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1.
iScience ; 26(5): 106525, 2023 May 19.
Article in English | MEDLINE | ID: mdl-37250326

ABSTRACT

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor central in the regulation of key cellular processes including cell metabolism, tissue differentiation, and regulation of the immune system. PPARγ is required for normal differentiation of the urothelium and is thought to be an essential driver of the luminal subtype of bladder cancer. However, the molecular components that regulate PPARG gene expression in bladder cancer remain unclear. Here, we developed an endogenous PPARG reporter system in luminal bladder cancer cells and performed genome-wide CRISPR knockout screening to identify bona fide regulators of PPARG gene expression. Functional validation of the dataset confirmed GATA3, SPT6, and the cohesin complex components SMC1A, and RAD21, as permissive upstream positive regulators of PPARG gene expression in luminal bladder cancer. In summary, this work provides a resource and biological insights to aid our understanding of PPARG regulation in bladder cancer.

2.
Nat Commun ; 14(1): 1394, 2023 03 13.
Article in English | MEDLINE | ID: mdl-36914633

ABSTRACT

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.


Subject(s)
Antibodies, Bispecific , Antineoplastic Agents , Breast Neoplasms , Humans , Female , Xenograft Model Antitumor Assays , Cell Line, Tumor , Trastuzumab/pharmacology , Trastuzumab/therapeutic use , Receptor, ErbB-2/metabolism , Antibody-Dependent Cell Cytotoxicity , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy
3.
Elife ; 82019 06 03.
Article in English | MEDLINE | ID: mdl-31157616

ABSTRACT

When B cells encounter antigens on the surface of an antigen-presenting cell (APC), B cell receptors (BCRs) are gathered into microclusters that recruit signaling enzymes. These microclusters then move centripetally and coalesce into the central supramolecular activation cluster of an immune synapse. The mechanisms controlling BCR organization during immune synapse formation, and how this impacts BCR signaling, are not fully understood. We show that this coalescence of BCR microclusters depends on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, in murine B cells, this dynamic spatial reorganization of BCR microclusters amplifies proximal BCR signaling reactions and enhances the ability of membrane-associated antigens to induce transcriptional responses and proliferation. Our finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound antigens, but not for soluble antigens, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound antigens.


Subject(s)
Actin-Related Protein 3/metabolism , Angiopoietin-like Proteins/metabolism , B-Lymphocytes/immunology , Immunological Synapses/metabolism , Lymphocyte Activation , Receptors, Antigen, B-Cell/metabolism , Signal Transduction , Actins/metabolism , Angiopoietin-Like Protein 2 , Animals , Mice, Inbred C57BL
4.
J Proteome Res ; 15(5): 1613-22, 2016 05 06.
Article in English | MEDLINE | ID: mdl-27018634

ABSTRACT

Enteropathogenic and enterohemorrhagic Escherichia coli cause enteric diseases resulting in significant morbidity and mortality worldwide. These pathogens remain extracellular and translocate a set of type III secreted effector proteins into host cells to promote bacterial virulence. Effectors manipulate host cell pathways to facilitate infection by interacting with a variety of host targets, yet the binding partners and mechanism of action of many effectors remain elusive. We performed a mass spectrometry screen to identify host targets for a library of effectors. We found five known effector targets and discovered four novel interactions. Interestingly, we identified multiple effectors that interacted with the microtubule associated protein, ensconsin. Using co-immunoprecipitations, we confirmed that NleB1 and EspL interacted with ensconsin in a region that corresponded to its microtubule binding domain. Ensconsin is an essential cofactor of kinesin-1 that is required for intracellular trafficking, and we demonstrated that intracellular trafficking was severely disrupted during wild type EPEC infections but not during infections with ΔnleB1 or ΔespL mutants. Our findings demonstrate the efficacy of quantitative proteomics for identifying effector-host protein interactions and suggest that vesicular trafficking is a crucial cellular process that may be targeted by NleB1 and EspL through their interaction with ensconsin.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/pathogenicity , Host-Pathogen Interactions , Type III Secretion Systems/metabolism , Virulence Factors/metabolism , Cell Line , Humans , Immunoprecipitation , Mass Spectrometry , Microtubule-Associated Proteins/metabolism , Protein Binding , Type III Secretion Systems/chemistry
5.
Nat Commun ; 6: 8777, 2015 Nov 03.
Article in English | MEDLINE | ID: mdl-26525107

ABSTRACT

Antigen receptor signalling activates the canonical NF-κB pathway via the CARD11/BCL10/MALT1 (CBM) signalosome involving key, yet ill-defined roles for linear ubiquitination. The paracaspase MALT1 cleaves and removes negative checkpoint proteins, amplifying lymphocyte responses in NF-κB activation and in B-cell lymphoma subtypes. To identify new human MALT1 substrates, we compare B cells from the only known living MALT1(mut/mut) patient with healthy MALT1(+/mut) family members using 10-plex Tandem Mass Tag TAILS N-terminal peptide proteomics. We identify HOIL1 of the linear ubiquitin chain assembly complex as a novel MALT1 substrate. We show linear ubiquitination at B-cell receptor microclusters and signalosomes. Late in the NF-κB activation cycle HOIL1 cleavage transiently reduces linear ubiquitination, including of NEMO and RIP1, dampening NF-κB activation and preventing reactivation. By regulating linear ubiquitination, MALT1 is both a positive and negative pleiotropic regulator of the human canonical NF-κB pathway-first promoting activation via the CBM--then triggering HOIL1-dependent negative-feedback termination, preventing reactivation.


Subject(s)
Caspases/genetics , Immunologic Deficiency Syndromes/genetics , Lymphocytes/immunology , NF-kappa B/metabolism , Neoplasm Proteins/genetics , Ubiquitin-Protein Ligases/metabolism , Adolescent , Adult , Animals , Antigen-Presenting Cells , B-Lymphocytes/immunology , Caspases/immunology , Caspases/metabolism , Family , Female , Fluorescent Antibody Technique , GTPase-Activating Proteins/metabolism , Gene Knock-In Techniques , Humans , I-kappa B Kinase/metabolism , Immunoblotting , Immunologic Deficiency Syndromes/immunology , Immunoprecipitation , In Vitro Techniques , Intracellular Signaling Peptides and Proteins/metabolism , Leukocytes, Mononuclear , Male , Mass Spectrometry , Mice , Microscopy, Confocal , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Mutation , NF-kappa B/immunology , Neoplasm Proteins/immunology , Neoplasm Proteins/metabolism , Nuclear Pore Complex Proteins/metabolism , Palatine Tonsil , Proteomics , RNA-Binding Proteins/metabolism , T-Lymphocytes/immunology , Tandem Mass Spectrometry , Transcription Factors , Ubiquitination/immunology
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