The Trk fused gene-product (Tfg) is part of a 600-700kDa CARMA1 complex

Grohmann, Markus; Steinmetz, Tobit; Jäck, Hans‐Martin; Mielenz, Dirk

doi:10.1101/857342

Cited by 3 publications

(6 citation statements)

References 37 publications

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“…Anti-TFG antibody was described previously [45]. Other antibodies were: mouse anti-ACTB/ACTIN (Sigma Aldrich, A1978), rabbit anti-ACTA1/ACTIN (Sigma Aldrich, A2066), rabbit anti-IRF4 (Cell Signaling Technology, 4964), goat antimouse IgM (Southern Biotech, 1021-01), goat anti-mouse IgM-horseradish peroxidase (HRP; Southern Biotech, 1020-05), goat anti-mouse IgG-HRP (Southern Biotech, 1030-05), rat anti-HSPA5/BIP (Santa Cruz Biotechnology, sc-13539), rabbit anti-MAP1LC3B/LC3 (Cell Signaling Technology, 2775S), rabbit anti-cleaved CASP3/CASPASE-3 (Cell Signaling Technology, 9661), goat anti-rabbit IgG-HRP (Jackson Immunoresearch, 111-035-046), goat anti-rat IgG-HRP (Jackson Immunoresearch, 112-035-071).…”

Section: Antibodiesmentioning

confidence: 99%

“…To visualize the ER, cells were incubated with ER-Tracker Blue-White (Invitrogen, E12353). Fixed cells were permeabilized with 0.1% Triton X-100 in PBS, blocked with 10% FCS in PBS and stained with a rabbit anti-TFG antibody [45], followed by Alexa Fluor 647-coupled goat anti-rabbit antibody (Dianova, 111-605-008). IgM was detected with goat anti-IgM antibody linked to Phycoerythrin (Southern Biotech, 1021-09).…”

Section: Immunofluorescence and Lc3 Turnover Assaysmentioning

confidence: 99%

“…In a proteomic screen for interaction partners of CARD11, an adaptor protein linking the BCR to NFKB and the MTOR (mechanistic target of rapamycin kinase) complex, both of which are involved in plasma cell physiology [20], we identified a 50-kDa protein named TFG (Trk-fused gene) in lysates from the mouse B lymphoma line CH27 [45]. TFG has been shown to support NFKB activation [46], to antagonize apoptosis, to promote cell size [47] and to control ER homeostasis [48], all of which accompany plasma cell differentiation.…”

Section: Introductionmentioning

confidence: 99%

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TFG is required for autophagy flux and to prevent endoplasmic reticulum stress in CH12 B lymphoma cells

Steinmetz

Schlötzer‐Schrehardt²,

Hearne

et al. 2020

Autophagy

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Plasma cells depend on quality control of newly synthesized antibodies in the endoplasmic reticulum (ER) via macroautophagy/autophagy and proteasomal degradation. The cytosolic adaptor protein TFG (Trkfused gene) regulates ER-Golgi transport, the secretory pathway and proteasome activity in non-immune cells. We show here that TFG is upregulated during lipopolysaccharide-and CpG-induced differentiation of B1 and B2 B cells into plasmablasts, with the highest expression of TFG in mature plasma cells. CRISPR-CAS9-mediated gene disruption of tfg in the B lymphoma cell line CH12 revealed increased apoptosis, which was reverted by BCL2 but even more by ectopic TFG expression. Loss of TFG disrupted ER structure, leading to an expanded ER and increased expression of ER stress genes. When compared to wild-type CH12 cells, tfg KO CH12 cells were more sensitive toward ER stress induced by tunicamycin, monensin and proteasome inhibition or by expression of an ER-bound immunoglobulin (Ig) μ heavy (µH) chain. CH12 tfg KO B cells displayed more total LC3, lower LC3-II turnover and increased numbers and size of autophagosomes. Tandem-fluorescent-LC3 revealed less accumulation of GFP-LC3 in starved and chloroquine-treated CH12 tfg KO B cells. The GFP:RFP ratio of tandem-fluorescent-LC3 was higher in tunicamycin-treated CH12 tfg KO B cells, suggesting less autophagy flux during induced ER stress. Based on these data, we suggest that TFG controls autophagy flux in CH12 B cells and propose that TFG is a survival factor that alleviates ER stress through the support of autophagy flux in activated B cells and mature plasma cells.

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Section: Antibodiesmentioning

confidence: 99%

Section: Immunofluorescence and Lc3 Turnover Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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TFG is required for autophagy flux and to prevent endoplasmic reticulum stress in CH12 B lymphoma cells

Steinmetz

Schlötzer‐Schrehardt²,

Hearne

et al. 2020

Autophagy

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“…In a proteomic screen for interaction partners of CARD11, we identified a 50 kDa protein named TRK-fused gene (TFG) in the mouse B lymphoma line CH27 . TFG consists of 397 amino acids structured into an N-terminal PB1 protein–protein interaction domain, a coiled-coil oligomerization domain, and a C-terminal proline-rich region.…”

Section: Introductionmentioning

confidence: 99%

Identification of TFG- and Autophagy-Regulated Proteins and Glycerophospholipids in B Cells

Steinmetz,

Thomas,

Reimann

et al. 2024

J. Proteome Res.

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Autophagy supervises the proteostasis and survival of B lymphocytic cells. Trk-f used gene (TFG) promotes autophagosome−lysosome flux in murine CH12 B cells, as well as their survival. Hence, quantitative proteomics of CH12tfgKO and WT B cells in combination with lysosomal inhibition should identify proteins that are prone to lysosomal degradation and contribute to autophagy and B cell survival. Lysosome inhibition via NH 4 Cl unexpectedly reduced a number of proteins but increased a large cluster of translational, ribosomal, and mitochondrial proteins, independent of TFG. Hence, we propose a role for lysosomes in ribophagy in B cells. TFG-regulated proteins include CD74, BCL10, or the immunoglobulin JCHAIN. Gene ontology (GO) analysis reveals that proteins regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane-bound organelles. Likewise, TFG regulates the abundance of metabolic enzymes, such as ALDOC and the fatty acid-activating enzyme ACOT9. To test consequently for a function of TFG in lipid metabolism, we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol is more abundant in CH12tfgKO B cells. Several glycerophospholipid species with similar acyl side chains, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, show a dysequilibrium. We suggest a role for TFG in lipid homeostasis, mitochondrial functions, translation, and metabolism in B cells.

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“…In a proteomics screen for interaction partners of CARD11 we identified a 50-kDa protein named TFG (TRK-fused gene) in the mouse B lymphoma line CH27 (Grohmann et al, 2019). TFG consists of 397 amino acids structured into an N-terminal PB1 protein-protein-interaction domain (Sumimoto et al, 2007), a coiled-coil oligomerization domain and a C-terminal proline-rich region.…”

Section: Introductionmentioning

confidence: 99%

Quantitative proteomics and lipidomics of TFG-deficient B cells provide insights into mechanisms of autophagic flux and plasma cell biology

Steinmetz

Reimann

Schulz

et al. 2022

Preprint

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The autophagy-flux-promoting protein TFG (Trk-fused gene) is up-regulated during B cell differentiation into plasma cells and supports survival of CH12 B cells. We hypothesized that quantitative proteomics analysis of CH12tfgKO B cells with intact or blocked autophagy-lysosome flux (via NH4Cl) will identify mechanisms of TFG-dependent autophagy, plasma cell biology and B cell survival. Analysis of CH12WT B cells in the presence of NH4Cl will identify proteins whose presence is continuously regulated by lysosomes independent of TFG. We determined hundreds of proteins to be controlled by TFG and/or NH4Cl. Notably, NH4Cl treatment alone increased the abundance of a cluster of cytosolic and mitochondrial translational proteins while it also reduced a number of proteins. Within the B cell relevant protein pool, BCL10 was reduced, while JCHAIN was increased in CH12tfgKO B cells. Furthermore, TFG regulated the abundance of transcription factors, such as JUNB, metabolic enzymes, such as the short-chain fatty acid activating enzyme ACOT9 or the glycolytic enzyme ALDOC. Gene ontology enrichment analysis revealed that TFG-regulated proteins localized to mitochondria and membrane-bounded organelles. Due to these findings we performed shotgun lipidomics of glycerophospholipids, uncovering that a particular phosphatidylethanolamine (PE) species, 32:0 PE, which lipidates LC3 most efficiently, was less abundant while phosphatidylglycerol (PG) was more abundant in CH12tfgKO B cells. In line with the role of PG as precursor for Cardiolipin (CL), the CL content was higher in CH12tfgKO B cells and addition of PG liposomes to B cells increased the amount of CL. We propose a role for TFG in B cell activation and plasma cell biology via regulation of proteins involved in germinal center and plasma cell development, such as BCL10 or JCHAIN, as well as in lipid homeostasis, mitochondria and metabolism.

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The Trk fused gene-product (Tfg) is part of a 600-700kDa CARMA1 complex

Cited by 3 publications

References 37 publications

TFG is required for autophagy flux and to prevent endoplasmic reticulum stress in CH12 B lymphoma cells

TFG is required for autophagy flux and to prevent endoplasmic reticulum stress in CH12 B lymphoma cells

Identification of TFG- and Autophagy-Regulated Proteins and Glycerophospholipids in B Cells

Quantitative proteomics and lipidomics of TFG-deficient B cells provide insights into mechanisms of autophagic flux and plasma cell biology

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