Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process

Gastrointestinal dysfunction in patients and mice expressing the autism-associated R451C mutation in neuroligin-3

Gastrointestinal (GI) problems constitute an important comorbidity in many patients with autism. Multiple mutations in the neuroligin family of synaptic adhesion molecules are implicated in autism, however whether they are expressed and impact GI function via changes in the enteric nervous system is unknown. We report the GI symptoms of two brothers with autism and an R451C mutation in Nlgn3 encoding the synaptic adhesion protein, neuroligin-3. We confirm the presence of an array of synaptic genes in the murine GI tract and investigate the impact of impaired synaptic protein expression in mice carrying the human neuroligin-3 R451C missense mutation (NL3R451C ). Assessing in vivo gut dysfunction, we report faster small intestinal transit in NL3R451C compared to wild-type mice. Using an ex vivo colonic motility assay, we show increased sensitivity to GABAA receptor modulation in NL3R451C mice, a well-established Central Nervous System (CNS) feature associated with this mutation. We further show increased numbers of small intestine myenteric neurons in NL3R451C mice. Although we observed altered sensitivity to GABAA receptor modulators in the colon, there was no change in colonic neuronal numbers including the number of GABA-immunoreactive myenteric neurons. We further identified altered fecal microbial communities in NL3R451C mice. These results suggest that the R451C mutation affects small intestinal and colonic function and alter neuronal numbers in the small intestine as well as impact fecal microbes. Our findings identify a novel GI phenotype associated with the R451C mutation and highlight NL3R451C mice as a useful preclinical model of GI dysfunction in autism. LAY SUMMARY: People with autism commonly experience gastrointestinal problems, however the cause is unknown. We report gut symptoms in patients with the autism-associated R451C mutation encoding the neuroligin-3 protein. We show that many of the genes implicated in autism are expressed in mouse gut. The neuroligin-3 R451C mutation alters the enteric nervous system, causes gastrointestinal dysfunction, and disrupts gut microbe populations in mice. Gut dysfunction in autism could be due to mutations that affect neuronal communication.This work was supported by an Idea Development Award from the United States Department of Defense’s Congressionally Directed Medical Research Programs (CDMRP) Autism Research Program (AR110134) to E.L.H.-Y. and J.C.B.; the Victorian Government through the Operational Infrastructure Scheme, National Health and Medical Research Council (NHMRC) project grants (APP566642 to J.C.B. and APP1047674 to E.L.H.-Y.) and the Royal Melbourne Hospital Neuroscience Foundation. E.L.H.-Y. also received an ARC Future Fellowship (FT160100126) and an RMIT Vice Chancellor’s Senior Research Fellowship which supported G.K.B. and S.H. T.S., P.U., and N.Y. were funded by grants RO1AI100914, P30-DK56338, and U01-AI24290 awards to Baylor College of Medicine funded from the National Institute of Allergy and Infectious Diseases and National Institute of Diabetes and Digestive and Kidney Diseases at the National Institutes of Health (T.C.S.)

Exploiting open source omics data to advance pancreas research

The “omics” revolution has transformed the biomedical research landscape by equipping scientists with the ability to interrogate complex biological phenomenon and disease processes at an unprecedented level. The volume of “big” data generated by the different omics studies such as genomics, transcriptomics, proteomics, and metabolomics has led to the concurrent development of computational tools to enable in silico analysis and aid data deconvolution. Considering the intensive resources and high costs required to generate and analyze big data, there has been centralized, collaborative efforts to make the data and analysis tools freely available as “Open Source,” to benefit the wider research community. Pancreatology research studies have contributed to this “big data rush” and have additionally benefitted from utilizing the open source data as evidenced by the increasing number of new research findings and publications that stem from such data. In this review, we briefly introduce the evolution of open source omics data, data types, the “FAIR” guiding principles for data management and reuse, and centralized platforms that enable free and fair data accessibility, availability, and provide tools for omics data analysis. We illustrate, through the case study of our own experience in mining pancreatitis omics data, the power of repurposing open source data to answer translationally relevant questions in pancreas research

Expression, purification, crystallization and preliminary X-ray diffraction analysis of Arabidopsis thaliana cyclophilin 38 (AtCyp38)

Crystallization of Arabidopsis thaliana cyclophilin 38. The crystal diffracts X-rays to 2.5 Å resolution

BECN1/Beclin 1 sorts cell-surface APP/amyloid β precursor protein for lysosomal degradation

<p>The regulation of plasma membrane (PM)-localized transmembrane protein/receptor trafficking has critical implications for cell signaling, metabolism and survival. In this study, we investigated the role of BECN1 (Beclin 1) in the degradative trafficking of PM-associated APP (amyloid β precursor protein), whose metabolism to amyloid-β, an essential event in Alzheimer disease, is dependent on divergent PM trafficking pathways. We report a novel interaction between PM-associated APP and BECN1 that recruits macroautophagy/endosomal regulatory proteins PIK3C3 and UVRAG. We found that BECN1 promotes surface APP internalization and sorting predominantly to endosomes and endolysosomes. BECN1 also promotes the targeting of a smaller fraction of internalized APP to LC3-positive phagophores, suggesting a role for BECN1-dependent PM macroautophagy in APP degradation. Furthermore, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites (soluble ectodomains, sAPP). The association between APP and BECN1 is dependent on the evolutionarily conserved domain (ECD) of BECN1 (amino acids 267–337). Deletion of a BECN1 ECD subregion (amino acids 285–299) did not impair BECN1- PIK3C3 interaction, PtdIns3K function or macroautophagy, but was sufficient to impair the APP-BECN1 interaction and BECN1's effects on surface APP internalization and degradation, resulting in increased secretion of sAPPs. Interestingly, both the BECN1-APP association and BECN1-dependent APP endocytosis and degradative trafficking were negatively regulated by active AKT. Our results further implicate phosphorylation of the BECN1 Ser295 residue in the inhibition of APP degradation by AKT. Our studies reveal a novel function for BECN1 in the sorting of a plasma membrane protein for endolysosomal and macroautophagic degradation.</p

Prolactin Inhibits Activity of Pyruvate Kinase M2 to Stimulate Cell Proliferation

Mitogenic and prosurvival effects underlie the tumorigenic roles of prolactin (PRL) in the pathogenesis of breast cancer. PRL signaling is mediated through its receptor (PRLr). A proteomics screen identified the pyruvate kinase M2 (PKM2), a glycolytic enzyme known to play an important role in tumorigenesis, as a protein that constitutively interacts with PRLr. Treatment of cells with PRL inhibited pyruvate kinase activity and increased the lactate content in human cells in a manner that was dependent on the abundance of PRLr, activation of Janus kinase 2, and tyrosine phosphorylation of the intracellular domain of PRLr. Knockdown of PKM2 attenuated PRL-stimulated cell proliferation. The extent of this proliferation was rescued by the knock-in of the wild-type PKM2 but not of its mutant insensitive to PRL-mediated inhibition. We discuss a hypothesis that the inhibition of PKM2 by PRL contributes to the PRL-stimulated cell proliferation