Modification Of Amino Acid Residues Research Articles

Engineered Nanobodies Bind Bismuth, Indium and Gallium for Applications in Theranostics.

Targeted theranostics heavily rely on metal isotopes conjugated to antibodies. Single-domain antibodies, known as nanobodies, are much smaller in size without compromising specificity and affinity. The conventional way of conjugating metals to nanobodies involves non-specific modification of amino acid residues with bifunctional chelating agents. We demonstrate that mutagenesis of a single residue in a nanobody creates a triple cysteine motif that selectively binds bismuth which is, for example, used in targeted alpha therapy. Two mutations create a quadruple cysteine mutant specific for gallium and indium used in positron emission tomography and single-photon emission computed tomography, respectively. Labelling is quantitative within a few minutes. The metal nanobodies maintain structural integrity and stability over weeks, resist competition from endogenous metal binders like glutathione, and retain functionality.

Engineered Nanobodies Bind Bismuth, Indium and Gallium for Applications in Theranostics

Targeted theranostics heavily rely on metal isotopes conjugated to antibodies. Single‐domain antibodies, known as nanobodies, are much smaller in size without compromising specificity and affinity. The conventional way of conjugating metals to nanobodies involves non‐specific modification of amino acid residues with bifunctional chelating agents. We demonstrate that mutagenesis of a single residue in a nanobody creates a triple cysteine motif that selectively binds bismuth which is, for example, used in targeted alpha therapy. Two mutations create a quadruple cysteine mutant specific for gallium and indium used in positron emission tomography and single‐photon emission computed tomography, respectively. Labelling is quantitative within a few minutes. The metal nanobodies maintain structural integrity and stability over weeks, resist competition from endogenous metal binders like glutathione, and retain functionality.

The effect of hypochlorite-induced fibrinogen oxidation on the protein structure, fibrin self-assembly and fibrinolysis

The article is dedicated to the structural-functional damage of fibrinogen treated with HOCl in the concentration range (10–100 µM). The MS/MS method detected 15 modified amino acid residues with a dose-dependent susceptibility to the oxidizing agent. Using turbidity measurements and confocal laser scanning microscopy, it has been shown that fibrinogen oxidation by 25–100 µM HOCl leads to the denser fibrin gel formation, as well as delayed polymerization onset and a decrease in the slope of the polymerization curve, presumably due to conformational changes of the protein. At lower HOCl concentration (10 µM), at least six amino acid residues were substantially modified (9–29%), but functionally such modified protein was not distinguishable from the native one. The detected amino acid residues are assumed to be ROS scavengers that prevent fibrinogen functions alteration.

Optimized carbonylation treatment of Litopenaeus vannamei matrix decreased its immunoreactivity and improved edible quality, simultaneously

The study aimed to investigate how carbonylation affects the immunoreactivity and edible quality of the Litopenaeus vannamei matrix. The carbonylation treatment conditions of the shrimp matrix were optimized. Firstly, the treatment condition is optimized with 1.0 mmol/L malonaldehyde at 37 °C, 12 h. The optimized carbonylated shrimp showed lower immunoreactivity, carbonyl group, and free amino acids. Then the edible quality was evaluated, optimized carbonylated shrimp matrix presented better digestibility and the continuous digestion products showed lower immunoreactivity. Optimized carbonylated shrimp for the other sensory indicators showed better texture properties and an inviting appearance. Looser microstructure by scanning electron microscopy contributed to the higher digestibility, lower immunoreactivity, and better edible quality for optimized carbonylated shrimp matrix. Besides, more potentially modified amino acid residues exposed on the allergen surface may be the other reason. In conclusion, optimized carbonylation treatment reduced the immunoreactivity and improved the edible quality of shrimp.

Engineering of Cas12a nuclease variants with enhanced genome-editing specificity.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system is a powerful tool in gene editing; however, crRNA-DNA mismatches might induce unwanted cleavage events, especially at the distal end of the PAM. To minimize this limitation, we engineered a hyper fidelity AsCas12a variant carrying the mutations S186A/R301A/T315A/Q1014A/K414A (termed HyperFi-As) by modifying amino acid residues interacting with the target DNA and crRNA strand. HyperFi-As retains on-target activities comparable to wild-type AsCas12a (AsCas12aWT) in human cells. We demonstrated that HyperFi-As has dramatically reduced off-target effects in human cells, and HyperFi-As possessed notably a lower tolerance to mismatch at the position of the PAM-distal region compared with the wild type. Further, a modified single-molecule DNA unzipping assay at proper constant force was applied to evaluate the stability and transient stages of the CRISPR/Cas ribonucleoprotein (RNP) complex. Multiple states were sensitively detected during the disassembly of the DNA-Cas12a-crRNA complexes. On off-target DNA substrates, the HyperFi-As-crRNA was harder to maintain the R-loop complex state compared to the AsCas12aWT, which could explain exactly why the HyperFi-As has low off-targeting effects in human cells. Our findings provide a novel version of AsCas12a variant with low off-target effects, especially capable of dealing with the high off-targeting in the distal region from the PAM. An insight into how the AsCas12a variant behaves at off-target sites was also revealed at the single-molecule level and the unzipping assay to evaluate multiple states of CRISPR/Cas RNP complexes might be greatly helpful for a deep understanding of how CRISPR/Cas behaves and how to engineer it in future.

Post-Translational Oxidative Modifications of Hemostasis Proteins: Structure, Function, and Regulation.

Reactive oxygen species (ROS) are constantly generated in a living organism. An imbalance between the amount of generated reactive species in the body and their destruction leads to the development of oxidative stress. Proteins are extremely vulnerable targets for ROS molecules, which can cause oxidative modifications of amino acid residues, thus altering structure and function of intra- and extracellular proteins. The current review considers the effect of oxidation on the structural rearrangements and functional activity of hemostasis proteins: coagulation system proteins such as fibrinogen, prothrombin/thrombin, factor VII/VIIa; anticoagulant proteins - thrombomodulin and protein C; proteins of the fibrinolytic system such as plasminogen, tissue plasminogen activator and plasminogen activator inhibitor-1. Structure and function of the proteins, oxidative modifications, and their detrimental consequences resulting from the induced oxidation or oxidative stress in vivo are described. Possible effects of oxidative modifications of proteins in vitro and in vivo leading to disruption of the coagulation and fibrinolysis processes are summarized and systematized, and the possibility of a compensatory mechanism in maintaining hemostasis under oxidative stress is analyzed.

Electrochemical diversification of cysteine derivatives and cysteine-containing peptides to phosphorothioates and sulfinates

This paper aims to explore the applications of modified cysteine in biomedicine, addressing the challenges as-sociated with modifying amino acid residues (cysteine). It provides new avenues for the functionalization of...

Efficient synthetic methods for α,β-dehydroamino acids as useful and environmentally benign building blocks in biological and materials science.

Both natural and unnatural amino acids, peptides, and proteins are widely recognized as green and sustainable organic chemicals, not only in the field of biological sciences but also in materials science. It has been discovered that artificially designed unnatural peptides and proteins exhibit advanced properties in medical and materials science. In this context, the development of precise chemical modification methods for amino acids and peptides is acknowledged as an important research project in the field of organic synthesis. While a wide variety of modification methods for amino acid residues have been developed to artificially modify peptides and proteins, the representative methods for modifying amino acid residues have traditionally relied on the nucleophilic properties of the functionalities on the residues. In this context, the development of different modification methods using an umpolung-like approach by utilizing the electrophilic nature of amino acid derivatives appears to be very attractive. One of the promising electrophilic amino acid compounds for realizing important modification methods of amino acid derivatives is α,β-dehydroamino acids, which possess an α,β-unsaturated carbonyl structure. This review article summarizes methods for the preparation of α,β-dehydroamino acids derived from natural and unnatural amino acid derivatives. The utilities of α,β-dehydroamino acid derivatives, including peptides and proteins containing dehydroalanine units, in bioconjugations are also discussed.

Insight into the mechanism of the decrease in mechanical strength and water-holding capacity of gels made from oxidized gelatin

The purpose of this study was to investigate the effect of oxidation on the physicochemical properties of gelatin and gelatin gels. Porcine skin gelatin was oxidized with different concentrations of H2O2 (0–30 mM). Upon oxidation of gelatin, a significant modification of amino acid residues including glycine, proline, hydroxyproline, and hydroxylysine occurred. Zeta-potential, ordered secondary structure and the fraction of triple-helix decreased, while particle size and surface hydrophobicity increased. Gels made from oxidized gelatin showed a looser network structure indicated by scanning electron microscope, and the gels had a weakened mechanical strength and water-holding as compared to non-oxidized gelatin gels. Based on these results, a mechanism of how oxidation affects the gelatin gel properties was proposed: Oxidation-induced increase of hydrophobicity and decrease of net charges promoted aggregation between gelatin molecules, thereby limiting the formation of triple-helix, which subsequently leads to a loose network structure and eventually a weakened gel strength and water-holding capacity.

Covalent Stapling of the Cereblon Sensor Loop Histidine Using Sulfur-Heterocycle Exchange.

Site-specific modification of amino acid residues in protein binding pockets using sulfonyl exchange chemistry expands the druggable proteome by enabling the development of covalent modulators that target residues beyond cysteine. Sulfonyl fluoride and triazole electrophiles were incorporated previously into the cereblon (CRBN) molecular glue degrader EM12, to covalently engage His353 within the CRBN sensor loop, but these probes had poor human plasma stability. Attenuation of intrinsic reactivity through the development of sulfonyl pyrazoles, imidazoles, and nucleobases enhanced plasma stability, and several compounds retained efficient labeling of His353. For example, sulfonyl imidazole EM12-SO2Im covalently blocked the CRBN binding site and possessed excellent metabolic stability in human plasma, liver microsomes, and hepatocytes. These results highlight the potential suitability of sulfonyl imidazole and related sulfur(VI)-diazole exchange (SuDEx) warheads for covalent drug development and further exemplify the therapeutic promise of site-specific histidine targeting.

Structural and biological characterization of shortened derivatives of the cathelicidin PMAP-36

Cathelicidins, a family of host defence peptides in vertebrates, play an important role in the innate immune response, exhibiting antimicrobial activity against many bacteria, as well as viruses and fungi. This work describes the design and synthesis of shortened analogues of porcine cathelicidin PMAP-36, which contain structural changes to improve the pharmacokinetic properties. In particular, 20-mers based on PMAP-36 (residues 12-31) and 13-mers (residues 12-24) with modification of amino acid residues at critical positions and introduction of lipid moieties of different lengths were studied to identify the physical parameters, including hydrophobicity, charge, and helical structure, required to optimise their antibacterial activity. Extensive conformational analysis, performed by CD and NMR, revealed that the substitution of Pro25-Pro26 with Ala25-Lys26 increased the α-helix content of the 20-mer peptides, resulting in broad-spectrum antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus epidermidis strains. Interestingly, shortening to just 13 residues resulted in only a slight decrease in antibacterial activity. Furthermore, two sequences, a 13-mer and a 20-mer, did not show cytotoxicity against HaCat cells up to 64 µM, indicating that both derivatives are not only effective but also selective antimicrobial peptides. In the short peptide, the introduction of the helicogenic α-aminoisobutyric acid forced the helix toward a prevailing 310 structure, allowing the antimicrobial activity to be maintained. Preliminary tests of resistance to Ser protease chymotrypsin indicated that this modification resulted in a peptide with an increased in vivo lifespan. Thus, some of the PMAP-36 derivatives studied in this work show a good balance between chain length, antibacterial activity, and selectivity, so they represent a good starting point for the development of even more effective and proteolysis-resistant active peptides.

Peptide Stapling Applied to Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are considered a promising therapeutic approach against multi-drug resistant microorganisms. Besides their advantages, there are limitations to be overcome so that these molecules can become market competitive. One of the biggest limitations is proteolytic susceptibility, which could be overcome by structural modifications such as cyclization, especially for helix-constraining strategies. Over the years, many helix stabilization techniques have arisen, such as lactam-bridging, triazole-based, N-alkylation and all-hydrocarbon stapling. All-hydrocarbon stapling takes advantage of modified amino acid residues and olefinic cross-linking to constrain peptide helices. Despite being a well-established strategy and presenting efficient stability results, there are different limitations especially related to toxicity. In this review, recent studies on stapled AMPs for antimicrobial usage are explored with the aim of understanding the future of these molecules as putative antimicrobial agents.

In Silico Analysis of Individual Fractions of Bovine Casein as Precursors of Bioactive Peptides—Influence of Post-Translational Modifications

Bovine casein is one of the most known precursors of bioactive peptides among food proteins. Thus far, in silico investigations addressing casein have taken no account of the impact of modifications of amino acid residues on the feasibility of bioactive peptide release. The present study aimed to determine the effect of such modification on the possibility of release of bioactive peptides from casein during simulated digestion. The αs1-, αs2-, β-, and κ-casein sequences were deposited in the BIOPEP-UWM protein database considering phosphorylated amino acids, cysteine residues forming disulfide bridges, and pyroglutamic acid residues. The frequency of occurrence of bioactive fragments and the frequency of their release by digestive enzymes were determined for the analyzed modified and unmodified proteins. Peptides found exclusively in the sequences of unmodified proteins were deemed as false-positive results. From 1.74% (β-casein A2) to 4.41% (αs2-casein B and D) of the false-positive results were obtained for the total frequency of occurrence of bioactive fragments (sums of frequencies computed for all activities). In turn, from 1.78% (κ-casein B) to 9.18% (β-casein A2 and A3) of false-positive results were obtained for the predicted total frequency of release of bioactive peptides by the system of digestive enzymes (pepsin, trypsin, and chymotrypsin).

Exploring Chemical Modifications of Aromatic Amino Acid Residues in Peptides

AbstractThe chemical diversification of biomolecules set forth a significant area of research that constitutes an important intersection between chemistry and biology. Amino acids and peptides are the fundamental building blocks of proteins and play essential roles in all living organisms. While significant efforts have been geared toward the chemical modification of amino acid residues, particularly the functionalization of reactive functional groups such as lysine NH2 and cysteine SH, the exploration of the aromatic amino acid residues of tryptophan, tyrosine, phenylalanine, and histidine has been relatively limited. Therefore, this review highlights strategies for the side-chain functionalization of these four aromatic amino acids in peptides, with a focus on elucidating the underlying mechanisms. We have also illustrated the use of these modifications in the chemical and biological realm.1 Introduction2 Tryptophan Modifications3 Tyrosine Modifications4 Phenylalanine Modifications5 Histidine Modifications6 Perspectives and Future Outlook

Y-mtPTM: Yeast mitochondrial posttranslational modification database.

One powerful strategy of how to increase the complexity of cellular proteomes is through posttranslational modifications (PTMs) of proteins. Currently, there are ∼400 types of PTMs, the different combinations of which yield a large variety of protein isoforms with distinct biochemical properties. Although mitochondrial proteins undergoing PTMs were identified nearly 6 decades ago, studies on the roles and extent of PTMs on mitochondrial functions lagged behind the other cellular compartments. The application of mass spectrometry for the characterization of the mitochondrial proteome as well as for the detection of various PTMs resulted in the identification of thousands of amino acid positions that can be modified by different chemical groups. However, the data on mitochondrial PTMs are scattered in several data sets, and the available databases do not contain a complete list of modified residues. To integrate information on PTMs of the mitochondrial proteome of the yeast Saccharomyces cerevisiae, we built the yeast mitochondrial posttranslational modification (y-mtPTM) database (http://compbio.fmph.uniba.sk/y-mtptm/). It lists nearly 20,000 positions on mitochondrial proteins affected by ∼20 various PTMs, with phosphorylated, succinylated, acetylated, and ubiquitylated sites being the most abundant. A simple search of a protein of interest reveals the modified amino acid residues, their position within the primary sequence as well as on its 3D structure, and links to the source reference(s). The database will serve yeast mitochondrial researchers as a comprehensive platform to investigate the functional significance of the PTMs of mitochondrial proteins.

Technologies for Direct Detection of Covalent Protein-Drug Adducts.

In the past two decades, drug candidates with a covalent binding mode have gained the interest of medicinal chemists, as several covalent anticancer drugs have successfully reached the clinic. As a covalent binding mode changes the relevant parameters to rank inhibitor potency and investigate structure-activity relationship (SAR), it is important to gather experimental evidence on the existence of a covalent protein-drug adduct. In this work, we review established methods and technologies for the direct detection of a covalent protein-drug adduct, illustrated with examples from (recent) drug development endeavors. These technologies include subjecting covalent drug candidates to mass spectrometric (MS) analysis, protein crystallography, or monitoring intrinsic spectroscopic properties of the ligand upon covalent adduct formation. Alternatively, chemical modification of the covalent ligand is required to detect covalent adducts by NMR analysis or activity-based protein profiling (ABPP). Some techniques are more informative than others and can also elucidate the modified amino acid residue or bond layout. We will discuss the compatibility of these techniques with reversible covalent binding modes and the possibilities to evaluate reversibility or obtain kinetic parameters. Finally, we expand upon current challenges and future applications. Overall, these analytical techniques present an integral part of covalent drug development in this exciting new era of drug discovery.

Murburn posttranslational modifications of proteins: Cellular redox processes and murzyme-mediated metabolo-proteomics.

Murburn concept constitutes the thesis that diffusible reactive species or DRS are obligatorily involved in routine metabolic and physiological activities. Murzymes are defined as biomolecules/proteins that generate/modulate/sustain/utilize DRS. Murburn posttranslational modifications (PTMs) result because murburn/murzyme functionalism is integral to cellular existence. Cells must incorporate the inherently stochastic nature of operations mediated by DRS. Due to the earlier/inertial stigmatic perception that DRS are mere agents of chaos, several such outcomes were either understood as deterministic modulations sponsored by house-keeping enzymes or deemed as unregulated nonenzymatic events resulting out of "oxidative stress". In the current review, I dispel the myths around DRS-functions, and undertake systematic parsing and analyses of murburn modifications of proteins. Although it is impossible to demarcate all PTMs into the classical or murburn modalities, telltale signs of the latter are evident from the relative inaccessibility of the locus, non-specificities and mechanistic details. It is pointed out that while many murburn PTMs may be harmless, some others could have deleterious or beneficial physiological implications. Some details of reversible/irreversible modifications of amino acid residues and cofactors that may be subjected to phosphorylation, halogenation, glycosylation, alkylation/acetylation, hydroxylation/oxidation, etc. are listed, along with citations of select proteins where such modifications have been reported. The contexts of these modifications and their significance in (patho)physiology/aging and therapy are also presented. With more balanced explorations and statistically verified data, a definitive understanding of normal versus pathological contexts of murburn modifications would be obtainable in the future.

A Program for Predicting the Retention Time of Peptides with Post-Translational Modifications

This paper describes the Retention Time Predictor (RTP) program and web service for predicting the retention time of peptides on a chromatographic column in mass spectrometry experiments. Taking into account post-translational modifications of peptides the program represents a modification of the well-known SSRCalc version 3 (Krokhin, Anal. Chem. 2006, 78(22), 7785-7795). The values of retention coefficients for modified amino acid residues and the algorithm for calculating the isoelectric point value were from the pIPredict program (Skvortsov et al., Biomed. Chem. Res. Meth. 2021, 4(4), e00161). Modifications described in the program include (i) Tandem Mass Tag (TMT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ) labels; (ii) acetylation, formylation, and methylation of the N-terminal residue and/or lysine side chain; (iii) carbamidomethylation of cysteine, asparagine, and glutamic acid residues; (iv) oxidation and double oxidation of methionine and proline residues; (v) phosphorylation of serine, threonine, and tyrosine residues; (vi) C-terminal amidation of lysine and arginine residues; (vii) formation of propionamide with a cysteine residue. Retention coefficient estimation was based on data from 25 mass spectrometry experiments for which identification was performed from the raw data deposited in the ProteomeXchange database. The RTP program and web service are freely available at http://lpcit.ibmc.msk.ru/RTP.

Contingent Synergistic Interactions between Non-Coding RNAs and DNA-Modifying Enzymes in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders with maturation and differentiation defects exhibiting morphological dysplasia in one or more hematopoietic cell lineages. They are associated with peripheral blood cytopenias and by increased risk for progression into acute myelogenous leukemia. Among their multifactorial pathogenesis, age-related epigenetic instability and the error-rate DNA methylation maintenance have been recognized as critical factors for both the initial steps of their pathogenesis and for disease progression. Although lower-risk MDS is associated with an inflammatory bone marrow microenvironment, higher-risk disease is delineated by immunosuppression and clonal expansion. "Epigenetics" is a multidimensional level of gene regulation that determines the specific gene networks expressed in tissues under physiological conditions and guides appropriate chromatin rearrangements upon influence of environmental stimulation. Regulation of this level consists of biochemical modifications in amino acid residues of the histone proteins' N-terminal tails and their concomitant effects on chromatin structure, DNA methylation patterns in CpG dinucleotides and the tissue-specific non-coding RNAs repertoire, which are directed against various gene targets. The role of epigenetic modifications is widely recognized as pivotal both in gene expression control and differential molecular response to drug therapies in humans. Insights to the potential of synergistic cooperations of epigenetic mechanisms provide new avenues for treatment development to comfort human diseases with a known epigenetic shift, such as MDS. Hypomethylating agents (HMAs), such as epigenetic modulating drugs, have been widely used in the past years as first line treatment for elderly higher-risk MDS patients; however, just half of them respond to therapy and are benefited. Rational outcome predictors following epigenetic therapy in MDS and biomarkers associated with disease relapse are of high importance to improve our efforts in developing patient-tailored clinical approaches.

One enzyme, many faces: urease is also canatoxin

Ureases catalyze the hydrolysis of urea into carbamate and ammonia. Well-conserved proteins, most plant ureases are hexamers of a single chain subunit, like the most abundant isoform of the jack bean (Canavalia ensiformis) urease (JBU). Canatoxin (CNTX) was originally isolated from these seeds as a neurotoxic protein, and later characterized as an isoform of JBU with lower molecular mass and enzyme activity. Inactive CNTX oligomers form upon storage and stabilization of CNTX was achieved by treatment with low concentration of formaldehyde, avoiding its oligomerization. Here, nano-LC-MS/MS-based peptide analysis of CNTX revealed 804 amino acids identical to those of JBU’s sequence (840 amino acids). De novo sequencing of CNTX revealed 15 different peptides containing substitution of amino acid residues, denoting CNTX as a product of a paralog gene of JBU. The MS/MS analysis of formaldehyde-treated CNTX showed that amino acid residues located at the trimer–trimer interface of JBU’s hexamer were modified. The data confirmed that CNTX is an isoform of JBU and elucidated that stabilization by formaldehyde treatment occurs by modification of amino acids at the protein’s surface that prevents the formation of the hexamer and of higher molecular mass inactive aggregates. HIGHLIGHTS Canatoxin (CNTX) is an isoform of jack bean urease (JBU, hexamer of 90 kDa chains) MS/MS sequencing of CNTX showed 804 amino acids identical in JBU (840 residues) Formaldehyde treatment of CNTX stabilizes its toxicity and avoids oligomerization Modified amino acid residues in CNTX are at the trimer–trimer interface of JBU Communicated by Ramaswamy H. Sarma