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

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.

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System-wide identification and prioritization of enzyme substrates by thermal analysis (SIESTA)

10.1101/423418 ◽

2018 ◽

Cited By ~ 3

Author(s):

Amir Ata Saei ◽

Christian M. Beusch ◽

Pierre Sabatier ◽

Juan Astorga Wells ◽

Alexey Chernobrovkin ◽

...

Keyword(s):

Thermal Stability ◽

Thermal Analysis ◽

Protein Kinase B ◽

Thioredoxin Reductase ◽

Post Translational Modification ◽

Enzyme Substrate ◽

Thioredoxin Reductase 1 ◽

Enzyme Substrates ◽

Substrate Proteins

AbstractDespite the immense importance of enzyme-substrate reactions, there is a lack of generic and unbiased tools for identifying and prioritizing substrate proteins which are modulated in the structural and functional levels through modification. Here we describe a high-throughput unbiased proteomic method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that enzymatic post-translational modification of substrate proteins might change their thermal stability. 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 in up to a depth of 7179 proteins. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, open new opportunities in investigating the effect of PTMs on signal transduction, and facilitate drug discovery.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22010323 ◽

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.

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

Biochemical Journal ◽

10.1042/bj20121632 ◽

2013 ◽

Vol 450 (3) ◽

pp. 433-442 ◽

Cited By ~ 8

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.

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Role of thioredoxin reductase 1 in dysplastic transformation of human breast epithelial cells triggered by chronic oxidative stress

Scientific Reports ◽

10.1038/srep36860 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 19

Author(s):

Chaoran Dong ◽

Lei Zhang ◽

Ruoxuan Sun ◽

Jianying Liu ◽

Hanwei Yin ◽

...

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Thioredoxin Reductase ◽

Human Breast ◽

Breast Epithelial Cells ◽

Chronic Oxidative Stress ◽

Thioredoxin Reductase 1 ◽

Breast Epithelial ◽

Human Breast Epithelial Cells

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The role of thioredoxin reductase 1 in cancer

Global Advances in Selenium Research from Theory to Application ◽

10.1201/b19240-31 ◽

2015 ◽

pp. 59-60

Author(s):

E Arnér

Keyword(s):

Thioredoxin Reductase ◽

Thioredoxin Reductase 1

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FOXO3a is stabilized by USP18-mediated de-ISGylation and inhibits TGF-β1-induced fibronectin expression

Journal of Investigative Medicine ◽

10.1136/jim-2019-001145 ◽

2019 ◽

Vol 68 (3) ◽

pp. 786-791 ◽

Cited By ~ 2

Author(s):

Ban Wang ◽

Yanhui Li ◽

Heather Wang ◽

Jing Zhao ◽

Yutong Zhao ◽

...

Keyword(s):

Half Life ◽

Transforming Growth Factor ◽

Fibroblast Cells ◽

Post Translational Modification ◽

Human Lung Fibroblast ◽

Post Translational Modifications ◽

Cellular Processes ◽

Fibronectin Expression ◽

Tgf Β1

FOXO3a belongs to a family of transcription factors characterized by a conserved forkhead box DNA-binding domain. It has been known to regulate various cellular processes including cell proliferation, apoptosis and differentiation. Post-translational modifications of FOXO3a and their roles in the regulation of FOXO3a activity have been well-documented. FOXO3a can be phosphorylated, acetylated and ubiquitinated, however, the ISGylation of FOXO3a has not been reported. Protein overexpression, ISGylation and half-life were measured to determine the post-translational modification of FOXO3a. Human fibroblast cells were treated with transforming growth factor (TGF)-β1 to determine the role of FOXO3a ISGylation in TGF-β1 signaling. FOXO3a’s half-life is around 3.7 hours. Inhibition of the proteasome, not lysosome, extends its half-life. ISGylation, but not ubiquitination of FOXO3a, is increased in the presence of the proteasome inhibitor. Overexpression of ISG15 increases FOXO3a degradation, while overexpression of USP18 stabilizes FOXO3a through de-ISGylation. These results suggest that FOXO3a is degraded in the ISGylation and proteasome system, which can be reversed by USP18, an ISG15-specific deubiquitinase. This study reveals a new molecular mechanism by which ISGylation regulates FOXO3a degradation. Furthermore, we show that the overexpression of FOXO3a attenuated TGF-β1-induced fibronectin expression in human lung fibroblast cells without altering Smad2/3 expression and activation. FOXO3a can be ISGylated, which can regulate FOXO3a stability. USP18/FOXO3a pathway is a potential target for treating TGF-β1-mediated fibrotic diseases such as idiopathic pulmonary fibrosis.

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Post-Translational Modification-Dependent Activity of Matrix Metalloproteinases

International Journal of Molecular Sciences ◽

10.3390/ijms20123077 ◽

2019 ◽

Vol 20 (12) ◽

pp. 3077 ◽

Cited By ~ 10

Author(s):

Elizabeta Madzharova ◽

Philipp Kastl ◽

Fabio Sabino ◽

Ulrich auf dem Keller

Keyword(s):

Matrix Metalloproteinases ◽

Transcriptional Control ◽

Proteolytic Processing ◽

Post Translational Modification ◽

Proteolytic Activation ◽

Control Of Gene Expression ◽

Post Translational Modifications ◽

Binding Partners ◽

Secreted Proteases

Due to their capacity to process different proteins of the extracellular matrix (ECM), matrix metalloproteinases (MMPs) were initially described as a family of secreted proteases, functioning as main ECM regulators. However, through proteolytic processing of various biomolecules, MMPs also modulate intra- and extracellular pathways and networks. Thereby, they are functionally implicated in the regulation of multiple physiological and pathological processes. Consequently, MMP activity is tightly regulated through a combination of epigenetic, transcriptional, and post-transcriptional control of gene expression, proteolytic activation, post-translational modifications (PTMs), and extracellular inhibition. In addition, MMPs, their substrates and ECM binding partners are frequently modified by PTMs, which suggests an important role of PTMs in modulating the pleiotropic activities of these proteases. This review summarizes the recent progress towards understanding the role of PTMs (glycosylation, phosphorylation, glycosaminoglycans) on the activity of several members of the MMP family.

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The Yin and Yang of Nrf2-Regulated Selenoproteins in Carcinogenesis

International Journal of Cell Biology ◽

10.1155/2012/486147 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 45

Author(s):

Regina Brigelius-Flohé ◽

Mike Müller ◽

Doris Lippmann ◽

Anna Patricia Kipp

Keyword(s):

Cancer Cells ◽

Thioredoxin Reductase ◽

Selenium Deficiency ◽

Related Factor ◽

Cellular Defense ◽

Thioredoxin Reductase 1 ◽

Cancer Initiation ◽

Yin And Yang ◽

Support Tumor Growth

The NF-E2-related factor-2 (Nrf2) is a transcription factor which regulates the major cellular defense systems and thereby contributes to the prevention of many diseases including cancer. Selenium deficiency is associated with a higher cancer risk making also this essential trace element a promising candidate for cancer prevention. Two selenoproteins, thioredoxin reductase-1 (TrxR1) and glutathione peroxidase-2 (GPx2), are targets for Nrf2. Selenium deficiency activates Nrf2 as does a TrxR1 knockout making a synergism between both systems plausible. Although this might hold true for healthy cells, the interplay may turn into the opposite in cancer cells. The induction of the detoxifying and antioxidant enzymes by Nrf2 will make cancer cells chemoresistant and will protect them against oxidative damage. The essential role of TrxR1 in maintaining proliferation makes its upregulation in cancer cells detrimental. The anti-inflammatory potential of GPx2 will help to inhibit cancer initiation and inflammation-triggered promotion, but its growth supporting potential will also support tumor growth. This paper considers beneficial and adverse consequences of the activation of Nrf2 and the selenoproteins which appear to depend on the cancer stage.

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