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We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum . In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance. The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 . Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum , we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent. IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum . In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.

CRISPR knockout (KO) screens have identified host factors regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Here, we conducted a meta-analysis of these screens, which showed a high level of cell-type specificity of the identified hits, highlighting the necessity of additional models to uncover the full landscape of host factors. Thus, we performed genome-wide KO and activation screens in Calu-3 lung cells and KO screens in Caco-2 colorectal cells, followed by secondary screens in four human cell lines. This revealed host-dependency factors, including AP1G1 adaptin and ATP8B1 flippase, as well as inhibitors, including mucins. Interestingly, some of the identified genes also modulate Middle East respiratory syndrome coronavirus (MERS-CoV) and seasonal human coronavirus (HCoV) (HCoV-NL63 and HCoV-229E) replication. Moreover, most genes had an impact on viral entry, with AP1G1 likely regulating TMPRSS2 activity at the plasma membrane. These results demonstrate the value of multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential targets for therapeutic interventions. Genome-wide CRISPR knockout and activation screens in Calu-3 lung cells and Caco-2 colorectal cells identify host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs.

Several studies have demonstrated the role of the oncogenic mutant p53 in promoting tumor progression; however, there is limited information on the effects of secreted oncogenic mutant p53 on the tumor microenvironment and tumor immune escape. In this study, we found that secretion of mutant p53, determined by exosome content, is dependent on its N-terminal dileucine motif via its binding to β-adaptin, and inhibited by the CHK2-mediated-Ser 20 phosphorylation. Moreover, we observed that the mutant p53 caused downregulation and dysfunction of CD4 + T lymphocytes in vivo and downregulated the levels and activities of rate-limiting glycolytic enzymes in vitro. Furthermore, inhibition of mutant p53 secretion by knocking down AP1B1 or mutation of dileucine motif could reverse the quantity and function of CD4 + T lymphocytes and restrain the tumor growth. Our study demonstrates that the tumor-derived exosome-mediated secretion of oncogenic mutant p53 inhibits glycolysis to alter the immune microenvironment via functional suppression of CD4 + T cells, which may be the underlying mechanism for tumor immune escape. Therefore, targeting TDE-mediated p53 secretion may serve as a potential therapeutic target for cancer treatment.

Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin‐5α and its putative partner γ 1 ‐adaptin in membrane recycling. We found that rabaptin‐5α forms a ternary complex with the γ 1 –σ 1 subcomplex of AP‐1, via a direct interaction with the γ 1 ‐subunit. The binding site for γ 1 ‐adaptin is in the hinge region of rabaptin‐5α, which is distinct from rab4‐ and rab5‐binding domains. Endogenous or ectopically expressed γ 1 ‐ adaptin localized to both the trans‐ Golgi network and endosomes. Co‐expressed rabaptin‐5α and γ 1 ‐adaptin, however, co‐localized in a rab4‐dependent manner on recycling endosomes. Transfection of rabaptin‐5α caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4‐GTP acts as a scaffold for a rabaptin‐5α–γ 1 ‐adaptin complex on recycling endosomes and that interactions between rab4, rabaptin‐5α and γ 1 ‐adaptin regulate membrane recycling.

Bidirectional trafficking between the trans -Golgi network (TGN) and endolysosomal compartments lies at the intersection of biosynthetic and degradative pathways. At the center of this trafficking route is the adaptor protein complex 1 (AP1), a heterotetramer essential for cargo recognition and vesicle budding. Here, we identified Male-Enhanced Antigen 1 (MEA1), a previously uncharacterized protein, as a critical AP1 regulator. Loss of MEA1 resulted in depletion of AP1 subunits and impaired trafficking of AP1-dependent cargoes. Mechanistically, MEA1 acts as a bi-handed chaperone, simultaneously engaging and stabilizing the μ1 and β1 subunits of AP1. The MEA1-stabilized μ1 and β1 collide with the γ and σ1 subunits stabilized by Alpha- and Gamma-Adaptin Binding Protein (AAGAB), another bi-handed chaperone, leading to formation of the tetrameric AP1 adaptor and release of both chaperones. These findings identify MEA1 as a key AP1 regulator and uncover a dual chaperone collision mechanism potentially generalizable to multiprotein complex assembly. Wan et al. identify MEA1 as a chaperone that promotes assembly of the AP1 adaptor complex and is essential for cargo transport. MEA1 and AAGAB cooperate through a dual-chaperone mechanism that may be broadly applicable to protein complex assembly.

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (V 2 R), agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the V 2 R (i.e., V 2 R T360A ) results in near-complete loss of βarr translocation to endosomes despite robust recruitment to the plasma membrane, and compromised ERK1/2 activation. Here, we discover that a synthetic intrabody (Ib30), which selectively recognizes activated βarr1, efficiently rescues the endosomal trafficking of βarr1 and ERK1/2 activation for V 2 R T360A . Molecular dynamics simulations reveal that Ib30 enriches active-like βarr1 conformation with respect to the inter-domain rotation, and cellular assays demonstrate that it also enhances βarr1-β 2 -adaptin interaction. Our data provide an experimental framework to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can be potentially integrated in the paradigm of GPCR-targeted drug discovery. G protein-coupled receptors (GPCRs) are integral membrane proteins and the largest class of drug targets in the human genome. Here, Baidya et al. show that a synthetic antibody can be used to modulate GPCR trafficking and signaling in live cells.

RAB23 is known to regulate signaling by several growth factors during organogenesis. RABs and other small GTPases function as molecular switches during cellular membrane trafficking. However, what has not been established is how RAB23 functions during cellular membrane trafficking and how this influences cell signaling. To address this, we characterized RAB23’s localization in the endocytic pathway and determined the route of endocytosis. We find that RAB23 interacts with β-adaptin (AP2β1) subunit of the clathrin adaptor protein 2 (AP-2) complex, suggesting RAB23’s involvement in clathrin-dependent endocytosis at the plasma membrane. Our results show that RAB23 might function at multiple steps during clathrin-coated nascent vesicle formation. We find that RAB23 interacts with clathrin assembly protein PICALM, vesicle curvature protein endophilin A2, and a protein linked with vesicle scission, cortactin. To understand the functionality of RAB23, we performed time-lapse live cell imaging of transferrin uptake, which showed that clathrin-dependent endocytosis is affected in RAB23 deficient osteoprogenitors with inefficient cargo internalization. We normalized transferrin uptake in RAB23 knockdown human osteosarcoma cells (MG-63) by overexpressing RAB23. Our results show that deficiency of RAB23 reduced the interaction between β-adaptin and clathrin. We demonstrate that vesicle formation upon BMP stimulation and subsequent signal transduction is aberrant in RAB23-deficient cells. We further show evidence by providing microarray data-driven hypergeometric test of differentially expressed genes in WT and RAB23-deficient samples which suggests RAB23’s participation in vesicle formation, endocytosis and cell signaling. Collectively, our data indicate a role for RAB23 in vesicle formation, membrane trafficking, and cell signaling.

Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin-binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homodimer. AAGAB dimerization is mediated by its C-terminal domain (CTD), which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the dimerization-mediating CTD, revealing an antiparallel dimer of bent helices. Interestingly, AAGAB uses the same CTD to recognize and stabilize the γ subunit in the AP1 complex and the α subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of CTD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.

Extracts of phytocannabinoids from Cannabis sativa have been studied for therapeutic purposes. Although nonpsychoactive CBD has been studied as a promising anticancer drug because it induces apoptosis in many cancer cells, it is also known to induce several physiological changes. In this study, we clarify the functional role it plays in the morphological characteristics of intracellular vesicle formation as well as apoptosis in A549 human lung cancer cells. CBD treatment shows growth inhibition at concentrations above 20 μM, but FACS analysis shows low efficacy in terms of cell death. Microscopic observations suggest that multiple vesicles were detected in the cytoplasmic region of CBD-treated A549 cells. CBD treatment upregulates apoptosis-related proteins, such as p53, PARP, RIP1, RIP3, Atg12, and Beclin, indicating that CBD regulates several types of cell death. CBD treatment also induced E-cadherin, PPARγ, clathrin, β-adaptin, and Tsg101, also known to be cellular-differentiation inducers or vesicle-formation components. Treatment combining CBD with GW9662, a PPARγ inhibitor, reduced CBD-induced cytoplasmic vesicle formation. This indicates that PPARγ regulates the vesicle-formation mechanism. However, CBD-treated E-cad KO clones did not show this regulatory mechanism. These results elucidate the pharmacological and molecular networks associated with CBD in PPARγ-dependent vesicle formation and the induction of apoptosis.

Hypoxic-ischemic encephalopathy (HIE) is a main cause of mortality and severe neurologic impairment in the perinatal and neonatal period. However, few satisfactory therapeutic strategies are available. Here, we reported that a rapid nuclear translocation of phosphatase and tensin homolog deleted on chromosome TEN (PTEN) is an essential step in hypoxic-ischemic brain damage (HIBD)- and oxygen-glucose deprivation (OGD)-induced neuronal injures both in vivo and in vitro. In addition, we found that OGD-induced nuclear translocation of PTEN is dependent on PTEN mono-ubiquitination at the lysine 13 residue (K13) that is mediated by neural precursor cell expressed developmentally downregulated protein 4-1 (NEDD4-1). Importantly, we for the first time identified α- and γ-adaptin binding protein (Aagab) as a novel NEDD4-1 regulator to regulate the level of NEDD4-1, subsequently mediating Pten nuclear translocation. Finally, we demonstrated that genetic upregulation of Aagab or application of Tat-K13 peptide (a short interference peptide that flanks K13 residue of PTEN) not only reduced Pten nuclear translocation, but also significantly alleviated the deficits of myodynamia, motor and spatial learning and memory in HIBD model rats. These results suggest that Aagab may serve as a regulator of NEDD4-1-mediated Pten nuclear translocation to promote functional recovery following HIBD in neonatal rats, and provide a new potential therapeutic target to guide the clinical treatment for HIE.