scholarly journals Identifying challenges and opportunities for histone peptides in protein engineering platforms

2021 ◽  
Author(s):  
Jenna N Meanor ◽  
Albert J Keung ◽  
Balaji M Rao
Keyword(s):  
Protein Engineering ◽  
De Novo ◽  
Cellular Functions ◽  
Experimental Conditions ◽  
Post Translational Modifications ◽  
Affinity Reagents ◽  
Cascading Effects ◽  
Challenges And Opportunities ◽  
Specificity Of Binding ◽  
Small Chemical

Histone post-translational modifications are small chemical changes to histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET based systems. High throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet published studies on their use with histones have been noticeably sparse. Here we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.

scholarly journals TFEB Signalling-Related MicroRNAs and Autophagy

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 985
Author(s):  
Davide Corà ◽  
Federico Bussolino ◽  
Gabriella Doronzo
Keyword(s):  
Transcriptional Regulation ◽  
Stress Factors ◽  
Cellular Functions ◽  
Post Translational Modifications ◽  
Cellular Processes ◽  
Factor Deficiency ◽  
Transcription Factor Eb ◽  
Important Regulator ◽  
Response To Stress ◽  
Post Transcriptional Regulation

The oncogenic Transcription Factor EB (TFEB), a member of MITF-TFE family, is known to be the most important regulator of the transcription of genes responsible for the control of lysosomal biogenesis and functions, autophagy, and vesicles flux. TFEB activation occurs in response to stress factors such as nutrient and growth factor deficiency, hypoxia, lysosomal stress, and mitochondrial damage. To reach the final functional status, TFEB is regulated in multimodal ways, including transcriptional rate, post-transcriptional regulation, and post-translational modifications. Post-transcriptional regulation is in part mediated by miRNAs. miRNAs have been linked to many cellular processes involved both in physiology and pathology, such as cell migration, proliferation, differentiation, and apoptosis. miRNAs also play a significant role in autophagy, which exerts a crucial role in cell behaviour during stress or survival responses. In particular, several miRNAs directly recognise TFEB transcript or indirectly regulate its function by targeting accessory molecules or enzymes involved in its post-translational modifications. Moreover, the transcriptional programs triggered by TFEB may be influenced by the miRNA-mediated regulation of TFEB targets. Finally, recent important studies indicate that the transcription of many miRNAs is regulated by TFEB itself. In this review, we describe the interplay between miRNAs with TFEB and focus on how these types of crosstalk affect TFEB activation and cellular functions.

Metabolism-driven post-translational modifications of H3K9 in early bovine embryos

Reproduction ◽  
2021 ◽  
Vol 162 (3) ◽  
pp. 181-191
Author(s):  
Jessica Ispada ◽  
Aldcejam Martins da Fonseca Junior ◽  
Otávio Luiz Ramos Santos ◽  
Camila Bruna de Lima ◽  
Erika Cristina dos Santos ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Developmental Stages ◽  
De Novo ◽  
Blastocyst Stage ◽  
Developmental Model ◽  
Bovine Embryos ◽  
Acetyl Coa ◽  
Post Translational Modifications ◽  
The Relationship ◽  
Different Levels

Metabolic and molecular profiles were reported as different for bovine embryos with distinct kinetics during the first cleavages. In this study, we used this same developmental model (fast vs slow) to determine if the relationship between metabolism and developmental kinetics affects the levels of acetylation or tri-methylation at histone H3 lysine 9 (H3K9ac and H3K9me3, respectively). Fast and slow developing embryos presented different levels of H3K9ac and H3K9me3 from the earliest stages of development (40 and 96 hpi) and up to the blastocyst stage. For H3K9me3, both groups of embryos presented a wave of demethylation and de novo methylation, although it was more pronounced in fast than slow embryos, resulting in blastocysts with higher levels of this mark. The H3K9ac reprogramming profile was distinct between kinetics groups. While slow embryos presented a wave of deacetylation, followed by an increase in this mark at the blastocyst stage, fast embryos reduced this mark throughout all the developmental stages studied. H3K9me3 differences corresponded to writer and eraser transcript levels, while H3K9ac patterns were explained by metabolism-related gene expression. To verify if metabolic differences could alter levels of H3K9ac, embryos were cultured with sodium-iodoacetate (IA) or dichloroacetate (DCA) to disrupt the glycolytic pathway or increase acetyl-CoA production, respectively. IA reduced H3K9ac while DCA increased H3K9ac in blastocysts. Concluding, H3K9me3 and H3K9ac patterns differ between embryos with different kinetics, the second one explained by metabolic pathways involved in acetyl-CoA production. So far, this is the first study demonstrating a relationship between metabolic differences and histone post-translational modifications in bovine embryos.

scholarly journals Allosteric changes in HDM2 by the ATM phosphomimetic S395D mutation: implications on HDM2 function

2019 ◽  
Vol 476 (21) ◽  
pp. 3401-3411 ◽  
Author(s):  
Lukas Uhrik ◽  
Lixiao Wang ◽  
Lucia Haronikova ◽  
Ixaura Medina-Medina ◽  
Yolanda Rebolloso-Gomez ◽  
...  
Keyword(s):  
Ataxia Telangiectasia ◽  
P53 Protein ◽  
Intrinsic Disorder ◽  
26S Proteasome ◽  
Ataxia Telangiectasia Mutated ◽  
Cellular Functions ◽  
Post Translational Modifications ◽  
Protein Protein Interaction ◽  
Positive Regulator ◽  
P53 Mrna

Allosteric changes imposed by post-translational modifications regulate and differentiate the functions of proteins with intrinsic disorder regions. HDM2 is a hub protein with a large interactome and with different cellular functions. It is best known for its regulation of the p53 tumour suppressor. Under normal cellular conditions, HDM2 ubiquitinates and degrades p53 by the 26S proteasome but after DNA damage, HDM2 switches from a negative to a positive regulator of p53 by binding to p53 mRNA to promote translation of the p53 mRNA. This change in activity is governed by the ataxia telangiectasia mutated kinase via phosphorylation on serine 395 and is mimicked by the S395D phosphomimetic mutant. Here we have used different approaches to show that this event is accompanied by a specific change in the HDM2 structure that affects the HDM2 interactome, such as the N-termini HDM2–p53 protein–protein interaction. These data will give a better understanding of how HDM2 switches from a negative to a positive regulator of p53 and gain new insights into the control of the HDM2 structure and its interactome under different cellular conditions and help identify interphases as potential targets for new drug developments.

scholarly journals The 3D architecture and molecular foundations of de novo centriole assembly via bicentrioles

2020 ◽  
Author(s):  
Sónia Gomes Pereira ◽  
Ana Laura Sousa ◽  
Catarina Nabais ◽  
Tiago Paixão ◽  
Alexander. J. Holmes ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
De Novo ◽  
Cellular Functions ◽  
Spindle Poles ◽  
3D Architecture ◽  
Centriole Assembly ◽  
Work Supports ◽  
Wall Components

Abstract/SummaryCentrioles are structurally conserved organelles, composing both centrosomes and cilia. In animal cycling cells, centrioles often form through a highly characterized process termed canonical duplication. However, a large diversity of eukaryotes form centrioles de novo through uncharacterized pathways. This unexplored diversity is key to understanding centriole assembly mechanisms and how they evolved to assist specific cellular functions. Here, combining electron microscopy and tomography, we show that during spermatogenesis of the moss Physcomitrium patens, centrioles are born as a co-axially oriented centriole pair united by a cartwheel. We observe that microtubules emanate from those bicentrioles, which localize to the spindle poles during cell division. Thereafter, each bicentriole breaks apart, and the two resulting sister centrioles mature asymmetrically, elongating specific microtubule triplets and a naked cartwheel. Subsequently, two cilia are assembled which are capable of beating asynchronously. We further show that conserved cartwheel and centriole wall components, SAS6, BLD10 and POC1 are expressed during spermatogenesis and are required for this de novo biogenesis pathway. Our work supports a scenario where centriole biogenesis is more diverse than previously thought and that conserved molecular modules underlie diversification of this essential pathway.

scholarly journals Eukaryotic systems broaden the scope of synthetic biology

2009 ◽  
Vol 187 (5) ◽  
pp. 589-596 ◽  
Author(s):  
Karmella A. Haynes ◽  
Pamela A. Silver
Keyword(s):  
Synthetic Biology ◽  
Nucleic Acids ◽  
Cell Biology ◽  
Cell Behavior ◽  
Cellular Functions ◽  
Complex Cells ◽  
Cellular Processes ◽  
Challenges And Opportunities ◽  
Foundational Work ◽  
Biology Research

Synthetic biology aims to engineer novel cellular functions by assembling well-characterized molecular parts (i.e., nucleic acids and proteins) into biological “devices” that exhibit predictable behavior. Recently, efforts in eukaryotic synthetic biology have sprung from foundational work in bacteria. Designing synthetic circuits to operate reliably in the context of differentiating and morphologically complex cells presents unique challenges and opportunities for progress in the field. This review surveys recent advances in eukaryotic synthetic biology and describes how synthetic systems can be linked to natural cellular processes in order to manipulate cell behavior and to foster new discoveries in cell biology research.

scholarly journals C-terminal tail polyglycylation and polyglutamylation alter microtubule mechanical properties

2019 ◽  
Author(s):  
Kathryn P. Wall ◽  
Harold Hart ◽  
Thomas Lee ◽  
Cynthia Page ◽  
Taviare L. Hawkins ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Molecular Mechanisms ◽  
High Stress ◽  
Resonance Spectroscopy ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Peptide Backbone ◽  
Post Translational Modifications ◽  
Intrinsic Stiffness ◽  
Insight Into

ABSTRACTMicrotubules are biopolymers that perform diverse cellular functions. The regulation of microtubule behavior occurs in part through post-translational modification of both the α- and β- subunits of tubulin. One class of modifications is the heterogeneous addition of glycine and glutamate residues to the disordered C-terminal tails of tubulin. Due to their prevalence in stable, high stress cellular structures such as cilia, we sought to determine if these modifications alter the intrinsic stiffness of microtubules. Here we describe the purification and characterization of differentially-modified pools of tubulin from Tetrahymena thermophila. We found that glycylation on the α-C-terminal tail is a key determinant of microtubule stiffness, but does not affect the number of protofilaments incorporated into microtubules. We measured the dynamics of the tail peptide backbone using nuclear magnetic resonance spectroscopy. We found that the spin-spin relaxation rate (R2) showed a pronounced decreased as a function of distance from the tubulin surface for the α-tubulin tail, indicating that the α-tubulin tail interacts with the dimer surface. This suggests that the interactions of the α-C-terminal tail with the tubulin body contributes to the stiffness of the assembled microtubule, providing insight into the mechanism by which glycylation and glutamylation can alter microtubule mechanical properties.SIGNIFICANCEMicrotubules are regulated in part by post-translational modifications including the heterogeneous addition of glycine and glutamate residues to the C-terminal tails. By producing and characterizing differentially-modified tubulin, this work provides insight into the molecular mechanisms of how these modifications alter intrinsic microtubule properties such as flexibility. These results have broader implications for mechanisms of how ciliary structures are able to function under high stress.

Artificial protein assemblies with well-defined supramolecular protein nanostructures

2021 ◽  
Author(s):  
Suyeong Han ◽  
Yongwon Jung
Keyword(s):  
Vaccine Development ◽  
De Novo ◽  
Building Blocks ◽  
Protein Assembly ◽  
Protein Assemblies ◽  
Cellular Processes ◽  
Wide Range ◽  
Array Structures ◽  
Small Chemical ◽  
Artificial Protein

Nature uses a wide range of well-defined biomolecular assemblies in diverse cellular processes, where proteins are major building blocks for these supramolecular assemblies. Inspired by their natural counterparts, artificial protein-based assemblies have attracted strong interest as new bio-nanostructures, and strategies to construct ordered protein assemblies have been rapidly expanding. In this review, we provide an overview of very recent studies in the field of artificial protein assemblies, with the particular aim of introducing major assembly methods and unique features of these assemblies. Computational de novo designs were used to build various assemblies with artificial protein building blocks, which are unrelated to natural proteins. Small chemical ligands and metal ions have also been extensively used for strong and bio-orthogonal protein linking. Here, in addition to protein assemblies with well-defined sizes, protein oligomeric and array structures with rather undefined sizes (but with definite repeat protein assembly units) also will be discussed in the context of well-defined protein nanostructures. Lastly, we will introduce multiple examples showing how protein assemblies can be effectively used in various fields such as therapeutics and vaccine development. We believe that structures and functions of artificial protein assemblies will be continuously evolved, particularly according to specific application goals.

Recurrent de novo missense variants in GNB2 can cause syndromic intellectual disability

2021 ◽  
pp. jmedgenet-2020-107462
Author(s):  
Natalie B Tan ◽  
Alistair T Pagnamenta ◽  
Matteo P Ferla ◽  
Jonathan Gadian ◽  
Brian HY Chung ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
G Proteins ◽  
De Novo ◽  
Neurodevelopmental Disorder ◽  
Missense Variant ◽  
Cellular Functions ◽  
Missense Variants ◽  
Neurodevelopmental Disease ◽  
Genes Encoding ◽  
International Data

PurposeBinding proteins (G-proteins) mediate signalling pathways involved in diverse cellular functions and comprise Gα and Gβγ units. Human diseases have been reported for all five Gβ proteins. A de novo missense variant in GNB2 was recently reported in one individual with developmental delay/intellectual disability (DD/ID) and dysmorphism. We aim to confirm GNB2 as a neurodevelopmental disease gene, and elucidate the GNB2-associated neurodevelopmental phenotype in a patient cohort.MethodsWe discovered a GNB2 variant in the index case via exome sequencing and sought individuals with GNB2 variants via international data-sharing initiatives. In silico modelling of the variants was assessed, along with multiple lines of evidence in keeping with American College of Medical Genetics and Genomics guidelines for interpretation of sequence variants.ResultsWe identified 12 unrelated individuals with five de novo missense variants in GNB2, four of which are recurrent: p.(Ala73Thr), p.(Gly77Arg), p.(Lys89Glu) and p.(Lys89Thr). All individuals have DD/ID with variable dysmorphism and extraneurologic features. The variants are located at the universally conserved shared interface with the Gα subunit, which modelling suggests weaken this interaction.ConclusionMissense variants in GNB2 cause a congenital neurodevelopmental disorder with variable syndromic features, broadening the spectrum of multisystem phenotypes associated with variants in genes encoding G-proteins.

scholarly journals MMHub, a database for the mulberry metabolome

Database ◽  
2020 ◽  
Vol 2020 ◽  
Author(s):  
Dong Li ◽  
Bi Ma ◽  
Xiaofei Xu ◽  
Guo Chen ◽  
Tian Li ◽  
...  
Keyword(s):  
Mass Spectra ◽  
Chemical Compounds ◽  
Bioactive Metabolites ◽  
Molecular Formula ◽  
Experimental Conditions ◽  
Chemical Structures ◽  
Pubchem Compound ◽  
Specific Distribution ◽  
Small Chemical ◽  
Metabolome Data

Abstract Mulberry is an important economic crop plant and traditional medicine. It contains a huge array of bioactive metabolites such as flavonoids, amino acids, alkaloids and vitamins. Consequently, mulberry has received increasing attention in recent years. MMHub (version 1.0) is the first open public repository of mass spectra of small chemical compounds (<1000 Da) in mulberry leaves. The database contains 936 electrospray ionization tandem mass spectrometry (ESI-MS2) data and lists the specific distribution of compounds in 91 mulberry resources with two biological duplicates. ESI-MS2 data were obtained under non-standardized and independent experimental conditions. In total, 124 metabolites were identified or tentatively annotated and details of 90 metabolites with associated chemical structures have been deposited in the database. Supporting information such as PubChem compound information, molecular formula and metabolite classification are also provided in the MS2 spectral tag library. The MMHub provides important and comprehensive metabolome data for scientists working with mulberry. This information will be useful for the screening of quality resources and specific metabolites of mulberry. Database URL: https://biodb.swu.edu.cn/mmdb/

Effects of tobacco smoke on IL-16 in CD8+ cells from human airways and blood: a key role for oxygen free radicals?

2011 ◽  
Vol 300 (1) ◽  
pp. L43-L55 ◽  
Author(s):  
Anders Andersson ◽  
Apostolos Bossios ◽  
Carina Malmhäll ◽  
Margareta Sjöstrand ◽  
Maria Eldh ◽  
...  
Keyword(s):  
Free Radicals ◽  
Human Blood ◽  
Tobacco Smoke ◽  
Oxygen Free Radicals ◽  
De Novo ◽  
Water Soluble ◽  
Cellular Functions ◽  
Short Term Exposure ◽  
Human Airways

Chronic exposure to tobacco smoke leads to an increase in the frequency of infections and in the number of CD8+ and CD4+ cells as well as the CD4+ chemoattractant cytokine IL-16 in the airways. Here, we investigated whether tobacco smoke depletes intracellular IL-16 protein and inhibits de novo production of IL-16 in CD8+ cells from human airways and blood while increasing extracellular IL-16 and whether oxygen free radicals (OFR) are involved. Intracellular IL-16 protein in CD8+ cells and mRNA in all cells was decreased in bronchoalveolar lavage (BAL) samples from chronic smokers. This was also the case in human blood CD8+ cells exposed to water-soluble tobacco smoke components in vitro, in which oxidized proteins were markedly increased. Extracellular IL-16 protein was increased in cell-free BAL fluid from chronic smokers and in human blood CD8+ cells exposed to water-soluble tobacco smoke components in vitro. This was not observed in occasional smokers after short-term exposure to tobacco smoke. A marker of activation (CD69) was slightly increased, whereas other markers of key cellular functions (membrane integrity, apoptosis, and proliferation) in human blood CD8+ cells in vitro were negatively affected by water-soluble tobacco smoke components. An OFR scavenger prevented these effects, whereas a protein synthesis inhibitor, a β-adrenoceptor, a glucocorticoid receptor agonist, a phosphodiesterase, a calcineurin phosphatase, and a caspase-3 inhibitor did not. In conclusion, tobacco smoke depletes preformed intracellular IL-16 protein, inhibits its de novo synthesis, and distorts key cellular functions in human CD8+ cells. OFR may play a key role in this context.

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