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Identification of TFG- and autophagy-regulated proteins and glycerophospholipids in B cells shows a role for TFG in metabolism

ABSTRACT: Autophagy supervises proteostasis and survival of plasma cells. TFG (Trk-fused gene) promotes autophagosome-lysosome flux in murine CH12 B cells, as well as their survival. Moreover, TFG is abundant in plasma cells. Hence, quantitative proteomics of CH12tfgKO and WT B cells in combination with lysosomal inhibition via NH4Cl should identify proteins that are prone to lysosomal degradation and contribute to autophagy, plasma cell biology and B cell survival. While lysosome inhibition via NH4Cl treatment unexpectedly reduced a number of proteins, it increased a large cluster of translational and ribosomal proteins, independent of TFG. The prosurvival protein BCL10 was decreased in CH12tfgKO B cells while the immunoglobulin JCHAIN was increased. Gene ontology analysis revealed that proteins 39 regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane 40 bounded organelles. Likewise, TFG regulated the abundance of metabolic enzymes, such as the glycolytic enzyme ALDOC and the short chain fatty acid activating enzyme ACOT9. To test for a function of TFG in lipid metabolism we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol was more abundant in CH12tfgKO B cells. In contrast, several glycerophospholipid species, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, showed a dysequilibrium. Addition of phosphatidylglycerol elevated Cardiolipin in B cells. Hence, we propose a role for TFG in the regulation of proteins involved in survival and plasma cell biology, as well as in lipid homeostasis, mitochondrial functions and metabolism.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): B Cell

SUBMITTER: Friedel Drepper

LAB HEAD: Bettina Warscheid

PROVIDER: PXD039180 | Pride | 2024-02-27

REPOSITORIES: Pride

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Project description:ABSTRACT Stimulating the commitment of implanted dystrophin+ muscle derived stem cells (MDSC) into myogenic, as opposed to lipofibrogenic, lineages is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). To examine whether counteracting myostatin, a negative regulator of muscle mass and a pro-lipofibrotic factor, would help this process, we compared the in vitro myogenic and fibrogenic capacity of MDSC from wild type (WT), myostatin knockout (Mst KO), and mdx (DMD model) (mdx) young mice under various modulators, the expression of key stem cell and myogenic genes, and the capacity of these MDSC to repair the injured gastrocnemius in aged mdx mice with exacerbated lipofibrosis. Surprisingly, the potent in vitro myotube formation by WT MDSC was refractory to modulators of myostatin expression or activity, and the Mst KO and mdx MDSC failed to form myotubes under any condition, despite all MDSC expressed Oct-4 and various stem cell genes and differentiated into other lineages. The genetic inactivation of myostatin or dystrophin in MDSC was associated with silencing of critical genes for early myogenesis (Actc1, Acta1, and MyoD). WT MDSC implanted into the injured gastrocnemius of old mdx mice significantly improved myofiber repair and reduced fat deposition and, to a lesser extent, fibrosis. In contrast to their in vitro behavior, Mst KO MDSC in vivo also significantly improved myofiber repair, but had no significant effects on lipofibrotic degeneration. In conclusion, while WT MDSC are considerably myogenic in culture and stimulate muscle repair after injury in the aged mdx mouse, myostatin genetic inactivation blocks myotube formation in vitro but the myogenic capacity is recovered in vivo under the influence of the host tissue environment, presumably by reactivation of key genes originally silenced in the Mst KO MDSC. Key words: dystrophin, mdx mouse, Duchenne, fibrosis, dystrophy One sample each of mouse wild type (WT), myostatin knockout (KO), muscular dystrophy model mdx, and the transgenic OCT4 promoter plus reporter were grown, RNA was isolated, and subjected to the SABiosciences mouse stem cell oligo array.

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Identification of TFG- and autophagy-regulated proteins and glycerophospholipids in B cells shows a role for TFG in metabolism

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