Evidence on differential role for alpha 1 and alpha 2 subtypes of AP-2 adaptin in the central nervous system.

Two subtypes of alpha (α)subunits, α1and α2, belonging to AP-2 complex have been described in the central nervous system (CNS). The specific role of each subtype is still unclear. In this study, we evaluated the expression and interaction with cell membranes of both subtypes in the postnatal developing cerebral cortex and cerebellum in two rat strains that display distinct developmental features. We observed that α2 displays higher variations than α1 during development, and at lesser extent in the rats with delayed rate of development. Additionally, by in vitro binding assays we evaluated the interaction of α subunits with bovine brain membranes. Both subtypes displayed clear differences in their performance, maximum binding of α1 was higher and α2 reached it faster than α1. In addition, both subtypes displayed different binding to membranes when bivalent cations or nucleotides were added. We conclude that both subtypes interact differently with membranes and that they may play different roles in clathrin-mediated endocytosis in the CNS.

Interfering with STING.

The clathrin-associated adaptor protein complex AP-1 stops STING signaling at the Golgi.

Alpha adaptins show isoform-specific association with neurofibrillary tangles in Alzheimer's disease.

AIMS: The heterotetrameric assembly protein complex 2 (AP-2) is a central hub for clathrin-dependent endocytosis. The AP-2 α-adaptin subunit has two major isoforms, encoded by two separate genes: AP2A1 and AP2A2. Endocytosis has been implicated in the pathogenesis of neurodegenerative disease, and recent studies linked α-adaptins (gene variants, splicing defects and altered expression) with late-onset Alzheimer's disease (LOAD) risk. Here, we used multiple antibodies to investigate α-adaptin isoforms and their localization in human brains. METHODS: The specificities of 10 different α-adaptin antibodies were evaluated using immunoblots after human AP2A1 and AP2A2 plasmid transfection in cultured cells. Additional immunoblot analyses were then performed on protein homogenates from control and LOAD subjects. Formalin-fixed, paraffin-embedded brain sections from control and LOAD subjects were immunohistochemically stained, and immunofluorescence experiments were performed for quantitation of colocalisation with digital image analysis. RESULTS: Eight of the 10 evaluated antibodies recognised transfected α-adaptin proteins on immunoblots. The α-adaptin subspecies were relatively uniformly expressed in five different human brain regions. The α-adaptins were present in the detergent-insoluble fraction from cognitively impaired, but less so in control, brains. Immunohistochemical analyses showed colocalisation of AP2A1 with tau pathology in LOAD brains. By contrast, AP2A2 colocalised with microglial cells. CONCLUSIONS: These observations provide evidence of isoform-specific changes of α-adaptins in the brains of LOAD subjects. Antibodies that were verified to recognise AP2A1, but not AP2A2, labelled neurofibrillary tangles of LOAD patients. The findings extend our understanding of AP-2 proteins in the human brain in healthy and diseased states.

Inhibiting endocytosis in CGRP+ nociceptors attenuates inflammatory pain-like behavior.

The advantage of locally applied anesthetics is that they are not associated with the many adverse effects, including addiction liability, of systemically administered analgesics. This therapeutic approach has two inherent pitfalls: specificity and a short duration of action. Here, we identified nociceptor endocytosis as a promising target for local, specific, and long-lasting treatment of inflammatory pain. We observed preferential expression of AP2α2, an α-subunit isoform of the AP2 complex, within CGRP+/IB4- nociceptors in rodents and in CGRP+ dorsal root ganglion neurons from a human donor. We utilized genetic and pharmacological approaches to inhibit nociceptor endocytosis demonstrating its role in the development and maintenance of acute and chronic inflammatory pain. One-time injection of an AP2 inhibitor peptide significantly reduced acute and chronic pain-like behaviors and provided prolonged analgesia. We evidenced sexually dimorphic recovery responses to this pharmacological approach highlighting the importance of sex differences in pain development and response to analgesics.

AP-2α-Mediated Activation of E2F and EZH2 Drives Melanoma Metastasis.

In melanoma metastasis, the role of the AP-2α transcription factor, which is encoded by TFAP2A, is controversial as some findings have suggested tumor suppressor activity while other studies have shown high TFAP2A expression in node-positive melanoma associated with poor prognosis. Here we demonstrate that AP-2α facilitates melanoma metastasis through transcriptional activation of genes within the E2F pathway including EZH2. A BioID screen found that AP-2α interacts with members of the nucleosome remodeling and deacetylase (NuRD) complex. Loss of AP-2α removed activating chromatin marks in the promoters of EZH2 and other E2F target genes through activation of the NuRD repression complex. In melanoma cells, treatment with tazemetostat, an FDA-approved and highly specific EZH2 inhibitor, substantially reduced anchorage-independent colony formation and demonstrated heritable antimetastatic effects, which were dependent on AP-2α. Single-cell RNA sequencing analysis of a metastatic melanoma mouse model revealed hyperexpansion of Tfap2a High/E2F-activated cell populations in transformed melanoma relative to progenitor melanocyte stem cells. These findings demonstrate that melanoma metastasis is driven by the AP-2α/EZH2 pathway and suggest that AP-2α expression can be used as a biomarker to predict responsiveness to EZH2 inhibitors for the treatment of advanced melanomas. SIGNIFICANCE: AP-2α drives melanoma metastasis by upregulating E2F pathway genes including EZH2 through inhibition of the NuRD repression complex, serving as a biomarker to predict responsiveness to EZH2 inhibitors.

N6-Methyladenosine-Related RNA Signature Predicting the Prognosis of Ovarian Cancer.

BACKGROUND: N6-Methyladenosine (m6A) RNA methylation is the most universal mRNA modification in eukaryotic cells. M6A mRNA modification affects almost every phases of RNA processing, including splicing, decay, export, translation and expression. Several patents have reported the application of m6A mRNA modification in cancer diagnosis and treatment. Ovarian cancer is the leading cause of death among all gynecological cancers. It is urgent to identify new biomarkers for early diagnosis and prognosis of ovarian cancer. OBJECTIVE: In the current study, we aimed to evaluate the m6A RNA methylation regulators and m6A related genes and establish a new gene signature panel for the prognosis of ovarian cancer. METHODS: We downloaded the mutations data, FPKM data and corresponding clinical information of 373 patients with Ovarian Cancer (OC) from the TCGA database. We performed LASSO regression analysis and multivariate cox regression analysis to develop a risk-identifying gene signature panel. RESULTS: A total of 317 candidate m6A RNA methylation related genes were obtained. Finally, 12 - genes (WTAP, LGR6, ZC2HC1A, SLC4A8, AP2A1, NRAS, CUX1, HDAC1, CD79A, ACE2, FLG2 and LRFN1) were selected to establish the signature panel. We analyzed the genetic alterations of the selected 12 -genes in OC using cBioPortal database. Among the 373 patients, 368 patients have mutations. The results showed that all queried genes were altered in 137 of 368 cases (37.23%). The 12-gene signature panel was confirmed as an independent prognostic indicator (P =2.29E-18, HR = 1.699, 95% CI = 1.508-1.913). CONCLUSION: We established an effective m6A-related gene signature panel consisted of 12 -genes, which can predict the outcome of patients with OC. The high risk score indicates unfavorable survival. Our study provided novel insights into the relationship between m6A and OC. This gene signature panel will be helpful in identifying poor prognostic patients with OC and could be a promising prognostic indicator in clinical practice.

An internally eGFP-tagged α-adaptin is a fully functional and improved fiduciary marker for clathrin-coated pit dynamics.

Clathrin mediated endocytosis (CME) has been extensively studied in living cells by quantitative total internal reflection fluorescence microscopy (TIRFM). Fluorescent protein fusions to subunits of the major coat proteins, clathrin light chains or the heterotetrameric adaptor protein (AP2) complexes, have been used as fiduciary markers of clathrin coated pits (CCPs). However, the functionality of these fusion proteins has not been rigorously compared. Here, we generated stable cells lines overexpressing mRuby-CLCa and/or µ2-eGFP, σ2-eGFP, two markers currently in use, or a novel marker generated by inserting eGFP into the unstructured hinge region of the α subunit (α-eGFP). Using biochemical and TIRFM-based assays, we compared the functionality of the AP2 markers. All of the eGFP-tagged subunits were efficiently incorporated into AP2 and displayed greater accuracy in image-based CCP analyses than mRuby-CLCa. However, overexpression of either µ2-eGFP or σ2-eGFP impaired transferrin receptor uptake. In addition, µ2-eGFP reduced the rates of CCP initiation and σ2-eGFP perturbed AP2 incorporation into CCPs and CCP maturation. In contrast, CME and CCP dynamics were unperturbed in cells overexpressing α-eGFP. Moreover, α-eGFP was a more sensitive and accurate marker of CCP dynamics than mRuby-CLCa. Thus, our work establishes α-eGFP as a robust, fully functional marker for CME.

The MUC6/AP2A2 Locus and Its Relevance to Alzheimer's Disease: A Review.

We recently reported evidence of Alzheimer's disease (AD)-linked genetic variation within the mucin 6 (MUC6) gene on chromosome 11p, nearby the adaptor-related protein complex 2 subunit alpha 2 (AP2A2) gene. This locus has interesting features related to human genomics and clinical research. MUC6 gene variants have been reported to potentially influence viral-including herpesvirus-immunity and the gut microbiome. Within the MUC6 gene is a unique variable number of tandem repeat (VNTR) region. We discovered an association between MUC6 VNTR repeat expansion and AD pathologic severity, particularly tau proteinopathy. Here, we review the relevant literature. The AD-linked VNTR polymorphism may also influence AP2A2 gene expression. AP2A2 encodes a polypeptide component of the adaptor protein complex, AP-2, which is involved in clathrin-coated vesicle function and was previously implicated in AD pathogenesis. To provide background information, we describe some key knowledge gaps in AD genetics research. The "missing/hidden heritability problem" of AD is highlighted. Extensive portions of the human genome, including the MUC6 VNTR, have not been thoroughly evaluated due to limitations of existing high-throughput sequencing technology. We present and discuss additional data, along with cautionary considerations, relevant to the hypothesis that MUC6 repeat expansion influences AD pathogenesis.

Flotillin and AP2A1/2 Promote IGF-1 Receptor Association with Clathrin and Internalization in Primary Human Keratinocytes.

IGF-1 receptor (IGF1R) signaling promotes keratinocyte proliferation, migration, and survival. However, the mechanism of IGF1R endocytosis in normal keratinocytes remains unclear. Confocal, super resolution structured illumination microscopy, total internal reflection fluorescence microscopy, and coimmunoprecipitation studies reveal that IGF1R associates with flotillin-1 (Flot-1), which currently has no known role in normal receptor tyrosine kinase endocytosis, under basal conditions in monolayer keratinocyte cultures. Ligand stimulation of IGF1R promotes its clathrin-dependent endocytosis, mediated by two distinct adaptors, Flot-1 in noncaveolar lipid rafts and the AP2A1/2 complex in clathrin vesicles. Concurrent, but not individual, short hairpin RNA knockdown of FLOT1/2 and AP2A1/2 reduced IGF1R association with clathrin, internalization, and pathway activation by more than 50% (of phosphorylated IGF1R, phosphorylated protein kinase B, and phosphorylated MAPK kinase), suggesting the complementarity of these two adaptor-specific pathways. The Flot-1 pathway is more responsive to low IGF-1 concentrations, whereas the AP2A1/2 pathway predominates at higher IGF-1 concentrations. Selective association of IGF1R-Flot-1-clathrin with Rab4, but IGF1R-AP2A1/2-clathrin with Rab11, implicates Flot-1 as the adaptor for faster recycling and AP2A1/2 as the adaptor for slower IGF1R recycling. These dual pathways, particularly flotillin-dependent, clathrin-mediated endocytosis, provide a new avenue for drug targeting in disorders with aberrant regulation of IGF1R signaling.

Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene.

We found evidence of late-onset Alzheimer disease (LOAD)-associated genetic polymorphism within an exon of Mucin 6 (MUC6) and immediately downstream from another gene: Adaptor Related Protein Complex 2 Subunit Alpha 2 (AP2A2). PCR analyses on genomic DNA samples confirmed that the size of the MUC6 variable number tandem repeat (VNTR) region was highly polymorphic. In a cohort of autopsied subjects with quantitative digital pathology data (n = 119), the size of the polymorphic region was associated with the severity of pTau pathology in neocortex. In a separate replication cohort of autopsied subjects (n = 173), more pTau pathology was again observed in subjects with longer VNTR regions (p = 0.031). Unlike MUC6, AP2A2 is highly expressed in human brain. AP2A2 expression was lower in a subset analysis of brain samples from persons with longer versus shorter VNTR regions (p = 0.014 normalizing with AP2B1 expression). Double-label immunofluorescence studies showed that AP2A2 protein often colocalized with neurofibrillary tangles in LOAD but was not colocalized with pTau proteinopathy in progressive supranuclear palsy, or with TDP-43 proteinopathy. In summary, polymorphism in a repeat-rich region near AP2A2 was associated with neocortical pTau proteinopathy (because of the unique repeats, prior genome-wide association studies were probably unable to detect this association), and AP2A2 was often colocalized with neurofibrillary tangles in LOAD.

The role of Ap2a2 in PPARα-mediated regulation of lipolysis in adipose tissue.

Adipose tissue plays a major role in the regulation of systemic metabolic homeostasis, with the AP2 adaptor complex being important in clathrin-mediated endocytosis (CME) of various cell surface receptors, including glucose transporter 4, the insulin receptor, and ß-adrenergic receptors (ARs). One of the AP2 subunits, adaptor-related protein complex 2, α2 subunit (Ap2a2), has recently been identified as a peroxisome proliferator-activated receptor (PPAR)α target gene. The effects of PPARα on the AP2 adaptor complex and CME are unknown. We generated adipocyte-specific Ap2a2 knockout mice and investigated their metabolism when fed a standard chow or high-fat diet, without and with supplementation with the PPARα-agonist WY-14643 (WY). Although Ap2a2 deletion had only minor effects on glycaemic control, it led to substantial impairment in ß-adrenergic activation of lipolysis, as evidenced by a loss of cAMP response, PKA activation, and glycerol/fatty acid release. These differences were related to increased cell surface localization of the ß2- and ß3-ARs. Lipolytic defects were accompanied by impaired WY-mediated loss of fat mass and whole-body fat oxidation. This study demonstrates a novel role for PPARα in ß-adrenergic regulation of adipose tissue lipolysis and for adipose tissue in supplying adequate substrate to other peripheral tissues to accommodate the increase in systemic fatty acid oxidation that occurs upon treatment with PPARα agonists.-Montgomery, M. K., Bayliss, J., Keenan, S., Rhost, S., Ting, S. B., Watt, M. J. The role of Ap2a2 in PPARα-mediated regulation of lipolysis in adipose tissue.

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.

Myosin VI Drives Clathrin-Mediated AMPA Receptor Endocytosis to Facilitate Cerebellar Long-Term Depression.

Myosin VI is an actin-based cytoskeletal motor implicated in various steps of membrane trafficking. Here, we investigated whether this myosin is crucial for synaptic function and plasticity in neurons. We find that myosin VI localizes at cerebellar parallel fiber to Purkinje cell synapses and that the myosin is indispensable for long-term depression of AMPA-receptor-mediated synaptic signal transmission at this synapse. Moreover, direct visualization of GluA2-containing AMPA receptors in Purkinje cells reveals that the myosin drives removal of AMPA receptors from the surface of dendritic spines in an activity-dependent manner. Co-immunoprecipitation and super-resolution microscopy indicate that specifically the interaction of myosin VI with the clathrin adaptor component α-adaptin is important during long-term depression. Together, these data suggest that myosin VI directly promotes clathrin-mediated endocytosis of AMPA receptors in Purkinje cells to mediate cerebellar long-term depression. Our results provide insights into myosin VI function and the molecular mechanisms underlying synaptic plasticity.

Whole Genome Sequencing of Giant Schnauzer Dogs with Progressive Retinal Atrophy Establishes NECAP1 as a Novel Candidate Gene for Retinal Degeneration.

Canine progressive retinal atrophies (PRA) are genetically heterogeneous diseases characterized by retinal degeneration and subsequent blindness. PRAs are untreatable and affect multiple dog breeds, significantly impacting welfare. Three out of seven Giant Schnauzer (GS) littermates presented with PRA around four years of age. We sought to identify the causal variant to improve our understanding of the aetiology of this form of PRA and to enable development of a DNA test. Whole genome sequencing of two PRA-affected full-siblings and both unaffected parents was performed. Variants were filtered based on those segregating appropriately for an autosomal recessive disorder and predicted to be deleterious. Successive filtering against 568 canine genomes identified a single nucleotide variant in the gene encoding NECAP endocytosis associated 1 (NECAP1): c.544G>A (p.Gly182Arg). Five thousand one hundred and thirty canids of 175 breeds, 10 cross-breeds and 3 wolves were genotyped for c.544G>A. Only the three PRA-affected GS were homozygous (allele frequency in GS, excluding proband family = 0.015). In addition, we identified heterozygotes belonging to Spitz and Dachshund varieties, demonstrating c.544G>A segregates in other breeds of German origin. This study, in parallel with the known retinal expression and role of NECAP1 in clathrin mediated endocytosis (CME) in synapses, presents NECAP1 as a novel candidate gene for retinal degeneration in dogs and other species.

A novel homozygous truncating variant of NECAP1 in early infantile epileptic encephalopathy: the second case report of EIEE21.

We report the second case of early infantile epileptic encephalopathy (EIEE) arising from a homozygous truncating variant of NECAP1. The boy developed infantile-onset tonic-clonic and tonic seizures, then spasms in clusters. His electroencephalogram (EEG) showed a burst suppression pattern, leading to the diagnosis of Ohtahara syndrome. Whole-exome sequencing revealed the canonical splice-site variant (c.301 + 1 G > A) in NECAP1. In rodents, Necap1 protein is enriched in neuronal clathrin-coated vesicles and modulates synaptic vesicle recycling. cDNA analysis confirmed abnormal splicing that produced early truncating mRNA. There has been only one previous report of a mutation in NECAP1 in a family with EIEE; this was a nonsense mutation (p.R48*) that was cited as EIEE21. Decreased mRNA levels and the loss of the WXXF motif in both the families suggests that loss of NECAP1 function is a common pathomechanism for EIEE21. This study provided additional support that synaptic vesicle recycling plays a key role in epileptogenesis.

AP2a enhanced the osteogenic differentiation of mesenchymal stem cells by inhibiting the formation of YAP/RUNX2 complex and BARX1 transcription.

OBJECTIVES: Bone regeneration by bone tissue engineering is a therapeutic option for bone defects. Improving the osteogenic differentiation of mesenchymal stem cells (MSCs) is essential for successful bone regeneration. We previously showed that AP2a enhances the osteogenic differentiation in MSCs. The present study investigated the mechanism of how AP2a regulates the direct differentiation. MATERIALS AND METHODS: Co-immunoprecipitation and ChIP assays were carried out to investigate the underlying mechanism in MSCs differentiation. The osteogenic differentiation potential was determined by mineralization ability and the expression of osteogenic marker in vitro and the in vivo bone-like tissue generation in nude mice. RESULTS: We show that AP2a can compete with RUNX2, a key transcription factor in osteogenic differentiation, to recruit YAP and release the inhibition of RUNX2 activity from YAP by forming YAP-AP2a protein complex. YAP-AP2a protein complex also interacts with the BARX1 promoter through AP2a, inhibit the transcription of BARX1. Moreover, BARX1 inhibits osteogenic differentiation of MSCs. CONCLUSIONS: Our discoveries revealed that AP2a may regulate the osteogenic differentiation in an indirect way through competing with RUNX2 to relieve the RUNX2 activity which inhibited by YAP, and also in a direct way via targeting the BARX1 and directly repressed its transcription. Thus, our discoveries shed new light on the mechanism of direct differentiation of MSCs and provide candidate targets for improving the osteogenic differentiation and enhancing bone tissue regeneration.

Prd1 associates with the clathrin adaptor α-Adaptin and the kinesin-3 Imac/Unc-104 to govern dendrite pruning in Drosophila.

Refinement of the nervous system depends on selective removal of excessive axons/dendrites, a process known as pruning. Drosophila ddaC sensory neurons prune their larval dendrites via endo-lysosomal degradation of the L1-type cell adhesion molecule (L1-CAM), Neuroglian (Nrg). Here, we have identified a novel gene, pruning defect 1 (prd1), which governs dendrite pruning of ddaC neurons. We show that Prd1 colocalizes with the clathrin adaptor protein α-Adaptin (α-Ada) and the kinesin-3 immaculate connections (Imac)/Uncoordinated-104 (Unc-104) in dendrites. Moreover, Prd1 physically associates with α-Ada and Imac, which are both critical for dendrite pruning. Prd1, α-Ada, and Imac promote dendrite pruning via the regulation of endo-lysosomal degradation of Nrg. Importantly, genetic interactions among prd1, α-adaptin, and imac indicate that they act in the same pathway to promote dendrite pruning. Our findings indicate that Prd1, α-Ada, and Imac act together to regulate discrete distribution of α-Ada/clathrin puncta, facilitate endo-lysosomal degradation, and thereby promote dendrite pruning in sensory neurons.

A genetic variant near adaptor-related protein complex 2 alpha 2 subunit gene is associated with coronary artery disease in a Chinese population.

BACKGROUND: Adaptor-related protein complex 2 alpha 2 subunit (AP2A2) gene encodes a protein-a subunit of the AP-2 adaptor protein complex. Evidence has revealed that benzodiazepine receptor-associated protein 1 (BZRAP1) is abundant in the hippocampus with potential effects on brain diseases. Recently, an epidemiological study reported that two variants (rs7396366 and rs2526378) closest to the AP2A2 and BZRAP1 genes are associated with higher plasma lipids and Alzheimer's disease. Whether the two single nucleotide polymorphisms (SNPs) are actually relevant to coronary artery disease (CAD) and CAD severity remains elusive. Our aim was to assess whether these two SNPs are relevant to CAD and its severity in a Chinese population. METHODS: Three hundred and thirty-five patients with documented CAD (282 stable CAD, 28 non-ST-segment elevation myocardial infarction, 25 ST-segment elevation myocardial infarction), and 372 non-CAD controls were included in the study. The participants were divided into two groups according to coronary angiography results. CAD patients were further demarcated into subgroups with one-, two-, or three-vessel stenosis. Genotypes at rs7396366 and rs2526378 were examined using polymerase chain reaction-ligase detection reaction. The association between these two SNPs with CAD and its severity were analyzed. RESULTS: The frequency of the rs7396366 TT genotype was significantly higher in CAD patients than in controls (13.7% vs. 7.8%, 95% CI: 1.15-3.07, P = 0.014). Subjects with a variant genotype T allele had an increased risk of CAD compared with G allele carriers (additive model: 95% CI: 1.21-3.35, P = 0.008). After adjustment for traditional cardiovascular risk factors, analysis of the dominant models involving rs7396366 also showed that T allele carriers had a significantly higher risk for CAD than G allele carriers had (dominant model: OR 1.48, 95% CI: 1.03-2.14, P = 0.035). Age, sex, type 2 diabetes mellitus, fasting plasma glucose, and the TT genotype in rs7396366 were significantly associated with three-vessel lesions. Despite these significant outcomes of rs7396366, information on rs2526378 showed no significant difference between CAD patients and non-CAD controls. CONCLUSION: Our results show that the T allele and TT genotype in rs7396366, closest to the AP2A2 gene, are linked to an increased risk of CAD and its severity in a Chinese population.

Molecular cloning of novel transcripts of the adaptor-related protein complex 2 alpha 1 subunit (AP2A1) gene, using Next-Generation Sequencing.

The adaptor-related protein (AP) complexes play important roles in cargo selection and vesicle formation, and hence in intracellular membrane trafficking. Five different AP complexes are currently known, each consisting of four subunits, known as adaptins. AP-2, the most thoroughly characterized of the five AP complexes, facilitates clathrin-mediated endocytosis. In this study, we describe the discovery and molecular cloning of seventy-seven novel alternatively spliced transcripts of the human AP2A1 gene, which encodes the αA adaptin of the AP-2 complex. For this purpose, we have used Next-Generation Sequencing (NGS), a powerful tool for studying alternative splicing. In brief, we subcultured fifty-five established human cell lines, originating from several distinct cancerous and normal tissues, extracted total RNA, and synthesized first-strand cDNA. Next, we used nested touchdown PCR to amplify the whole coding region of the AP2A1 transcripts of each cell line, mixed all PCR products, and proceeded to NGS library construction, template preparation, and semiconductor sequencing. Extensive bioinformatic analysis revealed thirteen novel splice junctions of previously annotated exons, as also verified via nested PCR with primers targeting these splice junctions. Moreover, consecutive nested PCRs led to the determination of the primary structure of seventy-seven novel AP2A1 transcripts, all of which were shown to comprise at least one premature translation termination codon, thus representing nonsense-mediated mRNA decay (NMD) candidates. NMD is a mechanism that cells use to control gene expression. Consequently, alterations in the levels of these potentially non-coding AP2A1 transcripts could lead to a decrease in the number of AP2A1 mRNA molecules, when needed. Undoubtedly, the exact role of these new APA1 splice variants merits elucidation.

The Listeriolysin O PEST-like Sequence Co-opts AP-2-Mediated Endocytosis to Prevent Plasma Membrane Damage during Listeria Infection.

Listeriolysin O (LLO) is a cholesterol-dependent cytolysin that mediates escape of Listeria monocytogenes from a phagosome, enabling growth of the bacteria in the host cell cytosol. LLO contains a PEST-like sequence that prevents it from killing infected cells, but the mechanism involved is unknown. We found that the LLO PEST-like sequence was necessary to mediate removal of LLO from the interior face of the plasma membrane, where it coalesces into discrete puncta. LLO interacts with Ap2a2, an adaptor protein involved in endocytosis, via its PEST-like sequence, and Ap2a2-dependent endocytosis is required to prevent LLO-induced cytotoxicity. An unrelated PEST-like sequence from a human G protein-coupled receptor (GPCR), which also interacts with Ap2a2, could functionally complement the PEST-like sequence in L. monocytogenes LLO. These data revealed that LLO co-opts the host endocytosis machinery to protect the integrity of the host plasma membrane during L. monocytogenes infection.