Characterization of an archaeal multidrug transporter with a unique amino acid composition

The Smr family of multidrug transporters consists of small membrane proteins that extrude various drugs in exchange with protons rendering cells resistant to these drugs. Smr proteins identified to date have been found only in Eubacteria. In this work we present the cloning and characterization of an Smr protein from the archaeon Halobacterium salinarum, the first Smr in the archaeal kingdom. The protein, named Hsmr, was identified through sequence similarity to the Smr family, and the DNA sequence was cloned into an Escherichia coli expression system. Hsmr is heterologously expressed in a functional form despite the difference in lipid composition of the membrane and the lower salt in the cell and its environment. Cells harboring the Hsmr plasmid transport ethidium bromide in an uncoupler-sensitive process and gain resistance to ethidium bromide and acriflavine. Hsmr binds tetraphenylphosphonium (TPP(+)) with a relatively low affinity (K(D) approximately 200 nm) at low salt concentration that increases (K(D) approximately 40 nm) upon the addition of 2 m of either NaCl or KCl. The Hsmr protein contains many of the signature sequence elements of the Smr family and also a high content of negative residues in the loops, characteristic of extreme halophiles. Strikingly, Hsmr is composed of over 40% valine and alanine residues. These residues are clustered at certain regions of the protein in domains that are not important for activity, as judged from lack of conservation and from previous studies with other Smr proteins. We suggest that this high content of alanine and valine residues is a reflection of a "natural" alanine and valine scanning necessitated by the high GC content of the gene. This phenomenon reveals significant sequence elements in small multidrug transporters.     

Suppressor of cytokine signaling-2 modulates the fibrogenic actions of GH and IGF-I in intestinal mesenchymal cells

Growth hormone (GH) and IGF-I play important roles in wound healing during intestinal injury and inflammation, but there is also indirect evidence that locally expressed IGF-I may act to induce excessive collagen deposition, which can lead to intestinal fibrosis. Factors that dictate the balance between normal wound healing and excessive healing responses are unknown. Using RNase protection assay and in situ hybridization, we determined whether GH and/or IGF-I increase type I collagen deposition in the intestine of rats fed by total parenteral nutrition (TPN), a feeding modality used for many patients following intestinal surgery and resection. We also used an in vitro model system to confirm our in vivo effects and to directly evaluate the relative potency of GH and IGF-I on DNA synthesis and collagen deposition in intestinal myofibroblasts. Both GH and IGF-I stimulated collagen production in vivo and in vitro, and IGF-I, but not GH, stimulated DNA synthesis in vitro. In collagen production, GH was less potent than IGF-I. Suppressors of cytokine signaling (SOC) are cytokine-inducible proteins that negatively feedback to inhibit the actions of cytokines and we recently found that GH selectively upregulates SOC-2 in the intestine of TPN-fed rats. We examined whether SOC-2 may be responsible for the difference in magnitude of action of GH and IGF-I on collagen accumulation. GH, but not IGF-I, induced SOC-2 in isolated myofibroblasts, and overexpression of SOC-2 led to a suppression of GH- and IGF-I-induced collagen accumulation. SOC-2 null mice infused with IGF-I showed greater collagen gene expression compared with wild-type (WT) mice. Myofibroblasts isolated from SOC-2 null mice showed increased IGF-I-stimulated DNA synthesis compared with WT cells. Taken together, these findings suggest that SOC-2 induced by GH may play an important role in suppressing collagen accumulation and mesenchymal cell proliferation induced by GH or GH-induced IGF-I, providing a mechanism for the differing potencies of GH and IGF-I on intestinal mesenchyme and collagen synthesis.     

Generation and analysis of a protein-protein interface data set with similar chemical and spatial patterns of interactions

Protein-protein interfaces are regions between 2 polypeptide chains that are not covalently connected. Here, we have created a nonredundant interface data set generated from all 2-chain interfaces in the Protein Data Bank. This data set is unique, since it contains clusters of interfaces with similar shapes and spatial organization of chemical functional groups. The data set allows statistical investigation of similar interfaces, as well as the identification and analysis of the chemical forces that account for the protein-protein associations. Toward this goal, we have developed I2I-SiteEngine (Interface-to-Interface SiteEngine) [Data set available at http://bioinfo3d.cs.tau.ac.il/Interfaces; Web server: http://bioinfo3d.cs.tau.ac.il/I2I-SiteEngine]. The algorithm recognizes similarities between protein-protein binding surfaces. I2I-SiteEngine is independent of the sequence or the fold of the proteins that comprise the interfaces. In addition to geometry, the method takes into account both the backbone and the side-chain physicochemical properties of the interacting atom groups. Its high efficiency makes it suitable for large-scale database searches and classifications. Below, we briefly describe the I2I-SiteEngine method. We focus on the classification process and the obtained nonredundant protein-protein interface data set. In particular, we analyze the biological significance of the clusters and present examples which illustrate that given constellations of chemical groups in protein-protein binding sites may be preferred, and are observed in proteins with different structures and different functions. We expect that these would yield further information regarding the forces stabilizing protein-protein interactions.     

Cloning and functional analysis of the rhesus macaque ABCG2 gene. Forced expression confers an SP phenotype among hematopoietic stem cell progeny in vivo

Hematopoietic cells can be highly enriched for repopulating ability based upon the efflux of the fluorescent Hoechst 33342 dye by sorting for SP (side population) cells, a phenotype attributed to expression of ABCG2, a member of the ABC transporter superfamily. Intriguingly, murine studies suggest that forced ABCG2 expression prevents hematopoietic differentiation. We cloned the full-length rhesus ABCG2 and introduced it into a retroviral vector. ABCG2-transduced human peripheral blood progenitor cells (PBPCs) acquired the SP phenotype but showed significantly reduced growth compared with control. Two rhesus macaques received autologous PBPCs split for transduction with the ABCG2 or control vectors. Marking levels were similar between fractions with no discrepancy between bone marrow and peripheral blood marking. Analysis for the SP phenotype among bone marrow and mature blood populations confirmed ABCG2 expression at levels predicted by vector copy number long term, demonstrating no block to differentiation in the large animal. In vitro studies showed selective protection against mitoxantrone among ABCG2-transduced rhesus PBPCs. Our results confirm the existence of rhesus ABCG2, establish its importance in conferring the SP phenotype, suggest no detrimental effect of its overexpression upon differentiation in vivo, and imply a potential role for its overexpression as an in vivo selection strategy for gene therapy applications.     

Successful cyclosporine treatment for atopic dermatitis in a rhesus macaque (Macaca mulatta)

A juvenile (1 year old ) female rhesus macaque (Macaca mulatta) developed a chronic active skin condition characterized by pruritus, erythema, alopecia, scaling, exfoliation, and lichenification. Lesions were limited to the ventrum, specifically rostral mandible and neck, axilla and inguinal regions, distal extremities, and interdigital regions. Differential diagnoses included infection, dietary deficiency, metabolic abnormality, endocrinopathy, and immunological injury. Diagnostic tests included complete hemogram, serum chemistry, skin scrapes for ectoparasite detection, hair plucks for dermatophyte culture, and a serum-based hypersensitivity panel. All results were within normal limits. Dermal biopsies revealed lesions consistent with active allergic dermatitis, and a diagnosis of atopic dermatitis was made. Oral cyclosporine (5 mg/kg daily) rapidly eliminated clinical evidence of dermatitis. Histologically, lesions resolved after 12 months of treatment. Atopic dermatitis is an inflammatory skin condition for which there are neither pathognomonic clinical or diagnostic features nor a single successful therapy. Basic criteria such as pruritus, lichenification, a chronic course, and history of allergies strongly support the diagnosis. One successful therapeutic agent is a macrolide calcineurin inhibitor, cyclosporine. It represents a safer class of immunomodulatory drugs than corticosteroids and provides targeted alteration of lymphocyte function. To our knowledge this case represents the first reported successful treatment of atopic dermatitis in a nonhuman primate utilizing cyclosporine.     

Listeria monocytogenes Multidrug Resistance Transporters and Cyclic Di-AMP,Which Contribute to Type I Interferon Induction,Play a Role in Cell Wall Stress

The intracellular bacterial pathogen Listeria monocytogenes activates a robust type I interferon response upon infection. This response is partially dependent on the multidrug resistance (MDR) transporter MdrM and relies on cyclic-di-AMP (c-di-AMP) secretion, yet the functions of MdrM and cyclic-di-AMP that lead to this response are unknown. Here we report that it is not MdrM alone but a cohort of MDR transporters that together contribute to type I interferon induction during infection. In a search for a physiological function of these transporters, we revealed that they play a role in cell wall stress responses. A mutant with deletion of four transporter genes (ΔmdrMTAC) was found to be sensitive to sublethal concentrations of vancomycin due to an inability to produce and shed peptidoglycan under this stress. Remarkably, c-di-AMP is involved in this phenotype, as overexpression of the c-di-AMP phosphodiesterase (PdeA) resulted in increased susceptibility of the ΔmdrMTAC mutant to vancomycin, whereas overexpression of the c-di-AMP diadenylate cyclase (DacA) reduced susceptibility to this drug. These observations suggest a physiological association between c-di-AMP and the MDR transporters and support the model that MDR transporters mediate c-di-AMP secretion to regulate peptidoglycan synthesis in response to cell wall stress.     

Regulation of cancer aggressive features in melanoma cells by microRNAs

MicroRNAs (miRNAs) are small non-coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer. A common strategy for identification of miRNAs involved in cell transformation is to compare malignant cells to normal cells. Here we focus on identification of miRNAs that regulate the aggressive phenotype of melanoma cells. To avoid differences due to genetic background, a comparative high-throughput miRNA profiling was performed on two isogenic human melanoma cell lines that display major differences in their net proliferation, invasion and tube formation activities. This screening revealed two major cohorts of differentially expressed miRNAs. We speculated that miRNAs up-regulated in the more-aggressive cell line contribute oncogenic features, while the down-regulated miRNAs are tumor suppressive. This assumption was further tested experimentally on five candidate tumor suppressive miRNAs (miR-31, -34a, -184, -185 and -204) and on one candidate oncogenic miRNA (miR-17-5p), all of which have never been reported before in cutaneous melanoma. Remarkably, all candidate Suppressive-miRNAs inhibited net proliferation, invasion or tube formation, while miR-17-5p enhanced cell proliferation. miR-34a and miR-185 were further shown to inhibit the growth of melanoma xenografts when implanted in SCID-NOD mice. Finally, all six candidate miRNAs were detected in 15 different metastatic melanoma specimens, attesting for the physiological relevance of our findings. Collectively, these findings may prove instrumental for understanding mechanisms of disease and for development of novel therapeutic and staging technologies for melanoma.     

Differential genetic susceptibility to child risk at birth in predicting observed maternal behavior

This study examined parenting as a function of child medical risks at birth and parental genotype (dopamine D4 receptor; DRD4). Our hypothesis was that the relation between child risks and later maternal sensitivity would depend on the presence/absence of a genetic variant in the mothers, thus revealing a gene by environment interaction (GXE). Risk at birth was defined by combining risk indices of children's gestational age at birth, birth weight, and admission to the neonatal intensive care unit. The DRD4-III 7-repeat allele was chosen as a relevant genotype as it was recently shown to moderate the effect of environmental stress on parental sensitivity. Mothers of 104 twin pairs provided DNA samples and were observed with their children in a laboratory play session when the children were 3.5 years old. Results indicate that higher levels of risk at birth were associated with less sensitive parenting only among mothers carrying the 7-repeat allele, but not among mothers carrying shorter alleles. Moreover, mothers who are carriers of the 7-repeat allele and whose children scored low on the risk index were observed to have the highest levels of sensitivity. These findings provide evidence for the interactive effects of genes and environment (in this study, children born at higher risk) on parenting, and are consistent with a genetic differential susceptibility model of parenting by demonstrating that some parents are inherently more susceptible to environmental influences, both good and bad, than are others.