Discovering the landscape of protein modifications.

Protein modifications modulate nearly every aspect of cell biology in organisms, ranging from Archaea to Eukaryotes. The earliest evidence of covalent protein modifications was found in the early 20th century by studying the amino acid composition of proteins by chemical hydrolysis. These discoveries challenged what defined a canonical amino acid. The advent and rapid adoption of mass-spectrometry-based proteomics in the latter part of the 20th century enabled a veritable explosion in the number of known protein modifications, with more than 500 discrete modifications counted today. Now, new computational tools in data science, machine learning, and artificial intelligence are poised to allow researchers to make significant progress in discovering new protein modifications and determining their function. In this review, we take an opportunity to revisit the historical discovery of key post-translational modifications, quantify the current landscape of covalent protein adducts, and assess the role that new computational tools will play in the future of this field.

Protein Termini 2022: central roles of protein ends.

Although locating at the protein ends, N- and C-termini are at the center of numerous cellular functions. This topic engages an increasing number of scientists, recently forming the International Society of Protein Termini (ISPT). Protein Termini 2022 gathered this interdisciplinary community to discuss how protein ends may steer protein functionality.

Co-translational, Post-translational, and Non-catalytic Roles of N-Terminal Acetyltransferases.

Recent studies of N-terminal acetylation have identified new N-terminal acetyltransferases (NATs) and expanded the known functions of these enzymes beyond their roles as ribosome-associated co-translational modifiers. For instance, the identification of Golgi- and chloroplast-associated NATs shows that acetylation of N termini also happens post-translationally. In addition, we now appreciate that some NATs are highly specific; for example, a dedicated NAT responsible for post-translational N-terminal acetylation of actin was recently revealed. Other studies have extended NAT function beyond Nt acetylation, including functions as lysine acetyltransferases (KATs) and non-catalytic roles. Finally, emerging studies emphasize the physiological relevance of N-terminal acetylation, including roles in calorie-restriction-induced longevity and pathological α-synuclein aggregation in Parkinson's disease. Combined, the NATs rise as multifunctional proteins, and N-terminal acetylation is gaining recognition as a major cellular regulator.

Biochemical and Structural Consequences of NEDD8 Acetylation.

Similar to ubiquitin, the ubiquitin-like protein NEDD8 is not only conjugated to other proteins but is itself subject to posttranslational modifications including lysine acetylation. Yet, compared to ubiquitin, only little is known about the biochemical and structural consequences of site-specific NEDD8 acetylation. Here, we generated site-specifically mono-acetylated NEDD8 variants for each known acetylation site by genetic code expansion. We show that, in particular, acetylation of K11 has a negative impact on the usage of NEDD8 by the NEDD8-conjugating enzymes UBE2M and UBE2F and that this is likely due to electrostatic and steric effects resulting in conformational changes of NEDD8. Finally, we provide evidence that p300 acts as a position-specific NEDD8 acetyltransferase.

Nonlinear Impact of Electrolyte Solutions on Protein Dynamics.

Halophilic organisms have adapted to multi-molar salt concentrations, their cytoplasmic proteins functioning despite stronger attraction between hydrophobic groups. These proteins, of interest in biotechnology because of decreasing fresh-water resources, have excess acidic amino acids. It has been suggested that conformational fluctuations - critical for protein function - decrease in the presence of a stronger hydrophobic effect, and that an acidic proteome would counteract this decrease. However, our understanding of the salt- and acidic amino acid dependency of enzymatic activity is limited. Here, using solution NMR relaxation and molecular dynamics simulations for in total 14 proteins, we show that salt concentration has a limited and moreover non-monotonic impact on protein dynamics. The results speak against the conformational-fluctuations model, instead indicating that maintaining protein dynamics to ensure protein function is not an evolutionary driving force behind the acidic proteome of halophilic proteins.

Conjugating Hemoglobin and Albumin by Strain-Promoted Azide- Alkyne Cycloaddition.

A one-to-one conjugate of cross-linked human hemoglobin and human serum albumin results from a strain-promoted alkyne-azide cycloaddition (SPAAC) of the modified proteins. Additions of a strained alkyne-substituted maleimide to the Cys-34 thiol of human serum albumin and an azide-containing cross-link between the amino groups of each ß-unit at Lys-82 of human hemoglobin provide sites for coupling by the SPAAC process. The coupled hemoglobin-albumin conjugate can be readily purified from unreacted hemoglobin. The oxygen binding properties of the two-protein bioconjugate demonstrate oxygen affinity and cooperativity that are suitable for use in an acellular oxygen carrier.

13 Cß-Valine and 13 Cγ-Leucine Methine Labeling To Probe Protein Ligand Interaction.

Precise information regarding the interaction between proteins and ligands at molecular resolution is crucial for effectively guiding the optimization process from initial hits to lead compounds in early stages of drug development. In this study, we introduce a novel aliphatic side chain isotope-labeling scheme to directly probe interactions between ligands and aliphatic sidechains using NMR techniques. To demonstrate the applicability of this method, we selected a set of Brd4-BD1 binders and analyzed 1 H chemical shift perturbation resulting from CH-π interaction of Hß -Val and Hγ -Leu as CH donors with corresponding ligand aromatic moieties as π acceptors.

The Plant PTM Viewer 2.0: in-depth exploration of plant protein modification landscapes.

Post-translational modifications (PTMs) greatly increase protein diversity and functionality. To help the plant research community interpret the ever-increasing number of reported PTMs, the Plant PTM Viewer (https://www.psb.ugent.be/PlantPTMViewer) provides an intuitive overview of plant protein PTMs and the tools to assess it. This update includes 62 novel PTM profiling studies, adding a total of 112 000 modified peptides reporting plant PTMs, including 14 additional PTM types and three species (moss, tomato, and soybean). Furthermore, an open modification re-analysis of a large-scale Arabidopsis thaliana mass spectrometry tissue atlas identified previously uncharted landscapes of lysine acylations predominant in seed and flower tissues and 3-phosphoglycerylation on glycolytic enzymes in plants. An extra 'Protein list analysis' tool was developed for retrieval and assessing the enrichment of PTMs in a protein list of interest. We conducted a protein list analysis on nuclear proteins, revealing a substantial number of redox modifications in the nucleus, confirming previous assumptions regarding the redox regulation of transcription. We encourage the plant research community to use PTM Viewer 2.0 for hypothesis testing and new target discovery, and also to submit new data to expand the coverage of conditions, plant species, and PTM types, thereby enriching our understanding of plant biology.

Emerging Roles of Vitamin B12 in Aging and Inflammation.

Vitamin B12 (cobalamin) is an essential nutrient for humans and animals. Metabolically active forms of B12-methylcobalamin and 5-deoxyadenosylcobalamin are cofactors for the enzymes methionine synthase and mitochondrial methylmalonyl-CoA mutase. Malfunction of these enzymes due to a scarcity of vitamin B12 leads to disturbance of one-carbon metabolism and impaired mitochondrial function. A significant fraction of the population (up to 20%) is deficient in vitamin B12, with a higher rate of deficiency among elderly people. B12 deficiency is associated with numerous hallmarks of aging at the cellular and organismal levels. Cellular senescence is characterized by high levels of DNA damage by metabolic abnormalities, increased mitochondrial dysfunction, and disturbance of epigenetic regulation. B12 deficiency could be responsible for or play a crucial part in these disorders. In this review, we focus on a comprehensive analysis of molecular mechanisms through which vitamin B12 influences aging. We review new data about how deficiency in vitamin B12 may accelerate cellular aging. Despite indications that vitamin B12 has an important role in health and healthy aging, knowledge of the influence of vitamin B12 on aging is still limited and requires further research.

Enzymatic Peptide and Protein Bromination: The BromoTrp Tag.

Bio-orthogonal reactions for modification of proteins and unprotected peptides are of high value in chemical biology. The combination of enzymatic halogenation with transition metal-catalyzed cross-coupling provides a feasible approach for the modification of proteins and unprotected peptides. By a semirational protein engineering approach, variants of the tryptophan 6-halogenase Thal were identified that enable efficient bromination of peptides with a C-terminal tryptophan residue. The substrate scope was explored using di-, tri-, and tetrapeptide arrays, leading to the identification of an optimized peptide tag we named BromoTrp tag. This tag was introduced into three model proteins. Preparative scale post-translational bromination was possible with only a single cultivation and purification step using the brominating E. coli coexpression system Brocoli. Palladium-catalyzed Suzuki-Miyaura cross-coupling of the bromoarene was achieved with Pd nanoparticle catalysts at 37 °C, highlighting the rich potential of this strategy for bio-orthogonal functionalization and conjugation.

Site-Selective Aryl Diazonium Installation onto Protein Surfaces at Neutral pH using a Maleimide-Functionalized Triazabutadiene.

Aryl diazonium cations are versatile bioconjugation reagents due to their reactivity towards electron-rich aryl residues and secondary amines, but historically their usage has been hampered by both their short lifespan in aqueous solution and the harsh conditions required to generate them in situ. Triazabutadienes address many of these issues as they are stable enough to endure multiple-step chemical syntheses and can persist for several hours in aqueous solution, yet upon UV-exposure rapidly release aryl diazonium cations under biologically-relevant conditions. This paper describes the synthesis of a novel maleimide-functionalized triazabutadiene suitable for site-selectively installing aryl diazonium cations into proteins at neutral pH; we show reaction with this molecule and a surface-cysteine of a thiol disulfide oxidoreductase. Through photoactivation of the site-selectively installed triazabutadiene motifs, we generate aryl diazonium functionality, which we further derivatize via azo-bond formation to electron-rich aryl species, showcasing the potential utility of this strategy for the generation of photoswitches or protein-drug conjugates.

Harnessing Split-Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells.

Biochemical studies of integral membrane proteins are often hampered by low purification yields and technical limitations such as aggregation causing in vitro manipulations to be challenging. The ability of controlling proteins in live cells bypasses these limitations while broadening the scope of accessible questions owing to the proteins being in their native environment. Here we take advantage of the intein biorthogonality to mammalian systems, site specificity, fast kinetics, and auto-processing nature as an attractive option for modifying surface proteins. Using EGFR as a model, we demonstrate that the split-intein pair AvaN /NpuC can be used to efficiently and specifically modify target membrane proteins with a synthetic adduct for downstream live cell application.

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.

Rapid On-Resin N-Formylation of Peptides as One-Pot Reaction.

N-formylation is a common pre- and post-translational modification of the N-terminus or the lysine side chain of peptides and proteins that plays a role in the initiation of immune responses, gene expression, or epigenetics. Despite its high biological relevance, protocols for the chemical N-formylation of synthetic peptides are scarce. The few available methods are elaborate in their execution and the yields are highly sequence-dependent. We present a rapid, easy-to-use one-pot procedure that runs at room temperature and can be used to formylate protected peptides at both the N-terminus and the lysine side chain on the resin in near-quantitative yields. Only insensitive, storage-stable standard chemicals - formic acid, acetic anhydride, pyridine and DMF - are used. Formylation works for both short and long peptides of up to 34 amino acids and over the spectrum of canonical amino acids.

Chemical Synthesis of Alpha-Synuclein Proteins via Solid-Phase Peptide Synthesis and Native Chemical Ligation.

Alpha-Synuclein (α-Synuclein) is a 140 amino acid protein implicated in neurodegenerative disorders known as synucleinopathies, where it accumulates in proteinaceous inclusions in the brain. The normal physiological function of α-Synuclein remains obscure, as it exists in several non-neuronal cells in which its function has not been studied. Given the tremendous interest in studying α-Synuclein, and the existing limitations in the production of modified forms of the protein, we developed a method for the chemical synthesis of α-Synuclein by combining peptide fragment synthesis via automated microwave-assisted solid-phase peptide synthesis and ligation strategies. Our synthetic pathway enables the synthesis of protein variants of interest, carrying either mutations or posttranslational modifications, for further investigations of the effects on the structure and aggregation behavior of the protein. Ultimately, our study forms the foundation for future syntheses and studies of other custom-made α-Synuclein variants with a single or several modifications, as necessary.

Histidine Photooxygenation Chemistry: Mechanistic Evidence and Elucidation.

Histidine photooxygenation has been the subject of extensive investigation for many years. The intricate nature of histidine distinguishes it from other amino acids, as its side chain readily undergoes changes in charge state and tautomerization in response to pH, and the polarity of the imidazole ring inverts upon oxidation. This complexity gives rise to a diverse range of oxidation products and mechanisms, posing challenges in their interpretation. This review aims to provide a thorough overview of the chemistry involved in histidine photooxygenation, encompassing a comprehensive analysis of resulting products, mechanisms engaged in their formation, and analytical techniques that have contributed to their identification. Additionally, it explores a wide range of applications stemming from this transformation, offering valuable insights into its practical implications in fields such as materials science, biomedical research, and drug development. By bridging the existing gap in literature, this review serves as a resource for understanding the intricacies of histidine photooxygenation and its diverse ramifications.

"Slight chemical damage due to drinking modest amount of sake, might induce beneficial effects" as a form of hormesis: an interview with Professor Sataro Goto.

Professor Sataro Goto is one of the pioneers of biological aging research in Japan. He is renowned for his work on the role of protein errors and modifications, the accumulation of abnormal proteins due to reduced protein turnover, and the modulation of aging and lifespan by adult-onset dietary restriction and regular exercise. Professor Goto is a remarkably intelligent, visionary, empathetic, humble, and wise man, who kindly agreed to this interview that I (Zsolt Radak) made with him during one of my frequent visits to his labs, in February 2023.

Deacetylation of Histones and Non-histone Proteins in Inflammatory Diseases and Cancer Therapeutic Potential of Histone Deacetylase Inhibitors.

Epigenetic changes play an important role in the pathophysiology of autoimmune diseases such as allergic asthma, multiple sclerosis, lung diseases, diabetes, cystic fibrosis, atherosclerosis, rheumatoid arthritis, and COVID-19. There are three main classes of epigenetic alterations: post-translational modifications of histone proteins, control by non-coding RNA and DNA methylation. Since histone modifications can directly affect chromatin structure and accessibility, they can regulate gene expression levels. Abnormal expression and activity of histone deacetylases (HDACs) have been reported in immune mediated diseases. Increased acetylated levels of lysine residues have been suggested to be related to the overexpression of inflammatory genes. This review focuses on the effect of HDAC modifications on histone and non-histone proteins in autoimmune diseases. Furthermore, we discuss the potential therapeutic effect of HDAC inhibitors (HDACi) used in these diseases.

Activity-based Tools for Interrogating Host Biology During Infection.

Host cells sense and respond to pathogens by dynamically regulating cell signaling. The rapid modulation of signaling pathways is achieved by post-translational modifications (PTMs) that can alter protein structure, function, and/or binding interactions. By using chemical probes to broadly profile changes in enzyme function or side-chain reactivity, activity-based protein profiling (ABPP) can reveal PTMs that regulate host-microbe interactions. While ABPP has been widely utilized to uncover microbial mechanisms of pathogenesis, in this review, we focus on more recent applications of this technique to the discovery of host PTMs and enzymes that modulate signaling within infected cells. Collectively, these advances underscore the importance of ABPP as a tool for interrogating the host response to infection and identifying potential targets for host-directed therapies.

Protein modifications due to homogenisation and heat treatment of cow milk.

This research paper aimed to locate protein modifications caused by treatment of milk and determine if the modification locations were consistent. The majority of milk for consumption is homogenised using pressure and heat, and this causes changes in the location of proteins in the milk as well as protein modifications. To investigate these proteomic changes, raw milk was pasteurised (72°C, 15 s), then, to separate the treatment for homogenisation, heated at these different pressures and temperatures: 45°C without no pressure applied, 45°C with 35 MPa, 80°C without pressure applied and 80°C, with 35 MPa. Proteomic analysis was done after separating the milk into three fractions: whey, casein and cream. Protein modifications in each fraction were examined and we found Maillard products as well as oxidation to be of interest. The proteins were also further identified and characterised to compare protein modification sites and differences in proteins present in the cream resulting from homogenisation and/or pasteurisation. This experiment showed that both heat and pressure during homogenisation can cause increases in protein modifications as a result of oxidation or the Maillard reaction. Total cysteine oxidation and total proline oxidation differed between treatments although this was only significantly different for cysteine. It was observed that protein modifications occurred in the same location in the protein sequence rather than in random locations which we highlighted by examining α-S1-casein, lactadherin and ß-lactoglobulin.