The Zebrafish Ortholog of Hemojuvelin Participates in Notochord and Somite Development, but Fails to Regulate Embryonic Hepcidin Expression

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 118-118 ◽  
Author(s):  
Yann Gibert ◽  
Victoria J. Lattanzi ◽  
Jason Holzheimer ◽  
Sarah F. Burnett ◽  
Paula G. Fraenkel
Keyword(s):  
Mammalian Cells ◽  
Zebrafish Embryos ◽  
Dependent Manner ◽  
Furin Cleavage ◽  
Hepcidin Expression ◽  
Morpholino Knockdown ◽  
Juvenile Hemochromatosis ◽  
Ancestral Function ◽  
Repulsive Guidance Molecule ◽  
And Function

Abstract Hemojuvelin (hjv), a member of the repulsive-guidance molecule (RGM) family upregulates the iron regulatory hormone hepcidin in a BMP-dependent manner in mammalian cells. A mutation interfering with hjv’s ability to bind neogenin has been identified in patients with juvenile hemochromatosis, while furin cleavage of hjv has also been implicated in its function. Previously, we demonstrated that hepcidin expression in zebrafish embryos increases in response to iron loading or activation of the BMP pathway. We hypothesized that hjv would regulate hepcidin expression in zebrafish embryos and used whole mount in situ hybridization and morpholino knockdowns to study the expression and function of hjv. We found that hjv is strongly and sequentially expressed in the notochord and somites and that knockdown of hjv resulted in severe defects in these structures. Hjv was not expressed in the liver and knockdown of hjv failed to affect the timing, intensity, or location of hepcidin expression. Furthermore, knockdown of hjv failed to prevent the upregulation of hepcidin expression caused by overexpression of BMP2b. Zebrafish hjv exhibits conservation at the site required for binding neogenin, however zebrafish hjv and all nonmammalian RGM’s lack the furin cleavage motif. We found that morpholino knockdown of the zebrafish orthologs of neogenin or furin failed to affect hepcidin expression. Taken together, these data indicate that regulation of hepcidin expression in the zebrafish embryo is BMP-responsive, but independent of hjv, furin, or neogenin zebrafish hjv participates in notochord and somite development, which we propose as the ancestral function of hjv.

Competitive regulation of hepcidin mRNA by soluble and cell-associated hemojuvelin

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2884-2889 ◽  
Author(s):  
Lan Lin ◽  
Y. Paul Goldberg ◽  
Tomas Ganz
Keyword(s):  
Mrna Expression ◽  
Iron Homeostasis ◽  
Iron Concentration ◽  
Protein Product ◽  
Dependent Manner ◽  
Hepcidin Expression ◽  
Juvenile Hemochromatosis ◽  
Repulsive Guidance Molecule ◽  
Hepcidin Mrna

Abstract Mutations in a recently identified gene HJV (also called HFE2, or repulsive guidance molecule C, RgmC) are the major cause of juvenile hemochromatosis (JH). The protein product of HJV, hemojuvelin, contains a C-terminal glycosylphosphatidylinositol anchor, suggesting that it can be present in either a soluble or a cell-associated form. Patients with HJV hemochromatosis have low urinary levels of hepcidin, the principal iron-regulatory hormone secreted by the liver. However, neither the specific role of hemojuvelin in maintaining iron homeostasis nor its relationship to hepcidin has been experimentally established. In this study we used hemojuvelin-specific siRNAs to vary hemojuvelin mRNA concentration and showed that cellular hemojuvelin positively regulated hepcidin mRNA expression, independently of the interleukin 6 pathway. We also showed that recombinant soluble hemojuvelin (rs-hemojuvelin) suppressed hepcidin mRNA expression in primary human hepatocytes in a log-linear dose-dependent manner, suggesting binding competition between soluble and cell-associated hemojuvelin. Soluble hemojuvelin was found in human sera at concentrations similar to those required to suppress hepcidin mRNA in vitro. In cells engineered to express hemojuvelin, soluble hemojuvelin release was progressively inhibited by increasing iron concentrations. We propose that soluble and cell-associated hemojuvelin reciprocally regulate hepcidin expression in response to changes in extracellular iron concentration. (Blood. 2005;106:2884-2889)

Knockdown of Matriptase-2 Increases Hepcidin Expression in a BMP-Dependent Manner in Zebrafish Embryos.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4050-4050
Author(s):  
Yann Gibert ◽  
Victoria J. Lattanzi ◽  
Paula G. Fraenkel
Keyword(s):  
In Situ Hybridization ◽  
Iron Overload ◽  
Zebrafish Embryos ◽  
Dependent Manner ◽  
Embryonic Liver ◽  
Rt Pcr ◽  
Hepcidin Expression ◽  
Juvenile Hemochromatosis ◽  
Membrane Bound

Abstract Abstract 4050 Poster Board III-985 Hepatocytes produce hepcidin, a transcriptionally regulated peptide hormone, in response to iron overload, resulting in decreased intestinal iron absorption. Hepcidin levels are inappropriately low in patients with thalassemia major and hemochromatosis. We have been developing the zebrafish embryo as a model to identify hepcidin regulators, which may be exploited to prevent and treat iron overload syndromes. Recently, it has been shown in mammalian models that the membrane-bound serine protease, matriptase-2 (also known as TMPRSS-6), inhibits hepcidin expression by cleaving membrane-bound hemojuvelin (hjv), a bone morphogenic protein (BMP) co-receptor, which is mutated in patients with juvenile hemochromatosis. Previously, we demonstrated that hepcidin expression increases in response to iron loading or activation of the BMP signaling pathway, but that hjv is not required for hepcidin expression in zebrafish embryos. We hypothesized that hepcidin expression in zebrafish embryos is normally hjv-independent, either because of low levels of hjv expression in the liver or because of the presence of matriptase-2. We used whole mount in situ hybridization, quantitative realtime RT-PCR, morpholino knockdowns and overexpression experiments to address these questions. As hjv was undetectable in the zebrafish embryonic liver by in situ hybridization, we used flow cytometry to sort zebrafish embryonic hepatocytes and assess hjv expression by RT-PCR. We found that hjv was weakly expressed in the embryonic liver, but strongly expressed in zebrafish adult liver. We overexpressed zebrafish hjv in human hepatocytes and found that it potentiated the effects of BMP6 to activate a human hepcidin promoter and a BMP response element. In contrast, overexpression of zebrafish hjv in zebrafish embryos failed to increase hepcidin expression. Knockdown of matriptase-2 in zebrafish embryos caused developmental delay and a smaller liver, but increased hepcidin expression, relative to liver size. Furthermore, treatment with the BMP inhibitor dorsomorphin abrogated the increased hepcidin expression associated with matriptase-2 knockdown. Experiments are underway to determine whether zebrafish hjv has little effect on hepcidin expression during embryonic development because of the activity of matriptase-2. Disclosures: No relevant conflicts of interest to declare.

Sensing Iron: Reciprocal Regulation of Hepcidin mRNA by Soluble and Cell-Associated Hemojuvelin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 512-512
Author(s):  
Lan Lin ◽  
Y. Paul Goldberg ◽  
Tomas Ganz
Keyword(s):  
Iron Homeostasis ◽  
Genomic Sequence ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Human Hepatoma Cell ◽  
Hepcidin Expression ◽  
Juvenile Hemochromatosis ◽  
Hepcidin Mrna

Abstract Human genetic studies identified HJV (also called HFE2) as the major cause for juvenile hemochromatosis (JH). Patients with HJV hemochromatosis have low urinary levels of hepcidin, the principal iron-regulatory hormone secreted by the liver. We attempted to establish the specific roles of HJV in iron metabolism, especially its relationship with hepcidin. Translation of the genomic sequence indicated a C-terminal GPI anchor for the protein product of HJV, hemojuvelin. This suggested that hemojuvelin may have either a soluble or a cell-associated form. In human hepatoma cell line Hep3B, knockdown of cellular HJV by siRNA decreased hepcidin expression, independently of the IL-6 pathway. Intriguingly, the addition of recombinant soluble hemojuvelin (rs-hemojuvelin) also suppressed hepcidin expression in primary human hepatocytes, in a log-linear dose-dependent manner, suggesting competition between soluble and cell-associated forms of hemojuvelin. Soluble hemojuvelin was found in human sera at concentrations similar to those required to suppress hepcidin mRNA in vitro. In cells engineered to express hemojuvelin, soluble hemojuvelin release was progressively inhibited by increasing iron or holotransferrin concentrations. Our study suggests that soluble and cell-associated hemojuvelin reciprocally regulate hepcidin mRNA levels, and that hemojuvelin may serve as a molecular messenger for iron homeostasis. Even in hepatocytes stimulated with IL-6, we observed strong suppression of hepcidin mRNA by rs-hemojuvelin. If rs-hemojuvelin or its active fragments also suppress hepcidin production in vivo, they could be used to alleviate anemia of inflammation.

scholarly journals Transferrin-a modulates hepcidin expression in zebrafish embryos

Blood ◽  
2009 ◽  
Vol 113 (12) ◽  
pp. 2843-2850 ◽  
Author(s):  
Paula G. Fraenkel ◽  
Yann Gibert ◽  
Jason L. Holzheimer ◽  
Victoria J. Lattanzi ◽  
Sarah F. Burnett ◽  
...  
Keyword(s):  
Large Scale ◽  
Critical Role ◽  
Hypochromic Anemia ◽  
Zebrafish Embryos ◽  
Genetic Screens ◽  
Hepcidin Expression ◽  
Morpholino Knockdown ◽  
Low Levels ◽  
Identification And Characterization

Abstract The iron regulatory hormone hepcidin is transcriptionally up-regulated in response to iron loading, but the mechanisms by which iron levels are sensed are not well understood. Large-scale genetic screens in the zebrafish have resulted in the identification of hypochromic anemia mutants with a range of mutations affecting conserved pathways in iron metabolism and heme synthesis. We hypothesized that transferrin plays a critical role both in iron transport and in regulating hepcidin expression in zebrafish embryos. Here we report the identification and characterization of the zebrafish hypochromic anemia mutant, gavi, which exhibits transferrin deficiency due to mutations in transferrin-a. Morpholino knockdown of transferrin-a in wild-type embryos reproduced the anemia phenotype and decreased somite and terminal gut iron staining, while coinjection of transferrin-a cRNA partially restored these defects. Embryos with transferrin-a or transferrin receptor 2 (TfR2) deficiency exhibited low levels of hepcidin expression, however anemia, in the absence of a defect in the transferrin pathway, failed to impair hepcidin expression. These data indicate that transferrin-a transports iron and that hepcidin expression is regulated by a transferrin-a–dependent pathway in the zebrafish embryo.

scholarly journals Sites of vulnerability on ricin B chain revealed through epitope mapping of toxin-neutralizing monoclonal antibodies

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0236538
Author(s):  
David J. Vance ◽  
Amanda Y. Poon ◽  
Nicholas J. Mantis
Keyword(s):  
Monoclonal Antibodies ◽  
Mammalian Cells ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Single Chain ◽  
Carbohydrate Recognition Domains ◽  
And Function ◽  
Golgi Network ◽  
Dose Dependent

Ricin toxin’s B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes uptake and intracellular trafficking of ricin’s ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, β, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (~90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB’s duplicative structure. To circumvent this problem, RTB-D1 and RTB-D2 were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as “bait” in mAb capture assays. We found that SylH3 captured RTB-D1 (but not RTB-D2) in a dose-dependent manner, while 24B11 captured RTB-D2 (but not RTB-D1) in a dose-dependent manner. We confirmed these domain assignments by competition studies with an additional 8 RTB-specific mAbs along with a dozen a single chain antibodies (VHHs). Collectively, these results demonstrate that type I and type II mAbs segregate on the basis of domain specificity and suggest that RTB’s two domains may contribute to distinct steps in the intoxication pathway.

scholarly journals Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin N. Nelson ◽  
Savannah G. Beakley ◽  
Sierra Posey ◽  
Brittney Conn ◽  
Emma Maritz ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Dose Dependent ◽  
Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

scholarly journals The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Alyssa N. Coyne ◽  
Jeffrey D. Rothstein
Keyword(s):  
Nuclear Pore Complex ◽  
Mammalian Cells ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Structured Illumination Microscopy ◽  
Neuronal Nuclei ◽  
Complex Injury ◽  
Sporadic Als ◽  
Human Neurons

AbstractNuclear pore complex injury has recently emerged as an early and significant contributor to familial and sporadic ALS disease pathogenesis. However, the molecular events leading to this pathological phenomenon characterized by the reduction of specific nucleoporins from neuronal nuclear pore complexes remain largely unknown. This is due in part to a lack of knowledge regarding the biological pathways and proteins underlying nuclear pore complex homeostasis specifically in human neurons. We have recently uncovered that aberrant nuclear accumulation of the ESCRT-III protein CHMP7 initiates nuclear pore complex in familial and sporadic ALS neurons. In yeast and non-neuronal mammalian cells, nuclear relocalization of CHMP7 has been shown to recruit the ESCRT-III proteins CHMP4B, CHMP2B, and VPS4 to facilitate nuclear pore complex and nuclear envelope repair and homeostasis. Here, using super resolution structured illumination microscopy, we find that neither CHMP4B nor CHMP2B are increased in ALS neuronal nuclei. In contrast, VPS4 expression is significantly increased in ALS neuronal nuclei prior to the emergence of nuclear pore injury in a CHMP7 dependent manner. However, unlike our prior CHMP7 knockdown studies, impaired VPS4 function does not mitigate alterations to the NPC and the integral transmembrane nucleoporin POM121. Collectively our data suggest that while alterations in VPS4 subcellular localization appear to be coincident with nuclear pore complex injury, therapeutic efforts to mitigate this pathogenic cascade should be targeted towards upstream events such as the nuclear accumulation of CHMP7 as we have previously described.

scholarly journals ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization

2002 ◽  
Vol 13 (9) ◽  
pp. 3078-3095 ◽  
Author(s):  
Annette L. Boman ◽  
Paul D. Salo ◽  
Melissa J. Hauglund ◽  
Nicole L. Strand ◽  
Shelly J. Rensink ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Carboxypeptidase Y ◽  
Minor Role ◽  
Temperature Sensitive ◽  
Adp Ribosylation ◽  
And Function ◽  
Adp Ribosylation Factor

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

scholarly journals Regulation of the tumour suppressor Fbw7α by PKC-dependent phosphorylation and cancer-associated mutations

2010 ◽  
Vol 432 (1) ◽  
pp. 77-87 ◽  
Author(s):  
Joanne Durgan ◽  
Peter J. Parker
Keyword(s):  
Mammalian Cells ◽  
Tumour Suppressor ◽  
Dependent Manner ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Pkc Protein Kinase C ◽  
Specific Regulation ◽  
The Relationship ◽  
Substrate Binding Domain

Fbw7 (F-box WD40 protein 7) is a major tumour suppressor, which mediates the degradation of several potent oncogenes. PKC (protein kinase C) comprises a serine/threonine kinase family that can promote transformation when dysregulated. In the present study, we investigated the relationship between Fbw7 and PKC. Multiple members of the PKC superfamily interact with the substrate-binding domain of Fbw7. However, we find no evidence for Fbw7-mediated degradation of PKC. Instead, we demonstrate that Fbw7 is a novel substrate for PKC. Two residues within the isoform-specific N-terminus of Fbw7α are phosphorylated in a PKC-dependent manner, both in vitro and in mammalian cells (Ser10 and Ser18). Mutational analyses reveal that phosphorylation of Fbw7α at Ser10 can regulate its nuclear localization. Cancer-associated mutations in nearby residues (K11R and the addition of a proline residue at position 16) influence Fbw7α localization in a comparable manner, suggesting that mislocalization of this protein may be of pathological significance. Together these results provide evidence for both physical and functional interactions between the PKC and Fbw7 families, and yield insights into the isoform-specific regulation of Fbw7α.

scholarly journals TCMacro: A Simple and Robust ImageJ-Based Method for Automated Measurement of Tail Coiling Activity in Zebrafish

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1133
Author(s):  
Kevin Adi Kurnia ◽  
Fiorency Santoso ◽  
Bonifasius Putera Sampurna ◽  
Gilbert Audira ◽  
Jong-Chin Huang ◽  
...  
Keyword(s):  
Pearson Correlation ◽  
Dynamic Change ◽  
Current Method ◽  
Previous Method ◽  
Third Party ◽  
Wilcoxon Test ◽  
Average Intensity ◽  
Zebrafish Embryos ◽  
Dependent Manner ◽  
Pixel Intensity

Tail coiling is a reflection response in fish embryos that can be used as a model for neurotoxic analysis. The previous method to analyze fish tail coiling is largely based on third-party software. In this study, we aim to develop a simple and cost-effective method called TCMacro by using ImageJ macro to reduce the operational complexity. The basic principle of the current method is based on the dynamic change of pixel intensity in the region of interest (ROI). When the fish tail is moving, the average intensity is increasing. In time when the fish freeze, the peak of mean intensity is maintaining at a relatively low level. By using the optimized macro settings and excel VBA scripts, all the tail coiling measurement processes can be archived with few operation steps with high precision. Three major endpoints of tail coiling counts, tail coiling duration and tail coiling intervals can be obtained in batch. To validate this established method, we tested the potential neurotoxic activity of Tricaine (methanesulfonate, MS-222) and psychoactive compound of caffeine. Zebrafish embryos after Tricaine exposure displayed significantly less tail coiling activity in a dose-dependent manner, and were comparable to manual counting through the Wilcoxon test and Pearson correlation double validation. Zebrafish embryos after caffeine exposure displayed significantly high tail coiling activity. In conclusion, the TCMacro method presented in this study provides a simple and robust method that is able to measure the relative tail coiling activities in zebrafish embryos in a high-throughput manner.

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