scholarly journals Protein CoAlation: a redox-regulated protein modification by coenzyme A in mammalian cells

2017 ◽  
Vol 474 (14) ◽  
pp. 2489-2508 ◽  
Author(s):  
Yugo Tsuchiya ◽  
Sew Yeu Peak-Chew ◽  
Clare Newell ◽  
Sheritta Miller-Aidoo ◽  
Sriyash Mangal ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protein Modification ◽  
Redox Regulation ◽  
Coenzyme A ◽  
Regulation Of Gene Expression ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
Wide Range

Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated in vitro and in vivo in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions.

scholarly journals Coenzyme A, protein CoAlation and redox regulation in mammalian cells

2018 ◽  
Vol 46 (3) ◽  
pp. 721-728 ◽  
Author(s):  
Ivan Gout
Keyword(s):  
Mammalian Cells ◽  
Redox Regulation ◽  
Coenzyme A ◽  
Regulation Of Gene Expression ◽  
Metabolic Stress ◽  
Thiol Group ◽  
Covalent Attachment ◽  
Current Status ◽  
Experimental Conditions ◽  
Diverse Range

In a diverse family of cellular cofactors, coenzyme A (CoA) has a unique design to function in various biochemical processes. The presence of a highly reactive thiol group and a nucleotide moiety offers a diversity of chemical reactions and regulatory interactions. CoA employs them to activate carbonyl-containing molecules and to produce various thioester derivatives (e.g. acetyl CoA, malonyl CoA and 3-hydroxy-3-methylglutaryl CoA), which have well-established roles in cellular metabolism, production of neurotransmitters and the regulation of gene expression. A novel unconventional function of CoA in redox regulation, involving covalent attachment of this coenzyme to cellular proteins in response to oxidative and metabolic stress, has been recently discovered and termed protein CoAlation (S-thiolation by CoA or CoAthiolation). A diverse range of proteins was found to be CoAlated in mammalian cells and tissues under various experimental conditions. Protein CoAlation alters the molecular mass, charge and activity of modified proteins, and prevents them from irreversible sulfhydryl overoxidation. This review highlights the role of a key metabolic integrator CoA in redox regulation in mammalian cells and provides a perspective of the current status and future directions of the emerging field of protein CoAlation.

scholarly journals Protein CoAlation and antioxidant function of coenzyme A in prokaryotic cells

2018 ◽  
Vol 475 (11) ◽  
pp. 1909-1937 ◽  
Author(s):  
Yugo Tsuchiya ◽  
Alexander Zhyvoloup ◽  
Jovana Baković ◽  
Naam Thomas ◽  
Bess Yi Kun Yu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Redox Regulation ◽  
Coenzyme A ◽  
Regulation Of Gene Expression ◽  
Pantothenic Acid ◽  
Metabolic Stress ◽  
Background Level ◽  
Catalytic Cysteine ◽  
Living Organisms

In all living organisms, coenzyme A (CoA) is an essential cofactor with a unique design allowing it to function as an acyl group carrier and a carbonyl-activating group in diverse biochemical reactions. It is synthesized in a highly conserved process in prokaryotes and eukaryotes that requires pantothenic acid (vitamin B5), cysteine and ATP. CoA and its thioester derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. A novel unconventional function of CoA in redox regulation has been recently discovered in mammalian cells and termed protein CoAlation. Here, we report for the first time that protein CoAlation occurs at a background level in exponentially growing bacteria and is strongly induced in response to oxidizing agents and metabolic stress. Over 12% of Staphylococcus aureus gene products were shown to be CoAlated in response to diamide-induced stress. In vitro CoAlation of S. aureus glyceraldehyde-3-phosphate dehydrogenase was found to inhibit its enzymatic activity and to protect the catalytic cysteine 151 from overoxidation by hydrogen peroxide. These findings suggest that in exponentially growing bacteria, CoA functions to generate metabolically active thioesters, while it also has the potential to act as a low-molecular-weight antioxidant in response to oxidative and metabolic stress.

scholarly journals Extensive Anti-CoA Immunostaining in Alzheimer’s Disease and Covalent Modification of Tau by a Key Cellular Metabolite Coenzyme A

2021 ◽  
Vol 15 ◽  
Author(s):  
Tammaryn Lashley ◽  
Maria-Armineh Tossounian ◽  
Neve Costello Heaven ◽  
Samantha Wallworth ◽  
Sew Peak-Chew ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Neurofibrillary Tangles ◽  
Cellular Response ◽  
Protein Modification ◽  
Coenzyme A ◽  
Neurodegenerative Disorder ◽  
Regulation Of Gene Expression ◽  
Covalent Modification

Alzheimer’s disease (AD) is a neurodegenerative disorder, accounting for at least two-thirds of dementia cases. A combination of genetic, epigenetic and environmental triggers is widely accepted to be responsible for the onset and development of AD. Accumulating evidence shows that oxidative stress and dysregulation of energy metabolism play an important role in AD pathogenesis, leading to neuronal dysfunction and death. Redox-induced protein modifications have been reported in the brain of AD patients, indicating excessive oxidative damage. Coenzyme A (CoA) is essential for diverse metabolic pathways, regulation of gene expression and biosynthesis of neurotransmitters. Dysregulation of CoA biosynthesis in animal models and inborn mutations in human genes involved in the CoA biosynthetic pathway have been associated with neurodegeneration. Recent studies have uncovered the antioxidant function of CoA, involving covalent protein modification by this cofactor (CoAlation) in cellular response to oxidative or metabolic stress. Protein CoAlation has been shown to both modulate the activity of modified proteins and protect cysteine residues from irreversible overoxidation. In this study, immunohistochemistry analysis with highly specific anti-CoA monoclonal antibody was used to reveal protein CoAlation across numerous neurodegenerative diseases, which appeared particularly frequent in AD. Furthermore, protein CoAlation consistently co-localized with tau-positive neurofibrillary tangles, underpinning one of the key pathological hallmarks of AD. Double immunihistochemical staining with tau and CoA antibodies in AD brain tissue revealed co-localization of the two immunoreactive signals. Further, recombinant 2N3R and 2N4R tau isoforms were found to be CoAlated in vitro and the site of CoAlation mapped by mass spectrometry to conserved cysteine 322, located in the microtubule binding region. We also report the reversible H2O2-induced dimerization of recombinant 2N3R, which is inhibited by CoAlation. Moreover, CoAlation of transiently expressed 2N4R tau was observed in diamide-treated HEK293/Pank1β cells. Taken together, this study demonstrates for the first time extensive anti-CoA immunoreactivity in AD brain samples, which occurs in structures resembling neurofibrillary tangles and neuropil threads. Covalent modification of recombinant tau at cysteine 322 suggests that CoAlation may play an important role in protecting redox-sensitive tau cysteine from irreversible overoxidation and may modulate its acetyltransferase activity and functional interactions.

scholarly journals Bacterial flavin homeostasis: the role of an FAD pyrophosphatase/FMN transferase

2014 ◽  
Vol 70 (a1) ◽  
pp. C311-C311
Author(s):  
Diana Tomchick ◽  
Ranjit Deka ◽  
Chad Brautigam ◽  
Wei Liu ◽  
Michael Norgard
Keyword(s):  
Pathogenic Bacteria ◽  
Treponema Pallidum ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
New Paradigm ◽  
Uptake Mechanism ◽  
Structural Investigations

Treponema pallidum, an obligate parasite of humans and the causative agent of syphilis, has evolved the capacity to exploit host-derived metabolites for its survival. Flavin-containing compounds are essential cofactors that are required for metabolic processes in all living organisms, and riboflavin is a direct precursor of the cofactors FMN and FAD. Unlike many pathogenic bacteria, Treponema pallidum cannot synthesize riboflavin; we recently described a flavin-uptake mechanism composed of an ABC-type transporter [1]. However, there is a paucity of information about flavin utilization in bacterial periplasms. We have identified the TP0796 lipoprotein as a previously uncharacterized Mg2+-dependent FAD pyrophosphatase/FMN transferase within the ApbE superfamily [2,3]. Biochemical and structural investigations revealed that the enzyme has a unique bimetal Mg2+ catalytic center. Furthermore, the pyrophosphatase activity is product-inhibited by AMP, indicating a possible role for this molecule in modulating FMN and FAD levels in the treponemal periplasm. The ApbE superfamily was previously thought to be involved in thiamine biosynthesis, but our characterization of TP0796 prompts a renaming of this superfamily as a periplasmic flavin-trafficking protein (Ftp). Treponemal Ftp (Ftp_Tp) is the first structurally and biochemically characterized metal-dependent FAD pyrophosphatase/FMN transferase in bacteria. We have shown in vitro and in vivo that Ftps from several types of pathogenic bacteria are capable of flavinylating proteins through covalent attachment of FMN via a phosphoester bond to threonine residues of an appropriate sequence signature. Progress on the structural characterization of a product of this post-translational modification will be presented. This new paradigm for a bacterial flavin utilization pathway may prove to be useful for future inhibitor design.

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 Isoaspartyl Post-translational Modification Triggers Anti-tumor T and B Lymphocyte Immunity

2006 ◽  
Vol 281 (43) ◽  
pp. 32676-32683 ◽  
Author(s):  
Hester A. Doyle ◽  
Jing Zhou ◽  
Martin J. Wolff ◽  
Bohdan P. Harvey ◽  
Robert M. Roman ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Tolerance ◽  
Protein Modification ◽  
B Lymphocyte ◽  
Post Translational Modification ◽  
B Cell Tolerance ◽  
Melanoma Antigen ◽  
Self Antigens

A hallmark of the immune system is the ability to ignore self-antigens. In attempts to bypass normal immune tolerance, a post-translational protein modification was introduced into self-antigens to break T and B cell tolerance. We demonstrate that immune tolerance is bypassed by immunization with a post-translationally modified melanoma antigen. In particular, the conversion of an aspartic acid to an isoaspartic acid within the melanoma antigen tyrosinase-related protein (TRP)-2 peptide-(181-188) makes the otherwise immunologically ignored TRP-2 antigen immunogenic. Tetramer analysis of iso-Asp TRP-2 peptide-immunized mice demonstrated that CD8+ T cells not only recognized the isoaspartyl TRP-2 peptide but also the native TRP-2 peptide. These CD8+ T cells functioned as cytotoxic T lymphocytes, as they effectively lysed TRP-2 peptide-pulsed targets both in vitro and in vivo. Potentially, post-translational protein modification can be utilized to trigger strong immune responses to either tumor proteins or potentially weakly immunogenic pathogens.

Tumor Reversion: Mesenchymal-Epithelial Transition as a Critical Step in Managing the Tumor-Microenvironment Cross-Talk

2017 ◽  
Vol 23 (32) ◽  
pp. 4705-4715 ◽  
Author(s):  
Mariano Bizzarri ◽  
Alessandra Cucina ◽  
Sara Proietti
Keyword(s):  
Conformational Changes ◽  
Cross Talk ◽  
Regulation Of Gene Expression ◽  
Pharmacological Intervention ◽  
Molecular Events ◽  
Wide Range ◽  
Cancer Types ◽  
Mesenchymal Epithelial Transition

Tumour reversion represents a promising field of investigation. The occurrence of cancer reversion both in vitro and in vivo has been ascertained by an increasing number of reports. The reverting process may be triggered in a wide range of different cancer types by both molecular and physical cues. This process encompasses mandatorily a change in the cell-stroma interactions, leading to profound modification in tissue architecture. Indeed, cancer reversion may be obtained by only resetting the overall burden of biophysical cues acting on the cell-stroma system, thus indicating that conformational changes induced by cell shape and cytoskeleton remodelling trigger downstream the cascade of molecular events required for phenotypic reversion. Ultimately, epigenetic regulation of gene expression (chiefly involving presenilin-1 and translationally controlled tumour protein) and modulation of a few critical biochemical pathways trigger the mesenchymal-epithelial transition, deemed to be a stable cancer reversion. As cancer can be successfully ‘reprogrammed’ by modifying the dynamical cross-talk with its microenvironment thus the cell-stroma interactions must be recognized as targets for pharmacological intervention. Yet, understanding cancer reversion remains challenging and refinement in modelling such processes in vitro as well as in vivo is urgently warranted. This new approach bears huge implications, from both a theoretical and clinical perspective, as it may facilitate the design of a novel anticancer strategy focused on mimicking or activating the tumour reversion pathway.

Regulatory Perspective on in Vitro Assays as Predictors of Phototoxicity and Photo Co-Carcinogenicity

1998 ◽  
Vol 17 (5) ◽  
pp. 571-575 ◽  
Author(s):  
Amy L. Ellis
Keyword(s):  
Mammalian Cells ◽  
Hairless Mouse ◽  
In Vitro Assays ◽  
In Vitro Tests ◽  
Drug Products ◽  
Drug Classes ◽  
Wide Range ◽  
Regulatory Perspective

Drugs from a variety of chemical classes used for a wide range of therapeutic indications can be photosensitizers in humans. Several drugs are phototoxic in animal models as well; there are no nonclinical data for many. In vitro tests have been developed as predictors of phototoxicity and although they have been used as screens, none have replaced the in vivo tests done in rodents (usually mice or guinea pigs) since these have been good predictors of clinical phototoxicity. Some phototoxic drug classes are co-carcinogens with ultraviolet radiation (UVA and/or UVB) in hairless mice, specifically psoralens, retinoids, and fluo-roquinolones. Treatment with 8-methoxypsoralen and ultraviolet A radiation for psoriasis is also carcinogenic in humans. It has been suggested that in vitro photogenotoxicity assays using microorganisms or mammalian cells may be predictive of photo co-carcinogenicity. Some attractions of these in vitro assays, compared to the hairless mouse photo co-carcinogenicity assay, are their generally shorter duration and lower cost as well as reducing the number of animals used in research. Currently, personnel at the Food and Drug Administration (FDA) are examining the available data on phototoxicity, photogenotoxicity, and photo co-carcinogenicity to determine how this information can best be used toregulate and label drug products, and considering which assays should be recommended under specific circumstances.

scholarly journals Mitochondria Can Cross Cell Boundaries: An Overview of the Biological Relevance, Pathophysiological Implications and Therapeutic Perspectives of Intercellular Mitochondrial Transfer

2021 ◽  
Vol 22 (15) ◽  
pp. 8312
Author(s):  
Daniela Valenti ◽  
Rosa Anna Vacca ◽  
Loredana Moro ◽  
Anna Atlante
Keyword(s):  
Mammalian Cells ◽  
Therapeutic Potential ◽  
Cell Metabolism ◽  
Immunological Response ◽  
Biological Relevance ◽  
Mitochondrial Transfer ◽  
Response To Chemotherapy ◽  
Wide Range

Mitochondria are complex intracellular organelles traditionally identified as the powerhouses of eukaryotic cells due to their central role in bioenergetic metabolism. In recent decades, the growing interest in mitochondria research has revealed that these multifunctional organelles are more than just the cell powerhouses, playing many other key roles as signaling platforms that regulate cell metabolism, proliferation, death and immunological response. As key regulators, mitochondria, when dysfunctional, are involved in the pathogenesis of a wide range of metabolic, neurodegenerative, immune and neoplastic disorders. Far more recently, mitochondria attracted renewed attention from the scientific community for their ability of intercellular translocation that can involve whole mitochondria, mitochondrial genome or other mitochondrial components. The intercellular transport of mitochondria, defined as horizontal mitochondrial transfer, can occur in mammalian cells both in vitro and in vivo, and in physiological and pathological conditions. Mitochondrial transfer can provide an exogenous mitochondrial source, replenishing dysfunctional mitochondria, thereby improving mitochondrial faults or, as in in the case of tumor cells, changing their functional skills and response to chemotherapy. In this review, we will provide an overview of the state of the art of the up-to-date knowledge on intercellular trafficking of mitochondria by discussing its biological relevance, mode and mechanisms underlying the process and its involvement in different pathophysiological contexts, highlighting its therapeutic potential for diseases with mitochondrial dysfunction primarily involved in their pathogenesis.

scholarly journals Use of Protein Biotinylation In Vivo for Immunoelectron Microscopic Localization of a Specific Protein Isoform

2008 ◽  
Vol 56 (10) ◽  
pp. 911-919 ◽  
Author(s):  
Antoine Viens ◽  
Francis Harper ◽  
Evelyne Pichard ◽  
Martine Comisso ◽  
Gérard Pierron ◽  
...  
Keyword(s):  
Immunoelectron Microscopy ◽  
Mammalian Cells ◽  
Protein Detection ◽  
Specific Protein ◽  
Covalent Attachment ◽  
Stable Cell Line ◽  
Single Experiment ◽  
Wide Range ◽  
Protein Isoform

Tagging of proteins in vivo by covalent attachment of a biotin moiety has emerged as a new prospective tool for protein detection and purification. Previously, we established a strategy for expression of in vivo biotinylated proteins in mammalian cells. It is based on coexpression of the protein of interest fused to a short biotin acceptor peptide and biotin ligase BirA cloned in the same vector. We show here that the in vivo biotinylation can be used for immunogold postembedding labeling in immunoelectron microscopy experiments. We show that immunoelectron microscopy with biotinylated nuclear proteins is compatible with a wide range of postembedding methods, facilitating combination of morphological and localization studies in a single experiment. We also show that the method works in both transient transfection and stable cell line expression protocols and can be used for colocalization studies. This manuscript contains online supplemental material at http://www.jhc.org . Please visit this article online to view these materials.

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