scholarly journals Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Roberta Noberini ◽  
Evelyn Oliva Savoia ◽  
Stefania Brandini ◽  
Francesco Greco ◽  
Francesca Marra ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Extraction Procedure ◽  
Clinical Samples ◽  
Epigenetic Mechanisms ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Epigenetic Biomarkers ◽  
Morphological Evaluation ◽  
Ms Imaging ◽  
Histone Ptms

Abstract Background Increasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples. Methods allowing the isolation of histones from patient samples followed by the accurate and comprehensive quantification of their PTMs by mass spectrometry (MS) have been developed. However, the applicability of these methods is limited by the requirement for substantial amounts of material. Results To address this issue, in this study we streamlined the protein extraction procedure from low-amount clinical samples and tested and implemented different in-gel digestion strategies, obtaining a protocol that allows the MS-based analysis of the most common histone PTMs from laser microdissected tissue areas containing as low as 1000 cells, an amount approximately 500 times lower than what is required by available methods. We then applied this protocol to breast cancer patient laser microdissected tissues in two proof-of-concept experiments, identifying differences in histone marks in heterogeneous regions selected by either morphological evaluation or MALDI MS imaging. Conclusions These results demonstrate that analyzing histone PTMs from very small tissue areas and detecting differences from adjacent tumor regions is technically feasible. Our method opens the way for spatial epi-proteomics, namely the investigation of epigenetic features in the context of tissue and tumor heterogeneity, which will be instrumental for the identification of novel epigenetic biomarkers and aberrant epigenetic mechanisms.

scholarly journals Histone Post-Translational Modifications and CircRNAs in Mouse and Human Spermatozoa: Potential Epigenetic Marks to Assess Human Sperm Quality

2020 ◽  
Vol 9 (3) ◽  
pp. 640 ◽  
Author(s):  
Teresa Chioccarelli ◽  
Riccardo Pierantoni ◽  
Francesco Manfrevola ◽  
Veronica Porreca ◽  
Silvia Fasano ◽  
...  
Keyword(s):  
Germ Cells ◽  
Sperm Quality ◽  
Critical Role ◽  
Human Sperm ◽  
Circular Rnas ◽  
Nuclear Condensation ◽  
Post Translational Modifications ◽  
Epigenetic Biomarkers ◽  
Histone Ptms ◽  
Non Coding Rnas

Spermatozoa (SPZ) are motile cells, characterized by a cargo of epigenetic information including histone post-translational modifications (histone PTMs) and non-coding RNAs. Specific histone PTMs are present in developing germ cells, with a key role in spermatogenic events such as self-renewal and commitment of spermatogonia (SPG), meiotic recombination, nuclear condensation in spermatids (SPT). Nuclear condensation is related to chromatin remodeling events and requires a massive histone-to-protamine exchange. After this event a small percentage of chromatin is condensed by histones and SPZ contain nucleoprotamines and a small fraction of nucleohistone chromatin carrying a landascape of histone PTMs. Circular RNAs (circRNAs), a new class of non-coding RNAs, characterized by a nonlinear back-spliced junction, able to play as microRNA (miRNA) sponges, protein scaffolds and translation templates, have been recently characterized in both human and mouse SPZ. Since their abundance in eukaryote tissues, it is challenging to deepen their biological function, especially in the field of reproduction. Here we review the critical role of histone PTMs in male germ cells and the profile of circRNAs in mouse and human SPZ. Furthermore, we discuss their suggested role as novel epigenetic biomarkers to assess sperm quality and improve artificial insemination procedure.

Dicarbonyl derived post-translational modifications: chemistry bridging biology and aging-related disease

2020 ◽  
Vol 64 (1) ◽  
pp. 97-110
Author(s):  
Christian Sibbersen ◽  
Mogens Johannsen
Keyword(s):  
Mass Spectrometry ◽  
Future Research ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Dicarbonyl Compounds ◽  
Protein Targets ◽  
Post Translational Modifications ◽  
Reactive Protein ◽  
Glycation End Products ◽  
Direct Mass Spectrometry

Abstract In living systems, nucleophilic amino acid residues are prone to non-enzymatic post-translational modification by electrophiles. α-Dicarbonyl compounds are a special type of electrophiles that can react irreversibly with lysine, arginine, and cysteine residues via complex mechanisms to form post-translational modifications known as advanced glycation end-products (AGEs). Glyoxal, methylglyoxal, and 3-deoxyglucosone are the major endogenous dicarbonyls, with methylglyoxal being the most well-studied. There are several routes that lead to the formation of dicarbonyl compounds, most originating from glucose and glucose metabolism, such as the non-enzymatic decomposition of glycolytic intermediates and fructosyl amines. Although dicarbonyls are removed continuously mainly via the glyoxalase system, several conditions lead to an increase in dicarbonyl concentration and thereby AGE formation. AGEs have been implicated in diabetes and aging-related diseases, and for this reason the elucidation of their structure as well as protein targets is of great interest. Though the dicarbonyls and reactive protein side chains are of relatively simple nature, the structures of the adducts as well as their mechanism of formation are not that trivial. Furthermore, detection of sites of modification can be demanding and current best practices rely on either direct mass spectrometry or various methods of enrichment based on antibodies or click chemistry followed by mass spectrometry. Future research into the structure of these adducts and protein targets of dicarbonyl compounds may improve the understanding of how the mechanisms of diabetes and aging-related physiological damage occur.

scholarly journals Immunological discrimination of β-tubulin isoforms in developing mouse brain. Post-translational modification of non-class-III β-tubulins

1992 ◽  
Vol 288 (3) ◽  
pp. 919-924 ◽  
Author(s):  
I Linhartová ◽  
P Dráber ◽  
E Dráberová ◽  
V Viklický
Keyword(s):  
Mouse Brain ◽  
Specific Class ◽  
Class Iii ◽  
Post Translational Modification ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Post Translational Modifications ◽  
Tubulin Isotypes ◽  
Tubulin Isoforms ◽  
Developing Mouse Brain

Individual beta-tubulin isoforms in developing mouse brain were characterized using immunoblotting, after preceding high-resolution isoelectric focusing, with monoclonal antibodies against different structural regions of beta-tubulin. Some of the antibodies reacted with a limited number of tubulin isoforms in all stages of brain development and in HeLa cells. The epitope for the TU-14 antibody was located in the isotype-defining domain and was present on the beta-tubulin isotypes of classes I, II and IV, but absent on the neuron-specific class-III isotype. The data suggest that non-class-III beta-tubulins in mouse brain are substrates for developmentally regulated post-translational modifications and that beta-tubulins of non-neuronal cells are also post-translationally modified.

scholarly journals Post translational modifications of milk proteins in geographically diverse goat breeds

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
P. K. Rout ◽  
M. Verma
Keyword(s):  
Protein Function ◽  
Whey Proteins ◽  
Milk Proteins ◽  
Goat Milk ◽  
Peptide Sequence ◽  
Cow Milk ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Functional Roles ◽  
Dpp Iv

AbstractGoat milk is a source of nutrition in difficult areas and has lesser allerginicity than cow milk. It is leading in the area for nutraceutical formulation and drug development using goat mammary gland as a bioreactor. Post translational modifications of a protein regulate protein function, biological activity, stabilization and interactions. The protein variants of goat milk from 10 breeds were studied for the post translational modifications by combining highly sensitive 2DE and Q-Exactive LC-MS/MS. Here we observed high levels of post translational modifications in 201 peptides of 120 goat milk proteins. The phosphosites observed for CSN2, CSN1S1, CSN1S2, CSN3 were 11P, 13P, 17P and 6P, respectively in 105 casein phosphopeptides. Whey proteins BLG and LALBA showed 19 and 4 phosphosites respectively. Post translational modification was observed in 45 low abundant non-casein milk proteins mainly associated with signal transduction, immune system, developmental biology and metabolism pathways. Pasp is reported for the first time in 47 sites. The rare conserved peptide sequence of (SSSEE) was observed in αS1 and αS2 casein. The functional roles of identified phosphopeptides included anti-microbial, DPP-IV inhibitory, anti-inflammatory and ACE inhibitory. This is first report from tropics, investigating post translational modifications in casein and non-casein goat milk proteins and studies their interactions.

scholarly journals System-wide identification and prioritization of enzyme substrates by thermal analysis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Amir Ata Saei ◽  
Christian M. Beusch ◽  
Pierre Sabatier ◽  
Juan Astorga Wells ◽  
Hassan Gharibi ◽  
...  
Keyword(s):  
Thermal Analysis ◽  
Thioredoxin Reductase ◽  
Structural Level ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Enzyme Substrate ◽  
Thioredoxin Reductase 1 ◽  
Enzyme Substrates ◽  
Substrate Proteins

AbstractDespite the immense importance of enzyme–substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery.

scholarly journals Role of Host-Mediated Post-Translational Modifications (PTMs) in RNA Virus Pathogenesis

2020 ◽  
Vol 22 (1) ◽  
pp. 323
Author(s):  
Ramesh Kumar ◽  
Divya Mehta ◽  
Nimisha Mishra ◽  
Debasis Nayak ◽  
Sujatha Sunil
Keyword(s):  
Rna Virus ◽  
Viral Proteins ◽  
Post Translational Modification ◽  
Host Proteins ◽  
Viral Protein Synthesis ◽  
Post Translational Modifications ◽  
Small Proteins ◽  
Cellular Machinery ◽  
Chemical Groups

Being opportunistic intracellular pathogens, viruses are dependent on the host for their replication. They hijack host cellular machinery for their replication and survival by targeting crucial cellular physiological pathways, including transcription, translation, immune pathways, and apoptosis. Immediately after translation, the host and viral proteins undergo a process called post-translational modification (PTM). PTMs of proteins involves the attachment of small proteins, carbohydrates/lipids, or chemical groups to the proteins and are crucial for the proteins’ functioning. During viral infection, host proteins utilize PTMs to control the virus replication, using strategies like activating immune response pathways, inhibiting viral protein synthesis, and ultimately eliminating the virus from the host. PTM of viral proteins increases solubility, enhances antigenicity and virulence properties. However, RNA viruses are devoid of enzymes capable of introducing PTMs to their proteins. Hence, they utilize the host PTM machinery to promote their survival. Proteins from viruses belonging to the family: Togaviridae, Flaviviridae, Retroviridae, and Coronaviridae such as chikungunya, dengue, zika, HIV, and coronavirus are a few that are well-known to be modified. This review discusses various host and virus-mediated PTMs that play a role in the outcome during the infection.

scholarly journals Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis

2009 ◽  
Vol 390 (2) ◽  
pp. 137-144 ◽  
Author(s):  
Yingmiao Liu ◽  
Chien-Tsun Kuan ◽  
Jing Mi ◽  
Xiuwu Zhang ◽  
Bryan M. Clary ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Rna Aptamers ◽  
Normal Brain ◽  
Post Translational Modification ◽  
Expression And Purification ◽  
Post Translational Modifications ◽  
Protein Expression And Purification ◽  
Epidermal Growth ◽  
Membrane Bound

Abstract Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein uniquely expressed in glioblastoma, but not in normal brain tissues. To develop targeted therapies for brain tumors, we selected RNA aptamers against the histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for protein expression and purification. Representative aptamer E21 has a dissociation constant (Kd) of 33×10-9 m, and exhibits high affinity and specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However, selected aptamers cannot bind the same protein expressed from eukaryotic cells because glycosylation, a post-translational modification present only in eukaryotic systems, significantly alters the structure of the target protein. By transfecting EGFRvIII aptamers into cells, we find that membrane-bound, glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis is increased. We postulate that transfected aptamers can interact with newly synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of mature EGFRvIII reaching the cell surface. Our work establishes the feasibility of disrupting protein post-translational modifications in situ with aptamers. This finding is useful for elucidating the function of proteins of interest with various modifications, as well as dissecting signal transduction pathways.

RegulatING chromatin regulators: post-translational modification of the ING family of epigenetic regulators

2013 ◽  
Vol 450 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Shankha Satpathy ◽  
Arash Nabbi ◽  
Karl Riabowol
Keyword(s):  
Biological Significance ◽  
Type Ii ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Chromatin Regulators ◽  
Cancer Types ◽  
Splicing Isoforms ◽  
Enrichment Techniques ◽  
Affect Cell Growth

The five human ING genes encode at least 15 splicing isoforms, most of which affect cell growth, differentiation and apoptosis through their ability to alter gene expression by epigenetic mechanisms. Since their discovery in 1996, ING proteins have been classified as type II tumour suppressors on the basis of reports describing their down-regulation and mislocalization in a variety of cancer types. In addition to their regulation by transcriptional mechanisms, understanding the range of PTMs (post-translational modifications) of INGs is important in understanding how ING functions are fine-tuned in the physiological setting and how they add to the repertoire of activities affected by the INGs. In the present paper we review the different PTMs that have been reported to occur on INGs. We discuss the PTMs that modulate ING function under normal conditions and in response to a variety of stresses. We also describe the ING PTMs that have been identified by several unbiased MS-based PTM enrichment techniques and subsequent proteomic analysis. Among the ING PTMs identified to date, a subset has been characterized for their biological significance and have been shown to affect processes including subcellular localization, interaction with enzymatic complexes and ING protein half-life. The present review aims to highlight the emerging role of PTMs in regulating ING function and to suggest additional pathways and functions where PTMs may effect ING function.

scholarly journals Evaluation of effective protein extraction procedure to profile petiole of Moringa oleifera

2020 ◽  
Vol 7 (2) ◽  
pp. 214
Author(s):  
Zetty Amirah Zulkifli ◽  
Zaidah Rahmat
Keyword(s):  
Moringa Oleifera ◽  
Protein Extraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Electrophoretic Pattern ◽  
Antioxidant Property ◽  
Extraction Methods ◽  
Protein Profiling ◽  
Sds Page

Moringa oleifera is widely known as multipurpose tree since all of its parts confer multiple functions. The leaf is highly favourable among consumers while the petiole is mostly wasted. There are numerous studies on the flavonoid and antioxidant property of the stem and twig. However, study on the petiole has never been done. There-upon, this study was conducted to develop protein profiling of the petiole. In this study, 6 different protein extraction methods were tested on the fresh petiole before its protein quantity and quality were checked via Bradford assay and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) respectively. The in-solution digestion was then done prior to LC-MS/MS analysis. The protein electrophoretic pattern from the SDS-PAGE proves that method 6 using Tris HCl buffer with incorporation of dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF) confers the best quality of protein. It produced the highest number of visible individual bands compared to other methods. Meanwhile, 93 proteins were successfully identified via LCMS analysis where the protein, signal response and carbohydrate metabolism categories confer the highest percentage. High quality and content of the protein extracted from the petiole including the antioxidant, anticancer and antidiabetic protein identified suggested that consuming this part of the plant could enhance nutrients of human body.

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