Publications by authors named "Daniel D Billadeau"

162 Publications

SNX27-FERM-SNX1 complex structure rationalizes divergent trafficking pathways by SNX17 and SNX27.

Proc Natl Acad Sci U S A 2021 Sep;118(36)

Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Paediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy and Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu 610041, China;

The molecular events that determine the recycling versus degradation fates of internalized membrane proteins remain poorly understood. Two of the three members of the SNX-FERM family, SNX17 and SNX31, utilize their FERM domain to mediate endocytic trafficking of cargo proteins harboring the NPxY/NxxY motif. In contrast, SNX27 does not recycle NPxY/NxxY-containing cargo but instead recycles cargo containing PDZ-binding motifs via its PDZ domain. The underlying mechanism governing this divergence in FERM domain binding is poorly understood. Here, we report that the FERM domain of SNX27 is functionally distinct from SNX17 and interacts with a novel DLF motif localized within the N terminus of SNX1/2 instead of the NPxY/NxxY motif in cargo proteins. The SNX27-FERM-SNX1 complex structure reveals that the DLF motif of SNX1 binds to a hydrophobic cave surrounded by positively charged residues on the surface of SNX27. The interaction between SNX27 and SNX1/2 is critical for efficient SNX27 recruitment to endosomes and endocytic recycling of multiple cargoes. Finally, we show that the interaction between SNX27 and SNX1/2 is critical for brain development in zebrafish. Altogether, our study solves a long-standing puzzle in the field and suggests that SNX27 and SNX17 mediate endocytic recycling through fundamentally distinct mechanisms.
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http://dx.doi.org/10.1073/pnas.2105510118DOI Listing
September 2021

The Trifecta of Single-Cell, Systems-Biology, and Machine-Learning Approaches.

Genes (Basel) 2021 Jul 20;12(7). Epub 2021 Jul 20.

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine and Science, Mayo Clinic, 200 First, Street SW, Rochester, MN 55905, USA.

Together, single-cell technologies and systems biology have been used to investigate previously unanswerable questions in biomedicine with unparalleled detail. Despite these advances, gaps in analytical capacity remain. Machine learning, which has revolutionized biomedical imaging analysis, drug discovery, and systems biology, is an ideal strategy to fill these gaps in single-cell studies. Machine learning additionally has proven to be remarkably synergistic with single-cell data because it remedies unique challenges while capitalizing on the positive aspects of single-cell data. In this review, we describe how systems-biology algorithms have layered machine learning with biological components to provide systems level analyses of single-cell omics data, thus elucidating complex biological mechanisms. Accordingly, we highlight the trifecta of single-cell, systems-biology, and machine-learning approaches and illustrate how this trifecta can significantly contribute to five key areas of scientific research: cell trajectory and identity, individualized medicine, pharmacology, spatial omics, and multi-omics. Given its success to date, the systems-biology, single-cell omics, and machine-learning trifecta has proven to be a potent combination that will further advance biomedical research.
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http://dx.doi.org/10.3390/genes12071098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8306972PMC
July 2021

A CVID-associated variant in the ciliogenesis protein CCDC28B disrupts immune synapse assembly.

Cell Death Differ 2021 Jul 22. Epub 2021 Jul 22.

Department of Life Sciences, University of Siena, Siena, Italy.

Ciliogenesis proteins orchestrate vesicular trafficking pathways that regulate immune synapse (IS) assembly in the non-ciliated T-cells. We hypothesized that ciliogenesis-related genes might be disease candidates for common variable immunodeficiency with impaired T-cell function (T-CVID). We identified a heterozygous, predicted pathogenic variant in the ciliogenesis protein CCDC28B present with increased frequency in a large CVID cohort. We show that CCDC28B participates in IS assembly by regulating polarized T-cell antigen receptor (TCR) recycling. This involves the CCDC28B-dependent, FAM21-mediated recruitment of the actin regulator WASH to retromer at early endosomes to promote actin polymerization. The CVID-associated CCDC28B variant failed to interact with FAM21, leading to impaired synaptic TCR recycling. CVID T cells carrying the ccdc28b 211 C > T allele displayed IS defects mapping to this pathway that were corrected by overexpression of the wild-type allele. These results identify a new disease gene in T-CVID and pinpoint CCDC28B as a new player in IS assembly.
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http://dx.doi.org/10.1038/s41418-021-00837-5DOI Listing
July 2021

All ways lead to Rome: assembly of retromer on membranes with different sorting nexins.

Signal Transduct Target Ther 2021 Mar 31;6(1):139. Epub 2021 Mar 31.

Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Paediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy and Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu, China.

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http://dx.doi.org/10.1038/s41392-021-00561-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011043PMC
March 2021

Targeting Endosomal Recycling Pathways by Bacterial and Viral Pathogens.

Front Cell Dev Biol 2021 4;9:648024. Epub 2021 Mar 4.

Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Paediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy, Sichuan University, Chengdu, China.

Endosomes are essential cellular stations where endocytic and secretory trafficking routes converge. Proteins transiting at endosomes can be degraded via lysosome, or recycled to the plasma membrane, trans-Golgi network (TGN), or other cellular destinations. Pathways regulating endosomal recycling are tightly regulated in order to preserve organelle identity, to maintain lipid homeostasis, and to support other essential cellular functions. Recent studies have revealed that both pathogenic bacteria and viruses subvert host endosomal recycling pathways for their survival and replication. Several host factors that are frequently targeted by pathogens are being identified, including retromer, TBC1D5, SNX-BARs, and the WASH complex. In this review, we will focus on the recent advances in understanding how intracellular bacteria, human papillomavirus (HPV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijack host endosomal recycling pathways. This exciting work not only reveals distinct mechanisms employed by pathogens to manipulate host signaling pathways, but also deepens our understanding of the molecular intricacies regulating endosomal receptor trafficking.
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http://dx.doi.org/10.3389/fcell.2021.648024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7970000PMC
March 2021

DOCK7 protects against replication stress by promoting RPA stability on chromatin.

Nucleic Acids Res 2021 04;49(6):3322-3337

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.

RPA is a critical factor for DNA replication and replication stress response. Surprisingly, we found that chromatin RPA stability is tightly regulated. We report that the GDP/GTP exchange factor DOCK7 acts as a critical replication stress regulator to promote RPA stability on chromatin. DOCK7 is phosphorylated by ATR and then recruited by MDC1 to the chromatin and replication fork during replication stress. DOCK7-mediated Rac1/Cdc42 activation leads to the activation of PAK1, which subsequently phosphorylates RPA1 at S135 and T180 to stabilize chromatin-loaded RPA1 and ensure proper replication stress response. Moreover, DOCK7 is overexpressed in ovarian cancer and depleting DOCK7 sensitizes cancer cells to camptothecin. Taken together, our results highlight a novel role for DOCK7 in regulation of the replication stress response and highlight potential therapeutic targets to overcome chemoresistance in cancer.
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http://dx.doi.org/10.1093/nar/gkab134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034614PMC
April 2021

Phosphorylation of SNX27 by MAPK11/14 links cellular stress-signaling pathways with endocytic recycling.

J Cell Biol 2021 04;220(4)

Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Paediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy, Sichuan University, Chengdu, China.

Endocytosed proteins can be delivered to lysosomes for degradation or recycled to either the trans-Golgi network or the plasma membrane. It remains poorly understood how the recycling versus degradation of cargoes is determined. Here, we show that multiple extracellular stimuli, including starvation, LPS, IL-6, and EGF treatment, can strongly inhibit endocytic recycling of multiple cargoes through the activation of MAPK11/14. The stress-induced kinases in turn directly phosphorylate SNX27, a key regulator of endocytic recycling, at serine 51 (Ser51). Phosphorylation of SNX27 at Ser51 alters the conformation of its cargo-binding pocket and decreases the interaction between SNX27 and cargo proteins, thereby inhibiting endocytic recycling. Our study indicates that endocytic recycling is highly dynamic and can crosstalk with cellular stress-signaling pathways. Suppression of endocytic recycling and enhancement of receptor lysosomal degradation serve as new mechanisms for cells to cope with stress and save energy.
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http://dx.doi.org/10.1083/jcb.202010048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901142PMC
April 2021

Regulation of murine copper homeostasis by members of the COMMD protein family.

Dis Model Mech 2021 01 7;14(1). Epub 2021 Jan 7.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Copper is an essential transition metal for all eukaryotes. In mammals, intestinal copper absorption is mediated by the ATP7A copper transporter, whereas copper excretion occurs predominantly through the biliary route and is mediated by the paralog ATP7B. Both transporters have been shown to be recycled actively between the endosomal network and the plasma membrane by a molecular machinery known as the COMMD/CCDC22/CCDC93 or CCC complex. In fact, mutations in COMMD1 can lead to impaired biliary copper excretion and liver pathology in dogs and in mice with liver-specific Commd1 deficiency, recapitulating aspects of this phenotype. Nonetheless, the role of the CCC complex in intestinal copper absorption in vivo has not been studied, and the potential redundancy of various COMMD family members has not been tested. In this study, we examined copper homeostasis in enterocyte-specific and hepatocyte-specific COMMD gene-deficient mice. We found that, in contrast to effects in cell lines in culture, COMMD protein deficiency induced minimal changes in ATP7A in enterocytes and did not lead to altered copper levels under low- or high-copper diets, suggesting that regulation of ATP7A in enterocytes is not of physiological consequence. By contrast, deficiency of any of three COMMD genes (Commd1, Commd6 or Commd9) resulted in hepatic copper accumulation under high-copper diets. We found that each of these deficiencies caused destabilization of the entire CCC complex and suggest that this might explain their shared phenotype. Overall, we conclude that the CCC complex plays an important role in ATP7B endosomal recycling and function.
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http://dx.doi.org/10.1242/dmm.045963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803461PMC
January 2021

Corrigendum: An FGFR/AKT/SOX2 Signaling Axis Controls Pancreatic Cancer Stemness.

Front Cell Dev Biol 2020 2;8:594589. Epub 2020 Oct 2.

School of Pharmaceutical Sciences and International Collaborative Center on Growth Factor Research, Wenzhou Medical University, Wenzhou, China.

[This corrects the article DOI: 10.3389/fcell.2020.00287.].
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http://dx.doi.org/10.3389/fcell.2020.594589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566215PMC
October 2020

Loss of MAGEL2 in Prader-Willi syndrome leads to decreased secretory granule and neuropeptide production.

JCI Insight 2020 09 3;5(17). Epub 2020 Sep 3.

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

Prader-Willi syndrome (PWS) is a developmental disorder caused by loss of maternally imprinted genes on 15q11-q13, including melanoma antigen gene family member L2 (MAGEL2). The clinical phenotypes of PWS suggest impaired hypothalamic neuroendocrine function; however, the exact cellular defects are unknown. Here, we report deficits in secretory granule (SG) abundance and bioactive neuropeptide production upon loss of MAGEL2 in humans and mice. Unbiased proteomic analysis of Magel2pΔ/m+ mice revealed a reduction in components of SG in the hypothalamus that was confirmed in 2 PWS patient-derived neuronal cell models. Mechanistically, we show that proper endosomal trafficking by the MAGEL2-regulated WASH complex is required to prevent aberrant lysosomal degradation of SG proteins and reduction of mature SG abundance. Importantly, loss of MAGEL2 in mice, NGN2-induced neurons, and human patients led to reduced neuropeptide production. Thus, MAGEL2 plays an important role in hypothalamic neuroendocrine function, and cellular defects in this pathway may contribute to PWS disease etiology. Moreover, these findings suggest unanticipated approaches for therapeutic intervention.
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http://dx.doi.org/10.1172/jci.insight.138576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526459PMC
September 2020

The septin cytoskeleton regulates natural killer cell lytic granule release.

J Cell Biol 2020 11;219(11)

Department of Immunology, College of Medicine, Mayo Clinic, Rochester, MN.

Natural killer (NK) cell-mediated killing involves the membrane fusion of preformed lytic granules. While the roles of actin and microtubules are well accepted during this process, the function of septins, another cytoskeletal component that associates with actin and microtubules, has not been investigated. Here we show that genetic depletion or pharmacologic stabilization of the septin cytoskeleton significantly inhibited NK cell cytotoxicity. Although the stabilization of septin filaments impaired conjugate formation, depletion of septin proteins had no impact on conjugate formation, lytic granule convergence, or MTOC polarization to the cytotoxic synapse (CS). Interestingly, septins copurify and accumulate near the polarized lytic granules at the CS, where they regulate lytic granule release. Mechanistically, we find that septin 7 interacts with the SNARE protein syntaxin 11 and facilitates its interaction with syntaxin binding protein 2 to promote lytic granule fusion. Altogether, our data identify a critical role for septins in regulating the release of lytic granule contents during NK cell-mediated killing.
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http://dx.doi.org/10.1083/jcb.202002145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594501PMC
November 2020

Inactivation of Rho GTPases by Burkholderia cenocepacia Induces a WASH-Mediated Actin Polymerization that Delays Phagosome Maturation.

Cell Rep 2020 06;31(9):107721

Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.

Burkholderia cenocepacia is an opportunistic bacterial pathogen that causes severe pulmonary infections in cystic fibrosis and chronic granulomatous disease patients. B. cenocepacia can survive inside infected macrophages within the B. cenocepacia-containing vacuole (BcCV) and to elicit a severe inflammatory response. By inactivating the host macrophage Rho GTPases, the bacterial effector TecA causes depolymerization of the cortical actin cytoskeleton. In this study, we find that B. cenocepacia induces the formation of large cytosolic F-actin clusters in infected macrophages. Cluster formation requires the nucleation-promoting factor WASH, the Arp2/3 complex, and TecA. Inactivation of Rho GTPases by bacterial toxins is necessary and sufficient to induce the formation of the cytosolic actin clusters. By hijacking WASH and Arp2/3 activity, B. cenocepacia disrupts interactions with the endolysosomal system, thereby delaying the maturation of the BcCV.
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http://dx.doi.org/10.1016/j.celrep.2020.107721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7315377PMC
June 2020

An FGFR/AKT/SOX2 Signaling Axis Controls Pancreatic Cancer Stemness.

Front Cell Dev Biol 2020 7;8:287. Epub 2020 May 7.

School of Pharmaceutical Sciences and International Collaborative Center on Growth Factor Research, Wenzhou Medical University, Wenzhou, China.

Cancer stemness is associated with high malignancy and low differentiation, as well as therapeutic resistance of tumors including pancreatic ductal adenocarcinoma (PDAC). Fibroblast growth factors (FGFs) exert pleiotropic effects on a variety of cellular processes and functions including embryonic stem cell pluripotency and cancer cell stemness via the activation of four tyrosine kinase FGF receptors (FGFRs). FGF ligands have been a major component of the cocktail of growth factors contained in the cancer stemness-inducing (CSI) and organoid culture medium. Although FGF/FGFR signaling has been hypothesized to maintain cancer stemness, its function in this process is still unclear. We report that inhibition of FGF/FGFR signaling impairs sphere-forming ability of PDAC , and knocking down and decreased their tumorigenesis abilities . Mechanistically, we demonstrated that SOX2 is down-regulated upon loss of FGFR signaling. The overexpression of in SOX2-negative cells, which normally do not display stemness capabilities, is sufficient to induce spheroid formation. Additionally, we found that AKT phosphorylation was reduced upon FGFR signaling inhibition. The inhibition of AKT using specific pharmacological inhibitors in the context of CSI medium leads to the loss of spheroid formation associated with loss of SOX2 nuclear expression and increased degradation. We demonstrate that an FGFR/AKT/SOX2 axis controls cancer stemness in PDAC and therefore may represent an important therapeutic target in the fight against this very aggressive form of cancer.
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http://dx.doi.org/10.3389/fcell.2020.00287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7221133PMC
May 2020

Structure of TBC1D23 N-terminus reveals a novel role for rhodanese domain.

PLoS Biol 2020 05 26;18(5):e3000746. Epub 2020 May 26.

Department of Pediatric Surgery and Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.

Members of the Tre2-Bub2-Cdc16 (TBC) family often function to regulate membrane trafficking and to control signaling transductions pathways. As a member of the TBC family, TBC1D23 is critical for endosome-to-Golgi cargo trafficking by serving as a bridge between Golgi-bound golgin-97/245 and the WASH/FAM21 complex on endosomal vesicles. However, the exact mechanisms by which TBC1D23 regulates cargo transport are poorly understood. Here, we present the crystal structure of the N-terminus of TBC1D23 (D23N), which consists of both the TBC and rhodanese domains. We show that the rhodanese domain is unlikely to be an active sulfurtransferase or phosphatase, despite containing a putative catalytic site. Instead, it packs against the TBC domain and forms part of the platform to interact with golgin-97/245. Using the zebrafish model, we show that impacting golgin-97/245-binding, but not the putative catalytic site, impairs neuronal growth and brain development. Altogether, our studies provide structural and functional insights into an essential protein that is required for organelle-specific trafficking and brain development.
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http://dx.doi.org/10.1371/journal.pbio.3000746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274447PMC
May 2020

The WW domains dictate isoform-specific regulation of YAP1 stability and pancreatic cancer cell malignancy.

Theranostics 2020 15;10(10):4422-4436. Epub 2020 Mar 15.

School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China.

YAP1 is a key mediator of the Hippo pathway capable of exerting a profound effect on organ size as well as tumorigenesis. Alternative mRNA splicing of human YAP1 results in at least 8 protein isoforms that differ within the 2 WW motif and the transcriptional activation domain. : To investigate the isoform-specific differences in their mRNA expression, transcriptional activity and tumor-promoting function, we cloned cDNA encoding all of the eight YAP1 protein isoforms. Then, we examined their mRNA expression, subcellular localization, transcriptional regulation properties, interactions with key regulatory partners, and protein stability in response to changes in cell density, as well as their effects on pancreatic cancer cell malignancy both and . : Multiple YAP1 mRNA isoforms are expressed in commonly used pancreatic cancer lines as well as human pancreatic cancer PDX lines. Based on the analysis of heterologous reporter and endogenous target genes, all YAP1 isoforms are capable of activating transcription, albeit to a different extent. Importantly, we unveiled a marked discrepancy between the mRNA and protein expression levels of the YAP1-1 and YAP1-2 isoforms. We further discovered that the YAP1-2 isoform, which contains two tandem WW motifs, is less stable at the protein level, particularly at high cell densities. Mechanistically, we found that the presence of the 2 WW motif in YAP1-2 facilitates the formation of the YAP1-2/AMOT/LATS1 complex and contributes to a stronger binding of YAP1-2 to LATS1 and subsequently increased YAP1-2 ubiquitination and degradation by β-TRCP. : Our data reveals a potent effect of YAP1-1 on pancreatic cancer malignancy and and provides novel mechanistic insight into isoform-specific and cell density-dependent regulation of YAP1 stability, as well as its impact on cancer malignancy.
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http://dx.doi.org/10.7150/thno.42795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150473PMC
April 2021

Endosome-to-TGN Trafficking: Organelle-Vesicle and Organelle-Organelle Interactions.

Front Cell Dev Biol 2020 18;8:163. Epub 2020 Mar 18.

Key Laboratory of Birth Defects and Related Diseases of Women and Children, State Key Laboratory of Biotherapy, Department of Paediatrics, West China Second University Hospital, Sichuan University, Chengdu, China.

Retrograde transport from endosomes to the -Golgi network (TGN) diverts proteins and lipids away from lysosomal degradation. It is essential for maintaining cellular homeostasis and signaling. In recent years, significant advancements have been made in understanding this classical pathway, revealing new insights into multiple steps of vesicular trafficking as well as critical roles of ER-endosome contacts for endosomal trafficking. In this review, we summarize up-to-date knowledge about this trafficking pathway, in particular, mechanisms of cargo recognition at endosomes and vesicle tethering at the TGN, and contributions of ER-endosome contacts.
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http://dx.doi.org/10.3389/fcell.2020.00163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093645PMC
March 2020

Glycogen synthase kinase-3β: a novel therapeutic target for pancreatic cancer.

Expert Opin Ther Targets 2020 05 21;24(5):417-426. Epub 2020 Mar 21.

The Division of Oncology Research, Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN, USA.

: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States with a single-digit 5-year survival rate despite advances in understanding the genetics and biology of the disease. Glycogen synthase kinase-3α (GSK-3α) and GSK-3β are serine/threonine kinases that localize to the cytoplasm, mitochondria and nucleus. Although they are highly homologous within their kinase domains and phosphorylate an overlapping set of target proteins, genetic studies have shown that GSK-3β regulates the activity of several proteins that promote neoplastic transformation. Significantly, GSK-3β is progressively overexpressed during PDAC development where it participates in tumor progression, survival and chemoresistance. Thus, GSK-3β has become an attractive target for treating PDAC.: This review summarizes the mechanisms regulating GSK-3β activity, including upstream translational and post-translational regulation, as well as the downstream targets and their functions in PDAC cell growth, metastasis and chemoresistance.: The activity of GSK-3 kinases are considered cell- and context-specific. In PDAC, oncogenic KRas drives the transcriptional expression of the GSK-3β gene, which has been shown to regulate cancer cell proliferation and survival, as well as resistance to chemotherapy. Thus, the combination of GSK-3 inhibitors with chemotherapeutic drugs could be a promising strategy for PDAC.
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http://dx.doi.org/10.1080/14728222.2020.1743681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539227PMC
May 2020

Mechanism of cargo recognition by retromer-linked SNX-BAR proteins.

PLoS Biol 2020 03 9;18(3):e3000631. Epub 2020 Mar 9.

Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Paediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy and Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu, China.

Endocytic recycling of internalized transmembrane proteins is essential for many important physiological processes. Recent studies have revealed that retromer-related Sorting Nexin family (SNX)-Bin/Amphiphysin/Rvs (BAR) proteins can directly recognize cargoes like cation-independent mannose 6-phosphate receptor (CI-MPR) and Insulin-like growth factor 1 receptor (IGF1R); however, it remains poorly understood how SNX-BARs select specific cargo proteins and whether they recognize additional ligands. Here, we discovered that the binding between SNX-BARs and CI-MPR or IGF1R is mediated by the phox-homology (PX) domain of SNX5 or SNX6 and a bipartite motif, termed SNX-BAR-binding motif (SBM), in the cargoes. Using this motif, we identified over 70 putative SNX-BAR ligands, many of which play critical roles in apoptosis, cell adhesion, signal transduction, or metabolite homeostasis. Remarkably, SNX-BARs could cooperate with both SNX27 and retromer in the recycling of ligands encompassing the SBM, PDZ-binding motif, or both motifs. Overall, our studies establish that SNX-BARs function as a direct cargo-selecting module for a large set of transmembrane proteins transiting the endosome, in addition to their roles in phospholipid recognition and biogenesis of tubular structures.
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http://dx.doi.org/10.1371/journal.pbio.3000631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082075PMC
March 2020

Molecular regulation of the plasma membrane-proximal cellular steps involved in NK cell cytolytic function.

J Cell Sci 2020 02 21;133(5). Epub 2020 Feb 21.

Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA

Natural killer (NK) cells, cytolytic lymphocytes of the innate immune system, play a crucial role in the immune response against infection and cancer. NK cells kill target cells through exocytosis of lytic granules that contain cytotoxic proteins, such as perforin and granzymes. Formation of a functional immune synapse, i.e. the interface between the NK cell and its target cell enhances lysis through accumulation of polymerized F-actin at the NK cell synapse, leading to convergence of lytic granules to the microtubule organizing center (MTOC) and its subsequent polarization along microtubules to deliver the lytic granules to the synapse. In this review, we focus on the molecular mechanisms regulating the cellular processes that occur after the lytic granules are delivered to the cytotoxic synapse. We outline how - once near the synapse - the granules traverse the clearings created by F-actin remodeling to dock, tether and fuse with the plasma membrane in order to secrete their lytic content into the synaptic cleft through exocytosis. Further emphasis is given to the role of Ca mobilization during degranulation and, whenever applicable, we compare these mechanisms in NK cells and cytotoxic T lymphocytes (CTLs) as adaptive immune system effectors.
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http://dx.doi.org/10.1242/jcs.240424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044456PMC
February 2020

Immunity to Influenza is dependent on MHC II polymorphism: study with 2 HLA transgenic strains.

Sci Rep 2019 12 13;9(1):19061. Epub 2019 Dec 13.

Department of Immunology Mayo Clinic, Rochester, MN, USA.

Major histocompatibility complex II (MHC II) molecules are involved in antigen presentation and the development of a functional adaptive immune response. Evolutionary selection for MHC molecules that effectively clear infectious agents provides an advantage to humans. However, certain class II molecules are associated with autoimmune diseases. In this study we infected autoimmune-susceptible DRB1*0401.AEo and non-susceptible *0402.AEo mice with H1N1 influenza and determined clearance and protective immunity to H3N2 virus. *0401 mice generated a robust TLR-triggered immune response and cleared H1N1 influenza virus infection. After vaccination and challenge with H1N1, *0401 mice, when challenged with H3N2, generated cross-protective immunity to heterosubtypic H3N2 influenza strain whereas *0402 mice cleared the H1N1 infection but did not generate cross-protective immunity against the H3N2 influenza strain. The intracellular trafficking route of MHCII revealed that *0401 molecules traffic through the late endosome/lysosomes while *0402 molecules traffic into early endosomes. This suggested that trafficking of MHCII could affect the functional output of the innate immune response and clearance of viral infections. Also, DRB1*0401 mice live longer than HLA-DRB1*0402 mice. The study provides a potential hypothesis for evolutionary selection of *0401 molecule, even though it is associated with autoreactivity, which may be dependent on the availability of peptide repertoire of self-antigens.
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http://dx.doi.org/10.1038/s41598-019-55503-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911063PMC
December 2019

NK cell defects in X-linked pigmentary reticulate disorder.

JCI Insight 2019 11 1;4(21). Epub 2019 Nov 1.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

X-linked reticulate pigmentary disorder (XLPDR, Mendelian Inheritance in Man #301220) is a rare syndrome characterized by recurrent infections and sterile multiorgan inflammation. The syndrome is caused by an intronic mutation in POLA1, the gene encoding the catalytic subunit of DNA polymerase-α (Pol-α), which is responsible for Okazaki fragment synthesis during DNA replication. Reduced POLA1 expression in this condition triggers spontaneous type I interferon expression, which can be linked to the autoinflammatory manifestations of the disease. However, the history of recurrent infections in this syndrome is as yet unexplained. Here we report that patients with XLPDR have reduced NK cell cytotoxic activity and decreased numbers of NK cells, particularly differentiated, stage V, cells (CD3-CD56dim). This phenotype is reminiscent of hypomorphic mutations in MCM4, which encodes a component of the minichromosome maintenance (MCM) helicase complex that is functionally linked to Pol-α during the DNA replication process. We find that POLA1 deficiency leads to MCM4 depletion and that both can impair NK cell natural cytotoxicity and show that this is due to a defect in lytic granule polarization. Altogether, our study provides mechanistic connections between Pol-α and the MCM complex and demonstrates their relevance in NK cell function.
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http://dx.doi.org/10.1172/jci.insight.125688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948767PMC
November 2019

Structural and functional studies of TBC1D23 C-terminal domain provide a link between endosomal trafficking and PCH.

Proc Natl Acad Sci U S A 2019 11 17;116(45):22598-22608. Epub 2019 Oct 17.

Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Paediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy and Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu 610041, China;

Pontocerebellar hypoplasia (PCH) is a group of neurological disorders that affect the development of the brain, in particular, the pons and cerebellum. Homozygous mutations of TBC1D23 have been found recently to lead to PCH; however, the underlying molecular mechanisms remain unclear. Here, we show that the crystal structure of the TBC1D23 C-terminal domain adopts a Pleckstrin homology domain fold and selectively binds to phosphoinositides, in particular, PtdIns(4)P, through one surface while binding FAM21 via the opposite surface. Mutation of key residues of TBC1D23 or FAM21 selectively disrupts the endosomal vesicular trafficking toward the Trans-Golgi Network. Finally, using the zebrafish model, we show that PCH patient-derived mutants, impacting either phosphoinositide binding or FAM21 binding, lead to abnormal neuronal growth and brain development. Taken together, our data provide a molecular basis for the interaction between TBC1D23 and FAM21, and suggest a plausible role for PtdIns(4)P in the TBC1D23-mediating endosome-to-TGN trafficking pathway. Defects in this trafficking pathway are, at least partially, responsible for the pathogenesis of certain types of PCH.
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http://dx.doi.org/10.1073/pnas.1909316116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842616PMC
November 2019

Endosomal PI(3)P regulation by the COMMD/CCDC22/CCDC93 (CCC) complex controls membrane protein recycling.

Nat Commun 2019 09 19;10(1):4271. Epub 2019 Sep 19.

Division of Oncology Research and Department of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic, Rochester, MN, 55905, USA.

Protein recycling through the endolysosomal system relies on molecular assemblies that interact with cargo proteins, membranes, and effector molecules. Among them, the COMMD/CCDC22/CCDC93 (CCC) complex plays a critical role in recycling events. While CCC is closely associated with retriever, a cargo recognition complex, its mechanism of action remains unexplained. Herein we show that CCC and retriever are closely linked through sharing a common subunit (VPS35L), yet the integrity of CCC, but not retriever, is required to maintain normal endosomal levels of phosphatidylinositol-3-phosphate (PI(3)P). CCC complex depletion leads to elevated PI(3)P levels, enhanced recruitment and activation of WASH (an actin nucleation promoting factor), excess endosomal F-actin and trapping of internalized receptors. Mechanistically, we find that CCC regulates the phosphorylation and endosomal recruitment of the PI(3)P phosphatase MTMR2. Taken together, we show that the regulation of PI(3)P levels by the CCC complex is critical to protein recycling in the endosomal compartment.
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http://dx.doi.org/10.1038/s41467-019-12221-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753146PMC
September 2019

Glycogen Synthase Kinase-3 Inhibition Sensitizes Pancreatic Cancer Cells to Chemotherapy by Abrogating the TopBP1/ATR-Mediated DNA Damage Response.

Clin Cancer Res 2019 11 18;25(21):6452-6462. Epub 2019 Sep 18.

The Division of Oncology Research, Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, Minnesota.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a predominantly fatal common malignancy with inadequate treatment options. Glycogen synthase kinase 3β (GSK-3β) is an emerging target in human malignancies including PDAC. Pancreatic cancer cell lines and patient-derived xenografts were treated with a novel GSK-3 inhibitor 9-ING-41 alone or in combination with chemotherapy. Activation of the DNA damage response pathway and S-phase arrest induced by gemcitabine were assessed in pancreatic tumor cells with pharmacologic inhibition or siRNA depletion of GSK-3 kinases by immunoblotting, flow cytometry, and immunofluorescence.

Results: 9-ING-41 treatment significantly increased pancreatic tumor cell killing when combined with chemotherapy. Inhibition of GSK-3 by 9-ING-41 prevented gemcitabine-induced S-phase arrest suggesting an impact on the ATR-mediated DNA damage response. Both 9-ING-41 and siRNA depletion of GSK-3 kinases impaired the activation of ATR leading to the phosphorylation and activation of Chk1. Mechanistically, depletion or knockdown of GSK-3 kinases resulted in the degradation of the ATR-interacting protein TopBP1, thus limiting the activation of ATR in response to single-strand DNA damage.

Conclusions: These data identify a previously unknown role for GSK-3 kinases in the regulation of the TopBP1/ATR/Chk1 DNA damage response pathway. The data also support the inclusion of patients with PDAC in clinical studies of 9-ING-41 alone and in combination with gemcitabine.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825568PMC
November 2019

-endocytosis elicited by nectins transfers cytoplasmic cargo, including infectious material, between cells.

J Cell Sci 2019 08 23;132(16). Epub 2019 Aug 23.

Department of Molecular Medicine, Mayo Clinic, Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN 55905, USA

Here, we show that cells expressing the adherens junction protein nectin-1 capture nectin-4-containing membranes from the surface of adjacent cells in a -endocytosis process. We find that internalized nectin-1-nectin-4 complexes follow the endocytic pathway. The nectin-1 cytoplasmic tail controls transfer: its deletion prevents -endocytosis, while its exchange with the nectin-4 tail reverses transfer direction. Nectin-1-expressing cells acquire dye-labeled cytoplasmic proteins synchronously with nectin-4, a process most active during cell adhesion. Some cytoplasmic cargo remains functional after transfer, as demonstrated with encapsidated genomes of measles virus (MeV). This virus uses nectin-4, but not nectin-1, as a receptor. Epithelial cells expressing nectin-4, but not those expressing another MeV receptor in its place, can transfer infection to nectin-1-expressing primary neurons. Thus, this newly discovered process can move cytoplasmic cargo, including infectious material, from epithelial cells to neurons. We name the process nectin-elicited cytoplasm transfer (NECT). NECT-related endocytosis processes may be exploited by pathogens to extend tropism. This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.235507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737912PMC
August 2019

NKG2D-DAP10 signaling recruits EVL to the cytotoxic synapse to generate F-actin and promote NK cell cytotoxicity.

J Cell Sci 2019 07 18;133(5). Epub 2019 Jul 18.

Department of Immunology, Schulze Center for Novel Therapeutics, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA

Natural killer (NK) cells eliminate abnormal cells through the release of cytolytic granule contents. In this process, NK cells must adhere to target cells through integrin-mediated adhesion, which is highly dependent on the generation of F-actin. Ena/VASP-like (EVL) is an actin regulatory protein previously shown to regulate integrin-mediated adhesion in other cell types, but its role in NK cell biology is not known. Herein, we show that EVL is recruited to the NK cell cytotoxic synapse and is required for NK cell cytotoxicity. Significantly, EVL is involved in the generation of F-actin at the cytotoxic synapse, antibody-stimulated spreading, and NK cell-target cell adhesion. EVL interacts with WASP (also known as WAS) and VASP and is required for localization of both proteins to the synapse. Recruitment of EVL to points of cellular activation occurs through the receptor NKG2D-DAP10 (also known as KLRK1 and HCST, respectively) via a binding site previously implicated in VAV1 and Grb2 recruitment. Taken together, this study implicates DAP10-mediated Grb2 and VAV1 signaling in the recruitment of an EVL-containing actin regulatory complex to the cytotoxic synapse where it can promote F-actin nucleation leading to NK cell-mediated killing.
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http://dx.doi.org/10.1242/jcs.230508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055393PMC
July 2019

The hepatic WASH complex is required for efficient plasma LDL and HDL cholesterol clearance.

JCI Insight 2019 06 6;4(11). Epub 2019 Jun 6.

Department of Pediatrics, University of Groningen, University Medical Center Groningen, Groningen, Netherlands.

The evolutionary conserved Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex is one of the crucial multiprotein complexes that facilitates endosomal recycling of transmembrane proteins. Defects in WASH components have been associated with inherited developmental and neurological disorders in humans. Here, we show that hepatic ablation of the WASH component Washc1 in chow-fed mice increases plasma concentrations of cholesterol in both LDLs and HDLs, without affecting hepatic cholesterol content, hepatic cholesterol synthesis, biliary cholesterol excretion, or hepatic bile acid metabolism. Elevated plasma LDL cholesterol was related to reduced hepatocytic surface levels of the LDL receptor (LDLR) and the LDLR-related protein LRP1. Hepatic WASH ablation also reduced the surface levels of scavenger receptor class B type I and, concomitantly, selective uptake of HDL cholesterol into the liver. Furthermore, we found that WASHC1 deficiency increases LDLR proteolysis by the inducible degrader of LDLR, but does not affect proprotein convertase subtilisin/kexin type 9-mediated LDLR degradation. Remarkably, however, loss of hepatic WASHC1 may sensitize LDLR for proprotein convertase subtilisin/kexin type 9-induced degradation. Altogether, these findings identify the WASH complex as a regulator of LDL as well as HDL metabolism and provide in vivo evidence for endosomal trafficking of scavenger receptor class B type I in hepatocytes.
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http://dx.doi.org/10.1172/jci.insight.126462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629119PMC
June 2019
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