Shushi Nagamori

Role of YidC in folding of polytopic membrane proteins

YidC of Echerichia coli, a member of the conserved Alb3/Oxa1/YidC family, is postulated to be important for biogenesis of membrane proteins. Here, we use as a model the lactose permease (LacY), a membrane transport protein with a known three-dimensional structure, to determine whether YidC plays a role in polytopic membrane protein insertion and/or folding. Experiments in vivo and with an in vitro transcription/translation/insertion system demonstrate that YidC is not necessary for insertion per se, but plays an important role in folding of LacY. By using the in vitro system and two monoclonal antibodies directed against conformational epitopes, LacY is shown to bind the antibodies poorly in YidC-depleted membranes. Moreover, LacY also folds improperly in proteoliposomes prepared without YidC. However, when the proteoliposomes are supplemented with purified YidC, LacY folds correctly. The results indicate that YidC plays a primary role in folding of LacY into its final tertiary conformation via an interaction that likely occurs transiently during insertion into the lipid phase of the membrane.

Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (NTCP).

Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. Although nucleos(t)ide analogs inhibiting viral reverse transcriptase are clinically available as anti‐HBV agents, emergence of drug‐resistant viruses highlights the need for new anti‐HBV agents interfering with other targets. Here we report that cyclosporin A (CsA) can inhibit HBV entry into cultured hepatocytes. The anti‐HBV effect of CsA was independent of binding to cyclophilin and calcineurin. Rather, blockade of HBV infection correlated with the ability to inhibit the transporter activity of sodium taurocholate cotransporting polypeptide (NTCP). We also found that HBV infection‐susceptible cells, differentiated HepaRG cells and primary human hepatocytes expressed NTCP, while nonsusceptible cell lines did not. A series of compounds targeting NTCP could inhibit HBV infection. CsA inhibited the binding between NTCP and large envelope protein in vitro. Evaluation of CsA analogs identified a compound with higher anti‐HBV potency, having a median inhibitory concentration <0.2 μM. Conclusion: This study provides a proof of concept for the novel strategy to identify anti‐HBV agents by targeting the candidate HBV receptor, NTCP, using CsA as a structural platform. (Hepatology 2014;59:1726–1737)

The Sec-independent Function of Escherichia coli YidC Is Evolutionary-conserved and Essential

YidC plays a role in the integration and assembly of many (if not all) Escherichia coli inner membrane proteins. Strikingly, YidC operates in two distinct pathways: one associated with the Sec translocon that also mediates protein translocation across the inner membrane and one independent from the Sec translocon. YidC is homologous to Alb3 and Oxa1 that function in the integration of proteins into the thylakoid membrane of chloroplasts and inner membrane of mitochondria, respectively. Here, we have expressed the conserved region of yeast Oxa1 in a conditional E. coli yidC mutant. We find that Oxa1 restores growth upon depletion of YidC. Data obtained from in vivo protease protection assays and in vitro cross-linking and folding assays suggest that Oxa1 complements the insertion of Sec-independent proteins but is unable to take over the Sec-associated function of YidC. Together, our data indicate that the Sec-independent function of YidC is conserved and essential for cell growth.

Prognostic significance of L-type amino-acid transporter 1 expression in surgically resected pancreatic cancer

Background:The expression of L-type amino-acid transporter 1 (LAT1) is tumour-specific and has been shown to have essential roles in cell growth and survival. However, little is known regarding the clinical significance of LAT1 expression in pancreatic cancer. This study was conducted to determine the prognostic significance of LAT1 expression.Methods:A total of 97 consecutive patients with surgically resected pathological stage I–IV pancreatic ductal adenocarcinoma were retrospectively reviewed. Tumour sections were stained by immunohistochemistry for LAT1, CD98, Ki-67 and vascular endothelial growth factor (VEGF), and microvessel density was determined by CD34 and p53.Results:L-type amino-acid transporter 1 and CD98 were highly expressed in 52.6% (51/97) and 56.7% (55/97) of cases, respectively (P=0.568). The expression of LAT1 within pancreatic cancer cells was significantly associated with disease stage, tumour size, Ki-67, VEGF, CD34, p53 and CD98. L-type amino-acid transporter 1 expression was confirmed to be a significant prognostic factor for predicting poor outcome by multivariate analysis.Conclusion:L-type amino-acid transporter 1 expression is a promising pathological marker for the prediction of outcome in patients with pancreatic cancer.

Transport of 3-Fluoro-l-α-Methyl-Tyrosine by Tumor-Upregulated L-Type Amino Acid Transporter 1: A Cause of the Tumor Uptake in PET

l-3-18F-α-methyl tyrosine (18F-FAMT) has been developed as a PET radiotracer for tumor imaging. Clinical studies have demonstrated the usefulness of 18F-FAMT PET for the prediction of prognosis and the differentiation of malignant tumors and benign lesions. 18F-FAMT exhibits higher cancer specificity in peripheral organs than other amino acid PET tracers and 18F-FDG. The accumulation of 18F-FAMT is strongly correlated with the expression of L-type amino acid transporter 1 (LAT1), an isoform of system L highly upregulated in cancers. In this study, we examined the interaction of 3-fluoro-l-α-methyl-tyrosine (FAMT) with amino acid transporters to assess the mechanisms of 18F-FAMT uptake in PET. Methods: We applied in vitro assays using established mammalian cell lines stably expressing LAT1 or a non–cancer-type system L isoform LAT2. The inhibitory effect on l-14C-leucine uptake and the induction effect on efflux of preloaded l-14C-leucine were examined for FAMT and other amino acid tracers. FAMT transport was compared among cell lines with varied LAT1 expression level. Results: FAMT prominently inhibited LAT1-mediated l-14C-leucine uptake in a competitive manner but had less of an effect on LAT2. In the efflux experiments, FAMT induced the efflux of preloaded l-14C-leucine through LAT1, indicating that FAMT is transported by LAT1 and not by LAT2. Among amino acid–related compounds examined in this study, including those used for PET tracers, the compounds with an α-methyl group such as FAMT, 2-fluoro-l-α-methyl-tyrosine, 3-iodo-l-α-methyl-tyrosine, and l-α-methyl-tyrosine were well transported by LAT1 but not by LAT2. However, l-methionine, l-tyrosine, 3-fluoro-l-tyrosine, 2-fluoro-l-tyrosine, and O-(2-fluoroethyl)-l-tyrosine were transported by both LAT1 and LAT2, suggesting that the α-methyl moiety is responsible for the LAT1 selectivity of FAMT. FAMT transport rate and LAT1 protein level were well correlated, supporting the importance of LAT1 for the cellular uptake of FAMT. Conclusion: Distinct from other amino acid PET tracers, because of its α-methyl moiety, FAMT is selective to LAT1 and not transported by LAT2. This property of FAMT is proposed to contribute to highly tumor-specific accumulation of 18F-FAMT in PET.

SecY alterations that impair membrane protein folding and generate a membrane stress

We report on a class of Escherichia coli SecY mutants that impair membrane protein folding. The mutants also up-regulate the Cpx/σE stress response pathways. Similar stress induction was also observed in response to a YidC defect in membrane protein biogenesis but not in response to the signal recognition particle–targeting defect or in response to a simple reduction in the abundance of the translocon. Together with the previous contention that the Cpx system senses a protein abnormality not only at periplasmic and outer membrane locations but also at the plasma membrane, abnormal states of membrane proteins are postulated to be generated in these secY mutants. In support of this notion, in vitro translation, membrane integration, and folding of LacY reveal that mutant membrane vesicles allow the insertion of LacY but not subsequent folding into a normal conformation recognizable by conformation-specific antibodies. The results demonstrate that normal SecY function is required for the folding of membrane proteins after their insertion into the translocon.

System L amino acid transporter inhibitor enhances anti-tumor activity of cisplatin in a head and neck squamous cell carcinoma cell line

LAT1, a subunit of heterodimeric system L transporter responsible for transporting neutral amino acids into cells, has been investigated in several cancers because of its onco-fetal nature. Based on the studies of its functional inhibition, LAT1 has been proposed to be a new molecular target of a cancer therapy. We have shown here that human head and neck cancer cell line, Hep-2, expresses both LAT1 and 4F2hc, another subunit of system L transporter. An inhibitor of system L, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), inhibited leucine uptake by the cells. BCH administration or restriction of essential amino acid leucine decreased viability of Hep-2 cells. Co-administration of cisplatin with BCH reduced the viability of the cells more than either agent alone. When BCH treatment preceded cisplatin administration, reduction in Hep-2 cell viability was additive. In contrast, when BCH was given after cisplatin treatment, synergistic effect in decreasing the number of viable cells was obtained. BCH treatment decreased the phosphorylation of mTOR, p70S6K and 4EBP1, suggesting that BCH enhanced anti-tumor action of cisplatin by inhibiting mTOR pathway. This potentiation may be used to reduce cisplatin exposure to alleviate many unwanted toxicity of the drug.

ASC amino-acid transporter 2 (ASCT2) as a novel prognostic marker in non-small cell lung cancer.

Background:ASC amino-acid transporter 2 (ASCT2) is a major glutamine transporter that has an essential role in tumour growth and progression. Although ASCT2 is highly expressed in various cancer cells, the clinicopathological significance of its expression in non-small cell lung cancer (NSCLC) remains unclear.Methods:One hundred and four patients with surgically resected NSCLC were evaluated as one institutional cohort. Tumour sections were stained by immunohistochemistry (IHC) for ASCT2, Ki-67, phospho-mTOR (mammalian target of rapamycin), and CD34 to assess the microvessel density. Two hundred and four patients with NSCLC were also validated by IHC from an independent cohort.Results:ASC amino-acid transporter 2 was expressed in 66% of patients, and was closely correlated with disease stage, lymphatic permeation, vascular invasion, CD98, cell proliferation, angiogenesis, and mTOR phosphorylation, particularly in patients with adenocarcinoma (AC). Moreover, two independent cohorts confirmed that ASCT2 was an independent marker for poor outcome in AC patients.Conclusions:ASC amino-acid transporter 2 expression has a crucial role in the metastasis of pulmonary AC, and is a potential molecular marker for predicting poor prognosis after surgery.

Prognostic significance of amino-acid transporter expression (LAT1, ASCT2, and xCT) in surgically resected tongue cancer.

Background:Amino-acid transporters are necessary for the tumour cell growth and survival, and have a crucial role in the development and invasiveness of cancer cells. But, it remains unclear about the prognostic significance of L-type amino-acid transporter 1 (LAT1), system ASC amino-acid transporter-2 (ASCT2), and xCT expression in patients with tongue cancer. We conducted the clinicopathological study to investigate the protein expression of these amino-acid transporters in tongue cancer.Methods:Eighty-five patients with surgically resected tongue cancer were evaluated. Tumour sections were stained by immunohistochemistry for LAT1, ASCT2, xCT, 4F2hc/CD98hc (4F2hc), Ki-67, and microvessel density (MVD) determined by CD34, and p53.Results:L-type amino-acid transporter 1 and 4F2hc were highly expressed in 61% (52 out of 85) and 45% (38 out of 47), respectively. ASC amino-acid transporter-2 and xCT were positively expressed in 59% (50 out of 85) and 21% (18 out of 85), respectively. The expression of both LAT1 and ASCT2 was significantly associated with disease staging, lymph-node metastasis, lymphatic permeation, 4F2hc expression and cell proliferation (Ki-67). xCT expression indicated a significant association with advanced stage and tumour factor. By univariate analysis, disease staging, lymphatic permeation, vascular invasion, LAT1, ASCT2, 4F2hc, and Ki-67 had a significant relationship with overall survival. Multivariate analysis confirmed that LAT1 was an independent prognostic factor for predicting poor prognosis.Conclusions:L-type amino-acid transporter 1 and ASCT2 can serve as a significant prognostic factor for predicting worse outcome after surgical treatment and may have an important role in the development and aggressiveness of tongue cancer.

Establishment of stable cell lines with high expression of heterodimers of human 4F2hc and human amino acid transporter LAT1 or LAT2 and delineation of their differential interaction with α-alkyl moieties.

Abstract System L is a major transport system for cellular uptake of neutral amino acids. Among system L transporters, L-type amino acid transporter 1 (LAT1) is responsible for the nutrient uptake in cancer cells, whereas L-type amino acid transporter 2 (LAT2) is a transporter for non-cancer cells. In this study, we have established HEK293 cell lines stably expressing high levels of human LAT1 and LAT2 forming heterodimers with native human 4F2hc of the cells. We have found that l-[14C]alanine is an appropriate substrate to examine the function of LAT2, whereas l-[14C]leucine is used for LAT1. By using l-[14C]alanine on LAT2, we have for the first time directly evaluated the function of human LAT2 expressed in mammalian cells and obtained its reliable kinetics. Using α-alkyl amino acids including α-methyl-alanine and α-ethyl-l-alanine, we have demonstrated that α-alkyl groups interfere with the interaction with LAT2. These cell lines with higher practical advantages would be useful for screening and analyzing compounds to develop LAT1-specific drugs that can be used for cancer diagnosis and therapeutics. The strategy that we took to establish the cell lines would also be applicable to the other heterodimeric transporters with important therapeutic implications.