Cargo selective vesicle tethering: The structural basis for binding of specific cargo proteins by the Golgi tether component TBC1D23.

The Golgi-localized golgins golgin-97 and golgin-245 capture transport vesicles arriving from endosomes via the protein TBC1D23. The amino-terminal domain of TBC1D23 binds to the golgins, and the carboxyl-terminal domain of TBC1D23 captures the vesicles, but how it recognizes specific vesicles was unclear. A search for binding partners of the carboxyl-terminal domain unexpectedly revealed direct binding to carboxypeptidase D and syntaxin-16, known cargo proteins of the captured vesicles. Binding is via a threonine-leucine-tyrosine (TLY) sequence present in both proteins next to an acidic cluster. A crystal structure reveals how this acidic TLY motif binds to TBC1D23. An acidic TLY motif is also present in the tails of other endosome-to-Golgi cargo, and these also bind TBC1D23. Structure-guided mutations in the carboxyl-terminal domain that disrupt motif binding in vitro also block vesicle capture in vivo. Thus, TBC1D23 attached to golgin-97 and golgin-245 captures vesicles by a previously undescribed mechanism: the recognition of a motif shared by cargo proteins carried by the vesicle.

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).

Engineering phosphatidylinositol-4,5-bisphosphate model membranes enriched in endocytic cargo: A neutron reflectometry, AFM and QCM-D structural study.

The combination of in vitro models of biological membranes based on solid-supported lipid bilayers (SLBs) and of surface sensitive techniques, such as neutron reflectometry (NR), atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D), is well suited to provide quantitative information about molecular level interactions and lipid spatial distributions. In this work, cellular plasma membranes have been mimicked by designing complex SLB, containing phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) lipids as well as incorporating synthetic lipo-peptides that simulate the cytoplasmic tails of transmembrane proteins. The QCM-D results revealed that the adsorption and fusion kinetics of PtdIns4,5P2 are highly dependent of Mg2+. Additionally, it was shown that increasing concentrations of PtdIns4,5P2 leads to the formation of SLBs with higher homogeneity. The presence of PtdIns4,5P2 clusters was visualized by AFM. NR provided important insights about the structural organization of the various components within the SLB, highlighting that the leaflet symmetry of these SLBs is broken by the presence of CD4-derived cargo peptides. Finally, we foresee our study to be a starting point for more sophisticated in vitro models of biological membranes with the incorporation of inositol phospholipids and synthetic endocytic motifs.

Subcutaneous delivery of a dendrimer-BH3 mimetic improves lymphatic uptake and survival in lymphoma.

As a malignant tumour of lymphatic origin, B-cell lymphoma represents a significant challenge for drug delivery, where effective therapies must access malignant cells in the blood, organs and lymphatics while avoiding off-target toxicity. Subcutaneous (SC) administration of nanomedicines allows preferential access to both the lymphatic and blood systems and may therefore provide a route to enhanced drug exposure to lymphomas. Here we examine the impact of SC dosing on lymphatic exposure, pharmacokinetics (PK), and efficacy of AZD0466, a small molecule dual Bcl-2/Bcl-xL inhibitor conjugated to a 'DEP®' G5 poly-l-lysine dendrimer. PK studies reveal that the plasma half-life of the dendrimer-drug conjugate is 8-times longer than that of drug alone, providing evidence of slow release from the circulating dendrimer nanocarrier. The SC dosed construct also shows preferential lymphatic transport, with over 50% of the bioavailable dose recovered in thoracic lymph. Increases in dose (up to 400 mg/kg) are well tolerated after SC administration and studies in a model of disseminated lymphoma in mice show that high dose SC treatment outperforms IV administration using doses that lead to similar total plasma exposure (lower peak concentrations but extended exposure after SC). These data show that the DEP® dendrimer can act as a circulating drug depot accessing both the lymphatic and blood circulatory systems. SC administration improves lymphatic exposure and facilitates higher dose administration due to improved tolerability. Higher dose SC administration also results in improved efficacy, suggesting that drug delivery systems that access both plasma and lymph hold significant potential for the treatment of haematological cancers where lymphatic and extranodal dissemination are poor prognostic factors.

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.

A BLOC-1-AP-3 super-complex sorts a cis-SNARE complex into endosome-derived tubular transport carriers.

Membrane transport carriers fuse with target membranes through engagement of cognate vSNAREs and tSNAREs on each membrane. How vSNAREs are sorted into transport carriers is incompletely understood. Here we show that VAMP7, the vSNARE for fusing endosome-derived tubular transport carriers with maturing melanosomes in melanocytes, is sorted into transport carriers in complex with the tSNARE component STX13. Sorting requires either recognition of VAMP7 by the AP-3δ subunit of AP-3 or of STX13 by the pallidin subunit of BLOC-1, but not both. Consequently, melanocytes expressing both AP-3δ and pallidin variants that cannot bind their respective SNARE proteins are hypopigmented and fail to sort BLOC-1-dependent cargo, STX13, or VAMP7 into transport carriers. However, SNARE binding does not influence BLOC-1 function in generating tubular transport carriers. These data reveal a novel mechanism of vSNARE sorting by recognition of redundant sorting determinants on a SNARE complex by an AP-3-BLOC-1 super-complex.

Architecture and mechanism of metazoan retromer:SNX3 tubular coat assembly.

Retromer is a master regulator of cargo retrieval from endosomes, which is critical for many cellular processes including signaling, immunity, neuroprotection, and virus infection. The retromer core (VPS26/VPS29/VPS35) is present on cargo-transporting, tubular carriers along with a range of sorting nexins. Here, we elucidate the structural basis of membrane tubulation and coupled cargo recognition by metazoan and fungal retromer coats assembled with the non-Bin1/Amphiphysin/Rvs (BAR) sorting nexin SNX3 using cryo-electron tomography. The retromer core retains its arched, scaffolding structure but changes its mode of membrane recruitment when assembled with different SNX adaptors, allowing cargo recognition at subunit interfaces. Thus, membrane bending and cargo incorporation can be modulated to allow retromer to traffic cargoes along different cellular transport routes.

Design and optimisation of dendrimer-conjugated Bcl-2/xL inhibitor, AZD0466, with improved therapeutic index for cancer therapy.

Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many haematological and solid malignancies, however, their use is limited by tolerability issues. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, has shown good efficacy however had dose limiting cardiovascular toxicity in preclinical species, coupled with challenging physicochemical properties, which prevented its clinical development. Here, we describe the design and development of AZD0466, a drug-dendrimer conjugate, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer. Mathematical modelling was employed to determine the optimal release rate of the drug from the dendrimer for maximal therapeutic index in terms of preclinical anti-tumour efficacy and cardiovascular tolerability. The optimised candidate is shown to be efficacious and better tolerated in preclinical models compared with AZD4320 alone. The AZD4320-dendrimer conjugate (AZD0466) identified, through mathematical modelling, has resulted in an improved therapeutic index and thus enabled progression of this promising dual Bcl-2/Bcl-xL inhibitor into clinical development.

Mechanism and evolution of the Zn-fingernail required for interaction of VARP with VPS29.

VARP and TBC1D5 are accessory/regulatory proteins of retromer-mediated retrograde trafficking from endosomes. Using an NMR/X-ray approach, we determined the structure of the complex between retromer subunit VPS29 and a 12 residue, four-cysteine/Zn++ microdomain, which we term a Zn-fingernail, two of which are present in VARP. Mutations that abolish VPS29:VARP binding inhibit trafficking from endosomes to the cell surface. We show that VARP and TBC1D5 bind the same site on VPS29 and can compete for binding VPS29 in vivo. The relative disposition of VPS29s in hetero-hexameric, membrane-attached, retromer arches indicates that VARP will prefer binding to assembled retromer coats through simultaneous binding of two VPS29s. The TBC1D5:VPS29 interaction is over one billion years old but the Zn-fingernail appears only in VARP homologues in the lineage directly giving rise to animals at which point the retromer/VARP/TBC1D5 regulatory network became fully established.

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.

Structure of the membrane-assembled retromer coat determined by cryo-electron tomography.

Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes1-3. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders4-7. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.

Reducing Dendrimer Generation and PEG Chain Length Increases Drug Release and Promotes Anticancer Activity of PEGylated Polylysine Dendrimers Conjugated with Doxorubicin via a Cathepsin-Cleavable Peptide Linker.

PEGylation typically improves the systemic exposure and tumor biodistribution of polymeric drug delivery systems, but may also restrict enzyme access to peptide-based drug linkers. The impact of dendrimer generation (G4 vs G5) and PEG length (570 vs 1100 Da) on the pharmacokinetics, tumor biodistribution, drug release kinetics, and anticancer activity of a series of PEGylated polylysine dendrimers conjugated with doxorubicin via a cathepsin-B cleavable valine-citrulline linker was therefore investigated in rodents. Although the smallest G4 PEG570 dendrimer showed the most efficient cathepsin-mediated doxorubicin release, systemic exposure and tumor uptake were limited. The largest G5 PEG1100 dendrimer showed good tumor uptake and retention but restricted drug liberation and therefore limited anticancer activity. Superior anticancer activity was achieved using an intermediate sized dendrimer that showed better drug release kinetics, systemic exposure, tumor uptake, and retention. The data suggest that balancing PEG molecular weight and dendrimer size is critical when designing chemotherapeutic dendrimers.

Doxorubicin Conjugation and Drug Linker Chemistry Alter the Intravenous and Pulmonary Pharmacokinetics of a PEGylated Generation 4 Polylysine Dendrimer in Rats.

PEGylated polylysine dendrimers have demonstrated potential as inhalable drug delivery systems that can improve the treatment of lung cancers. Their treatment potential may be enhanced by developing constructs that display prolonged lung retention, together with good systemic absorption, the capacity to passively target lung tumors from the blood and highly selective, yet rapid liberation in the tumor microenvironment. This study sought to characterize how the nature of cathepsin B-cleavable peptide linkers, used to conjugate doxorubicin (Dox) to a PEGylated (PEG570) G4 polylysine dendrimer, affects drug liberation kinetics and intravenous and pulmonary pharmacokinetics in rats. The construct bearing a self-emolative diglycolic acid-V-Citrulline linker exhibited faster Dox release kinetics compared to constructs bearing self-emolative diglycolic acid-glycine-leucine-phenylalanine-glycine (GLFG), or non-self-emolative glutaric acid-GLFG linkers. The V-Citrulline construct exhibited slower plasma clearance, but faster absorption from the lungs than a GLFG construct, although mucociliary clearance and urinary elimination were unchanged. Dox-conjugation enhanced localization in the bronchoalveolar lavage fluid compared to lung tissue, suggesting that projection of Dox from the dendrimer surface reduced tissue uptake. These data show that the linker chemistry employed to conjugate drugs to PEGylated carriers can affect drug release profiles and systemic and lung disposition.

Lymphatic transport and lymph node targeting of methotrexate-conjugated PEGylated dendrimers are enhanced by reducing the length of the drug linker or masking interactions with the injection site.

Drug conjugation to dendrimer-based delivery systems has been shown to enhance delivery to the lymphatic system after subcutaneous administration. Dendrimer interaction with components of the interstitium at the injection site, however, may prevent drainage from the injection site. The current study sought to vary the length of a linker employed to conjugate methotrexate (MTX) to a PEGylated dendrimer, in an attempt to reduce MTX interaction with interstitial binding sites and enhance lymphatic drainage. Dendrimers with shorter linkers resulted in higher lymphatic drainage, presumably via shielding of interaction sites by the PEG mantle, but were not retained in lymph nodes. Improved drainage of dendrimers with longer linkers was achieved through coadministration with dextran to mask interactions at the injection site while maintaining retention within the node. Enhanced drug exposure to the lymph node has the potential to enhance the treatment of lymph-node resident cancer metastases.

Contribution of the clathrin adaptor AP-1 subunit µ1 to acidic cluster protein sorting.

Acidic clusters act as sorting signals for packaging cargo into clathrin-coated vesicles (CCVs), and also facilitate down-regulation of MHC-I by HIV-1 Nef. To find acidic cluster sorting machinery, we performed a gene-trap screen and identified the medium subunit (µ1) of the clathrin adaptor AP-1 as a top hit. In µ1 knockout cells, intracellular CCVs still form, but acidic cluster proteins are depleted, although several other CCV components were either unaffected or increased, indicating that cells can compensate for long-term loss of AP-1. In vitro experiments showed that the basic patch on µ1 that interacts with the Nef acidic cluster also contributes to the binding of endogenous acidic cluster proteins. Surprisingly, µ1 mutant proteins lacking the basic patch and/or the tyrosine-based motif binding pocket could rescue the µ1 knockout phenotype completely. In contrast, these mutants failed to rescue Nef-induced down-regulation of MHC class I, suggesting a possible mechanism for attacking the virus while sparing the host cell.

Effect of increased surface hydrophobicity via drug conjugation on the clearance of inhaled PEGylated polylysine dendrimers.

PEGylated polylysine dendrimers are attractive and well tolerated inhalable drug delivery platforms that have the potential to control the release, absorption kinetics and lung retention time of conjugated drugs. The clinical application of these systems though, would likely require partial substitution of surface PEG groups with drug molecules that are anticipated to alter their lung clearance kinetics and clearance pathways. In the current study, we therefore evaluated the impact of increased surface hydrophobicity via substitution of 50% surface PEG groups with a model hydrophobic drug (α-carboxyl OtButylated methotrexate) on the lung clearance of a Generation 5 PEGylated polylysine dendrimer in rats. PEG substitution with OtBu-methotrexate accelerated lung clearance of the dendrimer by increasing polylysine scaffold catabolism, improving systemic absorption of the intact dendrimer and low molecular weight products of scaffold catabolism, and enhancing mucociliary clearance. These results suggest that the conjugation of hydrophobic drug on the surface of a PEGylated dendrimer is likely to accelerate lung clearance when compared to a fully PEGylated dendrimer.