scholarly journals Burkholderia PglL enzymes are Serine preferring oligosaccharyltransferases which target conserved proteins across the Burkholderia genus

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Andrew J. Hayes ◽  
Jessica M. Lewis ◽  
Mark R. Davies ◽  
Nichollas E. Scott
Keyword(s):  
Protein Modification ◽  
High Confidence ◽  
Protein Substrates ◽  
Glycosylation Sites ◽  
Bacterial Proteomes ◽  
Made In

AbstractGlycosylation is increasingly recognised as a common protein modification within bacterial proteomes. While great strides have been made in identifying species that contain glycosylation systems, our understanding of the proteins and sites targeted by these systems is far more limited. Within this work we explore the conservation of glycoproteins and glycosylation sites across the pan-Burkholderia glycoproteome. Using a multi-protease glycoproteomic approach, we generate high-confidence glycoproteomes in two widely utilized B. cenocepacia strains, K56-2 and H111. This resource reveals glycosylation occurs exclusively at Serine residues and that glycoproteins/glycosylation sites are highly conserved across B. cenocepacia isolates. This preference for glycosylation at Serine residues is observed across at least 9 Burkholderia glycoproteomes, supporting that Serine is the dominant residue targeted by PglL-mediated glycosylation across the Burkholderia genus. Combined, this work demonstrates that PglL enzymes of the Burkholderia genus are Serine-preferring oligosaccharyltransferases that target conserved and shared protein substrates.

scholarly journals Burkholderia PglL enzymes are serine preferring oligosaccharidetransferases which target conserved proteins across the Burkholderia genus

2021 ◽  
Author(s):  
Andrew J. Hayes ◽  
Jessica M. Lewis ◽  
Mark R. Davies ◽  
Nichollas E. Scott
Keyword(s):  
Protein Modification ◽  
Population Genomics ◽  
High Confidence ◽  
Dramatic Decrease ◽  
Protein Substrates ◽  
Glycosylation Sites ◽  
Model Protein ◽  
Bacterial Proteomes ◽  
Made In ◽  
Glycosylated Proteins

AbstractGlycosylation is increasingly recognised as a common protein modification within bacterial proteomes. While great strides have been made in identifying species that contain glycosylation systems, our understanding of the proteins and sites targeted by these enzymes is far more limited. Within this work we explore the conservation of glycoproteins and O-linked glycosylation sites across the pan-Burkholderia glycoproteome. Using a multi-protease glycoproteomic approach we generate high-confidence glycoproteomes and associated glycosylation sites in two widely utilized B. cenocepacia strains, K56-2 and H111. This resource reveals glycosylation occurs exclusively at serine residues and that glycoproteins/glycosylation sites are highly conserved across 294 publicly available B. cenocepacia genomes. Consistent with this we demonstrate that the substitution of Serine for Threonine residues in a model protein results in a dramatic decrease in glycosylation efficiency by the oligosaccharidetransferase pglLBC even when pglLBC is overexpressed. This preference for glycosylation at Serine residues is observed across at least 9 Burkholderia glycoproteomes supporting that Serine is the dominant residue targeted by pglL-mediated glycosylation across the Burkholderia genus. Using population genomics we observe that pglL targeted glycosylated proteins are common across Burkholderia species. Combined, this work demonstrates that PglL enzymes of the Burkholderia genus are Serine-preferring oligosaccharidetransferases that target conserved and shared protein substrates across the Burkholderia genus.

scholarly journals Targeting ubiquitin signaling for cancer immunotherapy

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Xiaofei Zhou ◽  
Shao-Cong Sun
Keyword(s):  
Cancer Immunotherapy ◽  
Immune Cells ◽  
Protein Modification ◽  
Antitumor Immunity ◽  
Checkpoint Inhibitors ◽  
Therapeutic Approaches ◽  
Protein Ubiquitination ◽  
Advanced Malignancies ◽  
Novel Targets ◽  
Made In

AbstractCancer immunotherapy has become an attractive approach of cancer treatment with tremendous success in treating various advanced malignancies. The development and clinical application of immune checkpoint inhibitors represent one of the most extraordinary accomplishments in cancer immunotherapy. In addition, considerable progress is being made in understanding the mechanism of antitumor immunity and characterizing novel targets for developing additional therapeutic approaches. One active area of investigation is protein ubiquitination, a post-translational mechanism of protein modification that regulates the function of diverse immune cells in antitumor immunity. Accumulating studies suggest that E3 ubiquitin ligases and deubiquitinases form a family of potential targets to be exploited for enhancing antitumor immunity in cancer immunotherapy.

scholarly journals ProMetheusDB: an in-depth analysis of the high-quality human methyl-proteome

2021 ◽  
Author(s):  
Enrico Massignani ◽  
Roberto Giambruno ◽  
Marianna Maniaci ◽  
Luciano Nicosia ◽  
Avinash Yadav ◽  
...  
Keyword(s):  
Histone Methylation ◽  
Rna Splicing ◽  
Biological Processes ◽  
Response Signal ◽  
Post Translational Modification ◽  
High Confidence ◽  
High Quality ◽  
False Discovery ◽  
Depth Analysis ◽  
Made In

Protein Arginine (R) methylation is a post-translational modification involved in various biological processes, such as RNA splicing, DNA repair, immune response, signal transduction, and tumour development. Although several advancements were made in the study of this modification by mass spectrometry, researchers still face the problem of a high false discovery rate. We present a dataset of high-quality methylations obtained from several different heavy methyl SILAC (hmSILAC) experiments analysed with a machine learning-based tool doublets and show that this model allows for improved high-confidence identification of real methyl-peptides. Overall, our results are consistent with the notion that protein R methylation modulates protein:RNA interactions and suggest a role in rewiring protein:protein interactions, for which we provide experimental evidence for a representative case (i.e. NONO:PSPC1). Upon intersecting our R-methyl-sites dataset with a phosphosites dataset, we observed that R methylation correlates differently with S/T-Y phosphorylation in response to various stimuli. Finally, we explored the application of hmSILAC to identify unconventional methylated residues and successfully identified novel histone methylation marks on Serine 28 and Threonine 32 of H3.

scholarly journals Protein substrates engage the lumen of O-GlcNac transferase’s tetratricopeptide repeat domain in different ways

2020 ◽  
Author(s):  
Cassandra M. Joiner ◽  
Forrest A. Hammel ◽  
John Janetzko ◽  
Suzanne Walker
Keyword(s):  
Tetratricopeptide Repeat ◽  
Substrate Selection ◽  
Post Translational Modification ◽  
Protein Substrates ◽  
Cytoplasmic Proteins ◽  
C Terminus ◽  
Glycosylation Sites ◽  
Repeat Domain ◽  
Tetratricopeptide Repeat Domain ◽  
Glcnac Transferase

ABSTRACTGlycosylation of nuclear and cytoplasmic proteins is an essential post-translational modification in mammals. O-GlcNAc transferase (OGT), the sole enzyme responsible for this modification, glycosylates over a thousand unique nuclear and cytoplasmic substrates. How OGT selects its substrates is a fundamental question that must be answered to understand OGT’s unusual biology. OGT contains a long tetratricopeptide repeat (TPR) domain that has been implicated in substrate selection, but there is almost no information about how changes to this domain affect glycosylation of individual substrates. Here, we used proteome-wide glycosylation profiling and probed glycosylation of selected purified substrates to show that asparagine and aspartate ladders that extend the full length of OGT’s TPR lumen control substrate glycosylation. We also found that substrates with glycosylation sites close to the C-terminus bypass lumenal binding. Our findings demonstrate that substrates can engage OGT in a variety of different ways for glycosylation.

scholarly journals Too good to be true: when overwhelming evidence fails to convince

2016 ◽  
Vol 472 (2187) ◽  
pp. 20150748 ◽  
Author(s):  
Lachlan J. Gunn ◽  
François Chapeau-Blondeau ◽  
Mark D. McDonnell ◽  
Bruce R. Davis ◽  
Andrew Allison ◽  
...  
Keyword(s):  
False Negative ◽  
False Negative Rate ◽  
Error Rates ◽  
Small Error ◽  
High Confidence ◽  
Primality Testing ◽  
Legal Evidence ◽  
Numerical Tests ◽  
Overwhelming Evidence ◽  
Made In

Is it possible for a large sequence of measurements or observations, which support a hypothesis, to counterintuitively decrease our confidence? Can unanimous support be too good to be true? The assumption of independence is often made in good faith; however, rarely is consideration given to whether a systemic failure has occurred. Taking this into account can cause certainty in a hypothesis to decrease as the evidence for it becomes apparently stronger. We perform a probabilistic Bayesian analysis of this effect with examples based on (i) archaeological evidence, (ii) weighing of legal evidence and (iii) cryptographic primality testing. In this paper, we investigate the effects of small error rates in a set of measurements or observations. We find that even with very low systemic failure rates, high confidence is surprisingly difficult to achieve; in particular, we find that certain analyses of cryptographically important numerical tests are highly optimistic, underestimating their false-negative rate by as much as a factor of 2 80 .

scholarly journals N-glycoproteomic analysis of Ginsenoside Rb1 on a hyperlipidemia rat model

2021 ◽  
Author(s):  
Yixin Ma ◽  
Shunyu Ning ◽  
Nan Song ◽  
Si Chen ◽  
Xue Leng ◽  
...  
Keyword(s):  
High Fat Diet ◽  
Protein Modification ◽  
Ion Homeostasis ◽  
Enrichment Analysis ◽  
Control Group ◽  
Pathway Enrichment Analysis ◽  
Hypolipidemic Effect ◽  
Ginsenoside Rb1 ◽  
High Fat ◽  
Glycosylation Sites

Abstract Background: Ginsenoside Rb1, known as Renshen in traditional Chinese medicine, is one of the major bioactive saponins isolated from Panax ginseng C.A.Mey. N-glycosylation is the most common type of post-translational modification in cells. The widespread localization of N-glycosylated proteins (N-glycoproteins) between extracellular spaces and on the cell surfaces give them unique advantages as disease biomarkers and drug targets. Previous study found that Ginsenoside Rb1 could potentially play a preventive role in hyperlipidemia. This study aims to reveal the hypolipidemic effect at the protein modification level. Methods: 24 male SD rats were randomly devided into 3 groups: control group (CON), high fat diet group (HFD) and Ginsenoside Rb1 group (Rb1). Both HFD and Rb1 groups were fed with high-fat diet for 12 weeks. The Rb1 group started intragastric administering Ginsenoside Rb1 200 mg·kg -1 ·d -1 at 5th week for 8 weeks, while the CON and HFD group the same amount of normal saline for the same amount of time. Lipid levels and liver histology were assayed to evaluate the effects of Ginsenoside Rb1 intake on hyperlipidemia rats. Furthermore, the workflow by combination of isotope TMT labeling, HILIC enrichment, and high-resolution LC-MS/MS analysis were employed to exploring the mechanisms of regulation role in hyperlipidemia rats.Results: The histopathologic characteristics and biochemical data shows that Ginsenoside Rb1 exhibited regulative effects on hyperlipidemia rats. After being analyzed by N-glycoproteomic, 98 differential N-glycosylation sites on 53 glycoproteins between 2 comparison groups (HFD: CON, Rb1: HFD) were identified. Analyses of N-glycosylation sites distribution found that albumin (Alb) and Serpinc1 were most heavily modified with 6 N-glycosylation sites changed in this work. GO enrichment analysis showed that differential modified glycoproteins were involved in inflammatory response, cellular iron ion homeostasis and positive regulation of cholesterol efflux etc. biosynthetic process. Complement and coagulation cascades was the most significant enriched in the KEGG pathway enrichment analysis. Conclusions: This study presents a comprehensive analysis of a new set of N-glycoproteins which are altered by Ginsenoside Rb1 and offers some valuable clues for novel mechanistic insights into the ragulative mechanism of Ginsenoside Rb1. Results from N-glycoproteomic suggest that to suppress hyperlipidemia, Rb1 may regulates N-glycosylation of Alb, Serpinc1, PON1, Lrp1, Cp and THBS1, as well as differentially modified glycoproteins in complement and coagulation cascades, which in turn improve the imbalance of lipid homeostasis.

N-terminal acetylation and other functions of Nα-acetyltransferases

2012 ◽  
Vol 393 (4) ◽  
pp. 291-298 ◽  
Author(s):  
Jolien Hollebeke ◽  
Petra Van Damme ◽  
Kris Gevaert
Keyword(s):  
Protein Modification ◽  
Recent Progress ◽  
Biological Role ◽  
Protein Acetylation ◽  
Terminal Protein ◽  
Made In

Abstract Protein N-terminal acetylation by Nα-acetyltransferases (NATs) is an omnipresent protein modification that affects a large number of proteins. The exact biological role of N-terminal acetylation has, however, remained enigmatic for the overall majority of affected proteins, and only for a rather small number of proteins, N-terminal acetylation was linked to various protein features including stability, localization, and interactions. This minireview tries to summarize the recent progress made in understanding the functionality of N-terminal protein acetylation and also focuses on noncanonical functions of the NATs subunits.

scholarly journals N-Terminal Acetyltransferases Are Cancer-Essential Genes Prevalently Upregulated in Tumours

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2631
Author(s):  
Costas Koufaris ◽  
Antonis Kirmizis
Keyword(s):  
Cancer Biology ◽  
Protein Modification ◽  
Patient Survival ◽  
Therapeutic Potential ◽  
Biological Processes ◽  
Auxiliary Subunits ◽  
Protein Substrates ◽  
Altered Gene Expression ◽  
Altered Gene ◽  
Omic Data

N-terminal acetylation (Nt-Ac) is an abundant eukaryotic protein modification, deposited in humans by one of seven N-terminal acetyltransferase (NAT) complexes composed of a catalytic and potentially auxiliary subunits. The involvement of NATs in cancers is being increasingly recognised, but a systematic cross-tumour assessment is currently lacking. To address this limitation, we conducted here a multi-omic data interrogation for NATs. We found that tumour genomic alterations of NATs or of their protein substrates are generally rare events, with some tumour-specific exceptions. In contrast, altered gene expression of NATs in cancers and their association with patient survival constitute a widespread cancer phenomenon. Examination of dependency screens revealed that (i), besides NAA60 and NAA80 and the NatA paralogues NAA11 and NAA16, the other ten NAT genes were within the top 80th percentile of the most dependent genes (ii); NATs act through distinct biological processes. NAA40 (NatD) emerged as a NAT with particularly interesting cancer biology and therapeutic potential, especially in liver cancer where a novel oncogenic role was supported by its increased expression in multiple studies and its association with patient survival. In conclusion, this study generated insights and data that will be of great assistance in guiding further research into the function and therapeutic potential of NATs in cancer.

scholarly journals SugarQuant: a streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

2020 ◽  
Author(s):  
Pan Fang ◽  
Yanlong Ji ◽  
Ivan Silbern ◽  
Carmen Doebele ◽  
Momchil Ninov ◽  
...  
Keyword(s):  
Database Search ◽  
High Confidence ◽  
Site Specific ◽  
Glycosylation Sites ◽  
Data Processing Tool ◽  
A Cell ◽  
One Step ◽  
Global Identification ◽  
Specific Regulation ◽  
Accurate Quantification

AbstractSite-specific regulation of protein N-glycosylation is essential in human cells. However, accurate quantification of glycosylation sites and their individual glycan moieties in a cell-wide manner is still technically challenging. Here, we introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising fast protein aggregation capture (PAC)-based sample preparation, optimized multi-notch MS3 LC-MS acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that allows for confident, global identification and quantification of intact glycopeptides in complex biological samples. PAC greatly reduces the overall samplehandling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and significantly lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search which increases the identification rates of intact glycopeptides by up to 22% when compared with one-step database search strategies. SugarQuant was applied to identify and quantify more than 5,000 unique glycoforms in Burkitt’s lymphoma cells, and determined complex site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence.

scholarly journals An Ultra-High-Throughput Screen for Catalytic Inhibitors of Serine/Threonine Protein Phosphatases Types 1 and 5 (PP1C and PP5C)

2016 ◽  
Vol 22 (1) ◽  
pp. 21-31 ◽  
Author(s):  
Mark Swingle ◽  
Claude-Henry Volmar ◽  
S. Adrian Saldanha ◽  
Peter Chase ◽  
Christina Eberhart ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Phosphatase ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Chemical Probes ◽  
Enzymatic Assays ◽  
Protein Substrates ◽  
Specific Inhibitors ◽  
Made In

Although there has been substantial success in the development of specific inhibitors for protein kinases, little progress has been made in the identification of specific inhibitors for their protein phosphatase counterparts. Inhibitors of PP1 and PP5 are desired as probes for research and to test their potential for drug development. We developed and miniaturized (1536-well plate format) nearly identical homogeneous, fluorescence intensity (FLINT) enzymatic assays to detect inhibitors of PP1 or PP5. The assays were used in an ultra-high-throughput screening (uHTS) campaign, testing >315,000 small-molecule compounds. Both assays demonstrated robust performance, with a Z′ of 0.92 ± 0.03 and 0.95 ± 0.01 for the PP1 and PP5 assays, respectively. Screening the same library with both assays aided the identification of class inhibitors and assay artifacts. Confirmation screening and hit prioritization assays used [32P/33P]-radiolabel protein substrates, revealing excellent agreement between the FLINT and radiolabel assays. This screening campaign led to the discovery of four novel unrelated small-molecule inhibitors of PP1 and ~30 related small-molecule inhibitors of PP5. The results suggest that this uHTS approach is suitable for identifying selective chemical probes that inhibit PP1 or PP5 activity, and it is likely that similar assays can be developed for other PPP-family phosphatases.

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