67 research outputs found

    Selective antagonism of cJun for cancer therapy

    Get PDF
    The activator protein-1 (AP-1) family of transcription factors modulate a diverse range of cellular signalling pathways into outputs which can be oncogenic or anti-oncogenic. The transcription of relevant genes is controlled by the cellular context, and in particular by the dimeric composition of AP-1. Here, we describe the evidence linking cJun in particular to a range of cancers. This includes correlative studies of protein levels in patient tumour samples and mechanistic understanding of the role of cJun in cancer cell models. This develops an understanding of cJun as a focal point of cancer-altered signalling which has the potential for therapeutic antagonism. Significant work has produced a range of small molecules and peptides which have been summarised here and categorised according to the binding surface they target within the cJun-DNA complex. We highlight the importance of selectively targeting a single AP-1 family member to antagonise known oncogenic function and avoid antagonism of anti-oncogenic function

    Understanding the coupling between DNA damage detection and UvrA’s ATPase using bulk and single molecule kinetics

    Get PDF
    Nucleotide excision repair (NER) protects cells against diverse types of DNA damage, principally UV irradiation. In Escherichia coli, damage is recognized by 2 key enzymes: UvrA and UvrB. Despite extensive investigation, the role of UvrA’s 2 ATPase domains in NER remains elusive. Combining single-molecule fluorescence microscopy and classic biochemical methods, we have investigated the role of nucleotide binding in UvrA’s kinetic cycle.Measurement of UvrA’s steady-state ATPase activity shows it is stimulated upon binding DNA (kcat 0.71–1.07/s). Despite UvrA’s ability to discriminate damage,we find UV-damagedDNA does not alter the steady-state ATPase. To understand how damage affects UvrA, we studied its binding to DNA under various nucleotide conditions at the single molecule level. We have found that both UV damage and nucleotide cofactors affect the attached lifetime of UvrA. In the presence of ATP and UV damage, the lifetime is significantly greater compared with undamaged DNA. To reconcile these observations, we suggest that UvrA uses negative cooperativity between its ATPase sites that is gated by damage recognition. Only in the presence of damage is the second site activated, most likely in a sequential manner.—Barnett, J. T., Kad, N. M. Understanding the coupling between DNA damage detection and UvrA’s ATPase using bulk and single molecule kinetics

    Single-cell mutagenic responses and cell death revealed in real time

    Get PDF

    A Branched Kinetic Scheme Describes the Mechanochemical Coupling of Myosin Va Processivity in Response to Substrate

    Get PDF
    Myosin Va is a double-headed cargo-carrying molecular motor that moves processively along cellular actin filaments. Long processive runs are achieved through mechanical coordination between the two heads of myosin Va, which keeps their ATPase cycles out of phase, preventing both heads detaching from actin simultaneously. The biochemical kinetics underlying processivity are still uncertain. Here we attempt to define the biochemical pathways populated by myosin Va by examining the velocity, processive run-length, and individual steps of a Qdot-labeled myosin Va in various substrate conditions (i.e., changes in ATP, ADP, and Pi) under zero load in the single-molecule total internal reflection fluorescence microscopy assay. These data were used to globally constrain a branched kinetic scheme that was necessary to fit the dependences of velocity and run-length on substrate conditions. Based on this model, myosin Va can be biased along a given pathway by changes in substrate concentrations. This has uncovered states not normally sampled by the motor, and suggests that every transition involving substrate binding and release may be strain-dependent. © 2012 Biophysical Society

    The effect of helix-inducing constraints and downsizing upon a transcription block survival-derived functional cJun antagonist

    Get PDF
    Inhibition of cJun is established as a promising therapeutic approach, particularly in cancer. We recently developed the "transcription block survival" (TBS) screening platform to derive functional peptide antagonists of transcription factor activity by ablating their ability to bind to cognate DNA. Using TBS, we screened a >131,000-member peptide library to select a 63-mer peptide that bound cJun and prevented 12-O-tetradecanoylphorbol-13-acetate response element (TRE) DNA binding. Iterative truncation was next combined with a systematic exploration of side-chain cyclization to derive a minimal active sequence. The resulting dual lactamized sequence was >40% smaller and retained low nM target affinity (equilibrium binding constant [K D ] = 0.2 versus 9.7 nM), with 8 residues at the acidic region required for functional antagonism. However, even modest C-terminal truncation resulted in functional loss. The peptide functionally antagonizes cJun (half-maximal inhibitory concentration [IC50] = 13 versus 45 μM) and is considerably more stable in human serum relative to its non-lactamized counterpart and HingeW

    An Approach to Derive Functional Peptide Inhibitors of Transcription Factor Activity

    Get PDF
    We report the development of a high-throughput, intracellular "transcription block survival" (TBS) screening platform to derive functional transcription factor antagonists. TBS is demonstrated using the oncogenic transcriptional regulator cJun, with the development of antagonists that bind cJun and prevent both dimerization and, more importantly, DNA binding remaining a primary challenge. In TBS, cognate TRE sites are introduced into the coding region of the essential gene, dihydrofolate reductase (DHFR). Introduction of cJun leads to TRE binding, preventing DHFR expression by directly blocking RNA polymerase gene transcription to abrogate cell proliferation. Peptide library screening identified a sequence that both binds cJun and antagonizes function by preventing DNA binding, as demonstrated by restored cell viability and subsequent in vitro hit validation. TBS is an entirely tag-free genotype-to-phenotype approach, selecting desirable attributes such as high solubility, target specificity, and low toxicity within a complex cellular environment. TBS facilitates rapid library screening to accelerate the identification of therapeutically valuable sequences

    Identification of the target and mode of action for the prokaryotic nucleotide excision repair inhibitor ATBC

    Get PDF
    In bacteria, nucleotide excision repair (NER) plays a major role in repairing DNA damage from a wide variety of sources. Therefore, its inhibition offers potential to develop a new antibacterial in combination with adjuvants, such as UV light. To date, only one known chemical inhibitor of NER is 2-(5-amino-1,3,4-thiadiazol-2-yl)benzo(f)chromen-3-one (ATBC) exists and targets Mycobacterium tuberculosis NER. To enable the design of future drugs, we need to understand its mechanism of action. To determine the mechanism of action, we used in silico structure-based prediction, which identified the ATP-binding pocket of Escherichia coli UvrA as a probable target. Growth studies in E. coli showed it was nontoxic alone, but able to impair growth when combined with DNA-damaging agents, and as we predicted, it reduced by an approximately 70% UvrA's ATPase rate. Since UvrA's ATPase activity is necessary for effective DNA binding, we used single-molecule microscopy to directly observe DNA association. We measured an approximately sevenfold reduction in UvrA molecules binding to a single molecule of dsDNA suspended between optically trapped beads. These data provide a clear mechanism of action for ATBC, and show that targeting UvrA's ATPase pocket is effective and ATBC provides an excellent framework for the derivation of more soluble inhibitors that can be tested for activity

    Developing novel antimicrobials by combining cancer chemotherapeutics with bacterial DNA repair inhibitors

    Get PDF
    Cancer chemotherapeutics kill rapidly dividing cells, which includes cells of the immune system. The resulting neutropenia predisposes patients to infection, which delays treatment and is a major cause of morbidity and mortality. To tackle this problem, we have isolated several compounds that inhibit bacterial DNA repair, alone they are non-toxic, however in combination with DNA damaging anti-cancer drugs, they prevent bacterial growth. These compounds were identified through screening of an FDA-approved drug library in the presence of the anti-cancer compound cisplatin. Using a series of triage tests, the screen was reduced to a handful of drugs that were tested for specific activity against bacterial nucleotide excision DNA repair (NER). Five compounds emerged, of which three possess promising antimicrobial properties including cell penetrance, and the ability to block replication in a multi-drug resistant clinically relevant E. coli strain. This study suggests that targeting NER could offer a new therapeutic approach tailor-made for infections in cancer patients, by combining cancer chemotherapy with an adjuvant that targets DNA repair

    The TFIIH components p44/p62 act as a damage sensor during nucleotide excision repair

    Get PDF
    Nucleotide excision repair (NER) protects the genome following exposure to diverse types of DNA damage, including UV light and chemotherapeutics. Mutations in human NER genes lead to diseases such as xeroderma pigmentosum and Cockayne syndrome. In eukaryotes, the major transcription factor TFIIH is the central hub of NER. The core components of TFIIH include the helicases XPB, XPD, and the five core structural subunits. Two of these core-TFIIH proteins, p44 and p62 remain relatively unstudied; although p44 is known to regulate the helicase activity of XPD during NER. p62's role is thought to be structural; however, a recent cryo-EM structure shows p44, p62, and XPD making contacts with each other, implying a more extensive role in DNA repair beyond the structural integrity of TFIIH. Here, we show that p44 stimulates XPD's ATPase, but upon encountering DNA damage further stimulation is only observed when p62 is in the ternary complex. More significantly, we show that the p44/p62 complex binds DNA independently of XPD and diffuses along its backbone, indicating a novel DNA-binding entity in TFIIH. These data support a role for p44/p62 in TFIIH's mechanism of damage detection. This revises our understanding of TFIIH and prompts more extensive investigation of all of the core subunits, for an active role during both DNA repair and transcription

    In vitro single molecule and bulk phase studies reveal the AP-1 transcription factor cFos binds to DNA without its partner cJun

    Get PDF
    The AP-1 transcription factor family crucially regulates progression of the cell cycle, as well as playing roles in proliferation, differentiation, and the stress response. The two best described AP-1 family members, cFos and cJun, are known to dimerize to form a functional AP-1 heterodimer that binds to a consensus response element sequence. Although cJun can also homodimerize and bind to DNA, the canonical view is that cFos cannot bind DNA without heterodimerizing with cJun. Here, we show that cFos can actually bind to DNA in the absence of cJun in vitro. Using dual color single molecule imaging of cFos alone, we directly visualize binding to and movement on DNA. Of all these DNA-bound proteins, detailed analysis suggested 30-46% were homodimers. Furthermore, we constructed fluorescent protein fusions of cFos and cJun for FRET experiments. These constructs indicated complete dimerization of cJun, but although cFos could dimerize, its extent was reduced. Finally, to provide orthogonal confirmation of cFos binding to DNA we performed bulk-phase circular dichroism experiments that showed clear structural changes in DNA; these were found to be specific to the AP-1 consensus sequence. Taken together our results clearly show cFos can interact with DNA both as monomers and dimers independently of its archetypal partner, cJun
    • …
    corecore