Proteomics and Beyond: Cell Decision-Making Shaped by Reactive Electrophiles.

Revolutionary proteomic strategies have enabled rapid profiling of the cellular targets of electrophilic small molecules. However, precise means to directly interrogate how these individual electrophilic modifications at low occupancy functionally reshape signaling networks have until recently been largely limited. We highlight here new methods that transcend proteomic platforms to forge a quantitative link between protein target-selective engagement and downstream signaling. We focus on recent progress in the study of non-enzyme-assisted signaling mechanisms and crosstalk choreographed by native reactive electrophilic species (RES). Using this as a model, we offer a long-term vision of how these toolsets together with fundamental biochemical knowledge of precision electrophile signaling may be harnessed to assist covalent ligand-target matching and ultimately amend disease-specific signaling dysfunction.

Evaluation and extension of the two-site, two-step model for binding and activation of the chemokine receptor CCR1.

Interactions between secreted immune proteins called chemokines and their cognate G protein-coupled receptors regulate the trafficking of leukocytes in inflammatory responses. The two-site, two-step model describes these interactions. It involves initial binding of the chemokine N-loop/ß3 region to the receptor's N-terminal region and subsequent insertion of the chemokine N-terminal region into the transmembrane helical bundle of the receptor concurrent with receptor activation. Here, we test aspects of this model with C-C motif chemokine receptor 1 (CCR1) and several chemokine ligands. First, we compared the chemokine-binding affinities of CCR1 with those of peptides corresponding to the CCR1 N-terminal region. Relatively low affinities of the peptides and poor correlations between CCR1 and peptide affinities indicated that other regions of the receptor may contribute to binding affinity. Second, we evaluated the contributions of the two CCR1-interacting regions of the cognate chemokine ligand CCL7 (formerly monocyte chemoattractant protein-3 (MCP-3)) using chimeras between CCL7 and the non-cognate ligand CCL2 (formerly MCP-1). The results revealed that the chemokine N-terminal region contributes significantly to binding affinity but that differences in binding affinity do not completely account for differences in receptor activation. On the basis of these observations, we propose an elaboration of the two-site, two-step model-the "three-step" model-in which initial interactions of the first site result in low-affinity, nonspecific binding; rate-limiting engagement of the second site enables high-affinity, specific binding; and subsequent conformational rearrangement gives rise to receptor activation.

Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding proteins HuR and AUF1.

The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling axis is a target of covalent drugs and bioactive native electrophiles. However, much of our understanding of Nrf2 regulation has been focused at the protein level. Here we report a post-transcriptional modality to directly regulate Nrf2-mRNA. Our initial studies focused on the effects of the key mRNA-binding protein (mRBP) HuR on global transcriptomic changes incurred upon oxidant or electrophile stimulation. These RNA-sequencing data and subsequent mechanistic analyses led us to discover a novel role of HuR in regulating Nrf2 activity, and in the process, we further identified the related mRBP AUF1 as an additional novel Nrf2 regulator. Both mRBPs regulate Nrf2 activity by direct interaction with the Nrf2 transcript. Our data showed that HuR enhances Nrf2-mRNA maturation and promotes its nuclear export, whereas AUF1 stabilizes Nrf2-mRNA. Both mRBPs target the 3'-UTR of Nrf2-mRNA. Using a Nrf2 activity-reporter zebrafish strain, we document that this post-transcriptional control of Nrf2 activity is conserved at the whole-vertebrate level.-Poganik, J. R., Long, M. J. C., Disare, M. T., Liu, X., Chang, S.-H., Hla, T., Aye, Y. Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding proteins HuR and AUF1.

The Structural Basis for Complement Inhibition by Gigastasin, a Protease Inhibitor from the Giant Amazon Leech.

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway.

Rapid additive-free selenocystine-selenoester peptide ligation.

We describe an unprecedented reaction between peptide selenoesters and peptide dimers bearing N-terminal selenocystine that proceeds in aqueous buffer to afford native amide bonds without the use of additives. The selenocystine-selenoester ligations are complete in minutes, even at sterically hindered junctions, and can be used in concert with one-pot deselenization chemistry. Various pathways for the transformation are proposed and probed through a combination of experimental and computational studies. Our new reaction manifold is also showcased in the total synthesis of two proteins.

Trifluoroethanethiol: an additive for efficient one-pot peptide ligation-desulfurization chemistry.

Native chemical ligation followed by desulfurization is a powerful strategy for the assembly of proteins. Here we describe the development of a high-yielding, one-pot ligation-desulfurization protocol that uses trifluoroethanethiol (TFET) as a novel thiol additive. The synthetic utility of this TFET-enabled methodology is demonstrated by the efficient multi-step one-pot syntheses of two tick-derived proteins, chimadanin and madanin-1, without the need for any intermediary purification.

Harmony of Protein Tags and Chimeric Molecules Empowers Targeted Protein Ubiquitination and Beyond.

Post-translational modifications (PTMs) are crucial mechanisms that underlie the intricacies of biological systems and disease mechanisms. This review focuses on the latest advancements in the design of heterobifunctional small molecules that hijack PTM machineries for target-specific modifications in living systems. A key innovation in this field is the development of proteolysis-targeting chimeras (PROTACs), which promote the ubiquitination of target proteins for proteasomal degradation. The past decade has seen several adaptations of the PROTAC concept to facilitate targeted (de)phosphorylation and acetylation. Protein fusion tags have been particularly vital in these proof-of-concept studies, aiding in the investigation of the functional roles of post-translationally modified proteins linked to diseases. This overview delves into protein-tagging strategies that enable the targeted modulation of ubiquitination, phosphorylation, and acetylation, emphasizing the synergies and challenges of integrating heterobifunctional molecules with protein tags in PTM research. Despite significant progress, many PTMs remain to be explored, and protein tag-assisted PTM-inducing chimeras will continue to play an important role in understanding the fundamental roles of protein PTMs and in exploring the therapeutic potential of manipulating protein modifications, particularly for targets not yet addressed by existing drugs.

Integrating Phenotypic and Chemoproteomic Approaches to Identify Covalent Targets of Dietary Electrophiles in Platelets.

A large variety of dietary phytochemicals has been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on the platelet activity and function remains unclear. This study explores the irreversible engagement of 23 electrophilic phytochemicals with platelets, unveiling the unique antiplatelet selectivity of sulforaphane (SFN). SFN impairs platelet responses to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist while not affecting thrombin and collagen-related peptide activation. It also substantially reduces platelet thrombus formation under arterial flow conditions. Using an alkyne-integrated probe, protein disulfide isomerase A6 (PDIA6) was identified as a rapid kinetic responder to SFN. Mechanistic profiling studies revealed SFN's nuanced modulation of PDIA6 activity and substrate specificity. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, aiming to enhance the clot-busting power of rtPA, currently the only approved therapeutic for stroke recanalization that has significant limitations.

SBHA: Sensitive Binary Hashing Autoencoder for Image Retrieval.

Binary hashing is an effective approach for content-based image retrieval, and learning binary codes with neural networks has attracted increasing attention in recent years. However, the training of hashing neural networks is difficult due to the binary constraint on hash codes. In addition, neural networks are easily affected by input data with small perturbations. Therefore, a sensitive binary hashing autoencoder (SBHA) is proposed to handle these challenges by introducing stochastic sensitivity for image retrieval. SBHA extracts meaningful features from original inputs and maps them onto a binary space to obtain binary hash codes directly. Different from ordinary autoencoders, SBHA is trained by minimizing the reconstruction error, the stochastic sensitive error, and the binary constraint error simultaneously. SBHA reduces output sensitivity to unseen samples with small perturbations from training samples by minimizing the stochastic sensitive error, which helps to learn more robust features. Moreover, SBHA is trained with a binary constraint and outputs binary codes directly. To tackle the difficulty of optimization with the binary constraint, we train the SBHA with alternating optimization. Experimental results on three benchmark datasets show that SBHA is competitive and significantly outperforms state-of-the-art methods for binary hashing.

Improving treatment for acute ischemic stroke-Clot busting innovation in the pipeline.

Acute ischemic stroke is a consequence of disrupted blood flow to the brain, caused by thrombosis-the pathological formation of occlusive clots within blood vessels, which can embolize distally to downstream tissues and microvasculature. The highest priority of stroke treatment is the rapid removal of occlusive clots and restoration of tissue perfusion. Intravenous thrombolysis is the pharmacological standard-of-care for the dissolution of blood clots, wherein thrombolytic drugs are administered to restore vessel patency. While the introduction of recombinant tissue-plasminogen activator (rtPA) in 1996 demonstrated the benefit of acute thrombolysis for clot removal, this was countered by severe limitations in terms of patient eligibility, lytic efficacy, rethrombosis and safety implications. Development of safer and efficacious treatment strategies to improve clot lysis has not significantly progressed over many decades, due to the challenge of maintaining the necessary efficacy-safety balance for these therapies. As such, rtPA has remained the sole approved acute therapeutic for ischemic stroke for over 25 years. Attempts to improve thrombolysis with coadministration of adjunct antithrombotics has demonstrated benefit in coronary vessels, but remain contraindicated for stroke, given all currently approved antithrombotics adversely impact hemostasis, causing bleeding. This Perspective provides a brief history of stroke drug development, as well as an overview of several groups of emerging drugs which have the potential to improve thrombolytic strategies in the future. These include inhibitors of the platelet receptor glycoprotein VI and the signaling enzyme PI3-Kinase, novel anticoagulants derived from hematophagous creatures, and proteolysis-targeting chimeras.

Black raspberry extract shifted gut microbe diversity and their metabolic landscape in a human colonic model.

Human gut microbiota is critical for human health, as their dysbiosis could lead to various diseases such as irritable bowel syndrome and obesity. Black raspberry (BRB) has been increasingly studied recently for its impact on gut microbiota as a rich source of phytochemicals (e.g., anthocyanin). To investigate the effect of BRB extract on the gut microbiota composition and their metabolism, an in-vitro human colonic model (HCM) was utilized to study the direct interaction between BRB and gut microbiome. Conditions (e.g., pH, temperature, anaerobic environment) in HCM were closely monitored and maintained to simulate the human intestinal system. Fresh fecal samples donated by three young healthy volunteers were used for gut microbiota inoculation in the HCM. 16S ribosomal DNA sequencing and liquid-chromatography mass spectrometry (LC/MS) based metabolomics were performed to study the impact of BRB on gut microbiota characteristics and their metabolism (fatty acids, polar metabolites, and phenolic compounds). Our data suggested that BRB intervention modulated gut microbiota at the genus level in different HCM sections mimicing ascending, transverse, and descending colons. Relative abundance of Enterococcus was commonly decreased in all colon sections, while modulations of other bacteria genera were mostly location-dependent. Meanwhile, significant changes in the metabolic profile of gut microbiota related to fatty acids, endogenous polar metabolites, and phenolic compounds were detected, in which arginine and proline metabolism, lysine degradation, and aminoacyl-tRNA biosynthesis were mostly regulated. Moreover, we identified several significant associations between altered microbial populations and changes in microbial metabolites. In summary, our study revealed the impact of BRB intervention on gut microbiota composition and metabolism change, which may exert physiological change to host metabolism and host health.

Fucosylated Chondroitin Sulfate against Parkinson's Disease through Inhibiting Inflammation Induced by Gut Dysbiosis.

Growing evidence for the importance of the gut-brain axis in Parkinson's disease (PD) has attracted researchers' interest in the possible application of microbiota-based treatment approaches. Using a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model, we looked into the prospect of treating PD with fucosylated chondroitin sulfate obtained from sea cucumbers Isostichopus badionotus (fCS-Ib). We showed that giving fCS-Ib polysaccharide orally greatly reduced the motor deficits, dopamine depletion, and alpha-synuclein increase caused by MPTP in the substantia nigra (SN). It appears that the anti-PD action of fCS-Ib polysaccharide could be attained by squelching inflammation. Glial cell hyperactivation in SN and overproduction of proinflammatory substances in serum could both be suppressed by fCS-Ib polysaccharide injection. The bacterial DNA in fresh colonic feces was submitted to 16S rRNA and untargeted metabolic analyses to confirm the participation of the microbiota-gut-brain axis in the aforementioned interpretation. The findings showed that the MPTP treatment-induced decrease in norank_f_Muribaculaceae and the increase in Staphylococcus were reversed by the administration of fCS-Ib polysaccharide. The NF-κB signaling pathway was shown to be involved in the fCS-Ib polysaccharide-induced anti-inflammation. In conclusion, our research demonstrated for the first time how fCS-Ib polysaccharide combats PD by reducing inflammation caused by gut microbial dysbiosis.

Z-REX uncovers a bifurcation in function of Keap1 paralogs.

Studying electrophile signaling is marred by difficulties in parsing changes in pathway flux attributable to on-target, vis-à-vis off-target, modifications. By combining bolus dosing, knockdown, and Z-REX-a tool investigating on-target/on-pathway electrophile signaling, we document that electrophile labeling of one zebrafish-Keap1-paralog (zKeap1b) stimulates Nrf2- driven antioxidant response (AR) signaling (like the human-ortholog). Conversely, zKeap1a is a dominant-negative regulator of electrophile-promoted Nrf2-signaling, and itself is nonpermissive for electrophile-induced Nrf2-upregulation. This behavior is recapitulated in human cells: (1) zKeap1b-expressing cells are permissive for augmented AR-signaling through reduced zKeap1b-Nrf2 binding following whole-cell electrophile treatment; (2) zKeap1a-expressing cells are non-permissive for AR-upregulation, as zKeap1a-Nrf2 binding capacity remains unaltered upon whole-cell electrophile exposure; (3) 1:1 ZKeap1a:zKeap1b-co-expressing cells show no Nrf2-release from the Keap1-complex following whole-cell electrophile administration, rendering these cells unable to upregulate AR. We identified a zKeap1a-specific point-mutation (C273I) responsible for zKeap1a's behavior during electrophilic stress. Human-Keap1(C273I), of known diminished Nrf2-regulatory capacity, dominantly muted electrophile-induced Nrf2-signaling. These studies highlight divergent and interdependent electrophile signaling behaviors, despite conserved electrophile sensing.

Synthetic Thiol and Selenol Derived Amino Acids for Expanding the Scope of Chemical Protein Synthesis.

Cells employ post-translational modifications (PTMs) as key mechanisms to expand proteome diversity beyond the inherent limitations of a concise genome. The ability to incorporate post-translationally modified amino acids into protein targets via chemical ligation of peptide fragments has enabled the access to homogeneous proteins bearing discrete PTM patterns and empowered functional elucidation of individual modification sites. Native chemical ligation (NCL) represents a powerful and robust means for convergent assembly of two homogeneous, unprotected peptides bearing an N-terminal cysteine residue and a C-terminal thioester, respectively. The subsequent discovery that protein cysteine residues can be chemoselectively desulfurized to alanine has ignited tremendous interest in preparing unnatural thiol-derived variants of proteogenic amino acids for chemical protein synthesis following the ligation-desulfurization logic. Recently, the 21st amino acid selenocysteine, together with other selenyl derivatives of amino acids, have been shown to facilitate ultrafast ligation with peptidyl selenoesters, while the advancement in deselenization chemistry has provided reliable bio-orthogonality to PTMs and other amino acids. The combination of these ligation techniques and desulfurization/deselenization chemistries has led to streamlined synthesis of multiple structurally-complex, post-translationally modified proteins. In this review, we aim to summarize the latest chemical synthesis of thiolated and selenylated amino-acid building blocks and exemplify their important roles in conquering challenging protein targets with distinct PTM patterns.

Precision Targeting of pten-Null Triple-Negative Breast Tumors Guided by Electrophilic Metabolite Sensing.

Off-target effects continue to impede disease interventions, particularly when targeting a specific protein within a family of similar proteins, such as kinase isoforms that play tumor-subtype-specific roles in cancers. Exploiting the specific electrophilic-metabolite-sensing capability of Akt3, versus moderate or no sensing, respectively, by Akt2 and Akt1, we describe a first-in-class functionally Akt3-selective covalent inhibitor [MK-H(F)NE], wherein the electrophilic core is derived from the native reactive lipid metabolite HNE. Mechanistic profiling and pathway interrogations point to retention of the metabolite's structure-as opposed to implicit electrophilicity-as being essential for biasing isoform preference, which we found translates to tumor-subtype specificity against pten-null triple-negative breast cancers (TNBCs). MK-H(F)NE further enables novel downstream target identification specific to Akt3-function in disease. In TNBC xenografts, MK-H(F)NE fares better than reversible pan-Akt-inhibitors and does not show commonly observed side-effects associated with Akt1-inhibition. Inhibitors derived from native-metabolite sensing are thus an enabling plan-of-action for unmasking kinase-isoform-biased molecular targets and tumor-subtype-specific interventions.

Genie in a bottle: controlled release helps tame natural polypharmacology?

Ability to faithfully report drug-target interactions constitutes a major critical parameter in preclinical/clinical settings. Yet the assessment of target engagement remains challenging, particularly for promiscuous and/or polypharmacologic ligands. Drawing from our improved insights into native electrophile signaling and emerging technologies that profile and interrogate these non-enzyme-assisted signaling subsystems, we posit that 'trained' polypharmocologic covalent inhibitors can be designed. Accumulating evidence indicates that electrophile-modified states at fractional occupancy can alter cell fate. Thus, by understanding sensing preferences and ligandable regions favored by the natural electrophilic signals at individual protein-ligand resolution, we can better evaluate target engagement and develop a function-guided understanding of polypharmacology.

Chemical Biology Gateways to Mapping Location, Association, and Pathway Responsivity.

Here we discuss, how by applying chemical concepts to biological problems, methods have been developed to map spatiotemporal regulation of proteins and small-molecule modulation of proteome signaling responses. We outline why chemical-biology platforms are ideal for such purposes. We further discuss strengths and weaknesses of chemical-biology protocols, contrasting them against classical genetic and biochemical approaches. We make these evaluations based on three parameters: occupancy; functional information; and spatial restriction. We demonstrate how the specific choice of chemical reagent and experimental set-up unite to resolve biological problems. Potential improvements/extensions as well as specific controls that in our opinion are often overlooked or employed incorrectly are also considered. Finally, we discuss some of the latest emerging methods to illuminate how chemical-biology innovations provide a gateway toward information hitherto inaccessible by conventional genetic/biochemical means. Finally, we also caution against solely relying on chemical-biology strategies and urge the field to undertake orthogonal validations to ensure robustness of results.

Mosquito-Derived Anophelin Sulfoproteins Are Potent Antithrombotics.

The anophelins are small protein thrombin inhibitors that are produced in the salivary glands of the Anopheles mosquito to fulfill a vital role in blood feeding. A bioinformatic analysis of anophelin sequences revealed the presence of conserved tyrosine residues in an acidic environment that were predicted to be post-translationally sulfated in vivo. To test this prediction, insect cell expression of two anophelin proteins, from Anopheles albimanus and Anopheles gambiae, was performed, followed by analysis by mass spectrometry, which showed heterogeneous sulfation at the predicted sites. Homogeneously sulfated variants of the two proteins were subsequently generated by chemical synthesis via a one-pot ligation-desulfurization strategy. Tyrosine sulfation of the anophelins was shown to significantly enhance the thrombin inhibitory activity, with a doubly sulfated variant of the anophelin from A. albimanus exhibiting a 100-fold increase in potency compared with the unmodified homologue. Sulfated anophelins were also shown to exhibit potent in vivo anticoagulant and antithrombotic activity.

Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors.

Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification-tyrosine sulfation-of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

Site-selective solid-phase synthesis of a CCR5 sulfopeptide library to interrogate HIV binding and entry.

Tyrosine (Tyr) sulfation is a common post-translational modification that is implicated in a variety of important biological processes, including the fusion and entry of human immunodeficiency virus type-1 (HIV-1). A number of sulfated Tyr (sTyr) residues on the N-terminus of the CCR5 chemokine receptor are involved in a crucial binding interaction with the gp120 HIV-1 envelope glycoprotein. Despite the established importance of these sTyr residues, the exact structural and functional role of this post-translational modification in HIV-1 infection is not fully understood. Detailed biological studies are hindered in part by the difficulty in accessing homogeneous sulfopeptides and sulfoproteins through biological expression and established synthetic techniques. Herein we describe an efficient approach to the synthesis of sulfopeptides bearing discrete sulfation patterns through the divergent, site-selective incorporation of sTyr residues on solid support. By employing three orthogonally protected Tyr building blocks and a solid-phase sulfation protocol, we demonstrate the synthesis of a library of target N-terminal CCR5(2-22) sulfoforms bearing discrete and differential sulfation at Tyr10, Tyr14, and Tyr15, from a single resin-bound intermediate. We demonstrate the importance of distinct sites of Tyr sulfation in binding gp120 through a competitive binding assay between the synthetic CCR5 sulfopeptides and an anti-gp120 monoclonal antibody. These studies revealed a critical role of sulfation at Tyr14 for binding and a possible additional role for sulfation at Tyr10. N-terminal CCR5 variants bearing a sTyr residue at position 14 were also found to complement viral entry into cells expressing an N-terminally truncated CCR5 receptor.