Apoptosis and Clearance of Apoptotic Cells.

The human body generates 10-100 billion cells every day, and the same number of cells die to maintain homeostasis in our body. Cells infected by bacteria or viruses also die. The cell death that occurs under physiological conditions mainly proceeds by apoptosis, which is a noninflammatory, or silent, process, while pathogen infection induces necroptosis or pyroptosis, which activates the immune system and causes inflammation. Dead cells generated by apoptosis are quickly engulfed by macrophages for degradation. Caspases are a large family of cysteine proteases that act in cascades. A cascade that leads to caspase 3 activation mediates apoptosis and is responsible for killing cells, recruiting macrophages, and presenting an "eat me" signal(s). When apoptotic cells are not efficiently engulfed by macrophages, they undergo secondary necrosis and release intracellular materials that represent a damage-associated molecular pattern, which may lead to a systemic lupus-like autoimmune disease.

Cryo-EM structures of human ABCA7 provide insights into its phospholipid translocation mechanisms.

Phospholipid extrusion by ABC subfamily A (ABCA) exporters is central to cellular physiology, although the specifics of the underlying substrate interactions and transport mechanisms remain poorly resolved at the molecular level. Here we report cryo-EM structures of lipid-embedded human ABCA7 in an open state and in a nucleotide-bound, closed state at resolutions between 3.6 and 4.0 Å. The former reveals an ordered patch of bilayer lipids traversing the transmembrane domain (TMD), while the latter reveals a lipid-free, closed TMD with a small extracellular opening. These structures offer a structural framework for both substrate entry and exit from the ABCA7 TMD and highlight conserved rigid-body motions that underlie the associated conformational transitions. Combined with functional analysis and molecular dynamics (MD) simulations, our data also shed light on lipid partitioning into the ABCA7 TMD and localized membrane perturbations that underlie ABCA7 function and have broader implications for other ABCA family transporters.

Mfsd2a utilizes a flippase mechanism to mediate omega-3 fatty acid lysolipid transport.

Major Facilitator Superfamily Domain containing 2a (Mfsd2a) is a sodium-dependent lysophosphatidylcholine (LPC) transporter expressed at the blood-brain barrier that constitutes the main pathway by which the brain obtains omega-3 fatty acids, such as docosahexanoic acid. Mfsd2a deficiency in humans results in severe microcephaly, underscoring the importance of LPC transport by Mfsd2a for brain development. Biochemical studies and recent cryo-electron microscopy (cryo-EM) structures of Mfsd2a bound to LPC suggest that Mfsd2a transports LPC via an alternating access mechanism between outward-facing and inward-facing conformational states in which the LPC inverts during transport between the outer and inner leaflet of a membrane. However, direct biochemical evidence of flippase activity by Mfsd2a has not been demonstrated and it is not understood how Mfsd2a could invert LPC between the outer and inner leaflet of the membrane in a sodium-dependent manner. Here, we established a unique in vitro assay using recombinant Mfsd2a reconstituted in liposomes that exploits the ability of Mfsd2a to transport lysophosphatidylserine (LPS) coupled with a small molecule LPS binding fluorophore that allowed for monitoring of directional flipping of the LPS headgroup from the outer to the inner liposome membrane. Using this assay, we demonstrate that Mfsd2a flips LPS from the outer to the inner leaflet of a membrane bilayer in a sodium-dependent manner. Furthermore, using cryo-EM structures as guides together with mutagenesis and a cell-based transport assay, we identify amino acid residues important for Mfsd2a activity that likely constitute substrate interaction domains. These studies provide direct biochemical evidence that Mfsd2a functions as a lysolipid flippase.

The DedA superfamily member PetA is required for the transbilayer distribution of phosphatidylethanolamine in bacterial membranes.

The sorting of phospholipids between the inner and outer leaflets of the membrane bilayer is a fundamental problem in all organisms. Despite years of investigation, most of the enzymes that catalyze phospholipid reorientation in bacteria remain unknown. Studies from almost half a century ago in Bacillus subtilis and Bacillus megaterium revealed that newly synthesized phosphatidylethanolamine (PE) is rapidly translocated to the outer leaflet of the bilayer [Rothman & Kennedy, Proc. Natl. Acad. Sci. U.S.A. 74, 1821-1825 (1977)] but the identity of the putative PE flippase has eluded discovery. Recently, members of the DedA superfamily have been implicated in flipping the bacterial lipid carrier undecaprenyl phosphate and in scrambling eukaryotic phospholipids in vitro. Here, using the antimicrobial peptide duramycin that targets outward-facing PE, we show that Bacillus subtilis cells lacking the DedA paralog PetA (formerly YbfM) have increased resistance to duramycin. Sensitivity to duramycin is restored by expression of B. subtilis PetA or homologs from other bacteria. Analysis of duramycin-mediated killing upon induction of PE synthesis indicates that PetA is required for efficient PE transport. Finally, using fluorescently labeled duramycin we demonstrate that cells lacking PetA have reduced PE in their outer leaflet compared to wildtype. We conclude that PetA is the long-sought PE transporter. These data combined with bioinformatic analysis of other DedA paralogs argue that the primary role of DedA superfamily members is transporting distinct lipids across the membrane bilayer.

Shapeshifting bullvalene-linked vancomycin dimers as effective antibiotics against multidrug-resistant gram-positive bacteria.

The alarming rise in superbugs that are resistant to drugs of last resort, including vancomycin-resistant enterococci and staphylococci, has become a significant global health hazard. Here, we report the click chemistry synthesis of an unprecedented class of shapeshifting vancomycin dimers (SVDs) that display potent activity against bacteria that are resistant to the parent drug, including the ESKAPE pathogens, vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), as well as vancomycin-resistant S. aureus (VRSA). The shapeshifting modality of the dimers is powered by a triazole-linked bullvalene core, exploiting the dynamic covalent rearrangements of the fluxional carbon cage and creating ligands with the capacity to inhibit bacterial cell wall biosynthesis. The new shapeshifting antibiotics are not disadvantaged by the common mechanism of vancomycin resistance resulting from the alteration of the C-terminal dipeptide with the corresponding d-Ala-d-Lac depsipeptide. Further, evidence suggests that the shapeshifting ligands destabilize the complex formed between the flippase MurJ and lipid II, implying the potential for a new mode of action for polyvalent glycopeptides. The SVDs show little propensity for acquired resistance by enterococci, suggesting that this new class of shapeshifting antibiotic will display durable antimicrobial activity not prone to rapidly acquired clinical resistance.

Cerebral Vascular Dysfunctions Detected in Human Small Vessel Disease and Implications for Preclinical Studies.

Cerebral small vessel disease (SVD) is highly prevalent and a common cause of ischemic and hemorrhagic stroke and dementia, yet the pathophysiology is poorly understood. Its clinical expression is highly varied, and prognostic implications are frequently overlooked in clinics; thus, treatment is currently confined to vascular risk factor management. Traditionally, SVD is considered the small vessel equivalent of large artery stroke (occlusion, rupture), but data emerging from human neuroimaging and genetic studies refute this, instead showing microvessel endothelial dysfunction impacting on cell-cell interactions and leading to brain damage. These dysfunctions reflect defects that appear to be inherited and secondary to environmental exposures, including vascular risk factors. Interrogation in preclinical models shows consistent and converging molecular and cellular interactions across the endothelial-glial-neural unit that increasingly explain the human macroscopic observations and identify common patterns of pathology despite different triggers. Importantly, these insights may offer new targets for therapeutic intervention focused on restoring endothelial-glial physiology.

Consensus, controversies, and conundrums of P4-ATPases: The emerging face of eukaryotic lipid flippases.

The cryo-EM resolution revolution has heralded a new era in our understanding of eukaryotic lipid flippases with a rapidly growing number of high-resolution structures. Flippases belong to the P4 family of ATPases (type IV P-type ATPases) that largely follow the reaction cycle proposed for the more extensively studied cation-transporting P-type ATPases. However, unlike the canonical P-type ATPases, no flippase cargos are transported in the phosphorylation half-reaction. Instead of being released into the intracellular or extracellular milieu, lipid cargos are transported to their destination at the inner leaflet of the membrane. Recent flippase structures have revealed multiple conformational states during the lipid transport cycle. Nonetheless, critical conformational states capturing the lipid cargo "in transit" are still missing. In this review, we highlight the amazing structural advances of these lipid transporters, discuss various perspectives on catalytic and regulatory mechanisms in the literature, and shed light on future directions in further deciphering the detailed molecular mechanisms of lipid flipping.

Inefficient development of syncytiotrophoblasts in the Atp11a-deficient mouse placenta.

The P4-ATPases ATP11A and ATP11C function as flippases at the plasma membrane to translocate phosphatidylserine from the outer to the inner leaflet. We herein demonstrated that Atp11a-deficient mouse embryos died at approximately E14.5 with thin-walled heart ventricles. However, the cardiomyocyte- or epiblast-specific Atp11a deletion did not affect mouse development or mortality. ATP11C may have compensated for the function of ATP11A in most of the cell types in the embryo. On the other hand, Atp11a, but not Atp11c, was expressed in the mouse placenta, and the Atp11a-null mutation caused poor development of the labyrinthine layer with an increased number of TUNEL-positive foci. Immunohistochemistry and electron microscopy revealed a disorganized labyrinthine layer with unfused trophoblasts in the Atp11a-null placenta. Human placenta-derived choriocarcinoma BeWo cells expressed the ATP11A and ATP11C genes. A lack of ATP11A and ATP11C eliminated the ability of BeWo cells to flip phosphatidylserine and fuse when treated with forskolin. These results indicate that flippases at the plasma membrane play an important role in the formation of syncytiotrophoblasts in placental development.

Structural insights into the activation of autoinhibited human lipid flippase ATP8B1 upon substrate binding.

SignificanceATP8B1 is a P4 ATPase that maintains membrane asymmetry by transporting phospholipids across the cell membrane. Disturbance of lipid asymmetry will lead to the imbalance of the cell membrane and eventually, cell death. Thus, defects in ATP8B1 are usually associated with severe human diseases, such as intrahepatic cholestasis. The present structures of ATP8B1 complexed with its auxiliary noncatalytic partners CDC50A and CDC50B reveal an autoinhibited state of ATP8B1 that could be released upon substrate binding. Moreover, release of this autoinhibition could be facilitated by the bile acids, which are key factors that alter the membrane asymmetry of hepatocytes. This enabled us to figure out a feedback loop of bile acids and lipids across the cell membrane.

Glucosylceramide flippases contribute to cellular glucosylceramide homeostasis.

Lipid transport is an essential cellular process with importance to human health, disease development, and therapeutic strategies. Type IV P-type ATPases (P4-ATPases) have been identified as membrane lipid flippases by utilizing nitrobenzoxadiazole (NBD)-labeled lipids as substrates. Among the 14 human type IV P-type ATPases, ATP10D was shown to flip NBD-glucosylceramide (GlcCer) across the plasma membrane. Here, we found that conversion of incorporated GlcCer (d18:1/12:0) to other sphingolipids is accelerated in cells exogenously expressing ATP10D but not its ATPase-deficient mutant. These findings suggest that 1) ATP10D flips unmodified GlcCer as well as NBD-GlcCer at the plasma membrane and 2) ATP10D can translocate extracellular GlcCer, which is subsequently converted to other metabolites. Notably, exogenous expression of ATP10D led to the reduction in cellular hexosylceramide levels. Moreover, the expression of GlcCer flippases, including ATP10D, also reduced cellular hexosylceramide levels in fibroblasts derived from patients with Gaucher disease, which is a lysosomal storage disorder with excess GlcCer accumulation. Our study highlights the contribution of ATP10D to the regulation of cellular GlcCer levels and maintaining lipid homeostasis.

The lipid flippase ATP8A1 regulates the recruitment of ARF effectors to the trans-Golgi Network.

Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.

Cryo-EM of the ATP11C flippase reconstituted in Nanodiscs shows a distended phospholipid bilayer inner membrane around transmembrane helix 2.

ATP11C is a member of the P4-ATPase flippase family that mediates translocation of phosphatidylserine (PtdSer) across the lipid bilayer. In order to characterize the structure and function of ATP11C in a model natural lipid environment, we revisited and optimized a quick procedure for reconstituting ATP11C into Nanodiscs using methyl-ß-cyclodextrin as a reagent for the detergent removal. ATP11C was efficiently reconstituted with the endogenous lipid, or the mixture of endogenous lipid and synthetic dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylserine (DOPS), all of which retained the ATPase activity. We obtained 3.4 Å and 3.9 Å structures using single-particle cryo-electron microscopy (cryo-EM) of AlF- and BeF-stabilized ATP11C transport intermediates, respectively, in a bilayer containing DOPS. We show that the latter exhibited a distended inner membrane around ATP11C transmembrane helix 2, possibly reflecting the perturbation needed for phospholipid release to the lipid bilayer. Our structures of ATP11C in the lipid membrane indicate that the membrane boundary varies upon conformational changes of the enzyme and is no longer flat around the protein, a change that likely contributes to phospholipid translocation across the membrane leaflets.

Still rocking in the structural era: A molecular overview of the small multidrug resistance (SMR) transporter family.

The small multidrug resistance (SMR) family is composed of widespread microbial membrane proteins that fulfill different transport functions. Four functional SMR subtypes have been identified, which variously transport the small, charged metabolite guanidinium, bulky hydrophobic drugs and antiseptics, polyamines, and glycolipids across the membrane bilayer. The transporters possess a minimalist architecture, with ∼100-residue subunits that require assembly into homodimers or heterodimers for transport. In part because of their simple construction, the SMRs are a tractable system for biochemical and biophysical analysis. Studies of SMR transporters over the last 25 years have yielded deep insights for diverse fields, including membrane protein topology and evolution, mechanisms of membrane transport, and bacterial multidrug resistance. Here, we review recent advances in understanding the structures and functions of SMR transporters. New molecular structures of SMRs representing two of the four functional subtypes reveal the conserved structural features that have permitted the emergence of disparate substrate transport functions in the SMR family and illuminate structural similarities with a distantly related membrane transporter family, SLC35/DMT.

The interaction of ATP11C-b with ezrin contributes to its polarized localization.

ATP11C, a member of the P4-ATPase family, translocates phosphatidylserine and phosphatidylethanolamine at the plasma membrane. We previously revealed that its C-terminal splice variant ATP11C-b exhibits polarized localization in motile cell lines, such as MDA-MB-231 and Ba/F3. In the present study, we found that the C-terminal cytoplasmic region of ATP11C-b interacts specifically with ezrin. Notably, the LLxY motif in the ATP11C-b C-terminal region is crucial for its interaction with ezrin as well as its polarized localization on the plasma membrane. A constitutively active, C-terminal phosphomimetic mutant of ezrin was colocalized with ATP11C-b in polarized motile cells. ATP11C-b was partially mislocalized in cells depleted of ezrin alone, and exhibited greater mislocalization in cells simultaneously depleted of the family members ezrin, radixin and moesin (ERM), suggesting that ERM proteins, particularly ezrin, contribute to the polarized localization of ATP11C-b. Furthermore, Atp11c knockout resulted in C-terminally phosphorylated ERM protein mislocalization, which was restored by exogenous expression of ATP11C-b but not ATP11C-a. These observations together indicate that the polarized localizations of ATP11C-b and the active form of ezrin to the plasma membrane are interdependently stabilized.

The role of lipid scramblases in regulating lipid distributions at cellular membranes.

Glycerophospholipids, sphingolipids and cholesterol assemble into lipid bilayers that form the scaffold of cellular membranes, in which proteins are embedded. Membrane composition and membrane protein profiles differ between plasma and intracellular membranes and between the two leaflets of a membrane. Lipid distributions between two leaflets are mediated by lipid translocases, including flippases and scramblases. Flippases use ATP to catalyze the inward movement of specific lipids between leaflets. In contrast, bidirectional flip-flop movements of lipids across the membrane are mediated by scramblases in an ATP-independent manner. Scramblases have been implicated in disrupting the lipid asymmetry of the plasma membrane, protein glycosylation, autophagosome biogenesis, lipoprotein secretion, lipid droplet formation and communications between organelles. Although scramblases in plasma membranes were identified over 10 years ago, most progress about scramblases localized in intracellular membranes has been made in the last few years. Herein, we review the role of scramblases in regulating lipid distributions in cellular membranes, focusing primarily on intracellular membrane-localized scramblases.

P4-ATPase subunit Cdc50 plays a role in yeast budding and cell wall integrity in Candida glabrata.

BACKGROUND: As highly-conserved types of lipid flippases among fungi, P4-ATPases play a significant role in various cellular processes. Cdc50 acts as the regulatory subunit of flippases, forming heterodimers with Drs2 to translocate aminophospholipids. Cdc50 homologs have been reported to be implicated in protein trafficking, drug susceptibility, and virulence in Saccharomyces cerevisiae, Candida albicans and Cryptococcus neoformans. It is likely that Cdc50 has an extensive influence on fungal cellular processes. The present study aimed to determine the function of Cdc50 in Candida glabrata by constructing a Δcdc50 null mutant and its complemented strain. RESULTS: In Candida glabrata, the loss of Cdc50 led to difficulty in yeast budding, probably caused by actin depolarization. The Δcdc50 mutant also showed hypersensitivity to azoles, caspofungin, and cell wall stressors. Further experiments indicated hyperactivation of the cell wall integrity pathway in the Δcdc50 mutant, which elevated the major cell wall contents. An increase in exposure of ß-(1,3)-glucan and chitin on the cell surface was also observed through flow cytometry. Interestingly, we observed a decrease in the phagocytosis rate when the Δcdc50 mutant was co-incubated with THP-1 macrophages. The Δcdc50 mutant also exhibited weakened virulence in nematode survival tests. CONCLUSION: The results suggested that the lipid flippase subunit Cdc50 is implicated in yeast budding and cell wall integrity in C. glabrata, and thus have a broad influence on drug susceptibility and virulence. This work highlights the importance of lipid flippase, and offers potential targets for new drug research.

Arabidopsis flippase ALA3 is required for adjustment of early subcellular trafficking in plant response to osmotic stress.

To compensate for their sessile lifestyle, plants developed several responses to exogenous changes. One of the previously investigated and not yet fully understood adaptations occurs at the level of early subcellular trafficking, which needs to be rapidly adjusted to maintain cellular homeostasis and membrane integrity under osmotic stress conditions. To form a vesicle, the membrane needs to be deformed, which is ensured by multiple factors, including the activity of specific membrane proteins, such as flippases from the family of P4-ATPases. The membrane pumps actively translocate phospholipids from the exoplasmic/luminal to the cytoplasmic membrane leaflet to generate curvature, which might be coupled with recruitment of proteins involved in vesicle formation at specific sites of the donor membrane. We show that lack of the AMINOPHOSPHOLIPID ATPASE3 (ALA3) flippase activity caused defects at the plasma membrane and trans-Golgi network, resulting in altered endocytosis and secretion, processes relying on vesicle formation and movement. The mentioned cellular defects were translated into decreased intracellular trafficking flexibility failing to adjust the root growth on osmotic stress-eliciting media. In conclusion, we show that ALA3 cooperates with ARF-GEF BIG5/BEN1 and ARF1A1C/BEX1 in a similar regulatory pathway to vesicle formation, and together they are important for plant adaptation to osmotic stress.

Phosphatidylserine exposure in living cells.

P4-ATPases, a subfamily of P-type ATPases, translocate cell membrane phospholipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet to generate and maintain membrane lipid asymmetry. Exposure of phosphatidylserine (PS) in the exoplasmic leaflet is well known to transduce critical signals for apoptotic cell clearance and platelet coagulation. PS exposure is also involved in many other biological processes, including myoblast and osteoclast fusion, and the immune response. Moreover, mounting evidence suggest that PS exposure is critical for neuronal regeneration and degeneration. In apoptotic cells, PS exposure is induced by irreversible activation of scramblases and inactivation of P4-ATPases. However, how PS is reversibly exposed and restored in viable cells during other biological processes remains poorly understood. In the present review, we discuss the physiological significance of reversible PS exposure in living cells, and the putative roles of flippases, floppases, and scramblases.

Lipid Transport by Candida albicans Dnf2 Is Required for Hyphal Growth and Virulence.

Candida albicans is a common cause of human mucosal yeast infections, and invasive candidiasis can be fatal. Antifungal medications are limited, but those targeting the pathogen cell wall or plasma membrane have been effective. Therefore, virulence factors controlling membrane biogenesis are potential targets for drug development. P4-ATPases contribute to membrane biogenesis by selecting and transporting specific lipids from the extracellular leaflet to the cytoplasmic leaflet of the bilayer to generate lipid asymmetry. A subset of heterodimeric P4-ATPases, including Dnf1-Lem3 and Dnf2-Lem3 from Saccharomyces cerevisiae, transport phosphatidylcholine (PC), phosphatidylethanolamine (PE), and the sphingolipid glucosylceramide (GlcCer). GlcCer is a critical lipid for Candida albicans polarized growth and virulence, but the role of GlcCer transporters in virulence has not been explored. Here, we show that the Candida albicans Dnf2 (CaDnf2) requires association with CaLem3 to form a functional transporter and flip fluorescent derivatives of GlcCer, PC, and PE across the plasma membrane. Mutation of conserved substrate-selective residues in the membrane domain strongly abrogates GlcCer transport and partially disrupts PC transport by CaDnf2. Candida strains harboring dnf2-null alleles (dnf2ΔΔ) or point mutations that disrupt substrate recognition exhibit defects in yeast-to-hypha growth transition, filamentous growth, and virulence in systemically infected mice. The influence of CaDNF1 deletion on the morphological phenotypes is negligible, although the dnf1ΔΔ dnf2ΔΔ strain was less virulent than the dnf2ΔΔ strain. These results indicate that the transport of GlcCer and/or PC by plasma membrane P4-ATPases is important for the pathogenicity of Candida albicans.

Phospholipid-flipping activity of P4-ATPase drives membrane curvature.

P4-ATPases are phospholipid flippases that translocate phospholipids from the exoplasmic/luminal to the cytoplasmic leaflet of biological membranes. All P4-ATPases in yeast and some in other organisms are required for membrane trafficking; therefore, changes in the transbilayer lipid composition induced by flippases are thought to be crucial for membrane deformation. However, it is poorly understood whether the phospholipid-flipping activity of P4-ATPases can promote membrane deformation. In this study, we assessed membrane deformation induced by flippase activity via monitoring the extent of membrane tubulation using a system that allows inducible recruitment of Bin/amphiphysin/Rvs (BAR) domains to the plasma membrane (PM). Enhanced phosphatidylcholine-flippase activity at the PM due to expression of ATP10A, a member of the P4-ATPase family, promoted membrane tubulation upon recruitment of BAR domains to the PM This is the important evidence that changes in the transbilayer lipid composition induced by P4-ATPases can deform biological membranes.