundwater levels and flow patterns on adjacent aquifer and surface water systems. Therefore it is of paramount importance to fully understand the flow of water and gases in the geological system on all scales. Flow in coal seams takes place on a wide range of scales from large faults and fractures to the micro-structure of a porous matrix intersected by a characteristic cleat network. On the micro-scale these cleats provide the principal source of permeability for fluid and gas flow. Description of the behaviour of the flow within the network is challenging due to the variations in number, sizing, orientation, aperture and connectivity at a given site. This paper presents a methodology to simulate flow and investigate the permeability of fractured media. A profound characterization of the geometry of the cleat network in micrometer resolution can be derived by CT-scans. The structural information is fed into a Lattice Boltzmann Method (LBM) based model that allows the implementation of virtual flow experiments. With the application of suitable hydraulic boundary conditions the full permeability tensor can be calculated in 3D.

Behavior as a window on physiology: a simple apparatus for recording caterpillar feeding.

1992 ◽  
Vol 262 (6) ◽  
pp. S9 ◽  
Author(s):  
E Bowdan
Keyword(s):  
Feeding Behavior ◽  
Control Mechanisms ◽  
Inexpensive Method ◽  
Simple Apparatus ◽  
Physiological Processes ◽  
Wide Range ◽  
Relationship Of ◽  
The Relationship

Regulation of feeding is a fundamental element of homeostasis. This is reflected in the similarity of control mechanisms in a wide range of animals, including insects and humans. A close examination of feeding behavior can illuminate the physiological processes driving regulation. A simple, inexpensive method for recording fine details of feeding by caterpillars is described. Possible experiments, interpretation of the data, and the relationship of observations to the underlying physiology, are outlined.

scholarly journals Mito Hacker: a set of tools to enable high-throughput analysis of mitochondrial network morphology

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ali Rohani ◽  
Jennifer A. Kashatus ◽  
Dane T. Sessions ◽  
Salma Sharmin ◽  
David F. Kashatus
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Data Science ◽  
Structural Information ◽  
Morphological Changes ◽  
Mitochondrial Morphology ◽  
Single Cell Level ◽  
Mitochondrial Network ◽  
Cell Level ◽  
Wide Range

Abstract Mitochondria are highly dynamic organelles that can exhibit a wide range of morphologies. Mitochondrial morphology can differ significantly across cell types, reflecting different physiological needs, but can also change rapidly in response to stress or the activation of signaling pathways. Understanding both the cause and consequences of these morphological changes is critical to fully understanding how mitochondrial function contributes to both normal and pathological physiology. However, while robust and quantitative analysis of mitochondrial morphology has become increasingly accessible, there is a need for new tools to generate and analyze large data sets of mitochondrial images in high throughput. The generation of such datasets is critical to fully benefit from rapidly evolving methods in data science, such as neural networks, that have shown tremendous value in extracting novel biological insights and generating new hypotheses. Here we describe a set of three computational tools, Cell Catcher, Mito Catcher and MiA, that we have developed to extract extensive mitochondrial network data on a single-cell level from multi-cell fluorescence images. Cell Catcher automatically separates and isolates individual cells from multi-cell images; Mito Catcher uses the statistical distribution of pixel intensities across the mitochondrial network to detect and remove background noise from the cell and segment the mitochondrial network; MiA uses the binarized mitochondrial network to perform more than 100 mitochondria-level and cell-level morphometric measurements. To validate the utility of this set of tools, we generated a database of morphological features for 630 individual cells that encode 0, 1 or 2 alleles of the mitochondrial fission GTPase Drp1 and demonstrate that these mitochondrial data could be used to predict Drp1 genotype with 87% accuracy. Together, this suite of tools enables the high-throughput and automated collection of detailed and quantitative mitochondrial structural information at a single-cell level. Furthermore, the data generated with these tools, when combined with advanced data science approaches, can be used to generate novel biological insights.

scholarly journals Novel Trifluoromethyl Pyrimidinone Compounds With Activity Against Mycobacterium tuberculosis

2021 ◽  
Vol 9 ◽  
Author(s):  
Erik Hembre ◽  
Julie V. Early ◽  
Joshua Odingo ◽  
Catherine Shelton ◽  
Olena Anoshchenko ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Major Change ◽  
Structural Features ◽  
Research Area ◽  
Biological Properties ◽  
Pyridyl Ring ◽  
Pyridyl Group ◽  
Gram Positive Bacteria ◽  
Wide Range ◽  
Cytotoxic Molecules

The identification and development of new anti-tubercular agents are a priority research area. We identified the trifluoromethyl pyrimidinone series of compounds in a whole-cell screen against Mycobacterium tuberculosis. Fifteen primary hits had minimum inhibitory concentrations (MICs) with good potency IC90 is the concentration at which M. tuberculosis growth is inhibited by 90% (IC90 < 5 μM). We conducted a structure–activity relationship investigation for this series. We designed and synthesized an additional 44 molecules and tested all analogs for activity against M. tuberculosis and cytotoxicity against the HepG2 cell line. Substitution at the 5-position of the pyrimidinone with a wide range of groups, including branched and straight chain alkyl and benzyl groups, resulted in active molecules. Trifluoromethyl was the preferred group at the 6-position, but phenyl and benzyl groups were tolerated. The 2-pyridyl group was required for activity; substitution on the 5-position of the pyridyl ring was tolerated but not on the 6-position. Active molecules from the series demonstrated low selectivity, with cytotoxicity against eukaryotic cells being an issue. However, there were active and non-cytotoxic molecules; the most promising molecule had an MIC (IC90) of 4.9 μM with no cytotoxicity (IC50 > 100 μM). The series was inactive against Gram-negative bacteria but showed good activity against Gram-positive bacteria and yeast. A representative molecule from this series showed rapid concentration-dependent bactericidal activity against replicating M. tuberculosis bacilli with ~4 log kill in <7 days. Overall the biological properties were promising, if cytotoxicity could be reduced. There is scope for further medicinal chemistry optimization to improve the properties without major change in structural features.

scholarly journals Capabilities of photoplethysmography as a method for screening of cardiovascular system pathology

Cardio-IT ◽  
2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Margarita A. Simonyan ◽  
Olga M. Posnenkova ◽  
Anton R. Kiselev
Keyword(s):  
Cardiac Activity ◽  
Vascular Wall ◽  
Smart Devices ◽  
Russian Science ◽  
Cardiovascular Pathology ◽  
Individual Health ◽  
Physiological Processes ◽  
Russian Science Citation Index ◽  
Wide Range ◽  
Diagnostic Capabilities

Currently, vegetative dysfunction considered to be one of principal mechanisms in the pathogenesis of cardiovascular pathology, which causes a cascade of events leading to changes in the properties and a structure of vascular wall. This review article contains literature from various databases (Russian science citation index, PubMed, Google Shcolar, Scopus). It presents the methods for assessing vegetative imbalance. In particular, the method of photoplethysmography (PPGV) is considered for recording periodic fluctuations at various frequencies in the distal vascular bed which characterize physiological processes (cardiac activity, respiratory influences, neurogenic, myogenic and endothelial activity). In addition, other diagnostic capabilities of PPGV such as heart rate (HR) assessment, determining the properties of vascular wall and the level of blood saturation are elucidated. This paper demonstrates a wide range of PPGV applications. The simplicity of PPGV reproduction and its cost-effectiveness make it feasible both in routine clinical practice for the purposes of screening for cardiovascular pathology, and for individual health monitoring incorporated in smart devices.

scholarly journals Inhibitors of Mycobacterium tuberculosis EgtD target both substrate binding sites to limit hercynine production

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Thanuja D. Sudasinghe ◽  
Michael T. Banco ◽  
Donald R. Ronning
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Binding Sites ◽  
Enzyme Inhibitor ◽  
Structural Information ◽  
Alkylating Agents ◽  
Enzyme Active Site ◽  
Treatment Regimens ◽  
Domains Of Life ◽  
Substrate Binding Sites

AbstractErgothioneine (EGT) is a low molecular weight histidine betaine essential in all domains of life but only synthesized by selected few organisms. Synthesis of EGT by Mycobacterium tuberculosis (M. tb) is critical for maintaining bioenergetic homeostasis and protecting the bacterium from alkylating agents, oxidative stress, and anti-tubercular drugs. EgtD, an S-adenosylmethionine-dependent methyltransferase (AdoMet), catalyzes the trimethylation of L-Histidine to initiate EGT biosynthesis and this reaction has been shown to be essential for EGT production in mycobacteria and for long-term infection of murine macrophages by M. tb. In this work, library screening and structure-guided strategies identified multiple classes of M. tb EgtD inhibitors that bind in various regions of the enzyme active site. X-ray crystal structures of EgtD-inhibitor complexes confirm that L-Histidine analogs bind solely to the L-Histidine binding site while drug-like inhibitors, such as TGX-221, and S-Glycyl-H-1152 span both the L-Histidine and AdoMet binding sites. These enzyme-inhibitor complexes provide detailed structural information of compound scaffolds useful for developing more potent inhibitors that could shorten Tuberculosis treatment regimens by weakening important bacterial defenses.

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