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

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.

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

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 to 46% were homodimers. Furthermore, we constructed fluorescent protein fusions of cFos and cJun for Förster resonance energy transfer 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.

An Approach to Derive Functional Peptide Inhibitors of Transcription Factor Activity.

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.

Selective antagonism of cJun for cancer therapy.

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.

Non-pathogenic Escherichia coli biofilms: effects of growth conditions and surface properties on structure and curli gene expression.

Biofilm formation is a harmful phenomenon in many areas, such as in industry and clinically, but offers advantages in the field of biocatalysis for the generation of robust biocatalytic platforms. In this work, we optimised growth conditions for the production of Escherichia coli biofilms by three strains (PHL644, a K-12 derivative with enhanced expression of the adhesin curli; the commercially-used strain BL21; and the probiotic Nissle 1917) on a variety of surfaces (plastics, stainless steel and PTFE). E. coli PHL644 and PTFE were chosen as optimal strain and substratum, respectively, and conditions (including medium, temperature, and glucose concentration) for biofilm growth were determined. Finally, the impact of these growth conditions on expression of the curli genes was determined using flow cytometry for planktonic and sedimented cells. We reveal new insights into the formation of biofilms and expression of curli in E. coli K-12 in response to environmental conditions.

Voluntary increase in latissimus dorsi muscle activity during the lat pull-down following expert instruction.

It has been observed anecdotally that while performing the multijoint lat pull-down exercise, novice strength trainers often rely on the elbow flexors to complete the movement rather than fully utilizing the relevant back muscles such as the latissimus dorsi (LD) and teres major (TM). The primary aim of the study was to determine whether specific technique instruction could result in a voluntary increase in LD and TM electromyographic (EMG) activity with a concurrent decrease in the activity of the biceps brachii (BB) during the front wide-grip lat pull-down exercise. Eight women with little or no background in strength training were asked to perform lat pull-down exercise with only basic instruction, performing 2 sets of 3 repetitions at 30% max. After a brief rest, subjects then performed the same 2 sets of 3 repetitions following verbal technique instruction on how to emphasize the latissimus while de-emphasizing the biceps. EMG activity of the LD, TM, and BB were recorded, converted to root mean square, and normalized to the maximum isometric EMG (NrmsEMG). A significant increase was seen in Nrms EMG in the LD (p = 0.005) from the average of preinstruction NrmsEMG to the average of postinstruction NrmsEMG. No significant differences were observed between pre- and postinstruction muscle activity in the BB or TM. The results show that untrained individuals can voluntarily increase the activity of a specified muscle group during the performance of a multijoint resistance exercise, but the increase probably does not represent "isolation" of the muscle group through voluntary reduction of activity in complementary agonist muscles.

Characterization of a Plasmodium falciparum mutant that has deleted the majority of the gametocyte-specific Pf11-1 locus

We identified a gametocyte-specific protein of Plasmodium falciparum called Pf11-1 and provide experimental evidence that this molecule is involved in the emergence of gametes of the infected erythrocyte (gametogenesis). A mutant parasite clone, which has deleted over 90% of the PF11-1 gene locus, was an important control to establish the gametocyte-specific expression of the Pf11-1. Molecular analysis of the Pf11-1 deletion indicates that it is presumably due a chromosome breakage with subsequent "healing" by the addition of telomeric heptanucleotides. Moreover, similar DNA rearrangements are observed in most of the laboratory isolates during asexual propagation in vitro