AP-1B regulates interactions of epithelial cells and intraepithelial lymphocytes in the intestine.

Intraepithelial lymphocytes (IELs) reside in the epithelial layer and protect against foreign pathogens, maintaining the epithelial barrier function in the intestine. Interactions between IEL and epithelial cells are required for IELs to function effectively; however, the underlying molecular machinery remains to be elucidated. In this study, we found that intestinal epithelium-specific deficiency of the clathrin adaptor protein (AP)-1B, which regulates basolateral protein sorting, led to a massive reduction in IELs. Quantitative proteomics demonstrated that dozens of proteins, including known IEL-interacting proteins (E-cadherin, butyrophilin-like 2, and plexin B2), were decreased in the basolateral membrane of AP-1B-deficient epithelial cells. Among these proteins, CD166 interacted with CD6 on the surface of induced IEL. CD166 knockdown, using shRNA in intestinal organoid cultures, significantly inhibited IEL recruitment to the epithelial layer. These findings highlight the essential role of AP-1B-mediated basolateral sorting in IEL maintenance and survival within the epithelial layer. This study reveals a novel function of AP-1B in the intestinal immune system.

Revising pathogenesis of AP1S1-related MEDNIK syndrome: a missense variant in the AP1S1 gene as a causal genetic lesion.

MEDNIK syndrome is a rare autosomal recessive disease characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma, and caused by variants in the adaptor-related protein complex 1 subunit sigma 1 (AP1S1) gene. This gene encodes the σ1A protein, which is a subunit of the adaptor protein complex 1 (AP-1), a key component of the intracellular protein trafficking machinery. Previous work identified three AP1S1 nonsense, frameshift and splice-site variants in MEDNIK patients predicted to encode truncated σ1A proteins, with consequent AP-1 dysfunction. However, two AP1S1 missense variants (c.269 T > C and c.346G > A) were recently reported in patients who presented with severe enteropathy but no additional symptoms of MEDNIK. This condition was described as a novel non-syndromic form of congenital diarrhea caused specifically by the AP1S1 missense variants. In this study, we report two patients with the same c.269 T > C variant, who, contrary to the previous cases, presented as complete MEDNIK syndrome. These data substantially revise the presentation of disorders associated with AP1S1 gene variants and indicate that all the identified pathogenic AP1S1 variants result in MEDNIK syndrome. We also provide a series of functional analyses that elucidate the impact of the c.269 T > C variant on σ1A function, contributing to a better understanding of the molecular pathogenesis of MEDNIK syndrome. KEY MESSAGES: A missense AP1S1 c.269 T > C (σ1A L90P) variant causes full MEDNIK syndrome. The σ1A L90P variant is largely unable to assemble into the AP-1 complex. The σ1A L90P variant fails to bind [DE]XXXL[LI] sorting motifs. The σ1A L90P variant results in loss-of-function of the protein.

The AP-1 adaptor complex is essential for intracellular trafficking of the ORF2 capsid protein and assembly of Hepatitis E virus.

Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.

AP-1 contributes to endosomal targeting of the ubiquitin ligase RNF13 via a secondary and novel non-canonical binding motif.

Cellular trafficking between organelles is typically assured by short motifs that contact carrier proteins to transport them to their destination. The ubiquitin E3 ligase RING finger protein 13 (RNF13), a regulator of proliferation, apoptosis and protein trafficking, localizes to endolysosomal compartments through the binding of a dileucine motif to clathrin adaptor protein complex AP-3. Mutations within this motif reduce the ability of RNF13 to interact with AP-3. Here, our study shows the discovery of a glutamine-based motif that resembles a tyrosine-based motif within the C-terminal region of RNF13 that binds to the clathrin adaptor protein complex AP-1, notably without a functional interaction with AP-3. Using biochemical, molecular and cellular approaches in HeLa cells, our study demonstrates that a RNF13 dileucine variant uses an AP-1-dependent pathway to be exported from the Golgi towards the endosomal compartment. Overall, this study provides mechanistic insights into the alternate route used by this variant of the dileucine sorting motif of RNF13.

Intestinal epithelium dysfunctions cause IgA deposition in the kidney glomeruli of intestine-specific Ap1m2-deficient mice.

BACKGROUND: Intestinal epithelial cells (IECs) serve as robust barriers against potentially hostile luminal antigens and commensal microbiota. Epithelial barrier dysfunction enhances intestinal permeability, leading to leaky gut syndrome (LGS) associated with autoimmune and chronic inflammatory disorders. However, a causal relationship between LGS and systemic disorders remains unclear. Ap1m2 encodes clathrin adaptor protein complex 1 subunit mu 2, which facilitates polarized protein trafficking toward the basolateral membrane and contributes to the establishment of epithelial barrier functions. METHODS: We generated IEC-specific Ap1m2-deficient (Ap1m2ΔIEC) mice with low intestinal barrier integrity as an LSG model and examined the systemic impact. FINDINGS: Ap1m2ΔIEC mice spontaneously developed IgA nephropathy (IgAN)-like features characterized by the deposition of IgA-IgG immune complexes and complement factors in the kidney glomeruli. Ap1m2 deficiency markedly enhanced aberrantly glycosylated IgA in the serum owing to downregulation and mis-sorting of polymeric immunoglobulin receptors in IECs. Furthermore, Ap1m2 deficiency caused intestinal dysbiosis by attenuating IL-22-STAT3 signaling. Intestinal dysbiosis contributed to the pathogenesis of IgAN because antibiotic treatment reduced aberrantly glycosylated IgA production and renal IgA deposition in Ap1m2ΔIEC mice. INTERPRETATION: IEC barrier dysfunction and subsequent dysbiosis by AP-1B deficiency provoke IgA deposition in the mouse kidney. Our findings provide experimental evidence of a pathological link between LGS and IgAN. FUNDING: AMED, AMED-CREST, JSPS Grants-in-Aid for Scientific Research, JST CREST, Fuji Foundation for Protein Research, and Keio University Program for the Advancement of Next Generation Research Projects.

RUSHing back: Kinetic analysis of adaptor protein complex-1 (AP-1)-mediated retrograde traffic.

Numerous biomedically important cargoes depend on adaptor protein complex-1 (AP-1) for their localization. However, controversy surrounds whether AP-1 mediates traffic from or to the Golgi. Robinson et al. (https://www.doi.org/10.1083/jcb.202310071) present compelling evidence that AP-1 mediates recycling to the Golgi.

Usmani-Riazuddin syndrome can have a recognizable phenotype: Report of a novel AP1G1 variant.

Usmani-Riazuddin syndrome (USRISR, MIM# 619548; USRISD, MIM#619467) is a very rare genetic condition. recently associated with deleterious variants in AP1G1 (MIM* 603533). It is characterized by multisystemic involvement including intellectual disability, speech and developmental delay, behavioral anomalies, muscular tone disorders, seizures, limb defects, and unspecified facial gestalt. In this report, we describe this syndrome for the second time, in association to a novel AP1G1 variant identified in a toddler with multisystemic involvement including intellectual disability, speech and developmental delay, behavioral anomalies, arrhythmias, hearing loss, skin changes, and limb defects. Next generation sequencing (NGS) analysis through clinical exome disclosed AP1G1: c.1969C>G (p.Leu657Val), de novo, likely pathogenic variant, according to ACMG classification criteria. Proband's facial features resembled the spectrum of chromatinopathies. Clinical pictures were analyzed and a clinical overlap was supported by DeepGestalt analysis (www.face2gene.com). The system identified 6 chromatin disorders out of 30 possible diagnoses. The remaining 24 included 9 miscellaneous cryptic chromosomal abnormalities (excluded due to normal microarray study). To the best of our knowledge, this is the first description of likely distinctive facial features in a patient with Usmani-Riazuddin syndrome. Further multicentric analyses are needed for a better definition of this aspect.

AP-1γ2 is an adaptor protein 1 variant required for endosome-to-Golgi trafficking of the mannose-6-P receptor (CI-MPR) and ATP7B copper transporter.

Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.

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.

Clinicopathologic Features of IDEDNIK (MEDNIK) Syndrome in a Term Infant: Histopathologic Features of the Gastrointestinal Tract and Report of a Novel AP1S1 Variant.

Inherited syndromes of congenital enteropathy are rare, with many genetic causes described. Mutations of the AP1S1 gene results in the syndrome of intellectual disability, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma (IDEDNIK, formerly in the medical literature as MEDNIK). The clinicopathologic features of the enteropathy in IDEDNIK syndrome have not been fully explored. We describe a female infant who presented with metabolic acidosis, lethargy, and 14 watery stools per day. In the intensive care unit she required parenteral nutrition. She was found to have a novel homozygous pathogenic variant in the AP1S1 gene c.186T>G (p.Y62*). Esophagogastroduodenoscopy and colonoscopy at 6 months of age were grossly normal. However, histologic sections of the duodenum showed mild villous blunting and enterocytes with cytoplasmic vacuoles. CD10 immunostaining highlighted the disrupted brush border. MOC31 immunostaining was wild-type with a membranous pattern of expression. Electron microscopy of the duodenum showed scattered enterocytes cells with shortened and disrupted apical microvilli. Although there is a mixed gap diarrhea and disrupted brush border, there are no significant inclusions typical of microvillus inclusion disease, nor tufted enterocytes typical of tufting enteropathy, making the clinical and histopathologic features for this syndrome unique.

The clathrin adaptor complex-1 and Rab12 regulate post-golgi trafficking of WT epidermal growth factor receptor (EGFR).

The epidermal growth factor receptor (EGFR) plays important roles in cancer progression and is one of the major drug targets for targeted cancer therapy. Although fundamentally important, how newly synthesized EGFR is delivered to the cell surface to perform its cellular functions remains to be further investigated. In this study, we found using the approaches of gene knockout, siRNA knockdown, streptavidin pull-down, and co-immunoprecipitation assays that the clathrin adaptor complex-1 (AP-1) and Rab12 interact with EGFR and regulate the export of EGFR out of the trans-Golgi network (TGN). In addition, the tyrosine residue at the 998 position on human EGFR is critical to bind to AP-1, and this residue is important for TGN export of EGFR. We demonstrate that AP-1 and Rab12 are important for epidermal growth factor-induced phosphorylation of EGFR, cell elongation, and proliferation, suggesting that AP-1-mediated and Rab12-mediated post-Golgi trafficking is important for EGFR signaling. Moreover, TGN export of the constitutively activated mutant form of EGFR (EGFRL858R) is independent of AP-1 and Rab12. Our results reveal insights into the molecular mechanisms that mediate the TGN-to-cell surface delivery of EGFR and indicate that TGN export of WT EGFR and EGFRL858R depends on different cellular factors.

mRNA Capture Sequencing and RT-qPCR for the Detection of Pathognomonic, Novel, and Secondary Fusion Transcripts in FFPE Tissue: A Sarcoma Showcase.

We assess the performance of mRNA capture sequencing to identify fusion transcripts in FFPE tissue of different sarcoma types, followed by RT-qPCR confirmation. To validate our workflow, six positive control tumors with a specific chromosomal rearrangement were analyzed using the TruSight RNA Pan-Cancer Panel. Fusion transcript calling by FusionCatcher confirmed these aberrations and enabled the identification of both fusion gene partners and breakpoints. Next, whole-transcriptome TruSeq RNA Exome sequencing was applied to 17 fusion gene-negative alveolar rhabdomyosarcoma (ARMS) or undifferentiated round cell sarcoma (URCS) tumors, for whom fluorescence in situ hybridization (FISH) did not identify the classical pathognomonic rearrangements. For six patients, a pathognomonic fusion transcript was readily detected, i.e., PAX3-FOXO1 in two ARMS patients, and EWSR1-FLI1, EWSR1-ERG, or EWSR1-NFATC2 in four URCS patients. For the 11 remaining patients, 11 newly identified fusion transcripts were confirmed by RT-qPCR, including COPS3-TOM1L2, NCOA1-DTNB, WWTR1-LINC01986, PLAA-MOB3B, AP1B1-CHEK2, and BRD4-LEUTX fusion transcripts in ARMS patients. Additionally, recurrently detected secondary fusion transcripts in patients diagnosed with EWSR1-NFATC2-positive sarcoma were confirmed (COPS4-TBC1D9, PICALM-SYTL2, SMG6-VPS53, and UBE2F-ALS2). In conclusion, this study shows that mRNA capture sequencing enhances the detection rate of pathognomonic fusions and enables the identification of novel and secondary fusion transcripts in sarcomas.

Characterization of 22q12 Microdeletions Causing Position Effect in Rare NF2 Patients with Complex Phenotypes.

Neurofibromatosis type 2 is an autosomal dominant tumor-prone disorder mainly caused by NF2 point mutations or intragenic deletions. Few individuals with a complex phenotype and 22q12 microdeletions have been described. The 22q12 microdeletions' pathogenic effects at the genetic and epigenetic levels are currently unknown. We here report on 22q12 microdeletions' characterization in three NF2 patients with different phenotype complexities. A possible effect of the position was investigated by in silico analysis of 22q12 topologically associated domains (TADs) and regulatory elements, and by expression analysis of 12 genes flanking patients' deletions. A 147 Kb microdeletion was identified in the patient with the mildest phenotype, while two large deletions of 561 Kb and 1.8 Mb were found in the other two patients, showing a more severe symptomatology. The last two patients displayed intellectual disability, possibly related to AP1B1 gene deletion. The microdeletions change from one to five TADs, and the 22q12 chromatin regulatory landscape, according to the altered expression levels of four deletion-flanking genes, including PIK3IP1, are likely associated with an early ischemic event occurring in the patient with the largest deletion. Our results suggest that the identification of the deletion extent can provide prognostic markers, predictive of NF2 phenotypes, and potential therapeutic targets, thus overall improving patient management.

Phenotypic spectrum of autosomal recessive Keratitis-Ichthyosis-Deafness Syndrome (KIDAR) due to mutations in AP1B1.

Inborn errors in copper metabolism result in a diverse set of abnormalities such as Wilson disease and MEDNIK syndrome. Homozygous pathogenic variants in AP1B1 lead to KIDAR (Keratitis-Ichthyosis-Deafness Syndrome). The main phenotypic features of KIDAR are ichthyosis, keratitis, erythroderma, and progressive hearing loss accompanied by developmental delay and failure to thrive. Herein, we describe a six-and-a-half-year-old boy with KIDAR caused by a novel pathogenic variant in AP1B1 (NM_001127.4:c.1263C > A, p.Tyr421*). The proband presented with ichthyosis, erythroderma, palmoplantar keratoderma, hearing loss, and corneal scarring. He also had hypotonia, global developmental delay, and photophobia. Lastly, we review all of the previously reported cases and the clinical features associated with KIDAR.

Interacting with AP1 complex mutated synergin gamma (SYNRG) reveals a novel coatopathy in the form of complicated hereditary spastic paraplegia.

BACKGROUND: Today, it is known that about 80 genes are involved in the etiology of hereditary spastic paraplegia. However, there are many cases whose etiology could not be determined by extensive genetic tests such as whole-exome sequencing, clinical exome. METHODS: Candidate genes were determined, since no clinically illuminating variant was detected in the whole-exome sequencing analysis of three patients, two of whom were siblings, with a complex hereditary spastic paraplegia phenotype. RESULTS: The p.Leu1202Pro variant in the SYNRG gene in the 1st and 2nd cases, and the p.Gly533* variant in the 3rd case were homozygous. DISCUSSION: We suggest that the SYNRG gene interacting with AP-1 (adaptor-related protein) from the AP complex family may cause the complex hereditary spastic paraplegia phenotype with extensive clinical spectrum. It may be important to evaluate SYNRG gene variants in patients with hereditary spastic paraplegia whose etiology has not been clarified.

AAGAB is an assembly chaperone regulating AP1 and AP2 clathrin adaptors.

Multimeric cargo adaptors such as AP2 play central roles in intracellular membrane trafficking. We recently discovered that the assembly of the AP2 adaptor complex, a key player in clathrin-mediated endocytosis, is a highly organized process controlled by alpha- and gamma-adaptin-binding protein (AAGAB, also known as p34). In this study, we demonstrate that besides AP2, AAGAB also regulates the assembly of AP1, a cargo adaptor involved in clathrin-mediated transport between the trans-Golgi network and the endosome. However, AAGAB is not involved in the formation of other adaptor complexes, including AP3. AAGAB promotes AP1 assembly by binding and stabilizing the γ and σ subunits of AP1, and its mutation abolishes AP1 assembly and disrupts AP1-mediated cargo trafficking. Comparative proteomic analyses indicate that AAGAB mutation massively alters surface protein homeostasis, and its loss-of-function phenotypes reflect the synergistic effects of AP1 and AP2 deficiency. Taken together, these findings establish AAGAB as an assembly chaperone for both AP1 and AP2 adaptors and pave the way for understanding the pathogenesis of AAGAB-linked diseases.

De novo and bi-allelic variants in AP1G1 cause neurodevelopmental disorder with developmental delay, intellectual disability, and epilepsy.

Adaptor protein (AP) complexes mediate selective intracellular vesicular trafficking and polarized localization of somatodendritic proteins in neurons. Disease-causing alleles of various subunits of AP complexes have been implicated in several heritable human disorders, including intellectual disabilities (IDs). Here, we report two bi-allelic (c.737C>A [p.Pro246His] and c.1105A>G [p.Met369Val]) and eight de novo heterozygous variants (c.44G>A [p.Arg15Gln], c.103C>T [p.Arg35Trp], c.104G>A [p.Arg35Gln], c.229delC [p.Gln77Lys∗11], c.399_400del [p.Glu133Aspfs∗37], c.747G>T [p.Gln249His], c.928-2A>C [p.?], and c.2459C>G [p.Pro820Arg]) in AP1G1, encoding gamma-1 subunit of adaptor-related protein complex 1 (AP1γ1), associated with a neurodevelopmental disorder (NDD) characterized by mild to severe ID, epilepsy, and developmental delay in eleven families from different ethnicities. The AP1γ1-mediated adaptor complex is essential for the formation of clathrin-coated intracellular vesicles. In silico analysis and 3D protein modeling simulation predicted alteration of AP1γ1 protein folding for missense variants, which was consistent with the observed altered AP1γ1 levels in heterologous cells. Functional studies of the recessively inherited missense variants revealed no apparent impact on the interaction of AP1γ1 with other subunits of the AP-1 complex but rather showed to affect the endosome recycling pathway. Knocking out ap1g1 in zebrafish leads to severe morphological defect and lethality, which was significantly rescued by injection of wild-type AP1G1 mRNA and not by transcripts encoding the missense variants. Furthermore, microinjection of mRNAs with de novo missense variants in wild-type zebrafish resulted in severe developmental abnormalities and increased lethality. We conclude that de novo and bi-allelic variants in AP1G1 are associated with neurodevelopmental disorder in diverse populations.

The MUDENG Augmentation: A Genesis in Anti-Cancer Therapy?

Despite multitudes of reports on cancer remedies available, we are far from being able to declare that we have arrived at that defining anti-cancer therapy. In recent decades, researchers have been looking into the possibility of enhancing cell death-related signaling pathways in cancer cells using pro-apoptotic proteins. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Mu-2/AP1M2 domain containing, death-inducing (MUDENG, MuD) have been established for their ability to bring about cell death specifically in cancer cells. Targeted cell death is a very attractive term when it comes to cancer, since most therapies also affect normal cells. In this direction TRAIL has made noteworthy progress. This review briefly sums up what has been done using TRAIL in cancer therapeutics. The importance of MuD and what has been achieved thus far through MuD and the need to widen and concentrate on applicational aspects of MuD has been highlighted. This has been suggested as the future perspective of MuD towards prospective progress in cancer research.