Cysteine Side Chains Research Articles

PTPN2 copper-sensing relays copper level fluctuations into EGFR/CREB activation and associated CTR1 transcriptional repression

Fluxes in human copper levels recently garnered attention for roles in cellular signaling, including affecting levels of the signaling molecule cyclic adenosine monophosphate. We herein apply an unbiased temporal evaluation of the signaling and whole genome transcriptional activities modulated by copper level fluctuations to identify potential copper sensor proteins responsible for driving these activities. We find that fluctuations in physiologically relevant copper levels modulate EGFR signal transduction and activation of the transcription factor CREB. Both intracellular and extracellular assays support Cu1+ inhibition of the EGFR phosphatase PTPN2 (and potentially PTPN1)–via ligation to the PTPN2 active site cysteine side chain–as the underlying mechanism. We additionally show i) copper supplementation drives weak transcriptional repression of the copper importer CTR1 and ii) CREB activity is inversely correlated with CTR1 expression. In summary, our study reveals PTPN2 as a physiological copper sensor and defines a regulatory mechanism linking feedback control of copper stimulated EGFR/CREB signaling and CTR1 expression.

Self-Promoted Glycosylation of Carbamoylated Peptides on Solid Phase.

Self-promoted glycosylations with trichloroacetimidate glycosyl donors are demonstrated on solid-phase-anchored peptides orthogonally deprotected and tosylcarbamoylated on the side-chains of cysteine and serine, respectively. The donor scope included glucosyl as well as mannosyl trichloroacetimidates, carrying benzyl, acetyl, or isopropylidene protecting groups. Isopropylidene groups were found to be removed under the acidic conditions used for release of the neoglycopeptides from the solid support, yielding neoglycopeptides with unprotected hydroxyl groups. Glycosylation of a peptide containing a carbamoylated tyrosine was attempted as well, but the desired neoglycopeptide could not be synthesized due to thermal instability of the carbamate.

THz spectroscopy on the amino acids L-serine and L-cysteine.

We present a detailed study on the temperature-dependent THz spectra of the polycrystalline amino acids, L-serine and L-cysteine, for wavenumbers from 20 to 120cm-1 and temperatures from 4 to 300K. Even though the structure of these two amino acids is very similar, with a sulfur atom in the side chain of cysteine instead of an oxygen atom in serine, the excitation spectra are drastically different. Obviously, the vibrational dynamics strongly depend on the ability of cysteine to form sulfur-hydrogen bonds. In addition, cysteine undergoes an order-disorder type phase transition close to 80K, documented by additional specific heat experiments, with accompanying anomalies in the THz results. On increasing temperatures, well-defined vibrational excitations exhibit significant shifts in the eigenfrequencies with concomitant line-broadening yielding partly overlapping modes. Interestingly, several modes completely lose all their dipolar strength and are unobservable under ambient conditions. Comparing the recent results to the published work utilizing THz, Raman, and neutron-scattering techniques, as well as with abinitio simulations, we aim at a consistent analysis of the results ascribing certain eigenfrequencies to distinct collective lattice modes. We document that THz spectra can be used to fine-tune the parameters of model calculations and as fingerprint properties of certain amino acids. In addition, we analyzed the low-temperature heat capacity of both the compounds and detected strong excess contributions compared to the canonical Debye behavior of crystalline solids, indicating soft excitations and a strongly enhanced phonon-density of states at low frequencies.

Structural determination of Zn2+, Cu2+, and Fe2+ complexed with glutathione by IRMPD spectroscopy and complimentary ab initio calculations.

Glutathione is a biologically abundant and redox active tripeptide that serves to protect cells from oxidative stress and rid the body of toxic heavy metals. The present study examines the coordination complexes of glutathione (GSH) with metals that are central to redox processes in biology, Zn, Cu, and Fe, using infrared multiple photon dissociation (IRMPD) action spectroscopy with a free electron laser. For all three metals, a complex between the metal dication and deprotonated GSH was formed, M(GSH-H)+. The experimental IRMPD spectra were compared to scaled harmonic vibrational spectra calculated at the MP2/6-311+G(d,p) level of theory after thorough exploration of conformational space using a simulated annealing protocol. Interestingly, spectra calculated at the B3LYP or ωB97XD level do not match experiment as well. These findings offer the first gas-phase spectroscopic evidence for how the biologically relevant metal ions coordinate with glutathione. There are spectral features that are common to all three metals, however, noting the differences in the strengths of the common features between the three metals enables an assessment of the preference or specificity that each individual metal has for a given coordination site. Additionally, all three metals form structures where the deprotonated thiol of the cysteine side chain coordinates with the metal center, which is consistent with the involvement of the thiol site in biologically relevant redox chemistry.

De-polymerisation and hydrolysis of seed storage protein is involved in the freezing tolerance of hydrated Brassica oleracea seeds

Hydrolysis of globulins might be involved in the freezing tolerance of hydrated lettuce seeds. The mechanism was further explored in hydrated brassica (Brassica oleracea) seeds, by application of 5,5'-dithiobis(2-nitro- benzoicacid) (DTNB), the Ellman's reagent, which can bind to the free sulphydryl side chain of cysteine. DTNB increased the viability of hydrated brassica seeds after slow cooling (-3°C hour-1 ) to -17°C. Two-dimensional electrophoresis and mass spectra analysis indicated the accumulation of small peptides derived from 12S globulins (cruciferin) in DTNB-treated seeds after slow cooling. The seed storage peptides with different molecular weight were cooled and the ice formation during cooling observed by cryomicroscopy, which proved that peptides of smaller molecular size produced smaller and rounder crystals. The smallest peptide in DTNB- treated seeds produced the smallest and roundest crystals during cooling. The levels of ASA, DHA and GSH were specifically up-regulated after DTNB treatment and following slow cooling, which indicates that DTNB enhanced the circulation of ASA and GSH to resist the oxidative stress. Consistent with this finding, RNA-seq and real time PCR indicated that glutathione reductase (GR) was up-regulated after DTNB treatment.

A simple method for developing lysine targeted covalent protein reagents

Peptide-based covalent probes can target shallow protein surfaces not typically addressable using small molecules, yet there is a need for versatile approaches to convert native peptide sequences into covalent binders that can target a broad range of residues. Here we report protein-based thio-methacrylate esters—electrophiles that can be installed easily on unprotected peptides and proteins via cysteine side chains, and react efficiently and selectively with cysteine and lysine side chains on the target. Methacrylate phosphopeptides derived from 14-3-3-binding proteins irreversibly label 14-3-3σ via either lysine or cysteine residues, depending on the position of the electrophile. Methacrylate peptides targeting a conserved lysine residue exhibit pan-isoform binding of 14-3-3 proteins both in lysates and in extracellular media. Finally, we apply this approach to develop protein-based covalent binders. A methacrylate-modified variant of the colicin E9 immunity protein irreversibly binds to the E9 DNAse, resulting in significantly higher thermal stability relative to the non-covalent complex. Our approach offers a simple and versatile route to convert peptides and proteins into potent covalent binders.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Substituted Cysteine Modification and Protection with n-Alkyl-MTS Reagents Quantifies Steric Changes Induced by a Mutation in Anesthetic Binding Sites on GABA Type A Receptors.

Multiple approaches, including cryogenic electron microscopy (cryo-EM), indicate that the anesthetics etomidate and propofol modulate α1β2/3γ2 GABAA receptors by binding in overlapping transmembrane inter-subunit sites near βM286 and αL232 sidechains. High-precision approaches in functional receptors are needed for comparisons with cryo-EM. We previously used substituted cysteine modification and protection (SCAMP) with n-alkyl-methanethiosulfonate (MTS) reagents and electrophysiology in α1β3M286Cγ2L receptors to estimate the distance from etomidate to β3M286 with precision near 1.3 Å. Here, we address three more aims using this approach: (i) SCAMP with etomidate was tested in α1L232Cβ3γ2L receptors; (ii) studies in α1L232Wβ3M286Cγ2L receptors assessed whether α1L232W displaces etomidate relative to β3M286C; and (iii) results with propofol were compared with those with etomidate. Voltage-clamp electrophysiology in Xenopus oocytes was used to assess persistent functional changes after exposing cysteine-substituted receptors to methyl-MTS through n-decyl-MTS. Overlap of modified cysteine sidechains with bound anesthetic was inferred when anesthetic co-application with alkyl-MTS reagent blocked the development of persistent effects. In α1L232Cβ3γ2L receptors, only pentyl-MTS and hexyl-MTS induced persistent effects that were unaltered by etomidate co-application, precluding a direct estimate of intermolecular distance. In α1L232Wβ3M286Cγ2L receptors, sidechain overlap with bound etomidate was inferred for modifications with ethyl-MTS through n-pentyl-MTS, with unambiguous cut-on and cut-off. Comparison with results in α1β3M286Cγ2L reveals that α1L232W, which increases maximal sidechain length by 2.1 Å, displaces etomidate closer to β3M286C by about 1.3 Å. Propofol results largely mirrored those with etomidate. These findings indicate that both etomidate and propofol bind within 1 Å of α1L232, consistent with cryo-EM structures. SIGNIFICANCE STATEMENT: We combined electrophysiology, cysteine substitutions, and n-alkyl-methanethiosulfonate modifiers in functional GABAA receptors to enable precise estimates of the distance between β3M286C sidechains and anesthetics (etomidate and propofol) bound in transmembrane β+/α- inter-subunit pockets. Comparing results in α1β3M286Cγ2L and α1L232Wβ3M286Cγ2L receptors reveals that α1L232W mutations displace both anesthetics toward β3M286C, indicating that these anesthetics bind within 1 Å of the α1L232 sidechain in functional receptors, consistent with cryogenic electron microscopy structures derived under nonphysiologic conditions.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

Structural characterisation and pH-dependent preference of pyrrole cross-link isoforms from reactions of oxoenal with cysteine and lysine side chains as model systems.

In this study, we subjected 5,5-diethoxy-4-oxopent-2-enal (DOPE), a model amino acids cross-linking reagent, to reactions with N-acetylcysteine (Ac-Cys) and Nα-acetyllysine (Ac-Lys), and identified three pyrrole cross-links. The compounds were isolated and their structures were rigorously determined by spectrometric and spectroscopic methods, including 2D NMR experiments. The use of 2D NMR spectroscopy was crucial to determine the position of the substituents in the pyrrole rings. The products were identified as 2,4-, 2,3-, and 2,5-substituted pyrroles. The data obtained from their structural characterisation can help similar studies on amino acids modifications induced by analogous bifunctional carbonyl compounds. Our results show that the study of pathways in which model electrophiles modify amino acids may be helpful for similar studies dealing with identification of structural changes in cysteine- and lysine-containing proteins associated with oxidative stress.

Front Cover: Constraining and Modifying Peptides Using Pd‐Mediated Cysteine Allylation (ChemBioChem 13/2023)

Pd-mediated cysteine allylation allows to constrain and rigidify peptides by linking two cysteine side chains with a large set of easily available stapling reagents. The staple itself can be further functionalized based on its inherent reactivity thereby allowing construction of a large variety of solid peptides. More information can be found in the Research Article by C. F. W. Becker et al.

Comparison of methods generating antibody-epitope conjugates for targeting cancer with virus-specific T cells.

Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab. Three approaches were taken: chemical conjugation (i.e. a thiol-maleimide reaction) to reduced cysteine side chains, heavy chain C-terminal enzymatic conjugation using sortase A, and genetic fusions, to the heavy chain (HC) C-terminus. All three conjugates were capable of T-cell activation and target-cell killing via proteolytic release of the EBV epitope and expression of the antibody target was a requirement for T-cell activation. Moreover, AECs generated with a second immunogenic epitope derived from CMV (NLV/A2) were able to deliver and redirect CMV specific T-cells, in which the amino sequence of the attached peptide appeared to influence the efficiency of epitope delivery. Therefore, screening of multiple protease cleavage sites and epitopes attached to the antibody is necessary. Taken together, our data demonstrated that multiple AECs could sensitize cancer cells to virus-specific T cells.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

HyperCys: A Structure- and Sequence-Based Predictor of Hyper-Reactive Druggable Cysteines.

The cysteine side chain has a free thiol group, making it the amino acid residue most often covalently modified by small molecules possessing weakly electrophilic warheads, thereby prolonging on-target residence time and reducing the risk of idiosyncratic drug toxicity. However, not all cysteines are equally reactive or accessible. Hence, to identify targetable cysteines, we propose a novel ensemble stacked machine learning (ML) model to predict hyper-reactive druggable cysteines, named HyperCys. First, the pocket, conservation, structural and energy profiles, and physicochemical properties of (non)covalently bound cysteines were collected from both protein sequences and 3D structures of protein-ligand complexes. Then, we established the HyperCys ensemble stacked model by integrating six different ML models, including K-nearest neighbors, support vector machine, light gradient boost machine, multi-layer perceptron classifier, random forest, and the meta-classifier model logistic regression. Finally, based on the hyper-reactive cysteines' classification accuracy and other metrics, the results for different feature group combinations were compared. The results show that the accuracy, F1 score, recall score, and ROC AUC values of HyperCys are 0.784, 0.754, 0.742, and 0.824, respectively, after performing 10-fold CV with the best window size. Compared to traditional ML models with only sequenced-based features or only 3D structural features, HyperCys is more accurate at predicting hyper-reactive druggable cysteines. It is anticipated that HyperCys will be an effective tool for discovering new potential reactive cysteines in a wide range of nucleophilic proteins and will provide an important contribution to the design of targeted covalent inhibitors with high potency and selectivity.

Modified Linear Peptides Effectively Silence STAT-3 in Breast Cancer and Ovarian Cancer Cell Lines

RNA interference (RNAi) has drawn enormous attention as a powerful tool because of its capability to interfere with mRNA and protein production. However, designing a safe and efficient delivery system in RNAi therapeutics remains challenging. Herein, we have designed and synthesized several linear peptides containing tryptophan (W) and arginine (R) residues separated by the β-alanine (βA) spacer and attached to a lipophilic fatty acyl chain, cholesterol, or PEG. The peptide backbone sequences were: Ac-C-βA-βA-W4-βA-βA-R4-CO-NH2 and Ac-K-βA-βA-W4-βA-βA-R4-CO-NH2, with only a difference in N-terminal amino acid. The cysteine side chain in the first sequence was used for the conjugation with PEG2000 and PEG550. Alternatively, the side chain of lysine in the second sequence was used for conjugation with cholesterol or oleic acid. We hypothesized that amphiphilic peptides and optimum fatty acyl chain or PEG could function as an effective siRNA carrier by complementing each structural component's self-assembly and membrane internalization properties. None of the designed peptides showed cytotoxicity up to 10 µM. Serum stability studies suggested that the newly designed peptides efficiently protected siRNA against early degradation by nucleases. Flow cytometry analysis indicated 50-90% cellular uptake of siRNA using the newly developed modified linear peptides (MLPs). Western blot results revealed more than 90% protein downregulation after targeting STAT3 in MDA-MB-231 and SKOV-3 cell lines. In summary, a new peptide class was developed to safely and efficiently deliver siRNA.

Local Hydration Control and Functional Implications Through S-Nitrosylation of Proteins: Kirsten Rat Sarcoma Virus (K-RAS) and Hemoglobin (Hb).

S-nitrosylation, the covalent addition of NO to the thiol side chain of cysteine, is an important post-transitional modification (PTM) that can affect the function of proteins. As such, PTMs extend and diversify protein function and thus characterizing consequences of PTM at a molecular level is of great interest. Although PTMs can be detected through various direct/indirect methods, they lack the capability to investigate the modifications with molecular detail. In the present work local and global structural dynamics, their correlation, the hydration structure, and the infrared spectroscopy for WT and S-nitrosylated Kirsten rat sarcoma virus (K-RAS) and hemoglobin (Hb) are characterized from molecular dynamics simulations. It is found that attaching NO to Cys118 in K-RAS rigidifies the protein in the Switch-I region which has functional implications, whereas for Hb, nitrosylation at Cys93 at the β1 chain increases the flexibility of secondary structural motives for Hb in its T0 and R4 conformational substates. Solvent water access decreased by 40% after nitrosylation in K-RAS, similar to Hb for which, however, local hydration of the R4SNO state is yet lower than for T0SNO. Finally, S-nitrosylation leads to detectable peaks for the NO stretch frequency, but the congested IR spectral region will make experimental detection of these bands difficult. Overall, S-nitrosylation in these two proteins is found to influence hydration, protein flexibility, and conformational dynamics which are all eventually involved in protein regulation and function at a molecular level.

A kinetic study of thiol addition to N-phenylchloroacetamide.

Irreversible enzyme inhibitors bind covalently to their target and permanently limit its function. The redox-sensitive thiol group on the side chain of cysteine (Cys) residues is often the nucleophilic group that is targeted for reaction with the electrophilic warhead of irreversible inhibitors. While the acrylamide group is the warhead applied most frequently currently in the design of inhibitors with therapeutic potential, the chloroacetamide group offers a comparable reactivity profile. In that context, we have studied the details of the mechanism of thiol addition to N-phenylchloroacetamide (NPC). A kinetic assay was developed to accurately monitor the reaction progress between NPC and a small library of thiols with varying pKa values. From these data, a Brønsted-type plot was constructed, from which a βnucRS- value of 0.22 ± 0.07 was derived, indicative of a relatively early transition state with respect to attack by the thiolate. The halide leaving group was also varied, for the reaction with one thiol, providing rate constants consistent with a transition state that is early with respect to leaving group departure. The effects of temperature and ionic strength were also studied, and all data are consistent with an early transition state for a concerted SN2 mechanism of addition. Molecular modelling was also performed, and these calculations confirm the concerted transition state and relative reactivity of the haloacetamides. Finally, this study allows a detailed comparison of the reactivity and reaction mechanisms of the chloroacetamide group with the benchmark acrylamides used in many irreversible inhibitor drugs.

Structural and biochemical analysis of a novel atypically split intein reveals a conserved histidine specific to cysteine-less inteins.

Protein trans-splicing mediated by a split intein reconstitutes a protein backbone from two parts. This virtually traceless autoprocessive reaction provides the basis for numerous protein engineering applications. Protein splicing typically proceeds through two thioester or oxyester intermediates involving the side chains of cysteine or serine/threonine residues. A cysteine-less split intein has recently attracted particular interest as it can splice under oxidizing conditions and is orthogonal to disulfide or thiol bioconjugation chemistries. Here, we report the split PolB16 OarG intein, a second such cysteine-independent intein. As a unique trait, it is atypically split with a short intein-N precursor fragment of only 15 amino acids, the shortest characterized to date, which was chemically synthesized to enable protein semi-synthesis. By rational engineering we obtained a high-yielding, improved split intein mutant. Structural and mutational analysis revealed the dispensability of the usually crucial conserved motif N3 (block B) histidine as an obvious peculiar property. Unexpectedly, we identified a previously unnoticed histidine in hydrogen-bond forming distance to the catalytic serine 1 as critical for splicing. This histidine has been overlooked so far in multiple sequence alignments and is highly conserved only in cysteine-independent inteins as a part of a newly discovered motif NX. The motif NX histidine is thus likely of general importance to the specialized environment in the active site required in this intein subgroup. Together, our study advances the toolbox as well as the structural and mechanistic understanding of cysteine-less inteins.

Gambogic acid and juglone inhibit RNase P through distinct mechanisms

The first step in transfer RNA (tRNA) maturation is the cleavage of the 5' end of precursor tRNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein (RNP) complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of PRORP for use as tools to study the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization (FP) cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 μM range were identified and analyzed. We found these compounds similarly inhibit human mtRNase P, a multi-subunit protein enzyme, and are 50-fold less potent against bacterial RNA-dependent RNase P. Our biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor targeting the PRORP1-substrate complex while juglone acts as a time-dependent PRORP1 inhibitor. Additionally, X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. Finally, we propose a model consistent with the kinetic data that involves juglone binding to PRORP1 rapidly to form an inactive enzyme-inhibitor (EI) complex, and then undergoing a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships.

Cysteine Oxidation in Proteins: Structure, Biophysics, and Simulation.

Cysteine side chains can exist in distinct oxidation states depending on the pH and redox potential of the environment, and cysteine oxidation plays important yet complex regulatory roles. Compared with the effects of post-translational modifications such as phosphorylation, the effects of oxidation of cysteine to sulfenic, sulfinic, and sulfonic acid on protein structure and function remain relatively poorly characterized. We present an analysis of the role of cysteine reactivity as a regulatory factor in proteins, emphasizing the interplay between electrostatics and redox potential as key determinants of the resulting oxidation state. A review of current computational approaches suggests underdeveloped areas of research for studying cysteine reactivity through molecular simulations.