Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies.

De novo peptides and proteins that switch state in response to chemical and physical cues would advance protein design and synthetic biology. Here we report two designed systems that disassemble and reassemble upon site-specific phosphorylation and dephosphorylation, respectively. As starting points, we use hyperthermostable de novo antiparallel and parallel coiled-coil heterotetramers, i.e., A2B2 systems, to afford control in downstream applications. The switches are incorporated by adding protein kinase A phosphorylation sites, R-R-X-S, with the phosphoacceptor serine residues placed to maximize disruption of the coiled-coil interfaces. The unphosphorylated peptides assemble as designed and unfold reversibly when heated. Addition of kinase to the assembled states unfolds them with half-lives of ≤5 min. Phosphorylation is reversed by Lambda Protein Phosphatase resulting in tetramer reassembly. We envisage that the new de novo designed coiled-coil components, the switches, and a mechanistic model for them will be useful in synthetic biology, biomaterials, and biotechnology applications.

De novo designed peptides for cellular delivery and subcellular localisation.

Increasingly, it is possible to design peptide and protein assemblies de novo from first principles or computationally. This approach provides new routes to functional synthetic polypeptides, including designs to target and bind proteins of interest. Much of this work has been developed in vitro. Therefore, a challenge is to deliver de novo polypeptides efficiently to sites of action within cells. Here we describe the design, characterisation, intracellular delivery, and subcellular localisation of a de novo synthetic peptide system. This system comprises a dual-function basic peptide, programmed both for cell penetration and target binding, and a complementary acidic peptide that can be fused to proteins of interest and introduced into cells using synthetic DNA. The designs are characterised in vitro using biophysical methods and X-ray crystallography. The utility of the system for delivery into mammalian cells and subcellular targeting is demonstrated by marking organelles and actively engaging functional protein complexes.

Resistance evolution can disrupt antibiotic exposure protection through competitive exclusion of the protective species.

Antibiotic degrading bacteria can reduce the efficacy of drug treatments by providing antibiotic exposure protection to pathogens. While this has been demonstrated at the ecological timescale, it is unclear how exposure protection might alter and be affected by pathogen antibiotic resistance evolution. Here, we utilised a two-species model cystic fibrosis (CF) community where we evolved the bacterial pathogen Pseudomonas aeruginosa in a range of imipenem concentrations in the absence or presence of Stenotrophomonas maltophilia, which can detoxify the environment by hydrolysing ß-lactam antibiotics. We found that P. aeruginosa quickly evolved resistance to imipenem via parallel loss of function mutations in the oprD porin gene. While the level of resistance did not differ between mono- and co-culture treatments, the presence of S. maltophilia increased the rate of imipenem resistance evolution in the four µg/ml imipenem concentration. Unexpectedly, imipenem resistance evolution coincided with the extinction of S. maltophilia due to increased production of pyocyanin, which was cytotoxic to S. maltophilia. Together, our results show that pathogen resistance evolution can disrupt antibiotic exposure protection due to competitive exclusion of the protective species. Such eco-evolutionary feedbacks may help explain changes in the relative abundance of bacterial species within CF communities despite intrinsic resistance to anti-pseudomonal drugs.

Super-resolution Imaging of the T cell Central Supramolecular Signaling Cluster Using Stimulated Emission Depletion Microscopy.

Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center interface of T cells activated by antigen presenting cells (APC). The adaptor protein linker for activation of T cells (LAT) is a key cSMAC component. The cSMAC has widely been studied using total internal reflection fluorescence microscopy of CD4+ T cells activated by planar APC substitutes. Here we provide a protocol to image the cSMAC in its cellular context at the interface between a T cell and an APC. Super resolution stimulated emission depletion microscopy (STED) was utilized to determine the localization of LAT, that of its active, phosphorylated form and its entire pool. Agonist peptide-loaded APCs were incubated with TCR transgenic CD4+ T cells for 4.5 min before fixation and antibody staining. Fixed cell couples were imaged using a 100x 1.4 NA objective on a Leica SP8 AOBS confocal laser scanning microscope. LAT clustered in multiple supramolecular complexes and their number and size distributions were determined. Using this protocol, cSMAC properties in its cellular context at the interface between a T cell and an APC could be quantified.

Transient protein accumulation at the center of the T cell antigen-presenting cell interface drives efficient IL-2 secretion.

Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center of the interface of T cells activated by antigen-presenting cells. We have determined that it is composed of multiple complexes of a supramolecular volume of up to 0.5 µm3 and associated with extensive membrane undulations. To determine cSMAC function, we have systematically manipulated the localization of three adaptor proteins, LAT, SLP-76, and Grb2. cSMAC localization varied between the adaptors and was diminished upon blockade of the costimulatory receptor CD28 and deficiency of the signal amplifying kinase Itk. Reconstitution of cSMAC localization restored IL-2 secretion which is a key T cell effector function as dependent on reconstitution dynamics. Our data suggest that the cSMAC enhances early signaling by facilitating signaling interactions and attenuates signaling thereafter through sequestration of a more limited set of signaling intermediates.

Cells receive dozens of signals at different times and in different places. Integrating incoming information and deciding how to respond is no easy task. Signaling molecules on the cell surface pass messages inwards using chemical messengers that interact in complicated networks within the cell. One way to unravel the complexity of these networks is to look at specific groups of signaling molecules in test tubes to see how they interact. But the interior of a living cell is a very different environment. Molecules inside cells are tightly packed and, under certain conditions, they interact with each other by the thousands. They form structures known as 'supramolecular complexes', which changes their behavior. One such supramolecular complex is the 'central supramolecular activation cluster', or cSMAC for short. It forms under the surface of immune cells called T cells when they are getting ready to fight an infection. Under the microscope, the cSMAC looks like the bullseye of a dartboard, forming a crowd of signaling molecules at the center of the interface between the T cell and another cell. Its exact role is not clear, but evidence suggests it helps to start and stop the signals that switch T cells on. The cSMAC contains two key protein adaptors called LAT and SLP-76 that help to hold the structure together. So, to find out what the cSMAC does, Clark et al. genetically modified these adaptors to gain control over when the cSMAC forms. Clark et al. examined mouse T cells using super-resolution microscopy and electron microscopy, watching as other immune cells delivered the signal to switch on. As the T cells started to activate, the composition of the cSMAC changed. In the first two minutes after the cells started activating, the cSMAC included a large number of different components. This made T cell activation more efficient, possibly because the supramolecular complex was helping the network of signals to interact. Later, the cSMAC started to lose many of these components. Separating components may have helped to stop the activation signals. Understanding how T cells activate could lead to the possibility of turning them on or off in immune-related diseases. But these findings are not just relevant to immune cells. Other cells also use supramolecular complexes to control their signaling. Investigating how these complexes change over time could help us to understand how other cell types make decisions.

PEGylated Bis-Sulfonamide Carbonic Anhydrase Inhibitors Can Efficiently Control the Growth of Several Carbonic Anhydrase IX-Expressing Carcinomas.

A series of aromatic/heterocyclic bis-sulfonamides were synthesized from three established aminosulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor pharmacophores, coupled with either ethylene glycol oligomeric or polymeric diamines to yield bis-sulfonamides with short or long (polymeric) linkers. Testing of novel inhibitors and their precursors against a panel of membrane-bound CA isoforms, including tumor-overexpressed CA IX and XII and cytosolic isozymes, identified nanomolar-potent inhibitors against both classes and several compounds with medium isoform selectivity in a detailed structure-activity relationship study. The ability of CA inhibitors to kill tumor cells overexpressing CA IX and XII was tested under normoxic and hypoxic conditions, using 2D and 3D in vitro cellular models. The study identified a nanomolar potent PEGylated bis-sulfonamide CA inhibitor (25) able to significantly reduce the viability of colon HT-29, breast MDA-MB231, and ovarian SKOV-3 cancer cell lines, thus revealing the potential of polymer conjugates in CA inhibition and cancer treatment.

Investigating the influence of haemodynamic stimuli on intracranial aneurysm inception.

We propose a novel method to reconstruct the hypothetical geometry of the healthy vasculature prior to intracranial aneurysm (IA) formation: a Frenet frame is calculated along the skeletonization of the arterial geometry; upstream and downstream boundaries of the aneurysmal segment are expressed in terms of the local Frenet frame basis vectors; the hypothetical healthy geometry is then reconstructed by propagating a closed curve along the skeleton using the local Frenet frames so that the upstream boundary is smoothly morphed into the downstream boundary. This methodology takes into account the tortuosity of the arterial vasculature and requires minimal user subjectivity. The method is applied to 22 clinical cases depicting IAs. Computational fluid dynamic simulations of the vasculature without IA are performed and the haemodynamic stimuli in the location of IA formation are examined. We observe that locally elevated wall shear stress (WSS) and gradient oscillatory number (GON) are highly correlated (20/22 for WSS and 19/22 for GON) with regions susceptible to sidewall IA formation whilst haemodynamic indices associated with the oscillation of the WSS vectors have much lower correlations.

Assessing remission in major depressive disorder and generalized anxiety disorder clinical trials with the discan metric of the Sheehan disability scale.

Relatively little research has focused on the relationship between functional remission and symptomatic remission in mood and anxiety disorders. This study investigates the relationship and synchrony between symptomatic and functional remission in outpatients with major depressive disorder (MDD) and generalized anxiety disorder (GAD). Using data from three MDD (N=1419) and four GAD (N=1847) randomized, placebo-controlled duloxetine studies, we calculated the percentages of patients meeting symptomatic, functional, and combined functional-symptomatic remission criteria for each disorder. We also calculated mean depression [17-item Hamilton depression rating scale (HAMD17), Montgomery-Asberg depression rating scale] scores and mean anxiety (Hamilton anxiety rating scale) scores for patients meeting Sheehan disability scale (SDS) functional remission and the mean SDS scores for patients with symptomatic remission. Among the patients with MDD, 38% achieved symptomatic remission (HAMD17 ≤ 7), 32% achieved functional remission (SDS ≤ 6), and 23% achieved combined functional-symptomatic remission. Mean HAMD17 and Montgomery-Asberg depression rating scale scores for patients with functional remission were approximately 6. Mean SDS total scores for patients with symptomatic remission were 7.1 (patients with HAMD17 ≤ 7) and 8.6 (patients with Montgomery-Asberg depression rating scale ≤ 10). Among the patients with GAD, 30% achieved symptomatic remission (Hamilton anxiety rating scale ≤ 7), 45% achieved functional remission (SDS ≤ 6), and 25% achieved combined symptomatic-functional remission. The mean Hamilton anxiety rating scale score in GAD was approximately 8 for patients with functional remission and the mean SDS total score was approximately 4 in patients with symptomatic remission. The study shows that functional remission does not always move in tandem with symptom remission and provides useful anchor points or rules of thumb for evaluating symptomatic and functional remission in MDD and GAD.