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1.
Cancer Immunol Res ; 11(5): 629-645, 2023 05 03.
Article in English | MEDLINE | ID: mdl-36881002

ABSTRACT

The composition of the tumor immune microenvironment (TIME) is considered a key determinant of patients' response to immunotherapy. The mechanisms underlying TIME formation and development over time are poorly understood. Glioblastoma (GBM) is a lethal primary brain cancer for which there are no curative treatments. GBMs are immunologically heterogeneous and impervious to checkpoint blockade immunotherapies. Utilizing clinically relevant genetic mouse models of GBM, we identified distinct immune landscapes associated with expression of EGFR wild-type and mutant EGFRvIII cancer driver mutations. Over time, accumulation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) was more pronounced in EGFRvIII-driven GBMs and was correlated with resistance to PD-1 and CTLA-4 combination checkpoint blockade immunotherapy. We determined that GBM-secreted CXCL1/2/3 and PMN-MDSC-expressed CXCR2 formed an axis regulating output of PMN-MDSCs from the bone marrow leading to systemic increase in these cells in the spleen and GBM tumor-draining lymph nodes. Pharmacologic targeting of this axis induced a systemic decrease in the numbers of PMN-MDSC, facilitated responses to PD-1 and CTLA-4 combination checkpoint blocking immunotherapy, and prolonged survival in mice bearing EGFRvIII-driven GBM. Our results uncover a relationship between cancer driver mutations, TIME composition, and sensitivity to checkpoint blockade in GBM and support the stratification of patients with GBM for checkpoint blockade therapy based on integrated genotypic and immunologic profiles.


Subject(s)
Brain Neoplasms , Glioblastoma , Myeloid-Derived Suppressor Cells , Animals , Mice , Glioblastoma/therapy , Glioblastoma/drug therapy , CTLA-4 Antigen/genetics , CTLA-4 Antigen/metabolism , Programmed Cell Death 1 Receptor , Cell Line, Tumor , Immunotherapy , Mutation , Tumor Microenvironment/genetics , Brain Neoplasms/genetics , Brain Neoplasms/therapy
2.
Nat Immunol ; 24(1): 55-68, 2023 01.
Article in English | MEDLINE | ID: mdl-36581713

ABSTRACT

The inhibitory receptor PD-1 suppresses T cell activation by recruiting the phosphatase SHP-2. However, mice with a T-cell-specific deletion of SHP-2 do not have improved antitumor immunity. Here we showed that mice with conditional targeting of SHP-2 in myeloid cells, but not in T cells, had diminished tumor growth. RNA sequencing (RNA-seq) followed by gene set enrichment analysis indicated the presence of polymorphonuclear myeloid-derived suppressor cells and tumor-associated macrophages (TAMs) with enriched gene expression profiles of enhanced differentiation, activation and expression of immunostimulatory molecules. In mice with conditional targeting of PD-1 in myeloid cells, which also displayed diminished tumor growth, TAMs had gene expression profiles enriched for myeloid differentiation, activation and leukocyte-mediated immunity displaying >50% overlap with enriched profiles of SHP-2-deficient TAMs. In bone marrow, GM-CSF induced the phosphorylation of PD-1 and recruitment of PD-1-SHP-2 to the GM-CSF receptor. Deletion of SHP-2 or PD-1 enhanced GM-CSF-mediated phosphorylation of the transcription factors HOXA10 and IRF8, which regulate myeloid differentiation and monocytic-moDC lineage commitment, respectively. Thus, SHP-2 and PD-1-SHP-2 signaling restrained myelocyte differentiation resulting in a myeloid landscape that suppressed antitumor immunity.


Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor , Neoplasms , Animals , Mice , Cell Differentiation , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Myeloid Cells , Programmed Cell Death 1 Receptor/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Signal Transduction
3.
J Vis Exp ; (177)2021 11 16.
Article in English | MEDLINE | ID: mdl-34866628

ABSTRACT

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.


Subject(s)
Immunity, Innate , Lung , Animals , Flow Cytometry/methods , Immunophenotyping , Mice
4.
Sci Immunol ; 5(43)2020 01 03.
Article in English | MEDLINE | ID: mdl-31901074

ABSTRACT

PD-1, a T cell checkpoint receptor and target of cancer immunotherapy, is also expressed on myeloid cells. The role of myeloid-specific versus T cell-specific PD-1 ablation on antitumor immunity has remained unclear because most studies have used either PD-1-blocking antibodies or complete PD-1 KO mice. We generated a conditional allele, which allowed myeloid-specific (PD-1f/fLysMcre) or T cell-specific (PD-1f/fCD4cre) targeting of Pdcd1 gene. Compared with T cell-specific PD-1 ablation, myeloid cell-specific PD-1 ablation more effectively decreased tumor growth. We found that granulocyte/macrophage progenitors (GMPs), which accumulate during cancer-driven emergency myelopoiesis and give rise to myeloid-derived suppressor cells (MDSCs), express PD-1. In tumor-bearing PD-1f/fLysMcre but not PD-1f/fCD4cre mice, accumulation of GMP and MDSC was prevented, whereas systemic output of effector myeloid cells was increased. Myeloid cell-specific PD-1 ablation induced an increase of T effector memory cells with improved functionality and mediated antitumor protection despite preserved PD-1 expression in T cells. In PD-1-deficient myeloid progenitors, growth factors driving emergency myelopoiesis induced increased metabolic intermediates of glycolysis, pentose phosphate pathway, and TCA cycle but, most prominently, elevated cholesterol. Because cholesterol is required for differentiation of inflammatory macrophages and DC and promotes antigen-presenting function, our findings indicate that metabolic reprogramming of emergency myelopoiesis and differentiation of effector myeloid cells might be a key mechanism of antitumor immunity mediated by PD-1 blockade.


Subject(s)
Colonic Neoplasms/immunology , Melanoma/immunology , Myeloid Cells/immunology , Programmed Cell Death 1 Receptor/immunology , Animals , Cell Differentiation , Cell Line, Tumor , Female , Male , Mice, Inbred C57BL , Mice, Transgenic , Programmed Cell Death 1 Receptor/genetics
5.
J Vis Exp ; (141)2018 11 21.
Article in English | MEDLINE | ID: mdl-30531720

ABSTRACT

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer's patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes. Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on TFH and GC B cell subsets.


Subject(s)
B-Lymphocytes/immunology , Flow Cytometry/methods , Immunity, Humoral/physiology , Peyer's Patches/cytology , Peyer's Patches/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Cell Differentiation/immunology , Germinal Center/immunology , Immunity, Mucosal/physiology , Mice
6.
Am J Physiol Heart Circ Physiol ; 311(6): H1392-H1408, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27694217

ABSTRACT

The HDL receptor SR-BI mediates the transfer of cholesteryl esters from HDL to cells and controls HDL abundance and structure. Depending on the genetic background, loss of SR-BI causes hypercholesterolemia, anemia, reticulocytosis, splenomegaly, thrombocytopenia, female infertility, and fatal coronary heart disease (CHD). The carboxy terminus of SR-BI (505QEAKL509) must bind to the cytoplasmic adaptor PDZK1 for normal hepatic-but not steroidogenic cell-expression of SR-BI protein. To determine whether SR-BI's carboxy terminus is also required for normal protein levels in steroidogenic cells, we introduced into SR-BI's gene a 507Ala/STOP mutation that produces a truncated receptor (SR-BIΔCT). As expected, the dramatic reduction of hepatic receptor protein in SR-BIΔCT mice was similar to that in PDZK1 knockout (KO) mice. Unlike SR-BI KO females, SR-BIΔCT females were fertile. The severity of SR-BIΔCT mice's hypercholesterolemia was intermediate between those of SR-BI KO and PDZK1 KO mice. Substantially reduced levels of the receptor in adrenal cortical cells, ovarian cells, and testicular Leydig cells in SR-BIΔCT mice suggested that steroidogenic cells have an adaptor(s) functionally analogous to hepatic PDZK1. When SR-BIΔCT mice were crossed with apolipoprotein E KO mice (SR-BIΔCT/apoE KO), pathologies including hypercholesterolemia, macrocytic anemia, hepatic and splenic extramedullary hematopoiesis, massive splenomegaly, reticulocytosis, thrombocytopenia, and rapid-onset and fatal occlusive coronary arterial atherosclerosis and CHD (median age of death: 9 wk) were observed. These results provide new insights into the control of SR-BI in steroidogenic cells and establish SR-BIΔCT/apoE KO mice as a new animal model for the study of CHD.


Subject(s)
Adrenal Cortex/metabolism , Hypercholesterolemia/genetics , Leydig Cells/metabolism , Liver/metabolism , Ovary/metabolism , Scavenger Receptors, Class B/genetics , Anemia, Macrocytic/genetics , Animals , Apolipoproteins E/genetics , Coronary Artery Disease/genetics , Coronary Artery Disease/mortality , Coronary Disease/genetics , Coronary Disease/mortality , Coronary Occlusion/genetics , Coronary Occlusion/mortality , Female , Gene Knock-In Techniques , Hematopoiesis, Extramedullary/genetics , Immunoblotting , Lipoproteins, HDL/genetics , Male , Mice , Mutation , Polymerase Chain Reaction , Receptors, Lipoprotein/genetics , Reticulocytosis/genetics , Splenomegaly/genetics , Thrombocytopenia/genetics , Transcriptome
7.
J Biol Chem ; 288(27): 19845-60, 2013 Jul 05.
Article in English | MEDLINE | ID: mdl-23720744

ABSTRACT

The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI's cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1's regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.


Subject(s)
Cell Membrane/metabolism , Hepatocytes/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , PDZ Domains/physiology , Scavenger Receptors, Class B/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Membrane/chemistry , Cell Membrane/genetics , Cells, Cultured , Deuterium Exchange Measurement , Hepatocytes/chemistry , Hepatocytes/cytology , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins , Mice , Mice, Knockout , Mutation, Missense , Protein Binding/physiology , Scavenger Receptors, Class B/chemistry , Scavenger Receptors, Class B/genetics , Sequence Deletion , Surface Plasmon Resonance
8.
PLoS One ; 8(2): e53819, 2013.
Article in English | MEDLINE | ID: mdl-23457445

ABSTRACT

The prostanoid prostacyclin, or prostaglandin I2, plays an essential role in many aspects of cardiovascular disease. The actions of prostacyclin are mainly mediated through its activation of the prostacyclin receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1) of PDZK1, we generated a nine residue peptide (KK(411)IAACSLC(417)) containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 µM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP ((411)IAACSLC(417)). Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 at least in vitro.


Subject(s)
Carrier Proteins/metabolism , PDZ Domains , Receptors, Epoprostenol/metabolism , Amino Acid Sequence , Carrier Proteins/chemistry , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Interaction Mapping , Receptors, Epoprostenol/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
9.
J Biol Chem ; 286(28): 25171-86, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21602281

ABSTRACT

The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1-PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI ("target peptide") binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr(20) → Ala) only partially, rather than completely, suppresses the ability of PDZK1 to control hepatic SR-BI. We used isothermal titration calorimetry to show that PDZ3, but not PDZ2 or PDZ4, can also bind the target peptide (K(d) = 37.0 µm), albeit with ∼10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr(253) → Ala substitution. Comparison of the 1.5-Å resolution crystal structure of PDZ3 with its bound target peptide ((505)QEAKL(509)) to that of peptide-bound PDZ1 indicated fewer target peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr(20) → Ala (PDZ1) + Tyr(253) → Ala (PDZ3)) substitution abrogated all target peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr(253) → Ala (PDZ3)) PDZK1 transgene (Tg) was able to correct all of the SR-BI-related defects in PDZK1 knock-out mice, whereas the doubly substituted [Tyr(20) → Ala (PDZ1) + Tyr(253) → Ala (PDZ3)]Tg was unable to correct these defects. Thus, we conclude that PDZK1-mediated control of hepatic SR-BI requires direct binding of the SR-BI C terminus to either the PDZ1 or PDZ3 domains, and that binding to both domains simultaneously is not required for PDZK1 control of hepatic SR-BI.


Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Liver/metabolism , Scavenger Receptors, Class B/metabolism , Amino Acid Substitution , Animals , Binding Sites , Gene Knock-In Techniques , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins , Mice , Mice, Knockout , Mutation, Missense , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Protein Binding , Scavenger Receptors, Class B/chemistry , Scavenger Receptors, Class B/genetics , Transgenes
10.
J Biol Chem ; 285(45): 34999-5010, 2010 Nov 05.
Article in English | MEDLINE | ID: mdl-20739281

ABSTRACT

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys(14)-Xaa(4)-Asn(19)-Tyr-Gly-Phe-Phe-Leu(24)), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI ((503)VLQEAKL(509)). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K(d)) of the K14A and F22A mutants were 3.2 and 4.0 µM, respectively, similar to 2.6 µM measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI ((505)QEAKL(509)) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10-20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.


Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Liver/metabolism , Scavenger Receptors, Class B/metabolism , Amino Acid Substitution , Animals , Cholesterol/genetics , Cholesterol/metabolism , Crystallography, X-Ray , Female , Gene Expression Regulation/genetics , Intracellular Signaling Peptides and Proteins/genetics , Lipoproteins/genetics , Lipoproteins/metabolism , Male , Membrane Proteins , Mice , Mice, Knockout , Mutation, Missense , Protein Structure, Tertiary , Scavenger Receptors, Class B/genetics
11.
PLoS One ; 4(12): e8103, 2009 Dec 01.
Article in English | MEDLINE | ID: mdl-19956623

ABSTRACT

BACKGROUND: PDZK1 is a four PDZ-domain containing protein that binds to the carboxy terminus of the HDL receptor, scavenger receptor class B type I (SR-BI), and regulates its expression, localization and function in a tissue-specific manner. PDZK1 knockout (KO) mice are characterized by a marked reduction of SR-BI protein expression ( approximately 95%) in the liver (lesser or no reduction in other organs) with a concomitant 1.7 fold increase in plasma cholesterol. PDZK1 has been shown to be atheroprotective using the high fat/high cholesterol ('Western') diet-fed murine apolipoprotein E (apoE) KO model of atherosclerosis, presumably because of its role in promoting reverse cholesterol transport via SR-BI. PRINCIPAL FINDINGS: Here, we have examined the effects of PDZK1 deficiency in apoE KO mice fed with the atherogenic 'Paigen' diet for three months. Relative to apoE KO, PDZK1/apoE double KO (dKO) mice showed increased plasma lipids (33% increase in total cholesterol; 49 % increase in unesterified cholesterol; and 36% increase in phospholipids) and a 26% increase in aortic root lesions. Compared to apoE KO, dKO mice exhibited substantial occlusive coronary artery disease: 375% increase in severe occlusions. Myocardial infarctions, not observed in apoE KO mice (although occasional minimal fibrosis was noted), were seen in 7 of 8 dKO mice, resulting in 12 times greater area of fibrosis in dKO cardiac muscle. CONCLUSIONS: These results show that Paigen-diet fed PDZK1/apoE dKO mice represent a new animal model useful for studying coronary heart disease and suggest that PDZK1 may represent a valuable target for therapeutic intervention.


Subject(s)
Apolipoproteins E/deficiency , Coronary Occlusion/complications , Coronary Occlusion/metabolism , Dietary Fats/pharmacology , Intracellular Signaling Peptides and Proteins/metabolism , Myocardial Infarction/complications , Myocardial Infarction/metabolism , Animals , Aorta/drug effects , Aorta/enzymology , Aorta/pathology , Apolipoproteins E/metabolism , Atherosclerosis/blood , Atherosclerosis/complications , Atherosclerosis/metabolism , Atherosclerosis/pathology , CD36 Antigens/metabolism , Cholesterol/blood , Coronary Occlusion/blood , Coronary Occlusion/pathology , Dietary Fats/administration & dosage , Immunoblotting , Intracellular Signaling Peptides and Proteins/deficiency , Lipoproteins/blood , Liver/drug effects , Liver/metabolism , Liver/pathology , Membrane Proteins , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardial Infarction/blood , Myocardial Infarction/pathology
12.
J Biol Chem ; 284(9): 5797-806, 2009 Feb 27.
Article in English | MEDLINE | ID: mdl-19116202

ABSTRACT

PDZK1 is a four PDZ domain-containing scaffold protein that binds to scavenger receptor class B, type I (SR-BI), the high density lipoprotein receptor, by its first PDZ domain (PDZ1). PDZK1 knock-out mice exhibit a >95% decrease in hepatic SR-BI protein and consequently an approximately 70% increase in plasma cholesterol in abnormally large high density lipoprotein particles. These defects are corrected by hepatic overexpression of full-length PDZK1 but not the PDZ1 domain alone, which partially restores SR-BI protein abundance but not cell surface expression or function. We have generated PDZK1 knock-out mice with hepatic expression of four PDZK1 transgenes encoding proteins with nested C-terminal truncations: pTEM, which lacks the three C-terminal residues (putative PDZ-binding motif), and PDZ1.2, PDZ1.2.3, or PDZ1.2.3.4, which contain only the first two, three, or four N-terminal PDZ domains, respectively, but not the remaining C-terminal sequences. Hepatic overexpression of pTEM restored normal hepatic SR-BI abundance, localization, and function. Hepatic overexpression of PDZ1.2 or PDZ1.2.3 partially restored SR-BI abundance ( approximately 12 or approximately 30% of wild type, respectively) but did not (PDZ1.2) or only slightly (PDZ1.2.3) restored hepatic SR-BI cell surface localization and function. Hepatic overexpression of PDZ1.2.3.4 completely restored SR-BI protein abundance, cell surface expression, and function (normalization of plasma cholesterol levels). Thus, all four PDZ domains in PDZK1, but not PDZ1-3 alone, are sufficient for its normal control of the abundance, localization, and therefore function of hepatic SR-BI, whereas the residues C-terminal to the PDZ4 domain, including the C-terminal putative PDZ-binding domain, are not required.


Subject(s)
CD36 Antigens/metabolism , Cell Membrane/metabolism , Hepatocytes/metabolism , Intracellular Signaling Peptides and Proteins/physiology , Liver/metabolism , PDZ Domains/physiology , Animals , CD36 Antigens/genetics , Immunoblotting , Lipoproteins/analysis , Male , Membrane Proteins , Mice , Mice, Knockout , Mice, Transgenic , Transgenes/physiology
13.
J Biol Chem ; 283(32): 22097-104, 2008 Aug 08.
Article in English | MEDLINE | ID: mdl-18544532

ABSTRACT

PDZK1 is a four-PDZ domain-containing scaffold protein that, via its first PDZ domain (PDZ1), binds to the C terminus of the high density lipoprotein (HDL) receptor scavenger receptor, class B, type I (SR-BI). Abolishing PDZK1 expression in PDZK1 knock-out (KO) mice leads to a post-transcriptional, tissue-specific decrease in SR-BI protein level and an increase in total plasma cholesterol carried in abnormally large HDL particles. Here we show that, although hepatic overexpression of PDZK1 restored normal SR-BI protein abundance and function in PDZK1 KO mice, hepatic overexpression of only the PDZ1 domain was not sufficient to restore normal SR-BI function. In wild-type mice, overexpression of the PDZ1 domain overcame the activity of the endogenous hepatic PDZK1, resulting in a 75% reduction in hepatic SR-BI protein levels and intracellular mislocalization of the remaining SR-BI. As a consequence, the plasma lipoproteins in PDZ1 transgenic mice resembled those in PDZK1 KO mice (hypercholesterolemia due to large HDL). These results indicate that the PDZ1 domain can control the abundance and localization, and therefore the function, of hepatic SR-BI and that structural features of PDZK1 in addition to its SR-BI-binding PDZ1 domain are required for normal hepatic SR-BI regulation.


Subject(s)
Gene Expression Regulation , Intracellular Signaling Peptides and Proteins/metabolism , PDZ Domains/physiology , Scavenger Receptors, Class B/metabolism , Animals , Cholesterol/blood , Cholesterol/metabolism , Glycosylation , Intracellular Signaling Peptides and Proteins/genetics , Lipoproteins/blood , Lipoproteins/metabolism , Male , Membrane Proteins , Mice , Mice, Knockout , PDZ Domains/genetics
14.
Biochim Biophys Acta ; 1782(5): 310-6, 2008 May.
Article in English | MEDLINE | ID: mdl-18342019

ABSTRACT

PDZK1 is a scaffold protein containing four PDZ protein interaction domains, which bind to the carboxy termini of a number of membrane transporter proteins, including ion channels (e.g., CFTR) and cell surface receptors. One of these, the HDL receptor, scavenger receptor class B type I (SR-BI), exhibits a striking, tissue-specific dependence on PDZK1 for its expression and activity. In PDZK1 knockout (KO) mice there is a marked reduction of SR-BI protein expression (approximately 95%) in the liver, but not in steroidogenic tissues or, as we show in this report, in bone marrow- or spleen-derived macrophages, or lung-derived endothelial cells. Because of hepatic SR-BI deficiency, PDZK1 KO mice exhibit dyslipidemia characterized by elevated plasma cholesterol carried in abnormally large HDL particles. Here, we show that inactivation of the PDZK1 gene promotes the development of aortic root atherosclerosis in apolipoprotein E (apoE) KO mice fed with a high fat/high cholesterol diet. However, unlike complete SR-BI-deficiency in SR-BI/apoE double KO mice, PDZK1 deficiency in PDZK1/apoE double knockout mice did not result in development of occlusive coronary artery disease or myocardial infarction, presumably because of their residual expression of SR-BI. These findings demonstrate that deficiency of an adaptor protein essential for normal expression of a lipoprotein receptor promotes atherosclerosis in a murine model. They also define PDZK1 as a member of the family of proteins that is instrumental in preventing cardiovascular disease by maintaining normal lipoprotein metabolism.


Subject(s)
Atherosclerosis/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Lipoproteins/metabolism , Animals , Aorta/metabolism , Aorta/pathology , Apolipoproteins E/deficiency , Apolipoproteins E/metabolism , Atherosclerosis/blood , CD36 Antigens/metabolism , Diet , Endothelial Cells/metabolism , Immunoblotting , Intracellular Signaling Peptides and Proteins/deficiency , Lipids/blood , Macrophages/metabolism , Membrane Proteins , Mice , Mice, Inbred C57BL
15.
J Biol Chem ; 281(39): 28975-80, 2006 Sep 29.
Article in English | MEDLINE | ID: mdl-16867981

ABSTRACT

PDZK1 is a multi-PDZ domain-containing adaptor protein that binds to the C terminus of the high density lipoprotein receptor, scavenger receptor, class B, type I (SR-BI), and controls the posttranscriptional, tissue-specific expression of this lipoprotein receptor. In the absence of PDZK1 (PDZK1(-/-) mice), murine hepatic SR-BI protein levels are very low (<5% of control). As a consequence, abnormal plasma lipoprotein metabolism ( approximately 1.5-1.7-fold increased total plasma cholesterol carried in both normal size and abnormally large high density lipoprotein particles) resembles, but is not as severely defective as, that in SR-BI(-/-) mice. Here we show that the total plasma cholesterol levels and size distribution of lipoproteins are virtually identical in SR-BI(-/-) and SR-BI(-/-)/PDZK1(-/-) mice, indicating that most, if not all of the effects of PDZK1 on lipoprotein metabolism are likely because of the effects of PDZK1 on SR-BI. Hepatic overexpression of wild-type SR-BI in PDZK1(-/-) mice restored near or greater than normal levels of cell surface-expressed, functional SR-BI protein levels in the livers of SR-BI(-/-)/PDZK1(-/-) mice and consequently restored apparently normal lipoprotein metabolism in the absence of PDZK1. Thus, PDZK1 is important for maintaining adequate steady state levels of SR-BI in the liver but is not essential for cell surface expression or function of hepatic SR-BI.


Subject(s)
CD36 Antigens/physiology , Carrier Proteins/physiology , Intracellular Signaling Peptides and Proteins/physiology , Animals , CD36 Antigens/metabolism , Carrier Proteins/metabolism , Cell Membrane/metabolism , Cholesterol/blood , Gene Expression Regulation , Genotype , Intracellular Signaling Peptides and Proteins/chemistry , Lipoproteins/metabolism , Membrane Proteins , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Transgenes
16.
Int J Syst Evol Microbiol ; 56(Pt 3): 667-670, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16514047

ABSTRACT

The sphingomonad group contains bacterial isolates that are quite diverse in terms of their phylogenetic, ecological and physiological properties. Thus, the genus Sphingomonas was divided into four distinct genera, Sphingomonas sensu stricto, Sphingobium, Novosphingobium and Sphingopyxis on the basis of 16S rRNA gene sequence phylogenetic analysis, signature nucleotides, fatty acid profiles and polyamine patterns and this classification is currently widely accepted. In this study, a complete analysis of the 16S rRNA gene sequences of all the members of the group of sphingomonads encompassed in the genera Sphingomonas sensu stricto, Sphingobium, Novosphingobium and Sphingopyxis was inferred by using tree-making algorithms. [Sphingomonas] xenophaga DSM 6383T was found to form a distinct clade with the members of the genus Sphingobium, whereas [Sphingomonas] taejonensis DSM 15583T forms a clade with the members of the genus Sphingopyxis. The respective positions of these strains were also supported by the data for signature nucleotides, 2-hydroxy fatty acid profiles, polyamine patterns and the nitrate reduction properties of the strains. We therefore propose the reclassification of [Sphingomonas] xenophaga and [Sphingomonas] taejonensis as Sphingobium xenophagum comb. nov. (type strain DSM 6383T = CIP 107206T) and Sphingopyxis taejonensis comb. nov. (type strain DSM 15583T = KCTC 2884T = KCCM 41068T), respectively.


Subject(s)
Sphingomonadaceae/classification , Sphingomonas/classification , DNA, Ribosomal/chemistry , Fatty Acids/analysis , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sphingomonadaceae/chemistry , Sphingomonadaceae/genetics , Sphingomonas/chemistry , Sphingomonas/genetics
17.
Trends Biotechnol ; 24(3): 121-30, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16473421

ABSTRACT

Two forms of hexachlorocyclohexane (HCH), gamma-HCH (lindane) and technical HCH (incorporating alpha-, beta-, gamma- and delta- isomers), have been used against agricultural pests and in health programs since the 1940s. Although all the isomers are present in the milieu, delta- and beta-HCH isomers are the most problematic and present a serious environmental problem. Bacteria that degrade HCH isomers have been isolated from HCH contaminated soils from different geographical locations around the world (from the family Sphingomonadaceae). Interestingly, all these bacteria contain nearly identical lin genes (responsible for HCH degradation), which are diverging to perform several catabolic functions. The organization and diversity of lin genes have been studied among several sphingomonads, and they have been found to be associated with plasmids and IS6100, both of which appear to have a significant role in their horizontal transfer. The knowledge of the molecular genetics, diversity and distribution of lin genes, and the potential of sphingomonads to degrade HCH isomers, can now be used for developing bioremediation techniques for the decontamination of HCH contaminated sites.


Subject(s)
Bacterial Proteins/genetics , Genetic Variation/genetics , Gram-Negative Bacteria/genetics , Hexachlorocyclohexane/metabolism , Insecticides/metabolism , Lyases/genetics , Bacterial Proteins/metabolism , Biodegradation, Environmental , Gene Transfer, Horizontal/genetics , Gram-Negative Bacteria/enzymology , Lyases/metabolism , Phylogeny , Plasmids/genetics , Stereoisomerism
18.
Int J Syst Evol Microbiol ; 55(Pt 5): 1965-1972, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16166696

ABSTRACT

Three strains of Sphingomonas paucimobilis, B90A, UT26 and Sp+, isolated from different geographical locations, were found to degrade hexachlorocyclohexane. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains do not fall in a clade that includes the type strain, Sphingomonas paucimobilis ATCC 29837(T), but form a coherent cluster with [Sphingomonas] chungbukensis IMSNU 11152(T) followed by Sphingobium chlorophenolicum ATCC 33790(T). The three strains showed low DNA-DNA relatedness values with Sphingomonas paucimobilis ATCC 29837(T) (8-25%), [Sphingomonas] chungbukensis IMSNU 11152(T) (10-17%), Sphingobium chlorophenolicum ATCC 33790(T) (23-54%) and Sphingomonas xenophaga DSM 6383(T) (10-28%), indicating that they do not belong to any of these species. Although the three strains were found to be closely related to each other based on 16S rRNA gene sequence similarity (99.1-99.4%), DNA-DNA relatedness (19-59%) and pulsed-field gel electrophoresis (PFGE) patterns indicated that they possibly represent three novel species of the genus Sphingobium. The three strains could also be readily distinguished by biochemical tests. The three strains showed similar polar lipid profiles and contained sphingoglycolipids. The strains differed from each other in fatty acid composition but contained the predominant fatty acids characteristic of other Sphingobium species. A phylogenetic study based on 16S rRNA gene sequences showed that [Sphingomonas] chungbukensis IMSNU 11152(T) formed a cluster with members of the genus Sphingobium. Based on these results, it is proposed that strains B90A, UT26 and Sp+, previously known as Sphingomonas paucimobilis, are the type strains of Sphingobium indicum sp. nov. (=MTCC 6364(T)=CCM 7286(T)), Sphingobium japonicum sp. nov. (=MTCC 6362(T)=CCM 7287(T)) and Sphingobium francense sp. nov. (=MTCC 6363(T)=CCM 7288(T)), respectively. It is also proposed that [Sphingomonas] chungbukensis be transferred to Sphingobium chungbukense comb. nov.


Subject(s)
Bacterial Proteins/genetics , Hexachlorocyclohexane/metabolism , Sphingomonadaceae/classification , Sphingomonas/classification , Bacterial Proteins/metabolism , Bacterial Typing Techniques , Biodegradation, Environmental , DNA, Bacterial/analysis , DNA, Ribosomal/analysis , Genes, rRNA , Molecular Sequence Data , Nucleic Acid Hybridization , Phenotype , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sphingomonadaceae/enzymology , Sphingomonadaceae/genetics , Sphingomonadaceae/physiology , Sphingomonas/enzymology , Sphingomonas/genetics , Sphingomonas/physiology
19.
Pflugers Arch ; 449(4): 392-402, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15517343

ABSTRACT

Inorganic phosphate (P(i)) is reabsorbed in the renal proximal tubule mainly via the type-IIa sodium-phosphate cotransporter (NaPi-IIa). This protein is regulated tightly by different factors, among them dietary P(i) intake and parathyroid hormone (PTH). A number of PDZ-domain-containing proteins have been shown to interact with NaPi-IIa in vitro, such as Na(+)/H(+) exchanger-3 regulatory factor-1 (NHERF1) and PDZK1. PDZK1 is highly abundant in kidney and co-localizes with NaPi-IIa in the brush border membrane of proximal tubules. Recently, a knock-out mouse model for PDZK1 (Pdzk1(-/-)) has been generated, allowing the role of PDZK1 in the expression and regulation of the NaPi-IIa cotransporter to be examined in in vivo and in ex vivo preparations. The localization of NaPi-IIa and other proteins interacting with PDZK1 in vitro [Na(+)/H(+) exchanger (NHE3), chloride-formate exchanger (CFEX)/putative anion transporter-1 (PAT1), NHERF1] was not altered in Pdzk1(-/-) mice. The abundance of NaPi-IIa adapted to acute and chronic changes in dietary P(i) intake, but steady-state levels of NaPi-IIa were reduced in Pdzk1(-/-) under a P(i) rich diet. This was paralleled by a higher urinary fractional P(i) excretion. The abundance of the anion exchanger CFEX/PAT1 (SLC26A6) was also reduced. In contrast, NHERF1 abundance increased in the brush border membrane of Pdzk1(-/-) mice fed a high-P(i) diet. Acute regulation of NaPi-IIa by PTH in vivo and by PTH and activators of protein kinases A, C and G (PKA, PKC and PKG) in vitro (kidney slice preparation) was not altered in Pdzk1(-/-) mice. In conclusion, loss of PDZK1 did not result in major changes in proximal tubule function or NaPi-IIa regulation. However, under a P(i)-rich diet, loss of PDZK1 reduced NaPi-IIa abundance indicating that PDZK1 may play a role in the trafficking or stability of NaPi-IIa under these conditions.


Subject(s)
Kidney/metabolism , Membrane Proteins/genetics , Membrane Proteins/physiology , Symporters/biosynthesis , Symporters/physiology , Animals , Blotting, Western , Cytoskeletal Proteins/metabolism , Diet , Female , Immunohistochemistry , In Vitro Techniques , Kidney Tubules, Proximal/metabolism , Male , Mice , Mice, Knockout , Microvilli/metabolism , Phosphates/pharmacology , Phosphoproteins/metabolism , Protein Kinases/metabolism , Sodium-Hydrogen Exchangers , Sodium-Phosphate Cotransporter Proteins , Sodium-Phosphate Cotransporter Proteins, Type IIa
20.
Mod Pathol ; 17(11): 1323-7, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15257314

ABSTRACT

p53 mutations have been implicated in the development of esophageal malignancies. The purpose of this study was to assess more accurately the incidence and types of p53 mutations in Barrett's esophagus (BE) with and without dysplasia and in esophageal adenocarcinoma, using pure preparations of epithelial cells obtained by laser capture microdissection (LCM). Assays were performed on paraffin-embedded tissue samples of normal antrum and premalignant and malignant esophageal samples from 57 patients, including 16 controls, 10 with BE metaplasia alone, 20 with BE-associated dysplasia, and 11 with BE-associated adenocarcinoma. All tissues were processed for LCM. DNA was extracted from isolated cells, and polymerase chain reaction (PCR) was performed using oligonucleutide primers for exons 5-8 of p53. PCR products were processed for DNA sequencing. p53 sequence abnormalities were identified in 2/16 cases of normal antrum and regenerative/chemical gastritis, 1/10 cases of BE, 1/20 cases of BE with dysplasia, and 2/11 cases of adenocarcinomas. The abnormalities occurred in exons 7 and 8 in the form of point mutations. Our results, using LCM, show that p53 gene mutations are relatively rare in esophageal preneoplastic and neoplastic conditions. Only point mutations were detected, but no deletions/insertions were identified.


Subject(s)
Adenocarcinoma/genetics , Barrett Esophagus/genetics , Esophageal Neoplasms/genetics , Genes, p53/genetics , Precancerous Conditions/genetics , Adenocarcinoma/pathology , Barrett Esophagus/pathology , Base Sequence , Biomarkers, Tumor/analysis , Esophageal Neoplasms/pathology , Humans , Lasers , Microdissection , Paraffin Embedding , Point Mutation , Polymerase Chain Reaction , Precancerous Conditions/pathology
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