Lamellar bodies (LBs) are lysosome-related organelles (LROs) of surfactant-producing alveolar type 2 (AT2) cells of the distal lung epithelium. Trafficking pathways to LBs have been understudied but ...are likely critical to AT2 cell homeostasis given associations between genetic defects of endosome to LRO trafficking and pulmonary fibrosis in Hermansky Pudlak syndrome (HPS). Our prior studies uncovered a role for AP-3, defective in HPS type 2, in trafficking Peroxiredoxin-6 to LBs. We now show that the P4-type ATPase ATP8A1 is sorted by AP-3 from early endosomes to LBs through recognition of a C-terminal dileucine-based signal. Disruption of the AP-3/ATP8A1 interaction causes ATP8A1 accumulation in early sorting and/or recycling endosomes, enhancing phosphatidylserine exposure on the cytosolic leaflet. This in turn promotes activation of Yes-activating protein, a transcriptional coactivator, augmenting cell migration and AT2 cell numbers. Together, these studies illuminate a mechanism whereby loss of AP-3-mediated trafficking contributes to a toxic gain-of-function that results in enhanced and sustained activation of a repair pathway associated with pulmonary fibrosis.
The Carcinoembryonic Antigen-Related Cell Adhesion Molecule (CEACAM) family of proteins plays a significant role in regulating peripheral insulin action by participating in the regulation of insulin ...metabolism and energy balance. In light of their differential expression, CEACAM1 regulates chiefly insulin extraction, whereas CEACAM2 appears to play a more important role in regulating insulin secretion and overall energy balance, including food intake, energy expenditure and spontaneous physical activity. We will focus this review on the role of CEACAM2 in regulating insulin metabolism and energy balance with an overarching goal to emphasize the importance of the coordinated regulatory effect of these related plasma membrane glycoproteins on insulin metabolism and action.
Abstract
We have discovered a way to cure type 1 diabetes in a mouse model that closely resembles the human disease. Specifically, we are able to genetically modify blood cells from affected ...individuals and re-infuse them back to rescue insulin-producing beta cells being destroyed by the immune system.When autoimmune diabetes affects the youth, it is known as Juvenile or type 1 diabetes (T1D). It is named Latent Autoimmune Diabetes of Adults (LADA) when it affects adults. T1D affects about 1 million people in the USA. LADA is estimated to affect at least 10% of the type 2 diabetes population (about 30 million patients in the USA). Combined, 4 million people live with the condition.Can you imagine reversing type 1 diabetes at once?Autoimmune diabetes as a whole is a disease without a cure that is chronically managed with insulin replacement. Even under best management, diabetes complications shorten life expectancy, including blindness, amputations, and kidney failure. These are the population that will benefit from our technology. Starting with new-onset diabetes, the technology can benefit almost any patient with autoimmune diabetes.Our group has developed two currently patented technologies that achieve the goal of reversing type 1 diabetes (our value proposition). One is a new spontaneous, humanized, transgenic mouse model of human T1D (1). The other one is an adoptive cell transfer (ACT) therapeutic approach for T1D (2). A 30-day pre-clinical study in which animals with diabetes were treated with this therapeutic approach provided the proof-of-concept that diabetes CAN BE CURED.Our technology involves a standard-of-care approach already Federal Drug Administration (FDA) approved for cancer treatment (ACT of Chimeric Antigen Receptor, CAR, T cells). We have the materials and know-how needed for the large-scale pre-clinical trial we are currently running. This will generate the necessary data for Investigational New Drug, IND, FDA filing so human clinical trials can be carried out.
References
1. Imam, S., Alfonso-Jaume, M., Jaume, J.C. (2019). Spontaneous Autoimmune Diabetes in Humanized Mice Carrying Human Type 1 Diabetes Susceptibility and Uses Therefor. United States Application No. 16530452, Publication Number. US20200037586. https://patentscope.wipo.int/search/en/detail.jsf?docId=US283198965&tab=NATIONALBIBLIO
2. Imam, S., Jaume, J.C. (2020). Immunosuppressive Antigen-Specific Chimeric Antigen Receptor Treg Cells for Prevention and/or Treatment of Autoimmune and Alloimmune Disorders. International Application No. PCT/US2019/060593, Publication Number WO/2020/097546. https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020097546&_cid=P10-KHXN2B-33433-1
Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.
Hermansky‐Pudlak syndrome (HPS) is a group of rare genetic diseases characterized by oculocutaneous albinism and bleeding diathesis, often associated with lung fibrosis. The symptoms of HPS reflect ...defects in membrane transport processes required to generate tissue‐specific lysosome‐related organelles (LROs), such as melanosomes in pigment cells, lamellar bodies (LB) in type 2 alveolar epithelial cells (AT2) and dense granules in platelets. Although the transport processes that are defective in HPS are shared among distinct LRO‐expressing cells, it is not clear whether the unique cargo proteins that confer distinct morphological and functional characteristics are targeted to their respective LROs by conserved or cell type‐specific recognition mechanisms. To determine whether LB and melanosome cargo proteins contain cell type‐independent LRO sorting information, we exploited the behavior of model cargo proteins, such as melanosomal protein, OCA2 and LB‐specific protein, ATP8A1 in AT2 cells and melanocytes. OCA2 is a pigment cell‐specific membrane protein and localizes to melanosomes via a cytoplasmic dileucine‐based sorting signal that engages both AP‐1 and AP‐3. By overexpression of wild‐type OCA2 in MLE15, we show that wild‐type OCA2 colocalized extensively with the LB marker, mCherry‐ABCA3 in MLE15, an AT2 cell line. OCA2‐AA23N bearing the double E96DP99S mutation that was unable to bind to AP‐1 and AP‐3 localized to the cell surface. To confirm the relative roles of AP‐1 and AP‐3 in transport of OCA2 to LB, we also expressed the AP‐3‐biased OCA2‐AA23‐P99S and AP‐1‐biased OCA2‐AA23‐E96D variants in MLE15. Mutants that preferentially bound either AP‐1 or AP‐3 each partially trafficked toward LB, but AP‐3 binding was necessary for steady‐state LB localization, as it is for delivery of OCA2 to melanosomes. ATP8A1, a member of the P4 subfamily of P‐type ATPases (P4‐ATPases), is highly enriched in LB but expressed at low levels in melanocytes. When expressed in immortalized mouse melan‐Ink4a−/− melanocytes, an EGFP‐tagged, catalytically inactive ATP8A1 partially overlapped with pigmented melanosomes. Overlap was substantially reduced by mutagenesis of a conserved dileucine‐based sorting signal or by expression in AP‐3‐deficient melanocytes. These data provide evidence that cellular machinery in AT2 or melanocytes can recognize a universal LRO targeting signal in a heterologous protein and deliver it to a cell type‐specific LRO. By contrast, two other AT2‐restricted LB cargoes – DC‐LAMP and ABCA3 ‐ failed to localize to pigmented melanosomes upon expression in melan‐Ink4a−/− cells. Together, these data suggest that LRO cargoes employ both universally conserved and cell type‐specific pathways for delivery to nascent LROs.
Support or Funding Information
NIH (GM108807/SG and MM). SG is the Julia Carell Stadler Professor of Pediatrics.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Alveolar type 2 cells (AT2) are involved in the maintenance of epithelial homeostasis and repair after injury. Lamellar bodies (LB) in AT2 play a critical role in lung health and disease as the site ...of surfactant synthesis, packaging and secretion. LB biogenesis is poorly understood, especially mechanisms that foster membrane and protein trafficking to LB. In this study, we found that ATP8a1, a member of the P4 subfamily of P‐;type ATPases (P4‐;ATPases), was highly enriched in the ABCA3‐positive LB fraction derived from human fetal lung explants. In AT2 from mice lacking the adaptor protein 3 complex (AP‐3), the pearl mouse model of Hermansky Pudlak syndrome type 2 (HPS2), ATP8a1 was largely depleted from ABCA3‐positive LB and accumulated in Rab11‐positive recycling endosomes (REs). Using targeted mutagenesis, we identified a di‐leucine‐based sorting signal (ExxxLL) present in the C‐terminal cytoplasmic domain of ATP8a1 that is necessary for its proper targeting to LB of AT2. To dissect the relative roles of AP‐1 and AP‐3 in the transport of ATP8a1 to LB, we have developed MLE‐15 cell lines in which a subunit of AP‐1 (Ap1γ1) and/or AP‐3 (Ap3β1) has been inactivated using CRISPR/Cas9 gene editing and tested the targeting of ATP8a1 in these mutated cell lines. The lack of AP‐3 in MLE‐15 reduced efficient LB delivery of ATP8a1, while deletion of the AP‐1 subunit had no effect on ATP8a1 delivery to LB. P4‐;ATPases typically flip aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of organelle membranes. PS is highly enriched in REs and is essential for endosomal membrane traffic. Here, we show that the cytoplasmic leaflet of LB in MLE‐15 and human AT2 are highly enriched in PS using a GFP‐LactC2 probe; in contrast, AP‐3‐deficient cells showed impaired distribution of PS, favoring RE locazliation. These data provide evidence that ATP8a1 is dependent on AP‐3 for trafficking from RE to LB in AT2, and suggest that loss of ATP8a1 targeting to LB in HPS2 impairs PS localization to LB in AT2 cells.
Support or Funding Information
National Institutes of Health (GM108807/SG, MM and LG). SG is the Julia Carell Stadler Professor of Pediatrics.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Glutamic acid decarboxylase 65kD autoantibody (GAD65Ab) is frequently detected in patients with refractory epilepsy and stiff person syndrome. In contrast to T1D, the pathological role of GAD65Ab in ...neurological disorders is still debatable. As a result, the implementation of possible immunotherapy is usually delayed. This report presents 2 cases of GAD65Ab-associated brain autoimmunity and their different management. We present clinical data and discuss management based on available evidence in the reviewed literature. Both cases presented with acute on chronic neurological symptoms and were GAD65Ab positive. Case 1, a 30-year-old man with a history of early-onset type 1 diabetes mellitus at 14 months, followed by cryptogenic temporal epilepsy at 11 years of age, presented with intractable seizures. Case 2, a 48-year-old woman, presented with a history of recurrent severe headaches, cognitive impairment, decreased memory, and behavioral symptoms. GAD65Ab was detected in both patients' sera. Cerebrospinal fluid GAD65Ab was only checked and positive in case 1. Case 2 was diagnosed with limbic encephalitis, treated with immunotherapy, and showed a remarkable clinical improvement. Case 1 with refractory epilepsy failed multiple antiepileptic drugs and responsive-stimulator system treatments. He was finally diagnosed with autoimmune epilepsy. The delay in diagnosis resulted in a lost opportunity for early immunotherapy. In conclusion, autoantibody screening and early initiation of immunotherapy should be considered to manage GAD65Ab-associated neurological disorders.
Abstract
The sequencing of aggressive pediatric solid tumors is revealing remarkably stable genomes. In the cases of malignant rhabdoid and retinoblastoma, there is a paucity of recurrently mutated ...genes, and oncogenesis appears to be driven, at least in part, by epigenetic deregulation. It has been suggested that pediatric tumors characterized by oncogenic fusions will exhibit relatively few additional somatic driver aberrancies. Ewing sarcoma, the second most common pediatric bone tumor, is characterized by rearrangements of the EWS gene and ETS-family transcription factor genes, most commonly FLI and ERG. In experimental models, Ewing sarcoma demonstrates dependency on the expression of the resulting chimeric fusion products. As such, Ewing sarcoma represents a paradigm for studying the genomic landscape of fusion-driven cancers. To this end, we performed whole-exome sequencing of 96 Ewing sarcoma tumors and 11 Ewing sarcoma cell lines, as well as whole-genome sequencing, transcriptome sequencing, and copy-number analysis of a subset of these samples. We found that Ewing sarcoma is one of the most genetically normal cancers sequenced to date, but that treatment, which generally employs genotoxic chemotherapy and radiation, is associated with an increase in mutation rate and single nucleotide substitutions associated with DNA damage. There was a marked absence of recurrent mutations in immediately druggable targets, such as tyrosine kinases, calling into question the feasibility of utilizing tumor sequencing to nominate targeted therapies for patients with Ewing sarcoma. Rather, these results highlight the importance of directly targeting the EWS/ETS fusion events or identifying synthetic lethal dependencies. To this end, we clarified a number of outstanding questions regarding the EWS/ETS fusions. We found that reciprocal ETS/EWS fusions are not expressed in Ewing sarcoma and therefore unlikely to play a role in Ewing pathogenesis as is seen with reciprocal fusions of PML-RARα in acute promyelocytic leukemia. We also found that wild-type FLI and wild-type ERG are not expressed in Ewing sarcoma tumors. However, there appears to be a role for ETS gene deregulation in this disease beyond the expression of EWS/ETS fusion proteins because we found recurrent somatic events in ERF and ETS1. We also identified a small number of other recurrently mutated genes that likely collaborate with EWS/ETS fusions in a minority of cases and confirmed that loss of STAG2 occurs in approximately 15% of Ewing sarcoma tumors. Thus, massively parallel sequencing of a large collection of Ewing sarcoma tumors supports the notion that fusion-driven pediatric malignancies bear quiet genomes, underscores the importance of identifying new treatment approaches targeting EWS/ETS fusions, and also identifies new genetic abnormalities that warrant further biological validation.
Citation Format: Brian Crompton, Chip Stewart, Amaro Taylor-Weiner, Gabriela Alexa, Kyle Kurek, Monica Calicchio, Adam Kiezun, Scott Carter, Sachet Shukla, Swapnil Mehta, Aaron Thorner, Carmen de Torres, Cinzia Lavarino, Mariona Sunol, Aaron McKenna, Andrey Sivachenko, Kristian Cibulskis, Michael Lawrence, Lauren Ambrogio, Daniel Auclair, Ivan Rosshandler, Angela Schwarz-Cruz y Celis, Miguel Rivera, Carlos Rodriguez-Galindo, Mark Fleming, Todd Golub, Gad Getz, Jaume Mora, Kimberly Stegmaier. The genomic landscape of pediatric Ewing sarcoma. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 999. doi:10.1158/1538-7445.AM2014-999
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