C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms

The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD) and an equivalent phage optimized peptide (12/1) were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.     

Association of peak aerobic power with capillary density but not oxidative potential in human vastus lateralis muscle

In this study, we have postulated that in healthy males, peak aerobic power ([Formula: see text]) would associate with muscle capillary density rather than oxidative potential, regardless of fibre type or subtype. To test this, active but untrained volunteers (n = 11) were separated into high (HI) and low (LO) groups based on [Formula: see text] obtained during a progressive cycle task to fatigue. The 26% higher (P?< 0.05) [Formula: see text] observed in HI (40.8?± 1.5?mL·kg(-1)·min(-1), mean?± SE) compared with LO ( 51.4?± 0.90?mL·kg(-1)·min(-1), mean?± SE) was not accompanied by differences in age (21.3?± 1.2 compared with 21.1?± 0.63?years, respectively) or body mass (72.4?± 4.6 compared with 71.6?± 1.9?kg, respectively). Tissue samples obtained from the vastus lateralis indicated greater (P?< 0.05) capillary counts per fibre (CC;?+24%) in HI compared with LO, regardless of fibre type (I, IIA, IIX, IIAX). Capillary density (CD) as measured in a field of defined area was also elevated (+22%; P?< 0.05), as was the number of capillaries per fibre (+22%; P?< 0.05). No differences were observed between the 2 groups in the distribution, area, and the CC/fibre area ratio in the different fibre types and subtypes. Similarly, there was no difference between the HI and LO groups in oxidative potential, as measured by succinic dehydrogenase activity in the different fibre types. It is concluded that the higher capillary density may contribute to improved vascular conductance and the elevated [Formula: see text] observed in the untrained participants.     

Optimal method for efficiently removing extracellular nanofilaments from Shewanella oneidensis MR-1

The identification, production, and potential electron conductivity of bacterial extracellular nanofilaments is an area of great study, specifically in Shewanella oneidensis MR-1. While some studies focus on nanofilaments attached to the cellular body, many studies require the removal of these nanofilaments for downstream applications. The removal of nanofilaments from S. oneidensis MR-1 for further study requires not only that the nanofilaments be detached, but also for the cell bodies to remain intact. This is a study to both qualitatively (AFM) and quantitatively (LC/MS-MS) assess several nanofilament shearing methods and determine the optimal procedure. The best method for nanofilament removal, as judged by maximizing extracellular filamentous proteins and minimizing membrane and intracellular proteins, is vortexing a washed cell culture for 10 min.     

Identification of residues in the heme domain of soluble guanylyl cyclase that are important for basal and stimulated catalytic activity

Nitric oxide signals through activation of soluble guanylyl cyclase (sGC), a heme-containing heterodimer. NO binds to the heme domain located in the N-terminal part of the β subunit of sGC resulting in increased production of cGMP in the catalytic domain located at the C-terminal part of sGC. Little is known about the mechanism by which the NO signaling is propagated from the receptor domain (heme domain) to the effector domain (catalytic domain), in particular events subsequent to the breakage of the bond between the heme iron and Histidine 105 (H105) of the β subunit. Our modeling of the heme-binding domain as well as previous homologous heme domain structures in different states point to two regions that could be critical for propagation of the NO activation signal. Structure-based mutational analysis of these regions revealed that residues T110 and R116 in the αF helix-β1 strand, and residues I41 and R40 in the αB-αC loop mediate propagation of activation between the heme domain and the catalytic domain. Biochemical analysis of these heme mutants allows refinement of the map of the residues that are critical for heme stability and propagation of the NO/YC-1 activation signal in sGC.     

The chemotaxis-like Che1 pathway has an indirect role in adhesive cell properties of Azospirillum brasilense

The Azospirillum brasilense chemotaxis-like Che1 signal transduction pathway was recently shown to modulate changes in adhesive cell surface properties that, in turn, affect cell-to-cell aggregation and flocculation behaviors rather than flagellar-mediated chemotaxis. Attachment to surfaces and root colonization may be functions related to flocculation. Here, the conditions under which A. brasilense wild-type Sp7 and che1 mutant strains attach to abiotic and biotic surfaces were examined using in vitro attachment and biofilm assays combined with atomic force microscopy and confocal microscopy. The nitrogen source available for growth is found to be a major modulator of surface attachment by A. brasilense and could be promoted in vitro by lectins, suggesting that it depends on interaction with surface-exposed residues within the extracellular matrix of cells. However, Che1-dependent signaling is shown to contribute indirectly to surface attachment, indicating that distinct mechanisms are likely underlying flocculation and attachment to surfaces in A. brasilense.     

ATR-mediated phosphorylation of DNA polymerase η is needed for efficient recovery from UV damage

DNA polymerase η (polη) belongs to the Y-family of DNA polymerases and facilitates translesion synthesis past UV damage. We show that, after UV irradiation, polη becomes phosphorylated at Ser601 by the ataxia-telangiectasia mutated and Rad3-related (ATR) kinase. DNA damage-induced phosphorylation of polη depends on its physical interaction with Rad18 but is independent of PCNA monoubiquitination. It requires the ubiquitin-binding domain of polη but not its PCNA-interacting motif. ATR-dependent phosphorylation of polη is necessary to restore normal survival and postreplication repair after ultraviolet irradiation in xeroderma pigmentosum variant fibroblasts, and is involved in the checkpoint response to UV damage. Taken together, our results provide evidence for a link between DNA damage-induced checkpoint activation and translesion synthesis in mammalian cells.     

Hyperstability masks declines in bumphead parrotfish (<Emphasis Type="Italic">Bolbometopon muricatum</Emphasis>) populations

Bolbometopon muricatum, the largest species of parrotfish, is a functionally important species that is characterised by the formation of aggregations for foraging, reproductive, and sleeping behaviours. Aggregations are restricted to shallow reef habitats, the locations of which are often known to local fishers. Bolbometopon muricatum fisheries are therefore vulnerable to overfishing and are likely to exhibit hyperstability, the maintenance of high catch per unit effort (CPUE) while population abundance declines. In this study, we provide a clear demonstration of hyperstable dynamics in a commercial B. muricatum fishery in Isabel Province, Solomon Islands. Initially, we used participatory mapping to demarcate the Kia fishing grounds into nine zones that had experienced different historic levels of fishing pressure. We then conducted comprehensive underwater visual census (UVC) and CPUE surveys across these zones over a 21-month period in 2012–2013. The individual sites for replicate UVC surveys were selected using a generalised random tessellation stratified variable probability design, while CPUE surveys involved trained provincial fisheries officers and local spearfishers. A comparison of fishery-independent abundance data and fishery-dependent CPUE data indicate extreme hyperstability, with CPUE maintained as B. muricatum abundance declines towards zero. Hyperstability may explain the sudden collapses of many B. muricatum spear fisheries across the Pacific and highlights the limitations of using data-poor fisheries assessment methods to evaluate the status of commercially valuable coral reef fishes that form predicable aggregations.     

The Toxoplasma gondii rhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

In mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity‐related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6‐specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18‐specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.