Protein modification and maintenance systems as biomarkers of ageing

Changes in the abundance and post-translational modification of proteins and accumulation of some covalently modified proteins have been proposed to represent hallmarks of biological ageing. Within the frame of the Mark-Age project, the workpackage dedicated to "markers based on proteins and their modifications" has been firstly focused on enzymatic and non-enzymatic post-translational modifications of serum proteins by carbohydrates. The second focus of the workpackage has been directed towards protein maintenance systems that are involved either in protein quality control (ApoJ/Clusterin) or in the removal of oxidatively damaged proteins through degradation and repair (proteasome and methionine sulfoxide reductase systems). This review describes the most relevant features of these protein modifications and maintenance systems, their fate during ageing and/or their implication in ageing and longevity

Controlled levels of protein modification through a chromatography-mediated bioconjugation.

Synthetically modified proteins are increasingly finding applications as well-defined scaffolds for materials. In practice it remains difficult to construct bioconjugates with precise levels of modification because of the limited number of repeated functional groups on proteins. This article describes a method to control the level of protein modification in cases where there exist multiple potential modification sites. A protein is first tagged with a handle using any of a variety of modification chemistries. This handle is used to isolate proteins with a particular number of modifications via affinity chromatography, and then the handle is elaborated with a desired moiety using an oxidative coupling reaction. This method results in a sample of protein with a well-defined number of modifications, and we find it particularly applicable to systems like protein homomultimers in which there is no way to discern between chemically identical subunits. We demonstrate the use of this method in the construction of a protein-templated light-harvesting mimic, a type of system which has historically been difficult to make in a well-defined manner

Protein modification by arginylation

The modification of protein by arginine catalyzed by arginyltransferases (ATE1) described by the Kashina group in this issue shows that arginylation of protein occurs widely in biology and is being recognized as a key regulatory reaction such as phosphorylation of proteins (Wang et al., 2011)

A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases

Robust circadian clocks from coupled protein modification and transcription-translation cycles

The cyanobacterium Synechococcus elongatus uses both a protein phosphorylation cycle and a transcription-translation cycle to generate circadian rhythms that are highly robust against biochemical noise. We use stochastic simulations to analyze how these cycles interact to generate stable rhythms in growing, dividing cells. We find that a protein phosphorylation cycle by itself is robust when protein turnover is low. For high decay or dilution rates (and co mpensating synthesis rate), however, the phosphorylation-based oscillator loses its integrity. Circadian rhythms thus cannot be generated with a phosphorylation cycle alone when the growth rate, and consequently the rate of protein dilution, is high enough; in practice, a purely post-translational clock ceases to function well when the cell doubling time drops below the 24 hour clock period. At higher growth rates, a transcription-translation cycle becomes essential for generating robust circadian rhythms. Interestingly, while a transcription-translation cycle is necessary to sustain a phosphorylation cycle at high growth rates, a phosphorylation cycle can dramatically enhance the robustness of a transcription-translation cycle at lower protein decay or dilution rates. Our analysis thus predicts that both cycles are required to generate robust circadian rhythms over the full range of growth conditions.Comment: main text: 7 pages including 5 figures, supplementary information: 13 pages including 9 figure

Site-selective protein modification via disulfide rebridging for fast tetrazine/trans-cyclooctene bioconjugation

An inverse electron demand Diels–Alder reaction between tetrazine and trans-cyclooctene (TCO) holds great promise for protein modification and manipulation. Herein, we report the design and synthesis of a tetrazine-based disulfide rebridging reagent, which allows the site-selective installation of a tetrazine group into disulfide-containing peptides and proteins such as the hormone somatostatin (SST) and the antigen binding fragment (Fab) of human immunoglobulin G (IgG). The fast and efficient conjugation of the tetrazine modified proteins with three different TCO-containing substrates to form a set of bioconjugates in a site-selective manner was successfully demonstrated for the first time. Homogeneous, well-defined bioconjugates were obtained underlining the great potential of our method for fast bioconjugation in emerging protein therapeutics. The formed bioconjugates were stable against glutathione and in serum, and they maintained their secondary structure. With this work, we broaden the scope of tetrazine chemistry for site-selective protein modification to prepare well-defined SST and Fab conjugates with preserved structures and good stability under biologically relevant conditions

Chemical protein modification through cysteine

The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chemically modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundanceboth allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chemical protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications

Advances in chemical protein modification.

Chemical protein modification has emerged as an invaluable tool for the development of modified proteins. The complementary use of both genetic and chemical methods has provided a large toolbox that allows the preparation of almost unlimited protein constructs with either natural or synthetically modified residues. Such a protein chemodiversity, usually achieved after translation and commonly referred to as post-translational protein modifications (PTMs), is often responsible for the vast biodiversity found in nature. These modifications include acylation, methylation, phosphorylation, sulfation, farnesylation, ubiquitination, and glycosylation, among others, and play a pivotal role in important cellular processes including trafficking, differentiation, migration, and signaling. Consequently, reproducing in a highly efficient and controlled way such natural modifications of proteins (by introducing natural PTMs) would provide an invaluable tool to study their precise function. Additionally, the possibility offered by the introduction and (bio)orthogonal modification of unnatural moieties/amino acids (usually improving the properties of natural PTMs during isolation, analysis, and processing) makes site-selective modification of proteins a key tool for interrogating and intervening biological systems both in vitro and in vivo. Given the range of chemical modification methods available, it is now possible to decide which residue to target and which modification to attach in order to confer the desired property/function (affinity probes, fluorophores, reactive tags, etc.). For example, increasing the circulation half-life of a therapeutic protein may be achieved by the addition of polyethylene glycol (PEG). On the other hand, the use of a spectroscopic label to monitor biomolecule distribution in vivo enables the construction of highly selective imaging agents. Despite the vast progress in the field of bioconjugation chemistry, scientists still face many challenges, not only synthetically but also from a processing, manufacturing, safety, and stability perspective. A number of methods have been developed and applied for the modification of particular proteins and therefore may not be applicable to any protein of interest. Thus, there remains a need for the development of complementary reactions for the site-selective chemical modification of proteins that are mild, efficient, and robust. Several reviews covering different aspects of the chemical synthesis of proteins, from general native chemical ligation strategies and the modification of endogenous amino acids to more specialized topics such as click modification protocols, the introduction of particular PTMs including glycosylation, PEGylation, and polymerization of protein-based initiators, and the challenging labeling of a specific protein of interest in a complex cellular mixture using the so-called “bioorthogonal” reactions, have been published during the past decade. While the later publications describe different protein syntheses/modifications in detail, the aim of the present review is not to be an exhaustive survey of all available bioconjugation methodologies but to discuss recent chemical strategies for the site-selective modification of proteins such as fast sulfur exchange or stable thioether formation, photo and metal-free cycloadditions, and other particularly challenging metal-mediated protocols. This review will be divided into two sections: transition metal-free and transition metal-mediated approaches. For clarity, we will use the following terminology throughout this manuscript: residue/amino acid/site-selective (or simply site-selective) reactions are those transformations that preferentially modify one amino acid residue over the others (e.g., cysteine versus lysine) and, thus, can be considered examples of chemoselective reactions; on the other hand transformations described as regioselective preferentially modify only one of a set of the same amino acid, in particular when more than one is present in the same molecule (e.g., solvent-exposed lysine versus internal lysine).O.B. thanks the European Commission (Marie Curie CIG) and Ministerio de Ciencia e Innovación, Spain (Juan de la Cierva Fellowship). G.J.L.B. thanks his generous sources of funding: Royal Society, FCT Portugal (FCT Investigator), European Commission (Marie Curie CIG), and the EPSRC. G.J.L.B. is a Royal Society University Research Fellow. The authors thank Paula Boutureira Regla and Francisco Pinteus da Cruz Lopes Bernardes for inspiration.This article was originally published in Chemical Reviews, 2015, 115 (5), pp 2174–2195 DOI: 10.1021/cr500399p. This is the final published version