P4-ATPases comprise a family of P-type ATPases that actively transport or flip phospholipids across cell membranes. This generates and maintains membrane lipid asymmetry, a property essential for a wide variety of cellular processes such as vesicle budding and trafficking, cell signaling, blood coagulation, apoptosis, bile and cholesterol homeostasis, and neuronal cell survival. Some P4-ATPases transport phosphatidylserine and phosphatidylethanolamine across the plasma membrane or intracellular membranes whereas other P4-ATPases are specific for phosphatidylcholine. The importance of P4-ATPases is highlighted by the finding that genetic defects in two P4-ATPases ATP8A2 and ATP8B1 are associated with severe human disorders. Recent studies have provided insight into how P4-ATPases translocate phospholipids across membranes. P4-ATPases form a phosphorylated intermediate at the aspartate of the P-type ATPase signature sequence, and dephosphorylation is activated by the lipid substrate being flipped from the exoplasmic to the cytoplasmic leaflet similar to the activation of dephosphorylation of Na+/K+-ATPase by exoplasmic K+. How the phospholipid is translocated can be understood in terms of a peripheral hydrophobic gate pathway between transmembrane helices M1, M3, M4, and M6. This pathway, which partially overlaps with the suggested pathway for migration of Ca2+ in the opposite direction in the Ca2+-ATPase, is wider than the latter, thereby accommodating the phospholipid head group. The head group is propelled along against its concentration gradient with the hydrocarbon chains projecting out into the lipid phase by movement of an isoleucine located at the position corresponding to an ion binding glutamate in the Ca2+- and Na+/K+-ATPases. Hence, the P4-ATPase mechanism is quite similar to the mechanism of these ion pumps, where the glutamate translocates the ions by moving like a pump rod. The accessory subunit CDC50 may be located in close association with the exoplasmic entrance of the suggested pathway, and possibly promotes the binding of the lipid substrate. This review focuses on properties of mammalian and yeast P4-ATPases for which most mechanistic insight is available. However, the structure, function and enigmas associated with mammalian and yeast P4-ATPases most likely extend to P4-ATPases of plants and other organisms.

Significance: Membranes of eukaryotic cells retain an essential asymmetric distribution of phospholipids in the two leaflets, which depends on the translocation by P4-ATPases (flippases) of specific phospholipids across the lipid bilayer. Severe diseases result from flippase mutations. Flippases are closely related to ion pumps like Na ⁺ ,K ⁺ -ATPase and Ca ²⁺ -ATPase, yet the large size of the lipid substrate compared with inorganic cations raises the fundamental issue of how the flippases accomplish lipid transport. We characterized the mammalian flippase ATP8A2, applying mutagenesis and specific functional assays in combination with computational studies. We identify a unique putative lipid transport pathway through the flippase protein and propose a plausible translocation mechanism, which is both distinct from and related to the well-characterized cation transport mechanisms.

ATP8A2 is a P ₄ -ATPase (“flippase”) located in membranes of retinal photoreceptors, brain cells, and testis, where it mediates transport of aminophospholipids toward the cytoplasmic leaflet. It has long been an enigma whether the mechanism of P ₄ -ATPases resembles that of the well-characterized cation-transporting P-type ATPases, and it is unknown whether the flippases interact directly with the lipid and with counterions. Our results demonstrate that ATP8A2 forms a phosphoenzyme intermediate at the conserved aspartate (Asp ⁴¹⁶ ) in the P-type ATPase signature sequence and exists in E ₁ P and E ₂ P forms similar to the archetypical P-type ATPases. Using the properties of the phosphoenzyme, the partial reaction steps of the transport cycle were examined, and the roles of conserved residues Asp ¹⁹⁶ , Glu ¹⁹⁸ , Lys ⁸⁷³ , and Asn ⁸⁷⁴ in the transport mechanism were elucidated. The former two residues in the A-domain T/D-G-E-S/T motif are involved in catalysis of E ₂ P dephosphorylation, the glutamate being essential. Transported aminophospholipids activate the dephosphorylation similar to K ⁺ activation of dephosphorylation in Na ⁺ ,K ⁺ -ATPase. Lys ⁸⁷³ mutants (particularly K873A and K873E) display a markedly reduced sensitivity to aminophospholipids. Hence, Lys ⁸⁷³ , located in transmembrane segment M5 at a “hot spot” for cation binding in Ca ²⁺ -ATPase and Na ⁺ ,K ⁺ -ATPase, appears to participate directly in aminophospholipid binding or to mediate a crucial interaction within the ATP8A2-CDC50 complex. By contrast, Lys ⁸⁶⁵ is unimportant for aminophospholipid sensitivity. Binding of Na ⁺ , H ⁺ , K ⁺ , Cl ^- , or Ca ²⁺ to the E ₁ form as a counterion is not required for activation of phosphorylation from ATP. Therefore, phospholipids could be the only substrate transported by ATP8A2.

Summary: In the last decade, there has been an explosion in both the number of and knowledge about miRNAs associated with both type 1 and type 2 diabetes. Even though we are presently in the initial stages of understanding how this novel class of posttranscriptional regulators are involved in diabetes, recent studies have demonstrated that miRNAs are important regulators of the islet transcriptome, controlling apoptosis, differentiation and proliferation, as well as regulating unique islet and beta‐cell functions and pathways such as insulin expression, processing and secretion. Furthermore, a large number of miRNAs have been linked to diabetogenic processes induced by elevated levels of glucose, free fatty acids and inflammatory cytokines. Thus, miRNAs are novel therapeutic targets with the potential of protecting the beta‐cell, and there is proof of principle that miRNA antagonists, so‐called antagomirs, are effective in vivo for other disorders. miRNAs are exported out of cells in exosomes, raising the intriguing possibility of cell‐to‐cell communication between distant tissues via miRNAs and that miRNAs can be used as biomarkers of beta‐cell function, mass and survival. The purpose of this review is to provide a status on how miRNAs control beta‐cell function and viability in health and disease. Copyright © 2015 John Wiley & Sons, Ltd.

The vertebrate 2-5A system is part of the innate immune system and central to cellular antiviral defense. Upon activation by viral double-stranded RNA, 5′-triphosphorylated, 2′–5′-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2′–5′-oligoadenylate synthetases. These unusual oligonucleotides activate RNase L, an unspecific endoribonuclease that mediates viral and cellular RNA breakdown. Subsequently, the 2-5As are removed by a 2′-phosphodiesterase (2′-PDE), an enzyme that apart from breaking 2′–5′ bonds also degrades regular, 3′–5′-linked oligoadenylates. Interestingly, 2′-PDE shares both functionally and structurally characteristics with the CCR4-type exonuclease–endonuclease–phosphatase family of deadenylases. Here we show that 2′-PDE locates to the mitochondrial matrix of human cells, and comprise an active 3′–5′ exoribonuclease exhibiting a preference for oligo-adenosine RNA like canonical cytoplasmic deadenylases. Furthermore, we document a marked negative association between 2′-PDE and mitochondrial mRNA levels following siRNA-directed knockdown and plasmid-mediated overexpression, respectively. The results indicate that 2′-PDE, apart from playing a role in the cellular immune system, may also function in mitochondrial RNA turnover.

Endometriosis displays some features that resemble malignant processes, including invasive growth, resistance to apoptosis and distant implantation. The objective of this study was to investigate whether gene alterations that are frequent in endometrial and/or ovarian cancers contribute to the pathogenesis of endometriosis. Biopsies were obtained from ectopic endometriosis lesions from 23 patients with revised American Fertility Score stage 1 (n= 1), 2 (n= 10), 3 (n= 11) or 4 (n= 1) endometriosis. Six genes (APC, CDKN2A, PYCARD, RARB, RASSF1 and ESR1) were analyzed for promoter hypermethylation using methylation-specific melting curve analysis, and 9 genes (BRAF, HRAS, NRAS, CTNNB1, CDK4, FGFR3, PIK3CA, TP53 and PTEN) were analyzed for mutations using denaturing gradient gel electrophoresis and direct sequencing. An oncogenic mutation in KRAS (c.34G > T; p.G12C) was detected in a single lesion. No gene alterations were found in the remaining samples. Our data suggest that genetic and epigenetic events contributing to endometrial and ovarian cancers are rare in endometriosis. However, other proto-oncogenes and tumor suppressor genes should be tested for alterations in order to identify the molecular basis of the susceptibility of endometriosis to malignant transformation.