The ATPase activity of ABCA1 is increased by cholesterol in the presence of anionic lipids.

High-density lipoprotein (HDL) transports excess cholesterol from peripheral tissues back to the liver, and plasma HDL levels are inversely related to cardiovascular disease incidence. ATP-binding cassette A1 (ABCA1) is a member of the ABC protein superfamily, and generates nascent HDL, which consists of several hundreds of phospholipids and cholesterol wrapped by apolipoprotein A-I (apoA-I). However, it remains unclear whether cholesterol is a transport substrate of ABCA1. Since ATP hydrolysis of ABC proteins is typically increased by their transport substrates, we characterized the effects of cholesterol on the ATPase activity of purified ABCA1 using liposomes of various lipid compositions. ABCA1 showed substantial ATPase activity (20-30 nmol$\cdot$min-1$\cdot$mg-1) only in liposomes containing anionic lipids, including phosphatidylserine. Cholesterol increased the ATPase activity by 1.6- to 3-fold in the presence of anionic lipids. Moreover, phosphatidylserine addition to BHK/ABCA1 cells increased phosphatidylcholine and cholesterol efflux to apoA-I. Next, we investigated the sterol specificity of ABCA1. The ATPase activity of ABCA1 was strongly enhanced by desmosterol and zymosterol, similar to cholesterol. In contrast, 7-dehydrocholesterol and lathosterol weakly increased the ATPase activity, and no increase was observed with stigmasterol or brassicasterol. These findings suggest that ABCA1 transports cholesterol and prefers cholesterol over plant sterols as a transport substrate.

The plasma membrane of focal adhesions has a high content of cholesterol and phosphatidylcholine with saturated acyl chains.

Cellular functions, such as differentiation and migration, are regulated by the extracellular microenvironment, including the extracellular matrix (ECM). Cells adhere to ECM through focal adhesions (FAs) and sense the surrounding microenvironments. Although FA proteins have been actively investigated, little is known about the lipids in the plasma membrane at FAs. In this study, we examine the lipid composition at FAs with imaging and biochemical approaches. Using the cholesterol-specific probe D4 with total internal reflection fluorescence microscopy and super-resolution microscopy, we show an enrichment of cholesterol at FAs simultaneously with FA assembly. Furthermore, we establish a method to isolate the lipid from FA-rich fractions, and biochemical quantification of the lipids reveals that there is a higher content of cholesterol and phosphatidylcholine with saturated fatty acid chains in the lipids of the FA-rich fraction than in either the plasma membrane fraction or the whole-cell membrane. These results demonstrate that plasma membrane at FAs has a locally distinct lipid composition compared to the bulk plasma membrane.

Cholesterol accumulation in ovarian follicles causes ovulation defects in Abca1a -/- Japanese medaka (Oryzias latipes).

ATP-binding cassette A1 (ABCA1) is a membrane protein, which exports excess cellular cholesterol to generate HDL to reduce the risk of the onset of cardiovascular diseases (CVD). In addition, ABCA1 exerts pleiotropic effects on such as inflammation, tissue repair, and cell proliferation and migration. In this study, we explored the novel physiological roles of ABCA1 using Japanese medaka (Oryzias latipes), a small teleost fish. Three Abca1 genes were found in the medaka genome. ABCA1A and ABCA1C exported cholesterol to generate nascent HDL as human ABCA1 when expressed in HEK293 cells. To investigate their physiological roles, each Abca1-deficient fish was generated using the CRISPR-Cas9 system. Abca1a -/- female medaka was found to be infertile, while Abca1b -/- and Abca1c -/- female medaka were fertile. In vitro ovarian follicle culture suggested that Abca1a deficiency causes ovulation defects. In the ovary, ABCA1A was expressed in theca cells, an outermost layer of the ovarian follicle. Total cholesterol content of Abca1a -/- ovary was significantly higher than that of the wild-type, while estrogen and progestin contents were compatible with those of the wild-type. Furthermore, cholesterol loading to the wild-type follicles caused ovulation defects. These results suggest that ABCA1A in theca cells regulates cholesterol content in the ovarian follicles and its deficiency inhibits successful ovulation through cholesterol accumulation in the ovarian follicle.

Targeted deletion of ecto-5'-nucleotidase results in retention of inosine monophosphate content in postmortem muscle of medaka (Oryzias latipes).

Inosine monophosphate (IMP) is an important indicator of meat freshness and contributes to its umami taste. An attractive strategy for enhancing umami is to suppress the IMP-degrading activity and increase the IMP content in the skeletal muscle through genome editing technology using the CRISPR-Cas9 system. However, the molecular mechanisms underlying IMP degradation remain unclear. We cloned two ecto-5'-nucleotidase genes, designated as ecto-5'-nucleotidase-a (nt5ea) and ecto-5'-nucleotidase-b (nt5eb), from medaka (Oryzias latipes), a vertebrate model organism. Expression analysis using embryos showed that nt5ea or nt5eb overexpression remarkably upregulated IMP degradation, and that the IMP-degrading activity was higher in Nt5ea than in Nt5eb. Furthermore, we established frame-shifted or large deletion (lacking nt5ea or nt5eb locus) mutant strains and assayed the effects of gene disruptions on the amount of IMP in skeletal muscle. The nt5ea-deficient medaka showed considerable higher levels of IMP at 48 h postmortem than did the wild-type fish. The nt5eb mutants also exhibited higher IMP contents than that in the wild types, but the increase was less than that in the nt5ea mutants. Our results demonstrated that nt5e is an important regulator of IMP levels in skeletal muscle and that its loss of function was effective in maintaining IMP content.

ABCA1 and cholesterol transfer protein Aster-A promote an asymmetric cholesterol distribution in the plasma membrane.

Cholesterol is a major and essential component of the mammalian cell plasma membrane (PM), and the loss of cholesterol homeostasis leads to various pathologies. Cellular cholesterol uptake and synthesis are regulated by a cholesterol sensor in the endoplasmic reticulum (ER). However, it remains unclear how changes in the cholesterol level of the PM are recognized. Here, we show that the sensing of cholesterol in the PM depends on ABCA1 and the cholesterol transfer protein Aster-A, which cooperatively maintain the asymmetric transbilayer cholesterol distribution in the PM. We demonstrate that ABCA1 translocates (flops) cholesterol from the inner leaflet of the PM to the outer leaflet of the PM to maintain a low inner leaflet cholesterol level. We also found that when inner cholesterol levels were increased, Aster-A was recruited to the PM-ER contact site to transfer cholesterol to the ER. These results suggest that ABCA1 could promote an asymmetric cholesterol distribution to suppress Aster-A recruitment to the PM-ER contact site to maintain intracellular cholesterol homeostasis.

Preoperative CA19-9 is a prognostic factor in pT3N0 gastric cancer patients undergoing curative resection.

BACKGROUND: The standard treatment for pT3N0 gastric cancer (GC) in Japanese guidelines is radical surgery without adjuvant chemotherapy. However, certain percentages of these patients develop recurrences; therefore, detecting the high-risk subgroup of recurrence may contribute to improve patient's outcome by adjuvant chemotherapy. In this study, we aimed to identify a predictive indicator of poor prognosis in pT3N0 GC. METHODS: Eighty-one patients who were diagnosed as pT3N0 GC after curative surgical resection and had not received adjuvant chemotherapy were included. The clinicopathological factors and laboratory parameters were evaluated by univariate and multivariate analyses to identify prognostic factors of tumor recurrence. Survival analysis was performed by Kaplan-Meier method. RESULTS: Male (P = 0.027), a high body mass index (BMI) (P = 0.031), a high CA19-9 value (P = 0.025), and a lower number of retrieved lymph nodes (P = 0.018) were found to be significantly associated with a shorter recurrence free survival (RFS). In a multivariate analysis, high CA19-9 value (> 37 U/ml) [(hazard ratio (HR): 3.326; 95% confidence interval (CI): 1.044 to 10.596; P = 0.042] was found to be an independent predictor of RFS. CONCLUSION: The preoperative high CA19-9 value is considered a useful prognostic marker for predicting cancer recurrence after curative surgery in pT3N0 GC patients. For those patients, adjuvant chemotherapy might be considered to improve the survival outcome.

Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein.

The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured cells. Although these in vitro experiments help illustrate details about the molecular mechanisms of P-gp, they do not reflect physiological membrane environments in terms of lateral pressure, curvature, constituent lipid species, etc. The protocol presented here includes a detailed guide for analyzing the conformational change of human P-gp in living HEK293 cells by using intramolecular fluorescence resonance energy transfer (FRET), in which excitation of the donor fluorophore is transferred to the acceptor without emission of a photon when two fluorescent proteins are in close proximity. Combining FRET analysis with membrane permeabilization, the contribution of small molecules such as nucleotides to the conformational change can be evaluated in living cells.

ABCA13 dysfunction associated with psychiatric disorders causes impaired cholesterol trafficking.

ATP-binding cassette subfamily A member 13 (ABCA13) is predicted to be the largest ABC protein, consisting of 5058 amino acids and a long N-terminal region. Mutations in the ABCA13 gene were reported to increase the susceptibility to schizophrenia, bipolar disorder, and major depression. However, little is known about the molecular functions of ABCA13 or how they associate with psychiatric disorders. Here, we examined the biochemical activity of ABCA13 using HEK293 cells transfected with mouse ABCA13. The expression of ABCA13 induced the internalization of cholesterol and gangliosides from the plasma membrane to intracellular vesicles. Cholesterol internalization by ABCA13 required the long N-terminal region and ATP hydrolysis. To examine the physiological roles of ABCA13, we generated Abca13 KO mice using CRISPR/Cas and found that these mice exhibited deficits of prepulse inhibition. Vesicular cholesterol accumulation and synaptic vesicle endocytosis were impaired in primary cultures of Abca13 KO cortical neurons. Furthermore, mutations in ABCA13 gene associated with psychiatric disorders disrupted the protein's subcellular localization and impaired cholesterol trafficking. These findings suggest that ABCA13 accelerates cholesterol internalization by endocytic retrograde transport in neurons and that loss of this function is associated with the pathophysiology of psychiatric disorders.

ABCB1/MDR1/P-gp employs an ATP-dependent twist-and-squeeze mechanism to export hydrophobic drugs.

ABCB1, also called MDR1 or P-glycoprotein, exports various hydrophobic compounds and plays an essential role as a protective physiological barrier in several organs, including the brain, testis, and placenta. However, little is known about the structural mechanisms that allow ABCB1 to recognize hydrophobic compounds of diverse structures or the coupling of ATP hydrolysis to uphill substrate export. High-resolution X-ray crystal structures of the pre- and post-transport states and FRET analyses in living cells have revealed that an aromatic hydrophobic network at the top of the inner cavity is key for the conformational change in ABCB1 that is triggered by a hydrophobic substrate. ATP binding, but not hydrolysis, induces a progressive network that results in a twisting motion of the whole protein, squeezing out the substrate directly to the extracellular space. This twist-and-squeeze mechanism by which ABCB1 exports hydrophobic substrates is distinct from those of other transporters.

Preoperative prognostic nutritional index predicts risk of recurrence after curative resection for stage IIA colon cancer.

BACKGROUND: Preoperative nutritional and inflammatory indices have been reported to be associated with the prognosis of patients with malignancy. We evaluated clinicopathological factors, including nutritional and inflammatory indices, and recurrence prognosis in patients with stage IIA colon cancer (CC) who underwent curative surgery. METHODS: This retrospective study included 197 patients with stage IIA CC who had undergone curative resection. We evaluated the association between prognostic nutritional index (PNI), neutrophil-lymphocyte ratio (NLR), and platelet-lymphocyte ratio (PLR) with clinicopathological factors and prognosis for recurrence. For the recurrence-free survival (RFS) analysis, receiver operating characteristic (ROC) curves were used to determine appropriate cutoff values for PNI, NLR, and PLR. RESULTS: Univariate analyses showed that PNI<44.8 (P = 0.028) was significantly associated with worse RFS in patients with stage IIA CC patients. In the multivariate analyses, PNI<44.8 (hazard ratio [HR] 2.082; 95% confidence interval [CI] 1.005-4.317; P = 0.049) independently and significantly predicted RFS. CONCLUSION: PNI is a useful marker for predicting recurrence prognosis in post-resection patients with stage IIA CC.

Predictive Effectiveness of the Glasgow Prognostic Score for Gastrointestinal Stromal Tumors.

The aim of this study was to evaluate the significance of the Glasgow prognostic score (GPS) in patients with resected gastrointestinal stromal tumors (GISTs). Forty-six GIST patients who underwent radical resection between January 2004 and December 2011 were enrolled in this retrospective study. The clinicopathological parameters examined included predictors of recurrence-free survival (RFS). Univariate and multivariate analysis of prognostic factors related to RFS were calculated using Cox proportional hazards model. The GPS classification system revealed 37 (80.4%), 6 (13.1%), and 3 (6.5%) patients with a GPS of 0, 1, and 2, respectively. Patients with GPS 1/2 had a significantly shorter RFS compared to those with GPS 0 (P = 0.01). The 3- and 5-year RFS rates for patients with GPS 0 were 94.0% and 90.9%, respectively, compared to 66.7% and 53.3%, respectively, for patients with GPS 1/2. Univariate analyses indicated that tumor size (P < 0.01), mitotic rate (P < 0.01), higher GPS (P < 0.01), and platelet count (P = 0.04) were prognostic factors for RFS; tumor size (P = 0.01) and GPS (P = 0.04) were independent prognostic factors in multivariate analysis. Preoperative high GPS were predictors of long-term prognosis in patients with resected GISTs.

CRISPR/Cas9 nickase-mediated efficient and seamless knock-in of lethal genes in the medaka fish Oryzias latipes.

The CRISPR/Cas system offers new opportunities for targeted gene modifications in a wide range of organisms. In medaka (Oryzias latipes), a vertebrate model organism, a wild-type Cas9-based approach is commonly used to establish desired strains, however, its use in lethal genes is still challenging due to excess gene disruptions triggered by DNA double strand breaks (DSBs). To overcome this problem, we aimed to develop a new knock-in system using Cas9 nickase (Cas9n) that can reduce DNA DSBs. We revealed that Cas9n allowed reduction of the DSB-induced unwanted mutagenesis via non-homologous end-joining at both on- and off- target sites. Further, with a new donor plasmid (p2BaitD) that provides a linear template through Cas9n-mediated nicks, we successfully integrated reporter cassettes via homology-directed repair (HDR) into all three loci tested, including a lethal gene. In the experiment targeting the lethal gene, the combination of p2BaitD and Cas9n achieved higher survival rates than the Cas9-based approach, which enabled the desired knock-in founders. Additionally, through a technical blend of our knock-in system with a recently developed One-step mating protocol, we successfully established a homozygous knock-in strain in one generation period. This study presents evidence of an effective method to generate an HDR-mediated gene knock-in in medaka and other organisms, which is useful for establishing screening platforms for genes or drugs toxicity or other applications.

ABC proteins in evolution.

ATP-binding cassette (ABC) proteins play diverse roles in all living organisms, making them an attractive model for evolution. Early evolution of ancestral unicellular organisms entailed the acquisition of at least three types of ABC proteins: type 1 ABC proteins to import nutrients, and type 2 and 3 ABC proteins to generate the outer cell membrane by flopping and loading lipids onto acceptors, respectively. To export various toxic lipophilic compounds, cells evolutionarily acquired a fourth type of ABC protein. This suggests that ABC proteins may have played an important role in evolution, especially when life became terrestrial, protecting plants and animals from water loss and pathogen infection. ABC proteins are also assumed to have accelerated the evolution of vertebrates by allowing cholesterol to function for intramembrane signaling. In this review, we discuss the roles of ABC proteins in the evolution of bacteria, plants, and animals.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

Stiffness of the extracellular matrix regulates differentiation into beige adipocytes.

Beige/brite adipocytes, which express high levels of uncoupling protein 1 (UCP1) to generate heat using stored triglycerides, are induced under specific stimuli such as cold exposure in inguinal white adipose tissue (iWAT). Although extracellular microenvironments such as extracellular matrix (ECM) stiffness are known to regulate cell behaviors, including cell differentiation into adipocytes, the effect on iWAT cells is unknown. In this study, we show that rigid ECM promotes the cell spreading of iWAT-derived preadipocytes. Furthermore, the expression of UCP1 and other thermogenic genes in iWAT cells is promoted when the cells are cultured on rigid ECM. The expression of mTOR, a kinase known to regulate the differentiation to beige adipocytes, is decreased on rigid substrates. These results suggest that ECM stiffness plays an important role in the differentiation to beige adipocytes.

In vivo FRET analyses reveal a role of ATP hydrolysis-associated conformational changes in human P-glycoprotein.

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

Cholesterol asymmetry at the tip of filopodia during cell adhesion.

During adhesion, cells develop filopodia to facilitate the attachment to the extracellular matrix. The small guanosine triphosphate (GTP)-binding protein, Cdc42, plays a central role in the formation of filopodia. It has been reported that Cdc42 activity is regulated by cholesterol (Chol). We examined Chol distribution in filopodia using Chol-binding domain 4 (D4) fragment of bacterial toxin, perfringolysin O that senses high membrane concentration of Chol. Our results indicate that fluorescent D4 was enriched at the tip of the outer leaflet of filopodia in the initiation phase of cell adhesion. This enrichment was accompanied by a defect of D4 labeling in the inner leaflet. Steady phase adhered cell experiment indicated that both Cdc42 and ATP-binding cassette transporter, ABCA1, were involved in the binding of D4 to the cell surface. Depletion of Chol activated Cdc42. Our results suggest that asymmetric distribution of Chol at the tip of filopodia induces activation of Cdc42, and thus, facilitates filopodia formation.

A case of esophageal cancer complicated with methotrexate-related lymphoproliferative disorder.

A 69-year-old woman had been undergoing treatment with methotrexate for rheumatoid arthritis for 9 years. Because fever and right hypochondriac pain continued, she visited the nearby hospital. The enlargement of intraabdominal multiple lymph nodes was detected through abdominal computed tomography. Esophagogastroduodenoscopy showed a depressed lesion in the lower intrathoracic esophagus, and squamous cell carcinoma was diagnosed by a biopsy. Positron emission tomography-computed tomography showed a highly abnormal accumulation of lymph nodes, mainly in the upper abdomen, and some lymph nodes around the aorta. Suspecting methotrexate-lymphoproliferative disorder, we discontinued the oral administration of methotrexate. Multiple lymphadenopathy reduced by the withdrawal of the oral administration of methotrexate. We operated for esophageal cancer, and she was discharged on the 17th postoperative day. The postoperative pathological result showed moderately differentiated squamous cell carcinoma. Metastasis to the lymph nodes around the esophagus was observed, and the patient was diagnosed with T1b (SM3), N2 M0, Stage II cancer. Immunostaining showed enlarged lymph nodes composed of CD20-positive cells and with cells positive for EBV-encoded small RNA in situ hybridization. This case shows that patients with rheumatoid arthritis who are being administered methotrexate may have enlarged lymph nodes due to methotrexate-lymphoproliferative disorder.

C-terminal of ABCA1 separately regulates cholesterol floppase activity and cholesterol efflux activity.

ATP-Binding Cassette A1 (ABCA1) is a key lipid transporter for cholesterol homeostasis. We recently reported that ABCA1 not only exports excess cholesterol in an apoA-I dependent manner, but that it also flops cholesterol from the inner to the outer leaflet of the plasma membrane. However, the relationship between these two activities of ABCA1 is still unclear. In this study, we analyzed the subcellular localization of ABCA1 by using a newly generated monoclonal antibody against its extracellular domain and the functions of eleven chimera proteins, in which the C-terminal domain of ABCA1 was replaced with those of the other ABCA subfamily members. We identified two motifs important for the functions of ABCA1. Three periodically repeated leucine residues were necessary for the cholesterol floppase activity but not the cholesterol efflux activity, while a VFVNFA motif was essential for both activities of ABCA1. These results suggest that the C-terminal of ABCA1 separately regulates the cholesterol floppase activity and the cholesterol efflux activity.

Hepatectomy for rapidly growing solitary liver metastasis from non-small cell lung cancer: a case report.

BACKGROUND: Patients with liver metastasis from non-small lung cancer (NSCLC) usually have multiple metastases at other sites and thus rarely undergo liver surgery. We present a case involving successful resection of rapidly growing liver metastasis from squamous cell carcinoma of the lung. CASE PRESENTATION: A 74-year-old man had undergone left lower lobectomy for squamous cell carcinoma of the lung, which was diagnosed pathologically as stage IA. A computed tomography (CT) scan that was taken 12 months after lung resection showed an irregularly shaped mass lesion (size, 8.3 cm) in segment five of the liver. Retrospectively, the mass was identifiable on CT 6 months before this initial recognition. Although the lesion showed rapid growth, positron emission tomography and brain magnetic resonance imaging ruled out the possibility of other metastatic lesions. Therefore, we performed right hepatectomy 14 months after the initial lung surgery. The patient was pathologically diagnosed with liver metastasis from lung cancer and has remained free from recurrence 41 months after the liver surgery, without receiving any adjuvant chemotherapy. CONCLUSIONS: Although there is no reliable clinical indicator for selecting oligo-recurrence, hepatectomy could be an option for solitary liver metastasis from NSCLC for patients who are in good health.