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Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

Curator: Stephen J. Williams, Ph.D.

The following paper in Cells describes the discovery of protein interactors of endoglin, which is recruited to membranes at the TGF-β receptor complex upon TGF-β signaling. Interesting a carbohydrate binding protein, galectin-3, and an E3-ligase, TRIM21, were found to be unique interactors within this complex.

Gallardo-Vara E, Ruiz-Llorente L, Casado-Vela J, Ruiz-Rodríguez MJ, López-Andrés N, Pattnaik AK, Quintanilla M, Bernabeu C. Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners. Cells. 2019 Sep 13;8(9):1082. doi: 10.3390/cells8091082. PMID: 31540324; PMCID: PMC6769930.

Abstract

Endoglin is a 180-kDa glycoprotein receptor primarily expressed by the vascular endothelium and involved in cardiovascular disease and cancer. Heterozygous mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1, a vascular disease that presents with nasal and gastrointestinal bleeding, skin and mucosa telangiectases, and arteriovenous malformations in internal organs. A circulating form of endoglin (alias soluble endoglin, sEng), proteolytically released from the membrane-bound protein, has been observed in several inflammation-related pathological conditions and appears to contribute to endothelial dysfunction and cancer development through unknown mechanisms. Membrane-bound endoglin is an auxiliary component of the TGF-β receptor complex and the extracellular region of endoglin has been shown to interact with types I and II TGF-β receptors, as well as with BMP9 and BMP10 ligands, both members of the TGF-β family. To search for novel protein interactors, we screened a microarray containing over 9000 unique human proteins using recombinant sEng as bait. We find that sEng binds with high affinity, at least, to 22 new proteins. Among these, we validated the interaction of endoglin with galectin-3, a secreted member of the lectin family with capacity to bind membrane glycoproteins, and with tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin-protein ligase. Using human endothelial cells and Chinese hamster ovary cells, we showed that endoglin co-immunoprecipitates and co-localizes with galectin-3 or TRIM21. These results open new research avenues on endoglin function and regulation.

Source: https://www.mdpi.com/2073-4409/8/9/1082/htm

Endoglin is an auxiliary TGF-β co-receptor predominantly expressed in endothelial cells, which is involved in vascular development, repair, homeostasis, and disease [1,2,3,4]. Heterozygous mutations in the human ENDOGLIN gene (ENG) cause hereditary hemorrhagic telangiectasia (HHT) type 1, a vascular disease associated with nasal and gastrointestinal bleeds, telangiectases on skin and mucosa and arteriovenous malformations in the lung, liver, and brain [4,5,6]. The key role of endoglin in the vasculature is also illustrated by the fact that endoglin-KO mice die in utero due to defects in the vascular system [7]. Endoglin expression is markedly upregulated in proliferating endothelial cells involved in active angiogenesis, including the solid tumor neovasculature [8,9]. For this reason, endoglin has become a promising target for the antiangiogenic treatment of cancer [10,11,12]. Endoglin is also expressed in cancer cells where it can behave as both a tumor suppressor in prostate, breast, esophageal, and skin carcinomas [13,14,15,16] and a promoter of malignancy in melanoma and Ewing’s sarcoma [17]. Ectodomain shedding of membrane-bound endoglin may lead to a circulating form of the protein, also known as soluble endoglin (sEng) [18,19,20]. Increased levels of sEng have been found in several vascular-related pathologies, including preeclampsia, a disease of high prevalence in pregnant women which, if left untreated, can lead to serious and even fatal complications for both mother and baby [2,18,19,21]. Interestingly, several lines of evidence support a pathogenic role of sEng in the vascular system, including endothelial dysfunction, antiangiogenic activity, increased vascular permeability, inflammation-associated leukocyte adhesion and transmigration, and hypertension [18,22,23,24,25,26,27]. Because of its key role in vascular pathology, a large number of studies have addressed the structure and function of endoglin at the molecular level, in order to better understand its mechanism of action.

 Galectin-3 Interacts with Endoglin in Cells

Galectin-3 is a secreted member of the lectin family with the capacity to bind membrane glycoproteins like endoglin and is involved in the pathogenesis of many human diseases [52]. We confirmed the protein screen data for galectin-3, as evidenced by two-way co-immunoprecipitation of endoglin and galectin-3 upon co-transfection in CHO-K1 cells. As shown in Figure 1A, galectin-3 and endoglin were efficiently transfected, as demonstrated by Western blot analysis in total cell extracts. No background levels of endoglin were observed in control cells transfected with the empty vector (Ø). By contrast, galectin-3 could be detected in all samples but, as expected, showed an increased signal in cells transfected with the galectin-3 expression vector. Co-immunoprecipitation studies of these cell lysates showed that galectin-3 was present in endoglin immunoprecipitates (Figure 1B). Conversely, endoglin was also detected in galectin-3 immunoprecipitates (Figure 1C).

Cells 08 01082 g001 550

Figure 1. Protein–protein association between galectin-3 and endoglin. (AC). Co-immunoprecipitation of galectin-3 and endoglin. CHO-K1 cells were transiently transfected with pcEXV-Ø (Ø), pcEXV–HA–EngFL (Eng) and pcDNA3.1–Gal-3 (Gal3) expression vectors. (A) Total cell lysates (TCL) were analyzed by SDS-PAGE under reducing conditions, followed by Western blot (WB) analysis using specific antibodies to endoglin, galectin-3 and β-actin (loading control). Cell lysates were subjected to immunoprecipitation (IP) with anti-endoglin (B) or anti-galectin-3 (C) antibodies, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin or anti-galectin-3 antibodies, as indicated. Negative controls with an IgG2b (B) and IgG1 (C) were included. (D) Protein-protein interactions between galectin-3 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 7.3 µM recombinant human galectin-3/6xHis at the C-terminus (LGALS3), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.

Cells 08 01082 g002 550

Figure 2.Galectin-3 and endoglin co-localize in human endothelial cells. Human umbilical vein-derived endothelial cell (HUVEC) monolayers were fixed with paraformaldehyde, permeabilized with Triton X-100, incubated with the mouse mAb P4A4 anti-endoglin, washed, and incubated with a rabbit polyclonal anti-galectin-3 antibody (PA5-34819). Galectin-3 and endoglin were detected by immunofluorescence upon incubation with Alexa 647 goat anti-rabbit IgG (red staining) and Alexa 488 goat anti-mouse IgG (green staining) secondary antibodies, respectively. (A) Single staining of galectin-3 (red) and endoglin (green) at the indicated magnifications. (B) Merge images plus DAPI (nuclear staining in blue) show co-localization of galectin-3 and endoglin (yellow color). Representative images of five different experiments are shown.

Endoglin associates with the cullin-type E3 ligase TRIM21
Cells 08 01082 g003 550

Figure 3.Protein–protein association between TRIM21 and endoglin. (AE) Co-immunoprecipitation of TRIM21 and endoglin. A,B. HUVEC monolayers were lysed and total cell lysates (TCL) were subjected to SDS-PAGE under reducing (for TRIM21 detection) or nonreducing (for endoglin detection) conditions, followed by Western blot (WB) analysis using antibodies to endoglin, TRIM21 or β-actin (A). HUVECs lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or negative control antibodies, followed by WB analysis with anti-endoglin (B). C,D. CHO-K1 cells were transiently transfected with pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (E) or pcDNA3.1–HA–hTRIM21 (T) expression vectors, as indicated. Total cell lysates (TCL) were subjected to SDS-PAGE under nonreducing conditions and WB analysis using specific antibodies to endoglin, TRIM21, and β-actin (C). Cell lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or anti-endoglin antibodies, followed by SDS-PAGE under reducing (upper panel) or nonreducing (lower panel) conditions and WB analysis with anti-TRIM21 or anti-endoglin antibodies. Negative controls of appropriate IgG were included (D). E. CHO-K1 cells were transiently transfected with pcDNA3.1–HA–hTRIM21 and pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (FL; full-length), pDisplay–HA–EngEC (EC; cytoplasmic-less) or pDisplay–HA–EngTMEC (TMEC; cytoplasmic-less) expression vectors, as indicated. Cell lysates were subjected to immunoprecipitation with anti-TRIM21, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin antibodies, as indicated. The asterisk indicates the presence of a nonspecific band. Mr, molecular reference; Eng, endoglin; TRIM, TRIM21. (F) Protein–protein interactions between TRIM21 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 5.4 µM recombinant human TRIM21/6xHis at the N-terminus (R052), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.

Table 1. Human protein-array analysis of endoglin interactors1.

Accession #Protein NameCellular Compartment
NM_172160.1Potassium voltage-gated channel, shaker-related subfamily, beta member 1 (KCNAB1), transcript variant 1Plasma membrane
Q14722
NM_138565.1Cortactin (CTTN), transcript variant 2Plasma membrane
Q14247
BC036123.1Stromal membrane-associated protein 1 (SMAP1)Plasma membrane
Q8IYB5
NM_173822.1Family with sequence similarity 126, member B (FAM126B)Plasma membrane, cytosol
Q8IXS8
BC047536.1Sciellin (SCEL)Plasma membrane, extracellular or secreted
O95171
BC068068.1Galectin-3Plasma membrane, mitochondrion, nucleus, extracellular or secreted
P17931
BC001247.1Actin-binding LIM protein 1 (ABLIM1)Cytoskeleton
O14639
NM_198943.1Family with sequence similarity 39, member B (FAM39B)Endosome, cytoskeleton
Q6VEQ5
NM_005898.4Cell cycle associated protein 1 (CAPRIN1), transcript variant 1Cytosol
Q14444
BC002559.1YTH domain family, member 2 (YTHDF2)Nucleus, cytosol
Q9Y5A9
NM_003141.2Tripartite motif-containing 21 (TRIM21)Nucleus, cytosol
P19474
BC025279.1Scaffold attachment factor B2 (SAFB2)Nucleus
Q14151
BC031650.1Putative E3 ubiquitin-protein ligase SH3RF2Nucleus
Q8TEC5
BC034488.2ATP-binding cassette, sub-family F (GCN20), member 1 (ABCF1)Nucleus
Q8NE71
BC040946.1Spliceosome-associated protein CWC15 homolog (HSPC148)Nucleus
Q9P013
NM_003609.2HIRA interacting protein 3 (HIRIP3)Nucleus
Q9BW71
NM_005572.1Lamin A/C (LMNA), transcript variant 2Nucleus
P02545
NM_006479.2RAD51 associated protein 1 (RAD51AP1)Nucleus
Q96B01
NM_014321.2Origin recognition complex, subunit 6 like (yeast) (ORC6L)Nucleus
Q9Y5N6
NM_015138.2RNA polymerase-associated protein RTF1 homolog (RTF1)Nucleus
Q92541
NM_032141.1Coiled-coil domain containing 55 (CCDC55), transcript variant 1Nucleus
Q9H0G5
BC012289.1Protein PRRC2B, KIAA0515Data not available
Q5JSZ5

1 Microarrays containing over 9000 unique human proteins were screened using recombinant sEng as a probe. Protein interactors showing the highest scores (Z-score ≥2.0) are listed. GeneBank (https://www.ncbi.nlm.nih.gov/genbank/) and UniProtKB (https://www.uniprot.org/help/uniprotkb) accession numbers are indicated with a yellow or green background, respectively. The cellular compartment of each protein was obtained from the UniProtKB webpage. Proteins selected for further studies (TRIM21 and galectin-3) are indicated in bold type with blue background.

Note: the following are from NCBI Genbank and Genecards on TRIM21

 From Genbank: https://www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=DetailsSearch&Term=6737

TRIM21 tripartite motif containing 21 [ Homo sapiens (human) ]

Gene ID: 6737, updated on 6-Sep-2022

Summary

Official Symbol TRIM21provided by HGNC Official Full Name tripartite motif containing 21provided by HGNC Primary source HGNC:HGNC:11312 See related Ensembl:ENSG00000132109MIM:109092;AllianceGenome:HGNC:11312 Gene type protein coding RefSeq status REVIEWED Organism Homo sapiens Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo Also known as SSA; RO52; SSA1; RNF81; Ro/SSA Summary This gene encodes a member of the tripartite motif (TRIM) family. The TRIM motif includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled-coil region. The encoded protein is part of the RoSSA ribonucleoprotein, which includes a single polypeptide and one of four small RNA molecules. The RoSSA particle localizes to both the cytoplasm and the nucleus. RoSSA interacts with autoantigens in patients with Sjogren syndrome and systemic lupus erythematosus. Alternatively spliced transcript variants for this gene have been described but the full-length nature of only one has been determined. [provided by RefSeq, Jul 2008] Expression Ubiquitous expression in spleen (RPKM 15.5), appendix (RPKM 13.2) and 24 other tissues See more Orthologs mouseall NEW Try the new Gene table
Try the new Transcript table

Genomic context

See TRIM21 in Genome Data Viewer Location:   11p15.4 Exon count:   7

Annotation releaseStatusAssemblyChrLocation
110currentGRCh38.p14 (GCF_000001405.40)11NC_000011.10 (4384897..4393702, complement)
110currentT2T-CHM13v2.0 (GCF_009914755.1)11NC_060935.1 (4449988..4458819, complement)
105.20220307previous assemblyGRCh37.p13 (GCF_000001405.25)11NC_000011.9 (4406127..4414932, complement)

Chromosome 11 – NC_000011.10Genomic Context describing neighboring genes

Bibliography

Related articles in PubMed

  1. TRIM21 inhibits the osteogenic differentiation of mesenchymal stem cells by facilitating K48 ubiquitination-mediated degradation of Akt.Xian J, et al. Exp Cell Res, 2022 Mar 15. PMID 35051432
  2. A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment.Chen G, et al. Biomolecules, 2021 Oct 14. PMID 34680146, Free PMC Article
  3. Induced TRIM21 ISGylation by IFN-β enhances p62 ubiquitination to prevent its autophagosome targeting.Jin J, et al. Cell Death Dis, 2021 Jul 13. PMID 34257278, Free PMC Article
  4. TRIM21 Polymorphisms are associated with Susceptibility and Clinical Status of Oral Squamous Cell Carcinoma patients.Chuang CY, et al. Int J Med Sci, 2021. PMID 34220328, Free PMC Article
  5. TRIM21 inhibits porcine epidemic diarrhea virus proliferation by proteasomal degradation of the nucleocapsid protein.Wang H, et al. Arch Virol, 2021 Jul. PMID 33900472, Free PMC Article

From GeneCard:https://www.genecards.org/cgi-bin/carddisp.pl?gene=TRIM21

Entrez Gene Summary for TRIM21 Gene

  • This gene encodes a member of the tripartite motif (TRIM) family. The TRIM motif includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled-coil region. The encoded protein is part of the RoSSA ribonucleoprotein, which includes a single polypeptide and one of four small RNA molecules. The RoSSA particle localizes to both the cytoplasm and the nucleus. RoSSA interacts with autoantigens in patients with Sjogren syndrome and systemic lupus erythematosus. Alternatively spliced transcript variants for this gene have been described but the full-length nature of only one has been determined. [provided by RefSeq, Jul 2008]

GeneCards Summary for TRIM21 Gene

TRIM21 (Tripartite Motif Containing 21) is a Protein Coding gene. Diseases associated with TRIM21 include Heart Block, Congenital and Sjogren Syndrome. Among its related pathways are Cytosolic sensors of pathogen-associated DNA and KEAP1-NFE2L2 pathway. Gene Ontology (GO) annotations related to this gene include identical protein binding and ligase activity. An important paralog of this gene is TRIM6.

UniProtKB/Swiss-Prot Summary for TRIM21 Gene

E3 ubiquitin-protein ligase whose activity is dependent on E2 enzymes, UBE2D1, UBE2D2, UBE2E1 and UBE2E2. Forms a ubiquitin ligase complex in cooperation with the E2 UBE2D2 that is used not only for the ubiquitination of USP4 and IKBKB but also for its self-ubiquitination. Component of cullin-RING-based SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes such as SCF(SKP2)-like complexes. A TRIM21-containing SCF(SKP2)-like complex is shown to mediate ubiquitination of CDKN1B (‘Thr-187’ phosphorylated-form), thereby promoting its degradation by the proteasome. Monoubiquitinates IKBKB that will negatively regulates Tax-induced NF-kappa-B signaling. Negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3. Mediates the ubiquitin-mediated proteasomal degradation of IgG1 heavy chain, which is linked to the VCP-mediated ER-associated degradation (ERAD) pathway. Promotes IRF8 ubiquitination, which enhanced the ability of IRF8 to stimulate cytokine genes transcription in macrophages. Plays a role in the regulation of the cell cycle progression. Enhances the decapping activity of DCP2. Exists as a ribonucleoprotein particle present in all mammalian cells studied and composed of a single polypeptide and one of four small RNA molecules. At least two isoforms are present in nucleated and red blood cells, and tissue specific differences in RO/SSA proteins have been identified. The common feature of these proteins is their ability to bind HY RNAs.2. Involved in the regulation of innate immunity and the inflammatory response in response to IFNG/IFN-gamma. Organizes autophagic machinery by serving as a platform for the assembly of ULK1, Beclin 1/BECN1 and ATG8 family members and recognizes specific autophagy targets, thus coordinating target recognition with assembly of the autophagic apparatus and initiation of autophagy. Acts as an autophagy receptor for the degradation of IRF3, hence attenuating type I interferon (IFN)-dependent immune responses (PubMed:26347139162978621631662716472766168805111802269418361920186413151884514219675099). Represses the innate antiviral response by facilitating the formation of the NMI-IFI35 complex through ‘Lys-63’-linked ubiquitination of NMI (PubMed:26342464). ( RO52_HUMAN,P19474 )

Molecular function for TRIM21 Gene according to UniProtKB/Swiss-Prot

Function:

  • E3 ubiquitin-protein ligase whose activity is dependent on E2 enzymes, UBE2D1, UBE2D2, UBE2E1 and UBE2E2.
    Forms a ubiquitin ligase complex in cooperation with the E2 UBE2D2 that is used not only for the ubiquitination of USP4 and IKBKB but also for its self-ubiquitination.
    Component of cullin-RING-based SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes such as SCF(SKP2)-like complexes.
    A TRIM21-containing SCF(SKP2)-like complex is shown to mediate ubiquitination of CDKN1B (‘Thr-187’ phosphorylated-form), thereby promoting its degradation by the proteasome.
    Monoubiquitinates IKBKB that will negatively regulates Tax-induced NF-kappa-B signaling.
    Negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3.
    Mediates the ubiquitin-mediated proteasomal degradation of IgG1 heavy chain, which is linked to the VCP-mediated ER-associated degradation (ERAD) pathway.
    Promotes IRF8 ubiquitination, which enhanced the ability of IRF8 to stimulate cytokine genes transcription in macrophages.
    Plays a role in the regulation of the cell cycle progression.

Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

Gallardo-Vara E, Ruiz-Llorente L, Casado-Vela J, Ruiz-Rodríguez MJ, López-Andrés N, Pattnaik AK, Quintanilla M, Bernabeu C. Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners. Cells. 2019 Sep 13;8(9):1082. doi: 10.3390/cells8091082. PMID: 31540324; PMCID: PMC6769930.

Abstract

Endoglin is a 180-kDa glycoprotein receptor primarily expressed by the vascular endothelium and involved in cardiovascular disease and cancer. Heterozygous mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1, a vascular disease that presents with nasal and gastrointestinal bleeding, skin and mucosa telangiectases, and arteriovenous malformations in internal organs. A circulating form of endoglin (alias soluble endoglin, sEng), proteolytically released from the membrane-bound protein, has been observed in several inflammation-related pathological conditions and appears to contribute to endothelial dysfunction and cancer development through unknown mechanisms. Membrane-bound endoglin is an auxiliary component of the TGF-β receptor complex and the extracellular region of endoglin has been shown to interact with types I and II TGF-β receptors, as well as with BMP9 and BMP10 ligands, both members of the TGF-β family. To search for novel protein interactors, we screened a microarray containing over 9000 unique human proteins using recombinant sEng as bait. We find that sEng binds with high affinity, at least, to 22 new proteins. Among these, we validated the interaction of endoglin with galectin-3, a secreted member of the lectin family with capacity to bind membrane glycoproteins, and with tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin-protein ligase. Using human endothelial cells and Chinese hamster ovary cells, we showed that endoglin co-immunoprecipitates and co-localizes with galectin-3 or TRIM21. These results open new research avenues on endoglin function and regulation.
 
 
Endoglin is an auxiliary TGF-β co-receptor predominantly expressed in endothelial cells, which is involved in vascular development, repair, homeostasis, and disease [1,2,3,4]. Heterozygous mutations in the human ENDOGLIN gene (ENG) cause hereditary hemorrhagic telangiectasia (HHT) type 1, a vascular disease associated with nasal and gastrointestinal bleeds, telangiectases on skin and mucosa and arteriovenous malformations in the lung, liver, and brain [4,5,6]. The key role of endoglin in the vasculature is also illustrated by the fact that endoglin-KO mice die in utero due to defects in the vascular system [7]. Endoglin expression is markedly upregulated in proliferating endothelial cells involved in active angiogenesis, including the solid tumor neovasculature [8,9]. For this reason, endoglin has become a promising target for the antiangiogenic treatment of cancer [10,11,12]. Endoglin is also expressed in cancer cells where it can behave as both a tumor suppressor in prostate, breast, esophageal, and skin carcinomas [13,14,15,16] and a promoter of malignancy in melanoma and Ewing’s sarcoma [17]. Ectodomain shedding of membrane-bound endoglin may lead to a circulating form of the protein, also known as soluble endoglin (sEng) [18,19,20]. Increased levels of sEng have been found in several vascular-related pathologies, including preeclampsia, a disease of high prevalence in pregnant women which, if left untreated, can lead to serious and even fatal complications for both mother and baby [2,18,19,21]. Interestingly, several lines of evidence support a pathogenic role of sEng in the vascular system, including endothelial dysfunction, antiangiogenic activity, increased vascular permeability, inflammation-associated leukocyte adhesion and transmigration, and hypertension [18,22,23,24,25,26,27]. Because of its key role in vascular pathology, a large number of studies have addressed the structure and function of endoglin at the molecular level, in order to better understand its mechanism of action.
 

 Galectin-3 Interacts with Endoglin in Cells

Galectin-3 is a secreted member of the lectin family with the capacity to bind membrane glycoproteins like endoglin and is involved in the pathogenesis of many human diseases [52]. We confirmed the protein screen data for galectin-3, as evidenced by two-way co-immunoprecipitation of endoglin and galectin-3 upon co-transfection in CHO-K1 cells. As shown in Figure 1A, galectin-3 and endoglin were efficiently transfected, as demonstrated by Western blot analysis in total cell extracts. No background levels of endoglin were observed in control cells transfected with the empty vector (Ø). By contrast, galectin-3 could be detected in all samples but, as expected, showed an increased signal in cells transfected with the galectin-3 expression vector. Co-immunoprecipitation studies of these cell lysates showed that galectin-3 was present in endoglin immunoprecipitates (Figure 1B). Conversely, endoglin was also detected in galectin-3 immunoprecipitates (Figure 1C).
Figure 1. Protein–protein association between galectin-3 and endoglin. (AC). Co-immunoprecipitation of galectin-3 and endoglin. CHO-K1 cells were transiently transfected with pcEXV-Ø (Ø), pcEXV–HA–EngFL (Eng) and pcDNA3.1–Gal-3 (Gal3) expression vectors. (A) Total cell lysates (TCL) were analyzed by SDS-PAGE under reducing conditions, followed by Western blot (WB) analysis using specific antibodies to endoglin, galectin-3 and β-actin (loading control). Cell lysates were subjected to immunoprecipitation (IP) with anti-endoglin (B) or anti-galectin-3 (C) antibodies, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin or anti-galectin-3 antibodies, as indicated. Negative controls with an IgG2b (B) and IgG1 (C) were included. (D) Protein-protein interactions between galectin-3 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 7.3 µM recombinant human galectin-3/6xHis at the C-terminus (LGALS3), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.
Figure 2. Galectin-3 and endoglin co-localize in human endothelial cells. Human umbilical vein-derived endothelial cell (HUVEC) monolayers were fixed with paraformaldehyde, permeabilized with Triton X-100, incubated with the mouse mAb P4A4 anti-endoglin, washed, and incubated with a rabbit polyclonal anti-galectin-3 antibody (PA5-34819). Galectin-3 and endoglin were detected by immunofluorescence upon incubation with Alexa 647 goat anti-rabbit IgG (red staining) and Alexa 488 goat anti-mouse IgG (green staining) secondary antibodies, respectively. (A) Single staining of galectin-3 (red) and endoglin (green) at the indicated magnifications. (B) Merge images plus DAPI (nuclear staining in blue) show co-localization of galectin-3 and endoglin (yellow color). Representative images of five different experiments are shown.
  
Endoglin associates with the cullin-type E3 ligase TRIM21
 
Figure 3. Protein–protein association between TRIM21 and endoglin. (AE) Co-immunoprecipitation of TRIM21 and endoglin. A,B. HUVEC monolayers were lysed and total cell lysates (TCL) were subjected to SDS-PAGE under reducing (for TRIM21 detection) or nonreducing (for endoglin detection) conditions, followed by Western blot (WB) analysis using antibodies to endoglin, TRIM21 or β-actin (A). HUVECs lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or negative control antibodies, followed by WB analysis with anti-endoglin (B). C,D. CHO-K1 cells were transiently transfected with pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (E) or pcDNA3.1–HA–hTRIM21 (T) expression vectors, as indicated. Total cell lysates (TCL) were subjected to SDS-PAGE under nonreducing conditions and WB analysis using specific antibodies to endoglin, TRIM21, and β-actin (C). Cell lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or anti-endoglin antibodies, followed by SDS-PAGE under reducing (upper panel) or nonreducing (lower panel) conditions and WB analysis with anti-TRIM21 or anti-endoglin antibodies. Negative controls of appropriate IgG were included (D). E. CHO-K1 cells were transiently transfected with pcDNA3.1–HA–hTRIM21 and pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (FL; full-length), pDisplay–HA–EngEC (EC; cytoplasmic-less) or pDisplay–HA–EngTMEC (TMEC; cytoplasmic-less) expression vectors, as indicated. Cell lysates were subjected to immunoprecipitation with anti-TRIM21, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin antibodies, as indicated. The asterisk indicates the presence of a nonspecific band. Mr, molecular reference; Eng, endoglin; TRIM, TRIM21. (F) Protein–protein interactions between TRIM21 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 5.4 µM recombinant human TRIM21/6xHis at the N-terminus (R052), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.
 
Table 1. Human protein-array analysis of endoglin interactors1.
Accession # Protein Name Cellular Compartment
NM_172160.1 Potassium voltage-gated channel, shaker-related subfamily, beta member 1 (KCNAB1), transcript variant 1 Plasma membrane
Q14722
NM_138565.1 Cortactin (CTTN), transcript variant 2 Plasma membrane
Q14247
BC036123.1 Stromal membrane-associated protein 1 (SMAP1) Plasma membrane
Q8IYB5
NM_173822.1 Family with sequence similarity 126, member B (FAM126B) Plasma membrane, cytosol
Q8IXS8
BC047536.1 Sciellin (SCEL) Plasma membrane, extracellular or secreted
O95171
BC068068.1 Galectin-3 Plasma membrane, mitochondrion, nucleus, extracellular or secreted
P17931
BC001247.1 Actin-binding LIM protein 1 (ABLIM1) Cytoskeleton
O14639
NM_198943.1 Family with sequence similarity 39, member B (FAM39B) Endosome, cytoskeleton
Q6VEQ5
NM_005898.4 Cell cycle associated protein 1 (CAPRIN1), transcript variant 1 Cytosol
Q14444
BC002559.1 YTH domain family, member 2 (YTHDF2) Nucleus, cytosol
Q9Y5A9
NM_003141.2 Tripartite motif-containing 21 (TRIM21) Nucleus, cytosol
P19474
BC025279.1 Scaffold attachment factor B2 (SAFB2) Nucleus
Q14151
BC031650.1 Putative E3 ubiquitin-protein ligase SH3RF2 Nucleus
Q8TEC5
BC034488.2 ATP-binding cassette, sub-family F (GCN20), member 1 (ABCF1) Nucleus
Q8NE71
BC040946.1 Spliceosome-associated protein CWC15 homolog (HSPC148) Nucleus
Q9P013
NM_003609.2 HIRA interacting protein 3 (HIRIP3) Nucleus
Q9BW71
NM_005572.1 Lamin A/C (LMNA), transcript variant 2 Nucleus
P02545
NM_006479.2 RAD51 associated protein 1 (RAD51AP1) Nucleus
Q96B01
NM_014321.2 Origin recognition complex, subunit 6 like (yeast) (ORC6L) Nucleus
Q9Y5N6
NM_015138.2 RNA polymerase-associated protein RTF1 homolog (RTF1) Nucleus
Q92541
NM_032141.1 Coiled-coil domain containing 55 (CCDC55), transcript variant 1 Nucleus
Q9H0G5
BC012289.1 Protein PRRC2B, KIAA0515 Data not available
Q5JSZ5
1 Microarrays containing over 9000 unique human proteins were screened using recombinant sEng as a probe. Protein interactors showing the highest scores (Z-score ≥2.0) are listed. GeneBank (https://www.ncbi.nlm.nih.gov/genbank/) and UniProtKB (https://www.uniprot.org/help/uniprotkb) accession numbers are indicated with a yellow or green background, respectively. The cellular compartment of each protein was obtained from the UniProtKB webpage. Proteins selected for further studies (TRIM21 and galectin-3) are indicated in bold type with blue background.
  

Note: the following are from NCBI Genbank and Genecards on TRIM21

TRIM21 tripartite motif containing 21 [ Homo sapiens (human) ]

Gene ID: 6737, updated on 6-Sep-2022

Summary
Official Symbol
TRIM21provided by HGNC
Official Full Name
tripartite motif containing 21provided by HGNC
Primary source
HGNC:HGNC:11312
See related
Ensembl:ENSG00000132109 MIM:109092; AllianceGenome:HGNC:11312
Gene type
protein coding
RefSeq status
REVIEWED
Organism
Homo sapiens
Lineage
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo
Also known as
SSA; RO52; SSA1; RNF81; Ro/SSA
Summary
This gene encodes a member of the tripartite motif (TRIM) family. The TRIM motif includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled-coil region. The encoded protein is part of the RoSSA ribonucleoprotein, which includes a single polypeptide and one of four small RNA molecules. The RoSSA particle localizes to both the cytoplasm and the nucleus. RoSSA interacts with autoantigens in patients with Sjogren syndrome and systemic lupus erythematosus. Alternatively spliced transcript variants for this gene have been described but the full-length nature of only one has been determined. [provided by RefSeq, Jul 2008]
Expression
Ubiquitous expression in spleen (RPKM 15.5), appendix (RPKM 13.2) and 24 other tissues See more
Orthologs
NEW
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Try the new Transcript table
Genomic context
 
See TRIM21 in Genome Data Viewer
Location:
11p15.4
Exon count:
7
Annotation release Status Assembly Chr Location
110 current GRCh38.p14 (GCF_000001405.40) 11 NC_000011.10 (4384897..4393702, complement)
110 current T2T-CHM13v2.0 (GCF_009914755.1) 11 NC_060935.1 (4449988..4458819, complement)
105.20220307 previous assembly GRCh37.p13 (GCF_000001405.25) 11 NC_000011.9 (4406127..4414932, complement)

Chromosome 11 – NC_000011.10Genomic Context describing neighboring genes

Neighboring gene olfactory receptor family 52 subfamily B member 4 Neighboring gene olfactory receptor family 52 subfamily B member 3 pseudogene Neighboring gene olfactory receptor family 51 subfamily R member 1 pseudogene Neighboring gene olfactory receptor family 52 subfamily P member 2 pseudogene

 

Entrez Gene Summary for TRIM21 Gene

  • This gene encodes a member of the tripartite motif (TRIM) family. The TRIM motif includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled-coil region. The encoded protein is part of the RoSSA ribonucleoprotein, which includes a single polypeptide and one of four small RNA molecules. The RoSSA particle localizes to both the cytoplasm and the nucleus. RoSSA interacts with autoantigens in patients with Sjogren syndrome and systemic lupus erythematosus. Alternatively spliced transcript variants for this gene have been described but the full-length nature of only one has been determined. [provided by RefSeq, Jul 2008]

GeneCards Summary for TRIM21 Gene

TRIM21 (Tripartite Motif Containing 21) is a Protein Coding gene. Diseases associated with TRIM21 include Heart Block, Congenital and Sjogren Syndrome. Among its related pathways are Cytosolic sensors of pathogen-associated DNA and KEAP1-NFE2L2 pathway. Gene Ontology (GO) annotations related to this gene include identical protein binding and ligase activity. An important paralog of this gene is TRIM6.

UniProtKB/Swiss-Prot Summary for TRIM21 Gene

E3 ubiquitin-protein ligase whose activity is dependent on E2 enzymes, UBE2D1, UBE2D2, UBE2E1 and UBE2E2. Forms a ubiquitin ligase complex in cooperation with the E2 UBE2D2 that is used not only for the ubiquitination of USP4 and IKBKB but also for its self-ubiquitination. Component of cullin-RING-based SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes such as SCF(SKP2)-like complexes. A TRIM21-containing SCF(SKP2)-like complex is shown to mediate ubiquitination of CDKN1B (‘Thr-187’ phosphorylated-form), thereby promoting its degradation by the proteasome. Monoubiquitinates IKBKB that will negatively regulates Tax-induced NF-kappa-B signaling. Negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3. Mediates the ubiquitin-mediated proteasomal degradation of IgG1 heavy chain, which is linked to the VCP-mediated ER-associated degradation (ERAD) pathway. Promotes IRF8 ubiquitination, which enhanced the ability of IRF8 to stimulate cytokine genes transcription in macrophages. Plays a role in the regulation of the cell cycle progression. Enhances the decapping activity of DCP2. Exists as a ribonucleoprotein particle present in all mammalian cells studied and composed of a single polypeptide and one of four small RNA molecules. At least two isoforms are present in nucleated and red blood cells, and tissue specific differences in RO/SSA proteins have been identified. The common feature of these proteins is their ability to bind HY RNAs.2. Involved in the regulation of innate immunity and the inflammatory response in response to IFNG/IFN-gamma. Organizes autophagic machinery by serving as a platform for the assembly of ULK1, Beclin 1/BECN1 and ATG8 family members and recognizes specific autophagy targets, thus coordinating target recognition with assembly of the autophagic apparatus and initiation of autophagy. Acts as an autophagy receptor for the degradation of IRF3, hence attenuating type I interferon (IFN)-dependent immune responses (PubMed:26347139162978621631662716472766168805111802269418361920186413151884514219675099). Represses the innate antiviral response by facilitating the formation of the NMI-IFI35 complex through ‘Lys-63’-linked ubiquitination of NMI (PubMed:26342464). ( RO52_HUMAN,P19474 )

Molecular function for TRIM21 Gene according to UniProtKB/Swiss-Prot

Function:
  • E3 ubiquitin-protein ligase whose activity is dependent on E2 enzymes, UBE2D1, UBE2D2, UBE2E1 and UBE2E2.
    Forms a ubiquitin ligase complex in cooperation with the E2 UBE2D2 that is used not only for the ubiquitination of USP4 and IKBKB but also for its self-ubiquitination.
    Component of cullin-RING-based SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes such as SCF(SKP2)-like complexes.
    A TRIM21-containing SCF(SKP2)-like complex is shown to mediate ubiquitination of CDKN1B (‘Thr-187’ phosphorylated-form), thereby promoting its degradation by the proteasome.
    Monoubiquitinates IKBKB that will negatively regulates Tax-induced NF-kappa-B signaling.
    Negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3.
    Mediates the ubiquitin-mediated proteasomal degradation of IgG1 heavy chain, which is linked to the VCP-mediated ER-associated degradation (ERAD) pathway.
    Promotes IRF8 ubiquitination, which enhanced the ability of IRF8 to stimulate cytokine genes transcription in macrophages.
    Plays a role in the regulation of the cell cycle progression.

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