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

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

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

Plastic Deformation in Laser-Induced Shock Compression of Monocrystalline Copper

Copper monocrystals were subjected to shock compression at pressures of 10–60 GPa by a short (3 ns initial) duration laser pulse. Transmission electron microscopy revealed features consistent with previous observations of shock-compressed copper, albeit at pulse durations in the µs regime. The results suggest that the defect structure is generated at the shock front. A mechanism for dislocation generation is presented, providing a realistic prediction of dislocation density as a function of pressure. The threshold stress for deformation twinning in shock compression is calculated from the constitutive equations for slip, twinning, and the Swegle-Grady relationship

MyBP-C: one protein to govern them all

The heart is an extraordinarily versatile pump, finely tuned to respond to a multitude of demands. Given the heart pumps without rest for decades its efficiency is particularly relevant. Although many proteins in the heart are essential for viability, the non-essential components can attract numerous mutations which can cause disease, possibly through alterations in pumping efficiency. Of these, myosin binding protein C is strongly over-represented with ~ 40% of all known mutations in hypertrophic cardiomyopathy. Therefore, a complete understanding of its molecular function in the cardiac sarcomere is warranted. In this review, we revisit contemporary and classical literature to clarify both the current standing of this fast-moving field and frame future unresolved questions. To date, much effort has been directed at understanding MyBP-C function on either thick or thin filaments. Here we aim to focus questions on how MyBP-C functions at a molecular level in the context of both the thick and thin filaments together. A concept that emerges is MyBP-C acts to govern interactions on two levels; controlling myosin access to the thin filament by sequestration on the thick filament, and controlling the activation state and access of myosin to its binding sites on the thin filament. Such affects are achieved through directed interactions mediated by phosphorylation (of MyBP-C and other sarcomeric components) and calcium

Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes

Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA?D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB? DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC?DNA complex, we investigated three UvrB mutants: Y96A, a b-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell. © 2013 The Author(s)

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

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

Culture media, DMSO and efflux affect the antibacterial activity of cisplatin and oxaliplatin

Cisplatin was originally discovered through its antibacterial action and subsequently has found use as a potent broad-spectrum anticancer agent. This study determines the effect of growth media and solvent on the antibacterial activity of cisplatin and its analogue, oxaliplatin. Escherichia coli MG1655 or MG1655 ΔtolC was treated with the platinum compounds under different conditions and susceptibility was determined. Our results showed that DMSO reduced the activity of cisplatin by fourfold (MIC 12·5 mg l-1 ) compared with 0·9% NaCl-solubilized cisplatin (MIC 3·12 mg l-1 ) when tested in MOPS. Surprisingly, complete loss of activity was observed in Mueller-Hinton Broth II (MHB II). By supplementing MOPS with individual components of MHB II such as the sulphur-containing amino acids, l-cysteine and l-methionine, individually or in combination reduced activity by ≥8-fold (MIC ≥25 mg l-1 ). Oxaliplatin was less active against E. coli (MIC 100 mg l-1 ) but exhibited similar inactivation in the presence of DMSO, MHBII or MOPS spiked with l-cysteine and l-methionine (MIC ≥400 mg l-1 ). Our data suggest that the antibacterial activity of cisplatin and oxaliplatin is modulated by both choice of solvent and composition of growth media. We demonstrate that this is primarily due to sulphur-containing amino acids cysteine and methionine, an essential component of the recommended media for testing antimicrobial susceptibility, MHBII

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