Repurposing an endogenous degradation domain for antibody-mediated disposal of cell-surface proteins.

The exquisite specificity of antibodies can be harnessed to effect targeted degradation of membrane proteins. Here, we demonstrate targeted protein removal utilising a protein degradation domain derived from the endogenous human protein Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9). Recombinant antibodies genetically fused to this domain drive the degradation of membrane proteins that undergo constitutive internalisation and recycling, including the transferrin receptor and the human cytomegalovirus latency-associated protein US28. We term this approach PACTAC (PCSK9-Antibody Clearance-Targeting Chimeras).

High-level compliance to opt-out HIV testing in the emergency department (ED) of a large teaching hospital using the biochemistry sample as the sample type for HIV screening.

INTRODUCTION: HIV remains a key public health issue. National Institute for Health and Care Excellence and British HIV Association guidance recommends that patients should be offered HIV testing when admitted to hospital or attending emergency departments (EDs) in areas with a prevalence ≥ 2 per 1000. We report a novel method of testing and the first 3-year results from our HIV ED testing programme utilizing biochemistry samples for HIV testing, with the aim of improving uptake while ensuring no changes to clinical practice in EDs. METHODS: Routine ED HIV testing was implemented on 1 October 2018; it was initially opt-in and was subsequently changed to opt-out on 1 February 2019. HIV testing was added to all ED blood test order sets and was performed on the biochemistry samples of those aged 18-59 years. The age range was extended to include those aged 16+ years on 1 March 2021 along with a move to notional consent. RESULTS: A total of 78 333 HIV tests were performed from an estimated 110 683 attendees who had bloods taken in the same age range, demonstrating an overall 69.5% testing coverage. On implementation of opt-out testing after the first 4 months, the proportion of tests increased (from 57.9% to 69%). After increase in age range to 16+ years and a move to notional consent, the overall testing coverage improved to 74.2%. Of 1054 reactive results, 728 (69%) were known people living with HIV, eight (0.8%) were not contactable, two (0.2%) re-tested elsewhere and three (0.3%) declined a re-test. A total of 259 false-positives were determined by follow-up testing and 50 (4.8%) were newly diagnosed with HIV. An HIV diagnosis was suspected in only 22%, and 48% had never previously tested for HIV. CONCLUSIONS: An opt-out HIV testing programme with notional consent and using biochemistry samples within the ED is feasible, acceptable and provides an excellent opportunity to diagnose patients who do not perceive themselves to be at risk or have never tested before.

FCHO controls AP2's initiating role in endocytosis through a PtdIns(4,5)P2-dependent switch.

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence-structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2's subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation.

Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles.

Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell's plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. Adaptor protein complex (AP2) is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here, we used cryo-electron microscopy, tomography, and subtomogram averaging to determine structures, interactions, and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P 2 (phosphatidylinositol 4,5-bisphosphate)-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 ß2 appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.

Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via AP2 Phosphorylation.

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the µ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. µ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with µ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.

Electromagnetic Chirps from Neutron Star-Black Hole Mergers.

We calculate the electromagnetic signal of a gamma-ray flare coming from the surface of a neutron star shortly before merger with a black hole companion. Using a new version of the Monte Carlo radiation transport code Pandurata that incorporates dynamic spacetimes, we integrate photon geodesics from the neutron star surface until they reach a distant observer or are captured by the black hole. The gamma-ray light curve is modulated by a number of relativistic effects, including Doppler beaming and gravitational lensing. Because the photons originate from the inspiraling neutron star, the light curve closely resembles the corresponding gravitational waveform: a chirp signal characterized by a steadily increasing frequency and amplitude. We propose to search for these electromagnetic chirps using matched filtering algorithms similar to those used in LIGO data analysis.

Prompt Electromagnetic Transients from Binary Black Hole Mergers.

Binary black hole (BBH) mergers provide a prime source for current and future interferometric GW observatories. Massive BBH mergers may often take place in plasma-rich environments, leading to the exciting possibility of a concurrent electromagnetic (EM) signal observable by traditional astronomical facilities. However, many critical questions about the generation of such counterparts remain unanswered. We explore mechanisms that may drive EM counterparts with magnetohydro-dynamic simulations treating a range of scenarios involving equal-mass black-hole binaries immersed in an initially homogeneous fluid with uniform, orbitally aligned magnetic fields. We find that the time development of Poynting luminosity, which may drive jet-like emissions, is relatively insensitive to aspects of the initial configuration. In particular, over a significant range of initial values, the central magnetic field strength is effectively regulated by the gas flow to yield a Poynting luminosity of 1045 - 1046 ρ -13 M 8 2 ergs-1, with BBH mass scaled to M 8 ≡ M/(108 M ⨀) and ambient density ρ -13 ≡ ρ/(10-13 g cm-3). We also calculate the direct plasma synchrotron emissions processed through geodesic ray-tracing. Despite lensing effects and dynamics, we find the observed synchrotron flux varies little leading up to merger.

Cellular and viral peptides bind multiple sites on the N-terminal domain of clathrin.

Short peptide motifs in unstructured regions of clathrin-adaptor proteins recruit clathrin to membranes to facilitate post-Golgi membrane transport. Three consensus clathrin-binding peptide sequences have been identified and structural studies show that each binds distinct sites on the clathrin heavy chain N-terminal domain (NTD). A fourth binding site for adaptors on NTD has been functionally identified but not structurally characterised. We have solved high resolution structures of NTD bound to peptide motifs from the cellular clathrin adaptors ß2 adaptin and amphiphysin plus a putative viral clathrin adaptor, hepatitis D virus large antigen (HDAg-L). Surprisingly, with each peptide we observe simultaneous peptide binding at multiple sites on NTD and viral peptides binding to the same sites as cellular peptides. Peptides containing clathrin-box motifs (CBMs) with the consensus sequence LΦxΦ[DE] bind at the 'arrestin box' on NTD, between ß-propeller blades 4 and 5, which had previously been thought to bind a distinct consensus sequence. Further, we structurally define the fourth peptide binding site on NTD, which we term the Royle box. In vitro binding assays show that clathrin is more readily captured by cellular CBMs than by HDAg-L, and site-directed mutagenesis confirms that multiple binding sites on NTD contribute to efficient capture by CBM peptides.

Using selenomethionyl derivatives to assign sequence in low-resolution structures of the AP2 clathrin adaptor.

Selenomethionine incorporation is a powerful technique for assigning sequence to regions of electron density at low resolution. Genetic introduction of methionine point mutations and the subsequent preparation and crystallization of selenomethionyl derivatives permits unambiguous sequence assignment by enabling the placement of the anomalous scatterers (Se atoms) thus introduced. Here, the use of this approach in the assignment of sequence in a part of the AP2 clathrin adaptor complex that is responsible for clathrin binding is described. AP2 plays a pivotal role in clathrin-mediated endocytosis, a tightly regulated process in which cell-surface transmembrane proteins are internalized from the plasma membrane by incorporation into lipid-enclosed transport vesicles. AP2 binds cargo destined for internalization and recruits clathrin, a large trimeric protein that helps to deform the membrane to produce the transport vesicle. By selenomethionine labelling of point mutants, it was shown that the clathrin-binding site is buried within a deep cleft of the AP2 complex. A membrane-stimulated conformational change in AP2 releases the clathrin-binding site from autoinhibition, thereby linking clathrin recruitment to membrane localization.

CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature.

The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.

Clathrin adaptors. AP2 controls clathrin polymerization with a membrane-activated switch.

Clathrin-mediated endocytosis (CME) is vital for the internalization of most cell-surface proteins. In CME, plasma membrane-binding clathrin adaptors recruit and polymerize clathrin to form clathrin-coated pits into which cargo is sorted. Assembly polypeptide 2 (AP2) is the most abundant adaptor and is pivotal to CME. Here, we determined a structure of AP2 that includes the clathrin-binding ß2 hinge and developed an AP2-dependent budding assay. Our findings suggest that an autoinhibitory mechanism prevents clathrin recruitment by cytosolic AP2. A large-scale conformational change driven by the plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate and cargo relieves this autoinhibition, triggering clathrin recruitment and hence clathrin-coated bud formation. This molecular switching mechanism can couple AP2's membrane recruitment to its key functions of cargo and clathrin binding.

Endocytic sorting of transmembrane protein cargo.

The accurate distribution and recycling of transmembrane proteins amongst the membrane-bound organelles of the cell is vital to ensure its correct functioning. Transmembrane protein cargo destined for clathrin-mediated endocytosis and transport along the endocytic pathway is sorted into transport vesicles by interactions with adaptors, which simultaneously link clathrin to the membrane. Clathrin adaptors recognize a variety of signals present in the cytoplasmic portions of cargo proteins; recent structural, biophysical and cell biological studies have elucidated new types of cargo-adaptor interactions and probed the molecular mechanisms regulating cargo selection and vesicle maturation. Here, we review this recent progress in the context of our existing knowledge of endocytic sorting mechanisms.

A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex.

The AP2 adaptor complex (alpha, beta2, sigma2, and mu2 subunits) crosslinks the endocytic clathrin scaffold to PtdIns4,5P(2)-containing membranes and transmembrane protein cargo. In the "locked" cytosolic form, AP2's binding sites for the two endocytic motifs, YxxPhi on the C-terminal domain of mu2 (C-mu2) and [ED]xxxL[LI] on sigma2, are blocked by parts of beta2. Using protein crystallography, we show that AP2 undergoes a large conformational change in which C-mu2 relocates to an orthogonal face of the complex, simultaneously unblocking both cargo-binding sites; the previously unstructured mu2 linker becomes helical and binds back onto the complex. This structural rearrangement results in AP2's four PtdIns4,5P(2)- and two endocytic motif-binding sites becoming coplanar, facilitating their simultaneous interaction with PtdIns4,5P(2)/cargo-containing membranes. Using a range of biophysical techniques, we show that the endocytic cargo binding of AP2 is driven by its interaction with PtdIns4,5P(2)-containing membranes.

Selective gene amplification.

We describe a system for directed evolution based on in vitro compartmentalisation in which amplification of a gene is coupled to the formation of product by the enzyme it encodes. This approach mimics the process of natural selection; 'fitter' genes--encoding more efficient enzymes--have more 'offspring'. It allows selection for any activity so long as a product-specific ligand (e.g. an antibody) is available.

Consistency of post-Newtonian waveforms with numerical relativity.

General relativity predicts the gravitational wave signatures of coalescing binary black holes. Explicit waveform predictions for such systems, required for optimal analysis of observational data, have so far been achieved primarily using the post-Newtonian (PN) approximation. The quality of this treatment is unclear, however, for the important late-inspiral portion. We derive late-inspiral waveforms via a complementary approach, direct numerical simulation of Einstein's equations. We compare waveform phasing from simulations of the last approximately 14 cycles of gravitational radiation from equal-mass, nonspinning black holes with the corresponding 2.5PN, 3PN, and 3.5PN orbital phasing. We find phasing agreement consistent with internal error estimates for either approach, suggesting that PN waveforms for this system are effective until the last orbit prior to final merger.

Miniaturizing chemistry and biology in microdroplets.

By compartmentalizing reactions in aqueous microdroplets of water-in-oil emulsions, reaction volumes can be reduced by factors of up to 10(9) compared to conventional microtitre-plate based systems. This allows massively parallel processing of as many as 10(10) reactions in a total volume of only 1 ml of emulsion. This review describes the use of emulsions for directed evolution of proteins and RNAs, and for performing polymerase chain reactions (PCRs). To illustrate these applications we describe certain specific experiments, each of which exemplifies a different facet of the technique, in some detail. These examples include directed evolution of Diels-Alderase and RNA ligase ribozymes and several classes of protein enzymes, including DNA polymerases, phosphotriesterases, beta-galactosidases and thiolactonases. We also describe the application of emulsion PCR to screen for rare mutations and for new ultra-high throughput sequencing technologies. Finally, we discuss the recent development of microfluidic tools for making and manipulating microdroplets and their likely impact on the future development of the field.

Droplets as microreactors for high-throughput biology.

Inspired by the principles of biological evolution, biologists--and others--have in recent decades harnessed the power of "natural" selection to sift through huge libraries of genes and find those with desirable properties. At the same time, the demand for high-throughput biochemical and genetic assays and screens has driven the development of increasingly miniaturised assay systems. An exciting synergy is now emerging between these two fields, whereby the tools of ultrahigh-throughput screening promise to open up new directions in molecular engineering.