PySupercharge: a python algorithm for enabling ABC transporter bacterial secretion of all proteins through amino acid mutation.

BACKGROUND: The process of producing proteins in bacterial systems and secreting them through ATP-binding cassette (ABC) transporters is an area that has been actively researched and used due to its high protein production capacity and efficiency. However, some proteins are unable to pass through the ABC transporter after synthesis, a phenomenon we previously determined to be caused by an excessive positive charge in certain regions of their amino acid sequence. If such an excessive charge is removed, the secretion of any protein through ABC transporters becomes possible. RESULTS: In this study, we introduce 'linear charge density' as the criteria for possibility of protein secretion through ABC transporters and confirm that this criterion can be applied to various non-secretable proteins, such as SARS-CoV-2 spike proteins, botulinum toxin light chain, and human growth factors. Additionally, we develop a new algorithm, PySupercharge, that enables the secretion of proteins containing regions with high linear charge density. It selectively converts positively charged amino acids into negatively charged or neutral amino acids after linear charge density analysis to enable protein secretion through ABC transporters. CONCLUSIONS: PySupercharge, which also minimizes functional/structural stability loss of the pre-mutation proteins through the use of sequence conservation data, is currently being operated on an accessible web server. We verified the efficacy of PySupercharge-driven protein supercharging by secreting various previously non-secretable proteins commonly used in research, and so suggest this tool for use in future research requiring effective protein production.

Probing the stability and interdomain interactions in the ABC transporter OpuA using single-molecule optical tweezers.

Transmembrane transporter proteins are essential for maintaining cellular homeostasis and, as such, are key drug targets. Many transmembrane transporter proteins are known to undergo large structural rearrangements during their functional cycles. Despite the wealth of detailed structural and functional data available for these systems, our understanding of their dynamics and, consequently, how they function is generally limited. We introduce an innovative approach that enables us to directly measure the dynamics and stability of interdomain interactions of transmembrane proteins using optical tweezers. Focusing on the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis, we examine the mechanical properties and potential interactions of its substrate-binding domains. Our measurements are performed in lipid nanodiscs, providing a native-mimicking environment for the transmembrane protein. The technique provides high spatial and temporal resolution and allows us to study the functionally relevant motions and interdomain interactions of individual transmembrane transporter proteins in real time in a lipid bilayer.

Emerging roles for ABC transporters as virulence factors in uropathogenic Escherichia coli.

Urinary tract infections (UTI) account for a substantial financial burden globally. Over 75% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which have demonstrated an extraordinarily rapid growth rate in vivo. This rapid growth rate appears paradoxical given that urine and the human urinary tract are relatively nutrient-restricted. Thus, we lack a fundamental understanding of how uropathogens propel growth in the host to fuel pathogenesis. Here, we used large in silico, in vivo, and in vitro screens to better understand the role of UPEC transport mechanisms and their contributions to uropathogenesis. In silico analysis of annotated transport systems indicated that the ATP-binding cassette (ABC) family of transporters was most conserved among uropathogenic bacterial species, suggesting their importance. Consistent with in silico predictions, we determined that the ABC family contributed significantly to fitness and virulence in the urinary tract: these were overrepresented as fitness factors in vivo (37.2%), liquid media (52.3%), and organ agar (66.2%). We characterized 12 transport systems that were most frequently defective in screening experiments by generating in-frame deletions. These mutant constructs were tested in urovirulence phenotypic assays and produced differences in motility and growth rate. However, deletion of multiple transport systems was required to achieve substantial fitness defects in the cochallenge murine model. This is likely due to genetic compensation among transport systems, highlighting the centrality of ABC transporters in these organisms. Therefore, these nutrient uptake systems play a concerted, critical role in pathogenesis and are broadly applicable candidate targets for therapeutic intervention.

Type 1 secretion necessitates a tight interplay between all domains of the ABC transporter.

Type I secretion systems (T1SS) facilitate the secretion of substrates in one step across both membranes of Gram-negative bacteria. A prime example is the hemolysin T1SS which secretes the toxin HlyA. Secretion is energized by the ABC transporter HlyB, which forms a complex together with the membrane fusion protein HlyD and the outer membrane protein TolC. HlyB features three domains: an N-terminal C39 peptidase-like domain (CLD), a transmembrane domain (TMD) and a C-terminal nucleotide binding domain (NBD). Here, we created chimeric transporters by swapping one or more domains of HlyB with the respective domain(s) of RtxB, a HlyB homolog from Kingella kingae. We tested all chimeric transporters for their ability to secrete pro-HlyA when co-expressed with HlyD. The CLD proved to be most critical, as a substitution abolished secretion. Swapping only the TMD or NBD reduced the secretion efficiency, while a simultaneous exchange abolished secretion. These results indicate that the CLD is the most critical secretion determinant, while TMD and NBD might possess additional recognition or interaction sites. This mode of recognition represents a hierarchical and extreme unusual case of substrate recognition for ABC transporters and optimal secretion requires a tight interplay between all domains.

Molecular insights into capsular polysaccharide secretion.

Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria1. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits1,2. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.

Preclinical Development of Antisense Oligonucleotides to Rescue Aberrant Splicing Caused by an Ultrarare ABCA4 Variant in a Child with Early-Onset Stargardt Disease.

Precision medicine is rapidly gaining recognition in the field of (ultra)rare conditions, where only a few individuals in the world are affected. Clinical trial design for a small number of patients is extremely challenging, and for this reason, the development of N-of-1 strategies is explored to accelerate customized therapy design for rare cases. A strong candidate for this approach is Stargardt disease (STGD1), an autosomal recessive macular degeneration characterized by high genetic and phenotypic heterogeneity. STGD1 is caused by pathogenic variants in ABCA4, and amongst them, several deep-intronic variants alter the pre-mRNA splicing process, generally resulting in the insertion of pseudoexons (PEs) into the final transcript. In this study, we describe a 10-year-old girl harboring the unique deep-intronic ABCA4 variant c.6817-713A>G. Clinically, she presents with typical early-onset STGD1 with a high disease symmetry between her two eyes. Molecularly, we designed antisense oligonucleotides (AONs) to block the produced PE insertion. Splicing rescue was assessed in three different in vitro models: HEK293T cells, fibroblasts, and photoreceptor precursor cells, the last two being derived from the patient. Overall, our research is intended to serve as the basis for a personalized N-of-1 AON-based treatment to stop early vision loss in this patient.

Inhibition of NEK2 Promotes Chemosensitivity and Reduces KSHV-positive Primary Effusion Lymphoma Burden.

Non-Hodgkin lymphoma (NHL) is a common cancer in both men and women and represents a significant cancer burden worldwide. Primary effusion lymphoma (PEL) is a subtype of NHL infected with Kaposi sarcoma-associated herpesvirus (KSHV). PEL is an aggressive and lethal cancer with no current standard of care, owing largely to its propensity to develop resistance to current chemotherapeutic regimens. Here, we report a reliance of KSHV-positive PEL on the mitotic kinase, NEK2, for survival. Inhibition of NEK2 with the inhibitor, JH295, resulted in caspase 3-mediated apoptotic cell death of PEL. Furthermore, NEK2 inhibition significantly prolonged survival and reduced tumor burden in a PEL mouse model. We also demonstrate that the ABC transporter proteins, MDR1 and MRP, are most active in PEL and that inhibition of NEK2 in PEL reduced the expression and activity of these ABC transporter proteins, which are known to mediate drug resistance in cancer. Finally, we report that JH295 treatment sensitized lymphomas to other chemotherapeutic agents such as rapamycin, resulting in enhanced cancer cell death. Overall, these data offer important insight into the mechanisms underlying PEL survival and drug resistance, and suggest that NEK2 is a viable therapeutic target for PEL. SIGNIFICANCE: The mitotic kinase, NEK2, is important for the survival of KSHV-positive PEL. NEK2 inhibition resulted in PEL apoptosis and reduced tumor burden in a mouse model. NEK2 inhibition also reduced drug resistance.

Adenosine triphosphate binding cassette transporters G5 and G8 early diagnostic tools for cardiovascular disease in human.

OBJECTIVE: Aim: The current study was designed to investigate the role of ABCG5 and ABCG5 in serum with normal and expected cardiac complaints with CVDs as individual early diagnostic tools. PATIENTS AND METHODS: Materials and Methods: Data was collected in paper form and recorded from 100 healthy personals and 100 personals suspected with CVS after take the case history and clinical signs in private clinical hospital and the serum was collected for measurements the activity of ABCG5 and ABCG5 by used ELISA reader and the results illustrated that activity of ABCG5 and ABCG5 in all aged groups. RESULTS: Results: Activity of ABCG5 and ABCG5 in all aged groups periods in patient person male and female significant decrease as compared with same age in same period of live, so that the researched depicted that can used the serum activity of ABCG5 and ABCG5 as a diagnostics tools for atherosclerotic cardiovascular disease. CONCLUSION: Conclusions: We identified areas of further exploration on cholesterol transport related with CVD risk and concluded that changes in the Adenosine Triphosphate Binding Cassette transporters mainly G5 and G8 early diagnostic tools for cardiovascular disease in Human. We correlated areas of farther disquisition on nutrient cholesterol and CVD threat, in the included trials, healthy grown-ups consumed high doses of dietary cholesterol.

Updated mutational spectrum and genotype-phenotype correlations in ichthyosis patients with ABCA12 pathogenic variants.

Autosomal recessive congenital ichthyoses (ARCI) is a genetically heterogeneous condition that can be caused by pathogenic variants in at least 12 genes, including ABCA12. ARCI mainly consists of congenital ichthyosiform erythroderma (CIE), lamellar ichthyosis (LI) and harlequin ichthyosis (HI). The objective was to determine previously unreported pathogenic variants in ABCA12 and to update genotype-phenotype correlations for patients with pathogenic ABCA12 variants. Pathogenic variants in ABCA12 were detected using Sanger sequencing or a combination of Sanger sequencing and whole-exome sequencing. To verify the pathogenicity of a previously unreported large deletion and intron variant, cDNA analysis was performed using total RNA extracted from hair roots. Genetic analyses were performed on the patients with CIE, LI, HI and non-congenital ichthyosis with unusual phenotypes (NIUP), and 11 previously unreported ABCA12 variants were identified. Sequencing of cDNA confirmed the aberrant splicing of the variant ABCA12 in the patients with the previously unreported large deletion and intron variant. Our findings expand the phenotype spectrum of ichthyosis patients with ABCA12 pathogenic variants. The present missense variants in ABCA12 are considered to be heterogenous in pathogenicity, and they lead to varying disease severities in patients with ARCI and non-congenital ichthyosis with unusual phenotypes (NIUP).

Mutations in BrABCG26, encoding an ATP-binding cassette transporter, are responsible for male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

KEY MESSAGE: The gene BrABCG26 responsible for male sterility of Chinese cabbage was confirmed by two allelic mutants. Male-sterile lines are an important way of heterosis utilization in Chinese cabbage. In this study, two allelic male-sterile mutants msm3-1 and msm3-2 were obtained from a Chinese cabbage double haploid (DH) line 'FT' by using EMS-mutagenesis. Compared to the wild-type 'FT,' the stamens of mutants were completely degenerated and had no pollen, and other characters had no obvious differences. Cytological observation revealed that the failure of vacuolation of the mononuclear microspore, accompanied by abnormal tapetal degradation, resulted in anther abortion in mutants. Genetic analysis showed that a recessive gene controlled the mutant trait. MutMap combined with kompetitive allele specific PCR genotyping analyses showed that BraA01g038270.3C, encoding a transporter ABCG26 that played a vital role in pollen wall formation, was the candidate gene for msm3-1, named BrABCG26. Compared with wild-type 'FT,' the mutations existed on the second exon (C to T) and the sixth exon (C to T) of BrABCG26 gene in mutants msm3-1 and msm3-2, leading to the loss-of-function truncated protein, which verified the BrABCG26 function in stamen development. Subcellular localization and expression pattern analysis indicated that BrABCG26 was localized in the nucleus and was expressed in all organs, with the highest expression in flower buds. Compared to the wild-type 'FT,' the expressions of BrABCG26 were significantly reduced in flower buds and anthers of mutants. Promoter activity analysis showed that a strong GUS signal was detected in flower buds. These results indicated that BrABCG26 is responsible for the male sterility of msm3 mutants in Chinese cabbage.

Intravitreal Delivery of PEGylated-ECO Plasmid DNA Nanoparticles for Gene Therapy of Stargardt Disease.

OBJECTIVE: Current gene therapy of inherited retinal diseases is achieved mainly by subretinal injection, which is invasive with severe adverse effects. Intravitreal injection is a minimally invasive alternative for gene therapy of inherited retinal diseases. This work explores the efficacy of intravitreal delivery of PEGylated ECO (a multifunctional pH-sensitive amphiphilic amino lipid) plasmid DNA (pGRK1-ABCA4-S/MAR) nanoparticles (PEG-ELNP) for gene therapy of Stargardt disease. METHODS: Pigmented Abca4-/- knockout mice received 1 µL of PEG-ELNP solution (200 ng/uL, pDNA concentration) by intravitreal injections at an interval of 1.5 months. The expression of ABCA4 in the retina was determined by RT-PCR and immunohistochemistry at 6 months after the second injection. A2E levels in the treated eyes and untreated controls were determined by HPLC. The safety of treatment was monitored by scanning laser ophthalmoscopy and electroretinogram (ERG). RESULTS: PEG-ELNP resulted in significant ABCA4 expression at both mRNA level and protein level at]6 months after 2 intravitreal injections, and a 40% A2E accumulation reduction compared with non-treated controls. The PEG-ELNP also demonstrated excellent safety as shown by scanning laser ophthalmoscopy, and the eye function evaluation from electroretinogram. CONCLUSIONS: Intravitreal delivery of the PEG-ELNP of pGRK1-ABCA4-S/MAR is a promising approach for gene therapy of Stargardt Disease, which can also be a delivery platform for gene therapy of other inherited retinal diseases.

Understanding and Rescuing the Splicing Defect Caused by the Frequent ABCA4 Variant c.4253+43G>A Underlying Stargardt Disease.

Pathogenic variants in ABCA4 are the underlying molecular cause of Stargardt disease (STGD1), an autosomal recessive macular dystrophy characterized by a progressive loss of central vision. Among intronic ABCA4 variants, c.4253+43G>A is frequently detected in STGD1 cases and is classified as a hypomorphic allele, generally associated with late-onset cases. This variant was previously reported to alter splicing regulatory sequences, but the splicing outcome is not fully understood yet. In this study, we attempted to better understand its effect on splicing and to rescue the aberrant splicing via antisense oligonucleotides (AONs). Wild-type and c.4253+43G>A variant-harboring maxigene vectors revealed additional skipping events, which were not previously detected upon transfection in HEK293T cells. To restore exon inclusion, we designed a set of 27 AONs targeting either splicing silencer motifs or the variant region and screened these in maxigene-transfected HEK293T cells. Candidate AONs able to promote exon inclusion were selected for further testing in patient-derived photoreceptor precursor cells. Surprisingly, no robust splicing modulation was observed in this model system. Overall, this research helped to adequately characterize the splicing alteration caused by the c.4253+43G>A variant, although future development of AON-mediated exon inclusion therapy for ABCA4 is needed.

Assimilation of phthalate esters in bacteria.

The massive usage of phthalate esters (PAEs) has caused serious pollution. Bacterial degradation is a potential strategy to remove PAE contamination. So far, an increasing number of PAE-degrading strains have been isolated, and the catabolism of PAEs has been extensively studied and reviewed. However, the investigation into the bacterial PAE uptake process has received limited attention and remains preliminary. PAEs can interact spontaneously with compounds like peptidoglycan, lipopolysaccharides, and lipids on the bacterial cell envelope to migrate inside. However, this process compromises the structural integrity of the cells and causes disruptions. Thus, membrane protein-facilitated transport seems to be the main assimilation strategy in bacteria. So far, only an ATP-binding-cassette transporter PatDABC was proven to transport PAEs across the cytomembrane in a Gram-positive bacterium Rhodococcus jostii RHA1. Other cytomembrane proteins like major facilitator superfamily (MFS) proteins and outer membrane proteins in cell walls like FadL family channels, TonB-dependent transporters, and OmpW family proteins were only reported to facilitate the transport of PAEs analogs such as monoaromatic and polyaromatic hydrocarbons. The functions of these proteins in the intracellular transport of PAEs in bacteria await characterization and it is a promising avenue for future research on enhancing bacterial degradation of PAEs. KEY POINTS: • Membrane proteins on the bacterial cell envelope may be PAE transporters. • Most potential transporters need experimental validation.

Erythrokeratodermia Variabilis-like Phenotype in Patients Carrying ABCA12 Mutations.

Erythrokeratodermia variabilis (EKV) is a rare genodermatosis characterized by well-demarcated erythematous patches and hyperkeratotic plaques. EKV is most often transmitted in an autosomal dominant manner. Until recently, only mutations in connexins such as GJB3 (connexin 31), GJB4 (connexin 30.3), and occasionally GJA1 (connexin 43) were known to cause EKV. In recent years, mutations in other genes have been described as rare causes of EKV, including the genes KDSR, KRT83, and TRPM4. Features of the EKV phenotype can also appear with other genodermatoses: for example, in Netherton syndrome, which hampers correct diagnosis. However, in autosomal recessive congenital ichthyosis (ARCI), an EKV phenotype has rarely been described. Here, we report on seven patients who clinically show a clear EKV phenotype, but in whom molecular genetic analysis revealed biallelic mutations in ABCA12, which is why the patients are classified in the ARCI group. Our study indicates that ARCI should be considered as a differential diagnosis in EKV.

Cry Toxins Use Multiple ATP-Binding Cassette Transporter Subfamily C Members as Low-Efficiency Receptors in Bombyx mori.

Recent studies have suggested that ABC transporters are the main receptors of Cry toxins. However, the receptors of many Cry toxins have not been identified. In this study, we used a heterologous cell expression system to identify Bombyx mori ABC transporter subfamily C members (BmABCCs) that function as receptors for five Cry toxins active in Lepidopteran insects: Cry1Aa, Cry1Ca, Cry1Da, Cry8Ca, and Cry9Aa. All five Cry toxins can use multiple ABCCs as low-efficiency receptors, which induce cytotoxicity only at high concentrations. Surface plasmon resonance analysis revealed that the KD values between the toxins and BmABCC1 and BmABCC4 were 10-5 to 10-9 M, suggesting binding affinities 8- to 10,000-fold lower than those between Cry1Aa and BmABCC2, which are susceptibility-determining receptors for Cry1Aa. Bioassays in BmABCC-knockout silkworm strains showed that these low-efficiency receptors are not involved in sensitivity to Cry toxins. The findings suggest that each family of Cry toxins uses multiple BmABCCs as low-efficiency receptors in the insect midgut based on the promiscuous binding of their receptor-binding regions. Each Cry toxin seems to have evolved to utilize one or several ABC transporters as susceptibility-determining receptors.

Mechanistic Insights Revealed by YbtPQ in the Occluded State.

Recently, several ATP-binding cassette (ABC) importers have been found to adopt the typical fold of type IV ABC exporters. Presumably, these importers would function under the transport scheme of "alternating access" like those exporters, cycling through inward-open, occluded, and outward-open conformations. Understanding how the exporter-like importers move substrates in the opposite direction requires structural studies on all the major conformations. To shed light on this, here we report the structure of yersiniabactin importer YbtPQ from uropathogenic Escherichia coli in the occluded conformation trapped by ADP-vanadate (ADP-Vi) at a 3.1 Å resolution determined by cryo-electron microscopy. The structure shows unusual local rearrangements in multiple helices and loops in its transmembrane domains (TMDs). In addition, the dimerization of the nucleotide-binding domains (NBDs) promoted by the vanadate trapping is highlighted by the "screwdriver" action at one of the two hinge points. These structural observations are rare and thus provide valuable information to understand the structural plasticity of the exporter-like ABC importers.

Towards Uncovering the Role of Incomplete Penetrance in Maculopathies through Sequencing of 105 Disease-Associated Genes.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Evaluation of the Copy Number Variants and Single-Nucleotide Polymorphisms of ABCA3 in Newborns with Respiratory Distress Syndrome-A Pilot Study.

Background and Objectives: Respiratory distress syndrome (RDS) in preterm infants commonly occurs due to the immaturity-related deficiency of pulmonary surfactant. Beyond prematurity, various environmental and genetic factors can influence the onset and progression of RDS. This study aimed to analyze three single-nucleotide polymorphisms (SNPs) of the ABCA3 gene to assess the ABCA3 gene as a candidate gene for susceptibility to RDS and overall survival in newborns and to evaluate the utility of MLPA in RDS neonatal patients. Materials and Methods: Three SNPs were chosen and genotyped in a cohort of 304 newborns. Data analysis and statistical tests were employed to examine allele frequencies, haplotypes, and measures of pairwise linkage disequilibrium. Results: There was no observed haplotype association with SNPs rs13332514 (c.1059G>A) and rs170447 (c.1741+33T>C) among newborns, both with and without RDS (p > 0.05). The minor C allele frequency of the ABCA3 rs323043 (c.1755G>C) SNP showed a significant increase in preterm infants with RDS. MLPA results indicated that the predominant findings were normal, revealing no CNVs in the genes ABCA3 and SFTPC that were investigated in our patients. Conclusions: The presence of the variant C allele in the rs323043 (c.1755G>C) SNP may be a risk factor for RDS in premature newborns.

Zosuquidar: An Effective Molecule for Intracellular Ca2+ Measurement in P-gp Positive Cells.

Intracellular calcium, as a second messenger, is involved in multilevel cellular regulatory pathways and plays a role (among other processes) in switching between survival and initiation of cell death in neoplastic cells. The development of multidrug resistance (MDR) in neoplastic cells is associated with the ability of cells to escape programmed cell death, in which dysregulation of intracellular calcium may play an important role. Therefore, reliable monitoring of intracellular calcium levels is necessary. However, such a role might be limited by a real obstacle since several fluorescent intracellular calcium indicators are substrates of membrane ABC drug transporters. For example, Fluo-3/AM is a substrate of P-glycoprotein (ABCB1 member of the ABC family), whose overexpression is the most frequent cause of MDR. The overexpression of ABCB1 prevents MDR cell variants from retaining this tracer in the intracellular space where it is supposed to detect calcium. The solution is to use a proper inhibitor of P-gp efflux activity to ensure the retention of the tracer inside the cells. The present study showed that Zosuquidar and Tariquidar (P-gp inhibitors) are suitable for monitoring intracellular calcium, either by flow cytometry or confocal microscopy, in cells overexpressing P-gp.

A systems framework for investigating the roles of multiple transporters and their impact on drug resistance.

Efflux transporters are a fundamental component of both prokaryotic and eukaryotic cells, play a crucial role in maintaining cellular homeostasis, and represent a key bridge between single cell and population levels. From a biomedical perspective, they play a crucial role in drug resistance (and especially multi-drug resistance, MDR) in a range of systems spanning bacteria and human cancer cells. Typically, multiple efflux transporters are present in these cells, and the efflux transporters transport a range of substrates (with partially overlapping substrates between transporters). Furthermore, in the context of drug resistance, the levels of transporters may be elevated either due to extra or intracellular factors (feedforward regulation) or due to the drug itself (feedback regulation). As a consequence, there is a real need for a transparent systems-level understanding of the collective functioning of a set of transporters and their response to one or more drugs. We develop a systems framework for this purpose and examine the functioning of sets of transporters, their interplay with one or more drugs and their regulation (both feedforward and feedback). Using computational and analytical work, we obtain transparent insights into the systems level functioning of a set of transporters arising from the interplay between the multiplicity of drugs and transporters, different drug-transporter interaction parameters, sequestration and feedback and feedforward regulation. These insights transparently arising from the most basic consideration of a multiplicity of transporters have broad relevance in natural biology, biomedical engineering and synthetic biology. Insight, Innovation, Integration: Innovation: creating a structured systems framework for evaluating the impact of multiple transporters on drug efflux and drug resistance. Systematic analysis allows us to evaluate the effect of multiple transporters on one/more drugs, and dissect associated resistance mechanisms. Integration allows for elucidation of key cause-and-effect relationships and a transparent systems-level understanding of the collective functioning of transporters and their impact on resistance, revealing the interplay of key underlying factors. Systems-level insights include the essentially different behaviour of transporters as part of a group; unintuitive effects of influx; effects of elevated transporter-levels by feedforward and drug-induced mechanisms. Relevance: a systems understanding of efflux, their role in MDR, providing a framework/platform for use in designing treatment, and in synthetic biology design.