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1.
Neurobiol Dis ; 198: 106537, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38772452

ABSTRACT

Hereditary spastic paraplegia (HSP) comprises a large group of neurogenetic disorders characterized by progressive lower extremity spasticity. Neurological evaluation and genetic testing were completed in a Malian family with early-onset HSP. Three children with unaffected consanguineous parents presented with symptoms consistent with childhood-onset complicated HSP. Neurological evaluation found lower limb weakness, spasticity, dysarthria, seizures, and intellectual disability. Brain MRI showed corpus callosum thinning with cortical and spinal cord atrophy, and an EEG detected slow background in the index patient. Whole exome sequencing identified a homozygous missense variant in the adaptor protein (AP) complex 2 alpha-2 subunit (AP2A2) gene. Western blot analysis showed reduced levels of AP2A2 in patient-iPSC derived neuronal cells. Endocytosis of transferrin receptor (TfR) was decreased in patient-derived neurons. In addition, we observed increased axon initial segment length in patient-derived neurons. Xenopus tropicalis tadpoles with ap2a2 knockout showed cerebral edema and progressive seizures. Immunoprecipitation of the mutant human AP-2-appendage alpha-C construct showed defective binding to accessory proteins. We report AP2A2 as a novel genetic entity associated with HSP and provide functional data in patient-derived neuron cells and a frog model. These findings expand our understanding of the mechanism of HSP and improve the genetic diagnosis of this condition.


Subject(s)
Adaptor Protein Complex 2 , Endocytosis , Spastic Paraplegia, Hereditary , Animals , Child , Child, Preschool , Female , Humans , Male , Adaptor Protein Complex 2/genetics , Endocytosis/genetics , Endocytosis/physiology , Mutation/genetics , Mutation, Missense , Neurons/metabolism , Neurons/pathology , Pedigree , Spastic Paraplegia, Hereditary/genetics , Spastic Paraplegia, Hereditary/pathology , Xenopus
2.
G3 (Bethesda) ; 14(7)2024 Jul 08.
Article in English | MEDLINE | ID: mdl-38696649

ABSTRACT

CED-1 (cell death abnormal) is a transmembrane receptor involved in the recognition of "eat-me" signals displayed on the surface of apoptotic cells and thus central for the subsequent engulfment of the cell corpse in Caenorhabditis elegans. The roles of CED-1 in engulfment are well established, as are its downstream effectors. The latter include the adapter protein CED-6/GULP and the ATP-binding cassette family homolog CED-7. However, how CED-1 is maintained on the plasma membrane in the absence of engulfment is currently unknown. Here, we show that CED-6 and CED-7 have a novel role in maintaining CED-1 correctly on the plasma membrane. We propose that the underlying mechanism is via endocytosis as CED-6 and CED-7 act redundantly with clathrin and its adaptor, the Adaptor protein 2 complex, in ensuring correct CED-1 localization. In conclusion, CED-6 and CED-7 impact other cellular processes than engulfment of apoptotic cells.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Cell Membrane , Clathrin , Endocytosis , Animals , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans/genetics , Clathrin/metabolism , Cell Membrane/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , Apoptosis , ATP-Binding Cassette Transporters/metabolism , ATP-Binding Cassette Transporters/genetics , Adaptor Protein Complex 2/metabolism , Protein Transport , Apoptosis Regulatory Proteins
3.
Cell Mol Life Sci ; 81(1): 218, 2024 May 17.
Article in English | MEDLINE | ID: mdl-38758395

ABSTRACT

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2ß and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued-AP-2ß complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued-AP-2ß interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued-AP-2ß interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued-AP-2ß interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued-AP-2ß interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.


Subject(s)
Autophagy , Dynactin Complex , Lysosomes , Animals , Humans , Mice , Adaptor Protein Complex 2/metabolism , Autophagosomes/metabolism , Dynactin Complex/metabolism , Lysosomal-Associated Membrane Protein 1/metabolism , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Neurons/metabolism , Protein Binding , Sirolimus/pharmacology
4.
Sci Adv ; 10(19): eadi9156, 2024 May 10.
Article in English | MEDLINE | ID: mdl-38718108

ABSTRACT

Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.


Subject(s)
Endocytosis , Exosomes , Tetraspanin 30 , Exosomes/metabolism , Humans , Tetraspanin 30/metabolism , Biomarkers/metabolism , Syntenins/metabolism , Syntenins/genetics , Tetraspanin 28/metabolism , Cell Membrane/metabolism , Adaptor Protein Complex 2/metabolism , Tetraspanin 29/metabolism
5.
Acta Otolaryngol ; 144(3): 198-206, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38662892

ABSTRACT

BACKGROUND: 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. OBJECTIVE: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSIONS AND SIGNIFICANCE: 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.


Subject(s)
Hair Cells, Auditory, Inner , Mice, Inbred CBA , Animals , Hair Cells, Auditory, Inner/metabolism , Mice , Adaptor Protein Complex 2/metabolism , Adaptor Protein Complex 2/genetics , Evoked Potentials, Auditory, Brain Stem , Nonmuscle Myosin Type IIB/metabolism , Nonmuscle Myosin Type IIB/genetics , Cochlea/metabolism
6.
Tumour Virus Res ; 17: 200279, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38485055

ABSTRACT

Multiple cellular pathways are affected by HPV E6 and E7 oncoproteins, including endocytic and cellular trafficking. HPV-16 E7 can target the adaptor protein (AP) complex, which contains proteins important during endocytosis transport. To further investigate the role of HPV E7 during this process, we analysed the expression of cell surface proteins in NIKS cells expressing HPV-16 E7. We show that different cell surface proteins are regulated by HPV-16 E7 via interaction with AP2. We observed that the expression of MET and CD109 membrane protein seems to be upregulated in cells expressing E7. Moreover, the interaction of MET and CD109 with AP2 proteins is disrupted by HPV-16 E7. In addition, in the absence of HPV-16 E7, there is a downregulation of the cell membrane expression of MET and CD109 in HPV-positive cell lines. These results expand our knowledge of the functions of E7 and open new potential cellular pathways affected by this oncoprotein.


Subject(s)
Antigens, CD , Human papillomavirus 16 , Papillomavirus E7 Proteins , Proto-Oncogene Proteins c-met , Humans , Papillomavirus E7 Proteins/metabolism , Papillomavirus E7 Proteins/genetics , Antigens, CD/metabolism , Antigens, CD/genetics , Proto-Oncogene Proteins c-met/metabolism , Proto-Oncogene Proteins c-met/genetics , Human papillomavirus 16/metabolism , Human papillomavirus 16/genetics , Adaptor Protein Complex 2/metabolism , Adaptor Protein Complex 2/genetics , Neoplasm Proteins/metabolism , Neoplasm Proteins/genetics , Cell Membrane/metabolism , Papillomavirus Infections/metabolism , Papillomavirus Infections/virology , Endocytosis , GPI-Linked Proteins
8.
J Plant Physiol ; 295: 154189, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38432037

ABSTRACT

Clathrin-mediated endocytosis (CME) is a highly conserved pathway that plays a crucial role in the endocytosis of plasma membrane proteins in eukaryotic cells. The pathway is initiated when the adaptor protein complex 2 (AP2) and TPLATE complex (TPC) work together to recognize cargo proteins and recruit clathrin. This review provides a concise overview of the functions of each subunit of AP2 and TPC, and highlights the involvement of CME in various biological processes, such as pollen development, root development, nutrient transport, extracellular signal transduction, auxin polar transport, hyperosmotic stress, salinity stress, high ammonium stress, and disease resistance. Additionally, the review explores the regulation of CME by phytohormones, clathrin-mediated exocytosis (CMX), and AP2M phosphorylation. It also suggests potential future research directions for CME.


Subject(s)
Biological Phenomena , Endocytosis , Endocytosis/physiology , Clathrin/metabolism , Adaptor Protein Complex 2/metabolism , Plant Development
9.
Sci Rep ; 14(1): 5959, 2024 03 12.
Article in English | MEDLINE | ID: mdl-38472335

ABSTRACT

In recent years, human umbilical cord mesenchymal stem cell (hUC-MSC) extracellular vesicles (EVs) have been used as a cell replacement therapy and have been shown to effectively overcome some of the disadvantages of cell therapy. However, the specific mechanism of action of EVs is still unclear, and there is no appropriate system for characterizing the differences in the molecular active substances of EVs produced by cells in different physiological states. We used a data-independent acquisition (DIA) quantitative proteomics method to identify and quantify the protein composition of two generations EVs from three different donors and analysed the function and possible mechanism of action of the proteins in EVs of hUC-MSCs via bioinformatics. By comparative proteomic analysis, we characterized the different passages EVs. Furthermore, we found that adaptor-related protein complex 2 subunit alpha 1 (AP2A1) and adaptor-related protein complex 2 subunit beta 1 (AP2B1) in hUC-MSC-derived EVs may play a significant role in the treatment of Alzheimer's disease (AD) by regulating the synaptic vesicle cycle signalling pathway. Our work provides a direction for batch-to-batch quality control of hUC-MSC-derived EVs and their application in AD treatment.


Subject(s)
Alzheimer Disease , Extracellular Vesicles , Mesenchymal Stem Cells , Humans , Alzheimer Disease/metabolism , Proteomics , Adaptor Protein Complex 2/metabolism , Extracellular Vesicles/metabolism
10.
Mol Psychiatry ; 28(11): 4766-4776, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37679472

ABSTRACT

Alcohol use disorder (AUD) is a life-threatening disease characterized by compulsive drinking, cognitive deficits, and social impairment that continue despite negative consequences. The inability of individuals with AUD to regulate drinking may involve functional deficits in cortical areas that normally balance actions that have aspects of both reward and risk. Among these, the orbitofrontal cortex (OFC) is critically involved in goal-directed behavior and is thought to maintain a representation of reward value that guides decision making. In the present study, we analyzed post-mortem OFC brain samples collected from age- and sex-matched control subjects and those with AUD using proteomics, bioinformatics, machine learning, and reverse genetics approaches. Of the 4,500+ total unique proteins identified in the proteomics screen, there were 47 proteins that differed significantly by sex that were enriched in processes regulating extracellular matrix and axonal structure. Gene ontology enrichment analysis revealed that proteins differentially expressed in AUD cases were involved in synaptic and mitochondrial function, as well as transmembrane transporter activity. Alcohol-sensitive OFC proteins also mapped to abnormal social behaviors and social interactions. Machine learning analysis of the post-mortem OFC proteome revealed dysregulation of presynaptic (e.g., AP2A1) and mitochondrial proteins that predicted the occurrence and severity of AUD. Using a reverse genetics approach to validate a target protein, we found that prefrontal Ap2a1 expression significantly correlated with voluntary alcohol drinking in male and female genetically diverse mouse strains. Moreover, recombinant inbred strains that inherited the C57BL/6J allele at the Ap2a1 interval consumed higher amounts of alcohol than those that inherited the DBA/2J allele. Together, these findings highlight the impact of excessive alcohol consumption on the human OFC proteome and identify important cross-species cortical mechanisms and proteins that control drinking in individuals with AUD.


Subject(s)
Alcoholism , Humans , Male , Female , Mice , Animals , Alcoholism/metabolism , Adaptor Protein Complex 2/metabolism , Proteome/metabolism , Mice, Inbred C57BL , Mice, Inbred DBA , Prefrontal Cortex/metabolism , Alcohol Drinking/genetics , Ethanol/metabolism
11.
Plant Cell ; 35(9): 3504-3521, 2023 09 01.
Article in English | MEDLINE | ID: mdl-37440281

ABSTRACT

ADAPTOR-ASSOCIATED PROTEIN KINASE1 (AAK1) is a known regulator of clathrin-mediated endocytosis in mammals. Human AAK1 phosphorylates the µ2 subunit of the ADAPTOR PROTEIN-2 (AP-2) complex (AP2M) and plays important roles in cell differentiation and development. Previous interactome studies discovered the association of AAK1 with AP-2 in Arabidopsis (Arabidopsis thaliana), but its function was unclear. Here, genetic analysis revealed that the Arabidopsis aak1 and ap2m mutants both displayed altered root tropic growth, including impaired touch- and gravity-sensing responses. In Arabidopsis, AAK1-phosphorylated AP2M on Thr-163, and expression of the phospho-null version of AP2M in the ap2m mutant led to an aak1-like phenotype, whereas the phospho-mimic forms of AP2M rescued the aak1 mutant. In addition, we found that the AAK1-dependent phosphorylation state of AP2M modulates the frequency distribution of endocytosis. Our data indicate that the phosphorylation of AP2M on Thr-163 by AAK1 fine-tunes endocytosis in the Arabidopsis root to control its tropic growth.


Subject(s)
Adaptor Protein Complex mu Subunits , Arabidopsis , Plant Roots , Animals , Humans , Adaptor Protein Complex 2/genetics , Adaptor Protein Complex 2/metabolism , Adaptor Protein Complex mu Subunits/metabolism , Arabidopsis/metabolism , Clathrin/metabolism , Endocytosis/genetics , Mammals/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Plant Roots/genetics , Plant Roots/metabolism
12.
Proc Natl Acad Sci U S A ; 120(2): e2205199120, 2023 01 10.
Article in English | MEDLINE | ID: mdl-36598941

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Vesicular Transport , Keratoderma, Palmoplantar , Humans , Adaptor Proteins, Vesicular Transport/metabolism , Carrier Proteins/genetics , Keratoderma, Palmoplantar/genetics , Keratoderma, Palmoplantar/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Clathrin/metabolism , Adaptor Protein Complex 2/genetics , Adaptor Protein Complex 2/metabolism
13.
Microbiol Spectr ; 11(1): e0386122, 2023 02 14.
Article in English | MEDLINE | ID: mdl-36533963

ABSTRACT

Protein palmitoylation, one of posttranslational modifications, is catalyzed by a group of palmitoyl transferases (PATs) and plays critical roles in the regulation of protein functions. However, little is known about the function and mechanism of PATs in plant pathogenic fungi. The present study reports the function and molecular mechanism of FonPATs in Fusarium oxysporum f. sp. niveum (Fon), the causal agent of watermelon Fusarium wilt. The Fon genome contains six FonPAT genes with distinct functions in vegetative growth, conidiation and conidial morphology, and stress response. FonPAT1, FonPAT2, and FonPAT4 have PAT activity and are required for Fon virulence on watermelon mainly through regulating in planta fungal growth within host plants. Comparative proteomics analysis identified a set of proteins that were palmitoylated by FonPAT2, and some of them are previously reported pathogenicity-related proteins in fungi. The FonAP-2 complex core subunits FonAP-2α, FonAP-2ß, and FonAP-2µ were palmitoylated by FonPAT2 in vivo. FonPAT2-catalyzed palmitoylation contributed to the stability and interaction ability of the core subunits to ensure the formation of the FonAP-2 complex, which is essential for vegetative growth, asexual reproduction, cell wall integrity, and virulence in Fon. These findings demonstrate that FonPAT1, FonPAT2, and FonPAT4 play important roles in Fon virulence and that FonPAT2-catalyzed palmitoylation of the FonAP-2 complex is critical to Fon virulence, providing novel insights into the importance of protein palmitoylation in the virulence of plant fungal pathogens. IMPORTANCE Fusarium oxysporum f. sp. niveum (Fon), the causal agent of watermelon Fusarium wilt, is one of the most serious threats for the sustainable development of the watermelon industry worldwide. However, little is known about the underlying molecular mechanism of pathogenicity in Fon. Here, we found that the palmitoyl transferase (FonPAT) genes play distinct and diverse roles in basic biological processes and stress response and demonstrated that FonPAT1, FonPAT2, and FonPAT4 have PAT activity and are required for virulence in Fon. We also found that FonPAT2 palmitoylates the core subunits of the FonAP-2 complex to maintain the stability and the formation of the FonAP-2 complex, which is essential for basic biological processes, stress response, and virulence in Fon. Our study provides new insights into the understanding of the molecular mechanism involved in Fon virulence and will be helpful in the development of novel strategies for disease management.


Subject(s)
Citrullus , Fusarium , Lipoylation , Stress, Physiological , Catalysis , Citrullus/microbiology , Fusarium/metabolism , Fusarium/physiology , Lipoylation/physiology , Plant Diseases/microbiology , Virulence , Adaptor Protein Complex 2/chemistry , Adaptor Protein Complex 2/metabolism , Stress, Physiological/physiology
14.
Traffic ; 24(1): 20-33, 2023 01.
Article in English | MEDLINE | ID: mdl-36412210

ABSTRACT

AP2S1 is the sigma 2 subunit of adaptor protein 2 (AP2) that is essential for endocytosis. In this study, we investigated the potential role of AP2S1 in intracellular processing of amyloid precursor protein (APP), which contributes to the pathogenesis of Alzheimer disease (AD) by generating the toxic ß-amyloid peptide (Aß). We found that knockdown or overexpression of AP2S1 decreased or increased the protein levels of APP and Aß in cells stably expressing human full-length APP695, respectively. This effect was unrelated to endocytosis but involved lysosomal degradation. Morphological studies revealed that silencing of AP2S1 promoted the translocalization of APP from RAB9-positive late endosomes (LE) to LAMP1-positive lysosomes, which was paralleled by the enhanced LE-lysosome fusion. In support, silencing of vacuolar protein sorting-associated protein 41 (VPS41) that is implicated in LE-lyso fusion prevented AP2S1-mediated regulation of APP degradation and translocalization. In APP/PS1 mice, an animal model of AD, AAV-mediated delivery of AP2S1 shRNA in the hippocampus significantly reduced the protein levels of APP and Aß, with the concomitant APP translocalization, LE-lyso fusion and the improved cognitive functions. Taken together, these data uncover a LE-lyso fusion mechanism in APP degradation and suggest a novel role for AP2S1 in the pathophysiology of AD.


Subject(s)
Adaptor Protein Complex sigma Subunits , Alzheimer Disease , Mice , Humans , Animals , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Amyloid beta-Peptides/metabolism , Alzheimer Disease/metabolism , Endosomes/metabolism , Lysosomes/metabolism , Amyloid Precursor Protein Secretases/metabolism , Adaptor Protein Complex 2/metabolism , Adaptor Protein Complex sigma Subunits/metabolism , rab GTP-Binding Proteins/metabolism
15.
J Cell Biol ; 221(12)2022 12 05.
Article in English | MEDLINE | ID: mdl-36374222

ABSTRACT

MR1 is a conserved molecule that binds microbial vitamin B metabolites and presents them to unconventional T cells. Lim and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202110125) uncover the role of AP2 in ensuring MR1 surface presentation, which relies on an atypical motif within the MR1 cytoplasmic tail.


Subject(s)
Adaptor Protein Complex 2 , Antigen Presentation , Histocompatibility Antigens Class I , Minor Histocompatibility Antigens , Histocompatibility Antigens Class I/metabolism , Minor Histocompatibility Antigens/metabolism , T-Lymphocytes , Humans , Vitamin B Complex/metabolism , Adaptor Protein Complex 2/metabolism
16.
J Cell Biol ; 221(12)2022 12 05.
Article in English | MEDLINE | ID: mdl-36129434

ABSTRACT

MR1 is a highly conserved microbial immune-detection system in mammals. It captures vitamin B-related metabolite antigens from diverse microbes and presents them at the cell surface to stimulate MR1-restricted lymphocytes including mucosal-associated invariant T (MAIT) cells. MR1 presentation and MAIT cell recognition mediate homeostasis through host defense and tissue repair. The cellular mechanisms regulating MR1 cell surface expression are critical to its function and MAIT cell recognition, yet they are poorly defined. Here, we report that human MR1 is equipped with a tyrosine-based motif in its cytoplasmic domain that mediates low affinity binding with the endocytic adaptor protein 2 (AP2) complex. This interaction controls the kinetics of MR1 internalization from the cell surface and minimizes recycling. We propose MR1 uses AP2 endocytosis to define the duration of antigen presentation to MAIT cells and the detection of a microbial metabolic signature by the immune system.


Subject(s)
Antigen Presentation , Endocytosis , Histocompatibility Antigens Class I , Minor Histocompatibility Antigens , Mucosal-Associated Invariant T Cells , Adaptor Protein Complex 2/genetics , Adaptor Protein Complex 2/metabolism , Histocompatibility Antigens Class I/genetics , Humans , Lymphocyte Activation , Minor Histocompatibility Antigens/genetics , Minor Histocompatibility Antigens/metabolism , Mucosal-Associated Invariant T Cells/metabolism , Tyrosine , Vitamins
17.
Mol Biol Cell ; 33(12): ar109, 2022 10 01.
Article in English | MEDLINE | ID: mdl-35976721

ABSTRACT

Adaptor protein 4 (AP-4) is a heterotetrameric complex composed of ε, ß4, µ4, and σ4 subunits that mediates export of a subset of transmembrane cargos, including autophagy protein 9A (ATG9A), from the trans-Golgi network (TGN). AP-4 has received particular attention in recent years because mutations in any of its subunits cause a complicated form of hereditary spastic paraplegia referred to as "AP-4-deficiency syndrome." The identification of proteins that interact with AP-4 has shed light on the mechanisms of AP-4-dependent cargo sorting and distribution within the cell. However, the mechanisms by which the AP-4 complex itself is assembled have remained unknown. Here, we report that the alpha- and gamma-adaptin-binding protein (AAGAB, also known as p34) binds to and stabilizes the AP-4 ε and σ4 subunits, thus promoting complex assembly. The physiological importance of these interactions is underscored by the observation that AAGAB-knockout cells exhibit reduced levels of AP-4 subunits and accumulation of ATG9A at the TGN like those in cells with mutations in AP-4-subunit genes. These findings demonstrate that AP-4 assembly is not spontaneous but AAGAB-assisted, further contributing to the understanding of an adaptor protein complex that is critically involved in development of the central nervous system.


Subject(s)
Adaptor Protein Complex Subunits , Membrane Proteins , Adaptor Protein Complex 1/metabolism , Adaptor Protein Complex 2/metabolism , Adaptor Protein Complex Subunits/metabolism , Adaptor Protein Complex gamma Subunits/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Membrane Proteins/metabolism , Protein Transport , trans-Golgi Network/metabolism
18.
Methods Mol Biol ; 2473: 195-212, 2022.
Article in English | MEDLINE | ID: mdl-35819768

ABSTRACT

Clathrin-coated vesicles mediate membrane cargo transportation from the plasma membrane, the trans-Golgi network, the endosome, and the lysosome. Heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) are bridges that link cargo-loaded membranes to clathrin coats. Assembly of AP2 was previously considered to be spontaneous; however, a recent study found AP2 assembly is a highly orchestrated process controlled by alpha and gamma adaptin binding protein (AAGAB). Evidence shows that AAGAB controls AP1 assembly in a similar way. Insights into the orchestrated assembly process and three-dimensional structures of assembly intermediates are only emerging. Here, we describe a protocol for reconstitution and purification of the complexes containing AAGAB and AP1 or AP2 subunits, known as AP1 and AP2 hemicomplexes. Our purification routinely yields milligrams of pure complexes suitable for structural analysis by X-ray crystallography and electron microscopy.


Subject(s)
Adaptor Protein Complex 2 , Adaptor Proteins, Vesicular Transport , Adaptor Protein Complex 1/metabolism , Adaptor Protein Complex 2/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Clathrin/metabolism , Clathrin-Coated Vesicles/metabolism
19.
Mol Cell Biol ; 42(7): e0007122, 2022 07 21.
Article in English | MEDLINE | ID: mdl-35727026

ABSTRACT

Host cell membrane-trafficking pathways are often manipulated by bacterial pathogens to gain cell entry, avoid immune responses, or to obtain nutrients. The 1,369-residue OtDUB protein from the obligate intracellular human pathogen Orientia tsutsugamushi bears a deubiquitylase (DUB) and additional domains. Here we show that OtDUB ectopic expression disrupts membrane trafficking through multiple mechanisms. OtDUB binds directly to the clathrin adaptor-protein (AP) complexes AP-1 and AP-2, and the OtDUB275-675 fragment is sufficient for binding to either complex. To assess the impact of OtDUB interactions with AP-1 and AP-2, we examined trans-Golgi trafficking and endocytosis, respectively. Endocytosis is reduced by two separate OtDUB fragments: one contains the AP-binding domain (OtDUB1-675), and the other does not (OtDUB675-1369). OtDUB1-675 disruption of endocytosis requires its ubiquitin-binding capabilities. OtDUB675-1369 also fragments trans- and cis-Golgi structures. Using a growth-based selection in yeast, we identified viable OtDUB675-1369 point mutants that also no longer caused Golgi defects in human cells. In parallel, we found OtDUB675-1369 binds directly to phosphatidylserine, and this lipid binding is lost in the same mutants. Together these results show that OtDUB contains multiple activities capable of modulating membrane trafficking. We discuss how these activities may contribute to Orientia infections.


Subject(s)
Orientia tsutsugamushi , Adaptor Protein Complex 1/metabolism , Adaptor Protein Complex 2/metabolism , Endocytosis , Golgi Apparatus/metabolism , Host-Pathogen Interactions , Humans , Orientia tsutsugamushi/metabolism , Protein Binding , Scrub Typhus/metabolism , Scrub Typhus/microbiology , Scrub Typhus/pathology
20.
J Mol Cell Biol ; 14(5)2022 09 19.
Article in English | MEDLINE | ID: mdl-35704676

ABSTRACT

High-altitude cerebral edema (HACE) is a potentially fatal encephalopathy associated with a time-dependent exposure to the hypobaric hypoxia of altitude. The formation of HACE is affected by both vasogenic and cytotoxic edema. The over-activated microglia potentiate the damage of blood-brain barrier (BBB) and exacerbate cytotoxic edema. In light with the activation of microglia in HACE, we aimed to investigate whether the over-activated microglia were the key turning point of acute mountain sickness to HACE. In in vivo experiments, by exposing mice to hypobaric hypoxia (7000 m above sea level) to induce HACE model, we found that microglia were activated and migrated to blood vessels. Microglia depletion by PLX5622 obviously relieved brain edema. In in vitro experiments, we found that hypoxia induced cultured microglial activation, leading to the destruction of endothelial tight junction and astrocyte swelling. Up-regulated nuclear respiratory factor 1 (NRF1) accelerated pro-inflammatory factors through transcriptional regulation on nuclear factor kappa B p65 (NF-κB p65) and mitochondrial transcription factor A (TFAM) in activated microglia under hypoxia. NRF1 also up-regulated phagocytosis by transcriptional regulation on caveolin-1 (CAV-1) and adaptor-related protein complex 2 subunit beta (AP2B1). The present study reveals a new mechanism in HACE: hypoxia over-activates microglia through up-regulation of NRF1, which both induces inflammatory response through transcriptionally activating NF-κB p65 and TFAM, and enhances phagocytic function through up-regulation of CAV-1 and AP2B1; hypoxia-activated microglia destroy the integrity of BBB and release pro-inflammatory factors that eventually induce HACE.


Subject(s)
Altitude Sickness , Brain Edema , Adaptor Protein Complex 2/metabolism , Altitude , Altitude Sickness/complications , Animals , Brain Edema/complications , Brain Edema/metabolism , Caveolin 1/metabolism , Hypoxia/complications , Mice , Microglia/metabolism , NF-kappa B/metabolism , Nuclear Respiratory Factor 1/metabolism
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