Chemical Modification Of Proteins Research Articles

Recent Advances towards the Reversible Chemical Modification of Proteins.

Proteins are intriguing biomacromolecules for all living systems, not only as essential building blocks of organisms, but also as participants in almost every aspect of cellular activity such as metabolism and gene transcription/expression. Developing chemical biology tools that are capable of labeling/modifying proteins is a powerful method for decoding their detailed structures and functions. However, most current approaches heavily rely on the installation of permanent tags or genetic engineering of unnatural amino acids. There has been slow development in reversible chemical labeling using small organic probes and bioorthogonal transformations to construct site-selectively modified proteins and conditionally restore their activities or structures. This review summarizes recent advances in the field of chemical regulation of proteins with reversible transformations towards distinct motifs, including amino acid residues, amide backbones and native post-translational lysine. Finally, current challenges and future perspectives are discussed.

Changes to Urinary Proteome in High-Fat-Diet ApoE-/- Mice.

Cardiovascular disease is currently the leading cause of death worldwide. Atherosclerosis is an important pathological basis of cardiovascular disease, and its early diagnosis is of great significance. Urine bears no need nor mechanism to be stable, so it accumulates many small changes and is therefore a good source of biomarkers in the early stages of disease. In this study, ApoE-/- mice were fed a high-fat diet for 5 months. Urine samples from the experimental group and control group (C57BL/6 mice fed a normal diet) were collected at seven time points. Proteomic analysis was used for comparison within the experimental group and for comparison between the experimental group and the control group. The results of the comparison within the experimental group showed a significant difference in the urinary proteome before and after a one-week high-fat diet, and several of the differential proteins have been reported to be associated with atherosclerosis and/or as biomarker candidates. The results of the comparison between the experimental group and the control group indicated that the biological processes enriched by the GO analysis of the differential proteins correspond to the progression of atherosclerosis. The differences in chemical modifications of urinary proteins have also been reported to be associated with the disease. This study demonstrates that urinary proteomics has the potential to sensitively monitor changes in the body and provides the possibility of identifying early biomarkers of atherosclerosis.

Emerging role of mRNA methylation in regulating the hallmarks of cancer

The dynamic chemical modifications of DNA, RNA, and proteins can transform normal cells into malignant ones. While the DNA and protein modifications in cancer have been described extensively in the literature, there are fewer reports about the role of RNA modifications in cancer. There are over 100 forms of RNA modifications and one of these, mRNA methylation, plays a critical role in the malignant properties of the cells. mRNA methylation is a reversible modification responsible for regulating protein expression at the post-transcriptional level. Despite being discovered in the 1970s, a complete understanding of the different proteins involved and the mechanism behind mRNA methylation remains largely unknown. However, these mRNA methylations have been shown to foster cancer hallmarks via specific cellular targets inside the cell. In this review, we provide a brief overview of mRNA methylation and its emerging role in regulating the various hallmarks of cancer.

The histone code of the fungal genus Aspergillus uncovered by evolutionary and proteomic analyses.

Chemical modifications of DNA and histone proteins impact the organization of chromatin within the nucleus. Changes in these modifications, catalysed by different chromatin-modifying enzymes, influence chromatin organization, which in turn is thought to impact the spatial and temporal regulation of gene expression. While combinations of different histone modifications, the histone code, have been studied in several model species, we know very little about histone modifications in the fungal genus Aspergillus, whose members are generally well studied due to their importance as models in cell and molecular biology as well as their medical and biotechnological relevance. Here, we used phylogenetic analyses in 94 Aspergilli as well as other fungi to uncover the occurrence and evolutionary trajectories of enzymes and protein complexes with roles in chromatin modifications or regulation. We found that these enzymes and complexes are highly conserved in Aspergilli, pointing towards a complex repertoire of chromatin modifications. Nevertheless, we also observed few recent gene duplications or losses, highlighting Aspergillus species to further study the roles of specific chromatin modifications. SET7 (KMT6) and other components of PRC2 (Polycomb Repressive Complex 2), which is responsible for methylation on histone H3 at lysine 27 in many eukaryotes including fungi, are absent in Aspergilli as well as in closely related Penicillium species, suggesting that these lost the capacity for this histone modification. We corroborated our computational predictions by performing untargeted MS analysis of histone post-translational modifications in Aspergillus nidulans. This systematic analysis will pave the way for future research into the complexity of the histone code and its functional implications on genome architecture and gene regulation in fungi.

Automated prediction of site and sequence of protein modification with ATRP initiators.

One of the most straightforward and commonly used chemical modifications of proteins is to react surface amino groups (lysine residues) with activated esters. This chemistry has been used to generate protein-polymer conjugates, many of which are now approved therapeutics. Similar conjugates have also been generated by reacting activated ester atom transfer polymerization initiators with lysine residues to create biomacromolecular initiators for polymerization reactions. The reaction between activated esters and lysine amino groups is rapid and has been consistently described in almost every publication on the topic as a “random reaction”. A random reaction implies that every accessible lysine amino group on a protein molecule is equally reactive, and as a result, that the reaction is indiscriminate. Nonetheless, the literature contradicts itself by also suggesting that some lysine amino groups are more reactive than others (as a function of pKa, surface accessibility, temperature, and local environment). If the latter assumption is correct, then the outcome of these reactions cannot be random at all, and we should be able to predict the outcome from the structure of the protein. Predicting the non-random outcome of a reaction between surface lysines and reactive esters could transform the speed at which active bioconjugates can be developed and engineered. Herein, we describe a robust integrated tool that predicts the activated ester reactivity of every lysine in a protein, thereby allowing us to calculate the non-random sequence of reaction as a function of reaction conditions. Specifically, we have predicted the intrinsic reactivity of each lysine in multiple proteins with a bromine-functionalised N-hydroxysuccinimide initiator molecule. We have also shown that the model applied to PEGylation. The rules-based analysis has been coupled together in a single Python program that can bypass tedious trial and error experiments usually needed in protein-polymer conjugate design and synthesis.

Fast Cysteine Bioconjugation Chemistry.

Cysteine bioconjugation serves as a powerful tool in biological research and has been widely used for chemical modification of proteins, constructing antibody-drug conjugates, and enabling cell imaging studies. Cysteine conjugation reactions with fast kinetics and exquisite selectivity have been under heavy pursuit as they would allow clean protein modification with just stoichiometric amounts of reagents, which minimizes side reactions, simplifies purification and broadens functional group tolerance. In this concept, we summarize the recent advances in fast cysteine bioconjugation, and discuss the mechanism and chemical principles that underlie the high efficiencies of the newly developed cysteine reactive reagents.

Selective Acylation of Proteins at Gly and Lys in His Tags.

The chemical modification of proteins is of great importance in chemical biology, biotechnology, and for the production of modified biopharmaceuticals, as it enables introduction of fluorophores, biotin, half-life extending moieties, and more. We have developed two methods that use poly-His sequences to direct the highly selective acylation of proteins, either at the N-terminus or at a specific Lys residue. For the former, we used an N-terminal Gly-His6 segment (Gly-His tag) that directed acylation of the N-terminal Nα -amine with 4-methoxyphenyl esters, resulting in stable conjugates. Next, we developed the peptide sequences Hisn -Lys-Hism (Lys-His tags) that direct the acylation of the designated Lys Nϵ -amine under mild conditions and with high selectivity over native Lys residues. Both the Gly-His and Lys-His tags maintain the capacity for immobilized metal ion affinity chromatography. We have demonstrated the robustness of these methods by attaching different moieties such as azides, fluorophores, and biotin to different proteins, including antibodies.

Superfast desulfurization for protein chemical synthesis and modification

Superfast desulfurization for protein chemical synthesis and modification

Deciphering the impact of whey protein powder storage on protein state and powder stability

Whey protein concentrate powders can be strongly affected by storage conditions inducing both chemical and structural protein modifications. In the present work, a combination of different storage conditions were evaluated, leading to the evaluation of 6 different batches that were chosen in order to evaluate the impact of the following parameters: storage temperature, storage duration and storage heat load (°C.days). In each set the relative humidity was maintained at 70%. To estimate the impact of the three parameters, whey protein lactosylation, denaturation and aggregation were followed. It was shown that the highest temperatures (60 vs 55.5 °C and 41 vs 36 °C) led to greater protein modification, whereas the duration (17 vs 14 days and 33 vs 30 days) and the total heat load (low vs high C°.days) did not affect protein modification. Lactose content is known to be an important parameter for powder browning. Here, we show that if lactose content is crucial for the level of initial process induced browning of the powder, the additional browning that occurs during storage is mostly independent of lactose content. In addition, during storage, detected lactosylated proteins were found to be inversely proportional to powder browning indicating the occurrence of advanced Maillard reaction steps ( e.g. dicarbonyl and AGEs formation and production of melanoidins). Protein aggregates contained both β-lactoglobulin and α-lactalbumin and were mostly formed by non-disulphide covalent bonds. Interestingly, despite protein structure being strongly impacted, good powder rehydration properties were obtained for the studied conditions. • Whey powder functionality is not impacted by storage. • Maillard-induced protein modification is repeatable between powder productions. • Initial protein lactosylation content drives powder aging. • Browning during storage is not related to lactose content.

Reactions

EPIGENETICS Reactions ShareShare onFacebookTwitterWechatLinked InRedditEmail C&EN, 2022, 100 (25), p 5July 18, 2022Cite this:C&EN 100, 25, 5Letters to the editorBeyond B-DNA for histone studiesI enjoyed reading the C&EN cover story “Expanding the Histone Code” (June 6, 2022, page 22). I fully agree with the overall characterization of the importance of locating new chemical modifications on histones that package our genes. Methylation and acetylation are the two most frequent modifications; they regulate gene expression by alternating between transcriptional activation and repression. Andrew J. Andrews is correct in saying that “we have to change our thinking from simple on-off switches and inhibitors like we’ve done for years to more complicated systems.” Additionally, we need to do the same with James Watson and Francis Crick’s canonical right-handed double-stranded (ds) B-DNA structure and histone interactions and expand our view on DNA structure to include non-B-DNA conformations, such as the alternative left-handed DNA (ds-Z-DNA), tightly bent DNA, and looped DNA.Additionally, other unusual nucleic acids, such as multistranded DNAs (e.g., triplex DNA, G-quadruplex DNA, and i-motif DNA molecules), need to be considered when discussing chemical modifications of histones. These noncanonical DNA structures play important roles in regulating transcriptional gene expression but are usually left out of the molecular biological conversation.Researchers need to place an emphasis on the relationship between chemical modifications of histones and not just canonical B-DNA but also alternative and multistranded DNA structures. Adding these noncanonical DNA-based structural factors will further expand our knowledge on how the histone code works, adding another layer to its complexity beyond just chemical modifications of proteins, canonical B-DNA, and epigenetics. And investigating the relationships between noncanonical B-DNA structures and histone modifications on gene expression will expand our knowledge of epigenetics.DNA can no longer be viewed as a static, one-dimensional molecule—namely, B-DNA—but rather as an extremely flexible and dynamic polymorphic polymer that can adopt many different structures on the basis of its sequence and length. This concept must be included in any conversations about expanding (and even cracking) the histone code.Claude E. GagnaBronxville, New York CorrectionJuly 4, 2022, page 26: The cover story on pharmaceutical outsourcing misstates the status of NP001, Neuvivo’s drug candidate for amyotrophic lateral sclerosis. Neuvivo is awaiting accelerated approval for all patients, not conditional approval for a subset of patients, from the US Food and Drug Administration. In addition, a photo of CordenPharma’s facility in Colorado (page 30) has the wrong credit. It is CordenPharma, not Merrick Chase Photography.

Single-Step N-Terminal Modification of Proteins via a Bio-Inspired Copper(II)-Mediated Aldol Reaction.

The chemical modification of proteins is an effective technique for manipulating the properties and functions of proteins, and for creating protein-based materials. The N-terminus is a promising target for single-site modification that provides modified proteins with uniform structures and properties. In this paper, a copper(II)-mediated aldol reaction with 2-pyridinecarboxaldehyde (2-PC) derivatives is proposed as an operationally simple method to selectively modify the N-terminus of peptides and proteins at room temperature and physiological pH. The copper(II) ion activates the N-terminal amino acids by complexation with an imine of the N-terminal amino acid and 2-PCs, realizing the selective formation of the nucleophilic intermediate at the N-terminus. This results in a stable carbon-carbon bond between the 2-PCs and the α-carbon of various N-terminal amino acids. The reaction is applied to four different proteins, including biopharmaceuticals such as filgrastim and trastuzumab. The modified trastuzumab retains the human epidermal growth factor receptor 2 recognition activity.

Many kinds of oxidized proteins are present more in the urine of the elderly

BackgroundMany studies have shown an association between aging and oxidation. To our knowledge, there have been no studies exploring aging-related urine proteome modifications. The purpose of this study was to explore differences in global chemical modifications of urinary protein at different ages.MethodsDiscovery (n=38) cohort MS data including children, young and old groups were downloaded from three published studies, and this data was analyzed using open-pFind for identifying modifications. Verification cohort human samples (n=28) including young, middle-aged, and old groups, rat samples (n=7) at three-time points after birth, adulthood, and old age were collected and processed in the laboratory simultaneously based on label-free quantification combined with pFind.ResultsDiscovery cohort: there were 28 kinds of differential oxidations in the old group that were higher than those in the young or children group in. Verification cohort: there were 17 kinds of differential oxidations of 49 oxidized proteins in the middle and old groups, which were significantly higher than those in the young group. Both oxidations and oxidized proteins distinguished different age groups well. There were also 15 kinds of differential oxidations in old age higher than others in the rat cohort. The results showed that the validation experiment was basically consistent with the results of the discovery experiment, showing that the level of oxidized proteins in urine increased significantly with age.ConclusionsOur study is the first to show that oxidative proteins occur in urine and that oxidations are higher in older than younger ages. Perhaps improving the degree of excretion of oxidative protein in vivo through the kidney is helpful for maintaining the homeostasis of the body’s internal environment, delaying aging and the occurrence of senile diseases.

Optimal Immunotherapy of Oncolytic Viruses and Adopted Cell Transfer in Cancer Treatment

We investigate therapeutic effects of monotherapy of oncolytic viruses, of adopted cell transfer, as well as the two combined therapies over a short time treatment period by applying optimal control techniques. The goal is to minimize the number of susceptible tumor cells and the costs associated with the therapy over the treatment period. We verify that there exists an optimal control pair and derive the necessary conditions. The optimality system is solved numerically to provide optimal protocols under different scenarios with respect to initial tumor sizes and parameter values. Although the two types of therapy do not work synergistically when the viral killing rate by immune cells is large, a small anti-viral killing can improve therapy success of either monotherapy of oncolytic viruses or combined therapy of oncolytic viruses and adopted T cell transfer. This finding can be accomplished either by manipulating certain genes of viruses via genetic engineering or by chemical modification of viral coat proteins to avoid detection by the immune cells.

Click Bioconjugation: Modifying Proteins Using Click-Like Chemistry.

Bioconjugation deals with the chemical modification of proteins. The reactions used exploit either the intrinsic chemical reactivity of the biomolecule or introduce functionalities that can then be subsequently reacted without interfering with other functional groups of the biological entity. Perfectly selective, high yielding chemical transformations are needed that can be run in aqueous environment under mild pH conditions. Requirements that have an obvious overlap with the definition of click chemistry. This review shows a selection of successfully applied click-type reactions in bioconjugation as well as some recent developments to broaden the chemical toolbox to meet the challenge of selective, bioorthogonal modification of biomolecules.

Deciphering the Structure and Formation of Amyloids in Neurodegenerative Diseases With Chemical Biology Tools.

Protein aggregation into highly ordered, regularly repeated cross-β sheet structures called amyloid fibrils is closely associated to human disorders such as neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases, or systemic diseases like type II diabetes. Yet, in some cases, such as the HET-s prion, amyloids have biological functions. High-resolution structures of amyloids fibrils from cryo-electron microscopy have very recently highlighted their ultrastructural organization and polymorphisms. However, the molecular mechanisms and the role of co-factors (posttranslational modifications, non-proteinaceous components and other proteins) acting on the fibril formation are still poorly understood. Whether amyloid fibrils play a toxic or protective role in the pathogenesis of neurodegenerative diseases remains to be elucidated. Furthermore, such aberrant protein-protein interactions challenge the search of small-molecule drugs or immunotherapy approaches targeting amyloid formation. In this review, we describe how chemical biology tools contribute to new insights on the mode of action of amyloidogenic proteins and peptides, defining their structural signature and aggregation pathways by capturing their molecular details and conformational heterogeneity. Challenging the imagination of scientists, this constantly expanding field provides crucial tools to unravel mechanistic detail of amyloid formation such as semisynthetic proteins and small-molecule sensors of conformational changes and/or aggregation. Protein engineering methods and bioorthogonal chemistry for the introduction of protein chemical modifications are additional fruitful strategies to tackle the challenge of understanding amyloid formation.

Proline reduces half‐life of homocysteine thiolactone

Homocysteine (Hcy) is a sulfur containing toxic amino acid synthesized from sulfur metabolism pathway. Elevated level of Hcy is associated with various cardiovascular complications including atherosclerosis. One of the likely mechanisms underlying harmful effects of Hcy is the chemical modification of proteins by homocysteine thiolactone (HTL), a highly reactive cyclic thioester of Hcy. HTL preferentially forms amide bonds with ɛ‐ amino group of protein lysine, arginine or histidine residues in a non‐enzymatic mechanism; a process referred to as “protein N‐homocysteinylation resulting in the impairment of protein functions. Therefore, novel approaches that could inhibit or delay protein N‐homocysteinylation would be therapeutically important to curb hyperhomocysteinemia. In the present communication, we screened for different cellular molecules produced under stressful insults for their ability to suppress covalent modification of proteins by HTL. We found that out of several molecules, proline has the potential to inhibit protein N‐homocysteinylation in a non‐enzymatic fashion. We also discovered that proline treatment increases Hcy levels in HeLa cells and induces cell cycle arrest. Our study highlights the importance of proline treatment in reducing toxicity load in hyperhomocysteinemic/homocystinuric patients.

Reversible Chemical Protein Modification to Endogenous Glutathione and Its Utilities in the Manufacture of Transcellular Pro-Enzymes

Proteins have been perceived as being an intriguing modality of therapeutics for the treatment of intractable diseases in view of their superlative precision and versatility. Nonetheless, proteins' intrinsic characters, particularly their being hydrophilic macromolecules with unmethodical charges, have imposed the exceeding challenge of seeking transcellular trafficking into cells' interiors. To circumvent this drawback, we have attempted to employ triple-functional amine-reactive 4-(2-((2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)disulfaneyl)ethoxy)-4-oxobutanoic acid for the efficient incorporation of the anionic carboxyl moiety into amine-enriched enzymes, resulting in overall negatively charged pro-enzymes. The resulting pro-enzymes could be readily electrostatically assembled with cationic species [for instance: block copolymers of poly(ethylene glycol)-polylysine] into core-shell architectural delivery nanoparticles for their facilitated endocytosis into cells. Noteworthy is the aforementioned carboxylation chemistry designed to allow facile reversal of the pro-enzymes to the original amine groups due to the thiolysis of intermediate disulfide linkage for subsequent cascade reactions in response to the cytosol-enriched glutathione. Therefore, cytosol-selective structural disassembly for the liberation and activation of the pro-enzymes was accomplished. Our subsequent investigations utilizing ribonuclease A and catalase as the model enzymes demonstrated appreciable transcellular transportation of the active enzymes to the cell interiors, exerting overwhelming cytotoxic potencies and H2O2 scavenging capacities, respectively. Hence, we reported an unprecedented redox-stimulated charge reversal strategy in engineering cytosol-activatable pro-enzymes, manifesting a simple and efficient approach in the manufacture of transcellular proteinic therapeutics, which should be highlighted to promote their wide availability for use with diverse functional proteins as molecular biological tools and precision therapeutics.

Chemical Modification of Silk Proteins: Current Status and Future Prospects

Chemical Modification of Silk Proteins: Current Status and Future Prospects

Site-Directed Chemical Modification of Amyloid by Polyoxometalates for Inhibition of Protein Misfolding and Aggregation.

Post-translational modification (PTM) of protein can significantly change protein conformation and function. Inspired by the natural PTM, we present a new approach to inhibit amyloid aggregation by chemical PTM modification. Polyoxometalates (POMs) were used as examples of inhibitors of β-amyloid peptide (Aβ) aggregation to illustrate the chemical PTM method. After the POMs were modified with thiazolidinethione (TZ), the resulting POMD-TZ acted as a chemical PTM agent and could covalently modify Aβ site-selectively at Lys16. Multiple biophysical techniques and biochemical assays have been employed to show the superiority of the chemical PTM method compared to traditional Aβ inhibitors. Since Aβ oligomers are more cytotoxic, we further functionalized POMD-TZ with an Aβ-targeted peptide and a fluorescent probe to obtain an "Aβ oligomer sensitive" probe. The use of PTM agents for the site-directed chemical modification of proteins provides a new way to regulate amyloid aggregation.

Site‐Directed Chemical Modification of Amyloid by Polyoxometalates for Inhibition of Protein Misfolding and Aggregation

AbstractPost‐translational modification (PTM) of protein can significantly change protein conformation and function. Inspired by the natural PTM, we present a new approach to inhibit amyloid aggregation by chemical PTM modification. Polyoxometalates (POMs) were used as examples of inhibitors of β‐amyloid peptide (Aβ) aggregation to illustrate the chemical PTM method. After the POMs were modified with thiazolidinethione (TZ), the resulting POMD‐TZ acted as a chemical PTM agent and could covalently modify Aβ site‐selectively at Lys16. Multiple biophysical techniques and biochemical assays have been employed to show the superiority of the chemical PTM method compared to traditional Aβ inhibitors. Since Aβ oligomers are more cytotoxic, we further functionalized POMD‐TZ with an Aβ‐targeted peptide and a fluorescent probe to obtain an “Aβ oligomer sensitive” probe. The use of PTM agents for the site‐directed chemical modification of proteins provides a new way to regulate amyloid aggregation.