scholarly journals Discovery of indole-modified aptamers for highly specific recognition of protein glycoforms

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alex M. Yoshikawa ◽  
Alexandra Rangel ◽  
Trevor Feagin ◽  
Elizabeth M. Chun ◽  
Leighton Wan ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Biological Function ◽  
Cultured Cells ◽  
Specific Protein ◽  
Biological Processes ◽  
Post Translational Modification ◽  
Affinity Reagents ◽  
Profound Impact ◽  
Wide Range ◽  
Protein Glycoforms

AbstractGlycosylation is one of the most abundant forms of post-translational modification, and can have a profound impact on a wide range of biological processes and diseases. Unfortunately, efforts to characterize the biological function of such modifications have been greatly hampered by the lack of affinity reagents that can differentiate protein glycoforms with robust affinity and specificity. In this work, we use a fluorescence-activated cell sorting (FACS)-based approach to generate and screen aptamers with indole-modified bases, which are capable of recognizing and differentiating between specific protein glycoforms. Using this approach, we were able to select base-modified aptamers that exhibit strong selectivity for specific glycoforms of two different proteins. These aptamers can discriminate between molecules that differ only in their glycan modifications, and can also be used to label glycoproteins on the surface of cultured cells. We believe our strategy should offer a generally-applicable approach for developing useful reagents for glycobiology research.

scholarly journals Discovery of indole-modified aptamers for highly specific recognition of protein glycoforms

2021 ◽  
Author(s):  
Alex M. Yoshikawa ◽  
Alexandra Rangel ◽  
Trevor Feagin ◽  
Elizabeth M. Chun ◽  
Leighton Wan ◽  
...  
Keyword(s):  
Clinical Research ◽  
Cell Sorting ◽  
Biological Processes ◽  
Post Translational Modification ◽  
Tertiary Structures ◽  
Affinity Reagents ◽  
Profound Impact ◽  
Specific Recognition ◽  
Wide Range ◽  
Protein Glycoforms

AbstractGlycosylation is one of the most abundant forms of post-translational modification, and can have a profound impact on a wide range of biological processes and diseases. Unfortunately, efforts to characterize such modifications in the context of basic and clinical research are severely hampered by the lack of affinity reagents that can differentiate protein glycoforms. This lack of reagents is largely due to the challenges associated with generating affinity reagents that can bind to particular glycan epitopes with robust affinity and specificity. In this work, we use a fluorescence-activated cell sorting (FACS)-based approach to generate and screen aptamers with indole-modified bases in an effort to isolate reagents that can differentiate between protein glycoforms. Using this approach, we were able to select multiple aptamers that exhibit strong selectivity for specific glycoforms of two different proteins, with the capacity to discriminate between molecules with identical tertiary structures that differ only in terms of their glycan modifications.

scholarly journals Developing Practical Therapeutic Strategies that Target Protein SUMOylation

2019 ◽  
Vol 20 (9) ◽  
pp. 960-969 ◽  
Author(s):  
Olivia F. Cox ◽  
Paul W. Huber
Keyword(s):  
Birth Defects ◽  
Inflammatory Disease ◽  
Intracellular Localization ◽  
Specific Protein ◽  
Therapeutic Strategies ◽  
Biological Processes ◽  
Post Translational Modification ◽  
Protein Activity ◽  
Multiple Components ◽  
Congenital Birth Defects

Post-translational modification by small ubiquitin-like modifier (SUMO) has emerged as a global mechanism for the control and integration of a wide variety of biological processes through the regulation of protein activity, stability and intracellular localization. As SUMOylation is examined in greater detail, it has become clear that the process is at the root of several pathologies including heart, endocrine, and inflammatory disease, and various types of cancer. Moreover, it is certain that perturbation of this process, either globally or of a specific protein, accounts for many instances of congenital birth defects. In order to be successful, practical strategies to ameliorate conditions due to disruptions in this post-translational modification will need to consider the multiple components of the SUMOylation machinery and the extraordinary number of proteins that undergo this modification.

scholarly journals Engineering and Characterisation of Bacterial Phosphopantetheinyl Transferases and their Peptide Substrates

2021 ◽  
Author(s):  
◽  
Jack Alexander Sissons
Keyword(s):  
Single Gene ◽  
Specific Protein ◽  
Rapid Identification ◽  
Phosphopantetheinyl Transferase ◽  
Post Translational Modification ◽  
Diverse Range ◽  
Peptide Motifs ◽  
Wide Range

<p>Throughout all domains of life, phosphopantetheinyl transferase (PPTase) enzymes catalyse a post-translational modification that is important in both primary and secondary metabolism; the transfer of a phosphopantetheine (PPant) group derived from Coenzyme A to specific protein domains within large, multi-modular biosynthetic enzymes, thereby activating each module for biosynthesis. The short peptide motif of the protein to which this group is attached is known as a ‘tag’, and can be fused to other proteins, making them also substrates for post-translational modification by a PPTase. Additionally, it has been demonstrated that PPTases can utilise a diverse range of CoA analogues, such as biotin-linked or click-chemistry capable CoA derivatives, as substrates for tag attachment. Together, these characteristics make post-translational modification by PPTases an attractive system for many different biotechnological applications. Perhaps the most significant application is in vivo and in vitro site-specific labelling of proteins, for which current technologies are hindered by cumbersome fusion protein requirements, toxicity of the process, or limited reporter groups that can be attached. Confoundingly, most PPTases exhibit a high degree of substrate promiscuity which limits the number of PPTase-tag pairs that can be used simultaneously, and therefore the number of protein targets that can be simultaneously labelled. To address this, directed evolution at a single gene level was used in an attempt to generate multiple PPTase variants that have non-overlapping tag specificity which have applications in orthogonal labelling. Furthermore, assays for the rapid identification, characterisation and evolution of short, novel peptide motifs that are recognised by PPTases has further diversified the labelling toolkit. These developments have enhanced the utility of the PPTase system and potentially have a wide range of applications in a number of fields.</p>

scholarly journals Large-scale analysis of post-translational modifications in E. coli under glucose-limiting conditions

2016 ◽  
Author(s):  
Colin W Brown ◽  
Viswanadham Sridhara ◽  
Daniel R Boutz ◽  
Maria D Person ◽  
Edward M Marcotte ◽  
...  
Keyword(s):  
Large Scale ◽  
Biological Function ◽  
Growth Conditions ◽  
Growth Cycle ◽  
Post Translational Modification ◽  
Proteomics Data ◽  
Proteomic Data ◽  
Large Scale Analysis ◽  
Wide Range ◽  
Domains Of Life

AbstractBackgroundPost-translational modification (PTM) of proteins is central to many cellular processes across all domains of life, but despite decades of study and a wealth of genomic and proteomic data the biological function of many PTMs remains unknown. This is especially true for prokaryotic PTM systems, many of which have only recently been recognized and studied in depth. It is increasingly apparent that a deep sampling of abundance across a wide range of environmental stresses, growth conditions, and PTM types, rather than simply cataloging targets for a handful of modifications, is critical to understanding the complex pathways that govern PTM deposition and downstream effects.ResultsWe utilized a deeply-sampled dataset of MS/MS proteomic analysis covering 9 timepoints spanning theEscherichia coligrowth cycle and an unbiased PTM search strategy to construct a temporal map of abundance for all PTMs within a 400 Da window of mass shifts. Using this map, we are able to identify novel targets and temporal patterns for N-terminal Nα acetylation, C-terminal glutamylation, and asparagine deamidation. Furthermore, we identify a possible relationship between N-terminal Na acetylation and regulation of protein degradation in stationary phase, pointing to a previously unrecognized biological function for this poorly-understood PTM.ConclusionsUnbiased detection of PTM in MS/MS proteomics data facilitates the discovery of novel modification types and previously unobserved dynamic changes in modification across growth timepoints.

scholarly journals Global Proteomic Analysis Reveals Widespread Lysine Succinylation in Rice Seedlings

2019 ◽  
Vol 20 (23) ◽  
pp. 5911 ◽  
Author(s):  
Kai Zhang ◽  
Yehui Xiong ◽  
Wenxian Sun ◽  
Guo-Liang Wang ◽  
Wende Liu
Keyword(s):  
Biological Function ◽  
Tricarboxylic Acid Cycle ◽  
Enrichment Analysis ◽  
Tca Cycle ◽  
Biological Processes ◽  
Rice Seedlings ◽  
Post Translational Modification ◽  
Lysine Succinylation ◽  
Rice Samples ◽  
Pathway Category

Lysine succinylation (Ksu) is a dynamic and reversible post-translational modification that plays an important role in many biological processes. Although recent research has analyzed Ksu plant proteomes, little is known about the scope and cellular distribution of Ksu in rice seedlings. Here, we report high-quality proteome-scale Ksu data for rice seedlings. A total of 710 Ksu sites in 346 proteins with diverse biological functions and subcellular localizations were identified in rice samples. About 54% of the sites were predicted to be localized in the chloroplast. Six putative succinylation motifs were detected. Comparative analysis with succinylation data revealed that arginine (R), located downstream of Ksu sites, is the most conserved amino acid surrounding the succinylated lysine. KEGG pathway category enrichment analysis indicated that carbon metabolism, tricarboxylic acid cycle (TCA) cycle, oxidative phosphorylation, photosynthesis, and glyoxylate and dicarboxylate metabolism pathways were significantly enriched. Additionally, we compared published Ksu data from rice embryos with our data from rice seedlings and found conserved Ksu sites between the two rice tissues. Our in-depth survey of Ksu in rice seedlings provides the foundation for further understanding the biological function of lysine-succinylated proteins in rice growth and development.

scholarly journals Starch biosynthesis in developing seeds

2010 ◽  
Vol 21 (1) ◽  
pp. 5-32 ◽  
Author(s):  
Ian J. Tetlow
Keyword(s):  
Seedling Establishment ◽  
Biological Function ◽  
Starch Synthesis ◽  
Industrial Applications ◽  
Starch Biosynthesis ◽  
Post Translational Modification ◽  
Developing Seeds ◽  
Starch Structure ◽  
Wide Range ◽  
Storage Starch

AbstractStarch is globally important as a source of food and, in addition, has a wide range of industrial applications. Much of this agriculturally produced starch is synthesized in developing seeds, where its biological function is to provide energy for seedling establishment. Storage starch in developing seeds is synthesized in heterotrophic plastids called amyloplasts and is distinct from the transient synthesis of starch in chloroplasts. This article reviews our current understanding of storage starch biosynthesis occurring in these organelles and discusses recent advances in research in this field. The review discusses starch structure and granule initiation, emerging ideas on the evolution of the pathway, the enzymes of starch synthesis, and the post-translational modification and regulation of key enzymes of amylopectin biosynthesis.

scholarly journals Therapeutic targeting of protein S-acylation for the treatment of disease

2019 ◽  
Vol 48 (1) ◽  
pp. 281-290 ◽  
Author(s):  
Niall J. Fraser ◽  
Jacqueline Howie ◽  
Krzysztof J. Wypijewski ◽  
William Fuller
Keyword(s):  
Critical Role ◽  
Cardiac Contractility ◽  
Protein S ◽  
Biological Processes ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Specific Target ◽  
Receptor Signalling ◽  
Therapeutic Drugs ◽  
Wide Range

The post-translational modification protein S-acylation (commonly known as palmitoylation) plays a critical role in regulating a wide range of biological processes including cell growth, cardiac contractility, synaptic plasticity, endocytosis, vesicle trafficking, membrane transport and biased-receptor signalling. As a consequence, zDHHC-protein acyl transferases (zDHHC-PATs), enzymes that catalyse the addition of fatty acid groups to specific cysteine residues on target proteins, and acyl proteins thioesterases, proteins that hydrolyse thioester linkages, are important pharmaceutical targets. At present, no therapeutic drugs have been developed that act by changing the palmitoylation status of specific target proteins. Here, we consider the role that palmitoylation plays in the development of diseases such as cancer and detail possible strategies for selectively manipulating the palmitoylation status of specific target proteins, a necessary first step towards developing clinically useful molecules for the treatment of disease.

scholarly journals Engineering and Characterisation of Bacterial Phosphopantetheinyl Transferases and their Peptide Substrates

2021 ◽  
Author(s):  
◽  
Jack Alexander Sissons
Keyword(s):  
Single Gene ◽  
Specific Protein ◽  
Rapid Identification ◽  
Phosphopantetheinyl Transferase ◽  
Post Translational Modification ◽  
Diverse Range ◽  
Peptide Motifs ◽  
Wide Range

<p>Throughout all domains of life, phosphopantetheinyl transferase (PPTase) enzymes catalyse a post-translational modification that is important in both primary and secondary metabolism; the transfer of a phosphopantetheine (PPant) group derived from Coenzyme A to specific protein domains within large, multi-modular biosynthetic enzymes, thereby activating each module for biosynthesis. The short peptide motif of the protein to which this group is attached is known as a ‘tag’, and can be fused to other proteins, making them also substrates for post-translational modification by a PPTase. Additionally, it has been demonstrated that PPTases can utilise a diverse range of CoA analogues, such as biotin-linked or click-chemistry capable CoA derivatives, as substrates for tag attachment. Together, these characteristics make post-translational modification by PPTases an attractive system for many different biotechnological applications. Perhaps the most significant application is in vivo and in vitro site-specific labelling of proteins, for which current technologies are hindered by cumbersome fusion protein requirements, toxicity of the process, or limited reporter groups that can be attached. Confoundingly, most PPTases exhibit a high degree of substrate promiscuity which limits the number of PPTase-tag pairs that can be used simultaneously, and therefore the number of protein targets that can be simultaneously labelled. To address this, directed evolution at a single gene level was used in an attempt to generate multiple PPTase variants that have non-overlapping tag specificity which have applications in orthogonal labelling. Furthermore, assays for the rapid identification, characterisation and evolution of short, novel peptide motifs that are recognised by PPTases has further diversified the labelling toolkit. These developments have enhanced the utility of the PPTase system and potentially have a wide range of applications in a number of fields.</p>

Emerging Principles for the Therapeutic Exploitation of Glycosylation

Science ◽  
2014 ◽  
Vol 343 (6166) ◽  
pp. 1235681 ◽  
Author(s):  
Martin Dalziel ◽  
Max Crispin ◽  
Christopher N. Scanlan ◽  
Nicole Zitzmann ◽  
Raymond A. Dwek
Keyword(s):  
Monoclonal Antibodies ◽  
Drug Design ◽  
Immune Regulation ◽  
Metabolic Disorders ◽  
Biological Function ◽  
Specific Drug ◽  
Biological Processes ◽  
Wide Range ◽  
Viral Vaccine ◽  
Novel Targets

Glycosylation plays a key role in a wide range of biological processes. Specific modification to a glycan’s structure can directly modulate its biological function. Glycans are not only essential to glycoprotein folding, cellular homeostasis, and immune regulation but are involved in multiple disease conditions. An increased molecular and structural understanding of the mechanistic role that glycans play in these pathological processes has driven the development of therapeutics and illuminated novel targets for drug design. This knowledge has enabled the treatment of metabolic disorders and the development of antivirals and shaped cancer and viral vaccine strategies. Furthermore, an understanding of glycosylation has led to the development of specific drug glycoforms, for example, monoclonal antibodies, with enhanced potency.

Review of Progress in Predicting Protein Methylation Sites

2019 ◽  
Vol 23 (15) ◽  
pp. 1663-1670 ◽  
Author(s):  
Chunyan Ao ◽  
Shunshan Jin ◽  
Yuan Lin ◽  
Quan Zou
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Transcriptional Activity ◽  
Regulation Of Gene Expression ◽  
Protein Methylation ◽  
Biological Processes ◽  
Post Translational Modification ◽  
Regulatory Enzymes ◽  
Lysine Residues ◽  
Arginine Residues

Protein methylation is an important and reversible post-translational modification that regulates many biological processes in cells. It occurs mainly on lysine and arginine residues and involves many important biological processes, including transcriptional activity, signal transduction, and the regulation of gene expression. Protein methylation and its regulatory enzymes are related to a variety of human diseases, so improved identification of methylation sites is useful for designing drugs for a variety of related diseases. In this review, we systematically summarize and analyze the tools used for the prediction of protein methylation sites on arginine and lysine residues over the last decade.

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