N-terminal Cysteine Research Articles

Macromolecular Function Emerging from Intramolecular Peptide Stapling of Synthetic Polymers.

Protein function results from the precise folding of polypeptides into bespoke architectures. Taking inspiration from nature, the field of single-chain nanoparticles (SCNPs), intramolecularly crosslinked synthetic polymers, emerged. In contrast to nature, the function of SCNPs is generally defined by the parent polymer or the applied crosslinker, rather than by the crosslinking process itself. This work explores the cyanopyridine-aminothiol click reaction to crosslink peptide-decorated polymers intra-macromolecularly to endow the resulting SCNPs with emerging functionality, resulting from the conversion of N-terminal cysteine units into pyridine-thiazolines. Dimethylacrylamide based polymers with different cysteine-terminated amino acid sequences tethered to their sidechains are investigated (P1 (C),P2 (GDHC),P3 (GDSC)) and intramolecularly crosslinked into SCNPs. Since the deprotection of the parent polymers yields disulfide-based SCNPs, a direct comparison between disulfide and pyridine-thiazolines crosslinked SCNPs is possible. This comparison revealed two emerging properties of the pyridine-thiazoline crosslinked SCNPs: 1) The formation of pyridine-thiazolines gave rise to metal binding sites within the SCNP, which complexed iron. 2) Depending on the peptide sequence in the precursor polymer, the hydrolytic activity of the peptide sequences is either increased (GDHC) or decreased (GDSC) uponpyridine-thiazoline formation compared to identical SCNPs based on disulfide crosslinks.

Thiazolidine Deprotection Using an Organic Solvent Extractable Aldehyde Scavenger for One-Pot Four-Segment Ligation.

We report a simple and convenient N-terminal thiazolidine (Thz) deprotection strategy and its application in one-pot multisegment ligation. In this strategy, O-benzylhydroxylamine (O-BHA) is used to efficiently and rapidly convert Thz into N-terminal cysteine. O-BHA can be easily separated from the ligation buffer by organic solvent extraction, avoiding the degradation of the peptide thioester by O-BHA. The utility of the O-BHA-based one-pot ligation strategy has been demonstrated in the assembly of CC chemokine ligand-2.

Structural and functional analysis of a bile salt hydrolase from the bison microbiome

The bile salt hydrolases (BSHs) are significant constituents of animal microbiomes. An evolving appreciation of their roles in health and disease has established them as targets of pharmacological inhibition. These bacterial enzymes belong to the N-terminal nucleophile superfamily and are best known to catalyze the deconjugation of glycine or taurine from bile salts to release bile acid substrates for transformation and or metabolism in the gastrointestinal tract. Here, we identify and describe the BSH from a common member of the Plains bison microbiome, Arthrobacter citreus (BSHAc). Steady-state kinetic analyses demonstrated that BSHAc is a broad-spectrum hydrolase with a preference for glycine-conjugates and deoxycholic acid (DCA). Second-order rate constants (kcat/KM) for BSHAc-catalyzed reactions of relevant bile salts-glyco- and tauro-conjugates of cholic acid and DCA- varied by ∼30-fold and measured between 1.4× 105 and 4.3× 106M-1s-1. Interestingly, a pan-BSH inhibitor named AAA-10 acted as a slow irreversible inhibitor of BSHAc with a rate of inactivation (kinact)of ∼2h-1 and a second order rate constant (kinact/KI) of ∼24M-1s-1 for the process. Structural characterization of BSHAc reacted with AAA-10 showed covalent modification of the N-terminal cysteine nucleophile, providing molecular details for an enzyme-stabilized product formed from this mechanism-based inhibitor's α-fluoromethyl ketone warhead. Structural comparison of the BSHs and BSH:inhibitor complexes highlighted the plasticity of the steroid-binding site, including a flexible loop that is variable across well-studied BSHs.

The enzymatic oxygen sensor cysteamine dioxygenase binds its protein substrates through their N-termini

The non-heme iron-dependent dioxygenase 2-aminoethanethiol (aka cysteamine) dioxygenase (ADO) has recently been identified as an enzymatic oxygen sensor that coordinates cellular changes to hypoxia by regulating the stability of proteins bearing an N-terminal cysteine (Nt-cys) through the N-degron pathway. It catalyzes O2-dependent Nt-cys sulfinylation, which promotes proteasomal degradation of the target. Only a few ADO substrates have been verified, including regulators of G-protein signaling (RGS) 4 and 5, and the proinflammatory cytokine interleukin-32, all of which exhibit cell and/or tissue specific expression patterns. ADO, in contrast, is ubiquitously expressed, suggesting it can regulate the stability of additional Nt-cys proteins in an O2-dependent manner. However, the role of individual chemical groups, active site metal, amino acid composition, and globular structure on protein substrate association remains elusive. To help identify new targets and examine the underlying biochemistry of the system, we conducted a series of biophysical experiments to investigate the binding requirements of established ADO substrates RGS5 and interleukin-32. We demonstrate, using surface plasmon response and enzyme assays, that a free, unmodified Nt-thiol and Nt-amine are vital for substrate engagement through active site metal coordination, with residues next to Nt-cys moderately impacting association and catalytic efficiency. Additionally, we show, through 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance titrations, that the globular portion of RGS5 has limited impact on ADO association, with interactions restricted to the N-terminus. This work establishes key features involved in ADO substrate binding, which will help identify new protein targets and, subsequently, elucidate its role in hypoxic adaptation.

The hydrophobicity of the CARD8 N-terminus tunes inflammasome activation

Mounting evidence indicates that proteotoxic stress is a primary activator of the CARD8 inflammasome, but the complete array of signals that control this inflammasome have not yet been established. Notably, we recently discovered that several hydrophobic radical-trapping antioxidants (RTAs), including JSH-23, potentiate CARD8 inflammasome activation through an unknown mechanism. Here, we report that these RTAs directly alkylate several cysteine residues in the N-terminal disordered region of CARD8. These hydrophobic modifications destabilize the repressive CARD8 N-terminal fragment and accelerate its proteasome-mediated degradation, thereby releasing the inflammatory CARD8 C-terminal fragment from autoinhibition. Consistently, we also found that unrelated (non-RTA) hydrophobic electrophiles as well as genetic mutation of the CARD8 cysteine residues to isoleucines similarly potentiate inflammasome activation. Overall, our results not only provide further evidence that protein folding stress is a key CARD8 inflammasome-activating signal, but also indicate that the N-terminal cysteines can play key roles in tuning the response to this stress.

Biocompatible Synthesis of Macrocyclic Thiazol(in)e Peptides.

Macrocyclic peptides containing a thiazole or thiazoline in the backbone are considered privileged structures in both natural compounds and drug discovery, owing to their enhanced bioactivity, stability, and permeability. Here, we present the biocompatible synthesis of macrocyclic peptides from N-terminal cysteine and C-terminal nitrile. While the N-terminal cysteine is incorporated during solid-phase peptide synthesis, the C-terminal nitrile is introduced during cleavage with aminoacetonitrile, utilizing a cleavable benzotriazole linker. This method directly yields the fully functionalized linear peptide precursor. The biocompatible cyclization reaction occurs in buffer at physiological pH and room temperature. The resulting thiazoline heterocycle remains stable in buffer but hydrolyzes under acidic conditions. While such hydrolysis enables access to macrocyclic peptides with a complete amide backbone, mild oxidation of the thiazoline leads to the stable thiazole macrocyclic peptide. While conventional oxidation strategies involve metals, we developed a protocol simply relying on alkaline salt and air. Therefore, we offer a rapid and metal-free pathway to macrocyclic thiazole peptides, featuring a biocompatible key cyclization step.

N-terminal cysteine acetylation and oxidation patterns may define protein stability

Oxygen homeostasis is maintained in plants and animals by O2-sensing enzymes initiating adaptive responses to low O2 (hypoxia). Recently, the O2-sensitive enzyme ADO was shown to initiate degradation of target proteins RGS4/5 and IL32 via the Cysteine/Arginine N-degron pathway. ADO functions by catalysing oxidation of N-terminal cysteine residues, but despite multiple proteins in the human proteome having an N-terminal cysteine, other endogenous ADO substrates have not yet been identified. This could be because alternative modifications of N-terminal cysteine residues, including acetylation, prevent ADO-catalysed oxidation. Here we investigate the relationship between ADO-catalysed oxidation and NatA-catalysed acetylation of a broad range of protein sequences with N-terminal cysteines. We present evidence that human NatA catalyses N-terminal cysteine acetylation in vitro and in vivo. We then show that sequences downstream of the N-terminal cysteine dictate whether this residue is oxidised or acetylated, with ADO preferring basic and aromatic amino acids and NatA preferring acidic or polar residues. In vitro, the two modifications appear to be mutually exclusive, suggesting that distinct pools of N-terminal cysteine proteins may be acetylated or oxidised. These results reveal the sequence determinants that contribute to N-terminal cysteine protein modifications, with implications for O2-dependent protein stability and the hypoxic response.

Identification and Characterization of the Lipoprotein N-acyltransferase in Bacteroides.

Members of the Bacteroidota compose a large portion of the human gut microbiota, contributing to overall gut health via the degradation of various polysaccharides. This process is facilitated by lipoproteins, globular proteins anchored to the cell surface by a lipidated N-terminal cysteine. Despite their importance, lipoprotein synthesis by these bacteria is understudied. In E. coli, the α-amino linked lipid of lipoproteins is added by the lipoprotein N-acyltransferase Lnt. Herein, we have identified a protein distinct from Lnt responsible for the same process in Bacteroides, named lipoprotein N-acyltransferase in Bacteroides (Lnb). Deletion of Lnb yields cells that synthesize diacylated lipoproteins, with impacts on cell viability and morphology, growth on polysaccharides, and protein composition of membranes and outer membrane vesicles (OMVs). Our results not only challenge the accepted paradigms of lipoprotein biosynthesis in Gram-negative bacteria, but also support the establishment of a new family of lipoprotein N-acyltransferases.

Cyclic Peptides from Graspetide Biosynthesis and Native Chemical Ligation.

The ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily of natural products includes many examples of cyclic peptides with diverse macrocyclization chemistries. The graspetides, one family of macrocyclized RiPPs, harbor side chain-side chain ester or amide linkages. We recently reported the structure and biosynthesis of the graspetide pre-fuscimiditide, a 22-amino-acid (aa) peptide with two ester cross-links forming a stem-loop structure. These cross-links are introduced by a single graspetide synthetase, the ATP-grasp enzyme ThfB. Here we show that ThfB can also catalyze the formation of amide or thioester cross-links in prefuscimiditide, with thioester formation being especially efficient. We further show that upon proteolysis to reveal an N-terminal cysteine residue, the thioester-linked peptide rapidly and quantitatively rearranges via native chemical ligation into an isopeptide-bonded head-to-tail cyclic peptide. The solution structure of this rearranged peptide was determined by using 2D NMR spectroscopy experiments. Our methodology offers a straightforward recombinant route to head-to-tail cyclic peptides.

Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

Biochemical characterization of cardiac α-actin mutations A21V and D26N implicated in hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (HCM) affects .2% of the world's population and is inherited in an autosomal dominant manner. Mutations in cardiac α-actin are the cause in 1%-5% of all observed cases. Here, we describe the recombinant production, purification, and characterization of the HCM-linked cardiac α-actin variants p.A21V and p.D26N. Mass spectrometric analysis of the initially purified recombinant cardiac α-actin variants and wild-type protein revealed improper N-terminal processing in the Spodoptera frugiperda (Sf-9) insect cell system, compromising the labeling of the protein with fluorescent probes for biochemical studies. Therefore, we produced N-terminal deletion mutants lacking the N-terminal cysteine (ΔC2). The ΔC2 wild-type construct behaved similar to porcine cardiac α-actin purified from native Sus scrofa heart tissue and all ΔC2 constructs showed improved fluorescent labeling. Further analysis of untruncated and ΔC2 constructs showed that while neither the A21V nor the D26N mutation affects nucleotide binding, they cause a similar slowing of the rate of filament formation as well as a reduction in the thermal stability of monomeric and filamentous cardiac α-actin. In vitro motility assays and transient-kinetic studies probing the interaction of the actin variants with cardiac β-myosin revealed perturbed actomyosin interactions and a reduced motile activity for the p.D26N variant. Addition of the small molecule effector EMD 57033, which targets cardiac β-myosin, rescued the approximately 40% drop in velocity observed with the p.D26N constructs and activated the motile activity of wild-type and p.D26N to the same level of 1100 nm s-1 .

Two-Layer Inkjet-Printed Microwave Split-Ring Resonators for Detecting Analyte Binding to the Gold Surface.

This work focuses on demonstrating the working principle of inkjet-printed Au nanoparticle (NP) two-layer Gigahertz (2.6 GHz) microwave split-ring resonators (SRRs) as a novel platform for the detection of analytes on flexible substrates. In contrast to the standard fabrication of split-ring resonator biosensors using printed circuit board technology, which results in a seven-layer system, the resonators in this work were fabricated using a two-layer system. A ground plane is embedded in the SRR measurement setup. In this method, a microwave electromagnetic wave is coupled into the Au SRR via an inkjet-printed Cu-NP stripline that is photonically sintered. This coupling mechanism facilitates the detection of analytes by inducing resonance shifts in the SRR. In this study, the functionality of the printed sensors was demonstrated using two different Au functionalization processes, firstly, with HS-PEG7500-COOH, and, secondly, with protein G with an N-terminal cysteine residue. The sensing capabilities of the printed structures are shown by the attachment of biomolecules to the SRR and the measurement of the resulting resonance shift. The experiments show a clear shift of the resonance frequency in the range of 20-30 MHz for both approaches. These results demonstrate the functionality of the simplified printed two-layer microwave split-ring resonator for use as a biosensor.

Evidence the Isc iron–sulfur cluster biogenesis machinery is the source of iron for [NiFe]-cofactor biosynthesis in Escherichia coli

[NiFe]-hydrogenases have a bimetallic NiFe(CN)2CO cofactor in their large, catalytic subunit. The 136 Da Fe(CN)2CO group of this cofactor is preassembled on a distinct HypC–HypD scaffold complex, but the intracellular source of the iron ion is unresolved. Native mass spectrometric analysis of HypCD complexes defined the [4Fe–4S] cluster associated with HypD and identified + 26 to 28 Da and + 136 Da modifications specifically associated with HypC. A HypCC2A variant without the essential conserved N-terminal cysteine residue dissociated from its complex with native HypD lacked all modifications. Native HypC dissociated from HypCD complexes isolated from Escherichia coli strains deleted for the iscS or iscU genes, encoding core components of the Isc iron–sulfur cluster biogenesis machinery, specifically lacked the + 136 Da modification, but this was retained on HypC from suf mutants. The presence or absence of the + 136 Da modification on the HypCD complex correlated with the hydrogenase enzyme activity profiles of the respective mutant strains. Notably, the [4Fe–4S] cluster on HypD was identified in all HypCD complexes analyzed. These results suggest that the iron of the Fe(CN)2CO group on HypCD derives from the Isc machinery, while either the Isc or the Suf machinery can deliver the [4Fe–4S] cluster to HypD.

Inferring co-expression networks of Arabidopsis thaliana genes during their interaction with Trichoderma spp.

Fungi of the Trichoderma genus are called "biostimulants" because they promote plant growth and development and induce disease resistance. We used conventional transcriptome and gene co-expression analyses to understand the molecular response of the plant Arabidopsis thaliana to inoculation with Trichoderma atroviride or Trichoderma virens. The transcriptional landscape of the plant during the interaction with these fungi showed a reduction in functions such as reactive oxygen species production, defense mechanisms against pathogens, and hormone signaling. T. virens, as opposed to T. atroviride, was more effective at downregulating genes related to terpenoid metabolism, root development, and chemical homeostasis. Through gene co-expression analysis, we found functional gene modules that closely link plant defense with hypoxia. Notably, we found a transcription factor (locus AT2G47520) with two functional domains of interest: a DNA-binding domain and an N-terminal cysteine needed for protein stability under hypoxia. We hypothesize that the transcription factor can bind to the promoter sequence of the GCC-box that is connected to pathogenesis by positioned weight matrix analysis.

Location-agnostic site-specific protein bioconjugation via Baylis Hillman adducts

Proteins labelled site-specifically with small molecules are valuable assets for chemical biology and drug development. The unique reactivity profile of the 1,2-aminothiol moiety of N-terminal cysteines (N-Cys) of proteins renders it highly attractive for regioselective protein labelling. Herein, we report an ultrafast Z-selective reaction between isatin-derived Baylis Hillman adducts and 1,2-aminothiols to form a bis-heterocyclic scaffold, and employ it for stable protein bioconjugation under both in vitro and live-cell conditions. We refer to our protein bioconjugation technology as Baylis Hillman orchestrated protein aminothiol labelling (BHoPAL). Furthermore, we report a lipoic acid ligase-based technology for introducing the 1,2-aminothiol moiety at any desired site within proteins, rendering BHoPAL location-agnostic (not limited to N-Cys). By using this approach in tandem with BHoPAL, we generate dually labelled protein bioconjugates appended with different labels at two distinct specific sites on a single protein molecule. Taken together, the protein bioconjugation toolkit that we disclose herein will contribute towards the generation of both mono and multi-labelled protein-small molecule bioconjugates for applications as diverse as biophysical assays, cellular imaging, and the production of therapeutic protein–drug conjugates. In addition to protein bioconjugation, the bis-heterocyclic scaffold we report herein will find applications in synthetic and medicinal chemistry.

The Cyanopyridine–Aminothiol Click Reaction: Expanding Horizons in Chemical Biology

AbstractBioorthogonal reactions hold significant promise for applications in chemical biology. Despite their potential, nitriles have often been overlooked as reactive functional groups for selective bioconjugation. The condensation reaction between cyanopyridines and 1,2-aminothiols stands out as a particularly favorable nitrile modification strategy that proceeds under biocompatible conditions. Cyanopyridines can be seamlessly incorporated into peptides and proteins through both chemical and biotechnological approaches. Similarly, the selective integration of 1,2-aminothiols into peptides and proteins is achievable, leveraging the uniquely reactive N-terminal cysteine functional group.1 Introduction2 The Biocompatible Condensation Reaction3 Peptide Macrocyclisation4 Orthogonality to Cysteine5 Combination with Genetic Encoding6 Conclusion

Formation and Properties of DNA Adducts Generated by Reactions of Abasic Sites with 1,2-Aminothiols Including Cysteamine, Cysteine Methyl Ester, and Peptides Containing N-Terminal Cysteine Residues.

The reaction of 1,2-aminothiol groups with aldehyde residues in aqueous solution generates thiazolidine products, and this process has been developed as a catalyst-free click reaction for bioconjugation. The work reported here characterized reactions of the biologically relevant 1,2-aminothiols including cysteamine, cysteine methyl ester, and peptides containing N-terminal cysteine residues with the aldehyde residue of apurinic/apyrimidinic (AP) sites in DNA oligomers. These 1,2-aminothiol-containing compounds rapidly generated adducts with AP sites in single-stranded and double-stranded DNA. NMR and MALDI-TOF-MS analyses provided evidence that the reaction generated a thiazolidine product. Conversion of an AP site to a thiazolidine-AP adduct protected against the rapid cleavage normally induced at AP sites by the endonuclease action of the enzyme APE1 and the AP-lyase activity of the biogenic amine spermine. In the presence of excess 1,2-aminothiols, the thiazolidine-AP adducts underwent slow strand cleavage via a β-lyase reaction that generated products with 1,2-aminothiol-modified sugar residues on the 3'-end of the strand break. In the absence of excess 1,2-aminothiols, the thiazolidine-AP adducts dissociated to release the parent AP-containing oligonucleotide. The properties of the thiazolidine-AP adducts described here mirror critical properties of SRAP proteins HMCES and YedK that capture AP sites in single-stranded regions of cellular DNA and protect them from cleavage.

Harnessing a bis-electrophilic boronic acid lynchpin for azaborolo thiazolidine (ABT) grafting in cyclic peptides.

Chemical modifications of native peptides have significantly advanced modern drug discovery in recent decades. On this front, the installation of multitasking molecular grafts onto macrocyclic peptides offers numerous opportunities in biomedical applications. Here, we showcase a new class of borono-cyclic peptides featuring an azaborolo thiazolidine (ABT) graft, which can be readily assembled utilizing a bis-electrophilic boronic acid lynchpin while harnessing the inherent reactivity difference (>103 M-1 s-1) between the N-terminal cysteine and backbone cysteine for rapid and highly regioselective macrocyclization (∼1 h) under physiological conditions. The ABT-crosslinked peptides are fairly stable in endogenous environments, but can provide the linear diazaborine peptides via treatment with α-nucleophiles. This efficient peptide crosslinking protocol was further extended for regioselective bicyclizations and engineering of α-helical structures. Finally, ABT-grafted peptides were exploited in biorthogonal conjugation, leading to highly effective intracellular delivery of an apoptotic peptide (KLA) in cancer cells. The mechanism of action by which ABT-grafted KLA peptide induces apoptosis was also explored.

Construction of photofunctional peptide conjugates through selective modification of N-terminal cysteine with cyclometallated iridium(III) 2-formylphenylboronic acid complexes for organelle-specific imaging, enzyme activity sensing and photodynamic therapy.

Luminescent cyclometallated iridium(III) complexes bearing a 2-formylphenylboronic acid moiety were designed; one of the complexes was utilised to modify peptides containing an N-terminal cysteine to afford luminescent conjugates with selective organelle-targeting or furin-responsive properties.

Azole reagents enabled ligation of peptide acyl pyrazoles for chemical protein synthesis.

Native chemical ligation (NCL) has been playing an increasingly important role in chemical protein synthesis (CPS). More efficient ligation methods that circumvent the requirement of a peptidyl thioester and thiol additive-which allow the following desulfurization or refolding in one pot-are urgently needed for the synthesis of more complex protein targets and in large quantities. Herein, we discover that the weak acyl donor peptidyl N-acyl pyrazole can be activated by azole reagents like 3-methylpyrazole or imidazole to facilitate its ligation directly with an N-terminal cysteine peptide. As it requires no thioester or thiol additive, this ligation strategy can be conveniently combined with metal-free desulfurization (MFD) or oxidative protein folding to allow various one-pot protocols. The utility and generality of the strategy are showcased by the total synthesis of ubiquitin via an N-to-C sequential ligation-MFD strategy, the semi-synthesis of the copper protein azurin, and the efficient assembly of a sulfated hirudin variant and the cyclotide kalata B1, all in a one-pot fashion.