scholarly journals Site-Specific Conversion of Cysteine in a Protein to Dehydroalanine Using 2-Nitro-5-thiocyanatobenzoic Acid

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2619
Author(s):  
Yuchen Qiao ◽  
Ge Yu ◽  
Sunshine Z. Leeuwon ◽  
Wenshe Ray Liu
Keyword(s):  
Functional Annotation ◽  
Chemical Reagent ◽  
Post Translational Modifications ◽  
Site Specific ◽  
Internal Region ◽  
Hydrolysis Products ◽  
Starting Point ◽  
Michael Acceptor ◽  
Formation Method ◽  
High Yields

Dehydroalanine exists natively in certain proteins and can also be chemically made from the protein cysteine. As a strong Michael acceptor, dehydroalanine in proteins has been explored to undergo reactions with different thiolate reagents for making close analogues of post-translational modifications (PTMs), including a variety of lysine PTMs. The chemical reagent 2-nitro-5-thiocyanatobenzoic acid (NTCB) selectively modifies cysteine to form S-cyano-cysteine, in which the S–Cβ bond is highly polarized. We explored the labile nature of this bond for triggering E2 elimination to generate dehydroalanine. Our results indicated that when cysteine is at the flexible C-terminal end of a protein, the dehydroalanine formation is highly effective. We produced ubiquitin and ubiquitin-like proteins with a C-terminal dehydroalanine residue with high yields. When cysteine is located at an internal region of a protein, the efficiency of the reaction varies with mainly hydrolysis products observed. Dehydroalanine in proteins such as ubiquitin and ubiquitin-like proteins can serve as probes for studying pathways involving ubiquitin and ubiquitin-like proteins and it is also a starting point to generate proteins with many PTM analogues; therefore, we believe that this NTCB-triggered dehydroalanine formation method will find broad applications in studying ubiquitin and ubiquitin-like protein pathways and the functional annotation of many PTMs in proteins such as histones.

The challenge of detecting modifications on proteins

2020 ◽  
Vol 64 (1) ◽  
pp. 135-153 ◽  
Author(s):  
Lauren Elizabeth Smith ◽  
Adelina Rogowska-Wrzesinska
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Chemical Properties ◽  
Lysine Acetylation ◽  
Mass Determination ◽  
Post Translational Modifications ◽  
Site Specific ◽  
The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

scholarly journals Phosphorylation of the Regulators, a Complex Facet of NF-κB Signaling in Cancer

Biomolecules ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 15
Author(s):  
Aishat Motolani ◽  
Matthew Martin ◽  
Mengyao Sun ◽  
Tao Lu
Keyword(s):  
Transcription Factor ◽  
Cardiovascular Diseases ◽  
Cancer Progression ◽  
Nuclear Factor Kappa B ◽  
Nuclear Factor ◽  
Malignant Diseases ◽  
Future Perspectives ◽  
Post Translational Modifications ◽  
Site Specific

The nuclear factor kappa B (NF-κB) is a ubiquitous transcription factor central to inflammation and various malignant diseases in humans. The regulation of NF-κB can be influenced by a myriad of post-translational modifications (PTMs), including phosphorylation, one of the most popular PTM formats in NF-κB signaling. The regulation by phosphorylation modification is not limited to NF-κB subunits, but it also encompasses the diverse regulators of NF-κB signaling. The differential site-specific phosphorylation of NF-κB itself or some NF-κB regulators can result in dysregulated NF-κB signaling, often culminating in events that induce cancer progression and other hyper NF-κB related diseases, such as inflammation, cardiovascular diseases, diabetes, as well as neurodegenerative diseases, etc. In this review, we discuss the regulatory role of phosphorylation in NF-κB signaling and the mechanisms through which they aid cancer progression. Additionally, we highlight some of the known and novel NF-κB regulators that are frequently subjected to phosphorylation. Finally, we provide some future perspectives in terms of drug development to target kinases that regulate NF-κB signaling for cancer therapeutic purposes.

scholarly journals First comprehensive proteome analysis of lysine crotonylation in Streptococcus agalactiae, a pathogen causing meningoencephalitis in teleosts

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Xinjin Chen ◽  
Bolin Fan ◽  
Chenlong Fan ◽  
Zhongliang Wang ◽  
Eakapol Wangkahart ◽  
...  
Keyword(s):  
Streptococcus Agalactiae ◽  
Proteome Analysis ◽  
Post Translational Modifications ◽  
Sensing System ◽  
Affinity Enrichment ◽  
Single Organism ◽  
Starting Point ◽  
Zoonotic Risk ◽  
Quorum Sensing System ◽  
First Time

Abstract Backgroud Streptococcus agalactiae is a common colonizer of the rectovaginal tract and lead to infectious diseases of neonatal and non-pregnant adults, which also causes infectious disease in fish and a zoonotic risk as well. Lysine crotonylation (Kcr) is a kind of histone post-translational modifications discovered in 2011. In yeast and mammals, Kcr function as potential enhancers and promote gene expression. However, lysine crotonylation in S. agalactiae has not been studied yet. Methods In this study, the crotonylation profiling of fish pathogen, S. agalactiae was investigated by combining affinity enrichment with LC MS/MS. The Kcr modification of several selected proteins were further validated by Western blotting. Results In the present study, we conducted the proteome-wide profiling of Kcr in S. agalactiae and identified 241 Kcr sites from 675 screened proteins for the first time. Bioinformatics analysis showed that 164 sequences were matched to a total of six definitively conserved motifs, and many of them were significantly enriched in metabolic processes, cellular process, and single-organism processes. Moreover, four crotonylation modified proteins were predicted as virulence factors or to being part of the quorum sensing system PTMs on bacteria. The data are available via ProteomeXchange with identifier PXD026445. Conclusions These data provide a promising starting point for further functional research of crotonylation in bacterial virulence in S. agalactiae.

scholarly journals The Hitchhiker's guide to glycoproteomics

2021 ◽  
Author(s):  
Tiago Oliveira ◽  
Morten Thaysen-Andersen ◽  
Nicolle H. Packer ◽  
Daniel Kolarich
Keyword(s):  
Functional Analysis ◽  
Cell Function ◽  
Prognostic Biomarker ◽  
Protein Glycosylation ◽  
Life Changes ◽  
Carrier Proteins ◽  
Post Translational Modifications ◽  
Starting Point ◽  
Domains Of Life ◽  
The Common

Protein glycosylation is one of the most common post-translational modifications that are essential for cell function across all domains of life. Changes in glycosylation are considered a hallmark of many diseases, thus making glycoproteins important diagnostic and prognostic biomarker candidates and therapeutic targets. Glycoproteomics, the study of glycans and their carrier proteins in a system-wide context, is becoming a powerful tool in glycobiology that enables the functional analysis of protein glycosylation. This ‘Hitchhiker's guide to glycoproteomics’ is intended as a starting point for anyone who wants to explore the emerging world of glycoproteomics. The review moves from the techniques that have been developed for the characterisation of single glycoproteins to technologies that may be used for a successful complex glycoproteome characterisation. Examples of the variety of approaches, methodologies, and technologies currently used in the field are given. This review introduces the common strategies to capture glycoprotein-specific and system-wide glycoproteome data from tissues, body fluids, or cells, and a perspective on how integration into a multi-omics workflow enables a deep identification and characterisation of glycoproteins — a class of biomolecules essential in regulating cell function.

scholarly journals Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

2020 ◽  
Author(s):  
Camille M. Le Gall ◽  
Johan M.S. van der Schoot ◽  
Iván Ramos-Tomillero ◽  
Melek Parlak Khalily ◽  
Floris J. van Dalen ◽  
...  
Keyword(s):  
Antibody Fragment ◽  
Therapeutic Index ◽  
Stable Cell Line ◽  
Hybridoma Cells ◽  
Target Cells ◽  
Site Specific ◽  
Drug Conjugates ◽  
High Yields ◽  
A Site

I.AbstractFunctionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theragnostics, and antibody-drug conjugates (ADC). Antibody functionalization is classically achieved by coupling payloads onto lysine or cysteine residues. However, such stochastic strategies typically lead to heterogenous products, bearing a varying number of payloads. This affects bioconjugate efficacy and stability, as well as its in vivo biodistribution, and therapeutic index, while potentially obstructing the binding sites and leading to off-target toxicity. In addition, therapeutic and theragnostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labelling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogenous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab’ fragments bearing two distinct site-specific labelling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab’ molecule (DTFab’), which can be easily isolated in high yields. To demonstrate feasibility, we functionalized the DTFab’ with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theragnostics, and next-generation ADCs.

scholarly journals Working with Proteins in silico: A Review of Online Available Tools for Basic Identification of Proteins

2017 ◽  
Vol 5 (1) ◽  
pp. 65
Author(s):  
Caner Yavuz ◽  
Zahide Neslihan Öztürk
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
In Silico ◽  
In Silico Analysis ◽  
Tips And Tricks ◽  
Post Translational Modifications ◽  
Starting Point ◽  
Protein Research ◽  
Experimental Approaches ◽  
Silico Analysis

Increase in online available bioinformatics tools for protein research creates an important opportunity for scientists to reveal characteristics of the protein of interest by only starting from the predicted or known amino acid sequence without fully depending on experimental approaches. There are many sophisticated tools used for diverse purposes; however, there are not enough reviews covering the tips and tricks in selecting and using the correct tools as the literature mainly state the promotion of the new ones. In this review, with the aim of providing young scientists with no specific experience on protein work a reliable starting point for in silico analysis of the protein of interest, we summarized tools for annotation, identification of motifs and domains, determination isoelectric point, molecular weight, subcellular localization, and post-translational modifications by focusing on the important points to be considered while selecting from online available tools.

scholarly journals A Peptidoform Matching Strategy in Bottom-Up Proteomics for Studying Functions of Post-Translational Modifications

2021 ◽  
Author(s):  
Yansheng Liu
Keyword(s):  
Quantitative Analysis ◽  
Relevant Information ◽  
Previous Literature ◽  
Biological Functions ◽  
Biological Investigation ◽  
Bottom Up ◽  
Post Translational Modifications ◽  
Site Specific ◽  
Specific Approach ◽  
Matching Strategy

Protein translational modifications (PTMs) generate an enormous, but as yet undetermined, expansion of the expressed proteoforms. In this Viewpoint, we firstly differentiate the concepts of proteoform and peptidoform by reviewing and discussing previous literature. We show that the current PTM biological investigation and annotation largely follow a PTM site-specific rather than proteoform-specific approach. We further illustrate a potentially useful matching strategy in which a particular “modified peptidoform” is matched to the corresponding “unmodified peptidoform” as a reference for the quantitative analysis between samples and conditions. We suggest this strategy could provide directly relevant information for learning the PTM site-specific biological functions. Accordingly, we advocate for the wider use of the nomenclature “peptidoform” in the future bottom-up proteomic studies.

The Process of Coming and Going in this World: Conversation About Interspecies Collaboration, Domestication, Sound

2021 ◽  
Vol 29 (7) ◽  
pp. 695-715
Author(s):  
Ruth K. Burke ◽  
Jessica Landau
Keyword(s):  
Sound Recording ◽  
Site Specific ◽  
Starting Point ◽  
Audio Recordings

Abstract The Process of Coming and Going in this World is a four-channel, site-specific installation by artist Ruth Burke. The work incorporates its audience, including nonhuman collaborators. While dependent on time and place, it has been preserved in audio recordings and photographs. In this interview between the artist and art historian Jessica Landau, they discuss the installation’s use of sound, time, and place to evoke interspecies relationships based on collaboration and co-constituted domestication. While using the installation and subsequent sound recording of it as a starting point, the conversation looks at the ways our relationships with animals on farms involve notions of time and place, companionship, and coevolution.

Estimation of the rate of slowly biodegradable COD (SBCOD) hydrolysis under anaerobic, anoxic and aerobic conditions by experiments using starch as model substrate

1995 ◽  
Vol 31 (2) ◽  
pp. 95-103 ◽  
Author(s):  
Takashi Mino ◽  
Delfin C. San Pedro ◽  
Tomonori Matsuo
Keyword(s):  
Activated Sludge ◽  
Biomass Concentration ◽  
Organic Substrate ◽  
Starch Hydrolysis ◽  
Hydrolysis Rate ◽  
Aerobic Conditions ◽  
Hydrolysis Products ◽  
Hydrolysis Rates ◽  
Pure Cultures ◽  
Formation Method

The hydrolysis rates under anaerobic, anoxic and aerobic conditions are studied by using starch as a model organic substrate representing the slowly biodegradable COD. An analytical method, the starch-iodine complex formation method, was introduced to distinguish the polymer starch and its hydrolysis products, and it was applied to estimate the hydrolysis rates in an activated sludge mixed culture, two bacterial pure cultures (Bacillus amyloliquefaciens and Aeromonas hydrophila) or a pure enzyme (α-amylase) system. The rate of starch hydrolysis was found to be independent of the electron acceptor conditions. The starch hydrolysis rate constant of the activated sludge was not affected by the biomass concentration, which indicates that the starch hydrolysis in the activated sludge process follows surface limited adsorption reaction kinetics.

scholarly journals Cultivation of low tetrahydrocannabinol (THC) Cannabis sativa L. cultivation in Victoria, Australia: Do we know enough?

2019 ◽  
pp. 911-919
Author(s):  
Talia Humphries ◽  
Singarayer Florentine
Keyword(s):  
Federal Government ◽  
Central Europe ◽  
Cannabis Sativa ◽  
Farming Practices ◽  
Dual Purpose ◽  
Site Specific ◽  
Grain Yields ◽  
Environmental Requirements ◽  
High Yields

Late 2017, the ban on the cultivation and consumption of low tetrahydrocannabinol (THC) Cannabis sativa L. in Victoria, was lifted by the Federal Government of Australia. Its legalization presents the opportunity for Victoria to become a leading producer and distributer of these economically valuable hemp products. However, as a novel crop to Victoria, there is little information available for obtaining economically viable yields. Therefore, the objectives of this review were to firstly, develop an understanding of the environmental requirements shared by C. sativa cultivars, and what conditions promote fibre and grain yields. Secondly, it seeks to identify what farming practices have been conducted throughout Europe, Canada and China, and to explore whether these practices could be adapted to Victoria. Thirdly, the review will assist in making recommendations regarding which cultivars would be ‘potential’ candidates for commencing trials under Victorian climates so to find out the varieties that can provide high yields for fibre, grain and dual-purpose production. This review notes that Victoria shares a similar climate to central Europe, and has an ideal climate for the development of a successful hemp industry, as it has suitable lengths of daylight throughout spring and summer months and meets the precipitation requirements. This review has thus strongly suggested that the properties and attributes of European varieties of C. sativa should be further researched for site-specific cultivation in Victoria for fibre, grain and dual-purpose production in order to maximise harvest yields.

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