LRRK2 directing ER‐to‐Golgi transport? (ER)yES!

Mutations in leucine‐rich repeat kinase 2 (LRRK2) represent the most frequent genetic lesions associated with Parkinson's disease (PD). LRRK2 function and the pathogenic mechanisms of LRRK2 genetic mutations remain poorly understood. Cho et al report in this issue of The EMBO Journal a new interaction between LRRK2 and the ER exit site (ERES) protein Sec16A, important for anterograde transport of secretory proteins. Moreover, a disease‐associated mutation disrupts LRRK2–Sec16A interaction and ERES function.     

ER sliding dynamics and ER–mitochondrial contacts occur on acetylated microtubules

The endoplasmic reticulum (ER) network is extremely dynamic in animal cells, yet little is known about the mechanism and function of its movements. The most common ER dynamic, termed ER sliding, involves ER tubule extension along stable microtubules (MTs). In this study, we show that ER sliding occurs on nocodazole-resistant MTs that are posttranslationally modified by acetylation. We demonstrate that high MT curvature is a good indicator of MT acetylation and show in live cells that ER sliding occurs predominantly on these curved, acetylated MTs. Furthermore, increasing MT acetylation by drug treatment increases the frequency of ER sliding. One purpose of the ER sliding on modified MT tracts could be to regulate its interorganelle contacts. We find that all mitochondria and many endosomes maintain contact with the ER despite the movements of each. However, mitochondria, but not endosomes, preferentially localize to acetylated MTs. Thus, different ER dynamics may occur on distinct MT populations to establish or maintain contacts with different organelles.     

The importance of ERα and ERβ gene polymorphisms in PCOS

Estrogens act through binding to estrogen receptor α (ERα) and β (ERβ). Studies in knockout mice have shown that the absence of ERα leads to the polycystic ovary syndrome (PCOS) phenotype. Furthermore, the expression of ERβ gene is lower in follicles derived from women with PCOS compared with healthy women. The aim of this study was to investigate the importance of ERα and ERβ gene polymorphisms in PCOS. A cohort of 180 women with PCOS and 140 healthy controls were recruited, and the PvuII and XbaI polymorphisms of ERα, as well as, the AluI and RsaI polymorphisms of ERβ were genotyped. No difference was found in the distribution of these polymorphisms between patients and healthy controls. However, in PCOS women, carriers of TC and TT genotypes of PvuII polymorphism had lower fasting glucose to insulin ratio compared with carriers of CC genotype (p = 0.029). In addition, the presence of AA genotype of XbaI polymorphism was associated with lower levels of follicle-stimulating hormone (FSH) compared with the presence of AG and GG genotypes (p = 0.03). The association of ERα polymorphisms with insulin resistance indices and FSH levels emphasizes the importance of ERα as a genetic modifier of the PCOS phenotype.     

LRP16与 ERα相互作用增强ERα介导的转录活性

LRP16是一个雌激素（E2）通过受体α（ERα）诱导表达的靶基因,LRP16不仅促进人乳腺癌MCF-7细胞的增殖,而且促进细胞的侵袭生长,但对LRP16作用的分子机制尚不清楚.首先,检测到LRP16表达缺陷明显削弱了MCF-7细胞对E2的反应性增殖能力;采用Northern 印迹与Western印迹法,进一步检测到抑制LRP16表达,明显损害了ERα靶基因对E2诱导上调的反应性,这些基因包括了cyclin D1, c-myc, c-fos,MTA3, pS2和维甲酸受体α基因（RARα）等;以上结果提示,LRP16参与了ERα介导的信号途径.将ERα模式启动子报告基因3×ERE-TATA-luc,ERα以及LRP16表达载体共转染MCF-7与HeLa细胞.结果发现,LRP16增强了ERα对报告基因的转录激活,并呈现对LRP16的剂量依赖性;进而采用GST-pull down以及免疫共沉淀方法证实了LRP16与ERα之间的直接相互作用,该作用不依赖于E2的存在,但可被E2增强;采用哺乳动物双杂交方法进一步证实了ERα与LRP16相互作用位点存在于A/B区的激活功能域-１（AF1）.以上结果表明,LRP16是ERα的一个共激活因子,通过相互作用,LRP16增强了ERα介导转录活性.该研究为LRP16促进ERα阳性乳腺癌细胞增殖与侵袭生长提供了合理的分子解释.     

α<Superscript>5</Superscript>β<Subscript>1</Subscript> integrins and fibronectin are involved in adherence of the<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ER97314 clinical strain to A549 cells

The role of fibronectin (Fn) and its natural receptors alpha5beta1 integrins in the interaction of P. aeruginosa with A549 epithelial cells was compared in the clinical isolate ER97314 and the reference PAK strain. Both strains expressed functional type IV pili, as shown by the results of the twitching motility assay. The ER97314 strain was highly adherent to immobilized Fn (640 000+/-20 000 CFU per well) while the PAK strain adhered less efficiently (70 000+/-10 000 CFU per well). Both strains adhered to A549 cells (33 400+/-1200 and 1200+/-100 CFU per well, for PAK and ER97314, respectively), only the PAK strain being significantly internalized (9430+/-2020 CFU per well). Cytochalasin D and genistein significantly decreased bacterial adherence of the 2 strains and caused also a significant decrease in PAK internalization. This inhibitory activity was not related to changes in the expression of alpha5beta1 integrins. Antibodies to Fn and alpha5beta1 integrins inhibited the adherence of the ER97314 strain but had no significant effect on PAK interaction with human cells. These findings suggest that only some P. aeruginosa strains can target Fn and their natural receptors alpha5beta1 integrins for adherence to A549 cells.     

Enhanced estrogen receptor beta (ERβ) selectivity of fluorinated carborane-containing ER modulators

We previously showed that fluorination of the carborane-containing selective estrogen receptor modulator (SERM) BE360 altered the agonist/antagonist activity balance and the estrogen receptor (ER) α/β subtype selectivity. Here, we designed and synthesized a series of fluorinated carboranyl phenols as candidate ERβ-selective ligands. Introduction of a fluorine atom onto the carborane cage commonly reduced the binding affinity for ERα, to an extent that depended on the other substituents present. The B-fluorinated m-carboranyl phenol 4a showed fourfold more potent ERβ-binding affinity than the parent non-fluorinated compound 7. 1-Iodo-9-fluoro-m-carboranyl phenol 4f showed high ERβ-binding affinity with an ERβ/ERα selectivity ratio of 8.2. Among the compounds tested, 6 showed the highest ERβ selectivity (10.1-fold) and the highest ER-agonistic activity (EC₅₀: 5.1 × 10^?10 M) in MCF-7 cell proliferation assay.     

Evaluation of <Emphasis Type="Italic">ERα</Emphasis> and <Emphasis Type="Italic">VDR</Emphasis> gene polymorphisms in relation to bone mineral density in Turkish postmenopausal women

It has been suggested that the estrogen receptor alpha (ERα) and vitamin D receptor (VDR) genes as possibly implicated in reduced bone mineral density (BMD) in osteoporosis. The present study investigated the relation of ERα PvuII/XbaI polymorphisms and VDR FokI/TaqI polymorphisms with BMD in Turkish postmenopausal women. Eighty-one osteoporotic and 122 osteopenic postmenopausal women were recruited. For detection of the polymorphisms, polymerase chain reaction-restriction fragment lenght polymorphism techniques have been used. BMD was measured at the lumbar spine and hip by dual-energy X-ray absorptiometry. Distributions of ERα (PvuII dbSNP: rs2234693, XbaI dbSNP: rs9340799) and VDR genotypes (FokI dbSNP rs10735810, TaqI dbSNP: rs731236) were similar in study population. Although overall prevalence of osteoporosis had no association with these genotypes, the prevalence of decreased femoral neck BMD values were higher in the subjects with ERα PvuII “PP” and ERα XbaI “XX” genotypes than in those with “Pp/pp” genotypes and “xx” genotype, respectively (P < 0.05). Furthermore, subjects with VDR FokI “FF” genotype had lower BMD values of femoral neck and total hip compared to those with “Ff” genotype (P < 0.05). In the logistic regression analysis, we confirmed the presence of relationships between the VDR FokI “FF” genotypes, BMI ≤ 27.5, age ≥ 55 and the increased risk of femoral neck BMD below 0.8 value in postmenopausal women. The present data suggests that the ERα PvuII/XbaI and VDR FokI polymorphisms may contribute to the determination of bone mineral density in Turkish postmenopausal women.     

A novel fractionation method of the rough ER integral membrane proteins; Resident proteins versus exported proteins?

We treated the high salt‐washed canine pancreatic rough ER (KRM) with 0.18% Triton X‐100, separated the extract from the residual membrane (0.18%Tx KRM), and processed the extract with SM‐2 beads to recover membrane proteins in proteoliposomes. To focus on integral membrane proteins, KRM, 0.18%Tx KRM and proteoliposomes were subjected to sodium carbonate treatment, and analyzed by 2‐D gel electrophoresis. Consequently we found that a distinct group of integral membrane protein of KRM preferentially extracted from the membrane and recovered in proteoliposomes did exist, while majority of KRM integral membrane proteins were fractionated in 0.18%Tx KRM, which retained the basic structure and functions of KRM. Protein identification showed that the former group was enriched with proteins exported from the ER and the latter group comprised mostly of ER resident proteins. This result will potentially affect the prevailing view of the ER membrane structure as well as protein sorting from the ER.     

ERβ相互作用蛋白PSMC5的分离及其在ERβ信号途径中的作用

雌激素受体β（ERβ）在乳腺癌发生发展中起着重要的作用,寻找与ERβ相互作用的共调节因子对阐明ER信号通路具有重要价值.应用酵母双杂交技术,以ERβ的AF2结构域为诱饵蛋白从人乳腺文库中筛选出了与之相互作用的蛋白26S蛋白酶的亚单位ATPase 5（PSMC5）.GST沉淀实验表明,PSMC5在体外特异地与ERβ相结合.转录活性实验表明,PSMC5以激素依赖的方式降低ERβ转录活性.上述结果提示,PSMC5可能通过影响ERβ信号途径在乳腺癌发生发展中起着重要的作用.     

新的ER下游靶基因BAP18的鉴定及其在ER阳性乳腺癌中的作用

雌激素受体(ER)/雌二醇(E2)调控其下游靶基因的转录及蛋白表达,在乳腺癌的发生发展过程中发挥至关重要的作用,寻找新的ER下游靶基因可以为乳腺癌的临床治疗提供新的治疗靶点。该研究通过Western blot和qPCR方法确定了一个新的ER下游靶基因BAP18,其表达量可以被E2和ER上调,同时使用拮抗E2和ER的药物如他莫昔芬或氟维斯群可以使BAP18表达量下降。通过生物信息学分析确定了BAP18转录起始位点前潜在的ER结合位点,构建了检测BAP18转录活性的质粒后,用荧光素酶双报告基因实验和染色质免疫共沉淀实验确定了BAP18启动子上ER的结合位点和上调转录的区域。凝胶迁移实验确定ER可以直接结合BAP18的启动子DNA。最后利用CRISPR-Cas9定向敲除BAP18,发现BAP18的敲除可以导致ER阳性乳腺癌细胞的生长和增殖减慢且凋亡增加。该研究鉴定了BAP18是ER下游靶基因,其在乳腺癌中有促癌作用。BAP18的发现有望为ER阳性乳腺癌的临床治疗提供理论基础和新的治疗靶点。     

比格犬卵巢子宫内雌激素受体ERα、ERβ的免疫组织化学定位

目的研究雌激素受体α,β在比格犬卵巢及子宫内的定位。方法采用免疫组化SP法DAB显色结合BCIP/NBT及AEC显色检测ERα、ERβ在比格犬子宫及卵巢内的表达。结果比格犬ERα主要表达于卵泡颗粒细胞、卵巢间质腺腺上皮细胞及子宫内膜腺体腺上皮细胞胞核内,胞质内仅有少量表达,而在卵泡膜内膜的间质细胞,腺体周围的基质细胞及小动脉血管内皮细胞和平滑肌细胞、小静脉内皮细胞的胞核内有少量表达。而ERβ则以相同的组织特异性主要表达于上述组织细胞的胞质内,在胞核内有微弱表达。ERα表达于膜黄体细胞的胞核内,而在黄体颗粒细胞胞核与胞质内均有表达。而ERβ则仍特异表达于不同生理阶段黄体细胞的胞质内。BCIP/NBT与AEC双染未见ERα、ERβ在子宫内有明显的共表达现象。结论比格犬ERα、ERβ在子宫与卵巢组织内定位不同,ERα主要定位于胞核,在胞质内有微弱表达,而ERβ主要定位于胞质,在胞核内有零星表达。     

NMR analysis reveals 17β-estradiol induced conformational change in ERβ ligand binding domain expressed in <Emphasis Type="Italic">E. coli</Emphasis>

Nuclear magnetic resonance (NMR) spectroscopy is a useful biophysical technique to study the ligand–protein interaction. In this report, we have used bacterially produced ERβ and its domains for studying the functional analysis of ligand–protein interaction. Briefly, ERβ and its transactivation domain (TAD) and ligand binding domain (LBD) were subcloned and overexpressed using a prokaryotic expression system. The recombinant proteins were purified using Ni⁺²-IDA affinity chromatography and analyzed by NMR. Purified ERβ and TAD show similar conformation in the absence or presence of 17β-estradiol. However, LBD shows altered conformation in the presence of 17β-estradiol. These findings suggest that ERβ produced in bacteria exhibits a conformation such that its LBD remains masked and consequently it binds less to 17β-estradiol. Such study may help to develop the therapeutic approaches for controlling the estradiol-mediated gene expression in hormone dependent diseases.     

ER subtype selectivity of m-carborane-containing phenols: C-alkyl groups on the m-carborane cage enhance ERα selectivity

Estrogen receptor (ER) exhibits two subtypes, ERα and ERβ, whose biological functions are quite different despite expression in the same tissues. We developed diiodo-m-carborane derivative 3a, which showed 14-fold selectivity for ERβ with high binding affinity toward ERβ. Interestingly, introduction of an alkyl group into the carbon atom of the m-carborane cage of 3a markedly enhanced the binding affinity toward ERα and decreased affinity toward ERβ. C-n-propyl derivative 3d showed 28-fold selectivity for ERα in an ER binding assay and promoted proliferation of MCF-7 breast cancer cells. Docking simulation studies suggest that the directions of the n-propyl group and the diiodo substituent introduced on the m-carborane cage play important roles for the control of ER subtype selectivity. As 3a and 3d showed ERβ and ERα selectivity with high binding affinity, respectively, these ligands may be useful as biological tools to aid in understanding the different roles of ER subtypes.     

兴国红鲤ER基因克隆表达及EE2暴露对肝中ER表达的影响

为评估环境内分泌干扰物对鱼类的影响,研究克隆了兴国红鲤雌激素受体（Estrogen receptor,ER）4种亚型ERα、ERβ、ERβ1、ERβ2的全长cDNA序列,氨基酸序列比对发现兴国红鲤ERs分别与3种鲤科鱼类相应亚型具有较高的同源性。实时荧光定量PCR（qRT-PCR）检测结果表明,4种ER亚型mRNA在雌雄成体组织中呈现差异表达,雌性个体中,肝、卵巢和肠中4种ERs的表达量均较高;在雄性个体中,ERα和ERβ主要在肝中表达,ERβ1、ERβ2分别在肠和精巢中的表达量最高。将150日龄的兴国红鲤幼鱼分别暴露在0.01、0.1和1 nmol/L的17α-乙炔基雌二醇（EE2）中4周,检测了雌性幼鱼肝中4种ER基因的表达变化情况。在EE2中暴露1-2周后,兴国红鲤雌性幼鱼肝中ERα基因的表达水平有极显著的提升;各浓度EE2能持续显著促进其肝中ERβ的表达;在1-2周内各浓度EE2对ERβ1表达有所抑制;第1周EE2能够抑制ERβ2基因mRNA的表达,并在0.01 nmol/L时抑制作用达到了显著的水平。上述研究结果表明,EE2暴露能诱导或抑制兴国红鲤雌性幼鱼肝中ER亚型的表达,相对于ERβ、ERβ1和ERβ2、ERα可作为EE2短期（1-2周）敏感性生物学标记。     

Leucine-rich repeat kinase 2 regulates Sec16A at ER exit sites to allow ER–Golgi export

Leucine-rich repeat kinase 2 (LRRK2) has been associated with Parkinson’s disease (PD) and other disorders. However, its normal physiological functions and pathogenic properties remain elusive. Here we show that LRRK2 regulates the anterograde ER–Golgi transport through anchoring Sec16A at the endoplasmic reticulum exit sites (ERES). LRRK2 interacted and co-localized with Sec16A, a key protein in the formation of ERES. Lrrk2 depletion caused a dispersion of Sec16A from ERES and impaired ER export. In neurons, LRRK2 and Sec16A showed extensive co-localization at the dendritic ERES (dERES) that locally regulate the transport of proteins to the dendritic spines. A loss of Lrrk2 affected the association of Sec16A with dERES and impaired the activity-dependent targeting of glutamate receptors onto the cell/synapse surface. Furthermore, the PD-related LRRK2 R1441C missense mutation in the GTPase domain interfered with the interaction of LRRK2 with Sec16A and also affected ER–Golgi transport, while LRRK2 kinase activity was not required for these functions. Therefore, our findings reveal a new physiological function of LRRK2 in ER–Golgi transport, suggesting ERES dysfunction may contribute to the pathogenesis of PD.     

α-Synuclein Delays Endoplasmic Reticulum (ER)-to-Golgi Transport in Mammalian Cells by Antagonizing ER/Golgi SNAREs

Toxicity of human α-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson''s disease has not been established. We tested elements of this hypothesis by expressing human α-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant α-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble α-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble α-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that α-synuclein antagonizes SNARE function. Ykt6 reversed α-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified α-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble α-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway.     

Mapping ERβ Genomic Binding Sites Reveals Unique Genomic Features and Identifies EBF1 as an ERβ Interactor

Considerable effort by numerous laboratories has resulted in an improved understanding of estrogen and SERM action mediated by the two estrogen receptors, ERα and ERβ. However, many of the targets for ERβ in cell physiology remain elusive. Here, the C4-12/Flag.ERβ cell line which stably expressed Flag.ERβ is used to study ERβ genomic functions without ERα interference. Mapping ERβ binding sites in these cells reveals ERβ unique distribution and motif enrichment patterns. Accompanying our mapping results, nascent RNA profiling is performed on cells at the same treatment time. The combined results allow the identification of ERβ target genes. Gene ontology analysis reveals that ERβ targets are enriched in differentiation, development and apoptosis. Concurrently, E2 treatment suppresses proliferation in these cells. Within ERβ binding sites, while the most prevalent binding motif is the canonical ERE, motifs of known ER interactors are also enriched in ERβ binding sites. Moreover, among enriched binding motifs are those of GFI, REST and EBF1, which are unique to ERβ binding sites in these cells. Further characterization confirms the association between EBF1 and the estrogen receptors, which favors the N-terminal region of the receptor. Furthermore, EBF1 negatively regulates ERs at the protein level. In summary, by studying ERβ genomic functions in our cell model, we confirm the anti-proliferative role of ERβ and discover the novel cross talk of ERβ with EBF1 which has various implications in normal physiology.