scholarly journals Association of PDZ-containing protein PDZD11 with the human sodium-dependent multivitamin transporter

2011 ◽  
Vol 300 (4) ◽  
pp. G561-G567 ◽  
Author(s):  
Svetlana M. Nabokina ◽  
Veedamali S. Subramanian ◽  
Hamid M. Said
Keyword(s):  
Cell Biology ◽  
Protein S ◽  
Confocal Imaging ◽  
Intestinal Epithelial ◽  
Sodium Dependent ◽  
Interfering Rna ◽  
First Time ◽  
Two Hybrid

Intestinal absorption of biotin is mediated via the sodium-dependent multivitamin transporter (SMVT). Studies from our laboratory and others have characterized different aspects of the human SMVT (hSMVT), but nothing is currently known about protein(s) that may interact with hSMVT and affect its physiology/biology. In this study, a PDZ-containing protein PDZD11 was identified as an interacting partner with hSMVT using yeast two-hybrid screen of a human intestinal cDNA library. The interaction between hSMVT and PDZD11 was confirmed by in vitro GST-pull-down assay and in vivo in a mammalian cell environment by a two-hybrid luciferase and coimmunoprecipitation assays. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells expressing hSMVT-GFP and DsRed-PDZD11 demonstrated colocalization of these two proteins. We also examined the functional consequence of the interaction between hSMVT and PDZD11 in HuTu-80 cells and observed significant induction in [3H]biotin uptake upon coexpression of hSMVT and PDZD11. In contrast, knocking down of PDZD11 with gene-specific small interfering RNA led to a significant decrease in biotin uptake; biotinylation assay showed this to be associated with a marked decrease in level of expression of hSMVT at the cell membrane. By truncation approach, we also demonstrated that the PDZ binding domain that is located in the COOH-terminal tail of hSMVT polypeptide is involved in the interaction with PDZD11. These results demonstrate for the first time that PDZD11 is an interacting partner with hSMVT in intestinal epithelial cells and that this interaction affects hSMVT function and cell biology.

scholarly journals Tspan-1 interacts with the thiamine transporter-1 in human intestinal epithelial cells and modulates its stability

2011 ◽  
Vol 301 (5) ◽  
pp. G808-G813 ◽  
Author(s):  
Svetlana M. Nabokina ◽  
Sundar Rajan Senthilkumar ◽  
Hamid M. Said
Keyword(s):  
Cell Biology ◽  
Intestinal Epithelial Cells ◽  
Protein S ◽  
Confocal Imaging ◽  
Intestinal Epithelial ◽  
Cell Type ◽  
Glutathione S Transferase ◽  
Human Intestinal Epithelial Cells ◽  
First Time ◽  
Two Hybrid

The human thiamine transporter-1 (hTHTR-1) contributes to intestinal thiamine uptake, and its function is regulated at both the transcriptional and posttranscriptional levels. Nothing, however, is known about the protein(s) that may interact with hTHTR-1 and affects its cell biology and physiology. We addressed this issue in the present investigation using a bacterial two-hybrid system to screen a human intestinal cDNA library with the complete coding sequence of hTHTR-1 as a bait. Our results showed that a member of the tetraspanin family of proteins, Tspan-1, interacts with hTHTR-1. Coimmunoprecipitation and glutathione S-transferase (GST)-pulldown assays confirmed the existence of such an interaction between hTspan-1 and hTHTR-1 in human intestinal epithelial Caco-2 cells. Furthermore, live cell confocal imaging demonstrated that hTspan-1 and hTHTR-1 colocalize in human intestinal epithelial HuTu-80 cells. The importance of the interaction between hTspan-1 and hTHTR-1 for cell biology of the thiamine transporter was examined in HuTu-80 cells stably expressing hTHTR-1. Coexpression of hTspan-1 in these cells led to a significant decrease in the rate of degradation of hTHTR-1 compared with cells expressing the hTHTR-1 alone; in fact the half-life of the hTHTR-1 protein was twice longer in the former cell type compared with the latter cell type (12 h vs. 6 h, respectively). This finding was also confirmed at the functional level when a significantly higher thiamine uptake was observed in cycloheximide-treated (6 h) cells expressing hTHTR-1 together with hTspan-1 compared with those expressing hTHTR-1 alone. These studies demonstrate for the first time that Tspan-1 is an interacting partner with hTHTR-1 and that this interaction affects hTHTR-1 stability.

scholarly journals Glyoxalate reductase/hydroxypyruvate reductase interacts with the sodium-dependent vitamin C transporter-1 to regulate cellular vitamin C homeostasis

2013 ◽  
Vol 304 (12) ◽  
pp. G1079-G1086 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Svetlana M. Nabokina ◽  
Joseph R. Patton ◽  
Jonathan S. Marchant ◽  
Hamid Moradi ◽  
...  
Keyword(s):  
Vitamin C ◽  
Cell Biology ◽  
Human Liver ◽  
Firefly Luciferase ◽  
Confocal Imaging ◽  
Hydroxypyruvate Reductase ◽  
Sodium Dependent ◽  
Two Hybrid

The human sodium-dependent vitamin C transporter 1 (hSVCT1) contributes to cellular uptake of ascorbic acid (AA). Although different aspects of hSVCT1 cell biology have been extensively studied, nothing is currently known about the broader hSVCT1 interactome that modulates its role in cellular physiology. Here, we identify the enzyme human glyoxalate reductase/hydroxypyruvate reductase (hGR/HPR) as an hSVCT1 associated protein by yeast two-hybrid (Y2H) screening of a human liver cDNA library. The interaction between hSVCT1 and hGR/HPR was further confirmed by in vitro GST pull-down assay, in vivo coimmunoprecipitation and mammalian two-hybrid firefly luciferase assays. This interaction had functional significance as coexpression of hGR/HPR with hSVCT1 led to an increase in AA uptake. Reciprocally, siRNA-mediated knockdown of endogenous hGR/HPR led to an inhibition of AA uptake. Given that oxalate is a degradation product of vitamin C and hGR/HPR acts to limit cellular oxalate levels, this association physically couples two independent regulators of cellular oxalate production. Furthermore, confocal imaging of human liver HepG2 cells coexpressing GFP-hSVCT1 and hGR/HPR-mCherry demonstrated that these two proteins colocalize within a subpopulation of intracellular organelles. This provides a possible molecular basis for organellar AA transport and regulation of local glyoxylate/glycolate concentration in the vicinity of organelle membranes.

scholarly journals A role for IL-34 in osteolytic disease of multiple myeloma

2019 ◽  
Vol 3 (4) ◽  
pp. 541-551 ◽  
Author(s):  
Muhammad Baghdadi ◽  
Kozo Ishikawa ◽  
Sayaka Nakanishi ◽  
Tomoki Murata ◽  
Yui Umeyama ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Osteoclast Differentiation ◽  
Neutralizing Antibody ◽  
Axial Skeleton ◽  
Osteoclast Formation ◽  
Interfering Rna ◽  
First Time ◽  
Stimulating Factor

AbstractMultiple myeloma (MM) is a hematological malignancy that grows in multiple sites of the axial skeleton and causes debilitating osteolytic disease. Interleukin-34 (IL-34) is a newly discovered cytokine that acts as a ligand of colony-stimulating factor-1 (CSF-1) receptor and can replace CSF-1 for osteoclast differentiation. In this study, we identify IL-34 as an osteoclastogenic cytokine that accelerates osteolytic disease in MM. IL-34 was found to be expressed in the murine MM cell line MOPC315.BM, and the expression of IL-34 was enhanced by stimulation with proinflammatory cytokines or by bone marrow (BM) stromal cells. MM-cell–derived IL-34 promoted osteoclast formation from mouse BM cells in vitro. Targeting Il34 by specific small interfering RNA impaired osteoclast formation in vitro and attenuated osteolytic disease in vivo. In BM aspirates from MM patients, the expression levels of IL-34 in CD138+ populations vary among patients from high to weak to absent. MM cell–derived IL-34 promoted osteoclast formation from human CD14+ monocytes, which was reduced by a neutralizing antibody against IL-34. Taken together, this study describes for the first time the expression of IL-34 in MM cells, indicating that it may enhance osteolysis and suggesting IL-34 as a potential therapeutic target to control pathological osteoclastogenesis in MM patients.

scholarly journals Innate lymphoid cells lacking surface expression of CD90 are functional

2021 ◽  
Author(s):  
J-H Schroeder ◽  
T Zabinski ◽  
J F Neves ◽  
GM Lord
Keyword(s):  
Steady State ◽  
Cell Biology ◽  
Surface Expression ◽  
Innate Lymphoid Cells ◽  
Lymphoid Cells ◽  
T Cell Biology ◽  
First Time ◽  
Made In

ABSTRACTHuge progress has been made in understanding the biology of innate lymphoid cells (ILC) by adopting several well-known concepts of T cell biology. As such flow cytometry gating strategies and markers, such as CD90, to identify ILC were discovered. Here we report that most non-NK intestinal ILC have a high expression of CD90 as expected, but surprisingly some have only a low or even no expression of this marker. CD90-negative CD127+ ILC were identified among all ILC subsets in the gut. CD90-negative cLP ILC2 were frequent at steady state. The frequency of CD90-negative CD127+ ILC was dependent on stimulatory cues in vitro and in vivo, and CD90-negative CD127+ ILC played a functional role as a source of IL-13, IFNγ and IL-17A at steady state and upon dextran sulphate sodium-elicited colitis. Hence, this study highlights for the first time that CD90 is not constitutively expressed by functional ILC in the gut.

scholarly journals The nuclear receptor LRH-1 critically regulates extra-adrenal glucocorticoid synthesis in the intestine

2006 ◽  
Vol 203 (9) ◽  
pp. 2057-2062 ◽  
Author(s):  
Matthias Mueller ◽  
Igor Cima ◽  
Mario Noti ◽  
Andrea Fuhrer ◽  
Sabine Jakob ◽  
...  
Keyword(s):  
Nuclear Receptor ◽  
Cell Renewal ◽  
Intestinal Epithelial ◽  
Steroidogenic Enzymes ◽  
Tissue Integrity ◽  
Immunological Stress ◽  
Important Regulator ◽  
First Time

The nuclear receptor liver receptor homologue-1 (LRH-1, NR5A2) is a crucial transcriptional regulator of many metabolic pathways. In addition, LRH-1 is expressed in intestinal crypt cells where it regulates the epithelial cell renewal and contributes to tumorigenesis through the induction of cell cycle proteins. We have recently identified the intestinal epithelium as an important extra-adrenal source of immunoregulatory glucocorticoids. We show here that LRH-1 promotes the expression of the steroidogenic enzymes and the synthesis of corticosterone in murine intestinal epithelial cells in vitro. Interestingly, LRH-1 is also essential for intestinal glucocorticoid synthesis in vivo, as LRH-1 haplo-insufficiency strongly reduces the intestinal expression of steroidogenic enzymes and glucocorticoid synthesis upon immunological stress. These results demonstrate for the first time a novel role for LRH-1 in the regulation of intestinal glucocorticoid synthesis and propose LRH-1 as an important regulator of intestinal tissue integrity and immune homeostasis.

scholarly journals Visfatin Induces Senescence of Human Dental Pulp Cells

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 193 ◽  
Author(s):  
Chang Youp Ok ◽  
Sera Park ◽  
Hye-Ock Jang ◽  
Takashi Takata ◽  
Moon-Kyoung Bae ◽  
...  
Keyword(s):  
Dental Pulp ◽  
Dental Pulp Cells ◽  
Human Dental Pulp Cells ◽  
Human Dental Pulp ◽  
Pulp Cells ◽  
Secretory Phenotype ◽  
Interfering Rna ◽  
First Time

Dental pulp plays an important role in the health of teeth. The aging of teeth is strongly related to the senescence of dental pulp cells. A novel adipokine, visfatin, is closely associated with cellular senescence. However, little is known about the effect of visfatin on the senescence of human dental pulp cells (hDPCs). Here, it was found that in vivo visfatin levels in human dental pulp tissues increase with age and are upregulated in vitro in hDPCs during premature senescence activated by H2O2, suggesting a correlation between visfatin and senescence. In addition, visfatin knockdown by small interfering RNA led to the reduction in hDPC senescence; however, treatment with exogenous visfatin protein induced the senescence of hDPCs along with increased NADPH consumption, which was reversed by FK866, a chemical inhibitor of visfatin. Furthermore, visfatin-induced senescence was associated with both the induction of telomere damage and the upregulation of senescence-associated secretory phenotype (SASP) factors as well as NF-κB activation, which were all inhibited by FK866. Taken together, these results demonstrate, for the first time, that visfatin plays a pivotal role in hDPC senescence in association with telomere dysfunction and the induction of SASP factors.

scholarly journals Salmonellainfection inhibits intestinal biotin transport: cellular and molecular mechanisms

2015 ◽  
Vol 309 (2) ◽  
pp. G123-G131 ◽  
Author(s):  
Abhisek Ghosal ◽  
Stefan Jellbauer ◽  
Rubina Kapadia ◽  
Manuela Raffatellu ◽  
Hamid M. Said
Keyword(s):  
Proinflammatory Cytokines ◽  
Molecular Mechanisms ◽  
Significant Inhibition ◽  
Intestinal Epithelial ◽  
Food Borne ◽  
Tnf Α ◽  
Sodium Dependent ◽  
Serovar Typhimurium

Infection with the nontyphoidal Salmonella is a common cause of food-borne disease that leads to acute gastroenteritis/diarrhea. Severe/prolonged cases of Salmonella infection could also impact host nutritional status, but little is known about its effect on intestinal absorption of vitamins, including biotin. We examined the effect of Salmonella enterica serovar Typhimurium ( S. typhimurium) infection on intestinal biotin uptake using in vivo (streptomycin-pretreated mice) and in vitro [mouse (YAMC) and human (NCM460) colonic epithelial cells, and human intestinal epithelial Caco-2 cells] models. The results showed that infecting mice with wild-type S. typhimurium, but not with its nonpathogenic isogenic invA spiB mutant, leads to a significant inhibition in jejunal/colonic biotin uptake and in level of expression of the biotin transporter, sodium-dependent multivitamin transporter. In contrast, infecting YAMC, NCM460, and Caco-2 cells with S. typhimurium did not affect biotin uptake. These findings suggest that the effect of S. typhimurium infection is indirect and is likely mediated by proinflammatory cytokines, the levels of which were markedly induced in the intestine of S. typhimurium-infected mice. Consistent with this hypothesis, exposure of NCM460 cells to the proinflammatory cytokines TNF-α and IFN-γ led to a significant inhibition of biotin uptake, sodium-dependent multivitamin transporter expression, and activity of the SLC5A6 promoter. The latter effects appear to be mediated, at least in part, via the NF-κB signaling pathway. These results demonstrate that S. typhimurium infection inhibits intestinal biotin uptake, and that the inhibition is mediated via the action of proinflammatory cytokines.

Posttranslational regulation of Abcc2 expression by SUMOylation system

2009 ◽  
Vol 296 (2) ◽  
pp. G406-G413 ◽  
Author(s):  
Satoko Minami ◽  
Kousei Ito ◽  
Masashi Honma ◽  
Yuki Ikebuchi ◽  
Naohiko Anzai ◽  
...  
Keyword(s):  
Protein S ◽  
Posttranslational Regulation ◽  
Linker Region ◽  
Atp Binding Cassette Transporter ◽  
Transporter Family ◽  
Membrane Spanning ◽  
Interfering Rna ◽  
First Time ◽  
Hybrid Screening

The ATP-binding cassette transporter family C 2 (Abcc2) is a member of efflux transporters involved in the biliary excretion of organic anions from hepatocytes. Posttranslational regulation of Abcc2 has been implicated, although the molecular mechanism is not fully understood. In the present study, we performed yeast two-hybrid screening to identify novel protein(s) that particularly interacts with the linker region of Abcc2 located between the NH2-terminal nucleotide binding domain and the last membrane-spanning domain. The screening resulted in the identification of a series of small ubiquitin-like modifier (SUMO)-related enzymes and their substrates. In yeast experiments, all of these interactions were abolished by substituting the putative SUMO consensus site in the linker region (IKKE) in Abcc2 to IRKE. In vitro SUMOylation experiments confirmed that the Abcc2 linker was a substrate of Ubc9-mediated SUMOylation. It was also found that the IKKE sequence is the target of SUMOylation, since a mutant with IKKE is substituted by IRKE was not SUMOylated. Furthermore, we demonstrated for the first time that Abcc2, endogenously expressed in rat hepatoma-derived McARH7777 cells, is SUMOylated. Suppression of endogenous Ubc9 by small interfering RNA resulted in a selective 30% reduction in Abcc2 protein expression in the postnuclear supernatant, whereas subcellular localization of Abcc2 confirmed by semiquantitative immunofluorescence analysis was minimally affected. This is the first demonstration showing the regulation of ABC transporter expression by SUMOylation.

scholarly journals Targeting of C-Terminal Binding Protein (CtBP) by ARF Results in p53-Independent Apoptosis

2006 ◽  
Vol 26 (6) ◽  
pp. 2360-2372 ◽  
Author(s):  
Seema Paliwal ◽  
Sandhya Pande ◽  
Ramesh C. Kovi ◽  
Norman E. Sharpless ◽  
Nabeel Bardeesy ◽  
...  
Keyword(s):  
Tumor Suppression ◽  
Negative Regulator ◽  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Induced Apoptosis ◽  
Interfering Rna ◽  
Two Hybrid ◽  
Potent Tumor

ABSTRACT ARF encodes a potent tumor suppressor that antagonizes MDM2, a negative regulator of p53. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF or p53 singly null mice, results in a broadened tumor spectrum and decreased tumor latency. To investigate the mechanism of p53-independent tumor suppression by ARF, potential interacting proteins were identified by yeast two-hybrid screen. The antiapoptotic transcriptional corepressor C-terminal binding protein 2 (CtBP2) was identified, and ARF interactions with both CtBP1 and CtBP2 were confirmed in vitro and in vivo. Interaction with ARF resulted in proteasome-dependent CtBP degradation. Both ARF-induced CtBP degradation and CtBP small interfering RNA led to p53-independent apoptosis in colon cancer cells. ARF induction of apoptosis was dependent on its ability to interact with CtBP, and reversal of ARF-induced CtBP depletion by CtBP overexpression abrogated ARF-induced apoptosis. CtBP proteins represent putative targets for p53-independent tumor suppression by ARF.

scholarly journals The melibiose-derived glycation product mimics a unique epitope present in human and animal tissues

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Magdalena Staniszewska ◽  
Agnieszka Bronowicka-Szydełko ◽  
Kinga Gostomska-Pampuch ◽  
Jerzy Szkudlarek ◽  
Arkadiusz Bartyś ◽  
...  
Keyword(s):  
Cell Biology ◽  
Resonance Spectroscopy ◽  
Open Chain ◽  
Glycation Product ◽  
Glycation End Products ◽  
Patients With Diabetes ◽  
Living Organisms ◽  
First Time

AbstractNon-enzymatic modification of proteins by carbohydrates, known as glycation, leads to generation of advanced glycation end-products (AGEs). In our study we used in vitro generated AGEs to model glycation in vivo. We discovered in vivo analogs of unusual melibiose-adducts designated MAGEs (mel-derived AGEs) synthesized in vitro under anhydrous conditions with bovine serum albumin and myoglobin. Using nuclear magnetic resonance spectroscopy we have identified MAGEs as a set of isomers, with open-chain and cyclic structures, of the fructosamine moiety. We generated a mouse anti-MAGE monoclonal antibody and show for the first time that the native and previously undescribed analogous glycation product exists in living organisms and is naturally present in tissues of both invertebrates and vertebrates, including humans. We also report MAGE cross-reactive auto-antibodies in patients with diabetes. We anticipate our approach for modeling glycation in vivo will be a foundational methodology in cell biology. Further studies relevant to the discovery of MAGE may contribute to clarifying disease mechanisms and to the development of novel therapeutic options for diabetic complications, neuropathology, and cancer.

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