M. Eugenio Vázquez

From transcription factors to designed sequence-specific DNA-binding peptides

Transcription factors are DNA-binding proteins responsible for initiating the transcription of particular genes upon interacting with specific DNA sequences located at their promoter or enhancer regions. The DNA recognition process, which is extremely selective, is mediated by non-covalent interactions between appropriately arranged structural motifs of the protein and exposed surfaces of the DNA bases and backbone. The great variability in DNA recognition by transcription factors has hampered the characterization of an amino acid-base step recognition code, making it very difficult to design non-natural peptides that can mimic the DNA-binding properties of these naturally occurring counterparts. However, in recent years, several transcription factor-based miniature proteins capable of tight interaction with specific DNA sites have been successfully constructed, most of them using bottom-up synthetic approaches.

A new environment-sensitive fluorescent amino acid for Fmoc-based solid phase peptide synthesis

A new 4-(N,N-dimethylamino) phthalimide-based environment-sensitive fluorescent building block for solid phase peptide synthesis, has been synthesized and incorporated into peptides. Peptides incorporating this residue show great potential for biological applications in sensing protein/protein interactions.

Highly Sensitive SERS Quantification of the Oncogenic Protein c‑Jun in Cellular Extracts

A surface-enhanced Raman scattering (SERS)-based sensor was developed for the detection of the oncoprotein c-Jun at nanomolar levels. c-Jun is a member of the bZIP (basic zipper) family of dimeric transcriptional activators, and its overexpression has been associated with carcinogenic mechanisms in several human cancers. For our sensing purpose, we exploited the ability of c-Jun to heterodimerize with its native protein partner, c-Fos, and therefore designed a c-Fos peptide receptor chemically modified to incorporate a thiophenol (TP) group at the N-terminal site. The TP functionality anchors the c-Fos protein onto the metal substrate and works as an effective SERS probe to sense the structural rearrangements associated with the c-Fos/c-Jun heterodimerization.

DNA Recognition by Synthetic Constructs

The interaction of transcription factors with specific DNA sites is key for the regulation of gene expression. Despite the availability of a large body of structural data on protein–DNA complexes, we are still far from fully understanding the molecular and biophysical bases underlying such interactions. Therefore, the development of non‐natural agents that can reproduce the DNA‐recognition properties of natural transcription factors remains a major and challenging goal in chemical biology. In this review we summarize the basics of double‐stranded DNA recognition by transcription factors, and describe recent developments in the design and preparation of synthetic DNA binders. We mainly focus on synthetic peptides that have been designed by following the DNA interaction of natural proteins, and we discuss how the tools of organic synthesis can be used to make artificial constructs equipped with functionalities that introduce additional properties to the recognition process, such as sensing and controllability.

Cyclin A Probes by Means of Intermolecular Sensitization of Terbium-Chelating Peptides

Intermolecular sensitization of lanthanide ions was effectively implemented in the development of fluorescent sensors targeting cyclin A. A chelating unit has been conjugated to peptides containing a known cyclin A binding motif (CBM). Upon interaction of the modified terbium-chelating peptides with the cyclin A substrate recruitment groove, the Tb3+ ion is placed in the vicinity of the Trp217 side chain, which results in efficient intermolecular terbium sensitization and specific long wavelength fluorescent emission from the metal center.

A Synthetic Miniprotein that Binds Specific DNA Sequences by Contacting Both the Major and the Minor Groove

Attachment of a slightly modified basic region of a bZIP protein (GCN4) to a distamycin-related tripyrrole provides a bivalent system capable of binding with high affinity to specific DNA sequences. Appropriate adjustment of the linker between the two units has led to a hybrid that binds a 9 base-pair-long DNA site (TTTTATGAC) with low nanomolar affinity at 4 degrees C. Circular dichroism and gel retardation studies indicate that the binding occurs by simultaneous insertion of the bZIP basic region into the DNA major groove and the tripyrrole moiety into the minor groove of the flanking sequence. Analysis of hybrids bearing alternative linkers revealed that tight, specific binding is strongly dependent on the length and nature of the connecting unit.

Straightforward access to bisbenzamidine DNA binders and their use as versatile adaptors for DNA-promoted processes

Bisbenzamidines are an important family of minor groove DNA-binding agents. We present a one-step synthesis of aromatic aza-bisbenzamidines that allows straightforward and versatile access to a large number of these molecules. One of them, the azide-aza-bisbenzamidine 13, can be readily modified via click-chemistry with a variety of functionalities that can, therefore, be delivered to the vicinity of an A/T-rich DNA minor groove. This strategy, therefore, provides a simple means for triggering site selective, DNA-promoted biochemical and physicochemical processes.

Reversible Supramolecular Assembly at Specific DNA Sites: Nickel-Promoted Bivalent DNA Binding with Designed Peptide and Bipyridyl–Bis(benzamidine) Components†

At specific DNA sites, nickel(II) salts promote the assembly of designed components, namely a bis(histidine)-modified peptide that is derived from a bZIP transcription factor and a bis(benzamidine) unit that is equipped with a bipyridine. This programmed supramolecular system with emergent properties reproduces some key characteristics of naturally occurring DNA-binding proteins, such as bivalence, selectivity, responsiveness to external agents, and reversibility.

Temporary Electrostatic Impairment of DNA Recognition: Light-Driven DNA Binding of Peptide Dimers†

Gene expression relies on a myriad of carefully orchestrated interactions between specialized proteins called transcription factors (TFs) and regulatory DNA sequences. In general, such interactions are subtly regulated in time and space, so that many TFs remain inactive until receiving an appropriate activation signal. It is well-established that the DNA readout by TFs largely relies on interactions between amino acid side chains and the DNA bases and phosphates. Among these contacts, those involving positively charged basic amino acids are critical for the thermodynamic stability of their DNA complexes. We reasoned that the temporary electrostatic deactivation of such contacts might provide for the development of TF-based systems where DNA binding activity could be externally controllable, for instance by light. These systems could be useful methods for transcriptional control, or for probing spatiotemporal patterns of gene expression in living organisms. It is curious that despite the well-established use of light-activated compounds in chemical biology, examples of photocontrolled DNA binding are certainly scarce. These include reversible switches based on the photostationary equilibrium of azo-modified DNA binders, special chromophores with poor DNA binding affinity and/or specificity, and single-use caging strategies for triggering minor groove binding or intercalation. Thus, inspired by the use of negatively charged elements to modulate cell internalization, we sought to develop a general strategy for photocontrolling the sequencespecific DNA binding of TF peptide mimics. Herein we demonstrate that tethering polyanionic tails to basic DNA-binding bZIP peptides through a light-sensitive linker suppresses the DNA interaction. Upon irradiation, the negatively charged appendages are released, and the DNAbinding activity is thus restored. As reference system for implementing the strategy we chose the GCN4 transcription factor, an archetypical bZIP TF that specifically binds to ATF/CREB (5’-ATGA(c/g)TCAT-3’) or AP1 (5’-ATGA(c)TCAT-3’) sites as a leucine zipper-mediated dimer of uninterrupted a helices. The N-terminal basic regions feature many positively charged amino acids that are key for the DNA recognition (10 Lys or Arg out of 31 residues in the basic region; Figure 1, GCN4br). It has been shown that the leucine zipper itself can be substituted by a number of dimerizing units without significant loss in the DNA binding properties. Therefore, in our first iteration for the design of the electrostatically impaired DNA-binding peptides, we selected the minimum sequence of the bZIP basic region (br) that it is known to retain the DNA binding ability when engineered as a disulfide dimer. This minimal peptide was extended at the N-terminus by adding acidic extensions with four or eight Glu residues linked to the core br sequence

Advances in lanthanide-based luminescent peptide probes for monitoring the activity of kinase and phosphatase

Signaling pathways based on protein phosphorylation and dephosphorylation play critical roles in the orchestration of complex biochemical events and form the core of most signaling pathways in cells (i.e. cell cycle regulation, cell motility, apoptosis, etc.). The understanding of these complex signaling networks is based largely on the biochemical study of their components, i.e. kinases and phosphatases. The development of luminescent sensors for monitoring kinase and phosphatase activity is therefore an active field of research. Examples in the literature usually rely on the modulation of the fluorescence emission of organic fluorophores. However, given the exceptional photophysical properties of lanthanide ions, there is an increased interest in their application as emissive species for monitoring kinase and phosphatase activity. This review summarizes the advances in the development of lanthanide‐based luminescent peptide sensors as tools for the study of kinases and phosphatases and provides a critical description of current examples and synthetic approaches to understand these lanthanide‐based luminescent peptide sensors.