Determinants of Bacteriophage 933W Repressor DNA Binding Specificity

We reported previously that 933W repressor apparently does not cooperatively bind to adjacent sites on DNA and that the relative affinities of 933W repressor for its operators differ significantly from that of any other lambdoid bacteriophage. These findings indicate that the operational details of the lysis-lysogeny switch of bacteriophage 933W are unique among lambdoid bacteriophages. Since the functioning of the lysis-lysogeny switch in 933W bacteriophage uniquely and solely depends on the order of preference of 933W repressor for its operators, we examined the details of how 933W repressor recognizes its DNA sites. To identify the specificity determinants, we first created a molecular model of the 933W repressor-DNA complex and tested the predicted protein-DNA interactions. These results of these studies provide a picture of how 933W repressor recognizes its DNA sites. We also show that, opposite of what is normally observed for lambdoid phages, 933W operator sequences have evolved in such a way that the presence of the most commonly found base sequences at particular operator positions serves to decrease, rather than increase, the affinity of the protein for the site. This finding cautions against assuming that a consensus sequence derived from sequence analysis defines the optimal, highest affinity DNA binding site for a protein

Pneumoproteins and biomarkers of inflammation and coagulation do not predict rapid lung function decline in people living with HIV

Chronic obstructive pulmonary disease (COPD) is among the leading causes of death worldwide and HIV is an independent risk factor for the development of COPD. However, the etiology of this increased risk and means to identify persons with HIV (PWH) at highest risk for COPD have remained elusive. Biomarkers may reveal etiologic pathways and allow better COPD risk stratification. We performed a matched case:control study of PWH in the Strategic Timing of Antiretoviral Treatment (START) pulmonary substudy. Cases had rapid lung function decline (> 40 mL/year FEV1 decline) and controls had stable lung function (+ 20 to − 20 mL/year). The analysis was performed in two distinct groups: (1) those who were virally suppressed for at least 6 months and (2) those with untreated HIV (from the START deferred treatment arm). We used linear mixed effects models to test the relationship between case:control status and blood concentrations of pneumoproteins (surfactant protein-D and club cell secretory protein), and biomarkers of inflammation (IL-6 and hsCRP) and coagulation (d-dimer and fibrinogen); concentrations were measured within ± 6 months of first included spirometry. We included an interaction with treatment group (untreated HIV vs viral suppression) to test if associations varied by treatment group. This analysis included 77 matched case:control pairs in the virally suppressed batch, and 42 matched case:control pairs in the untreated HIV batch (n = 238 total) who were followed for a median of 3 years. Median (IQR) CD4 + count was lowest in the controls with untreated HIV at 674 (580, 838). We found no significant associations between case:control status and pneumoprotein or biomarker concentrations in either virally suppressed or untreated PWH. In this cohort of relatively young, recently diagnosed PWH, concentrations of pneumoproteins and biomarkers of inflammation and coagulation were not associated with subsequent rapid lung function decline. Trial registration: NCT00867048 and NCT01797367

IQGAP1 regulates cell proliferation through a novel CDC42-mTOR pathway

Cell proliferation requires close coordination of cell growth and division to ensure constant cell size through the division cycles. IQGAP1, an effector of CDC42 GTPase has been implicated in the modulation of cell architecture, regulation of exocytosis and in human cancers. The precise mechanism underlying these activities is unclear. Here, we show that IQGAP1 regulates cell proliferation, which requires phosphorylation of IQGAP1 and binding to CDC42. Expression of the C-terminal region of IQGAP1 enhanced cellular transformation and migration, but reduced the cell size, whereas expression of the N-terminus increased the cell size, but inhibited cell transformation and migration. The N-terminus of IQGAP1 interacts with mTOR, which is required for IQGAP1-mediated cell proliferation. These findings are consistent with a model where IQGAP1 serves as a phosphorylation-sensitive conformation switch to regulate the coupling of cell growth and division through a novel CDC42-mTOR pathway, dysregulation of which generates cellular transformation

Model of the 933W repressor-operator complex.

<p><i>(A)</i> 933W repressor in complex with the 933W consensus DNA site. The complex is oriented looking down the C-terminal portion of the DNA contacting (“recognition") to the N-terminal portions. The identity of the putative DNA contacting residues is indicated and highlighted in purple. The DNA is modeled on the right with the “top" strand (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#pone-0034563-g001" target="_blank">Figure 1C</a>) shown in grey and the complementary strand in black. <i>(B)</i> Schematic representation of the contacts identified from the model built structure between residues in the recognition helix of 933W repressor and bases in the 933W consensus half site DNA sequence. Predicted interactions are depicted by arrows.</p

Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis.

Antimicrobial peptides (AMPs) are a diverse group of molecules which play an important role in the innate immune response. Bovine NK-lysins, a type of AMP, have been predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Bovine NK-lysin-derived peptides demonstrate antimicrobial activity against various bacterial pathogens, including several involved in bovine respiratory disease complex (BRDC) in cattle; however, such studies are yet to be performed with one important contributor to the BRDC, Mycoplasma bovis. Therefore, the goal of this study was to assess the antimicrobial activity of bovine NK-lysin-derived peptides on M. bovis. Thirty-mer synthetic peptides corresponding to the functional region helices 2 and 3 of bovine NK-lysins NK1, NK2A, NK2B, and NK2C were evaluated for killing activity on M. bovis isolates. Among four peptides, NK2A and NK2C showed the highest antimicrobial activity against the M. bovis isolates tested. All four NK-lysin peptides induced rapid plasma membrane depolarization in M. bovis at two concentrations tested. However, based on propidium iodide uptake, only NK2A and NK2C appeared capable of causing structural damage to M. bovis plasma membrane. Confocal microscopy, flow cytometry, and transmission electron microscopy further suggested NK-lysin-induced damage to the plasma membrane. Taken together, the findings in this study suggest that plasma membrane depolarization alone was insufficient to induce lethality, but disruption/permeabilization of the M. bovis plasma membrane was the cause of lethality

Affinities of wild-type 933W repressor protein for synthetic 933W operators.

<p>The affinities of 933W repressor for the substituted DNAs are expressed as the dissociation constants of these operators for 933W repressor. NS-DNA that does not contain a DNA sequence to which 933W repressor binds specifically. The affinity of 933W repressor for non-specific DNA is give as an intrinsic ‘per-site’ dissociation constant (K_D^NS = K_D^OBS[2L-n], where L is DNA length and n is site size <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#pone.0034563-JenJacobson1" target="_blank">[28]</a>). The protein concentrations used were corrected for activity.</p

Naturally occurring 933W operator site sequences.

<p><i>(A)</i> The sequences and affinity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#pone.0034563-Bullwinkle1" target="_blank">[12]</a> (dissociation constants, K_D) of 933W repressor binding for naturally occurring 933W binding sites. <i>(B)</i> Logo sequence alignment of the ten ‘half-sites’ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#pone.0034563-Schneider1" target="_blank">[20]</a> found within the five repressor operator sites and the consensus 933W repressor binding site sequence constructed from <i>in vitro</i> selection <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#pone.0034563-Koudelka1" target="_blank">[11]</a>.</p

Role of Position 4 base substituents in determining 933W repressor affinity for DNA.

<p>(A) The base sequence at position 4 in one half site of the 933W binding site was changed as indicated. Dissociation constants were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#s2" target="_blank">Methods and Materials</a>. (B) DNase I footprinting analysis of complexes between 933W repressor and 933W consensus sequence or its position 4 variants shown in (A) were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#s2" target="_blank">Methods and Materials</a>. Shown is a Phosphoromager scan of the gel. The sequence of the operators are shown together with the positions of the bands resulting from DNase I cleavages in the absence (−) and presence (+) of saturating concentrations of 933W repressor.</p