Regulated secretion of mutant p53 negatively affects T lymphocytes in the tumor microenvironment.

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

AP1/2ß-mediated exocytosis of tapetum-specific transporters is required for pollen development in Arabidopsis thaliana.

AP-1 and AP-2 adaptor protein (AP) complexes mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 is required for trafficking to plasma membrane and vacuoles, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (ß1 and γ for AP-1 and ß2 and α for AP-2), a medium subunit µ, and a small subunit σ. In general, adaptins are unique to each AP complex, with the exception of ß subunits that are shared by AP-1 and AP-2 in some invertebrates. Here, we show that the two putative Arabidopsis thaliana AP1/2ß adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization at the TGN and plasma membrane. Deletion of both ß adaptins is lethal in plants. We identified a critical role of ß adaptins in pollen wall formation and reproduction, involving the regulation of membrane trafficking in the tapetum and pollen germination. In tapetal cells, ß adaptins localize almost exclusively to the TGN and mediate exocytosis of the plasma membrane transporters such as ATP-binding cassette (ABC)G9 and ABCG16. This study highlights the essential role of AP1/2ß adaptins in plants and their specialized roles in specific cell types.

Germline variants in UNC13D and AP3B1 are enriched in COVID-19 patients experiencing severe cytokine storms.

Critically ill coronavirus disease 2019 (COVID-19) is characterized by severe cytokine storms, a hyperinflammatory condition intimately related to the development of fatal outcomes. Why some individuals seem particularly vulnerable to severe cytokine storms is still unknown. Primary immunodeficiency (PID)-related genes are inherited factors that dysregulate host inflammatory responses to infection, especially hemophagocytic lymphohistiocytosis (HLH)-related genes, established as contributors to the development of excessive cytokine storms. We analyzed the association between PID gene variants with severe cytokine storms in COVID-19. We conducted whole-exome sequencing in 233 hospitalized COVID-19 patients and identified four PID gene (UNC13D, AP3B1, RNF168, DHX58) variants were significantly enriched in COVID-19 patients experiencing severe cytokine storms. The total percentage of COVID-19 patients with variants in UNC13D or AP3B1, two typical HLH genes, was dramatically higher in high-level cytokine group than in low-level group (33.3 vs. 5.7%, P < 0.001). Germline variants in UNC13D and AP3B1 were associated with the development of severe cytokine storms, fatal outcomes in COVID-19. These findings advance the understanding of individual susceptibility to severe cytokine storms and help optimize the current management of COVID-19.

Novel function for AP-1B during cell migration.

The epithelial cell-specific clathrin adaptor protein (AP)-1B has a well-established role in polarized sorting of cargos to the basolateral membrane. Here we show that ß1 integrin was dependent on AP-1B and its coadaptor, autosomal recessive hypercholesterolemia protein (ARH), for sorting to the basolateral membrane. We further demonstrate an unprecedented role for AP-1B at the basal plasma membrane during collective cell migration of epithelial sheets. During wound healing, expression of AP-1B (and ARH in AP-1B-positive cells) slowed epithelial-cell migration. We show that AP-1B colocalized with ß1 integrin in focal adhesions during cell migration using confocal microscopy and total internal reflection fluorescence microscopy on fixed specimens. Further, AP-1B labeling in cell protrusions was distinct from labeling for the endocytic adaptor complex AP-2. Using stochastic optical reconstruction microscopy we identified numerous AP-1B-coated structures at or close to the basal plasma membrane in cell protrusions. In addition, immunoelectron microscopy showed AP-1B in coated pits and vesicles at the plasma membrane during cell migration. Lastly, quantitative real-time reverse transcription PCR analysis of human epithelial-derived cell lines revealed a loss of AP-1B expression in highly migratory metastatic cancer cells suggesting that AP-1B's novel role at the basal plasma membrane during cell migration might be an anticancer mechanism.

The interactome of the prostate-specific protein Anoctamin 7.

BACKGROUND: Elevated Anoctamin 7 (ANO7) expression is associated with poor survival in prostate cancer patients. OBJECTIVE: The aim was to discover proteins that interact with ANO7 to understand its functions and regulatory mechanisms. METHODS: The proximity-dependent biotin identification (BioID) method was utilized. ANO7 fused to biotin ligase was transiently transfected into LNCaP cells, and the biotinylated proteins were collected and analysed by mass spectrometry. Four identified proteins were stained with dual fluorescent immunostaining and visualized using Stimulated emission depletion microscopy (STED). RESULTS: After bioinformatic filtering steps, 64 potentially ANO7-interacting proteins were identified and analysed with the GO enrichment analysis tool. One of the most prominently enriched cellular components was cellular vesicle. Co-localization was showed for staphylococcal nuclease and tudor domain containing 1 (SND1), heat shock protein family A (Hsp70) member 1A (HSPA1A), adaptor related protein complex 2 subunit beta 1 (AP2B1) and coatomer protein complex subunit gamma 2 (COPG2). CONCLUSIONS: This is the first study in which ANO7 interacting proteins have been identified. Although further studies are needed, the findings reported here expand our understanding of the role and regulation of ANO7 in prostate cancer cells. Furthermore, these results are likely to introduce new targets for the novel cancer therapies.

Adaptor protein complex 4 deficiency: a paradigm of childhood-onset hereditary spastic paraplegia caused by defective protein trafficking.

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3-5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.

The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry.

Influenza A virus (IAV) coopts numerous host factors to complete its replication cycle. Here, we identify free fatty acid receptor 2 (FFAR2) as a cofactor for IAV entry into host cells. We found that downregulation of FFAR2 or Ffar2 expression significantly reduced the replication of IAV in A549 or RAW 264.7 cells. The treatment of A549 cells with small interfering RNA (siRNA) targeting FFAR2 or the FFAR2 pathway agonists 2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide (4-CMTB) and compound 58 (Cmp58) [(S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide] dramatically inhibited the nuclear accumulation of viral nucleoprotein (NP) at early time points postinfection, indicating that FFAR2 functions in the early stage of the IAV replication cycle. FFAR2 downregulation had no effect on the expression of sialic acid (SA) receptors on the cell membrane, the attachment of IAV to the SA receptors, or the activity of the viral ribonucleoprotein (vRNP) complex. Rather, the amount of internalized IAVs was significantly reduced in FFAR2-knocked-down or 4-CMTB- or Cmp58-treated A549 cells. Further studies showed that FFAR2 associated with ß-arrestin1 and that ß-arrestin1 interacted with the ß2-subunit of the AP-2 complex (AP2B1), the essential adaptor of the clathrin-mediated endocytosis pathway. Notably, siRNA knockdown of either ß-arrestin1 or AP2B1 dramatically impaired IAV replication, and AP2B1 knockdown or treatment with Barbadin, an inhibitor targeting the ß-arrestin1/AP2B1 complex, remarkably decreased the amount of internalized IAVs. Moreover, we found that FFAR2 interacted with three G protein-coupled receptor (GPCR) kinases (i.e., GRK2, GRK5, and GRK6) whose downregulation inhibited IAV replication. Together, our findings demonstrate that the FFAR2 signaling cascade is important for the efficient endocytosis of IAV into host cells.IMPORTANCE To complete its replication cycle, IAV hijacks the host endocytosis machinery to invade cells. However, the underlying mechanisms of how IAV is internalized into host cells remain poorly understood, emphasizing the need to elucidate the role of host factors in IAV entry into cells. In this study, we identified FFAR2 as an important host factor for the efficient replication of both low-pathogenic and highly pathogenic IAV. We revealed that FFAR2 facilitates the internalization of IAV into target cells during the early stage of infection. Upon further characterization of the role of FFAR2-associated proteins in virus replication, we found that the FFAR2-ß-arrestin1-AP2B1 signaling cascade is important for the efficient endocytosis of IAV. Our findings thus further our understanding of the biological details of IAV entry into host cells and establish FFAR2 as a potential target for antiviral drug development.

Circular RNA NF1-419 enhances autophagy to ameliorate senile dementia by binding Dynamin-1 and Adaptor protein 2 B1 in AD-like mice.

Recent studies have demonstrated circular RNAs (circRNAs) to be widely expressed and to have important physiological functions. However, the expression, regulation, and function of circRNAs in neuroglial cells are unknown. Herein, we characterized the expression, regulation, and function of circRNAs in astrocytes. Astrocyte circRNAs were identified by computational analysis of newborn SD rat primary astrocytes cultured with 20 g/L D-galactose. In this manner, 7376 circRNAs were identified, among which most circRNAs (5754) were derived from annot_exons, whereas 27 were antisense, 853 were exon/intron, 329 were intergenic, 41 were intronic, and 372 were one exon. Among these, circNF1-419 was demonstrated to regulate autophagy, in over-expressing circNF1-419 transfected astrocytes, through the PI3K-I/Akt-AMPK-mTOR and PI3K-I/Akt-mTOR signaling pathways. An adenovirus associated virus packaging system (virus titer 1 ×1012), over-expressing circNF1-419 and injected into mouse cerebral cortex, showed autophagy enhancing activity by binding the proteins Dynamin-1 and Adaptor protein 2 B1 (AP2B1). This binding regulated aging markers (p21, p35/25, and p16) and inflammatory factors (TNF-α and NF-κB), and reduced the expression of Alzheimer's disease marker proteins (Tau, p-Tau, Aß1-42, and APOE), which delayed senile dementia. Transcriptome analysis of the brain showed that circNF1-419 improved other signaling pathways, especially those related to the synapses of SAMP8 mice. These findings provide novel insights into circNF1-419 and its potential usefulness for the diagnosis and treatment of dementia by regulating Dynamin-1 and AP2B1 mediated autophagy.

Autoimmune gait disturbance accompanying adaptor protein-3B2-IgG.

OBJECTIVE: To describe phenotypes, treatment response, and outcomes of autoimmunity targeting a synaptic vesicle coat protein, the neuronal (B2) form of adaptor protein-3 (AP3). METHODS: Archived serum and CSF specimens (from 616,025 screened) harboring unclassified synaptic antibodies mimicking amphiphysin-immunoglobulin G (IgG) on tissue-based indirect immunofluorescence assay (IFA) were re-evaluated for novel IgG staining patterns. Autoantigens were identified by western blot and mass spectrometry. Recombinant western blot and cell-binding assay (CBA) were used to confirm antigen specificity. Clinical data were obtained retrospectively. RESULTS: Serum (10) and CSF (6) specimens of 10 patients produced identical IFA staining patterns throughout mouse nervous system tissues, most prominently in cerebellum (Purkinje neuronal perikarya, granular layer synapses, and dentate regions), spinal cord gray matter, dorsal root ganglia, and sympathetic ganglia. The antigen revealed by mass spectrometry analysis and confirmed by recombinant assays (western blot and CBA) was AP3B2 in all. Of 10 seropositive patients, 6 were women; median symptom onset age was 42 years (range 24-58). Clinical information was available for 9 patients, all with subacute onset and rapidly progressive gait ataxia. Neurologic manifestations were myeloneuropathy (3), peripheral sensory neuropathy (2), cerebellar ataxia (2), and spinocerebellar ataxia (2). Five patients received immunotherapy; none improved, but they did not worsen over the follow-up period (median 36 months; range 3-94). Two patients (both with cancer) died. One of 50 control sera was positive by western blot only (but not by IFA or CBA). CONCLUSION: AP3B2 (previously named ß-neuronal adaptin-like protein) autoimmunity appears rare, is accompanied by ataxia (sensory or cerebellar), and is potentially treatable.

PTENα regulates endocytosis and modulates olfactory function.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) α is the first identified isoform of the well-known tumor suppressor PTEN. PTENα has an evolutionarily conserved 173-aa N terminus compared with canonical PTEN. Recently, PTENα has been shown to play roles in multiple biologic processes including learning and memory, cardiac homeostasis, and antiviral immunity. Here, we report that PTENα maintains mitral cells in olfactory bulb (OB), regulates endocytosis in OB neurons, and controls olfactory behaviors in mice. We show that PTENα directly dephosphorylates the endocytic protein amphiphysin and promotes its binding to adaptor-related protein complex 2 subunit ß1 (Ap2b1). In addition, we identified mutations in the N terminus of PTENα in patients with Parkinson disease and Lewy-body dementia, which are neurodegenerative disorders with early olfactory loss. Overexpression of PTENα mutant H169N in mice OB reduces odor sensitivity. Our data demonstrate a role of PTENα in olfactory function and provide insight into the mechanism of olfactory dysfunction in neurologic disorders.-Yuan, Y., Zhao, X., Wang, P., Mei, F., Zhou, J., Jin, Y., McNutt, M. A., Yin, Y. PTENα regulates endocytosis and modulates olfactory function.

Different functions of biogenesis of lysosomal organelles complex 3 subunit 1 (Hps1) and adaptor-related protein complex 3, beta 1 subunit (Ap3b1) genes on spermatogenesis and male fertility.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder in humans and mice. Pale ear (ep) and pearl (pe) mice, bearing mutations in the biogenesis of lysosomal organelles complex 3 subunit 1 (Hps1) and adaptor-related protein complex 3, beta 1 subunit (Ap3b1) genes respectively, are mouse models of human HPS Type 1 (HPS1) and Type 2 (HPS2) respectively. In the present study we investigated and compared the reduced fertilities of ep and pe male mice. Both ep and pe males exhibited lower abilities to impregnate C57BL/6J (B6) females, and B6 females mated with ep males produced smaller litters than those mated with pe males. Delayed testis development, reduced sperm count and lower testosterone concentrations were observed in the pe but not ep male mice. However, the reduction in sperm motility was greater in ep than pe males, likely due to the mitochondrial and fibrous sheath abnormalities observed by electron microscopy in the sperm tails of ep males. Together, the results indicate that the Hps1 and Ap3b1 genes play distinct roles in male reproductive system development and spermatogenesis in mice, even though ep and pe males share common phenotypes, including reduced lysosomes in Sertoli cells and dislocated Zn2+ in sperm heads.

Defective AP-3-dependent VAMP8 trafficking impairs Weibel-Palade body exocytosis in Hermansky-Pudlak Syndrome type 2 blood outgrowth endothelial cells.

Weibel-Palade bodies are endothelial secretory organelles that contain von Willebrand factor, P-selectin and CD63. Release of von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the adaptor protein complex 3 complex. Mutations in the AP3B1 gene, which encodes the adaptor protein complex 3 ß1 subunit, result in Hermansky-Pudlak syndrome 2, a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study, we investigated the role of adaptor protein complex 3-dependent mechanisms in trafficking of proteins during Weibel-Palade body maturation in endothelial cells. An ex vivo patient-derived endothelial model of Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in AP3B1 Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered AP3B1-/- endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking. Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2+-mediated and cAMP-mediated exocytosis. Whole proteome analysis revealed that, apart from adaptor protein complex 3 ß1, also the µ1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8-/-endothelial cells. Our data show that defects in adaptor protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8.

AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis.

Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1), from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here ß1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells. Incubating these peptides with human MNT-1 cells or 3D-reconstructed pigmented epidermis decreases pigmentation by impacting the maturation of melanosomes in fully pigmented organelles. This study highlights that peptides targeting the intracellular trafficking of melanocytes are candidate molecules to tune pigmentation in health and disease.

A Novel ß-adaptin/c-Myc Complex Formation Modulated by Oxidative Stress in the Control of the Cell Cycle in Macrophages and its Implication in Atherogenesis.

Our study tested the proposal that c-Myc activation in macrophages is differentially carried out dependent on the intracellular oxidative state of cells and potentially associated to the process of atherogenesis. Under our experimental conditions, the generation of reactive oxygen species carried out by the presence of oxidized low density lipoproteins (oxLDL) or Gram negative bacterial lipopolysaccharides (LPS) modifies the expression of cellular adhesion molecules such as c-Abl, calcium transport proteins such as the plasma membrane Ca2+-ATPase (PMCA), CD47, procaspase-7, CASP7, CHOP, transcriptional activators such as c-Jun and c-Myc and molecules that participate in the process of endocytosis like α- and ß-adaptin. We present the first evidence showing that a state of oxidative stress alters c-Myc-dependent activity pathways in macrophages through binding to molecules such as ß-adaptin promoting the reversible formation of a complex that presents the ability to regulate the development of the cell cycle. We propose that the subtle regulation carried out through the formation of this c-Myc/ß-adaptin complex when cells change from a normal physiological condition to a state of oxidative stress, represents a defense mechanism against the deleterious effects caused by the loss of cell homeostasis.

The Zic family homologue Odd-paired regulates Alk expression in Drosophila.

The Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) plays a critical role in the specification of founder cells (FCs) in the Drosophila visceral mesoderm (VM) during embryogenesis. Reporter gene and CRISPR/Cas9 deletion analysis reveals enhancer regions in and upstream of the Alk locus that influence tissue-specific expression in the amnioserosa (AS), the VM and the epidermis. By performing high throughput yeast one-hybrid screens (Y1H) with a library of Drosophila transcription factors (TFs) we identify Odd-paired (Opa), the Drosophila homologue of the vertebrate Zic family of TFs, as a novel regulator of embryonic Alk expression. Further characterization identifies evolutionarily conserved Opa-binding cis-regulatory motifs in one of the Alk associated enhancer elements. Employing Alk reporter lines as well as CRISPR/Cas9-mediated removal of regulatory elements in the Alk locus, we show modulation of Alk expression by Opa in the embryonic AS, epidermis and VM. In addition, we identify enhancer elements that integrate input from additional TFs, such as Binou (Bin) and Bagpipe (Bap), to regulate VM expression of Alk in a combinatorial manner. Taken together, our data show that the Opa zinc finger TF is a novel regulator of embryonic Alk expression.

A new inhibitor of the ß-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, ß-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of ß-arrestin recruitment to the receptor and ß-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between ß-arrestin and the ß2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/ß-arrestin complexes. This selective ß-arrestin/ß2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical ß2-adrenergic (ß2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect ß-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and ß2AR, supporting the concept of ß-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

Several adaptor proteins promote intracellular localisation of the transporter MRP4/ABCC4 in platelets and haematopoietic cells.

The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit ß3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.

Human Immunodeficiency Virus Type 2 (HIV-2) Gag Is Trafficked in an AP-3 and AP-5 Dependent Manner.

Although human immunodeficiency virus (HIV) types 1 and 2 are closely related lentiviruses with similar replication cycles, HIV-2 infection is associated with slower progression to AIDS, a higher proportion of long term non-progressors, and lower rates of transmission than HIV-1, likely as a consequence of a lower viral load during HIV-2 infection. A mechanistic explanation for the differential viral load remains unclear but knowledge of differences in particle production between HIV-1 and HIV-2 may help to shed light on this issue. In contrast to HIV-1, little is known about the assembly of HIV-2 particles, and the trafficking of HIV-2 Gag, the structural component of the virus, within cells. We have established that HIV-2 Gag accumulates in intracellular CD63 positive compartments, from which it may be delivered or recycled to the cell surface, or degraded. HIV-2 particle release was dependent on the adaptor protein complex AP-3 and the newly identified AP-5 complex, but much less so on AP-1. In contrast, HIV-1 particle release required AP-1 and AP-3, but not AP-5. AP-2, an essential component of clathrin-mediated endocytosis, which was previously shown to be inhibitory to HIV-1 particle release, had no effect on HIV-2. The differential requirement for adaptor protein complexes confirmed that HIV-1 and HIV-2 Gag have distinct cellular trafficking pathways, and that HIV-2 particles may be more susceptible to degradation prior to release.

Mycobacterium avium MAV_2941 mimics phosphoinositol-3-kinase to interfere with macrophage phagosome maturation.

Mycobacterium avium subsp hominissuis (M. avium) is a pathogen that infects and survives in macrophages. Previously, we have identified the M. avium MAV_2941 gene encoding a 73 amino acid protein exported by the oligopeptide transporter OppA to the macrophage cytoplasm. Mutations in MAV_2941 were associated with significant impairment of M. avium growth in THP-1 macrophages. In this study, we investigated the molecular mechanism of MAV_2941 action and demonstrated that MAV_2941 interacts with the vesicle trafficking proteins syntaxin-8 (STX8), adaptor-related protein complex 3 (AP-3) complex subunit beta-1 (AP3B1) and Archain 1 (ARCN1) in mononuclear phagocytic cells. Sequencing analysis revealed that the binding site of MAV_2941 is structurally homologous to the human phosphatidylinositol 3-kinase (PI3K) chiefly in the region recognized by vesicle trafficking proteins. The ß3A subunit of AP-3, encoded by AP3B1, is essential for trafficking cargo proteins, including lysosomal-associated membrane protein 1 (LAMP-1), to the phagosome and lysosome-related organelles. Here, we show that while the heat-killed M. avium when ingested by macrophages co-localizes with LAMP-1 protein, transfection of MAV_2941 in macrophages results in significant decrease of LAMP-1 co-localization with the heat-killed M. avium phagosomes. Mutated MAV_2941, where the amino acids homologous to the binding region of PI3K were changed, failed to interact with trafficking proteins. Inactivation of the AP3B1 gene led to alteration in the trafficking of LAMP-1. These results suggest that M. avium MAV_2941 interferes with the protein trafficking within macrophages altering the maturation of phagosome.

Absence of a stable secondary structure is not a limitation for photoswitchable inhibitors of ß-arrestin/ß-Adaptin 2 protein-protein interaction.

Many protein-protein interactions (PPIs) are mediated by short, often helical, linear peptides. Molecules mimicking these peptides have been used to inhibit their PPIs. Recently, photoswitchable peptides with little secondary structure have been developed as modulators of clathrin-mediated endocytosis. Here we perform a systematic analysis of a series of azobenzene-crosslinked peptides based on a ß-arrestin P-long 20-mer peptide (BAP-long) sequence to assess the relevance of secondary structure in their interaction with ß-adaptin 2 and to identify the design requirements for photoswitchable inhibitors of PPI (PIPPIs). We observe that flexible structures show a greater inhibitory capacity and enhanced photoswitching ability and that the absence of helical structures in free inhibitor peptide is not a limitation for PIPPI candidates. Therefore, our PIPPIs expand the field of potential inhibitors of PPIs to the wide group of flexible peptides, and we argue against using a stable secondary structure as a sole criterion when designing PIPPI candidates.