A time-resolved Förster resonance energy transfer assay to investigate drug and inhibitor binding to ABCG2.

The human ATP-binding cassette (ABC) transporter, ABCG2, is responsible for multidrug resistance in some tumours. Detailed knowledge of its activity is crucial for understanding drug transport and resistance in cancer, and has implications for wider pharmacokinetics. The binding of substrates and inhibitors is a key stage in the transport cycle of ABCG2. Here, we describe a novel binding assay using a high affinity fluorescent inhibitor based on Ko143 and time-resolved Förster resonance energy transfer (TR-FRET) to measure saturation binding to ABCG2. This binding is displaced by Ko143 and other known ABCG2 ligands, and is sensitive to the addition of AMP-PNP, a non-hydrolysable ATP analogue. This assay complements the arsenal of methods for determining drug:ABCG2 interactions and has the possibility of being adaptable for other multidrug pumps.

Mechanistic insight into the role of AUXIN RESISTANCE4 in trafficking of AUXIN1 and LIKE AUX1-2.

AUXIN RESISTANCE4 (AXR4) regulates the trafficking of auxin influx carrier AUXIN1 (AUX1), a plasma-membrane protein that predominantly localizes to the endoplasmic reticulum (ER) in the absence of AXR4. In Arabidopsis (Arabidopsis thaliana), AUX1 is a member of a small multigene family comprising 4 highly conserved genes-AUX1, LIKE-AUX1 (LAX1), LAX2, and LAX3. We report here that LAX2 also requires AXR4 for correct localization to the plasma membrane. AXR4 is a plant-specific protein and contains a weakly conserved α/ß hydrolase fold domain that is found in several classes of lipid hydrolases and transferases. We have previously proposed that AXR4 may either act as (i) a post-translational modifying enzyme through its α/ß hydrolase fold domain or (ii) an ER accessory protein, which is a special class of ER protein that regulates targeting of their cognate partner proteins. Here, we show that AXR4 is unlikely to act as a post-translational modifying enzyme as mutations in several highly conserved amino acids in the α/ß hydrolase fold domain can be tolerated and active site residues are missing. We also show that AUX1 and AXR4 physically interact with each other and that AXR4 reduces aggregation of AUX1 in a dose-dependent fashion. Our results suggest that AXR4 acts as an ER accessory protein. A better understanding of AXR4-mediated trafficking of auxin transporters in crop plants will be crucial for improving root traits (designer roots) for better acquisition of water and nutrients for sustainable and resilient agriculture.

Extracellular Vesicles Potentiate Medulloblastoma Metastasis in an EMMPRIN and MMP-2 Dependent Manner.

Extracellular vesicles (EVs) have emerged as pivotal mediators of communication in the tumour microenvironment. More specifically, nanosized extracellular vesicles termed exosomes have been shown to contribute to the establishment of a premetastatic niche. Here, we sought to determine what role exosomes play in medulloblastoma (MB) progression and elucidate the underlying mechanisms. Metastatic MB cells (D458 and CHLA-01R) were found to secrete markedly more exosomes compared to their nonmetastatic, primary counterparts (D425 and CHLA-01). In addition, metastatic cell-derived exosomes significantly enhanced the migration and invasiveness of primary MB cells in transwell migration assays. Protease microarray analysis identified that matrix metalloproteinase-2 (MMP-2) was enriched in metastatic cells, and zymography and flow cytometry assays of metastatic exosomes demonstrated higher levels of functionally active MMP-2 on their external surface. Stable genetic knockdown of MMP-2 or extracellular matrix metalloproteinase inducer (EMMPRIN) in metastatic MB cells resulted in the loss of this promigratory effect. Analysis of serial patient cerebrospinal fluid (CSF) samples showed an increase in MMP-2 activity in three out of four patients as the tumour progressed. This study demonstrates the importance of EMMPRIN and MMP-2-associated exosomes in creating a favourable environment to drive medulloblastoma metastasis via extracellular matrix signalling.

Drug Resistance in Medulloblastoma Is Driven by YB-1, ABCB1 and a Seven-Gene Drug Signature.

Therapy resistance represents an unmet challenge in the treatment of medulloblastoma. Accordingly, the identification of targets that mark drug-resistant cell populations, or drive the proliferation of resistant cells, may improve treatment strategies. To address this, we undertook a targeted approach focused on the multi-functional transcription factor YB-1. Genetic knockdown of YB-1 in Group 3 medulloblastoma cell lines diminished cell invasion in 3D in vitro assays and increased sensitivity to standard-of-care chemotherapeutic vincristine and anti-cancer agents panobinostat and JQ1. For vincristine, this occurred in part by YB-1-mediated transcriptional regulation of multi-drug resistance gene ABCB1, as determined by chromatin immunoprecipitation. Whole transcriptome sequencing of YB-1 knockdown cells identified a role for YB-1 in the regulation of tumourigenic processes, including lipid metabolism, cell death and survival and MYC and mTOR pathways. Stable cisplatin- and vincristine-tolerant Group 3 and SHH cell lines were generated to identify additional mechanisms driving resistance to standard-of-care medulloblastoma therapy. Next-generation sequencing revealed a vastly different transcriptomic landscape following chronic drug exposure, including a drug-tolerant seven-gene expression signature, common to all sequenced drug-tolerant cell lines, representing therapeutically targetable genes implicated in the acquisition of drug tolerance. Our findings provide significant insight into mechanisms and genes underlying therapy resistance in medulloblastoma.

Identifying new biomarkers of aggressive Group 3 and SHH medulloblastoma using 3D hydrogel models, single cell RNA sequencing and 3D OrbiSIMS imaging.

The most common malignant brain tumour in children, medulloblastoma (MB), is subdivided into four clinically relevant molecular subgroups, although targeted therapy options informed by understanding of different cellular features are lacking. Here, by comparing the most aggressive subgroup (Group 3) with the intermediate (SHH) subgroup, we identify crucial differences in tumour heterogeneity, including unique metabolism-driven subpopulations in Group 3 and matrix-producing subpopulations in SHH. To analyse tumour heterogeneity, we profiled individual tumour nodules at the cellular level in 3D MB hydrogel models, which recapitulate subgroup specific phenotypes, by single cell RNA sequencing (scRNAseq) and 3D OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) imaging. In addition to identifying known metabolites characteristic of MB, we observed intra- and internodular heterogeneity and identified subgroup-specific tumour subpopulations. We showed that extracellular matrix factors and adhesion pathways defined unique SHH subpopulations, and made up a distinct shell-like structure of sulphur-containing species, comprising a combination of small leucine-rich proteoglycans (SLRPs) including the collagen organiser lumican. In contrast, the Group 3 tumour model was characterized by multiple subpopulations with greatly enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle activity. Extensive TCA cycle metabolite measurements revealed very high levels of succinate and fumarate with malate levels almost undetectable particularly in Group 3 tumour models. In patients, high fumarate levels (NMR spectroscopy) alongside activated stress response pathways and high Nuclear Factor Erythroid 2-Related Factor 2 (NRF2; gene expression analyses) were associated with poorer survival. Based on these findings we predicted and confirmed that NRF2 inhibition increased sensitivity to vincristine in a long-term 3D drug treatment assay of Group 3 MB. Thus, by combining scRNAseq and 3D OrbiSIMS in a relevant model system we were able to define MB subgroup heterogeneity at the single cell level and elucidate new druggable biomarkers for aggressive Group 3 and low-risk SHH MB.

Vinca alkaloid binding to P-glycoprotein occurs in a processive manner.

A mechanistic understanding of how P-glycoprotein (Pgp) is able to bind and transport its astonishing range of substrates remains elusive. Pharmacological data demonstrated the presence of at least four distinct binding sites, but their locations have not been fully elucidated. The combination of biochemical and structural data suggests that initial binding may occur in the central cavity or at the lipid-protein interface. Our objective was to define the binding sites for two transported substrates of Pgp; the anticancer drug vinblastine and the fluorescent probe rhodamine 123. A series of mutations was generated in positions proximal to previously defined drug-interacting residues on Pgp. The protein was purified and reconstituted into styrene-maleic acid lipid particles (SMALPs) to measure the apparent drug binding constant or into liposomes for assessment of drug-stimulated ATP hydrolysis. The biochemical data were reconciled with structural models of Pgp using molecular docking. The data indicated that the binding of rhodamine 123 occurred predominantly within the central cavity of Pgp. In contrast, the significantly more hydrophobic vinblastine bound to both the lipid-protein interface and within the central cavity. The data suggest that the initial interaction of vinca alkaloids with Pgp occurs at the lipid interface followed by internalisation into the central cavity, which also provides the transport conduit. This model is supported by recent structural observations with Pgp and early biophysical and cross-linking approaches. Moreover, the proposed model illustrates that the broad substrate profile for Pgp is underpinned by a combination of multiple initial interaction sites and an accommodating transport conduit.

Detergent-Free Membrane Protein Purification Using SMA Polymer.

One of the big challenges for the study of structure and function of membrane proteins is the need to extract them from the membrane. Traditionally this was achieved using detergents which disrupt the membrane and form a micelle around the protein, but this can cause issues with protein function and/or stability. In 2009 an alternative approach was reported, using styrene maleic acid (SMA) copolymer to extract small discs of lipid bilayer encapsulated by the polymer and termed SMALPs (SMA lipid particles). Since then this approach has been shown to work for a range of different proteins from many different expression systems. It allows the extraction and purification of a target protein while maintaining a lipid bilayer environment. Recently this has led to several new high-resolution structures and novel insights to function. As with any method there are some limitations and issues to be aware of. Here we describe a standard protocol for preparation of the polymer and its use for membrane protein purification, and also include details of typical challenges that may be encountered and possible ways to address those.

Determination of the Rab27-Effector Binding Affinity Using a High-Throughput FRET-Based Assay.

Thus far, two Rab27 isoforms (Rab27a and Rab27b) have been identified that interact with their eleven downstream effectors proteins, preferentially in their GTP-bound state. In recent years, a number of studies has suggested roles for Rab27-effector protein interactions in the development of cancer cell invasion and metastasis, and immune and inflammatory responses. Here we develop an in vitro fluorescence resonance energy transfer (FRET)-based protein-protein interaction assay to report Rab27 protein interactions with their effectors. We particularly focus on determining the interaction of mouse (m) Synaptotagmin-like protein (Slp)1 and mSlp2 effector proteins with human (h)Rab27. Green fluorescent protein (GFP)-N-terminus Rab27 binding domains (m-Slp1 and m-Slp2) recombinant proteins were used as donor fluorophores, whereas mCherry-hRab27a/b recombinant proteins were used as acceptor fluorophores. The conditions of this assay were validated and optimized, and the specificity of the assay was confirmed. Accordingly, this assay can be used to assess and identify key determinants and/or candidate inhibitors of Rab27-effector interactions.

ABCB1 inhibition provides a novel therapeutic target to block TWIST1-induced migration in medulloblastoma.

BACKGROUND: Therapeutic intervention in metastatic medulloblastoma is dependent on elucidating the underlying metastatic mechanism. We investigated whether an epithelial-mesenchymal transition (EMT)-like pathway could drive medulloblastoma metastasis. METHODS: A 3D Basement Membrane Extract (3D-BME) model was used to investigate medulloblastoma cell migration. Cell line growth was quantified with AlamarBlue metabolic assays and the morphology assessed by time-lapse imaging. Gene expression was analyzed by qRT-PCR and protein expression by immunohistochemistry of patient tissue microarrays and mouse orthotopic xenografts. Chromatin immunoprecipitation was used to determine whether the EMT transcription factor TWIST1 bound to the promoter of the multidrug pump ABCB1. TWIST1 was overexpressed in MED6 cells by lentiviral transduction (MED6-TWIST1). Inhibition of ABCB1 was mediated by vardenafil, and TWIST1 expression was reduced by either Harmine or shRNA. RESULTS: Metastatic cells migrated to form large metabolically active aggregates, whereas non-tumorigenic/non-metastatic cells formed small aggregates with decreasing metabolic activity. TWIST1 expression was upregulated in the 3D-BME model. TWIST1 and ABCB1 were significantly associated with metastasis in patients (P = .041 and P = .04, respectively). High nuclear TWIST1 expression was observed in the invasive edge of the MED1 orthotopic model, and TWIST1 knockdown in cell lines was associated with reduced cell migration (P < .05). TWIST1 bound to the ABCB1 promoter (P = .03) and induced cell aggregation in metastatic and TWIST1-overexpressing, non-metastatic (MED6-TWIST1) cells, which was significantly attenuated by vardenafil (P < .05). CONCLUSIONS: In this study, we identified a TWIST1-ABCB1 signaling axis during medulloblastoma migration, which can be therapeutically targeted with the clinically approved ABCB1 inhibitor, vardenafil.

An investigation into possible factors that may impact on the potential for inappropriate prescriptions of antibiotics: a survey of general dental practitioners' approach to treating adults with acute dental pain.

Objective To investigate factors that might influence inappropriate prescriptions of antibiotics (ABs) by UK-based general dental practitioners (GDPs) in their management of acute dental pain in adults in primary dental care.Methods A questionnaire was circulated via social media to UK-based GDPs. The questionnaire examined GDPs' likelihood of issuing an inappropriate AB in two hypothetical clinical scenarios.Results A total of 205 questionnaires were completed, of which 198 were included for analysis. The resulting data were analysed to try and identify factors that correlated with an increased likelihood of an inappropriate AB prescription being issued for each clinical scenario. The results suggested the following factors as being associated with a statistically greater chance of the survey respondent issuing an inappropriate AB prescription: no postgraduate qualification; received their primary dental qualification from a non-UK university; scheduled appointments of less than 20 minutes; and low confidence in their ability to provide adequate local anaesthesia for the patients in the clinical scenario.Conclusions Four factors were shown to be associated with dentists' stated intention to prescribe ABs for acute dental pain, not in accordance with guidance. These results should guide further research to understand the significance of appropriate AB prescribing on the quality of urgent dental care. While the total number of respondents was too low to enable the results to be generalised, it is hoped that the results may help guide future research. Further studies could focus on these factors to understand more about their impact on the urgent care of adult patients in pain. The impact of the restrictions caused by the response to COVID-19 needs to be considered.

BLBP Is Both a Marker for Poor Prognosis and a Potential Therapeutic Target in Paediatric Ependymoma.

Paediatric ependymomas are aggressive, treatment-resistant tumours with a tendency towards relapse, consistent with a sub-population of therapy-resistant cancer stem cells. These cells are believed to derive from brain lipid binding protein (BLBP)-expressing radial glia, hence we proposed that BLBP may be a marker for ependymoma therapy resistance. BLBP protein expression correlated with reduced overall survival (OS) in patients from two trials (CNS9204, a chemotherapy-led infant trial-5 y OS 45% vs. 80%, p = 0.011-and CNS9904, a radiotherapy-led trial-OS 38% vs. 85%, p = 0.002). All ependymoma cell lines examined by qRT-PCR expressed BLBP, with expression elevated in stem cell-enriched neurospheres. Modulation of BLBP function in 2D and 3D assays, using either peroxisome proliferator activated receptor (PPAR) antagonists or BLBP's fatty acid substrate docosahexaneoic acid (DHA), potentiated chemotherapy response and reduced cell migration and invasion in ependymoma cell lines. BLBP is therefore an independent predictor of poor survival in paediatric ependymoma, and treatment with PPAR antagonists or DHA may represent effective novel therapies, preventing chemotherapy resistance and invasion in paediatric ependymoma patients.

Analysis of Sequence Divergence in Mammalian ABCGs Predicts a Structural Network of Residues That Underlies Functional Divergence.

The five members of the mammalian G subfamily of ATP-binding cassette transporters differ greatly in their substrate specificity. Four members of the subfamily are important in lipid transport and the wide substrate specificity of one of the members, ABCG2, is of significance due to its role in multidrug resistance. To explore the origin of substrate selectivity in members 1, 2, 4, 5 and 8 of this subfamily, we have analysed the differences in conservation between members in a multiple sequence alignment of ABCG sequences from mammals. Mapping sets of residues with similar patterns of conservation onto the resolved 3D structure of ABCG2 reveals possible explanations for differences in function, via a connected network of residues from the cytoplasmic to transmembrane domains. In ABCG2, this network of residues may confer extra conformational flexibility, enabling it to transport a wider array of substrates.

Function of the pseudo phosphotransfer proteins has diverged between rice and Arabidopsis.

The phytohormone cytokinin plays a significant role in nearly all aspects of plant growth and development. Cytokinin signaling has primarily been studied in the dicot model Arabidopsis, with relatively little work done in monocots, which include rice (Oryza sativa) and other cereals of agronomic importance. The cytokinin signaling pathway is a phosphorelay comprised of the histidine kinase receptors, the authentic histidine phosphotransfer proteins (AHPs) and type-B response regulators (RRs). Two negative regulators of cytokinin signaling have been identified: the type-A RRs, which are cytokinin primary response genes, and the pseudo histidine phosphotransfer proteins (PHPs), which lack the His residue required for phosphorelay. Here, we describe the role of the rice PHP genes. Phylogenic analysis indicates that the PHPs are generally first found in the genomes of gymnosperms and that they arose independently in monocots and dicots. Consistent with this, the three rice PHPs fail to complement an Arabidopsis php mutant (aphp1/ahp6). Disruption of the three rice PHPs results in a molecular phenotype consistent with these elements acting as negative regulators of cytokinin signaling, including the induction of a number of type-A RR and cytokinin oxidase genes. The triple php mutant affects multiple aspects of rice growth and development, including shoot morphology, panicle architecture, and seed fill. In contrast to Arabidopsis, disruption of the rice PHPs does not affect root vascular patterning, suggesting that while many aspects of key signaling networks are conserved between monocots and dicots, the roles of at least some cytokinin signaling elements are distinct.

Corrigendum to: ABCB1 inhibition provides a novel therapeutic target to block TWIST1-induced migration in medulloblastoma.

[This corrects the article DOI: 10.1093/noajnl/vdab030.].

A randomized, controlled pilot study of cognitive analytic therapy for stressed pregnant women with underlying anxiety and depression in a routine health service setting.

A pilot study of cognitive analytic therapy (CAT) plus treatment as usual (TAU), versus TAU in stressed pregnant women with anxiety and depression, was undertaken as an essential preliminary to any definitive, randomized controlled trial (RCT). The trial was pragmatic, multicentre, parallel, randomized, controlled, and unblinded. Participants were pregnant women screened using the Hospital Anxiety and Depression Scale (HADS). Treatment was standard 16-session CAT. Main outcome measures were Spielberger State/Trait Anxiety Inventory (STAI) (primary outcome measure) at 24 weeks after randomization, therefore 1 month after therapy for the CAT group; HADS; Clinical Outcomes in Routine Evaluation-Outcome Measure (CORE-OM); Edinburgh Postnatal Depression Scale (EPDS); 36-item Short Form Health Survey (SF-36); and a brief Experiences of Therapy Questionnaire, completed at baseline and on average at 12, 24, 40, and 82 weeks after randomization. Thirty-nine patients (CAT + TAU, n = 20; TAU, n = 19) were randomized with mean baseline STAI State scores of 50.8 (SD 11.4) and 51.1 (SD 13.3), respectively. Sixteen patients had missing primary outcome data leaving 23 (n = 11 and n = 12) patients for analysis. The mean STAI State score was 38.5 (SD 13.8) and 45.7 (SD 16.8) in the CAT and TAU groups respectively at 24 weeks after randomization, with an adjusted difference in means of 7.2 (95% confidence interval [CI]: -7.9 to 20.6). No safety issues were reported. Patient retention for the CAT group was high (18/20; 90% of patients completed therapy). Ten out of 11 (90.9%) respondents 'agreed' or 'strongly agreed' that having CAT had been 'very helpful'. The study demonstrated the feasibility of safely undertaking CAT in this setting. Outcomes showed positive trends compatible with a clinically important effect, although statistically definitive conclusions cannot be drawn in such a study.

3D hydrogels reveal medulloblastoma subgroup differences and identify extracellular matrix subtypes that predict patient outcome.

Medulloblastoma (MB) is the most common malignant brain tumour in children and is subdivided into four subgroups: WNT, SHH, Group 3, and Group 4. These molecular subgroups differ in their metastasis patterns and related prognosis rates. Conventional 2D cell culture methods fail to recapitulate these clinical differences. Realistic 3D models of the cerebellum are therefore necessary to investigate subgroup-specific functional differences and their role in metastasis and chemoresistance. A major component of the brain extracellular matrix (ECM) is the glycosaminoglycan hyaluronan. MB cell lines encapsulated in hyaluronan hydrogels grew as tumour nodules, with Group 3 and Group 4 cell lines displaying clinically characteristic laminar metastatic patterns and levels of chemoresistance. The glycoproteins, laminin and vitronectin, were identified as subgroup-specific, tumour-secreted ECM factors. Gels of higher complexity, formed by incorporation of laminin or vitronectin, revealed subgroup-specific adhesion and growth patterns closely mimicking clinical phenotypes. ECM subtypes, defined by relative levels of laminin and vitronectin expression in patient tissue microarrays and gene expression data sets, were able to identify novel high-risk MB patient subgroups and predict overall survival. Our hyaluronan model system has therefore allowed us to functionally characterize the interaction between different MB subtypes and their environment. It highlights the prognostic and pathological role of specific ECM factors and enables preclinical development of subgroup-specific therapies. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Mammalian ABCG-transporters, sterols and lipids: To bind perchance to transport?

Members of the ATP binding cassette (ABC) transporter family perform a critical function in maintaining lipid homeostasis in cells as well as the transport of drugs. In this review, we provide an update on the ABCG-transporter subfamily member proteins, which include the homodimers ABCG1, ABCG2 and ABCG4 as well as the heterodimeric complex formed between ABCG5 and ABCG8. This review focusses on progress made in this field of research with respect to their function in health and disease and the recognised transporter substrates. We also provide an update on post-translational regulation, including by transporter substrates, and well as the involvement of microRNA as regulators of transporter expression and activity. In addition, we describe progress made in identifying structural elements that have been recognised as important for transport activity. We furthermore discuss the role of lipids such as cholesterol on the transport function of ABCG2, traditionally thought of as a drug transporter, and provide a model of potential cholesterol binding sites for ABCG2.

Y-Box Binding Protein-1: A Neglected Target in Pediatric Brain Tumors?

Brain and central nervous system tumors represent the most common childhood solid tumors. Comprising 21% of all pediatric cancers, they remain the leading cause of cancer-related mortality and morbidity in childhood. Due to advances in neurosurgical technique, radiotherapy and the use of combination therapy, survival rates have generally increased. However, by cause of the lesion itself, its surgical removal and subsequent treatment, survivors are at high risk of long-term neurocognitive sequelae and secondary cancer. Clearly, improvements in diagnosis and treatment are needed. Accordingly, current treatment is evolving away from conventional, uniform therapy and towards risk-stratified regimens and molecularly-targeted therapies, with the aim of diminishing adverse side effects while minimizing the risk of disease recurrence. The multifunctional oncoprotein Y-box binding protein 1 (YB-1) may serve as one such molecular target. Increased YB-1 levels have been reported in a number of pediatric brain tumors, where YB-1 appears to facilitate the advancement of malignant phenotypes. These include proliferation, invasion, and resistance to therapy, as well as the maintenance of brain tumor-initiating cells. Here we evaluate the current literature and show how YB-1 modulates signaling pathways driving each of these phenotypes. We also review the regulation of YB-1 at a transcriptional, translational, posttranslational and subcellular level and argue that there is strong and sufficient evidence to support the development of YB-1 as a biomarker and future therapeutic target in childhood brain tumors.

Picky ABCG5/G8 and promiscuous ABCG2 - a tale of fatty diets and drug toxicity.

Structural data on ABCG5/G8 and ABCG2 reveal a unique molecular architecture for subfamily G ATP-binding cassette (ABCG) transporters and disclose putative substrate-binding sites. ABCG5/G8 and ABCG2 appear to use several unique structural motifs to execute transport, including the triple helical bundles, the membrane-embedded polar relay, the re-entry helices, and a hydrophobic valve. Interestingly, ABCG2 shows extreme substrate promiscuity, whereas ABCG5/G8 transports only sterol molecules. ABCG2 structures suggest a large internal cavity, serving as a binding region for substrates and inhibitors, while mutational and pharmacological analyses support the notion of multiple binding sites. By contrast, ABCG5/G8 shows a collapsed cavity of insufficient size to hold substrates. Indeed, mutational analyses indicate a sterol-binding site at the hydrophobic interface between the transporter and the lipid bilayer. In this review, we highlight key differences and similarities between ABCG2 and ABCG5/G8 structures. We further discuss the relevance of distinct and shared structural features in the context of their physiological functions. Finally, we elaborate on how ABCG2 and ABCG5/G8 could pave the way for studies on other ABCG transporters.

In Vitro Fluorescence Resonance Energy Transfer-Based Assay Used to Determine the Rab27-Effector-Binding Affinity.

The Rab27 subfamily consists of Rab27a/b isoforms that have similar but not identical functions. Those functions include the regulation of trafficking, docking, and fusion of various lysosome-related organelles and secretory granules; such as melanosomes in melanocytes and lytic granules in cytotoxic T lymphocytes. Rab27a/b exert their specific and versatile functions by interacting with 11 effector proteins, preferentially in their GTP-bound state. In recent years, a number of studies have identified roles for Rab27 proteins and their effectors in cancer cell invasion and metastasis, immune response, inflammation, and allergic responses. These findings suggest that Rab27-effector protein interaction inhibitors could contribute to the development of effective strategies to treat these diseases. To facilitate inhibitor identification, in this study we developed a fluorescence resonance energy transfer-based protein-protein interaction assay that reports Rab27-effector interactions. Green fluorescent protein (GFP)-mouse (m) synaptotagmin-like protein (Slp)1 and GFP-mSlp2 (N-terminus Rab27-binding domains) recombinant proteins were used as donor fluorophores, whereas mCherry-human (h) Rab27a/b recombinant proteins were used as acceptor fluorophores. The in vitro binding affinity of mSlp2 to Rab27 was found to be higher compared with mSlp1 and was evidenced by the effective concentration 50 value differences (mSlp2-hRab27b = 0.15 µM < mSlp2-hRab27a = 0.2 µM < mSlp1-hRab27a = 0.32 µM < mSlp1-hRab27b = 0.33 µM). The specificity of the assay was assessed using unlabeled rat (r) Rab27a and hRab27b recombinant proteins as typical competitive inhibitors for Rab27-effector interactions and was evidenced by the inhibitory concentration 50 value differences. Accordingly, this in vitro assay can be employed in identification of candidate inhibitors of Rab27-effector interactions.