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Neurons are dependent on efficient quality control mechanisms to maintain cellular homeostasis and function due to their polarization and long-life span. Autophagy is a lysosomal degradative pathway that provides nutrients during starvation and recycles damaged and/or aged proteins and organelles. In neurons, autophagosomes constitutively form in distal axons and at synapses and are trafficked retrogradely to the cell soma to fuse with lysosomes for cargo degradation. How the neuronal autophagy pathway is organized and controlled remains poorly understood. Several presynaptic endocytic proteins have been shown to regulate both synaptic vesicle recycling and autophagy. Here, by combining electron, fluorescence, and live imaging microscopy with biochemical analysis, we show that the neuron-specific protein APache, a presynaptic AP-2 interactor, functions in neurons as an important player in the autophagy process, regulating the retrograde transport of autophagosomes. We found that APache colocalizes and co-traffics with autophagosomes in primary cortical neurons and that induction of autophagy by mTOR inhibition increases LC3 and APache protein levels at synaptic boutons. APache silencing causes a blockade of autophagic flux preventing the clearance of p62/SQSTM1, leading to a severe accumulation of autophagosomes and amphisomes at synaptic terminals and along neurites due to defective retrograde transport of TrkB-containing signaling amphisomes along the axons. Together, our data identify APache as a regulator of the autophagic cycle, potentially in cooperation with AP-2, and hypothesize that its dysfunctions contribute to the early synaptic impairments in neurodegenerative conditions associated with impaired autophagy. [ABSTRACT FROM AUTHOR]

Activating enhancer-binding protein 2 (AP-2) is a family of transcription factors (TFs) that play crucial roles in regulating embryonic and oncogenic development. In addition to splice isoforms, five major family members encoded by the TFAP2A/B/C/D/E genes have been identified in humans, i.e., AP-2α/β/γ/δ/ε. In general, the first three TFs have been studied more thoroughly than AP-2δ or AP-2ε. Currently, there is a relatively limited body of literature focusing on the AP-2 family in the context of gastroenterological research, and a comprehensive overview of the existing knowledge and recommendations for further research directions is lacking. Herein, we have collected available gastroenterological data on AP-2 TFs, discussed the latest medical applications of each family member, and proposed potential future directions. Research on AP-2 in gastrointestinal tumors has predominantly been focused on the two best-described family members, AP-2α and AP-2γ. Surprisingly, research in the past decade has highlighted the importance of AP-2ε in the drug resistance of gastric cancer (GC) and colorectal cancer (CRC). While numerous questions about gastroenterological disorders await elucidation, the available data undoubtedly open avenues for anti-cancer targeted therapy and overcoming chemotherapy resistance. In addition to gastrointestinal cancers, AP-2 family members (primarily AP-2β and marginally AP-2γ) have been associated with other health issues such as obesity, type 2 diabetes, liver dysfunction, and pseudo-obstruction. On the other hand, AP-2δ has been poorly investigated in gastroenterological disorders, necessitating further research to delineate its role. In conclusion, despite the limited attention given to AP-2 in gastroenterology research, pivotal functions of these transcription factors have started to emerge and warrant further exploration in the future. [ABSTRACT FROM AUTHOR]

Recycling of synaptic vesicles plays an important role in vesicle pool replenishment, neurotransmitter release and synaptic plasticity. Clathrin-mediated endocytosis (CME) is considered to be the main mechanism for synaptic vesicle replenishment. AP-2 (adaptor-related protein complex 2) and myosin Ⅵ are known as key proteins that regulate the structure and dynamics of CME. This study aims to reveal the spatiotemporal expression of AP-2/myosin Ⅵ in inner hair cells (IHCs) of the mouse cochlea and its correlation with auditory function. Immunofluorescence was used to detect the localization and expression of AP-2 and myosin Ⅵ in cochlear hair cells (HCs) of CBA/CaJ mice of various ages. qRT-PCR was used to verify the differential expression of AP-2 and myosin Ⅵ mRNA in the mouse cochlea, and ABR tests were administered to mice of various ages. A preliminary analysis of the correlation between AP-2/myosin Ⅵ levels and auditory function was conducted. AP-2 was located in the cytoplasmic region of IHCs and was mainly expressed in the basal region of IHCs and the area near ribbon synapses, while myosin Ⅵ was expressed in the cytoplasmic region of IHCs and OHCs. Furthermore, AP-2 and myosin Ⅵ were not significant detected in the cochleae of P7 mice; the expression level reached a peak at P35 and then decreased significantly with age. The expression patterns and expression levels of AP-2 and myosin Ⅵ in the cochleae of the mice were consistent with the development of the auditory system. AP-2 and myosin Ⅵ protein expression may differ in mice of different ages, and this variation probably leads to a difference in the efficiency in CME; it may also cause a defect in IHC function. [ABSTRACT FROM AUTHOR]

Since their discovery, the Hox genes, with their incredible power to reprogram the identity of complete body regions, a phenomenon called homeosis, have captured the fascination of many biologists. Recent research has provided new insights into the function of Hox proteins in different germ layers and the mechanisms they employ to control tissue morphogenesis. We focus in this review on the ectoderm and mesoderm to highlight new findings and discuss them with regards to established concepts of Hox target gene regulation. Furthermore, we highlight the molecular mechanisms involved the transcriptional repression of specific groups of Hox target genes, and summarize the role of Hox mediated gene silencing in tissue development. Finally, we reflect on recent findings identifying a large number of tissue-specific Hox interactor partners, which open up new avenues and directions towards a better understanding of Hox function and specificity in different tissues. [ABSTRACT FROM AUTHOR]

Activating enhancer-binding protein 2 (AP-2) is a family of transcription factors (TFs) that play crucial roles in regulating embryonic and oncogenic development. In addition to splice isoforms, five major family members encoded by the TFAP2A/B/C/D/E genes have been identified in humans, i.e., AP-2α/β/γ/δ/ε. In general, the first three TFs have been studied more thoroughly than AP-2δ or AP-2ε. Currently, there is a relatively limited body of literature focusing on the AP-2 family in the context of gastroenterological research, and a comprehensive overview of the existing knowledge and recommendations for further research directions is lacking. Herein, we have collected available gastroenterological data on AP-2 TFs, discussed the latest medical applications of each family member, and proposed potential future directions. Research on AP-2 in gastrointestinal tumors has predominantly been focused on the two best-described family members, AP-2α and AP-2γ. Surprisingly, research in the past decade has highlighted the importance of AP-2ε in the drug resistance of gastric cancer (GC) and colorectal cancer (CRC). While numerous questions about gastroenterological disorders await elucidation, the available data undoubtedly open avenues for anti-cancer targeted therapy and overcoming chemotherapy resistance. In addition to gastrointestinal cancers, AP-2 family members (primarily AP-2β and marginally AP-2γ) have been associated with other health issues such as obesity, type 2 diabetes, liver dysfunction, and pseudo-obstruction. On the other hand, AP-2δ has been poorly investigated in gastroenterological disorders, necessitating further research to delineate its role. In conclusion, despite the limited attention given to AP-2 in gastroenterology research, pivotal functions of these transcription factors have started to emerge and warrant further exploration in the future.

Members of the activator protein 2 (AP-2) transcription factor (TF) family are known to play a role in both physiological processes and cancer development. The family comprises five DNA-binding proteins encoded by the TFAP2A to TFAP2E genes. Numerous scientific reports describe differential expression of these TF and their genes in various types of cancer, identifying among them a potential oncogene or suppressor like TFAP2A or TFAP2C. Other reports suggest their influence on disease development and progression, as well as response to treatment. Not all members of this AP-2 family have been comprehensively studied thus far. The aim of the present article is to gather and discuss knowledge available in bioinformatics databases regarding all five members of this family and to differentiate them in relation to the two most common lung cancer subtypes: adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). In addition, to assess the difference in levels depending on a number of clinicopathological factors, the impact on patient survival and interactions with tumor-infiltrating immune cells. This article may help to identify the target for further original research that may contribute to the discovery of new diagnostic biomarkers and define the molecular differences between LUAD and LUSC, which may affect the therapy effectiveness improvement and longer survival. [ABSTRACT FROM AUTHOR]

Formerly hailed as "undruggable" proteins, transcription factors (TFs) are now under investigation for targeted therapy. In cancer, this may alter, inter alia, immune evasion or replicative immortality, which are implicated in genome organization, a process that accompanies multi-step tumorigenesis and which frequently develops in a non-random manner. Still, targeting-related research on some TFs is scarce, e.g., among AP-2 proteins, which are known for their altered functionality in cancer and prognostic importance. Using public repositories, bioinformatics tools, and RNA-seq data, the present study examined the ligandability of all AP-2 members, selecting the best one, which was investigated in terms of mutations, targets, co-activators, correlated genes, and impact on genome organization. AP-2 proteins were found to have the conserved "TF_AP-2" domain, but manifested different binding characteristics and evolution. Among them, AP-2δ has not only the highest number of post-translational modifications and extended strands but also contains a specific histidine-rich region and cleft that can receive a ligand. Uterine, colon, lung, and stomach tumors are most susceptible to AP-2δ mutations, which also co-depend with cancer hallmark genes and drug targets. Considering AP-2δ targets, some of them were located proximally in the spatial genome or served as co-factors of the genes regulated by AP-2δ. Correlation and functional analyses suggested that AP-2δ affects various processes, including genome organization, via its targets; this has been eventually verified in lung adenocarcinoma using expression and immunohistochemistry data of chromosomal conformation-related genes. In conclusion, AP-2δ affects chromosomal conformation and is the most appropriate target for cancer therapy focused on the AP-2 family. [ABSTRACT FROM AUTHOR]

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Nef hijacks the clathrin adaptor complex 2 (AP-2) to downregulate the viral receptor CD4 and the antiviral multipass transmembrane proteins SERINC3 and SERINC5, which inhibit the infectivity of progeny virions when incorporated. In Jurkat Tag T lymphoid cells lacking SERINC3 and SERINC5, Nef is no longer required for full progeny virus infectivity and for efficient viral replication. However, in MOLT-3 T lymphoid cells, HIV-1 replication remains highly dependent on Nef even in the absence of SERINC3 and SERINC5. Using a knockout (KO) approach, we now show that the Nef-mediated enhancement of HIV-1 replication in MOLT-3 cells does not depend on the Nef-interacting kinases LCK and PAK2. Furthermore, Nef substantially enhanced HIV-1 replication even in triple-KO MOLT-3 cells that simultaneously lacked the three Nef/AP-2 targets, SERINC3, SERINC5, and CD4, and were reconstituted with a Nef-resistant CD4 to permit HIV-1 entry. Nevertheless, the ability of Nef mutants to promote HIV-1 replication in the triple-KO cells correlated strictly with the ability to bind AP-2. In addition, knockdown and reconstitution experiments confirmed the involvement of AP-2. These observations raise the possibility that MOLT-3 cells express a novel antiviral factor that is downregulated by Nef in an AP-2-dependent manner. IMPORTANCE The HIV-1 Nef protein hijacks a component of the cellular endocytic machinery called AP-2 to downregulate the viral receptor CD4 and the antiviral cellular membrane proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 are taken up into viral particles, which reduces their infectivity. Surprisingly, in a T cell line called MOLT-3, Nef remains crucial for HIV-1 spreading in the absence of SERINC3 and SERINC5. We now show that this effect of Nef also does not depend on the cellular signaling molecules and Nef interaction partners LCK and PAK2. Nef was required for efficient HIV-1 spreading even in triple-knockout cells that completely lacked Nef/AP-2-sensitive CD4, in addition to the Nef/AP-2 targets SERINC3 and SERINC5. Nevertheless, our results indicate that the enhancement of HIV-1 spreading by Nef in the triple-knockout cells remained AP-2 dependent, which suggests the presence of an unknown antiviral factor that is sensitive to Nef/AP-2-mediated downregulation.

The facial surface ectoderm is essential for normal development of the underlying cranial neural crest cell populations, providing signals that direct appropriate growth, patterning, and morphogenesis. Despite the importance of the ectoderm as a signaling center, the molecular cues and genetic programs implemented within this tissue are understudied. Here, we show that removal of two members of the AP-2 transcription factor family, AP-2α and AP-2ß, within the early embryonic ectoderm of the mouse leads to major alterations in the craniofacial complex. Significantly, there are clefts in both the upper face and mandible, accompanied by fusion of the upper and lower jaws in the hinge region. Comparison of ATAC-seq and RNA-seq analyses between controls and mutants revealed significant changes in chromatin accessibility and gene expression centered on multiple AP-2 binding motifs associated with enhancer elements within these ectodermal lineages. In particular, loss of these AP-2 proteins affects both skin differentiation as well as multiple signaling pathways, most notably the WNT pathway. We also determined that the mutant clefting phenotypes that correlated with reduced WNT signaling could be rescued by Wnt1 ligand overexpression in the ectoderm. Collectively, these findings highlight a conserved ancestral function for AP-2 transcription factors in ectodermal development and signaling, and provide a framework from which to understand the gene regulatory network operating within this tissue that directs vertebrate craniofacial development.

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/adaptor protein complex 2 (AP-2)-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in mouse hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a revised model for the endocytosis of SV membranes at mammalian central synapses.

Members of the activator protein 2 (AP-2) transcription factor (TF) family are known to play a role in both physiological processes and cancer development. The family comprises five DNA-binding proteins encoded by the TFAP2A to TFAP2E genes. Numerous scientific reports describe differential expression of these TF and their genes in various types of cancer, identifying among them a potential oncogene or suppressor like TFAP2A or TFAP2C. Other reports suggest their influence on disease development and progression, as well as response to treatment. Not all members of this AP-2 family have been comprehensively studied thus far. The aim of the present article is to gather and discuss knowledge available in bioinformatics databases regarding all five members of this family and to differentiate them in relation to the two most common lung cancer subtypes: adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). In addition, to assess the difference in levels depending on a number of clinicopathological factors, the impact on patient survival and interactions with tumor-infiltrating immune cells. This article may help to identify the target for further original research that may contribute to the discovery of new diagnostic biomarkers and define the molecular differences between LUAD and LUSC, which may affect the therapy effectiveness improvement and longer survival.

AP-2 transcription factors regulate ectodermal development, but their roles in epidermal homeostasis in adult skin are unknown. We find that AP-2α is the predominant AP-2 family member in adult epidermis, followed by AP-2β. Through inactivation of AP-2α, AP-2β, or both in keratinocytes, we assessed the effects of a gradient of epidermal AP-2 activity on skin function. We find that (i) loss of AP-2β in keratinocytes is compensated for by AP-2α, (ii) loss of AP-2α impairs terminal keratinocyte differentiation and hair morphogenesis, and (iii) the combined loss of AP-2α/AP-2β results in more severe skin and hair abnormalities. Keratinocyte differentiation defects precede progressive neutrophilic skin inflammation. Inducible inactivation of AP-2α/AP-2β in the adult phenocopies these manifestations. Transcriptomic analyses of epidermis lacking AP-2α or AP-2α/AP-2β in keratinocytes demonstrate a terminal keratinocyte differentiation defect with upregulation of alarmin keratins and of several immune pathway regulators. Moreover, our analyses suggest a key role of reduced AP-2α-dependent gene expression of CXCL14 and the keratin 15 gene K15 as an early pathogenic event toward the manifestation of skin inflammation. Thus, AP-2α and AP-2β are critical regulators of epidermal homeostasis in adult skin., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

AP-2 is a family of transcription factors involved in many aspects of development, cell differentiation, and regulation of cell growth and death. AP-2δ is a member of this group and specific gene expression patterns are required in the adult mouse brain for the development of parts of the inferior colliculus (IC), as well as the cortex, dorsal thalamus, and superior colliculus. The midbrain is one of the central areas in the brain where multimodal integration, i.e., integration of information from different senses, occurs. Previous data showed that AP-2δ-deficient mice are viable but due to increased apoptosis at the end of embryogenesis, lack part of the posterior midbrain. Despite the absence of the IC in AP-2δ-deficient mice, these animals retain at least some higher auditory functions. Neuronal responses to tones in the neocortex suggest an alternative auditory pathway that bypasses the IC. While sufficient data are available in mammals, little is known about AP-2δ in chickens, an avian model for the localization of sounds and the development of auditory circuits in the brain. Here, we identified and localized AP-2δ expression in the chicken midbrain during embryogenesis. Our data confirmed the presence of AP-2δ in the inferior colliculus and optic tectum (TeO), specifically in shepherd's crook neurons, which are an essential component of the midbrain isthmic network and involved in multimodal integration. AP-2δ expression in the chicken midbrain may be related to the integration of both auditory and visual afferents in these neurons. In the future, these insights may allow for a more detailed study of circuitry and computational rules of auditory and multimodal networks. [ABSTRACT FROM AUTHOR]

AP-2 is a family of transcription factors involved in many aspects of development, cell differentiation, and regulation of cell growth and death. AP-2δ is a member of this group and specific gene expression patterns are required in the adult mouse brain for the development of parts of the inferior colliculus (IC), as well as the cortex, dorsal thalamus, and superior colliculus. The midbrain is one of the central areas in the brain where multimodal integration, i.e., integration of information from different senses, occurs. Previous data showed that AP-2δ-deficient mice are viable but due to increased apoptosis at the end of embryogenesis, lack part of the posterior midbrain. Despite the absence of the IC in AP-2δ-deficient mice, these animals retain at least some higher auditory functions. Neuronal responses to tones in the neocortex suggest an alternative auditory pathway that bypasses the IC. While sufficient data are available in mammals, little is known about AP-2δ in chickens, an avian model for the localization of sounds and the development of auditory circuits in the brain. Here, we identified and localized AP-2δ expression in the chicken midbrain during embryogenesis. Our data confirmed the presence of AP-2δ in the inferior colliculus and optic tectum (TeO), specifically in shepherd’s crook neurons, which are an essential component of the midbrain isthmic network and involved in multimodal integration. AP-2δ expression in the chicken midbrain may be related to the integration of both auditory and visual afferents in these neurons. In the future, these insights may allow for a more detailed study of circuitry and computational rules of auditory and multimodal networks.

Mice possess two types of teeth that differ in their cusp patterns; incisors have one cusp and molars have multiple cusps. The patterning of these two types of teeth relies on fine-tuning of the reciprocal molecular signaling between dental epithelial and mesenchymal tissues during embryonic development. The AP-2 transcription factors, particularly Tfap2a and Tfap2b , are essential components of such epithelial-mesenchymal signaling interactions that coordinate craniofacial development in mice and other vertebrates, but little is known about their roles in the regulation of tooth development and shape. Here we demonstrate that incisors and molars differ in their temporal and spatial expression of Tfap2a and Tfap2b. At the bud stage, Tfap2a is expressed in both the epithelium and mesenchyme of the incisors and molars, but Tfap2b expression is restricted to the molar mesenchyme, only later appearing in the incisor epithelium. Tissue-specific deletions show that loss of the epithelial domain of Tfap2a and Tfap2b affects the number and spatial arrangement of the incisors, notably resulting in duplicated lower incisors. In contrast, deletion of these two genes in the mesenchymal domain has little effect on tooth development. Collectively these results implicate epithelial expression of Tfap2a and Tfap2b in regulating the extent of the dental lamina associated with patterning the incisors and suggest that these genes contribute to morphological differences between anterior (incisor) and posterior (molar) teeth within the mammalian dentition. • Tfap2a and Tfap2b are expressed in spatially and temporally dynamic patterns and differ between incisor and molar tooth germs. • Epithelial expression of Tfap2a and Tfap2b is necessary for incisor development. • Expression of Tfap2a and Tfap2b in the mesenchyme is not required for incisor or molar development. [ABSTRACT FROM AUTHOR]

Focal adhesion kinase (FAK) regulates key biological processes downstream of G protein-coupled receptors (GPCRs) in normal and cancer cells, but the modes of kinase activation by these receptors remain unclear. We report that after GPCR stimulation, FAK activation is controlled by a sequence of events depending on the scaffolding proteins β-arrestins and G proteins. Depletion of β-arrestins results in a marked increase in FAK autophosphorylation and focal adhesion number. We demonstrate that β-arrestins interact directly with FAK and inhibit its autophosphorylation in resting cells. Both FAK–β-arrestin interaction and FAK inhibition require the FERM domain of FAK. Following the stimulation of the angiotensin receptor AT1AR and subsequent translocation of the FAK–β-arrestin complex to the plasma membrane, β-arrestin interaction with the adaptor AP-2 releases inactive FAK from the inhibitory complex, allowing its activation by receptor-stimulated G proteins and activation of downstream FAK effectors. Release and activation of FAK in response to angiotensin are prevented by an AP-2-binding deficient β-arrestin and by a specific inhibitor of β-arrestin/AP-2 interaction; this inhibitor also prevents FAK activation in response to vasopressin. This previously unrecognized mechanism of FAK regulation involving a dual role of β-arrestins, which inhibit FAK in resting cells while driving its activation at the plasma membrane by GPCR-stimulated G proteins, opens new potential therapeutic perspectives in cancers with up-regulated FAK. [ABSTRACT FROM AUTHOR]

Besides AP-2 and clathrin triskelia, clathrin coat inception depends on a group of early-arriving proteins including Fcho1/2 and Eps15/R. Using genome-edited cells, we described the role of the unstructured Fcho linker in stable AP-2 membrane deposition. Here, expanding this strategy in combination with a new set of llama nanobodies against EPS15 shows an FCHO1/2–EPS15/R partnership plays a decisive role in coat initiation. A nanobody containing an Asn-Pro-Phe peptide within the complementarity-determining region 3 loop is a function-blocking pseudoligand for tandem EPS15/R EH domains. Yet, in living cells, EH domains gathered at clathrin-coated structures are poorly accessible, indicating residence by endogenous NPF-bearing partners. Forcibly sequestering cytosolic EPS15 in genome-edited cells with nanobodies tethered to early endosomes or mitochondria changes the subcellular location and availability of EPS15. This combined approach has strong effects on clathrin coat structure and function by dictating the stability of AP-2 assemblies at the plasma membrane.

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The Amyloid Precursor Protein (APP) has been extensively studied for its role as the precursor of the β-amyloid protein (Aβ) in Alzheimer’s disease (AD). However, our understanding of the normal function of APP is still patchy. Emerging evidence indicates that a dysfunction in APP trafficking and degradation can be responsible for neuronal deficits and progressive degeneration in humans. We recently reported that the Y682 mutation in the 682YENPTY687 domain of APP affects its binding to specific adaptor proteins and leads to its anomalous trafficking, to defects in the autophagy machinery and to neuronal degeneration. In order to identify adaptors that influence APP function, we performed pull-down experiments followed by quantitative mass spectrometry (MS) on hippocampal tissue extracts of three month-old mice incubated with either the 682YENPTY687 peptide, its mutated form, 682GENPTY687 or its phosphorylated form, 682pYENPTY687. Our experiments resulted in the identification of two proteins involved in APP internalization and trafficking: Clathrin heavy chain (hc) and its Adaptor Protein 2 (AP-2). Overall our results consolidate and refine the importance of Y682 in APP normal functions from an animal model of premature aging and dementia. Additionally, they open the perspective to consider Clathrin hc and AP-2 as potential targets for the design and development of new therapeutic strategies.

The evolution of a hinged moveable jaw with variable morphology is considered a major factor behind the successful expansion of the vertebrates. DLX homeobox transcription factors are critical to establish the positional code that patterns the mandible, maxilla and intervening hinge domain, but how these genes are regulated remains unclear. Herein, we demonstrate that the concerted action of the AP-2α and AP-2β transcription factors within the mouse neural crest is essential for jaw patterning. In the absence of these two genes the hinge domain is lost and there are alterations in the size and patterning of the jaws correlating with dysregulation of homeobox gene expression, with reduced levels of Emx, Msx and Dlx paralogs accompanied by an expansion of Six1. Moreover, detailed analysis of morphological features and gene expression changes indicate significant overlap with various compound Dlx mutants. Together, these findings reveal that the AP-2 genes have a major function in mammalian neural crest development, influencing patterning of the craniofacial skeleton via the DLX-code, a result that has implications for vertebrate facial evolution as well as for human craniofacial disorders. [ABSTRACT FROM AUTHOR]

It is increasingly evident that carcinogenesis, in the vast majority of cancers, cannot be explained simply through an accumulation of somatic mutations, or epigenetics, the stem cell theory, or the Warburg effect. Here, decades of thinking based on incorrect assumptions has resulted in an incorrect hypothesis on the origin of cancer. Many papers studying DNA, genetics, RNA, miRNA, proteomics, and epigenetics have increased our understanding of biology. Our paradigm, though more complex, is more reliable and plausible. It states that cancer originates from a disruption of homeostasis. This essential biological phenomenon, homeostasis, maintains the interrelationships of various signaling pathways and induced crosstalk which modify cellular functions together with the interactions of surrounding cells and structures such that the equilibrium lies towards the optimal health of the organism. This Special Issue “Disruption of signaling homeostasis induced crosstalk in the carcinogenesis paradigm Epistemology of the origin of cancer” provides compelling evidence that carcinogenesis is explained by a six-step sequence of events for the vast majority of cancers. These six steps include, (1) a pathogenic stimulus followed by (2) chronic inflammation, from which develops (3) fibrosis with associated remodeling in the cellular microenvironment. From these changes a (4) pre-cancerous niche develops which triggers the deployment of (5) a chronic stress escape strategy, and when this fails to resolve, and (6) the transition of a normal cell to a cancer cell occurs. This paradigm provides opportunities to move away from a symptom-oriented understanding of cancer and is much closer to a cause-based understanding, which opens the door for early preventative strategies to mitigate cancer as a disease, and to interdict metastases. This is underpinned by the fact that an independent recently published proof of this paradigm showed how a stimulus trigger the proposed multi-sequence cascade of events as abrupt involution-induced chronic inflammation, followed by fibrosis with remodeling, which describes the pre-cancerous niche followed by hyperplasia, metaplasia, and cancer.

Acquired Immune Deficiency Syndrome is characterized by a decline in CD4 + T cells. Here, we elucidated the mechanism underlying apoptosis in Human Immunodeficiency Virus-1 (HIV-1) infection by examining host apoptotic pathways hijacked by the HIV-1 Nef protein in the CD4 + T-cell line Sup-T1. Using a panel of Nef mutants unable to bind specific host proteins we uncovered that Nef generates pro- and anti-apoptotic signals. Apoptosis increased upon mutating the motifs involved in the interaction of Nef:AP-1 (Nef M20A or Nef EEEE62-65AAAA ) or Nef:AP-2 (Nef LL164/165AA ), implying these interactions limit Nef-mediated apoptosis. In contrast, disrupting the Nef:PAK2 interaction motifs (Nef H89A or Nef F191A ) reduced apoptosis. To validate further, apoptosis was measured after short-hairpin RNA knock-down of AP-1, AP-2 and PAK2. AP-2α depletion enhanced apoptosis, demonstrating that disrupting the Nef:AP-2α interaction limits Nef-mediated apoptosis. Collectively, we describe a mechanism by which HIV-1 regulates cell survival and demonstrate the consequence of interfering with Nef:host protein interactions. [ABSTRACT FROM AUTHOR]

The neuron-enriched, endosomal protein Calcyon (Caly) regulates endocytosis and vesicle sorting, and is important for synaptic plasticity and brain development. In the current investigation of Caly interacting proteins in brain, the microtubule retrograde motor subunit, cytoplasmic dynein 1 heavy chain (DYNC1H), and microtubule structural proteins, α and β tubulin, were identified as Caly associated proteins by MALDI-ToF/ToF. Direct interaction of the Caly-C terminus with dynein and tubulin was further confirmed in in vitro studies. In Cos-7 cells, mCherry-Caly moved along the microtubule network in organelles largely labeled by the late endosome marker Rab7. Expression of the dynein inhibitor CC1, produced striking alterations in Caly distribution, consistent with retrograde motors playing a prominent role in Caly localization and movement. In axons of cultured adult rat sensory neurons, Caly-positive organelles co-localized with dynein intermediate chain (DYNC1I1-isoform IC-1B) and the dynein regulator, lissencephaly 1 (LIS1), both of which co-precipitated from brain with the Caly C-terminus. Manipulation of dynein function in axons altered the motile properties of Caly indicating that Caly vesicles utilize the retrograde motor. Altogether, the current evidence for association with dynein motors raises the possibility that the endocytic and cargo sorting functions of Caly in neurons could be regulated by interaction with the microtubule transport system. [ABSTRACT FROM AUTHOR]

To date, five AP-2 genes that encode AP-2α, β, γ, δ and ε have been identified in vertebrates and they have been reported to be key regulators of embryonic development. However, the role of AP-2 family members in the development of central nervous system (CNS) has not been characterized. In the present study, we systematically examined the spatiotemporal expression pattern of AP-2 genes in the developing spinal cord of mouse and chick embryos and found that AP-2α and AP-2β are specifically expressed in post-mitotic dorsal interneurons. Loss-of-function analysis using in ovo electroporation in embryonic chick spinal cord preliminarily demonstrated that cAP-2α and cAP-2β regulates dorsal Class A and Class B interneuron specification, respectively. Gain-of-function experiments further revealed that misexpression of cAP-2α, but not cAP-2β, was able to induce the ectopic generation of Class A interneurons. Together, our studies indicated that AP-2 family members, AP-2α and AP-2β, have distinct functions in the regulation of dorsal interneuron development. [ABSTRACT FROM AUTHOR]

The horizontal transfer of genes between distantly related organisms is undoubtedly a major factor in the evolution of novel traits. Because genes are functionless without expression, horizontally transferred genes must acquire appropriate transcriptional regulations in their recipient organisms, although the evolutionary mechanism is not known well. The defining characteristic of tunicates is the presence of a cellulose containing tunic covering the adult and larval body surface. Cellulose synthase was acquired by horizontal gene transfer from Actinobacteria. We found that acquisition of the binding site of AP-2 transcription factor was essential for tunicate cellulose synthase to gain epidermal-specific expression. Actinobacteria have very GC-rich genomes, regions of which are capable of inducing specific expression in the tunicate epidermis as the AP-2 binds to a GCrich region. Therefore, the actinobacterial cellulose synthase could have been potentiated to evolve its new function in the ancestor of tunicates with a higher probability than the evolution depending solely on a spontaneous event. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs), the largest family of cell receptors, act as important regulators of diverse signaling pathways. Our understanding of the impact of GPCRs in tumors is emerging, yet there is no therapeutic platform based on GPCR driver genes. As cancer progresses, it disrupts normal epithelial organization and maintains the cells outside their normal niche. The dynamic and flexible microenvironment of a tumor contains both soluble and matrix-immobilized proteases that contribute to the process of cancer advancement. An example is the activation of cell surface protease-activated receptors (PARs). Mammalian PARs are a subgroup of GPCRs that form a family of four members, PAR1⁻4, which are uniquely activated by proteases found in the microenvironment. PAR1 and PAR2 play central roles in tumor biology, and PAR3 acts as a coreceptor. The significance of PAR4 in neoplasia is just beginning to emerge. PAR1 has been shown to be overexpressed in malignant epithelia, in direct correlation with tumor aggressiveness, but there is no expression in normal epithelium. In this review, the involvement of key transcription factors such as Egr1, p53, Twist, AP2, and Sp1 that control PAR1 expression levels specifically, as well as hormone transcriptional regulation by both estrogen receptors (ER) and androgen receptors (AR) are discussed. The cloning of the human protease-activated receptor 2; Par2 (hPar2) promoter region and transcriptional regulation of estrogen (E2) via binding of the E2⁻ER complex to estrogen response elements (ERE) are shown. In addition, evidence that TEA domain 4 (TEAD4) motifs are present within the hPar2 promoter is presented since the YAP oncogene, which plays a central part in tumor etiology, acts via the TEAD4 transcription factor. As of now, no information is available on regulation of the hPar3 promoter. With regard to hPar4, only data showing CpG methylation promoter regulation is available. Characterization of the PAR transcriptional landscape may identify powerful targets for cancer therapies.

Activating enhancer-binding protein 2γ (AP-2γ) is a member of the developmentally regulated AP-2 transcription factor family that regulates the expression of many downstream genes. Whereas the effects of AP-2α overexpression on cell growth are fairly well established, the cellular effects of AP-2γ overexpression are less well studied. Our new findings show that AP-2γ significantly upregulates p21 mRNA and proteins, inhibits cell growth, decreases clonogenic survival. Cell cycle analysis revealed that forced AP-2γ expression induced G1-phase arrest, decreased DNA synthesis, decreased the fraction of cells in S phase. AP-2γ expression also led to cyclin D1 repression, decreased Rb phosphorylation, decreased E2F activity in breast carcinoma cells. AP-2γ binding to the p21 promoter was observed in vivo, the absence of growth inhibition in response to AP-2γ expression in p21 (-/-) cells demonstrated that p21 caused, at least in part, AP-2-induced cell cycle arrest. Finally, the tumor growth of human breast carcinoma cells in vivo was inhibited by the expression of AP-2γ relative to empty vector-infected cells, suggesting that AP-2γ acts as a tumor suppressor. In summary, expression of either AP-2γ or AP-2α inhibited breast carcinoma cell growth; thus, these genes may be therapeutic targets for breast cancer.

Background: The AP-2 transcription factor APTF-1 is crucially required for developmentally controlled sleep behavior in Caenorhabditis elegans larvae. Its human ortholog, TFAP-2beta, causes Char disease and has also been linked to sleep disorders. These data suggest that AP-2 transcription factors may be highly conserved regulators of various types of sleep behavior. Here, we tested the idea that AP-2 controls adult sleep in Drosophila. Results: Drosophila has one AP-2 ortholog called TfAP-2, which is essential for viability. To investigate its potential role in sleep behavior and neural development, we specifically downregulated TfAP-2 in the nervous system. We found that neuronal TfAP-2 knockdown almost completely abolished night sleep but did not affect day sleep. TfAP-2 insufficiency affected nervous system development. Conditional TfAP-2 knockdown in the adult also produced a modest sleep phenotype, suggesting that TfAP-2 acts both in larval as well as in differentiated neurons. Conclusions: Thus, our results show that AP-2 transcription factors are highly conserved regulators of development and sleep. [ABSTRACT FROM AUTHOR]

Endocytosis is the process by which cells regulate extracellular fluid uptake and internalize molecules bound to their plasma membrane. This process requires the generation of protein-coated vesicles. In clathrin-mediated endocytosis (CME) the assembly polypeptide 2 (AP-2) adaptor facilitates rapid endocytosis of some plasma membrane receptors by mediating clathrin recruitment to the endocytic site and by connecting cargoes to the clathrin coat. While this adaptor is essential for early embryonic development in mammals, initial results suggested that it is dispensable for endocytosis in unicellular eukaryotes. The drastic effect of depleting AP-2 in metazoa and the mild effect of deleting AP-2 subunits in Saccharomyces cerevisiae have prevented a detailed analysis of the dynamics of endocytic patches in the absence of this adaptor. Using live-cell imaging of Schizosaccharomyces pombe endocytic sites we have shown that eliminating AP-2 perturbs the dynamics of endocytic patches beyond the moment of coat assembly. These perturbations affect the cell growth pattern and cell wall synthesis. Our results highlight the importance of using different model organisms to address the study of conserved aspects of CME. [ABSTRACT FROM AUTHOR]

Autophagy is an intracellular degradation pathway conserved in eukaryotes. Among core autophagy-related (Atg) proteins, mammalian Atg9A is the sole multi-spanning transmembrane protein, and both of its N- and C-terminal domains are exposed to the cytoplasm. It is known that Atg9A travels through the trans-Golgi network (TGN) and the endosomal system under nutrient-rich conditions, and transiently localizes to the autophagosome upon autophagy induction. However, the significance of Atg9A trafficking for autophagosome formation remains elusive. Here, we identified sorting motifs in the N-terminal cytosolic stretch of Atg9A that interact with the adaptor protein AP-2. Atg9A with mutations in the sorting motifs could not execute autophagy and was abnormally accumulated at the recycling endosomes. The combination of defects in autophagy and Atg9A accumulation in the recycling endosomes was also found upon the knockdown of TRAPPC8, a specific subunit of the TRAPPIII complex. These results show directly that the trafficking of Atg9A through the recycling endosomes is an essential step for autophagosome formation. [ABSTRACT FROM AUTHOR]

Background: AP-2δ is the most divergent member of the Activating Protein-2 (TFAP2) family of transcription factors. AP-2δ is restricted to specific regions of the CNS, including a subset of ganglion cells in the retina. Retinal ganglion cells (RGCs), the only output neurons of the retina, are responsible for transmitting the visual signal to the brain. Results: AP-2δ knockout results in loss of Brn3c (Pou4f3) expression in AP-2δ -positive RGCs. While AP-2δ-/- mice have morphologically normal retinas at birth, there is a significant reduction in retinal ganglion cell numbers by P21, after eye opening. Chromatin immunoprecipitation indicates that Brn3c is a target of AP-2δ in the retina. Using fluorochrome-conjugated cholera toxin subunit B to trace ganglion cell axons from the eye to the major visual pathways in the brain, we found 87 % and 32 % decreases in ipsilateral and contralateral projections, respectively, to the superior colliculus in AP-2δ-/- mice. In agreement with anatomical data, visually evoked responses recorded from the brain confirmed that retinal outputs to the brain are compromised. Conclusions: AP-2δ is important for the maintenance of ganglion cell numbers in the retina. Loss of AP-2δ alters retinal axonal projections to visual centers of the brain, with ipsilaterial projections to the superior colliculus being the most dramatically affected. Our results have important implications for integration of the visual signal at the superior colliculus. [ABSTRACT FROM AUTHOR]

The Amyloid Precursor Protein (APP) has been extensively studied for its role as the precursor of the β-amyloid protein (Aβ) in Alzheimer's disease (AD). However, our understanding of the normal function of APP is still patchy. Emerging evidence indicates that a dysfunction in APP trafficking and degradation can be responsible for neuronal deficits and progressive degeneration in humans. We recently reported that the Y682 mutation in the 682YENPTY687 domain of APP affects its binding to specific adaptor proteins and leads to its anomalous trafficking, to defects in the autophagy machinery and to neuronal degeneration. In order to identify adaptors that influence APP function, we performed pull-down experiments followed by quantitative mass spectrometry (MS) on hippocampal tissue extracts of three month-old mice incubated with either the 682YENPTY687 peptide, its mutated form, 682GENPTY687 or its phosphorylated form, 682pYENPTY687. Our experiments resulted in the identification of two proteins involved in APP internalization and trafficking: Clathrin heavy chain (hc) and its Adaptor Protein 2 (AP-2). Overall our results consolidate and refine the importance of Y682 in APP normal functions from an animal model of premature aging and dementia. Additionally, they open the perspective to consider Clathrin hc and AP-2 as potential targets for the design and development of new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type 1 (HIV-1) Nef hijacks the clathrin adaptor complex 2 (AP-2) to downregulate the viral receptor CD4 and the antiviral multipass transmembrane proteins SERINC3 and SERINC5, which inhibit the infectivity of progeny virions when incorporated. In Jurkat Tag T lymphoid cells lacking SERINC3 and SERINC5, Nef is no longer required for full progeny virus infectivity and for efficient viral replication. However, in MOLT-3 T lymphoid cells, HIV-1 replication remains highly dependent on Nef even in the absence of SERINC3 and SERINC5. Using a knockout (KO) approach, we now show that the Nef-mediated enhancement of HIV-1 replication in MOLT-3 cells does not depend on the Nef-interacting kinases LCK and PAK2. Furthermore, Nef substantially enhanced HIV-1 replication even in triple-KO MOLT-3 cells that simultaneously lacked the three Nef/AP-2 targets, SERINC3, SERINC5, and CD4, and were reconstituted with a Nef-resistant CD4 to permit HIV-1 entry. Nevertheless, the ability of Nef mutants to promote HIV-1 replication in the triple-KO cells correlated strictly with the ability to bind AP-2. In addition, knockdown and reconstitution experiments confirmed the involvement of AP-2. These observations raise the possibility that MOLT-3 cells express a novel antiviral factor that is downregulated by Nef in an AP-2-dependent manner. IMPORTANCE The HIV-1 Nef protein hijacks a component of the cellular endocytic machinery called AP-2 to downregulate the viral receptor CD4 and the antiviral cellular membrane proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 are taken up into viral particles, which reduces their infectivity. Surprisingly, in a T cell line called MOLT-3, Nef remains crucial for HIV-1 spreading in the absence of SERINC3 and SERINC5. We now show that this effect of Nef also does not depend on the cellular signaling molecules and Nef interaction partners LCK and PAK2. Nef was required for efficient HIV-1 spreading even in triple-knockout cells that completely lacked Nef/AP-2-sensitive CD4, in addition to the Nef/AP-2 targets SERINC3 and SERINC5. Nevertheless, our results indicate that the enhancement of HIV-1 spreading by Nef in the triple-knockout cells remained AP-2 dependent, which suggests the presence of an unknown antiviral factor that is sensitive to Nef/AP-2-mediated downregulation.

Adaptor protein (AP)-1/σ1B mice have reduced synaptic-vesicle (SV) recycling and increased endosomes. Mutant mice have impaired spatial memory, and σ1B-deficient humans have a severe mental retardation. In order to define these σ1B 'bulk' endosomes and to determine their functions in SV recycling, we developed a protocol to separate them from the majority of the neuronal endosomes. The σ1B 'bulk' endosomes proved to be classic early endosomes with an increase in the phospholipid phosphatidylinositol 3-phosphate (PI-3-P), which recruits proteins mediating protein sorting out of early endosomes into different routes. σ1B deficiency induced alterations in the endosomal proteome reveals two major functions: SV protein storage and sorting into endolysosomes. Alternative endosomal recycling pathways are not up-regulated, but certain SV proteins are misrouted. Tetraspanins are enriched in σ1B synaptosomes, but not in their endosomes or in their clathrin-coated-vesicles (CCVs), indicating AP-1/σ1B-dependent sorting. Synapses contain also more AP-2 CCV, although it is expected that they contain less due to reduced SV recycling. Coat composition of these AP-2 CCVs is altered, and thus, they represent a subpopulation of AP-2 CCVs. Association of calmodulin-dependent protein kinase (CaMK)-IIα, −δ and casein kinase (CK)-IIα with the endosome/SV pool is altered, as well as 14-3-3η, indicating changes in specific signalling pathways regulating synaptic plasticity. The accumulation of early endosomes and endocytotic AP-2 CCV indicates the regulation of SV recycling via early endosomes by the interdependent regulation of AP-2-mediated endocytosis and AP-1/σ1B-mediated SV reformation. [ABSTRACT FROM AUTHOR]

Epithelial apicobasal polarity has fundamental roles in epithelial physiology and morphogenesis. The PAR complex, comprising PAR-3, PAR-6 and atypical protein kinase C (aPKC), is involved in determining cell polarity in various biological contexts, including in epithelial cells. However, it is not fully understood how the PAR complex induces apicobasal polarity. In this study, we found that PAR-3 regulates the protein expression of Girdin (also known as GIV or CCDC88A), a guanine-nucleotide-exchange factor (GEF) for heterotrimeric Gai subunits, at the transcriptional level by cooperating with the AP-2 transcription factor. In addition, we confirmed that PAR-3 physically interacts with Girdin, and show that Girdin, together with the Gai3 (also known as GNAI3), controls tight junction formation, apical domain development and actin organization downstream of PAR-3. Taken together, our findings suggest that transcriptional upregulation of Girdin expression and Girdin-Gai3 signaling play crucial roles in regulating epithelial apicobasal polarity through the PAR complex. [ABSTRACT FROM AUTHOR]