scholarly journals Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function

2012 ◽  
Vol 20 (1) ◽  
pp. 46-52 ◽  
Author(s):  
Rachael Baker ◽  
Steven M Lewis ◽  
Atsuo T Sasaki ◽  
Emily M Wilkerson ◽  
Jason W Locasale ◽  
...  
Keyword(s):  
Protein Function ◽  
Gtpase Activating Protein ◽  
Site Specific

scholarly journals Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function

Small GTPases ◽  
2013 ◽  
Vol 4 (3) ◽  
pp. 186-192 ◽  
Author(s):  
G Aaron Hobbs ◽  
Harsha P Gunawardena ◽  
Rachael Baker ◽  
Xian Chen ◽  
Sharon L Campbell
Keyword(s):  
Protein Function ◽  
Gtpase Activating Protein ◽  
Site Specific

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 Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine

2019 ◽  
Author(s):  
Junetha Syed ◽  
Saravanan Palani ◽  
Scott T. Clarke ◽  
Zainab Asad ◽  
Andrew R. Bottrill ◽  
...  
Keyword(s):  
Genetic Code ◽  
Protein Function ◽  
Fluorescent Protein ◽  
Trna Synthetase ◽  
Glutathione S Transferase ◽  
Base Pairs ◽  
Site Specific ◽  
Green Fluorescent ◽  
Natural Amino Acids ◽  
Sense Codon

AbstractSite-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function [1-4]. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA [5, 6]. Here, we report expansion of the zebrafish genetic code to incorporate the UNAAs, Azido-lysine (AzK), bicyclononyne-lysine (BCNK), and Diazirine-lysine (AbK) into green fluorescent protein (GFP) and Glutathione-S-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of UNAA mutagenesis to investigate and engineer protein function in zebrafish.

scholarly journals Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jugal Mohapatra ◽  
Kyuto Tashiro ◽  
Ryan L Beckner ◽  
Jorge Sierra ◽  
Jessica A Kilgore ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Protein Function ◽  
Post Translational Modification ◽  
Site Specific ◽  
Precise Control ◽  
Protein Ligation ◽  
Adp Ribosylation ◽  
Nuclear Extracts ◽  
Synthesis Strategy ◽  
The Impact

Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto peptides with precise control over chain length and modification site. Importantly, the enzymatically mono- and poly-ADP-ribosylated peptides are fully compatible with protein ligation technologies. This chemoenzymatic protein synthesis strategy was employed to assemble a series of full-length, ADP-ribosylated histones and show that ADPr at H2BS6 or H3S10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. We found ALC1 preferentially remodels 'activated' substrates within heterogeneous mononucleosome populations and asymmetrically ADP-ribosylated dinucleosome substrates, and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a new, highly modular approach to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.

scholarly journals Salicylaldehyde ester-mediated protein semi-synthesis enables studies on the tetra-acetylation of HMGB1

2021 ◽  
Author(s):  
Tongyao Wei ◽  
Jiamei Liu ◽  
Yi Tan ◽  
Ruohan Wei ◽  
Jinzheng Wang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
High Purity ◽  
Protein Function ◽  
Protein A ◽  
High Mobility ◽  
High Mobility Group ◽  
Biological Study ◽  
Post Translational Modifications ◽  
Site Specific ◽  
Synthesis Strategy

To answer how protein post-translational modifications (PTMs) affect protein function, conformation, sta-bility, localization and interaction with binders remains important in the biological study. However, the re-lated study has been dramatically hindered by the difficulty in obtaining homogenous proteins with site-specific PTMs of interest. Herein, we introduce a protein semi-synthesis strategy via salicylaldehyde ester-mediated chemical ligations (Ser/Thr ligation and Cys/Pen ligation). This methodology has enabled us to generate Lys (2/6/7/11) tetra-acetylated HMGB1 (high-mobility group box 1) protein, a 25 kDa proin-flammatory protein, in high purity. Further studies revealed that the tetra-acetylation may represent a regu-latory switch to control the HMGB1 signaling pathway by abolishing its interaction with lipopolysaccha-ride (LPS) and accelerating its degradation, consequently preventing cells from pyroptosis and lethality upon infectious injury.

scholarly journals Glycoproteomic measurement of site-specific polysialylation

2019 ◽  
Author(s):  
Ruby Pelingon ◽  
Cassandra L. Pegg ◽  
Lucia F. Zacchi ◽  
Toan K. Phung ◽  
Christopher B. Howard ◽  
...  
Keyword(s):  
Protein Function ◽  
Neurological Diseases ◽  
Polysialic Acid ◽  
Monosaccharide Composition ◽  
Degree Of Polymerization ◽  
Site Specific ◽  
Esi Ms ◽  
Therapeutic Drugs ◽  
Protease Digestion ◽  
Analytical Approaches

AbstractPolysialylation is the enzymatic addition of a highly negatively charged sialic acid polymer to the non-reducing termini of glycans. Polysialylation plays an important role in development, and is involved in neurological diseases, neural tissue regeneration, and cancer. Polysialic acid (PSA) is also a biodegradable and non-immunogenic conjugate to therapeutic drugs to improve their pharmacokinetics. PSA chains vary in length, composition, and linkages, while the specific sites of polysialylation are important determinants of protein function. However, PSA is difficult to analyse by mass spectrometry (MS) due to its high negative charge and size. Most analytical approaches for analysis of PSA measure its degree of polymerization and monosaccharide composition, but do not address the key questions of site specificity and occupancy. Here, we developed a high-throughput LC-ESI-MS/MS glycoproteomics method to measure site-specific polysialylation of glycoproteins. This method measures site-specific PSA modification by using mild acid hydrolysis to eliminate PSA and sialic acids while leaving the glycan backbone intact, together with protease digestion followed by LC-ESI-MS/MS glycopeptide detection. PSA-modified glycopeptides are not detectable by LC-ESI-MS/MS, but become detectable after desialylation, allowing measurement of site-specific PSA occupancy. This method is an efficient analytical workflow for the study of glycoprotein polysialylation in biological and therapeutic settings.Graphical Abstract

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