Bovine RNase MRP cleaves the divergent bovine mitochondrial RNA sequence at the displacement-loop region

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves mitochondrial RNA from the origin of leading-strand DNA synthesis contained within the displacement-loop region. Bovine mitochondrial DNA maintains the typical gene content and order of mammalian mitochondrial DNAs but differs in the nature of sequence conservation within this displacement-loop regulatory region. This markedly different sequence arrangement raises the issue of the degree to which a bovine RNase MRP would reflect the physical and functional properties ascribed to the enzymes previously characterized from mouse and human. We find that bovine RNase MRP exists as a ribonucleoprotein, with an RNA component of 279 nucleotides that is homologous to that of mouse or human RNase MRP RNA. Characterization of the nuclear gene for bovine RNase MRP RNA showed conservation of sequence extending 5 of the RNase MRP RNA coding sequence, including the presence of a cis-acting element known to be important for the expression of some mitochondrial protein-coding nuclear genes. Bovine or mouse RNase MRP cleaves a standard mouse mitochondrial RNA substrate in the same manner; each also cleaves a bovine mitochondrial RNA substrate identically. Since bovine and mouse RNase MRPs process both bovine and mouse substrates, we conclude that the structural features of the mitochondrial RNA substrate required for enzymatic cleavage have been well conserved despite significant overall primary sequence divergence. Inspection of the bovine RNA substrate reveals conservation of only the most critical portion of the primary sequence as indicated by earlier studies with mouse and human RNase MRPs. Interestingly, a principal cleavage site in the bovine mitochondrial RNA substrate is downstream of the promoter located at the leading-strand mitochondrial DNA replication origin. Correspondence to: D.J. Dairaghi     

Cyclohexyl-linked tricyclic isoxazoles are potent and selective modulators of the multidrug resistance protein (MRP1)

Structure–activity relationship (SAR) studies on the tricyclic isoxazole series of MRP1 modulators have resulted in the identification of potent and selective inhibitors containing cyclohexyl-based linkers. These studies ultimately identified compound 21b, which reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC₅₀ = 0.093 μM), while showing no inherent cytotoxicity. Additionally, 21b inhibits ATP-dependent, MRP1-mediated LTC₄ uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC₅₀ = 0.064 μM) and shows selectivity (1115-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, 21b showed tumor regression and growth delay in MRP1-overexpressing tumors in vivo.     

Misregulation of membrane trafficking processes in human nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) remodels the expression and function of genes and proteins that are critical for drug disposition. This study sought to determine whether disruption of membrane protein trafficking pathways in human NASH contributes to altered localization of multidrug resistance‐associated protein 2 (MRP2). A comprehensive immunoblot analysis assessed the phosphorylation, membrane translocation, and expression of transporter membrane insertion regulators, including several protein kinases (PK), radixin, MARCKS, and Rab11. Radixin exhibited a decreased phosphorylation and total expression, whereas Rab11 had an increased membrane localization. PKCδ, PKCα, and PKA had increased membrane activation, whereas PKCε had a decreased phosphorylation and membrane expression. Radixin dephosphorylation may activate MRP2 membrane retrieval in NASH; however, the activation of Rab11/PKCδ and PKA/PKCα suggest an activation of membrane insertion pathways as well. Overall these data suggest an altered regulation of protein trafficking in human NASH, although other processes may be involved in the regulation of MRP2 localization.     

The ABC gene family in arthropods: Comparative genomics and role in insecticide transport and resistance

About a 100 years ago, the Drosophila white mutant marked the birth of Drosophila genetics. The white gene turned out to encode the first well studied ABC transporter in arthropods. The ABC gene family is now recognized as one of the largest transporter families in all kingdoms of life. The majority of ABC proteins function as primary-active transporters that bind and hydrolyze ATP while transporting a large diversity of substrates across lipid membranes. Although extremely well studied in vertebrates for their role in drug resistance, less is known about the role of this family in the transport of endogenous and exogenous substances in arthropods. The ABC families of five insect species, a crustacean and a chelicerate have been annotated in some detail. We conducted a thorough phylogenetic analysis of the seven arthropod and human ABC protein subfamilies, to infer orthologous relationships that might suggest conserved function. Most orthologous relationships were found in the ABCB half transporter, ABCD, ABCE and ABCF subfamilies, but specific expansions within species and lineages are frequently observed and discussed. We next surveyed the role of ABC transporters in the transport of xenobiotics/plant allelochemicals and their involvement in insecticide resistance. The involvement of ABC transporters in xenobiotic resistance in arthropods is historically not well documented, but an increasing number of studies using unbiased differential gene expression analysis now points to their importance. We give an overview of methods that can be used to link ABC transporters to resistance. ABC proteins have also recently been implicated in the mode of action and resistance to Bt toxins in Lepidoptera. Given the enormous interest in Bt toxicology in transgenic crops, such findings will provide an impetus to further reveal the role of ABC transporters in arthropods.     

Therapeutic options for triple-negative breast cancers with defective homologous recombination

Breast cancer is the most common malignancy among women in developed countries, affecting more than a million women per year worldwide. Over the last decades, our increasing understanding of breast cancer biology has led to the development of endocrine agents against hormone receptor-positive tumors and targeted therapeutics against HER2-expressing tumors. However, no targeted therapy is available for patients with triple-negative breast cancer, lacking expression of hormone receptors and HER2. Overlap between BRCA1-mutated breast cancers and triple-negative tumors suggests that an important part of the triple-negative tumors may respond to therapeutics targeting BRCA1-deficient cells. Here, we review the features shared between triple-negative, basal-like and BRCA1-related breast cancers. We also discuss the development of novel therapeutic strategies to target BRCA1-mutated tumors and triple-negative tumors with BRCA1-like features. Finally, we highlight the utility of mouse models for BRCA1-mutated breast cancer to optimize (combination) therapy and to understand drug resistance.     

Deconstructing breast cancer cell biology and the mechanisms of multidrug resistance

Cancer complexity constantly challenges the way that clinicians manage breast cancer therapy. Tumor heterogeneity and intratumoral stroma characteristics allow cells with different phenotypes and deregulated apoptotic, proliferative and migration abilities to co-exist contributing to a disappointing therapeutic response. While new approaches are being associated with conventional chemotherapy, such as hormonal therapy or target monoclonal antibodies, recurrence and metastasization are still observed. Membrane transporters are the cell's first line of contact with anticancer drugs having a major role in multidrug resistance events. This structural-based activity enables the cell to be drug-resistant by decreasing drug intracellular concentration through an efflux-transport mechanism, mainly associated with overexpression of ATP-binding cassette (ABC) proteins. This review focuses on some of the important structural and biological properties of the malignant cell and tumor microenvironment, addressing the role of the membrane ABC transporters in therapeutic outcomes, and highlighting related molecular pathways that may represent meaningful target therapies.     

Involvement of NHERF1 in apical membrane localization of MRP4 in polarized kidney cells

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette protein superfamily, confers resistance to nucleoside and nucleotide analogs as well as camptothecin derivatives. MRP4 also mediates the efflux of certain cyclic nucleotides, eicosanoids, conjugated steroids, and uric acid. Depending on the cell type, MRP4 may localize to either apical or basolateral membranes in polarized cells. The adaptor protein NHERF1 has previously been implicated in MRP4 internalization in non-polarized cells. We have now found that NHERF1 levels are very low in polarized MDCKI cells which express MRP4 on basolateral membranes relative to polarized LLC-PK1 cells which express MRP4 on apical membranes. Furthermore, ectopic expression of FLAG-tagged NHERF1 in MDCKI cells and in MDCKI cells stably expressing eGFP-tagged MRP4 causes endogenous MRP4 and eGFP-MRP4, respectively, to traffic to the apical membranes. These data establish NHERF1 as a major determinant of MRP4 trafficking to apical membranes of mammalian kidney cells.