Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity.

Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-γ signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7-H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta-subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock-out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM-3, restored the NK cell ability to eliminate TMEM30A-mutated cells. The key role of the TIM-3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM-3 in the realm of cancer immunotherapy.

Knockdown of TMEM30A in renal tubular epithelial cells leads to reduced glucose absorption.

The kidney reabsorbs large amounts of glucose through Na+-glucose cotransporter 2 (SGLT2). P4-ATPase acts together with the ß-subunit TMEM30A to mediate the asymmetric distribution of phosphatidylserine (PS), phosphatidylethanolamine (PE), and other amino phospholipids, promoting plasma membrane and internal vesicle fusion, and facilitating vesicle protein transport. We observed reduced TMEM30A expression in renal tubules of DKD and IgA patients, suggesting a potential role of TMEM30A in renal tubular cells. To investigate the role of TMEM30A in renal tubules, we constructed a TMEM30A knockdown cell model by transfecting mouse kidney tubular epithelium cells (TCMK-1) with TMEM30A shRNA. Knockdown of TMEM30A in TCMK-1 cells attenuated vesicle transporter protein synthesis, resulting in reduced transport and expression of SGLT2, which in turn reduced glucose absorption. These data suggested that TMEM30A plays a crucial role in renal tubules.

The phosphatidylserine flippase ß-subunit Tmem30a is essential for normal insulin maturation and secretion.

The processing, maturation, and secretion of insulin are under precise regulation, and dysregulation causes profound defects in glucose handling, leading to diabetes. Tmem30a is the ß subunit of the phosphatidylserine (PS) flippase, which maintains the membrane asymmetric distribution of PS. Tmem30a regulates cell survival and the localization of subcellular structures and is thus critical to the normal function of multiple physiological systems. Here, we show that conditional knockout of Tmem30a specifically in pancreatic islet ß cells leads to obesity, hyperglycemia, glucose intolerance, hyperinsulinemia, and insulin resistance in mice, due to insufficient insulin release. Moreover, we reveal that Tmem30a plays an essential role in clathrin-mediated vesicle transport between the trans Golgi network (TGN) and the plasma membrane (PM), which comprises immature secretory granule (ISG) budding at the TGN. We also find that Tmem30a deficiency impairs clathrin-mediated vesicle budding and thus blocks both insulin maturation in ISGs and the transport of glucose-sensing Glut2 to the PM. Collectively, these disruptions compromise both insulin secretion and glucose sensitivity, thus contributing to impairments in glucose-stimulated insulin secretion. Taken together, our data demonstrate an important role of Tmem30a in insulin maturation and glucose metabolic homeostasis and suggest the importance of membrane phospholipid distribution in metabolic disorders.

TMEM30A deficiency in endothelial cells impairs cell proliferation and angiogenesis.

Phosphatidylserine (PS) asymmetry in the eukaryotic cell membrane is maintained by a group of proteins belonging to the P4-ATPase family, namely, PS flippases. The folding and transporting of P4-ATPases to their cellular destination requires a ß-subunit member of the TMEM30 protein family. Loss of Tmem30a has been shown to cause multiple disease conditions. However, its roles in vascular development have not been elucidated. Here, we show that TMEM30A plays critical roles in retinal vascular angiogenesis, which is a fundamental process in vascular development. Our data indicate that knockdown of TMEM30A in primary human retinal endothelial cells led to reduced tube formation. In mice, endothelial cell (EC)-specific deletion of Tmem30a led to retarded retinal vascular development with a hyperpruned vascular network as well as blunted-end, aneurysm-like tip ECs with fewer filopodia at the vascular front and a reduced number of tip cells. Deletion of Tmem30a also impaired vessel barrier integrity. Mechanistically, deletion of TMEM30A caused reduced EC proliferation by inhibiting VEGF-induced signaling. Our findings reveal essential roles of TMEM30A in angiogenesis, providing a potential therapeutic target.

Tmem30a deficiency leads to retinal rod bipolar cell degeneration.

Phospholipids are asymmetrically distributed across the mammalian plasma membrane, with phosphatidylserine (PS) and phosphatidylethanolamine concentrated in the cytoplasmic leaflet of the membrane bilayer and phosphatidylcholine in the exoplasmic leaflet. This asymmetric distribution is dependent on a group of P4 ATPases called PS flippases. The proper transport and function of PS flippases require a ß-subunit transmembrane protein 30A (TMEM30A). Disruption of PS flippases leads to several human diseases. Tmem30a is essential for photoreceptor survival. However, the roles of Tmem30a in the retinal rod bipolar cells (RBC) remain elusive. To investigate the role of Tmem30a in the RBCs, we generated a RBC-specific Tmem30a knockout (cKO) mouse model using PCP2-Cre line. The Tmem30a cKO mice exhibited defect in RBC function and progressive RBC death. PKCα staining of retinal cryosections from cKO mice revealed a remarkable dendritic sprouting of rod bipolar cells during the early degenerative process. Immunostaining analysis of PSD95 and mGluT6 expression demonstrated that rod bipolar cells in Tmem30a cKO retinas exhibited aberrant dendritic sprouting as a result of impaired synaptic efficacy, which implied a crucial role for Tmem30a in synaptic transmission in the retina. In addition, loss of Tmem30a led to reactive gliosis with increased expression of glial fibrillary acidic protein and CD68. TUNEL staining suggested that apoptotic cell death occurred in the retinal inner nuclear layer (INL). Our data show that loss of Tmem30a in RBCs results in dendritic sprouting of rod bipolar cells, increased astrogliosis and RBC death. Taken together, our studies demonstrate an essential role for Tmem30a in the retinal bipolar cells. Cover Image for this issue: doi: 10.1111/jnc.14492.

Loss of phosphatidylserine flippase ß-subunit Tmem30a in podocytes leads to albuminuria and glomerulosclerosis.

The asymmetric distribution of phosphatidylserine (PS) in the cytoplasmic leaflet of eukaryotic cell plasma membranes is regulated by a group of P4-ATPases (named PS flippases) and the ß-subunit TMEM30A. Podocytes in the glomerulus form a filtration barrier to prevent the traversing of large cellular elements and macromolecules from the blood into the urinary space. Damage to podocytes can disrupt the filtration barrier and lead to proteinuria and podocytopathy. We observed reduced TMEM30A expression in patients with minimal change disease and membranous nephropathy, indicating potential roles of TMEM30A in podocytopathy. To investigate the role of Tmem30a in the kidney, we generated a podocyte-specific Tmem30a knockout (KO) mouse model using the NPHS2-Cre line. Tmem30a KO mice displayed albuminuria, podocyte degeneration, mesangial cell proliferation with prominent extracellular matrix accumulation and eventual progression to focal segmental glomerulosclerosis. Our data demonstrate a critical role of Tmem30a in maintaining podocyte survival and glomerular filtration barrier integrity. Understanding the dynamic regulation of the PS distribution in the glomerulus provides a unique perspective to pinpointing the mechanism of podocyte damage and potential therapeutic targets.

The biology of the tumor microenvironment in DLBCL: Targeting the "don't eat me" signal.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of malignant lymphoma with biologically and clinically heterogeneous features. Recently, the tumor microenvironment of this disease has been recognized as an important biological aspect of tumor development and therapeutic targets. Recurrent genetic alterations play significant roles in immune recognition of lymphoma cells. In particular, novel genetic alterations promoting phagocytosis were identified, suggesting a potential therapeutic strategy targeting the "don't eat me" signal.

Deletion of phosphatidylserine flippase ß-subunit Tmem30a in satellite cells leads to delayed skeletal muscle regeneration.

Phosphatidylserine (PS) is distributed asymmetrically in the plasma membrane of eukaryotic cells. Phosphatidylserine flippase (P4-ATPase) transports PS from the outer leaflet of the lipid bilayer to the inner leaflet of the membrane to maintain PS asymmetry. The ß subunit TMEM30A is indispensable for transport and proper function of P4-ATPase. Previous studies have shown that the ATP11A and TMEM30A complex is the molecular switch for myotube formation. However, the role of Tmem30a in skeletal muscle regeneration remains elusive. In the current study, Tmem30a was highly expressed in the tibialis anterior (TA) muscles of dystrophin-null ( mdx) mice and BaCl 2-induced muscle injury model mice. We generated a satellite cell (SC)-specific Tmem30a conditional knockout (cKO) mouse model to investigate the role of Tmem30a in skeletal muscle regeneration. The regenerative ability of cKO mice was evaluated by analyzing the number and diameter of regenerated SCs after the TA muscles were injured by BaCl 2-injection. Compared to the control mice, the cKO mice showed decreased Pax7 + and MYH3 + SCs, indicating diminished SC proliferation, and decreased expression of muscular regulatory factors (MYOD and MYOG), suggesting impaired myoblast proliferation in skeletal muscle regeneration. Taken together, these results demonstrate the essential role of Tmem30a in skeletal muscle regeneration.