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Scramblase Xk-related protein 8 (Xkr8) regulates the externalization of phosphatidylserine (PS) during apoptosis and holds a pivotal role in fostering tumor immunosuppression. Targeting Xkr8 in conjunction with chemotherapy demonstrated a novel avenue for amplifying antitumor immune response and overcoming chemo-immune resistance. Here we further evaluated this strategy by using a clinically relevant orthotopic model and elucidated the mechanism through in-depth single-cell RNA sequencing (scRNA-seq). We found that Xkr8 knockdown exhibited the potential to lead to immunogenic cell death (ICD) by impeding the normal clearance of apoptotic cells. Co-delivery of Xkr8 small interference RNA (siRNA) and a prodrug conjugate of 5-fluorouracil (5-Fu) and oxoplatin (FuOXP) showed remarkable therapeutic efficacy in an orthotopic pancreatic tumor model with increased infiltration of proliferative NK cells and activated macrophages in the tumor microenvironment (TME). Single-cell trajectory analysis further unveiled that tumor infiltrating CD8+ T cells are differentiated favorably to cytotoxic over exhausted phenotype after combination treatment. Our study sheds new light on the impact of Xkr8 knockdown on TME and solidifies the rationale of combining Xkr8 knockdown with chemotherapy to treat various types of cancers. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Background Auditory neuropathy is an unusual type of hearing loss. At least 40% of patients with this disease have underlying genetic causes. However, in many hereditary auditory neuropathy cases, etiology remains undetermined. Methods We collected data and blood samples from a four-generation Chinese family. After excluding relevant variants in known deafness-related genes, exome sequencing was conducted. Candidate genes were verified by pedigree segregation, transcript/protein expression in the mouse cochlea, and plasmid expression studies in HEK 293T cells. Moreover, a mutant mouse model was generated and underwent hearing evaluations; protein localization in the inner ear was also assessed. Results The clinical features of the family were diagnosed as auditory neuropathy. A novel variant c.710G > A (p.W237X) in apoptosis-related gene XKR8 was identified. Genotyping of 16 family members confirmed the segregation of this variant with the deafness phenotype. Both XKR8 mRNA and XKR8 protein were expressed in the mouse inner ear, predominantly in regions of spiral ganglion neurons; Moreover, this nonsense variant impaired the surface localization of XKR8 in cells. Transgenic mutant mice exhibited late-onset auditory neuropathy, and their altered XKR8 protein localization in the inner ear confirmed the damaging effects of this variant. Conclusions We identified a variant in the XKR8 gene that is relevant to auditory neuropathy. The essential role of XKR8 in inner ear development and neural homeostasis should be explored.

Background: Auditory neuropathy is an unusual type of hearing loss. At least 40% of patients with this disease have underlying genetic causes. However, in many hereditary auditory neuropathy cases, etiology remains undetermined. Methods: We collected data and blood samples from a four-generation Chinese family. After excluding relevant variants in known deafness-related genes, exome sequencing was conducted. Candidate genes were verified by pedigree segregation, transcript/protein expression in the mouse cochlea, and plasmid expression studies in HEK 293T cells. Moreover, a mutant mouse model was generated and underwent hearing evaluations; protein localization in the inner ear was also assessed. Results: The clinical features of the family were diagnosed as auditory neuropathy. A novel variant c.710G > A (p.W237X) in apoptosis-related gene XKR8 was identified. Genotyping of 16 family members confirmed the segregation of this variant with the deafness phenotype. Both XKR8 mRNA and XKR8 protein were expressed in the mouse inner ear, predominantly in regions of spiral ganglion neurons; Moreover, this nonsense variant impaired the surface localization of XKR8 in cells. Transgenic mutant mice exhibited late-onset auditory neuropathy, and their altered XKR8 protein localization in the inner ear confirmed the damaging effects of this variant. Conclusions: We identified a variant in the XKR8 gene that is relevant to auditory neuropathy. The essential role of XKR8 in inner ear development and neural homeostasis should be explored. [ABSTRACT FROM AUTHOR]

The asymmetric distribution of lipids, maintained by flippases/floppases and scramblases, plays a pivotal role in various physiologic processes. Scramblases are proteins that move phospholipids between the leaflets of the lipid bilayer of the cellular membrane in an energy‐independent manner. Recent studies have indicated that viral infection is closely related to cellular lipid distribution. The level and distribution of phosphatidylserine (PtdSer) in cells have been demonstrated to be critical regulators of viral infections. Previous studies have supported that the infection of human immunodeficiency virus (HIV), Zika virus, Ebola virus (EBOV), influenza virus, and dengue fever virus require the externalization of phospholipids mediated by scramblases, which are also involved in the pathogenicity of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). In this review, we review the relationship of scramblases with viruses and the potential viral effector proteins that might utilize host scramblases. [ABSTRACT FROM AUTHOR]

ADAM17, a prominent member of the “Disintegrin and Metalloproteinase” (ADAM) family, controls vital cellular functions through the cleavage of transmembrane substrates, including epidermal growth factor receptor (EGFR) ligands such as transforming growth factor (TGF)-alpha and Epiregulin (EREG). Several ADAM17 substrates are relevant to oncogenesis and tumor growth. We have presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. The scramblase Xkr8 is instrumental for calcium-independent exposure of PS in apoptotic cells. Xkr8 can be dually activated by caspase-3 and by kinases. In this investigation, we examined whether Xkr8 would modulate ADAM17 activity under apoptotic and non-apoptotic conditions. Overexpression of Xkr8 in HEK293T cells led to significantly increased caspase-dependent as well as PMA-induced release of EREG and TGF-alpha. Conversely, siRNA-mediated downregulation of Xkr8 in colorectal Caco-2 cancer cells led to decreased PS externalization upon induction of apoptosis, which was accompanied by reduced shedding of endogenously expressed EREG and reduced cell survival. We conclude that Xkr8 shares with conventional scramblases the propensity to upmodulate the ADAM-sheddase function. Liberation of growth factors could serve a rescue function in cells on the pathway to apoptotic death.

Ebola virus (EBOV) attaches to target cells using two categories of cell surface receptors: C-type lectins and phosphatidylserine (PS) receptors. PS receptors typically bind to apoptotic cell membrane PS and orchestrate the uptake and clearance of apoptotic debris. Many enveloped viruses also contain exposed PS and can therefore exploit these receptors for cell entry. Viral infection can induce PS externalization in host cells, resulting in increased outer PS levels on budding virions. Scramblase enzymes carry out cellular PS externalization; thus, we targeted these proteins in order to manipulate viral envelope PS levels. We investigated two scramblases previously identified to be involved in EBOV PS levels, transmembrane protein 16F and Xk-related protein 8 (XKR8), as possible mediators of cellular and viral envelope surface PS levels during the replication of recombinant vesicular stomatitis virus containing its native glycoprotein (rVSV/G) or the EBOV glycoprotein (rVSV/EBOV-GP). We found that rVSV/G and rVSV/EBOV-GP virions produced in XKR8 knockout cells contain decreased levels of PS on their surfaces, and the PS-deficient rVSV/EBOV-GP virions are 70% less efficient at infecting cells through PS receptors. We also observed reduced rVSV and EBOV virus-like particle (VLP) budding in DXKR8 cells. Deletion of XKR8 in HAP1 cells reduced rVSV/G and rVSV/EBOV-GP budding by 60 and 65%, respectively, and reduced Ebola VLP budding more than 60%. We further demonstrated that caspase cleavage of XKR8 is required to promote budding. This suggests that XKR8, in addition to mediating virion PS levels, may also be critical for enveloped virus budding at the plasma membrane. [ABSTRACT FROM AUTHOR]

During spermatogenesis, up to 75% of germ cells in the testes undergo apoptosis and are cleared by Sertoli cells. XKR8 is a plasma membrane protein that scrambles phospholipids in response to apoptotic signals, exposing phosphatidylserine (PtdSer). Here, we found that Xkr8-/- male mice were infertile due to reduced sperm counts in their epididymides. Apoptotic stimuli could not induce PtdSer exposure in Xkr8-/- germ cells. Consistent with the hypothesis that PtdSer functions as an "eat me" signal to phagocytes, cells expressing phosphatidylserine receptor TIM4 and MER tyrosine kinase receptor efficiently engulfed apoptotic wild-type male germ cells, but not Xkr8-/- germ cells. Fluorescence and electron microscopy revealed Sertoli cells carrying engulfed and degenerated dead cells. However, many unengulfed apoptotic cells, residual bodies, and cell debris were present in Xkr8-/- testes and epididymides. These results indicate that XKR8-mediated PtdSer exposure is essential for the clearance of apoptotic germ cells by Sertoli cells. There was no apparent inflammation in Xkr8-/- testes, suggesting that the unengulfed apoptotic cells may have undergone secondary necrosis, releasing noxious materials that affected the germ cells. Alternatively, failure to engulf the apoptotic germ cells may have caused the Sertoli cells to starve and lose their ability to support spermatogenesis. [ABSTRACT FROM AUTHOR]

Ebola virus (EBOV) interacts with cells using multiple categories of cell-surface receptors, including C-type lectins and phosphatidylserine (PS) receptors. PS receptors typically bind to apoptotic cell membrane PS and orchestrate the uptake and clearance of apoptotic bodies. Many viruses coated with PS-containing lipid envelopes, acquired during budding from host cells, can also exploit these receptors for internalization. PS is restricted to the inner leaflet of the plasma membrane in homeostatic cells, an orientation that would be unfavorable for PS receptor-mediated uptake if conserved on the viral envelope. Therefore, it is theorized that viral infection induces host-cell PS externalization to the outer leaflet during replication. Cells have several membrane scramblase enzymes that enrich outer leaflet PS when activated. Here, we investigate the role of two scramblases, TMEM16F and XKR8, as possible mediators of cellular and viral envelope surface PS levels during recombinant vesicular stomatitis virus (VSV) in which the VSV glycoprotein was replaced with the Ebola glycoprotein (rVSV/EBOV-GP) replication and EBOV virus-like particle (VLP) production. We find that rVSV/EBOV-GP and EBOV VLPs produced in XKR8 knockout cells contain two- to threefold less PS in their outer leaflets. Consequently, rVSV/EBOV-GP produced in deltaXKR8 is 70% less efficient at infecting cells through apoptotic mimicry as compared to the viruses produced by parental cells. In addition, the budding efficiency of both recombinant VSV particles and VLPs was significantly reduced in cells lacking XKR8. Our data suggest that virion surface PS acquisition requires XKR8 activity, whereas the deletion of TMEM16F did not affect EBOV-GP-mediated entry of VLP production. Unexpectedly, we observed an additional role of XKR8 in rVSV/G, rVSV/EBOV-GP, and EBOV VLP budding.

ADAM17, a prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, controls vital cellular functions through the cleavage of transmembrane substrates, including epidermal growth factor receptor (EGFR) ligands such as transforming growth factor (TGF)-alpha and Epiregulin (EREG). Several ADAM17 substrates are relevant to oncogenesis and tumor growth. We have presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. The scramblase Xkr8 is instrumental for calcium-independent exposure of PS in apoptotic cells. Xkr8 can be dually activated by caspase-3 and by kinases. In this investigation, we examined whether Xkr8 would modulate ADAM17 activity under apoptotic and non-apoptotic conditions. Overexpression of Xkr8 in HEK293T cells led to significantly increased caspase-dependent as well as PMA-induced release of EREG and TGF-alpha. Conversely, siRNA-mediated downregulation of Xkr8 in colorectal Caco-2 cancer cells led to decreased PS externalization upon induction of apoptosis, which was accompanied by reduced shedding of endogenously expressed EREG and reduced cell survival. We conclude that Xkr8 shares with conventional scramblases the propensity to upmodulate the ADAM-sheddase function. Liberation of growth factors could serve a rescue function in cells on the pathway to apoptotic death.

Xk-related protein (Xkr) 8, a protein carrying 10 transmembrane regions, is essential for scrambling phospholipids during apoptosis. Here, we found Xkr8 as a complex with basigin (BSG) or neuroplastin (NPTN), type I membrane proteins in the Ig superfamily. In BSG−/−NPTN−/− cells, Xkr8 localized intracellularly, and the apoptosis stimuli failed to expose phosphatidylserine, indicating that BSG and NPTN chaperone Xkr8 to the plasma membrane to execute its scrambling activity. Mutational analyses of BSG showed that the atypical glutamic acid in the transmembrane region is required for BSG’s association with Xkr8. In cells exposed to apoptotic signals, Xkr8 was cleaved at the C terminus and the Xkr8/BSG complex formed a higher-order complex, likely to be a heterotetramer consisting of two molecules of Xkr8 and two molecules of BSG or NPTN, suggesting that this cleavage causes the formation of a larger complex of Xkr8-BSG/NPTN for phospholipid scrambling. [ABSTRACT FROM AUTHOR]

In "healthy" tumor cells, phosphatidylserine (PS) is predominately localized in the inner plasma membrane leaflet. During apoptosis, PS relocates to the outer leaflet. Herein, we established PSout tumor models with tumor cells lacking PS flippase component CDC50A, constantly exposing PS but alive. PSout tumors developed bigger than wild-type (WT) tumors, featuring M2 polarized tumor-associated macrophages (TAMs) and fewer tumor-antigen-specific T cells. The PS receptor TIM-3 is responsible for PS recognition. Employing an opposite tumor model, PSin, with tumor cells lacking the PS scramblase Xkr8 and unable to expose PS during otherwise normal apoptosis, we find that the accumulated apoptotic tumor cells produce and release cyclic GAMP (cGAMP) to immune cells to activate the STING pathway, leading to TAM M1 polarization, suppressed interleukin (IL)-10 secretion, and natural killer (NK) cell cytotoxicity. Silencing Xkr8 in vivo by either short hairpin RNA (shRNA) or small interfering RNA (siRNA) to achieve a PS externalization blockade provides robust therapeutic anti-tumor efficiency. [Display omitted] • PSout tumors grow larger than WT, with an immune-suppressive tumor microenvironment • TIM-3 is responsible for PS sensing in PSout tumors • PSin tumors activate STING, leading to TAM M1 polarization and NK cell cytotoxicity • Silencing Xkr8 in vivo to block PS externalization provides anti-tumor effects in mice Wang et al. show that externalized phosphatidylserine (PS) on cancer cells induces an immune-suppressive microenvironment to promote tumor growth, which is dependent on the PS receptor TIM-3. Blocking PS externalization by silencing the PS scramblase Xkr8 enhances anti-tumor immunity, suggesting Xkr8 as a potential therapeutic target. [ABSTRACT FROM AUTHOR]

During spermatogenesis, up to 75% of germ cells in the testes undergo apoptosis and are cleared by Sertoli cells. X-linked XK blood group-related 8 (Xkr8) is a plasma membrane protein that scrambles phospholipids in response to apoptotic signals, exposing phosphatidylserine (PtdSer). Here, we found that Xkr8 -/- male mice were infertile due to reduced sperm counts in their epididymides. Apoptotic stimuli could not induce PtdSer exposure in Xkr8 -/- testes and epididymides. These results indicate that Xkr8-mediated PtdSer exposure is essential for the clearance of apoptotic germ cells by Sertoli cells. There was no apparent inflammation in Xkr8 -/- germ cells. Fluorescence and electron microscopy revealed Sertoli cells carrying engulfed and degenerated dead cells. However, many unengulfed apoptotic cells and residual bodies and much cell debris were present in Xkr8 -/- testes and epididymides. These results indicate that Xkr8-mediated PtdSer exposure is essential for the clearance of apoptotic germ cells by Sertoli cells. There was no apparent inflammation in Xkr8 -/- testes, suggesting that the unengulfed apoptotic cells may have undergone secondary necrosis, releasing noxious materials that affected the germ cells. Alternatively, failure to engulf the apoptotic germ cells may have caused the Sertoli cells to starve and lose their ability to support spermatogenesis., (Copyright © 2020 American Society for Microbiology.)

Xk-related protein (Xkr) 8, a protein carrying 10 transmembrane regions, is essential for scrambling phospholipids during apoptosis. Here, we found Xkr8 as a complex with basigin (BSG) or neuroplastin (NPTN), type I membrane proteins in the Ig superfamily. In BSG(-/-)NPTN(-/-) cells, Xkr8 localized intracellularly, and the apoptosis stimuli failed to expose phosphatidylserine, indicating that BSG and NPTN chaperone Xkr8 to the plasma membrane to execute its scrambling activity. Mutational analyses of BSG showed that the atypical glutamic acid in the transmembrane region is required for BSG's association with Xkr8. In cells exposed to apoptotic signals, Xkr8 was cleaved at the C terminus and the Xkr8/BSG complex formed a higher-order complex, likely to be a heterotetramer consisting of two molecules of Xkr8 and two molecules of BSG or NPTN, suggesting that this cleavage causes the formation of a larger complex of Xkr8-BSG/NPTN for phospholipid scrambling., Competing Interests: The authors declare no conflict of interest.

The distribution of phospholipid types between the two leaflets of a membrane bilayer is a controlled feature of membrane structure. One of the two membrane catalytic activities governing this distribution randomizes the composition of the two leaflets-the phospholipid scramblases. Two proteins (Xkr8 and TMEM16F) required for the activation of these activities have been identified. One of these proteins (TMEM16F) is quite clearly a scramblase itself and provides insight into the mechanism by which transbilayer phospholipid movement is facilitated.