RESUMEN
Transcription factors (TFs) intricately navigate the vast genomic landscape to locate and bind specific DNA sequences for the regulation of gene expression programs. These interactions occur within a dynamic cellular environment, where both DNA and TF proteins experience continual chemical and structural perturbations, including epigenetic modifications, DNA damage, mechanical stress, and post-translational modifications (PTMs). While many of these factors impact TF-DNA binding interactions, understanding their effects remains challenging and incomplete. This review explores the existing literature on these dynamic changes and their potential impact on TF-DNA interactions.
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Chemical protein synthesis provides a powerful means to prepare novel modified proteins with precision down to the atomic level, enabling an unprecedented opportunity to understand fundamental biological processes. Of particular interest is the process of gene expression, orchestrated through the interactions between transcription factors (TFs) and DNA. Here, we combined chemical protein synthesis and high-throughput screening technology to decipher the role of post-translational modifications (PTMs), e.g., Lys-acetylation on the DNA binding activity of Max TF. We synthesized a focused library of singly, doubly, and triply modified Max variants including site-specifically acetylated and fluorescently tagged analogs. The resulting synthetic analogs were employed to decipher the molecular role of Lys-acetylation on the DNA binding activity and sequence specificity of Max. We provide evidence that the acetylation sites at Lys-31 and Lys-57 significantly inhibit the DNA binding activity of Max. Furthermore, by utilizing high-throughput binding measurements, we assessed the binding activities of the modified Max variants across diverse DNA sequences. Our results indicate that acetylation marks can alter the binding specificities of Max toward certain sequences flanking its consensus binding sites. Our work provides insight into the hidden molecular code of PTM-TFs and DNA interactions, paving the way to interpret gene expression regulation programs.
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Site-selective functionalization strategies are in high demand to prepare well-defined homogeneous proteins for basic research and biomedical applications. In this regard, cysteine-based reactions have enabled a broad set of transformations to produce modified proteins for various applications. However, these approaches were mainly employed to modify a single reactive site with a specific transformation. Achieving site selectivity or multiple transformations, essential for preparing complex biomolecules, remains challenging. Herein we demonstrate the power of combining palladium(II)-mediated C-S bond formation and C-S bond cleavage reactions to selectively edit desired cysteine sites in complex and uniquely modified proteins. We developed an orthogonal palladium(II) strategy for rapid and effective diversification of multiple cysteine sites (3-6 residues) with various transformations. Importantly, we employed our approach to prepare 10 complex analogues, including modified, stapled, and multimeric proteins on a milligram scale. Furthermore, we also synthesized a focused library of stabilized artificial transcription factors that displayed enhanced stability and potent DNA binding activity. Our approach enables rapid and effective protein editing and opens new avenues to engineer new biomolecules for fundamental research and therapeutic applications.
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Cisteína , Factores de Transcripción , Cisteína/química , Paladio/química , Ingeniería Química , CatálisisRESUMEN
Histone post-translational modifications promote a chromatin environment that controls transcription, DNA replication and repair, but surprisingly few phosphorylations have been documented. We report the discovery of histone H3 serine-57 phosphorylation (H3S57ph) and show that it is implicated in different DNA repair pathways from fungi to vertebrates. We identified CHK1 as a major human H3S57 kinase, and disrupting or constitutively mimicking H3S57ph had opposing effects on rate of recovery from replication stress, 53BP1 chromatin binding, and dependency on RAD52. In fission yeast, mutation of all H3 alleles to S57A abrogated DNA repair by both non-homologous end-joining and homologous recombination, while cells with phospho-mimicking S57D alleles were partly compromised for both repair pathways, presented aberrant Rad52 foci and were strongly sensitised to replication stress. Mechanistically, H3S57ph loosens DNA-histone contacts, increasing nucleosome mobility, and interacts with H3K56. Our results suggest that dynamic phosphorylation of H3S57 is required for DNA repair and recovery from replication stress, opening avenues for investigating the role of this modification in other DNA-related processes.
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Histonas , Virus de la Influenza A , Humanos , Animales , Fosforilación , Procesamiento Proteico-Postraduccional , Reparación del ADN , CromatinaRESUMEN
The chemical synthesis of site-specifically modified transcription factors (TFs) is a powerful method to investigate how post-translational modifications (PTMs) influence TF-DNA interactions and impact gene expression. Among these TFs, Max plays a pivotal role in controlling the expression of 15 % of the genome. The activity of Max is regulated by PTMs; Ser-phosphorylation at the N-terminus is considered one of the key regulatory mechanisms. In this study, we developed a practical synthetic strategy to prepare homogeneous full-length Max for the first time, to explore the impact of Max phosphorylation. We prepared a focused library of eight Max variants, with distinct modification patterns, including mono-phosphorylated, and doubly phosphorylated analogues at Ser2/Ser11 as well as fluorescently labeled variants through native chemical ligation. Through comprehensive DNA binding analyses, we discovered that the phosphorylation position plays a crucial role in the DNA-binding activity of Max. Furthermore, in vitro high-throughput analysis using DNA microarrays revealed that the N-terminus phosphorylation pattern does not interfere with the DNA sequence specificity of Max. Our work provides insights into the regulatory role of Max's phosphorylation on the DNA interactions and sequence specificity, shedding light on how PTMs influence TF function.
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Regulación de la Expresión Génica , Factores de Transcripción , Factores de Transcripción/metabolismo , Fosforilación , Secuencia de Aminoácidos , Procesamiento Proteico-Postraduccional , ADN/metabolismoRESUMEN
Synthetic strategies to assemble peptide fragments are in high demand to access homogeneous proteins for various applications. Here, we combined native chemical ligation (NCL) and Pd-mediated Cys arylation to enable practical peptide ligation at aromatic junctions. The utility of one-pot NCL and S-arylation at the Phe and Tyr junctions was demonstrated and employed for the rapid chemical synthesis of the DNA-binding domains of the transcription factors Myc and Max. Organometallic palladium reagents coupled with NCL enabled a practical strategy to assemble peptides at aromatic junctions.
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Cisteína , Paladio , Paladio/química , Cisteína/química , Péptidos/química , Proteínas/química , Fragmentos de PéptidosRESUMEN
Nature has developed a plethora of protein machinery to operate and maintain nearly every task of cellular life. These processes are tightly regulated via post-expression modifications-transformations that modulate intracellular protein synthesis, folding, and activation. Methods to prepare homogeneously and precisely modified proteins are essential to probe their function and design new bioactive modalities. Synthetic chemistry has contributed remarkably to protein science by allowing the preparation of novel biomacromolecules that are often challenging or impractical to prepare via common biological means. The ability to chemically build and precisely modify proteins has enabled the production of new molecules with novel physicochemical properties and programmed activity for biomedical research, diagnostic, and therapeutic applications. This minireview summarizes recent developments in chemical protein synthesis to produce bioactive proteins, with emphasis on novel analogs with promising in vitro and in vivo activity.
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Biosíntesis de Proteínas , Proteínas , Proteínas/químicaRESUMEN
Posttranslational modifications (PTMs) dramatically expand the functional diversity of the proteome. The precise addition and removal of PTMs appears to modulate protein structure and function and control key regulatory processes in living systems. Deciphering how particular PTMs affect protein activity is a current frontier in biology and medicine. The large number of PTMs which can appear in several distinct positions, states, and combinations makes preparing such complex analogs using conventional biological and chemical tools challenging. Strategies to access homogeneous and precisely modified proteins with desired PTMs at selected sites and in feasible quantities are critical to interpreting their molecular code. Here, we summarize recent advances in posttranslational chemical mutagenesis and late-stage functionalization chemistry to transfer novel PTM mimicry into recombinant proteins with emphasis on novel transformations.
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Procesamiento Proteico-Postraduccional , Proteoma , Mutagénesis , Proteoma/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Dysregulation of the transcription factor MYC is involved in many human cancers. The dimeric transcription factor complexes of MYC/MAX and MAX/MAX activate or inhibit, respectively, gene transcription upon binding to the same enhancer box DNA. Targeting these complexes in cancer is a long-standing challenge. Inspired by the inhibitory activity of the MAX/MAX dimer, we engineered covalently linked, synthetic homo- and heterodimeric protein complexes to attenuate oncogenic MYC-driven transcription. We prepared the covalent protein complexes (â¼20 kDa, 167-231 residues) in a single shot via parallel automated flow synthesis in hours. The stabilized covalent dimers display DNA binding activity, are intrinsically cell-penetrant, and inhibit cancer cell proliferation in different cell lines. RNA sequencing and gene set enrichment analysis in A549 cancer cells confirmed that the synthetic dimers interfere with MYC-driven transcription. Our results demonstrate the potential of automated flow technology to rapidly deliver engineered synthetic protein complex mimetics that can serve as a starting point in developing inhibitors of MYC-driven cancer cell growth.
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Transcription factors (TF), such as Myc, are proteins implicated in disease pathogenesis, with dysregulation of Myc expression in 50% of all human cancers. Still, targeting Myc remains a challenge due to the lack of small molecule binding pockets in the tertiary structure. Here, we report synthetic covalently linked TF mimetics that inhibit oncogenic Myc-driven transcription by antagonistic binding of the target DNA-binding site. We combined automated flow peptide chemistry with palladium(II) oxidative addition complexes (OACs) to engineer covalent protein dimers derived from the DNA-binding domains of Myc, Max, and Omomyc TF analogs. Palladium-mediated cross-coupling of synthesized protein monomers resulted in milligram quantities of seven different covalent homo- and heterodimers. The covalent helical dimers were found to bind DNA and exhibited improved thermal stability. Cell-based studies revealed the Max-Max covalent dimer is cell-penetrating and interfered with Myc-dependent gene transcription resulting in reduced cancer cell proliferation (EC50 of 6 µM in HeLa). RNA sequencing and gene analysis of extracted RNA from treated cancer cells confirmed that the covalent Max-Max homodimer interferes with Myc-dependent transcription. Flow chemistry, combined with palladium(II) OACs, has enabled a practical strategy to generate new bioactive compounds to inhibit tumor cell proliferation.
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Indicadores y Reactivos/química , Paladio/química , Ingeniería de Proteínas , Proteínas Proto-Oncogénicas c-myc/síntesis química , Proliferación Celular/efectos de los fármacos , ADN/química , Células HeLa , Humanos , Indicadores y Reactivos/farmacología , Modelos Moleculares , Paladio/farmacología , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
Organometallic reagents enable practical strategies for bioconjugation. Innovations in the design of water-soluble ligands and the enhancement of reaction rates have allowed for chemoselective cross-coupling reactions of peptides and proteins to be carried out in water. There are currently no organometallic-based methods for oligonucleotide bioconjugation to other biomolecules. Here we report bifunctional palladium(II)-oxidative addition complexes (OACs) as reagents for high-yielding oligonucleotide bioconjugation reactions. These bifunctional OACs react chemoselectively with amine-modified oligonucleotides to generate the first isolable, bench stable oligonucleotide-palladium(II) OACs. These complexes undergo site-selective C-S arylation with a broad range of native thiol-containing biomolecules at low micromolar concentrations in under one hour. This approach provided oligonucleotide-peptide, oligonucleotide-protein, oligonucleotide-small molecule, and oligonucleotide-oligonucleotide conjugates in >80 % yield and afforded conjugation of multiple copies of oligonucleotides onto a monoclonal antibody.
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Reactivos de Enlaces Cruzados/química , Oligonucleótidos/química , Compuestos Organometálicos/química , Reactivos de Enlaces Cruzados/síntesis química , Oligonucleótidos/síntesis química , Compuestos Organometálicos/síntesis química , Paladio/química , Péptidos/química , Trastuzumab/químicaRESUMEN
The ß-coronavirus SARS-CoV-2 has caused a global pandemic. Affinity reagents targeting the SARS-CoV-2 spike protein are of interest for the development of therapeutics and diagnostics. We used affinity selection-mass spectrometry for the rapid discovery of synthetic high-affinity peptide binders for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. From library screening with 800 million synthetic peptides, we identified three sequences with nanomolar affinities (dissociation constants K d = 80-970 nM) for RBD and selectivity over human serum proteins. Nanomolar RBD concentrations in a biological matrix could be detected using the biotinylated lead peptide in ELISA format. These peptides do not compete for ACE2 binding, and their site of interaction on the SARS-CoV-2-spike-RBD might be unrelated to the ACE2 binding site, making them potential orthogonal reagents for sandwich immunoassays. These findings serve as a starting point for the development of SARS-CoV-2 diagnostics or conjugates for virus-directed delivery of therapeutics.
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Chemists have been interested in the N-alkylation of a peptide bond because such a modification alters the conformation of the amide bond, interferes with hydrogen bond formation, and changes other properties of the peptide (e.g., solubility). This modification also opens the door for attaching functional groups for various applications. Nonetheless, the irreversibility of some of these modifications and the harsh conditions required for their removal currently limits the wide utility of this approach. Herein, we report applying a propargyl group for peptide bond modification at diverse junctions, which can be removed under mild and aqueous conditions via treatment with gold(I). Considering the straightforward conditions for both the installation and removal of this group, the propargyl group provides access to the benefits of backbone N-alkylation, while preserving the ability for on-demand depropargylation and full recovery of the native amide bond. This reversible modification was found to improve solid-phase peptide synthesis as demonstrated in the chemical synthesis of NEDD8 protein, without the use of special dipeptide analogues. Also, the reported approach was found to be useful in decaging a broad range of propargyl-based protecting groups used in chemical protein synthesis. Remarkably, reversing the order of the two residues in the propargylation site resulted in rapid amide bond cleavage, which extends the applicability of this approach beyond a removable backbone modification to a cleavable linker. The easy attach/detach of this functionality was also examined in loading and releasing of biotinylated peptides from streptavidin beads.
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Dipéptidos/química , Oro/química , Proteína NEDD8/síntesis química , Dipéptidos/síntesis química , Humanos , Enlace de Hidrógeno , Estructura Molecular , Proteína NEDD8/química , Agua/químicaRESUMEN
Posttranslational modifications of histone proteins regulate all biological processes requiring access to DNA. Monoubiquitination of histone H2B is a mark of actively transcribed genes in all eukaryotes that also plays a role in DNA replication and repair. Solution and structural studies of the mechanism by which histone ubiquitination modulates these processes depend on the ability to generate homogeneous preparations of nucleosomes containing ubiquitin conjugated to a specific lysine residue. We describe here methods for generating milligram quantities of histone H2B with ubiquitin (Ub) conjugated to Lys 120 via either a nonhydrolyzable, dichloroacetone linkage or a cleavable isopeptide bond. H2B-Ub with an isopeptide linkage is generated by a combination of intein-fusion protein derivatization and native chemical ligation, yielding a fully native ubiquitinated lysine that can be cleaved by Ub isopeptidases. We also describe how to reconstitute nucleosomes containing ubiquitinated H2B.
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Histonas/síntesis química , Ubiquitina/síntesis química , Proteínas de Xenopus/síntesis química , Xenopus laevis , Animales , Histonas/química , Histonas/genética , Hidrólisis , Lisina/síntesis química , Lisina/química , Lisina/genética , Modelos Moleculares , Ubiquitina/química , Ubiquitina/genética , Ubiquitinación , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
Monoubiquitination of histone H2B (H2B-Ub) plays a role in transcription and DNA replication, and is required for normal localization of the histone chaperone, FACT. In yeast, H2B-Ub is deubiquitinated by Ubp8, a subunit of SAGA, and Ubp10. Although they target the same substrate, loss of Ubp8 and Ubp10 cause different phenotypes and alter the transcription of different genes. We show that Ubp10 has poor activity on yeast nucleosomes, but that the addition of FACT stimulates Ubp10 activity on nucleosomes and not on other substrates. Consistent with a role for FACT in deubiquitinating H2B in vivo, a FACT mutant strain shows elevated levels of H2B-Ub. Combination of FACT mutants with deletion of Ubp10, but not Ubp8, confers increased sensitivity to hydroxyurea and activates a cryptic transcription reporter, suggesting that FACT and Ubp10 may coordinate nucleosome assembly during DNA replication and transcription. Our findings reveal unexpected interplay between H2B deubiquitination and nucleosome dynamics.
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Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Elongación Transcripcional/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación , Alelos , Replicación del ADN/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Hidroxiurea/farmacología , Mutación/genética , Nucleosomas/efectos de los fármacos , Fenotipo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Transcripción Genética/efectos de los fármacos , Ubiquitina/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
Organic chemistry allows for the modification and chemical preparation of protein analogues for various studies. The thiolate side chain of the Cys residue has been a key functionality in these ventures. In order to generate complex molecular targets, there is a particular need to incorporate orthogonal protecting groups of the thiolated amino acids to control the directionality of synthesis and modification site. Here, we demonstrate the tuning of palladium chemoselectivity in aqueous medium for on-demand deprotection of several Cys-protecting groups that are useful in protein synthesis and modification. These tools allow the preparation of highly complex analogues as we demonstrate in the synthesis of the copper storage protein and selectively modified peptides with multiple Cys residues. We also report the synthesis of an activity-based probe comprising ubiquitinated histone H2A and its incorporation into nucleosomes and demonstrate its reactivity with deubiquitinating enzyme to generate a covalent nucleosome-enzyme complex.
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Cisteína/química , Paladio/química , Procesamiento Proteico-Postraduccional , Proteínas/síntesis química , Secuencia de Aminoácidos , Aminoácidos , Cobre/química , Enzimas Desubicuitinizantes/metabolismo , Histonas/síntesis química , Nucleosomas/química , Coloración y Etiquetado , Tiazolidinas/química , Proteínas Ubiquitinadas/síntesis químicaRESUMEN
An efficient native chemical ligation approach at Asp and Glu sites is reported applying a hydrazide precursor, as a peptide thioester, and allyl protection at the side chain of Asp and Glu. The allyl protection was efficiently removed, after the ligation step, using the water-soluble palladium complex [Pd(allyl)Cl]2 and glutathione within a few minutes under fully aqueous conditions.
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Histone modifications dynamically regulate chromatin structure and function, thereby mediating many processes that require access to DNA. Chemical protein synthesis has emerged as a powerful approach for generating homogeneously modified histone analogues in workable amounts for subsequent incorporation into nucleosome arrays for biochemical, functional and structural studies. This short review focuses on the strength of total chemical protein synthesis and semisynthetic approaches to generate ubiquitylated histones in their native or non-native forms and the utility of these analogues to decode the role of ubiquitylation in epigenetics.
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Cromatina/química , Histonas/química , Ubiquitinación , Animales , Técnicas de Química Sintética/métodos , Epigénesis Genética , Histonas/síntesis química , Humanos , Modelos Moleculares , Nucleosomas/químicaRESUMEN
Chemical synthesis of histones allows precise control of the installation of post-translational modifications via the coupling of derivatized amino acids. Shortcomings of other approaches for obtaining modified histones for epigenetic studies include heterogeneity of the obtained product and difficulties in incorporating multiple modifications on the same histone. In this protocol, unprotected peptide fragments are prepared by Fmoc solid-phase synthesis and coupled in aqueous buffers via native chemical ligation (NCL; in NCL, a peptide bond is formed between a peptide with an N-terminal Cys and another peptide having a C-terminal thioester). This task is challenging, with obstacles relating to the preparation and ligation of hydrophobic peptides, as well as the requirement for multiple purification steps due to protecting-group manipulations during the polypeptide assembly process. To address this, our approach uses an easily removable solubilizing tag for the synthesis and ligation of hydrophobic peptides, as well as a more efficient and better-yielding method to remove Cys-protecting groups that uses palladium chemistry (specifically [Pd(allyl)Cl]2 and PdCl2 complexes). The utility of this approach is demonstrated in the syntheses of ubiquitinated H2B at Lys34, phosphorylated H2A at Tyr57 and unmodified H4. Each of these analogs can be prepared in milligram quantities within â¼20-30 d.
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Técnicas de Química Sintética/métodos , Histonas/síntesis química , Paladio/química , Fragmentos de Péptidos/síntesis química , Aminoácidos , Fluorenos , Histonas/química , Fragmentos de Péptidos/químicaRESUMEN
Histone H3 methylation plays an important role in regulating gene expression. In histones in general, this mark is dynamically regulated via various demethylases, which found to control cell fate decisions as well as linked to several diseases, including neurological and cancer. Despite major progress in studying methylation mark at various positions in H3 histone proteins, less is known about the regulation of methylated H3 at Lys79. Methylation at this site is known to have direct cross-talk with monoubiquitination of histone H2B at positions Lys120 and 34, as well as with acetylated H3 at Lys9. Herein we applied convergent total chemical protein synthesis to prepare trimethylated H3 at Lys79 to perform initial studies related to the regulation of this mark. Our study enabled us to identify KDM4D lysine demethylase as a potential regulator for trimethylated H3 at Lys79.